Advances in Experimental Medicine and Biology Volume 705
Editorial Board: IRUN R. COHEN, The Weizmann Institute of Science ABEL LAJTHA, N. S. Kline Institute for Psychiatric Research JOHN D. LAMBRIS, University of Pennsylvania RODOLFO PAOLETTI, University of Milan
For other titles published in this series, go to www.springer.com/series/5584
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Albert M. Wu Editor
The Molecular Immunology of Complex Carbohydrates-3
Editor Albert M. Wu Glyco-Immunochemistry Research Laboratory Institute of Molecular and Cellular Biology College of Medicine Chang-Gung University Kwei-Shan, Tao-Yuan 333 Taiwan
[email protected] ISSN 0065-2598 ISBN 978-1-4419-7876-9 e-ISBN 978-1-4419-7877-6 DOI 10.1007/978-1-4419-7877-6 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011926797 © Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)
Winifred M. Watkins August 1924-October 2003 On 3 October 2003, Winifred Watkins died, and even now, I can remember the sadness I felt when I heard the news. Three weeks before her death, I met her at a meeting in Cambridge organised by the Lister Institute in honour of Walter Morgan, who had died earlier in the year. Despite being wheelchair-bound following an earlier stroke, Winifred was still enjoying talking about science. Winifred was not well enough to give her own tribute to Walter but did correct others who strayed from the facts. During lunch, I had the feeling she was saying goodbye to those of us who had worked with her and Walter. I remember laughing as she persuaded Marcela Contreras to push her wheelchair back to the car by the shortest but, for Marcela, most dangerous route, given the height of Marcela’s stilettos! That was the last time I saw Winifred. News of Winifred’s death, a few weeks later, did not come as a surprise. In many ways, Walter and Winifred were an inseparable scientific team, and their passing within months of each other brought to the end a golden era of research into carbohydrate blood-group antigen structure. Winifred Watkins was born in London, on 6 August 1924, the younger daughter of a process engraver who was an accomplished amateur painter. She began her schooling in London and, in 1935, was awarded a scholarship at Godolphin and Latymer School, where she was able to pursue her studies of the sciences. During the war, the school was evacuated to the Berkshire. Newbury was a boys’ school that had teaching laboratories in which Winifred gained permission to study sciences alongside her male counterparts. In 1940, she returned to London and graduated in 1942 with her Higher School Certificate. The war precluded her entry to university, and she joined the Biochemistry Department of the Lister Institute in Chelsea Bridge Road, London, as a technician. At the time, the Lister was a “hot bed” of scientific ideas and research, and this stimulated her to study at Chelsea College in the evenings to gain her honours degree in chemistry in 1947. During this time, she worked with Walter Morgan on blood-group substances, and in 1944 they produced enough data for her first publication. This seems to have been a
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contentious event as she had no degree and was required to have written permission from the directors of the Lister Institute before the paper was accepted. Having learnt from her own earlier experiences, she encouraged her younger technicians to undertake research projects and publish papers, realising the benefit this can bring; many went on to gain their own Ph.D.s. Indeed, my first-ever publication with Winifred was whilst I was a sandwich student, a year before I gained my own undergraduate degree. In 1947, she began her post-graduate studies at St. Bartholomew’s Hospital Medical School, London, with Arthur Wormall, a renowned immunochemist. She studied the action of nitrogen mustards on the immunological properties of proteins. In 1950, she returned as a post-doctoral fellow to work with Walter Morgan at the Lister Institute, and this was the start of their lifelong scientific partnership. Between 1957 and 1958, she and Walter uncovered the relationships between the H antigen and the ABO system and, using serological and biochemical techniques, described the structure of the ABH and Lewis blood-group antigens. In the late 1950s, Walter and Winifred speculated on the mechanisms involved in the biosynthesis of the blood-group antigens, and this led to the proposal that the products of the ABO, H, and Le genes were glycosyltransferases that transferred terminal sugars from nucleotide sugar donors onto growing oligosaccharide chains. In 1960, Winifred was awarded a Henry Wellcome Travel Fellowship and spent a sabbatical year in Zev Hassid’s laboratory at the University of California, Berkeley, where pioneering work on glycosyltransferases was underway. During that time, she was involved with work leading to the discovery of the enzyme lactose synthetase. In 1966, Winifred proposed, without experimental evidence, that glycosyltransferases were responsible for the production of A, B, H, and Le antigens. The pathways predicted helped explain the variation in H antigen levels in individuals with A, B, AB, and O blood types and predicted that individuals from Bombay would lack the α-2-fucosyltransferase product of the H gene. The scheme also explained why secretors of H substance who possessed the Le gene would always produce Lebb, whereas non-secretors would produce Leaa. Over the next 5 years in Winifred’s laboratory and in Vic Ginsburg’s laboratory in the USA, the appropriate enzymes were found to prove the hypothesis, and by the early 1970s, glycosyltransferase assays were carried out routinely within laboratories. This allowed analyses of rare blood groups and chimeras, tissue distribution studies, and the onco-developmental nature of the antigens. This period was the heyday of the Lister Institute, which was also home to Rob Race, Ruth Sanger, Pat Tippett, and Marcela Contreras—with whom Winifred had many lively discussions and for whom she had an enduring respect. In 1975, the Lister Institute closed, and after much discussion, Winifred moved with her group to the MRC Clinical Research Centre in Harrow, where she was the only non-medically qualified departmental head, to lead the new Division of Immunochemical Genetics. Their work on glycosyltransferases continued, focusing on localisation, characterisation, purification, and, finally, gene cloning. The underlying causes of blood group ABO, H, and Le anomalies and weak subgroups
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were unravelled. Support for the allelic basis of the A and B genes was found when the group demonstrated the overlapping specificity of the A and B transferases. Work on fucosyltransferases supported the idea put forward by Rafael Oriol that H and Se genes were responsible for the production of H antigen on red cells and in secretions, respectively, and demonstrated relationships between some but not all fucosyltransferases. Further research demonstrated specificities of a range of fucosyltransferases and highlighted their roles in leukaemias. Following the production of polyclonal and monoclonal antibodies against the purified A transferase and the fucosyltransferases, the work moved into molecular biology, using polyclonal antibodies to screen expression libraries. In 1984, Winifred’s 60th birthday was celebrated with a Biochemical Society meeting in London, which many of her colleagues and friends attended. However, following a very positive quinquennial review, the decision was made to close the Division of Immunochemical Genetics when Winifred retired from the MRC in 1989. The work was curtailed, and the group disbanded. The closure of the Division at the MRC was a bitter blow, particularly as it followed such an excellent review of the research. I am sure she deeply regretted that the work into blood-group antigen biosynthesis did not continue in the UK. Winifred then moved to the Hammersmith Hospital, where she was able to follow up work on fucosyltransferase and sialyltransferase expression in normal white cell maturation and leukaemia. This work continued until the millennium, when Winifred stopped laboratory based research and concentrated on more academic work, until a stroke prevented her from continuing. During her lifetime, the impact she made in glycobiology and transfusion science can be demonstrated by the awards she received: In 1965, she was awarded the Oliver Memorial Fund award for her work in transfusion science; in 1967, she received the Karl Landsteiner Award of the American Association of Blood Banks; and in 1969, the Paul Ehrlich-Ludwig Darmstadter Medal and Prize. In the same year, she was elected to the Fellowship of the Royal Society of London, and in 1970, she was awarded the William Julius Mickle Fellowship at the University of London. She was presented with the Kenneth Goldsmith Award of the British Blood Transfusion Society in 1986, the Royal Medal of the Royal Society in 1988, the Franz Oehleckler Medal of the German Society of Transfusion Medicine and Immunohaematology in 1989, and the Phillip Levine Award of the American Society of Chemical Pathologists in 1990. She also received an Honorary DSc in chemistry from the University of Utrecht in 1990. She was elected as a member or a fellow to the Royal Society of London (Fellow, 1969), The Royal College of Pathologists (Fellow, 1983), the International Society of Blood Transfusion (Honorary Member, 1984), the Polish Academy of Sciences (Foreign Member, 1988), The Royal College of Physicians (Fellow, 1990), the Japanese Biochemical Society (Honorary Member, 1990), the British Blood Transfusion Society (Honorary Member, 1996), The Royal Swedish Academy of Sciences (Member, 1988), the Academy of Medical Sciences (Fellow, 1998), and the Biochemical Society (Honorary Member, 2000).
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On a personal level, Winifred expected absolute commitment to work, and she was both supportive and protective of her staff. I well remember how, when I was a very young doctoral student on my first trip to Canada, she paid for my lunch at the Hilton for the whole meeting to make sure that I wasn’t short of money and made sure to keep a “motherly eye” on me. Twenty years later, when her laboratory was closing, she called me over and gave me equipment and materials “knowing I would make good use of them”. Winifred had a love of both good food and wine. When she died, I helped clear her flat and found a collection of labels from wine bottles neatly inscribed with the occasion and date with a comment about both the quality of the wine (and the food) and the occasion. These included labels from the meeting when she heard the Lister Institute was closing (“unbelievable” and “frustrating”) to one celebrating her 60th birthday (“memorable”). Winifred will always be remembered as a pioneer in glycobiology and bloodgroup antigen research, one of the most respected scientists in the UK, and one of the most talented women scientists. During her working life, Winifred gave many memorable lectures and was highly respected in her field. With her enthusiasm for her work, she had the ability to make glycobiology fascinating to those with little interest in biochemistry. Winifred was an extraordinary scientist in many respects, succeeding in what was, at a time, essentially a male-dominated area. She was intelligent, determined, and self-motivated. She enjoyed interacting with other scientists, and she had particularly strong ties with Poland through her friendships with Jerzy Koscielak and a number of Polish students who came on sabbatical to the laboratory. She also had friendly links with Albert M. Wu. She participated in two meetings organised by him: Molecular Immunology of Complex Carbohydrates (MICC)-1 (College Station, TX, 1985) and MICC-2 (Taipei, Taiwan, 1999). Winifred contributed with excellent meeting lectures and articles at MICC-2, and moreover, Winifred and Albert published several papers together. Winifred also had strong links with scientists in Sweden, USA, Israel, Japan, and France. Until her first stroke, Winifred continued to attend meetings, thoroughly enjoying simply being part of the science scene and engaging in discussions and debate. Although it is difficult to separate “Watkins and Morgan”, Winifred was, in her own right, a brilliant researcher. When Walter was in his late 80s and worked at the CRC characterising the Sda antigen and elucidating its relationship with the Tamm– Horsfall protein, Winifred was always mindful of his needs. Walter saw Winifred as his protégé and as a highly talented scientist in her own right. He was always anxious that she be recognised as an independent researcher and valued both their academic and personal relationships. For those of us who worked with Winifred, she could be a hard taskmaster; visits to the library, for example, were done on our own time, and holidays were fitted around work, not vice versa. Lectures were practised endlessly, and mistakes were not allowed; preparation was key to the accomplished speaker. Papers were written and re-written until they were perfect, and doctoral theses did not contain a single mistake. On the other hand, she was inspirational, easy to talk to, and happy to share her knowledge and experience. There are still occasions when I realise what a font of knowledge she had. I learnt
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much from Winifred, and in many ways, I wish she had lived to see the upsurge in glycobiology research that has taken place over the past 5 years. Those of us who worked with her, knew her professionally, and shared her friendship will always be grateful for the experience. Pamela Greenwell School of Biosciences University of Westminster 115 New Cavendish Street London, UK
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Four glyco-immune chemists met at the XIII International Symposium on Glycoconjugates in Seattle, WA, on 23 August 1995. From left: W.T.J. Morgan, W.M. Watkins, J. Koscielak, and R.W. Jeanloz
Left: Dr. W.M. Watkins commented on her talk at the MICC-1, 14 September 1985: “No one was assigned to give a historical perspective of the past except me” Right: Two cyst glycoprotein pioneers, Drs. E. Kabat (left) and W.M. Watkins (right), met at a party honoring Dr. Kabat in Seattle, WA, on 31 August 1995
Left: Dr. W.M. Watkins as a chair at the MICC-2, 29 August 1999 Right: Dr. W.M. Watkins (left) and Dr. A.M. Wu (right) in front of Wu’s poster at the XV International Carbohydrate Symposium, Yokohama, Japan, 14 August 1990
European and Taiwanese glyco-immunologists met at the MICC-2 symposium on 31 August 1999. From left: Drs. E. Lisowska, M. Lin (Mackay Memorial Hospital), W.M. Watkins, and M. Duk
Dr. P.W. Cheng ( far left) was teaching Dr. W.M. Watkins (second from left) to use chopsticks at the MICC-2 symposium dinner on 29 August 1999. Also pictured: Dr. N. Sharon (second from right) and Mrs. N. Sharon ( far right)
Dr. W.M. Watkins (left) with microbial lectin experts, Drs. N. Gilboa-Garber (middle) and N.C. Garber (right), from Bar-Ilan University, Israel, at the MICC-2 symposium on 30 August 1999
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, 8–12 July 2007
國際 醣類 分子免疫學 學術會議-3
Participants of the Third International Symposium on Molecular Immunology of Complex Carbohydrates (MICC-3)
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Preface
This is the third time I have organized the international symposium on Molecular Immunology of Complex Carbohydrates (MICC), which was held at the Institute of Biological Chemistry (IBC), Academia Sinica, Taipei, Taiwan, 8–12 July 2007, as a satellite meeting of the 19th International Glycoconjugate Organization (IGO) meeting held 15–20 July 2007 in Cairns, Australia. MICC-2 was held at the same place from 28 August to 2 September 1999, after the 15th IGO meeting in Tokyo, Japan. We have arranged two other glyco symposia and three workshops since MICC-2. MICC-2 (Adv Exp Med Biol [2001]; 491) was selected as “an excellent textbook” from Kluwer Academic Publishers in 2002. In this MICC-3 book, three quarters of the content are based on lectures and posters of the MICC-3 symposium, and one quarter is from workshops and promotional materials. The book is divided into an Introduction, eight sections, and an appendix. The Introduction (Part I), “Glyco Experiences”, shares in one essay the serendipity of scientific discoveries in the glycosciences obtained from the lifetime experiences of Dr. Y.C. Lee (Department of Biology, Johns Hopkins University, MD, USA). In the second essay, Noriko Takahashi (Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan) describes 30 years of work devoted to determining the structures of N-glycans. The main text starts with Part II, “Blood Group ABH/Le-related Antigens,” to pay a tribute to the work of three pioneers in this field: Walter T.J. Morgan, Elvin A. Kabat, and – especially – Winifred M. Watkins, to whom this book is dedicated. This section presents the advanced concepts concerning the structure and functions of these antigens. In my comprehensive review on human blood group ABH/Ii, Lea,b,x,y, and sialyl Lea,x glycotopes of human ovarian cyst glycoproteins, I begin with the history of the most important findings and continue to our contemporary view. The interactions of plant, bacterial, and animal lectins with carbohydrates (and proteins) are thoroughly presented in eight contributions to Part III. Recognition intensities of mammalian structural units, ligand clusters, and polyvalency in the lectin–glycan interaction are some of the important issues covered in this section. The review on the ligand selectivity of adhesion/growth-regulatory galectins is a report of our decade-long collaboration with two glyco labs in Munich, Germany. The evidence for various regulatory roles of glycolipids is continuously increasing. Six contributions in Part IV describe the structures and functions of gangliosides xv
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and microbial glycolipids. The physiological roles of other glycoconjugates (glycoproteins, glycans, cellular receptors) in humans, animals, sponges, and bacteria, as well as aging-related alterations of the glycosylation profile in humans, are the subject of eight articles in Part V. The role of carbohydrates as antigens and regulators of the immune response is widely studied in the four reviews included in Part VI. Malignant transformation of cells is associated with profound changes of glycosylation, and some cancer-related glycoforms play a role in cancer development and metastasis. These aspects of the glycobiology of cancer are presented in five articles in Part VII. New methodologies in glycosynthesis and lectin–carbohydrate binding assays, and strategies for treatment as crucial applications, are described in four articles in Part VIII. Three of them present results obtained by Taiwanese research groups. An appendix is added at the end of the proceedings to correct typographical and structural errors found in MICC-1 (Adv Exp Med Biol [1988]; 228) and MICC-2 (Adv Exp Med Biol [2001]; 491). After the MICC-2 symposium, we lost three blood-group pioneers: Drs. Walter T.J. Morgan (October 1900–February 2003), Elvin A. Kabat (September 1914–June 2000), and Winifred M. Watkins (August 1924–October 2003). Drs. Kabat and Watkins have contributed timeless works to the MICC series. Dr. Kabat’s obituary was included in the MICC-2 issue, and Dr. Watkins’s obituary is included in this issue. To this day, this series continues to provide valuable knowledge in the field of glycotopes, structures and functions of complex carbohydrates, recognition factors of lectins, biomolecular interactions, and other glycosciences. I hope these proceedings reflect our worldwide connections, including the friendly collaboration of our lab with colleagues in Taiwan, and other parts of the world, especially Poland, Germany, Israel, Belgium, USA, Japan, Italy, England, India, and China. As a symposium chair, I owe special thanks to our three co-chairs: Drs. S.H. Wu, C.H. Lin, and K.H. Khoo from IBC, who were the actual driving forces for this symposium and to Drs. Y.C. Lee, R. Schauer, and A. Kobata for their important comments and suggestions. I also thank many members of our lab and staff of IBC, as they worked from early morning to late evening for several months. I also wish to thank the director of IBC, Dr. M.T. Tsai, and the president, C.H. Wong, who provided excellent facilities; the National Science Council (NSC), the Ministry of Education, the Foundation for Research and Education of Glycoscience, and the Research Promotion Center for Life Science of NSC for their financial support; and Chang Gung University for Workshops. I also appreciate the help of Amvo Publishing Company in Taiwan, and Drs. E. Lisowska, H.J. Gabius, Z. Yang, and Ms. Y.P. Gong for their assistance in editing this book. Without their efforts, the symposiums, workshops, and publication of these proceedings would be impossible. Thanks again to all contributors and participants. Tao-yuan, Taiwan 10 September 2011
Albert M. Wu
Contents
Part I Introduction to Glyco-Experiences 1 Serendipity in Scientific Discoveries: Some Examples in Glycosciences.......................................................................................... Yuan-Chuan Lee
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2 My 30-Year Devotion to the N-Linked Oligosaccharide Structures....................................................................... Noriko Takahashi
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Part II Blood Group ABH/Le-Related Antigens 3 Human Blood Group ABH/Ii, Lea,b,x,y, and Sialyl Lea,x Glycotopes; Internal Structures; and Immunochemical Roles of Human Ovarian Cyst Glycoproteins......................................... Albert M. Wu 4 Lewis Glyco-Epitopes: Structure, Biosynthesis, and Functions............ Hui-Li Chen 5 A Unique Endo-b-Galactosidase that Cleaves Both Blood Group A and B Glycotopes................................................... Su-Chen Li, Kimberly M. Anderson, and Yu-Teh Li
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Part III Lectins 6 Recognition Roles of Mammalian Structural Units and Polyvalency in Lectin–Glycan Interactions...................................... Albert M. Wu, Jia-Haw Liu, Tanuja Singh, and Zhangung Yang
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7 Adhesion/Growth-Regulatory Galectins: Insights into Their Ligand Selectivity Using Natural Glycoproteins and Glycotopes......................................................................................... 117 Albert M. Wu, Tanuja Singh, Jia-Hau Liu, Sabine André, Martin Lensch, Hans-Christian Siebert, Mickael Krzeminski, Alexandre M.J.J. Bonvin, Herbert Kaltner, June H. Wu, and Hans-Joachim Gabius 8 Glycotope Structures and Intramolecular Affinity Factors of Plant Lectins for Tn/T Antigens........................................... 143 Pierre Rougé, Willy J. Peumans, Els J.M. Van Damme, Annick Barre, Tanuja Singh, June H. Wu, and Albert M. Wu 9 The Five Bacterial Lectins (PA-IL, PA-IIL, RSL, RS-IIL, and CV-IIL): Interactions with Diverse Animal Cells and Glycoproteins.................................................................................... 155 Nechama Gilboa-Garber, Keren D. Zinger-Yosovich, Dvora Sudakevitz, Batya Lerrer, Anne Imberty, Michaela Wimmerova, Albert M. Wu, and Nachman C. Garber 10 On the Differential Sialic Acid Specificity of Lectins from Different Parts of Saraca indica.................................................... 213 Bishnu P. Chatterjee and Mainak Majumder 11 Regulation of Lectin Production by the Human Pathogens Pseudomonas aeruginosa and Chromobacterium violaceum: Effects of Choline, Trehalose, and Ethanol............................................ 229 Nachman C. Garber, Keren D. Zinger-Yosovich, Dvora Sudakevitz, Itschak Axelrad, and Nechama Gilboa-Garber 12 Non-carbohydrate-Mediated Interaction of Lectins with Plant Proteins................................................................................... 257 Jared Q. Gerlach, Michelle Kilcoyne, Seron Eaton, Veer Bhavanandan, and Lokesh Joshi 13 Novel Concepts About the Role of Lectins in the Plant Cell................ 271 Els J.M. Van Damme, Elke Fouquaert, Nausicaä Lannoo, Gianni Vandenborre, Dieter Schouppe, and Willy J. Peumans Part IV Structures and Functions of Glycolipids 14 Role of Gangliosides and Plasma Membrane-Associated Sialidase in the Process of Cell Membrane Organization.................... 297 Sandro Sonnino, Vanna Chigorno, Massimo Aureli, Anie Priscilla Masilamani, Manuela Valsecchi, Nicoletta Loberto, Simona Prioni, Laura Mauri, and Alessandro Prinetti
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15 9-O-Acetyl GD3 in Lymphoid and Erythroid Cells.............................. 317 Kankana Mukherjee, Suchandra Chowdhury, Susmita Mondal, Chandan Mandal, Sarmila Chandra, and Chitra Mandal 16 GM3 Upregulation of Matrix Metalloproteinase-9 Possibly Through PI3K, AKT, RICTOR, RHOGDI-2, and TNF-A Pathways in Mouse Melanoma B16 Cells.............................................. 335 Pu Wang, Xiaodong Wang, Peixing Wu, Jinghai Zhang, Toshinori Sato, Sadako Yamagata, and Tatsuya Yamagata 17 Pathological Roles of Ganglioside Mimicry in Guillain–Barré Syndrome and Related Neuropathies..................................................... 349 Robert K. Yu, Toshio Ariga, Seigo Usuki, and Ken-ichi Kaida 18 Structure and Function of Glycolipids in Thermophilic Bacteria........................................................................ 367 Feng-Ling Yang, Yu-Liang Yang, and Shih-Hsiung Wu 19 Lipooligosaccharides of Neisseria Species: Similarity Between N. polysaccharea and N. meningitidis LOSs........................................... 381 Chao-Ming Tsai Part V Structures and Functions of Complex Carbohydrates 20 Roles for N- and O-Glycans in Early Mouse Development.................. 397 Suzannah A. Williams and Pamela Stanley 21 Glycobiology in the Field of Gerontology (Glycogerontology)............ 411 Akira Kobata 22 Galectins in Regulation of Apoptosis..................................................... 431 Fu-Tong Liu, Ri-Yao Yang, Jun Saegusa, Huan-Yuan Chen, and Daniel K. Hsu 23 Avian and Human Influenza Virus Receptors and Their Distribution............................................................................. 443 Yasuo Suzuki 24 Importance of a Factor VIIIc-Like Glycoprotein Expressed in Capillary Endothelial Cells (eFactor VIIIc) in Angiogenesis......................................................................................... 453 Dipak K. Banerjee, Caroline M. Oliveira, José J. Tavárez, Viswa N. Katiyar, Subiman Saha, Juan A. Martínez, Aditi Banerjee, Aurymar Sánchez, and Krishna Baksi
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25 Mucin O-Glycan Branching Enzymes: Structure, Function, and Gene Regulation.............................................................. 465 Pi-Wan Cheng and Prakash Radhakrishnan 26 Studying Carbohydrate Self-Recognition in Marine Sponges Using Synthetic Aggregation Factor Epitopes........................ 493 Johannis P. Kamerling and Adriana Carvalho de Souza 27 Docking of Chitin Oligomers and Nod Factors on Lectin Domains of the LysM-RLK Receptors in the Medicago-Rhizobium Symbiosis................................................... 511 Pierre Rougé, Wim Nerinckx, Clare Gough, Jean-Jacques Bono, and Annick Barre Part VI Glycoimmunology 28 O-Acetylated Sialic Acids and Their Role in Immune Defense........... 525 Roland Schauer, G. Vinayaga Srinivasan, Dirk Wipfler, Bernhard Kniep, and Reinhard Schwartz-Albiez 29 Sialylated and Sulfated Carbohydrate Ligands for Selectins and Siglecs: Involvement in Traffic and Homing of Human Memory T and B Lymphocytes............................................ 549 Reiji Kannagi, Katsuyuki Ohmori, Guo-Yun Chen, Keiko Miyazaki, Mineko Izawa, and Keiichiro Sakuma 30 Diversity of Natural Anti-a-Galactosyl Antibodies in Human Serum...................................................................................... 571 Elwira Lisowska and Maria Duk 31 Significance of Serum Glycoprotein Profiles in Spontaneous Tolerance After Liver Allograft Transplantation................................. 585 Pei-Weng Wang and Tai-Long Pan Part VII Glycobiology of Cancer 32 Hematogenous Metastasis: Roles of CD44v and Alternative Sialofucosylated Selectin Ligands............................... 601 Konstantinos Konstantopoulos and Susan N. Thomas 33 Regulation of Glycosyltransferase Genes in Apoptotic Breast Cancer Cells Induced by l-PPMP and Cisplatin...................... 621 Rui Ma, Elizabeth A. Hopp, N. Matthew Decker, Audrey Loucks, James R. Johnson, Joseph Moskal, Manju Basu, Sipra Banerjee, and Subhash Basu
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34 Aberrant Glycosphingolipid Expression and Membrane Organization in Tumor Cells: Consequences on Tumor–Host Interactions................................................................... 643 Alessandro Prinetti, Simona Prioni, Nicoletta Loberto, Massimo Aureli, Valentina Nocco, Giuditta Illuzzi, Laura Mauri, Manuela Valsecchi, Vanna Chigorno, and Sandro Sonnino 35 Human KDN (Deaminated Neuraminic Acid) and Its Elevated Expression in Cancer Cells: Mechanism and Significance................................................................... 669 Sadako Inoue, Ken Kitajima, Chihiro Sato, and Shinji Go 36 Polysialic Acid Bioengineering of Cancer and Neuronal Cells by N-Acyl Sialic Acid Precursor Treatment................................. 679 Robert A. Pon, Wei Zou, and Harold J. Jennings Part VIII Glyco Applications 37 Synthesis of Hemagglutinin-Binding Trisaccharides............................ 691 Cheng-Chung Wang, Suvarn S. Kulkarni, Medel Manuel L. Zulueta, and Shang-Cheng Hung 38 Fabrication and Applications of Glyconanomaterials.......................... 727 Po-Chiao Lin, Avijit Kumar Adak, and Chun-Cheng Lin 39 Glycan Arrays to Decipher the Specificity of Plant Lectins................. 757 Els J.M. Van Damme, David F. Smith, Richard Cummings, and Willy J. Peumans 40 Targeting C-Type Lectin for the Treatment of Flavivirus Infections............................................................................ 769 Szu-Ting Chen, Yi-Ling Lin, Ming-Ting Huang, Ming-Fang Wu, and Shie-Liang Hsieh Appendix........................................................................................................... 777 Index.................................................................................................................. 787
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Contributors
Avijit Kumar Adak Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan Kimberly M. Anderson Department of Biochemistry, Tulane University Health Sciences Center, School of Medicine, New Orleans, LA 70112, USA Sabine André Faculty of Veterinary Medicine, Institute of Physiological Chemistry, Ludwig-Maximilians-University Munich, Veterinärstr. 13, 80539 Munich, Germany Toshio Ariga Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912, USA; Institute of Neuroscience, Medical College of Georgia, Augusta, GA 30912, USA Massimo Aureli Department of Medical Chemistry, Biochemistry and Biotechnology, Center of Excellence on Neurodegenerative Diseases, University of Milan, Via Fratelli Cervi 93, 20090 Segrate, Milano, Italy Itschak Axelrad The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel Krishna Baksi Department of Anatomy and Cell Biology, School of Medicine, Universidad Central del Caribe, Bayamón 00960-6032, Puerto Rico Aditi Banerjee Department of Biochemistry, School of Medicine, University of Puerto Rico, Medical Sciences Campus, San Juan 00936-5067, Puerto Rico Dipak K. Banerjee Department of Biochemistry, School of Medicine, University of Puerto Rico, Medical Sciences Campus, San Juan 00936-5067, Puerto Rico xxiii
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Contributors
Sipra Banerjee Department of Cancer Biology, Cleveland Clinic Foundation, Cleveland, OH 44129, USA Annick Barre Surfaces Cellulaires et Signalisation chez les Végétaux, UMR UPS-CNRS 5546, Pôle de Biotechnologie Végétale, 24 Chemin de Borde Rouge, 31326 Castanet Tolosan, France Manju Basu Department of Chemistry and Biochemistry, The University of Notre Dame, Notre Dame, IN 46556, USA Subhash Basu Department of Chemistry and Biochemistry, The University of Notre Dame, Notre Dame, IN 46556, USA Veer Bhavanandan Department of Bioengineering, Center for Glycosciences and Technology, The Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA Jean-Jacques Bono Surfaces Cellulaires et Signalisation chez les Végétaux, UMR UPS-CNRS 5546, 24 Chemin de Borde Rouge, 31326 Castanet Tolosan, France Alexandre M. J. J. Bonvin Department of NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands Sarmila Chandra Kothari Medical Centre, 8/3, Alipore Road, Kolkata, 700027, India Bishnu P. Chatterjee West Bengal University of Technology, Salt Lake, 700064, Kolkata, India Guo-Yun Chen Department of Molecular Pathology, Aichi Cancer Center, Nagoya 464-8681, Japan Huan-Yuan Chen Department of Dermatology, University of California, 3301 C Street, Suite 1400, Sacramento, CA 95816, USA Hui-Li Chen Key Laboratory of Glycoconjugate Research, Ministry of Health, Department of Biochemistry, Shanghai Medical College, Fudan University, Shanghai 200032, China Szu-Ting Chen Department of Microbiology and Immunology, Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan
Contributors
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Pi-Wan Cheng Department of Biochemistry and Molecular Biology, College of Medicine and Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198-5870, USA Vanna Chigorno Department of Medical Chemistry, Biochemistry and Biotechnology, Center of Excellence on Neurodegenerative Diseases, University of Milan, Via Fratelli Cervi 93, 20090 Segrate, Milano, Italy Suchandra Chowdhury Infectious Disease and Immunology Division, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 700032, India Richard Cummings Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA Adriana Carvalho de Souza Department of Bio-Organic Chemistry, Bijvoet Center, Utrecht University, Padualaan 8, CH Utrecht NL-3584, The Netherlands N. Matthew Decker Department of Chemistry and Biochemistry, The University of Notre Dame, Notre Dame, IN 46556, USA Maria Duk Department of Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 53-114 Wroclaw, Poland Seron Eaton Department of Molecular and Cellular Biology, Center for Biosensors and Bioelectronics, The Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA Elke Fouquaert Department of Molecular Biotechnology, Laboratory of Biochemistry and Glycobiology, Ghent University, Coupure Links 653, 9000 Gent, Belgium Hans-Joachim Gabius Faculty of Veterinary Medicine, Institute of Physiological Chemistry, LudwigMaximilians-University Munich, Veterinärstr. 13, 80539 Munich, Germany Nachman C. Garber The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel Jared Q. Gerlach Glycoscience Group, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland
xxvi
Contributors
Nechama Gilboa-Garber The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel Shinji Go Institute of Molecular Biomembranes and Glycobiology, Tohoku Pharmaceutical University, Sendai, Japan 981-8558 Clare Gough Laboratoire des Interactions Plantes-Microorganismes, UMR CNRS-INRA, 2594/441, 24 Chemin de Borde Rouge, 31326 Castanet Tolosan, France Pamela Greenwell (Obituary) School of Biosciences, University of Westminster, 115 New Cavendish Street, London, UK Elizabeth A. Hopp Department of Chemistry and Biochemistry, The University of Notre Dame, Notre Dame, IN 46556, USA Shie-Liang Hsieh Department and Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan Daniel K. Hsu Department of Dermatology, University of California, 3301 C Street, Suite 1400, Sacramento, CA 95816, USA Ming-Ting Huang Department of Microbiology and Immunology, Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan Shang-Cheng Hung Genomics Research Center, Academia Sinica, 128, Section 2, Academia Road, Taipei 115, Taiwan Giuditta Illuzzi Department of Medical Chemistry, Biochemistry and Biotechnology, Center of Excellence on Neurodegenerative Diseases, University of Milan, Via Fratelli Cervi 93, 20090 Segrate, Milano, Italy Anne Imberty CERMAV-CNRS (Joseph Fourier University), BP 53, 38041 Grenoble, Cedex 09, France Sadako Inoue Bioscience and Biotechnology Center, Nagoya University, Nagoya 464-8601, Japan Mineko Izawa Department of Molecular Pathology, Aichi Cancer Center, Nagoya 464-8681, Japan
Contributors
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Harold J. Jennings Institute for Biological Sciences, National Research Council of Canada, Ottawa, ON K1A 0R6, Canada James R. Johnson Department of Chemistry and Biochemistry, The University of Notre Dame, Notre Dame, IN 46556, USA Lokesh Joshi National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland; Department of Bioengineering, The Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA Ken-ichi Kaida Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912, USA; Institute of Neuroscience, Medical College of Georgia, Augusta, GA 30912, USA Herbert Kaltner Faculty of Veterinary Medicine, Institute of Physiological Chemistry, Ludwig-Maximilians-University Munich, Veterinärstr. 13, 80539 Munich, Germany Johannis P. Kamerling Department of Bio-Organic Chemistry, Bijvoet Center, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands Reiji Kannagi Research Complex for the Medical Frontiers, Aichi Medical University, Aichi 480-1195, Japan Viswa N. Katiyar Department of Chemistry, InterAmerican University, Metro Campus, San Juan 00919-1293, Puerto Rico Nava Katri The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel Michelle Kilcoyne National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland; Department of Bioengineering, Center for Glycosciences and Technology, The Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA Ken Kitajima Bioscience and Biotechnology Center, Nagoya University, Nagoya, Japan 464-8601
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Contributors
Bernhard Kniep Institut für Immunologie, Medizinische Fakultät “Carl Gustav Carus”, Technische Universität Dresden, Fetscherstr. 74, D-01307 Dresden, Germany Akira Kobata The Noguchi Institute, 1-8-1 Kaga, Itabashi-ku, Tokyo 173-0003, Japan Konstantinos Konstantopoulos Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, MD 21218, USA Mickael Krzeminski Department of NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands Suvarn S. Kulkarni Genomics Research Center, Academia Sinica, Taipei 115, Taiwan Nausicaä Lannoo Department of Molecular Biotechnology, Laboratory of Biochemistry and Glycobiology, Ghent University, Coupure Links 653, 9000 Gent, Belgium Yuan-Chuan Lee Biology Department, Johns Hopkins University, 3400 N. Charles St, Baltimore, MD 21218, USA Martin Lensch Faculty of Veterinary Medicine, Institute of Physiological Chemistry, Ludwig-Maximilians-University Munich, Veterinärstr. 13, 80539 Munich, Germany Batya Lerrer The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel Su-Chen Li Department of Biochemistry, Tulane University Health Sciences Center, School of Medicine, New Orleans, LA 70112, USA Yu-Teh Li Department of Biochemistry, Tulane University Health Sciences Center, School of Medicine, New Orleans, LA 70112, USA Chun-Cheng Lin Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan; Chemical Biology and Molecular Biophysics, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan
Contributors
xxix
Po-Chiao Lin Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan; Chemical Biology and Molecular Biophysics, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan Yi-Ling Lin Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan; Genomics Research Center, Academia Sinica, Taipei, Taiwan Elwira Lisowska Department of Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 53-114 Wroclaw, Poland Jia-Hau Liu Glyco-Immunochemistry Research Laboratory, Institute of Molecular and Cellular Biology, College of Medicine, Chang-Gung University, Kwei-San, Tao-Yuan, 333, Taiwan Jia-Haw Liu Glyco-Immunochemistry Research Laboratory, Institute of Molecular and Cellular Biology, College of Medicine, Chang-Gung University, Kwei-San, Tao-Yuan 333, Taiwan Fu-Tong Liu Department of Dermatology, University of California, 3301 C Street, Suite 1400, Sacramento, CA 95816, USA Nicoletta Loberto Department of Medical Chemistry, Biochemistry and Biotechnology, Center of Excellence on Neurodegenerative Diseases, University of Milan, Via Fratelli Cervi 93, 20090 Segrate, Milano, Italy Audrey Loucks Department of Chemistry and Biochemistry, The University of Notre Dame, Notre Dame, IN 46556, USA Mainak Majumder Lab India Ltd., Gurgaon, India Rui Ma Department of Chemistry and Biochemistry, The University of Notre Dame, Notre Dame, IN 46556, USA Chandan Mandal Infectious Disease and Immunology Division, Indian Institute of Chemical Biology, A Unit of Council of Scientific and Industrial Research (CSIR), 4, Raja S. C. Mullick Road, Kolkata 700032, India
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Contributors
Chitra Mandal Infectious Disease and Immunology Division, Indian Institute of Chemical Biology, A Unit of Council of Scientific and Industrial Research (CSIR), 4, Raja S. C. Mullick Road, Kolkata 700032, India Juan A. Martínez Department of Biochemistry, School of Medicine, University of Puerto Rico, Medical Sciences Campus, San Juan 00936-5067, Puerto Rico Anie Priscilla Masilamani Department of Medical Chemistry, Biochemistry and Biotechnology, Center of Excellence on Neurodegenerative Diseases, University of Milan, Via Fratelli Cervi 93, 20090 Segrate, Milano, Italy Laura Mauri Department of Medical Chemistry, Biochemistry and Biotechnology, Center of Excellence on Neurodegenerative Diseases, University of Milan, Via Fratelli Cervi 93, 20090 Segrate, Milano, Italy Keiko Miyazaki Department of Molecular Pathology, Aichi Cancer Center, Nagoya 464-8681, Japan Susmita Mondal Infectious disease and Immunology Division, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 700032, India Joseph Moskal Department of Biomedical Engineering, The Falk Center for Molecular Therapeutics, McCormick School of Engineering and Applied Sciences, Northwestern University, Evanston, IL 60201, USA Kankana Mukherjee Infectious Disease and Immunology Division, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 700032, India Wim Nerinckx Laboratorium voor Eiwitbiochemie en Eiwitengineering, Ghent University, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium Valentina Nocco Department of Medical Chemistry, Biochemistry and Biotechnology, Center of Excellence on Neurodegenerative Diseases, University of Milan, Via Fratelli Cervi 93, 20090 Segrate, Milano, Italy Katsuyuki Ohmori Department of Clinical Pathology, Kyoto University School of Medicine, Kyoto 606-8501, Japan Caroline M. Oliveira Department of Biochemistry, School of Medicine, University of Puerto Rico, Medical Sciences Campus, San Juan 00936-5067, Puerto Rico
Contributors
xxxi
Tai-Long Pan School of Traditional Chinese Medicine, Chang Gung University, Tao-Yuan, Taiwan Willy J. Peumans Laboratory of Biochemistry and Glycobiology, Department of Molecular Biotechnology, Ghent University, Coupure Links 653, 9000 Gent, Belgium Robert A. Pon Institute for Biological Sciences, National Research Council of Canada, Ottawa, ON KIA 0R6, Canada Alessandro Prinetti Department of Medical Chemistry, Biochemistry and Biotechnology, Center of Excellence on Neurodegenerative Diseases, University of Milan, Via Fratelli Cervi 93, 20090 Segrate, Milano, Italy Simona Prioni Department of Medical Chemistry, Biochemistry and Biotechnology, Center of Excellence on Neurodegenerative Diseases, University of Milan, Via Fratelli Cervi 93, 20090 Segrate, Milano, Italy Prakash Radhakrishnan Department of Biochemistry and Molecular Biology, College of Medicine Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198-5870, USA Pierre Rougé Surfaces Cellulaires et Signalisation chez les Végétaux, UMR UPS-CNRS 5546, 24 Chemin de Borde Rouge, 31326 Castanet, Tolosan, France Jun Saegusa Department of Dermatology, University of California, 3301 C Street, Suite 1400 Sacramento, CA 95816, USA Subiman Saha Department of Biochemistry, School of Medicine, University of Puerto Rico, Medical Sciences Campus, San Juan 00936-5067, Puerto Rico Keiichiro Sakuma Department of Molecular Pathology, Aichi Cancer Center, Nagoya 464-8681, Japan Aurymar Sánchez Department of Anatomy and Cell Biology, School of Medicine, Universidad Central del Caribe, Bayamón 00960-6032, Puerto Rico Chihiro Sato Bioscience and Biotechnology Center, Nagoya University, Nagoya, Japan 464-8601 Toshinori Sato Department of Biosciences and Informatics, Keio University, Hiyoshi, Yokohama, Japan 223-8522
xxxii
Contributors
Roland Schauer Biochemisches Institut, Christian-Albrechts-Universität, Olshausenstr. 40, D-24098 Kiel, Germany Dieter Schouppe Department of Molecular Biotechnology, Laboratory of Biochemistry and Glycobiology, Ghent University, Coupure Links 653, 9000 Gent, Belgium Reinhard Schwartz-Albiez Tumor Immunologie Programm, Deutsches Krebsforschungszentrum Heidelberg, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany Hans-Christian Siebert Faculty of Veterinary Medicine, Institute of Physiological Chemistry, Ludwig-Maximilians-University Munich, Veterinärstr. 13, 80539 Munich, Germany Tanuja Singh Glyco-Immunochemistry Research Laboratory, Institute of Molecular and Cellular Biology, College of Medicine, Chang-Gung University, Kwei-San, Tao-Yuan 333, Taiwan; Faculty of Veterinary Medicine, Institute of Physiological Chemistry, LudwigMaximilians-University Munich, Veterinärstr. 13, 80539 Munich, Germany David F. Smith Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA Sandro Sonnino Department of Medical Chemistry, Biochemistry and Biotechnology, Center of Excellence on Neurodegenerative Diseases, University of Milan, Via Fratelli Cervi 93, Segrate, Milano 20090, Italy G. Vinayaga Srinivasan Biochemisches Institut, Christian-Albrechts-Universität, Olshausenstr. 40, D-24098 Kiel, Germany Pamela Stanley Department of Cell Biology, Albert Einstein College of Medicine, New York, NY 1046, USA Dvora Sudakevitz The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel Yasuo Suzuki Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, 1200 Matsumoto-cho, Kasugai-shi 487-8501, Aichi, Japan
Contributors
xxxiii
Noriko Takahashi Graduate School of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori 3-1, Mizuho-ku, Nagoya 467-8603, Japan José J. Tavárez Department of Biochemistry, School of Medicine, University of Puerto Rico, Medical Sciences Campus, San Juan 00936-5067, Puerto Rico Susan N. Thomas Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, MD 21218, USA Chao-Ming Tsai Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, MD, USA Seigo Usuki Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912, USA; Institute of Neuroscience, Medical College of Georgia, Augusta, GA 30912, USA Manuela Valsecchi Department of Medical Chemistry, Biochemistry and Biotechnology, Center of Excellence on Neurodegenerative Diseases, University of Milan, Via Fratelli Cervi 93, 20090 Segrate, Milano, Italy Els J.M. Van Damme Laboratory of Biochemistry and Glycobiology, Department of Molecular Biotechnology, Ghent University, Coupure Links 653, 9000 Gent, Belgium Gianni Vandenborre Laboratory of Biochemistry and Glycobiology, Department of Molecular Biotechnology, Ghent University, Coupure Links 653, 9000 Gent, Belgium Cheng-Chung Wang Department of Chemistry, National Tsing Hua University, Hsinchu 300, Taiwan; Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei 115, Taiwan Pei-Weng Wang Chang Gung Molecular Medicine Center, Chang Gung University, Tao-Yuan, Taiwan Pu Wang Laboratory of Tumor Biology and Glycobiology, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China; Department of Life Sciences, Laboratory of Biochemistry, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China
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Contributors
Xiaodong Wang Laboratory of Tumor Biology and Glycobiology, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China Suzannah A. Williams Department of Physiology, Anatomy, and Genetics, University of Oxford, Le Gros Clark Building, South Parks Road, Oxford, OX1 3QX, UK Michaela Wimmerova Department of Biochemistry, Masaryk University, Kotlarska 2, Brno 61137, Czech Republic Dirk Wipfler Tumor Immunologie Programm, Deutsches Krebsforschungszentrum Heidelberg, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany Albert M. Wu Glyco-Immunochemistry Research Laboratory, Institute of Molecular and Cellular Biology, College of Medicine, Chang Gung University, Kwei-san, Tao-yuan 333, Taiwan June H. Wu Glyco-Immunochemistry Research Laboratory, Institute of Molecular and Cellular Biology, College of Medicine, Chang-Gung University, Kwei-Shan, Tao-Yuan 333, Taiwan Ming-Fang Wu Department and Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan Peixing Wu Department of Biosciences and Informatics, Keio University, Hiyoshi, Yokohama, Japan 223-8522 Shih-Hsiung Wu Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan Sadako Yamagata Laboratory of Tumor Biology and Glycobiology, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China Tatsuya Yamagata Laboratory of Tumor Biology and Glycobiology, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China Feng-Ling Yang Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan Ri-Yao Yang Department of Dermatology, University of California, 3301 C Street, Suite 1400, Sacramento, CA 95816, USA
Contributors
xxxv
Yu-Liang Yang Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan Zhangung Yang Glyco-Immunochemistry Research Laboratory, Institute of Molecular and Cellular Biology, College of Medicine, Chang-Gung University, Kwei-San, Tao-Yuan 333, Taiwan Robert K. Yu Institute of Molecular Medicine and Genetics and Institute of Neuroscience, Medical College of Georgia, Augusta, GA 30912, USA Jinghai Zhang Department of Life Sciences, Laboratory of Biochemistry, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China Keren D. Zinger-Yosovich The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel Wei Zou Institute for Biological Sciences, National Research Council of Canada, Ottawa, ON KIA 0R6, Canada Medel Manuel L. Zulueta Department of Chemistry, National Tsing Hua University, Hsinchu 300, Taiwan
Part I
Introduction to Glyco-Experiences
Chapter 1
Serendipity in Scientific Discoveries: Some Examples in Glycosciences Yuan-Chuan Lee
Keywords Serendipity • Penicillin • Saccharine • Splenda • Sialic acid According to the Merriam-Webster Online Dictionary, serendipity is “the faculty or phenomenon of finding valuable or agreeable things not sought for.” Serendip (or Serendib) was the name for the island of ancient Sri Lanka and was thought to be a corruption of the Sanskrit compound Simhaladvipa (“Dwelling-Place-of-Lions Island”). The Arabs are thought to have borrowed the name from Indians with whom they traded. The word “serendipity” was coined by an eighteenth-century English author/politician, Horace Wolpole, who wrote a novel, The Three Princes of Serendip, based on a purported Persian fairy tale. Actually, the first book of The Three Princes of Serendip was published in 1557 by M. Tramezzino and was perhaps a collection of ancient Indian fables. Tennett wrote [1]: “In ancient times there existed in the country of Serendippo, in the Far East, a great and powerful king by the name of Giaffer. He had three sons who were very dear to him. And being a good father and very concerned about their education, he decided that he had to leave them endowed not only with great power, but also with all kinds of virtues of which princes are particularly in need.” Thus, began the voyage of the three princes of Serendip. At any rate, in the Wolpole book, the heroes in the novel (the princes) often made discoveries by chance and sagacity. The novel was a great success, and the expression of “serendipity” has become quite popular since then. One of the better known stories in the novel is the case of a camel blinded in one eye. During their trip, the princes were stopped by a camel driver who asked them if they had seen one of his camels, which was missing. Although they had not seen the camel, they noticed the signs of the camel and mystified the camel driver by asking him if his missing camel was blind in one eye (because the princes saw that the grass was eaten on one side of the road), missing one tooth (the cuds of grass on the ground Y.-C. Lee (*) Biology Department, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA e-mail:
[email protected] A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_1, © Springer Science+Business Media, LLC 2011
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indicated a tooth gap), and lame (the traces of a dragged hoof). The camel driver was tremendously impressed by their astute observations and prudence and immediately hurried off to pursue the missing camel. Perhaps the most celebrated case of serendipity, although not directly related to glycosciences, is the “discovery” of America by Christopher Columbus in 1492. Columbus firmly believed that the earth was round and thus one could reach China or India by westward voyage from Europe over what was presumed to be an open sea. By attempting to find a sea route to the rich continent of Cathay (China), India, and the fabled gold and spice islands of the East, Columbus hoped to gain riches for his family and to join the ranks of the nobility of Spain. He was extremely fortunate, after many abortive trials for presenting his plans to various monarchs in Europe at the time (Portugal, Genoa, Venice, and England), to finally be able to gain support from the Spanish monarchy. Isabella and Ferdinando II (who were just married and had just conquered Granada) also felt a need to find an alternative to the land route to the Orient, which was becoming increasingly restricted because of the rise of Islamic power. As we know, Columbus never actually reached Cathay or India, although he at first believed that he did (the name of West Indies is a reminder for such a misconception). Nevertheless, he is accredited with the “discovery” of America. One relatively unknown factor that helped Columbus to secure the support of Isabella and Ferdinando was that Columbus was led to believe that the distance from the Canary Islands to Cathay was merely 3,400 miles while in reality it was greater than 9,000 miles (some think he willfully misrepresented the data). Had he shown the real distance in his proposal to the Spanish monarchy, he might not have been granted the financial support for the voyage. His ships were furnished only for the 3,400-mile voyage. If the New World did not exist, Columbus and his crew would have perished somewhere in the Atlantic Ocean. Many scientific discoveries are the result of serendipity, and glycosciences are no exception. In this article, I cite only those serendipitous discoveries in the glycosciences that are close to my interests and environ. The classic artificial sweetener, saccharin, was serendipitously discovered in 1879 when a couple of Johns Hopkins chemists were studying the influence of substituents on the oxidation of toluene-2-sulfonamide. While Constantine Fahlberg (a visiting scientist) was working in the laboratory of Ira Remsen (who eventually became the second president of the Johns Hopkins University), he spilled a chemical on his hand. Later in the evening, while eating dinner, Fahlberg noticed that the bread he was eating tasted sweeter than usual. He traced the sweetness back to the chemical by tasting various residues on his hands and clothes to finally conclude that certain chemicals in the lab were the cause. (In the late nineteenth century, during the early stages of the chemistry discipline, laboratory procedures and precautions were not nearly as strict as they are now). The sweet-tasting chemical was found to be orthotoluenesulfonamide (Fig. 1.1) produced by oxidation of toluene-2-sulfonamide. Interestingly, Fahlberg foresaw the commercial potential of the unusually sweet compound and patented the process for commercial manufacture in 1885 (without involving Remsen). Remsen was reported to be quite upset by Fahlberg’s action because he felt it was inappropriate to benefit from such a scientific discovery.
1 Serendipity in Glycosciences
5 O
CH3
Oxidation
NH S O2
SO2NH2
Toluene-2-sulfonamide
Saccharine
Fig. 1.1 Oxidation of toluene-2-sulfonamide to produce “saccharin”
Fig. 1.2 Structure of sucralose (Splenda)
Another sweetener discovery was also the result of serendipity. Sucralose, also known as Splenda® (Fig. 1.2), was discovered in 1976 by Leslie Hough and Shashikant Phadnis (in Queen Elizabeth College, London), who were trying to develop chlorinated sugars as chemical intermediates [2, 3]. During the course of their research, Phadnis was asked by Hough to “test” the powder, presumably in a certain chemical reaction. Phadnis thought that Hough asked him to “taste” it, so he did. He found the compound to be exceptionally sweet (hundreds of times sweeter than sucrose), and they worked with the sugar business Tate & Lyle for a year before settling down on the final formula. If Phadnis had listened to Hough more carefully, we may not have Splenda so readily today. Discovery of the best known anticoagulant, heparin, is widely credited, at least in part, to a second-year medical student, Jay McLean, who was working in the laboratory of William Henry Howell, a physiology professor at the Johns Hopkins Medical School. McLean and Howell were originally searching for thromboplastic (procoagulant) factors in tissues. In 1916, McLean found, in addition to the known thromboplastic cephaline (phosphatidylethanolamine), a fat-soluble phosphatide that was thromboplastic. McLean’s discovery of a fat-soluble phosphatide was arguably the result of attempting to thoroughly purify cephaline, thus removing the thromboplastic effect that was masking the new anticoagulant. The term heparin was actually coined by Howell, first in 1918 [4] in reference to the fat-soluble phosphatide, and then much later in 1925 in reference to the polysaccharide that we now know as heparin [5] (Fig. 1.3). This is an entirely different substance from the phosphatide once named heparin (for example, not fat-soluble, not containing phosphorus). There is some controversy as to who really discovered heparin and should
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get the credit. The detailed account by Marcum [6] of the events surrounding the Howell laboratory in that period indicates that Howell should be credited for the discovery of heparin, as we know now. In 1927, Alexander Fleming accidentally discovered (actually, rediscovered) penicillin while he was in search of a bacteria-dissolving “wonder drug.” One summer, when he took off for a vacation, he left a pile of Petri dishes on the bench to make room for a coworker. Upon his return, he was cleaning up the accumulated Petri dishes while complaining to his coworker about how insufficient funding had led to the pile up and his having to do the cleaning himself. As he was cleaning the Petri dishes, he noticed one of the dishes was contaminated by a mold that appeared to have dissolved the bacterial layer (Fig. 1.4). Further pursuit of the mystery identified the mold to be Penicillium notatum, and this mold secreted something that can dissolve bacteria. Thus, the first wellcharacterized antibiotic, penicillin, was born. Although it is not as well publicized, Fleming is also credited for the accidental discovery of lysozyme, a bacteriolytic enzyme that cleaves the glycosidic linkage of muramic acid (a derivative of GlcNAc) (Fig. 1.5). CH2SO3−
COO−
CH2SO3−
OH
OH
OH
O
O NHSO3−
O OH
O NHSO3−
CH2SO3− COO− OH
OSO3−
OH O
O NHSO3−
Fig. 1.3 Stylized structure of heparin
Fig. 1.4 Illustration of Penicillium dissolving Staphylococcus
CH2SO3− COO− OH
OSO3−
OH O
NHSO3−
1 Serendipity in Glycosciences
7
Lysozyme
Penicillin Fig. 1.5 Differential actions of penicillin and lysozyme on microbial mureins
While working in an unheated laboratory, he was suffering from nasal congestion, and a drop of nasal mucus fell into a Petri dish. The lysozyme in the nasal mucus dissolved the bacteria in the Petri dish. If Fleming did not have this prior observation of nasal mucus dissolving bacteria, he might not have suspected that mold could also dissolve bacteria. A more recent case was the discovery of the hepatic carbohydrate receptor by Ashwell and Morell [7, 8] that has its origin in an investigation of the metabolism of ceruloplasmin (a copper-carrying protein). Thinking that carbohydrate is a totally innocuous appendage on ceruloplasmin, they attempted to label ceruloplasmin by selectively oxidizing the exocyclic chain of sialic acid, thus generating aldehyde, which can be reduced with sodium borotritide to introduce tritium label. Alternatively, they removed the sialic acid with sialidase, and the galactose thus exposed was oxidized with galactose oxidase to produce 6-oxo-galactose, which can also be labeled in a similar way (Fig. 1.6). A totally unexpected result was observed when these ceruloplasmin derivatives labeled on the different sugar residues that were injected into the blood stream. If the label was on the trimmed sialic acid, ceruloplasmin behaved normally. However, when the label was on galactose (6-3H-Gal), which was not masked by sialic acid, ceruloplasmin was quickly removed from the circulation, and the protein (or the radioactivity) removed from circulation was found to accumulate in the liver. These observations led to the discovery of the hepatic carbohydrate receptor, which opened the gate of modern glycobiology. When it comes to sialic acid, Saul Roseman can claim the first case of serendipity. While he was an assistant professor at the University of Michigan, he and Donald Comb (who later became the founder of New England BioLab) established the correct structure by overhauling the wrong structure “established” by other workers, including some Nobelites. This was a well-known case of serendipity, and Saul Roseman is often referred to as the Prince of Serendipity among glycoscientists. In the late 1950s, the laboratories of Blix (Sweden), Klenk (Germany), Zilliken (USA), and Gottschalk (Germany) were all working, independently, on what is now
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OH O
OH
HO H N
OH
O HO
O
O O
OH
HO
HO
NaIO4
O
NaB3H4 OH
O OH
HO
3
H O
O
O OH
O
O
Gal oxidase
OH OH
H N
R
R
NaB3H4 H
O OH
O OH
3
OHOH
sialidase
OH O
R
O
HO OH
R
Fig. 1.6 Oxidation of NeuAc or Gal for labeling ceruloplasmin
known as sialic acid (but was known as lactamic acid or neuraminic acid) [9]. Eventually, the works from the laboratories of Kuhn (chemical degradation [10]) and Cornforth (chemical synthesis and X-ray diffraction [11]) appeared to indicate that sialic acid was made of pyruvic acid and N-acetyl-glucosamine. Comb and Roseman (now a professor of biology at Johns Hopkins University) were interested in metabolism of sialic acid and studied the action of NANA aldolase (NANA stood for N-acetylneuraminic acid in those days). The aldolase from Vibrio cholerae was known to split sialic acid into pyruvic acid and an N-acetylhexosamine (HexNAc), which was identified as GlcNAc by others [12]. Most aldolases known then could also catalyze condensation reaction (for example, aldolase can condense dihydroxyacetone with glyceraldehydes to form fructose, and fructose can be split into those two 3-carbon components). However, Comb and Roseman could never condense pyruvic acid and GlcNAc with the aldolase. Moreover, in their hands, Comb and Roseman could never obtain the correct stoichiometry of pyruvic acid and HexNAc after splitting sialic acid with this aldolase, always yielding only half as much “GlcNAc” for 1 mol of pyruvic acid. Interestingly, when they took the HexNAc component produced from the cleavage of natural sialic acid with the aldolase and used it in the condensation reaction with pyruvic acid, the reaction then occurred smoothly. As it turned out, the HexNAc component generated from the natural sialic acid was not identical to N-acetylglucosamine, and critical examination of this HexNAc – by elemental analysis, optical rotation, chromatography, colorimetry, and X-ray diffraction – finally revealed that it was the 2-epimer of GlcNAc, namely, ManNAc [13]. At that time, ManNAc hardly appeared in the lexicon of carbohydrate
1 Serendipity in Glycosciences
9 O
O
O
O
H N
H
H
HO
H
HO
H
OH
H
OH
ManNAc
Pyruvic acid HO
O
NH H
H
OH
H
OH
CH2OH
CH2OH
GlcNAc
O
O
O
OH
O
O
OH
O
H
H
H
H
H
OH
H
OH
H N
H
H
NH
HO
H
Sialic Acid
HO
H
H
OH
H
OH
H
OH
H
OH
CH2OH
O
CH 2OH
Fig. 1.7 Confusion on the configuration of sialic acid
chemistry or biochemistry. So the question is, how could other investigators have mistaken ManNAc for GlcNAc? (Fig. 1.7) In the chemical synthesis [11], they used oxaloacetic acid as a donor of pyruvic acid and carried out its condensation (aldol condensation) with GlcNAc under alkaline conditions. Chemical degradation, which yielded GlcNAc from sialic acid, also was in alkaline condition. In retrospect, it was the alkali-catalyzed epimerization of GlcNAc into ManNAc that eventually resulted in the formation of real sialic acid, which gave the same X-ray diffraction pattern as the naturally derived sialic acid. How could Comb and Roseman have suspected that the aldolase action yielded not GlcNAc but a related N-acetyl-sugar? Being young and zealous, they used a short heating time (3 min) for the analysis of HexNAc (Morgan–Elson reaction [14]) rather than the standard 12-min heating. Their serendipity was that ManNAc gives the same color yield as GlcNAc under the standard conditions of 12-min heating, but when 3-min heating is employed (Fig. 1.8), ManNAc gives only half the color yield of GlcNAc. However, the fact that Comb and Roseman could not condense pyruvic acid with GlcNAc but were successful with the HexNAc obtained from the natural sialic acid was a big factor in their elucidation of the correct sialic acid structure.
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100 80 60 40 12 min
20
3 min
0 GlcNAc
ManNAc
Fig. 1.8 Differential color yields of ManNAc and GlcNAc by Morgan–Elson reaction
In my own experience, serendipity manifested itself while we were testing the efficacy of neoglycoproteins. Neoglycoproteins are proteins modified with structurally well-characterized carbohydrate derivatives by chemical or enzymatic means (Fig. 1.9). We had prepared BSA derivatives modified with many different sugars by many different methods [15]. The most frequent form of linkage we used was the amidino linkage, which was formed from methyl imidate of thioglycosides reacting with amino groups of BSA or other proteins (Fig. 1.9). One of the advantages of neoglycoproteins is that one can change the density of sugars on protein with considerably more flexibility than what nature provides. When we were comparing the inhibitory power of Man–BSA, Gal–BSA, and others with respect to the inhibition of binding of 125I-labeled asialoorosomucoid (also known as a-1-acid glycoprotein) by rabbit hepatic membranes [16], it was abundantly clear that the liver membranes very much preferred Gal-derivatives over any other sugar (Fig. 1.10), thus the neoglycoprotein confirmed the known binding specificity of rabbit hepatic membrane. However, the more exciting result was that when we examined the inhibitory power of Gal–BSA with respect to the Gal density (number of Gal residues on BSA), we realized that linear increase in the Gal density on BSA resulted in logarithmic increase in the binding activity [16]. I believe this was our first observation of the “cluster effect.” Then, when we used a series of synthetic branched oligosaccharides (chemically synthesized by Dr. Löngrenn and his coworkers in Uppsala, Sweden), which are precise partial structures of various N-glycans, the effect was more dramatic [17], and we found that the progression of valency with respect to galactose from 1 to 2 and 3 yielded ca. a 1,000-fold and 1,000,000-fold increase in the binding affinity, respectively (Fig. 1.11). The increase in the binding affinity from the trivalent to the tetravalent oligosacchrides was marginal, suggesting that the receptor was organized in trimeric form. The hidden subtlety, however, was that the valency is not the only requirement for the manifestation of a “cluster effect,” as among the di- and trivalent structures, there were considerable differences in binding strengths, indicating that it was the exact special arrangement of the terminal target sugars that was important.
1 Serendipity in Glycosciences HO
11
OH S
HO
HO
NaOMe
O OH
R: Protein R-NH2
O S
N
CH2C
HO
HO
NH CH2C
OH
OMe
OH O
NH
S
pH 8-9
OH
HO
CH2C
OH
NH
R
Fig. 1.9 Outline of preparation of amidine-type neoglycoprotein (bearing Gal) Inhibition of Binding by Hepatic Membranes
12 11 10
−log(IC50)
9 Gal-BSA 8 7 6 5 Man-BSA 4 3
0
5
10
15
20
25
30
35
40
Sugar/BSA (mol /mol) Fig. 1.10 Discovery of the “cluster effect” with neoglycoproteins
The great pioneer of bioorganic chemistry, Emil Fischer, established the configuration of several common sugars (d-glucose, d-mannose, d-galactose, and l-arabinose) by a few simple chemical reactions (including oxidation, condensation, and reduction) in combination with marvelous dexterity and impeccable deductive reasoning, thus clearly establishing the relationship among all these sugars as well as lower sugars starting from glyceraldehydes. Fisher called all sugars derivable from d-glyceraldehyde “d-sugars” and those extended from l-glyceraldehyde “l-sugars” (Fig. 1.12), irrespective of their optical rotation values. As such, this
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Y.-C. Lee Inhibition of rat hepatocyte binding
Bound ligand (pM)
0.3
Di
0.2
Mono
Tri Tetra
0.1
0.0 −11 −10
−9
−8
−7
−6
−5
−4
−3
−2
−1
Log [Inhibitor, M] Fig. 1.11 Cluster effects by synthetic oligosaccharide mimetics
CHO H
CHO
OH
HO
H
H HO
H
OH
H
H
OH
H
CH2OH
OH H OH
*
OH
CH2OH
D-Glucose
CHO
CHO H
OH CH2OH
D-Glyceraldehyde
HOH CH2OH
L-Glyceraldehyde
Fig. 1.12 Absolute configuration of sugar series
work may not be described as the result of serendipity. Fisher chose to write d-glyceraldehyde with the hydroxyl group of the lowest chiral carbon on the righthand side of the carbon backbone [18], most likely using a left-to-right stroke. This decision presumably was made without any theoretical or experimental ground but because it was simply a common habit for any European language practitioners to draw a line from left to right, especially when it was branching out to the right side of a backbone. The chance of assigning the structure on the right in Fig. 1.8 as d-glyceraldehyde was 50%, all things being equal. This was extremely fortunate because many decades later, Bijvoet et al. [19] in Utrecht (remember: van¢t Hoff
1 Serendipity in Glycosciences
13
was in Utrecht when he proposed the tetrahedral theory) determined that Fisher’s choice was perfectly correct: the d-sugars indeed had the correct configuration as depicted in Fig. 1.12. This incident may not be classified as a serendipity, unless the scope of serendipity is expanded to include those in which the discoverer does not actually see the results of serendipity. Scientific discoveries obviously contain a significant serendipitous aspect. We cannot plan serendipity. John Barth (a Johns Hopkins graduate and teacher), for example, wrote in his extraordinary retelling of the Sindbad saga in Serendip, The Last Voyage of Somebody the Sailor (New York, 1991): “You don’t reach Serendip by plotting a course for it. You have to set out in good faith for elsewhere and lose your bearings serendipitously.” However, we should also remember what Louis Pasteur once said: “Chance favors the prepared mind.” What may look like an accidental discovery perhaps is just another expression of an alert mind.
References 1. Tennett JE (1861) Sketches of the natural history of Ceylon. Purana Books, India 2. Khan RA, Hough L, Phadnis SP (1979) Chloro derivatives of sucrose. Division of Brit 1,543,167. Research Corp, USA. Application: GB 3. Hough L, Phadnis SP, Khan RA, Jenner MR (1977) Chlorinated sucrose sweeteners. Tate and Lyle Ltd., UK. Application: DE 4. Howell WH, Holt E (1918) Two new factors in blood coagulation – heparin and proantithrombin. Am J Physiol 47:328–341 5. Howell WH (1925) The purification of heparin and its presence in blood. Am J Physiol 71:553–562 6. Marcum JA (2000) The origin of the dispute over the discovery of heparin. J Hist Med Allied Sci 55:37–66 7. Morell AG, Irvine RA, Sternlieb I, Scheinberg IH, Ashwell G (1968) Physical and chemical studies on ceruloplasmin. V. Metabolic studies on sialic acid-free ceruloplasmin in vivo. J Biol Chem 243:155–159 8. Morell AG, Van den Hamer CJ, Scheinberg IH, Ashwell G (1966) Physical and chemical studies on ceruloplasmin. IV. Preparation of radioactive, sialic acid-free ceruloplasmin labeled with tritium on terminal d-galactose residues. J Biol Chem 241:3745–3749 9. Rosenberg A (1994) The beginning of sialic acid. In: Rosenberg A (ed) Biology of sialic acids. Plenum, New York 10. Kuhn R, Brossmer R (1956) Degradation of lactaminic acid to N-acetyl-d-glucosamine. Chem Ber 89:2471–2475 11. Cornforth JW, Firth ME, Gottschalk A (1958) The synthesis of N-acetyl-neuraminic acid. Biochem J 68:57–61 12. Heimer R, Meyer K (1956) Studies on sialic acid of submaxillary mucoid. Proc Natl Acad Sci USA 42:728–734 13. Comb DG, Roseman S (1960) The sialic acids I. The structure and enzymatic synthesis of N-acetylneuraminic acid. J Biol Chem 235:2529–2537 14. Reissig JL, Storminger JL, Leloir LF (1955) A modified colorimetric method for the estimation of N-acetylamino sugars. J Biol Chem 217:959–966 15. Lennarz W, Lane D (eds) (2004) Neoglycoproteins in Encyclopedia of Biochemistry. Elsevier, Amsterdam 16. Stowell CP, Lee RT, Lee YC (1980) Studies on the specificity of rabbit hepatic carbohydratebinding protein using neoglycoproteins. Biochemistry 19:4904–4908
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17. Lee YC, Townsend RR, Hardy MR, Lönngren J, Arnarp J, Haraldsson M, Lönn H (1983) Binding of synthetic oligosaccharides to the hepatic Gal/GalNAc lectin. J Biol Chem 258:199–202 18. Hudson CS (1948) Historical aspects of Emil Fischer’s fundamental conventions for writing stereo-formulas in a plane. Adv Carbohydr Chem 3:1–22 19. Bijvoet JM, Peerdeman AF, Bommel V (1951) Determination of the absolute configuration of optically active compounds by means of X-rays. Nature 168:271–272
Chapter 2
My 30-Year Devotion to the N-Linked Oligosaccharide Structures Noriko Takahashi
Keywords Glycoamidase A • 2-D map • 3-D map • GALAXY Thirty years ago, in 1977, I unexpectedly discovered a new enzyme, glycoamidase A [1–3] that cleaves intact carbohydrate moiety from the original glycoproteins without affecting either the carbohydrate or peptide structures. This new enzyme was readily approved by the International Enzyme Committee and was assigned a new number: EC.3.5.1.52. At that time, I had been working on determining the primary amino acid sequence of stem bromelain, a pineapple proteolytic enzyme, which my professor had shown to be a glycoprotein. The presence of the sugar moieties was really an obstruction in sequencing bromelain. Therefore, I dreamed of having an enzyme that would cleave the carbohydrate chains without affecting the peptide structure. The dream came true as, all of a sudden, this enzyme appeared in front of my eyes. I was convinced that this was a gift from the Goddess of Science to a humble female scientist struggling and being frustrated in a male-dominant Japanese academic world. The new enzyme had been hiding in the crude b-glucosidase, a product supplied by Sigma Ltd. This development encouraged me to commit myself to the comprehensive analysis of N-linked oligosaccharide structures in glycoproteins. Under normal circumstances, in those days, the freedom to conduct independent research would have been difficult for a novice junior faculty member of the medical school. Fortunately, because of the sudden move of my professor out of the school, I had the opportunity to completely switch my research direction to determine the carbohydrate structures that were cleaved from several glycoproteins by the enzyme glycoamidase A. The first target was the oligosaccharide moiety of stem bromelain [4], followed by ovalbumin [5], human fibrinogen [6], proteoglycan core molecule [7], human placenta and umbilical cord [8], Taka-amylase A apoprotein [9], and xylose-containing common structural unit in N-linked oligosaccharides of laccase from sycamore cells [10]. N. Takahashi (*) Graduate School of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori 3-1, Mizuho-ku, Nagoya 467-8603, Japan e-mail:
[email protected] A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_2, © Springer Science+Business Media, LLC 2011
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In 1988 (11 years after the discovery of glycoamidase A), I published the accumulated oligosaccharide structural data in Analytical Biochemistry. This paper, titled “A two-dimensional (2-D) mapping technique,” included the structures of 113 different neutral oligosaccharides [11]. The aim of the 2-D mapping technique was to separate and determine closely related structures of N-linked oligosaccharides. Using this new method in 1988, my coworkers and I analyzed the carbohydrate of urinary and recombinant human erythropoietins [12] and found that they contained more than eight and 13 different N-linked oligosaccharides, respectively. Continuing this line of research, I published in 1995 a further methodological paper introducing a new 3-D mapping technique [13]. This paper documented 220 different neutral and 42 acidic oligosaccharide structures, the latter consisting of 26 mono-, seven di-, seven tri-, and two tetrasialylated oligosaccharides. Thirty years ago, there were few simple and effective methods for isolating and determining the detailed structure of N-glycans. Since the discovery of the first deglycosylating enzyme, I have analyzed and placed on the 2-D and 3-D maps more than 500 data of pyridylaminated N-glycan structures, which include not only sialic acid-containing, but also sulfuric acid-containing oligosaccharides [14]. Based on these accumulated data, the Web application “GALAXY” (glyco analysis by the three axes of MS and chromatography) (http://www.glycoanalysis.info/) was developed [15].
2.1 Development of the Methods 2.1.1 Glycoamidase A The reaction mechanism of glycoamidase A is shown in Fig. 2.1. Studies done so far indicate that this enzyme can cleave all N-linked oligosaccharides from the glycopeptides at the linkage indicated by the arrow, whether they are neutral, sialylated, sulfated, or phosphorylated. The more than 500 oligosaccharides recorded in the GALAXY [15] database were all obtained by the use of the glycoamidase.
Fig. 2.1 Reaction mechanism of glycoamidase A
2 N-Linked Oligosaccharide Structures
17
2.1.2 Two-Dimensional Mapping In 1988, I proposed a 2-D mapping technique involving the use of two different high-performance liquid chromatography (HPLC) columns [11] for the structural analysis of pyridylaminated neutral oligosaccharides. In this method, the reducing oligosaccharide mixture is released from the glycopeptide by glycoamidase digestion. The oligosaccharides are then derivatized with 2-aminopyridine using Dr. Hase’s improved method for PA-derivatization that works well with both sialylated and neutral oligosaccharides. Because of the fluorescent nature of the 2-aminopyridine derivative, the sensitivity of detection of PA-oligosaccharides is in the picomole range. Before analysis of the PA-derivatized oligosaccharides, both HPLC columns are calibrated with isomalto-oligosaccharide mixtures. The first separation of the PA-derivatized oligosaccharides is done on the calibrated octadecylsilyl (ODS)silica column (for example, 14.7 glucose unit (GU) peak on the top left of Fig. 2.2). The second separation is done on the amide-silica column (6.7 GU peak on the bottom left of Fig. 2.2). Finally, the pair of calibrated elution time factors are plotted on a 2-D map (Fig. 2.2, right) and compared to the elution times of previously analyzed structures. This method is not only an oligosaccharide separation technique, but it can also identify the structure of the target oligosaccharide by its unique position on the 2-D map.
a
ODS 20.0
Relative fluorescence intensity
4 567
8
9
GU 14.7
10 11
0
b
12
13 14
20
18
Amide
40
10.0
Amide-silica GU 6.7 4 5 6 7 8 9 10 11
0
15 16 17
20
12 13 14 15 16
40
Elution time (min)
ODS = 14.7 AMIDE = 6.7
1718
0.0 0.0
14.0
28.0
ODS
Fig. 2.2 Two-dimensional mapping of glycoamidase-released PA-derivatized neutral o ligosaccharides
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N. Takahashi
2.1.3 Three-Dimensional Mapping In 1995, we introduced a third dimension to the 2-D map to analyze oligosaccharides with acidic groups, that is, sialylated [13] or sulfated [14] (Fig. 2.3). Today, more than 500 N-linked oligosaccharides are distributed on this 3-D map. The present 3-D mapping technique involves the following four steps: first, separation of the PA-oligosaccharide mixture by HPLC on a diethylaminoethyl (DEAE) column, based on the acidity of the oligosaccharide, that is its sialic acid [13] or sulfuric acid [14] content. This step produces neutral, mono-, di-, tri-, tetrasialyl, and/or sulfated oligosaccharides. In the second and third steps, each of these classes is further separated sequentially on ODS-silica and amide-silica columns. The data obtained for each acidic oligosaccharide are plotted separately on a 2-D map, with ODS elution units on the X-axis and amide units on the Y-axis. Finally, in the fourth step, those individual 2-D maps are combined into a 3-D map, with the sample’s acidity on the Z-axis. Such a 3-D map method can clearly differentiate even closely related sialic a2,3- and a2,6-containing oligosaccharides.
Fig. 2.3 Three-dimensional mapping
2 N-Linked Oligosaccharide Structures
19
2.1.4 The Unit Contribution Concept From 1990 to 1993, we published a new concept: “parameterization of contribution of sugar units” [16–18]. The “unit” denotes a monosaccharide component at a specific position in the whole N-linked oiligosaccharide [16]. For example, Fig. 2.4 shows a popular high-mannose oligosaccharide containing nine mannose residues, which has an elution time of 5.2 GU on the ODS column. This value can be expressed as a total of the unit contributions (UC) specific for each of the participating units. In the case of the amide column, this is more or less obvious, since the elution time on an amide column is proportional to the molecular size, at least for neutral oligosaccharides. Surprisingly, we found that the same is true in the case of the ODS column, indicating that the elution times on the ODS column can also be expressed as a total of UCs. Comparison of calculated and measured GU values is useful in suggesting the structure of glycans resulting from glycosidase treatments. For example, Man8GlcNAc2 produced by the a-mannosidase digestion of the Man9GlcNAc2 can result in three possible structures, depending on which nonreducing terminal mannose residue is cleaved. The actual structure produced can be identified from the observed GU on the ODS column. If the experimental ODS GU value of a- mannosidase digestion product is 6.4, one can conclude that the uppermost terminal mannose (the one with unit contribution −1.07) was cleaved. The other two possibilities would have resulted in ODS GUs of 4.8 and 5.7, respectively, and can therefore be excluded from consideration.
Fig. 2.4 The unit contribution values for high-mannose type N-glycans
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2.1.5 The Glyco Tree Diagram of N-Glycans The approximately 500 N-glycan structures so far documented can be depicted by a diagram in which branches spread from a trunk of a trimannosyl core structure: the “glyco tree” diagram (Fig. 2.5). This diagram consists of 63 units of sugar residues, with their UC values expressed in calibrated elution times (GUs). The glyco tree diagram can also track and explain glycosidase digestion by various enzymes used for structural identification purposes. The most widely used enzymes are a-sialidases (a2,3 or a2,6), a-fucosidase, b-galactosidase, b-GlcNAcase, and a-manno-sidase.
2.1.6 Comparison of Predicted and Observed Coordinates for Trisialyl and Triantennary N-Glycans Figure 2.6 shows a comparison of the predicted and observed coordinates for enzymatically prepared trisialyl and triantennary N-glycans. The shaded and clear dots indicate calculated and measured values, respectively. The agreement between calculated and measured values is very good.
Fig. 2.5 The glyco tree diagram of N-glycans
2 N-Linked Oligosaccharide Structures
21
8.8
S-G-GN2-M6 S-G-GN4 S-G-GN2
M-GN-GN M
3
Amide-Silica (Glc Unit)
8.4
0 0 0
3 3 6
6 6 3
3 6 3
6 3 3
7.2 3 3 3
6.8
6.4 12
Predicted Observed
3 6 6
6 3 6
8.0
7.6
6 6 6
Neutral
13
14
15
16
17
18
19
20
ODS-Silica (Glc Unit)
Fig. 2.6 Predicted and observed coordinates for trisialyl and triantennary N-glycans
2.1.7 Sulfooligosaccharides on the Map [14] Although the biological importance of sulfated oligosaccharides has been widely recognized, there are only a few reports describing detailed structures of sulfated N-glycans. This is largely due to the lack of a convenient method for identifying structures of sulfated glycans, which are usually found in low abundance. Figure 2.7 shows HPLC data of desialylated sulfooligosaccharides derived from LS12 cells. We have recently reported the coordinates of 40 sulfated oligosaccharides. This has resulted in new 2-D monosulfate, disulfate, etc., layers, in addition to the previously established neutral and sialic acid layers on our 3-D map.
2.2 Examples of Oligosaccharide Structures In this section, we describe several interesting results of detailed N-glycan structures that were analyzed using the 2-D and 3-D mapping methods.
2.2.1 N-Glycan Structures from Human Immunoglobulin G (IgG) First, we discuss N-glycans from human IgG. The N-glycans of IgG from healthy individuals exhibit complex microheterogeneity with 16 neutral components, as
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Fig. 2.7 HPLC data of the desialylated sulfooligosaccharides derived from LS12 cells
shown in Fig. 2.8. In particular, components F and G have the same molecular weight and very similar structures. Therefore, reliable separation of the peaks corresponding to these complicated N-glycans requires very high-resolution elution profiles. This was achieved by fine control of the buffer conditions on the HPLC. Figure 2.9 demonstrates the change of IgG N-glycan oligosaccharide with age by comparing profiles from two healthy males, 81 and 29-years-old. Even with the limited data shown (only neutral HPLC profiles), the difference is well pronounced. In the older person, peak E (agalactosyl, G0) is predominant. By contrast, in the younger person, peak H (digalactosyl, G2) is predominant, and the contents of G0 and G1 are characteristically lower. Note the clear separation of F and G components on the ODS column of HPLC, in spite of their very similar structures. Similarly, male and female IgG oligosaccharide profiles are also clearly different, as determined by comparing profiles of a healthy 27-year-old male and a healthy 24-year-old female (not shown here). The difference was characterized by a predominant peak H (digalactosyl, Gal2) in the female, in contrast to a relatively lower content of it in the male. Another study showed for the first time the difference between normal and pathological human IgG oligosaccharides using HPLC [19].
2.2.2 Chlamydia Trachomatis Oligosaccharides It is generally believed that N-linked oligosaccharides are rarely present in bacteria. However, Dr. Kuo and coworkers found that these oligosaccharides are involved in
2 N-Linked Oligosaccharide Structures
23 ± Fuc1α1
±Galβ1琊4GlcNAcβ1琊2Manα1
6 6 ±GlcNAc1琊4Manβ1琊4GlcNAcβ1琊4GlcNAc 3 ±Galβ1琊4GlcNAcβ1琊2Manα1
A
Fab
GN M M GN GN GN M
B G GN M M GN GN GN M
GN M M GN GN G GN M
D G GN M M GN GN G GN M
E
F GN M M GN GN GN M
F F G GN M M GN GN GN M
G
F GN M M GN GN G GN M
C
Asn297
Fc
J G GN M GN M GN GN GN M
GN M GN M GN GN G GN M F M GN M GN M GN GN GN M
L G GN M GN M GN GN G GN M F N G GN M GN M GN GN GN M
O
P
K
Immunoglobulin G (IgG)
F G GN M M GN GN G GN M
GN M GN M GN GN GN M
I
N-glycan
H
F GN M GN M GN GN G GN M
F G GN M GN M GN GN G GN M
Fig. 2.8 Microheterogeneity of the desialylated N-linked oligosaccharides derived from healthy human IgG
E
FLUORESCENCE INTENSITY
81 years old G0
F G2 H G
AC
D
M N O
B
P
G2
27 years old G0
10
20
30
40
ELUTION TIME (min) Fig. 2.9 Comparison of HPLC profiles of PA-oligosaccharides of IgG purified from an older male (81 years) and a younger male (27 years). Structures of oligosaccharides are the same as those in Fig. 2.8
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N. Takahashi
the infection of HeLa cells by Chlamydia trachomatis [20]. The N-linked o ligosaccharides from the membrane protein of C. trachomatis are of the highmannose type only, from M-5 to M-9 (Fig. 2.10). It is interesting to note that on an ODS column, the shortest elution time is not M-9, but M-8, followed by M-9, M-7, M-6, and lastly, M-5.
M2M6 M3 R M2M2
M M GN GN R = M
M2M6 M2M3 R M2M2
Fluorescence
M6 M3 R M2 M2
M6 M3 R M2 M6 M3 R
G4GN6 G4GN2 R G4GN2 G4GN2 R G4GN2
0
10
20
Elution time (min) Fig. 2.10 Oligosaccharides of outer membrane protein of Chlamydia trachomatis
2 N-Linked Oligosaccharide Structures
25
2.2.3 Oligosaccharide Structures of Squid Rhodopsin Rhodopsin exists in photoreceptor cells, and the rhodopsin N-terminal segment is N-glycosylated. We have analyzed the major N-glycan structure of squid rhodopsin and found that the characteristic sequence Gal b(1,4) Fuca(1,6), previously found in octopus, was also found in squid rhodopsin [21]. Mana(1,6) Galb(1,4) Fuca(1,6) |⋅| Man b(1,4)GlcNAc b(1,4)GlcNAc |⋅| Mana(1,3) Fuca(1,3) Furthermore, for the first time, we demonstrated in mollusks the occurrence of an insect-specific N-glycan sequence, in which the innermost N-acetylglucosamine residue is difucosylated.
2.2.4 High-Mannose Oligosaccharides (M7) in Golgi Bodies and Cell Membrane In insect cells, the proportion of high-mannose type oligosaccharides (M7) in Golgi bodies and cell membrane appears almost the same when analyzed on an amide column (Fig. 2.11, left). However, on the ODS column, the M7 peak splits into two, M71 and M72, and their ratio is very different between membrane and Golgi bodies. This demonstrates the importance of the 2-D mapping method (that is, successive analysis on two kinds of HPLC columns) for the structure differentiation of N-glycans.
2.2.5 Differentiation of a 2,3 and a 2,6 Sialic Acids in N-linked Oligosaccharides The differentiation of structures containing a2,3 and a2,6 sialic acids is rather difficult and, to my knowledge, has not been done previously. Figure 2.12 shows an example of such a differentiation in the N-linked oligosaccharides of chicken epithelial cells. The data for all N-glycan structures analyzed so far have been collected in our GALAXY database, made available online. Figure 2.13 shows the title screen of the GALAXY system. The position of each white dot corresponds to one N-linked oligosaccharide with a specific structure.
26
N. Takahashi M M
M7.2 Amide-HPLC
ODS-HPLC
Golgi
uV
Complex type
High-mannose type
M
5
10
15
M GN GN
M7.1
M9 M
M8 M5 M6
M
M M
M
M
M GN GN
M M M
M7
20
25
5.0
30 min
ODS-HPLC
7.5
M7.2
Cell membrane
Amide-HPLC uV
Complex type
High-mannose type M7.1 M5 M6
5
10
15
M7 M8 M9 20
25
5.0
30 min
7.5
37%
30.997
63%
a 2,3
28.817 29.377
a 2,6
24.506 25.636 26.639
50
21.769 22.275
19.500 19.664
100
1Det.A Ch1 17.308
12.713
150
14.181 14.949
Fig. 2.11 Comparative analysis of the proportion of high-mannose oligosaccharides (M7) in Golgi bodies and cell membrane
0 0
5
10
15
20
25
30
min
Fig. 2.12 Differentiation between a2,3 and a2,6 sialic acids in N-linked oligosaccharides of chicken epithelial cellsTHE GALAXY DATABASE
This application lets the user search for candidate structures satisfying 2-D or 3-D HPLC and/or mass spectrometric data. For glycosidase treatments, one can use GALAXY to predict coordinates of candidate PA-glycans and to trace the effects of the treatment in a graphical manner.
2 N-Linked Oligosaccharide Structures
27
Fig. 2.13 The title screen of the GALAXY database (http://www.glycoanalysis.info)
2.3 Summary Most N-linked oligosaccharides can be completely released from the original glycopeptides by glycoamidase A digestion, regardless of origin, size, acidity, or structure. After the pyridylamination of the newly produced reducing ends of the released oligosaccharides, their structural assignment becomes easy not only in the case of 2-D and 3-D mapping methods, but also in the entire process of purification and identification. The sensitivity of the analysis is at the femtomole level. The identification of N-linked oligosaccharide structures by 2-D and 3-D mapping methods is very reproducible and reliable. Using the above procedures, one can easily identify the structure of newly found oligosaccharides. Presently, we have data for more than 500 N-linked oligosaccharides in the GALAXY database. Acknowledgments I would like to acknowledge the work of those who directly helped to develop the N-glycan structure determination technique described here, in particular the contribution of Dr. Kay-Hooi Khoo (Taipei), Dr. Noboru Tomiya, Hiroaki Nakagawa, and Hirokazu Yagi, as well as the insightful advice of Professors Yuan-Chuan Lee (USA), Takashi Muramatsu, Yoji Arata, and Koichi Kato.
28
N. Takahashi
References 1. Takahashi N (1977) Demonstration of a new amidase acting on glycopeptides. Biochem Biophys Res Commun 76:1194–1201 2. Takahashi N, Nishibe H (1978) Some characteristics of a new glycopeptidase acting on aspartylgly-cosylamine linkages. J Biochem 84:1467–1473 3. Takahashi N, Nishibe H (1981) Almond glycopeptidas acting on aspartylglycosylamine linkages. Multiplicity and substrate specificity. Biochim Biophys Acta 657:457–467 4. Ishihara H, Takahashi N, Takeuchi N, Oguri S, Tejima S (1979) Complete structure of the carbohydrate moiety of stem bromelain: an application of the almond glycopeptidase for structural studies of glycopeptides. J Biol Chem 254:10715–10719 5. Ishihara H, Takahashi N, Ito J, Takeuchi E, Tejima S (1981) Either high-mannose-type or hybrid type oligosaccharide is linked to the same asparagine residue in ovalbumin. Biochim Biophys Acta 669:216–221 6. Nishibe H, Takahashi N (1981) The release of carbohydrate moieties from human fibrinogen by almond glycopeptidase without alteration in fibrinogen clottability. Biochim Biophys Acta 661:274–279 7. Oike Y, Kimata K, Shinomura T, Suzyki S, Takahashi N, Tanabe K (1982) A mapping technique for probing the structure of proteoglycan core molecules. J Biol Chem 257:9751–9752 8. Takahashi N, Shimizu S, Yamada K (1982) Asparagine-linked oligosaccharides in human placenta and umbilical cord as demonstrated by almond glycopeptidase. FEBS Lett 146:139–142 9. Takahashi N, Toda H, Nishibe H, Yamamoto K (1982) Isolation and characterization of Takaamylase A apoprotein deglycosylated by digestion with almond glycopeptidase immobilized on sepharose. Biochim Biophys Acta 707:236–242 10. Takahashi N, Hotta T, Ishihara H, Mori M, Tejima S, Bligny R, Akazawa T, Endo S, Arata Y (1986) Xylose-containing common structural unit in N-linked oligosaccharides of laccase from sycamore cells. Biochemistry 25:388–395 11. Tomiya N, Awaya J, Kurono M, Endo S, Arata Y, Takahashi N (1988) Analyses of N-linked oligosaccharides using a two-dimensional mapping technique. Anal Biochem 171:73–90 12. Tsuda E, Goto M, Murakami A, Akai K, Ueda M, Kawanishi G, Takahashi N, Sasaki R, Chiba H, Ishihara H, Mori M, Tejima S, Endo S, Arata Y (1988) Comparative structural study of N-linked oligosaccharides of urinary and recombinant erythropoitins. Biochemistry 27:5646–5654 13. Takahashi N, Nakagawa H, Fujikawa K, Kawamura Y, Tomiya N (1995) Three-dimensional elution mapping of pyridylaminated N-linked neutral and sialyl oligosaccharides. Anal Biochem 226:139–146 14. Yagi H, Takahashi N, Yamaguchi Y, Kimura N, Uchimura K, Kannagi R, Kato K (2005) Development of structural analysis of sulfated N-glycans by multidimensional high performance liquid chromatography mapping methods. Glycobiology 15:1051–1060 15. Takahashi N, Kato K (2003) GALAXY (glycoanalysis by the three axes of MS and chromatography). Trends Glycosci Glyc 15(84):235–251 16. Lee YC, Lee BI, Tomiya N, Takahashi N (1990) Parameterization of contribution of sugar units to elution volumes in reversed-phase HPLC of 2-pyridylaminated oligosaccharides. Anal Biochem 188:259–66 17. Tomiya N, Lee YC, Yoshida T, Wada Y, Awaya J, Kurono M, Takahashi N (1991) Calculated two-dimensional sugar map of pyridylaminated oligosaccharides: elucidation of the jack bean alpha-mannosidase digestion pathway of Man9GlcNAc2. Anal Biochem 193:90–100 18. Takahashi N, Wada Y, Awaya J, Kurono M, Tomiya N (1993) Two-dimensional elution map of GalNAc-containing N-linked oligosaccharides. Anal Biochem 208:96–109 19. Takahashi N, Ishii I, Ishihara H, Mori M, Tejima F, Jefferis R, Endo S, Arata Y (1987) Comparative structural study of the N-linked oligosaccharides of human normal and pathological immuno-globulin G. Biochemistry 26:1137–1144
2 N-Linked Oligosaccharide Structures
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20. Kuo C, Takahashi N, Swanson AF, Ozeki Y, Hakomori S (1996) An N-linked high-mannose type oligosaccharide, expressed at the major outer membrane protein of Chlamydia trachomatis, mediates attachment and infectivity of the microorganism to HeLa cells. J Clin Invest 98:2813–2818 21. Takahashi N, Masuda K, Hiraki K, Yoshihara K, Huang HH, Khoo KH, Kato K (2003) N-Glycan structures of squid rhodopsin (existence of the 1-3 and 1-6 difucosylated innermost GlcNAc residue in a molluscan glycoprotein). Eur J Biochem 270:2627–2632
Part II
Blood Group ABH/Le-Related Antigens
Chapter 3
Human Blood Group ABH/Ii, Lea,b,x,y, and Sialyl Lea,x Glycotopes; Internal Structures; and Immunochemical Roles of Human Ovarian Cyst Glycoproteins Albert M. Wu Keywords Human ovarian cyst glycoproteins • Human blood group • Glycotopes • Structure
3.1 Introduction: Human Blood Group ABH/Lewis Antigens Antigenic determinants (glycotopes) of human blood group ABH antigens are located on the complex glycoconjugates. These ABH glycotope-containing glycans are generally conjugated with polypeptides or ceramides to form glycoproteins and glycosphingolipids, respectively [15, 35]. As shown in Fig. 3.1 and Table 3.1, blood group A (GalNAca1→3[lFuca1→2]Gal) and B (Gala1→3[lFuca1→2]Gal) trisaccharide units, differing by the presence of a terminal GalNAc and Gal residue, respectively, are linked to either Type 1 (Galb1→3GlcNAcb, Ib) or Type 2 (Galb1→4GlcNAcb, IIb) precursor disaccharides [3, 4, 11, 14, 34, 35, 38]. Another form is the Type 3 structure, which carries short ABH sequences attached to Galb1→3GalNAca (core 1, Ta), which in turn is linked via GalNAc to Ser or Thr residues of the peptide backbone [7]. The ABH blood group system was originally defined in terms of the antigenic substances occurring on red blood cells (RBCs) [15, 19, 34, 35]. However, blood group substances exhibiting similar serological specificities also occur in various mucous secretions of the human body, mostly as glycoproteins [2, 13, 26]. The ABH antigens in secretions are O-linked glycoproteins, in which oligosaccharide chains are attached to a protein moiety by O-glycosidic linkage to Ser or Thr
This chapter is dedicated to the memory of Dr. E.A. Kabat (Columbia University College of Physicians and Surgeons, New York, USA) and Dr. W.M. Watkins (Royal Postgraduate Medical School, London, UK), who were pioneers in the field of human blood active glycotopes. A.M. Wu () Glyco-Immunochemistry Research Laboratory, Institute of Molecular and Cellular Biology, College of Medicine, Chang-Gung University, Kwei-shan, Tao-Yuan 333, Taiwan e-mail:
[email protected] A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_3, © Springer Science+Business Media, LLC 2011
33
A.M. Wu
34
b
Gal 1 4GlcNAc 1 R Human blood group Type II precursor (II ) sequence
Gal 1 4GlcNAc 1 R 1,2 LFuc Type II,H determinant
1,2 LFuctrans
2,3 NeuAc
1,3 LFuctrans
4GlcNAc 1 R 1,3 LFuc Lex determinant
Gal 1 4GlcNAc 1 1,2 LFuc
Gal 1 4GlcNAc 1 R 2,3 1,3 LFuc NeuAc Sialyl Lex determinant
R
Type II,H determinant
1,3 Galtrans Gal 1 3Gal 1 4GlcNAc 1 1,2
1,3 LFuctrans
3Gal 1 4GlcNAc 1 R 1,2 LFuc Type II,A determinant 3Gal 1 4GlcNAc 1 1,2 LFuc
1,3 GalNActrans
1,3 Galtrans
R
R
LFuc Type II,B determinant
1,3 GalNActrans
GalNAc 1
Gal 1
1,3 LFuctrans
1,2 LFuctrans
Gal 1
Gal 1 4GlcNAc 1 R 1,2 1,3 LFuc LFuc Ley determinant
2,3 SAtrans Gal 1 4GlcNAc 1 R
Galc 1
GalNAc 1
3Gal 1 4GlcNAc 1 R 1,2 LFuc
1,3 LFuctrans
3Gal 1 4GlcNAc 1 R 1,2 1,3 LFuc LFuc B,Ley determinant
Type II,A determinant
Type II,B determinant
1,3 LFuctrans
GalNAc 1
3Gal 1 4GlcNAc 1 1,2 1,3 LFuc LFuc
R
A,Ley determinant
Fig. 3.1 Generalized structures of carbohydrate chains of blood group active and sialyl glycoproteins, isolated from human ovarian cyst fluid. (i) The four-branched structure (1–4) shown above represents the internal portion of the carbohydrate moiety of blood group substances, to which the residues responsible for A, B, H, Lea, Leb, Lex, and Ley (unknown glycotopes in 1988) activities are attached [21, 38, 39]. The numbers in parentheses ([1] to [12]) indicate the site of attachment for the human blood group A, B, H, Lea, Leb, Lex, and Ley determinants (Table 3.1). Most of the carbohydrate chains isolated are parts of this structure, including short chains of T and Tn determinants. (ii) The core structure of a Lea, Lex, sLea, and sLex active glycoprotein isolated from human ovarian cyst fluid (HOC 350) is proposed to be a simple (short) type chain, which is composed of a linear tetra-core 1 of I/IIb1→3Ta (one unit of Ib/IIb chain and the Ta determinant sequence), and a longer Branch 3 with an additional Ib/IIb unit and Branch 4, with NeuAca2→3/6 linked to Gal at the nonreducing end and lFuca1→3/4 linked to GlcNAc [41]. Compared to the structure in (i), this structure may contain both Branches 3 and 4 with attachment of lFuc in [12], [16], and [18], and NeuAca2→3/6 in [11], [15], and [17], representing the site of attachment for the sLea and sLex determinants (Table 3.1)
3 Human Blood Group ABH/Ii, Lea,b,x,y, and Sialyl Lea,x Glycotopes
35
Table 3.1 Identification of 12 blood group active cyst glycoproteins from Fig. 3.1 Blood group active glycoprotein purified from Human blood group or human ovarian cyst fluid antigenic determinant Sugar added Site of addition None Ii MSS 1st Smith Pneumococcus type 14 Beach P-1 polysaccharide (II) Tighe P-1 Cyst N-1 Lea 20% 2×
Lea Lex (*)
lFuca1→4 lFuca1→3
[6] and/or [10] [8], [10] and/or [12]
Cyst JS phenol insoluble Tighe phenol insoluble
H, Leb/Ley (*) H, Ley (*) H, Lex (*) > Leb
lFuca1→2 lFuca1→2 and others as in Lea
[5], [7], [9], [11] [5], [7], [9] and/or [11] [6], [8], [12]
MSS, native MSM Cyst 9 Cyst 14
A1 or A2 Leb Leb/Ley > Lea Leb/Ley (*)
GalNAca1→3 and others as in H, Leb and Ley (*)
[1], [2], [3] and/or [4] in addition to [5] to [12]
Cyst Beach phenol insoluble
B Leb Ley (*)
Gala1→3 and same as H, Leb and Ley (*)
[1], [2], [3] and/or [4] in addition to [5] to [12]
HOC 350[44]
sLea sLex Lea
[15], [17] NeuAca2→3/6 [16], [18] and lFuca1→4 [11], [15], [17] NeuAca2→3/6 [12], [16], [18] and lFuca1→3 and other as in Lea and Lex (*) (*) considered as unknown genes and unknown glycotopes in the early 1980s
r esidues of peptide chains (O-glycans) [35, 36, 38]. They are structurally different from the ABH-active glycoproteins (N-linked glycoproteins, Band 3) on the RBC membrane [9, 11].
3.2 Biosynthesis, Expression, and Roles of the ABH, Lewis, and Sialyl Lewis Antigens The precursor oligosaccharides of glycosphingolipid protein glycans are sequentially modified by concerted actions of several glycosyltransferases to form the ABH and Lewis blood group determinants (Fig. 3.2a, b). The genes encoding these glycosyltransferases show polymorphism in their loci [47]. The H blood group determinant is synthesized by the action of an a1,2 fucosyltransferase (a1,2 lFuc-T). In blood group A, B, or AB individuals, the blood group H determinant acts as an acceptor substrate to form blood group A or B determinant. Blood groups A and B result from adding aGalNAc or aGal to H determinant by a1,3-N-acetylgalactosaminyltransferase (a1,3 GalNAc-T) or a,3-galactosyltransferase (a,3 GalT), respectively (Fig. 3.2a, b). The ABH determinants are also expressed in the
A.M. Wu
36
epithelial cells of the secretory glands [29]. However, the presence of ABH g lycotopes in secreted glycoproteins is determined by the expression of alleles at the Se gene locus close to the H gene. The H and Se genes code for a1,2 lFuc-T, now called FUT1 and FUT2, respectively, which are different gene products expressed in tissue-specific manner. The expression of FUT1 in erythrocyte progenitors and FUT2 in secretory cells is responsible for the expression of ABH determinants in these two different tissues, respectively. Both can act on several types of the human blood group type I and II precursors of the mucin-type O-glycans. The Lea determinant is formed by a1→4 linkage of lFuc to a subterminal GlcNAc of Ib precursor chains. The Lex determinant contains the Fuc residue-linked a1-3 to the subterminal GlcNAc of IIb chains (Fig. 3.2a, b). The Lewis glycotopes
a
Gal 1 3GlcNAc 1
R
Human blood group TypeIprecursor (I ) sequence
Gal 1 3GlcNAc 1 R 1,2 LFuc TypeI,H determinant
1,2 L Fuctrans
2,3 NeuAc
1,4 LFuctrans
3GlcNAc 1 R 1,4 LFuc a Le determinant
GalNAc 1
3Gal 1 3GlcNAc 1 1,2 LFuc Type I,A determinant Gal 1
1,4 LFuctrans
1,2 LFuctrans
Gal 1
Gal 1 3GlcNAc 1 R 1,2 1,4 LFuc LFuc b Le determinant
2,3 SAtrans Gal 1 3GlcNAc 1 R
Gal 1 3GlcNAc 1 1,2 LFuc
Gal 1 3GlcNAc 1 R 2,3 1,4 LFuc NeuAc a Sialyl Le determinant
R
Type I,H determinant
1,3 Galtrans Gal 1 3Gal 1 3GlcNAc 1 1,2
1,4 LFuctrans
LFuc Type I,B determinant
1,3 GalNActrans
R
3Gal 1 3GlcNAc 1 1,2 LFuc
1,3 GalNActrans
1,3 Galtrans
R
R
Galc 1
GalNAc 1
3Gal 1 3GlcNAc 1 R 1,2 LFuc
1,4 LFuctrans
3Gal 1 3GlcNAc 1 R 1,2 1,4 LFuc LFuc B,Leb determinant
Type I,A determinant
Type I,B determinant
1,4 LFuctrans
GalNAc 1
3Gal 1 3GlcNAc 1 1,2 1,4 LFuc LFuc
R
A,Leb determinant
Fig. 3.2 (a) Proposed biosynthetic pathways for the formation of Type I A, Type I B, Type I H, Lea, Leb, sialyl Lea, ALeb, and BLeb determinant structures on Type I core structures of human ovarian cyst glycoproteins
3 Human Blood Group ABH/Ii, Lea,b,x,y, and Sialyl Lea,x Glycotopes
b
37
Gal 1 4GlcNAc 1 R Human blood group Type II precursor (II ) sequence
Gal 1 4GlcNAc 1 R 1,2 LFuc Type II,H determinant
1,2 LFuctrans
2,3 NeuAc
1,3 LFuctrans
4GlcNAc 1 R 1,3 LFuc Lex determinant
Gal 1 4GlcNAc 1 1,2 LFuc
Gal 1 4GlcNAc 1 R 2,3 1,3 LFuc NeuAc Sialyl Lex determinant
R
Type II,H determinant
1,3 Galtrans Gal 1 3Gal 1 4GlcNAc 1 1,2
1,3 LFuctrans
3Gal 1 4GlcNAc 1 R 1,2 LFuc Type II,A determinant 3Gal 1 4GlcNAc 1 1,2 LFuc
1,3 GalNActrans
1,3 Galtrans
R
R
LFuc Type II,B determinant
1,3 GalNActrans
GalNAc 1
Gal 1
1,3 LFuctrans
1,2 LFuctrans
Gal 1
Gal 1 4GlcNAc 1 R 1,2 1,3 LFuc LFuc Ley determinant
2,3 SAtrans Gal 1 4GlcNAc 1 R
Galc 1
GalNAc 1
3Gal 1 4GlcNAc 1 R 1,2 LFuc
1,3 LFuctrans
3Gal 1 4GlcNAc 1 R 1,2 1,3 LFuc LFuc B,Ley determinant
Type II,A determinant
Type II,B determinant
1,3 LFuctrans
GalNAc 1
3Gal 1 4GlcNAc 1 1,2 1,3 LFuc LFuc
R
A,Ley determinant
Fig. 3.2 (b) Proposed biosynthetic pathways for the formation of Type II A, Type II B, Type II H, Lex, Ley, sialyl Lex, ALey, and BLey determinant structures on Type II core structures of human ovarian cyst glycoproteins
are synthesized by an a1,4/1,3 fucosyltransferase (a1,4/1,3 lFuc-T). There is a relationship between the Le gene and Se gene, which is involved in the formation of Lewis glycotopes in an individual [20]. An individual Le/Le or Le/le, who belongs to nonsecretors (se/se), but does not synthesize a1,2 lFuc linkage (blood group H epitope) but can synthesize a1,4/1,3-linked lFuc, has in the secretions glycoproteins containing Lea/x determinant (Fig. 3.2a, b). On the other hand, an individual (Se/Se or Se/se, Le/Le or Le/le) who expresses a1,2 lFuc-T can form blood group H determinant that is followed by the action of a1,4/1,3 Fuc-T to synthesize Leb/y by adding the second aFuc to the 4/3 position of the penultimate GlcNAc. The Lex and Ley glycotopes were already found in early studies by Kabat’s group and were suggested to be coded by new genes of unknown functions [8, 38].
Table 3.2 Interactions of human ovarian cyst fluid glycoproteins with anti-A, -B, and -H monoclonal antibodies by ELISAa Maximum A405c Glycoprotein Absorbance Binding required for 1.5 reading intensity (A405) unit (ng) Human ovarian cyst glycoproteins A B H A B H A B H (glycotopesb; blood group specificity) I. Blood group A active glycoproteins a. Cyst MSS 10% 2× (Ah [A1]) 0.5 – – 3.9 0.0 0.0 5+ − − b. Cyst 9 (Ah [A1]) 0.5 – – 3.9 0.0 0.2 5+ − ± c. Cyst MSM 10% ppt (Ah [A1]) 5.1 – – 3.0 0.0 0.0 5+ − − d. HOC 88 2.3 – – 3.3 0.0 0.1 5+ − − e. Cyst 14 phenol insoluble (Ah 60.0 3.9 0.0 2.5 5+ − 5+ 0.7 –d [A2]) f. HOC 338 native 75.0 – 180.0 3.4 0.0 3.2 5+ − 5+ 0.7 – 80.0 3.3 0.0 2.3 5+ − 4+ g. Cyst Mcdon (Ah) h. Cyst 11 phenol insoluble 9.5 – 260.0 3.1 0.0 1.6 5+ − 3+ II. Blood group B active glycoproteins a. HOC 413 b. Cyst Beach phenol insoluble (Bh) c. Cyst Tij II 20% 2× (I/II, Bh) d. HOC 302 e. HOC 531
– –
16.0 0.5
– –
0.1 0.0
2.9 3.8
0.6 0.3
− −
5+ 1+ 5+ ±
– – –
3.5 5.5 8.0
– – –
0.4 0.1 0.0
3.0 2.9 3.0
0.0 0.0 0.1
± − −
5+ − 5+ − 5+ −
1.5 20.0
– –
3.1 3.9
3.1 2.6
0.0 0.0
5+ 5+
5+ − 5+ −
III. Blood group A and B active glycoproteins a. Cyst 19 2.5 b. HOC 89 1.2 IV. Blood group H active glycoproteins a. Cyst 12 b. Cyst Tighe phenol insoluble (H, Leb) c. Cyst JS phenol insoluble (H)
– –
– –
18.0 10.0
0.3 0.0
0.0 0.0
3.4 3.8
± −
− −
5+ 5+
–
–
4.0
0.0
0.0
4.2
−
−
5+
–
0.8
0.0
0.0
1+
−
−
– – – – – –
0.0 0.3 0.0 0.0 0.0 0.0
0.8 0.0 0.3 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0
− ± − − − −
1+ − ± − − −
− − − − − −
V. Blood group ABH inactive or weak glycoproteins a. HOC 350 (sialyl T/Tn, I/II, – – Lea/x) b. Cyst OG 10% 2× ppt (I/II) – – – – c. HOC 408 native (Lea) d. HOC 484 precursor (I/II) – – e. Cyst N-1 20% 2× (Lea) – – f. HOC F1 precursor (I/II) – – – – g. HOC 502 precursor (I/II)
a Dilution ratio of 1st and 2nd monoclonal antibodies: (1) anti-A (1:100), Anti-IgM (1:20,000); (2) anti-B (1:100), Anti-IgM (1:20,000); (3) anti-H (1:500), Anti-IgM (1:3,000) were added to various glycoproteins ranging from 0.1 ng to 1 mg b The symbol in parentheses indicates the human blood group activity and/or glycotopes. Expressed in bold are: A or Ah (GalNAca1-3[lFuca1-2]Gal); B or Bh (Gala1→3[lFuca1→2]Gal); H (lFuca1-2Gal); T (Galb1-3GalNAc); Tn (GalNAca1-Ser/Thr); I/II (Galb1-3/4GlcNAc) c Each well of the plate was coated with 50 ml of untreated or desialylated glycoproteins, etc. The plates were incubated with alkaline phosphates-conjugated goat antibodies against mouse IgM. All incubations were performed at room temperature. The results were interpreted according to the measured A450 after 4 h incubation with the substrate (PNPP) at room temperature in the dark as follows: 5+ (OD ³ 2.5), 4+ (2.5 > OD ³ 2.0), 3+ (2.0 > OD ³ 1.5), 2+ (1.5 > OD ³ 1.0), 1+ (1.0 > OD ³ 0.5), ± (0.5 > OD ³ 0.2), and − (OD sLea Leb/Ley Leb/Ley Leb/Ley
Lea/Lex Lex
sLea/Leb/Lex/Ley sLea/Lea/Leb/Ley sLea/Leb/Ley > Lea sLea/Leb/Ley > Lea sLea/Leb/Ley sLea/Leb > Ley sLea/Lea > Lex sLea
Table 3.3 Interactions of human ovarian cyst fluid glycoproteins with various anti-Lewis monoclonal antibodies by ELISAa Maximum A405c Human ovarian cyst glycoproteins Glycoprotein required for 1.5 (A405) b unit (ng) Absorbance reading Binding intensity (glycotopes ; blood group specificity) sLea sLex Lea Leb Lex Ley sLea sLex Lea Leb Lex Ley sLea sLex Lea Leb Lex Ley Lewis activities I. Sialyl Lea and sialyl Lex active glycoproteins a. HOC 350 (sialyl T/Tn, – – 4.4 3.5 2.4 0.0 0.1 0.0 5+ 5+ 4+ − − − sLea/sLex > Lea 10.0 90.0 160.0 –d I/II, Lea/x)
Table 3.3 (continued)
– –
80.0 – –
18.0
1.4
0.8 0.1
0.0 0.0 0.2
0.0
0.0
0.1 0.0
0.0 0.0 0.0
0.0
0.1 2.8 −
4.0 −
0.1 0.0 1.3 0.1 0.0 0.9
0.5 1+ 0.0 −
2.8 3.4 1.8 1.8 − 0.2 3.0 0.0 1.3 − 0.1 3.6 0.0 0.9 ±
0.1 3.1 0.0
0.5 4.1 0.0
− −
− − −
−
−
− −
5+ ± −
−
1+
− −
5+ 5+ 5+
5+
5+
2+ 1+
3+ − −
−
−
1+ −
3+ 2+ 1+
5+
5+
– –
Lea/Leb > Lex/Ley Leb Leb
Leb/Ley
Leb/Ley
Maximum A405c Absorbance reading Binding intensity sLea sLex Lea Leb Lex Ley sLea sLex Lea Leb Lex Ley Lewis activities
a
Dilution ratio of 1st and 2nd monoclonal antibodies: (1) anti-sialy Lea (1:500), anti-IgG (1:8,000); (2) anti-sialy Lex (1:500), anti-IgM (1:4,000); (3) anti-Lea (1: 1,000), anti-IgG (1:10,000); (4) anti-Leb (1:200), anti-IgM (1:10,000); (5) anti-Lex (1:250), anti-IgM (1:5,000); (6) anti-Ley (1:125), anti-IgM (1:5,000) were added to various glycoproteins ranging from 0.1 ng to 1 mg b The symbol in parentheses indicates the human blood group activity and/or glycotopes. Expressed in bold are: A or Ah (GalNAca1-3[lFuca1-2]Gal); B or Bh (Gala1→3[lFuca1→2]Gal); H (lFuca1-2Gal); T (Galb1-3GalNAc); Tn (GalNAca1-Ser/Thr); I/II (Galb1-3/4GlcNAc) c Each well of the plate was coated with 50 ml of untreated or desialylated glycoproteins, etc. The plates were incubated with alkaline phosphates-conjugated goat antibodies against mouse IgM. All incubations were performed at room temperature. The results were interpreted according to the measured A450 after 4 h incubation with the substrate (PNPP) at room temperature in the dark as follows: 5+ (OD ³ 2.5), 4+ (2.5 > OD ³ 2.0), 3+ (2.0 > OD ³ 1.5), 2+ (1.5 > OD ³ 1.0), 1+ (1.0 > OD ³ 0.5), ± (0.5 > OD ³ 0.2), and – (OD Lex
MWCO molecular weight (mass) cutoff a Dilution ratio of 1st and 2nd monoclonal antibodies: (1) anti-A (1:100), anti-IgM (1:20,000); (2) anti-B (1:100), anti-IgM (1:20,000); (3) anti-H (1:500), anti-IgM (1:3,000) were added to various glycoproteins ranging from 0.1 ng to 1 mg b The symbol in parentheses indicates the human blood group activity and/or glycotopes. Expressed in bold are: A or Ah (GalNAca1-3[lFuca1-2]Gal); B or Bh (Gala1→3[lFuca1→2] Gal); H (lFuca1-2Gal); T (Galb1-3GalNAc); Tn (GalNAca1-Ser/Thr); I/II (Galb1-3/4GlcNAc) c Each well of the plate was coated with 50 ml of untreated or desialylated glycoproteins, etc. The plates were incubated with alkaline phosphates-conjugated goat antibodies against mouse IgM. All incubations were performed at room temperature. The results were interpreted according to the measured A450 after 4 h incubation with the substrate (PNPP) at room temperature in the dark as follows: 5 + (OD ³ 2.5), 4 + (2.5 > OD ³ 2.0), 3 + (2.0 > OD ³ 1.5), 2 + (1.5 > OD ³ 1.0), 1 + (1.0 > OD ³ 0.5), ± (0.5 > OD ³ 0.2), and – (OD 3.0 × 104) mild acid hydrolysis-I at (pH 2.0, 80°C for 90 min) a. Asialo HOC 350 (T/Tn, – – –d – – 39.0 – 420.0 – 1.4 0.0 0.0 0.1 0.0 2.5 0.0 1.6 0.0 2+ I/II, Lea/x) b. Asialo HOC 484 (I/II) – – – – – – – 67.0 – 0.2 0.3 0.1 0.0 0.0 0.0 0.0 2.6 0.0 ± c. Asialo HOC 408 – – – – – – – – – 0.2 0.0 0.0 0.0 0.0 1.2 0.0 0.0 0.0 ± d. Asialo HOC 502 (I/II) – – – – – – – – – 0.0 0.0 0.0 0.0 0.0 0.3 0.0 0.7 0.1 − e. Asialo HOC F1 (I/II) – – – – – – – – – 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.2 −
Human ovarian cyst glycoproteins Glycoprotein required for 1.5 (A405) unit (ng) (glycotopesb; blood group specificity) A B H sLea sLex Lea Leb Lex
Table 3.4 Interactions of mild acid hydrolyzed cyst glycoproteins, P-1, and Smith degraded glycoproteins from human ovarian cyst fluid with various monoclonal antibodies by ELISAa
3 Human Blood Group ABH/Ii, Lea,b,x,y, and Sialyl Lea,x Glycotopes
a
45
Gal/GalNAc 1,3 B, A or Fuc 1 2Gal B-Leb/y, A-Leb/y 1,3/4 + Fuc 1 4/3GlcNAc 1,6 GalNAc/Gal 1 3Gal 1 3GalNAc 1 Ser/Thr 1,2 Fuc A, B
b
GalNAc/Gal 1,3 Fuc 1 2Gal 1,3/4 + Fuc 1 4/3GlcNAc 1,6 + NeuAc 2 3Gal 1 3GalNAc 1 + 1,2 Fuc
GalNAc/Gal 1 3Gal 1 3/4GlcNAc 1 3Gal 1 3GalNAc 1 + 1,4/3 1,2 Fuc Fuc
A, B or A-Leb/y, B-Leb/y
Ser/Thr
Ser/Thr
(i)
(ii)
A, B or A-Leb/y, B-Leb/y
Fig. 3.3 O-glycan structures from human HOC 89 and Cyst 19 that may carry blood group A and B antigens. (a) Structures that carry both A and B antigens. A minimal structure identified in this study carries the B determinant on the 6-arm and A determinant directly attached to the C3 of GalNAc, although the full repertoire most likely includes both A and B on either arm of the core 2 structure. Further heterogeneity arises from (1) LacNAc extension from the core before termination with the A/B determinants and (2) internal a3/4-fucosylation to give B-Leb/y and A-Leb/y. (b) Structures that carry only A or B antigens. This subset corresponds to those core 2 structures, where the Gal on the Galb1-3GalNAc core can either be directly sialylated or a2-fucosylated to give H antigen but not further substituted to give either A or B determinant (i) or core 1 structures with only one terminus (ii). Boxed and shaded areas represent the blood group A and B active glycotopes
Despite distinctively different O-glycosylation profiles for the two HOC, their interaction profiles with human blood group A and B active lectins and monoclonal antibodies against AB glycotopes were similar. They also showed similar profiles with respect to IIb, Tn, and Man-specific lectins. It thus suggests that the primary recognition events do not extend beyond the terminal ABH determinants to additional internal sites (Tables 3.5 and 3.6).
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A.M. Wu
Table 3.5 Binding profiles of HOC 89 and Cyst 19 with various human blood group A and B, Galb1→, GalNAc b1→ and Man-specific lectins by ELLSA Maximum A405 Binding Amount 1.5 (A405) Determinantsa absorbanceb intensityb of lectin unit (ng) (carbohydrate (ng) specificities) Lectin HOC 89 Cyst 19 HOC 89 Cyst 19 HOC 89 Cyst 19 50 100.0 15.0 GSI-A4 A > Ah >> B 3.5 4.1 5+ 5+ GSI-B4 B > E > A 100 300.0 15.0 2.5 4.3 5+ 5+ HPL F > A(>Ah) Tn, T 20 11.0 31.0 2.8 2.2 5+ 4+ RCA1 II > I > B > T >> Tn 5 1900.0 70.0 1.7 2.5 3+ 5+ WGA mGlcNAcb1 → >mTn 5 15.0 13.0 3.3 2.9 5+ 5+ VVL-B4 mTn >> T 5 − − 0.1 0.4 − ± 20 Morniga Mb1 → 4C − − 0.1 0.3 − ± M Carbohydrate specificity of lectins as expressed by lectin determinants — F, GalNAca1→3GalNAc; A, GalNAca1→3Gal; Ah, GalNAca1→3[lFuca1→2]Gal; Tn, GalNAcal→Ser/Thr; T, Galb1→3GalNAc; I/II, Galb1→3/4GlcNAc; m, multivalent; B, Gala1→3Gal; E, Gala1→4Gal; M, the trimannosidic core structure in N-linked glycoprotein; C, GlcNAcb1→4GlcNAc (chitin disaccharide) b Results were interpreted according to the measured A405 after 4 h of incubation as follows: 5+ (O.D. ≥ 2.5), 4+ (2.5 > O.D. ≥ 2.0), 3+ (2.0 > O.D. ≥ 1.5), 2+ (1.5 > O.D. ≥ 1.0), 1+ (1.0 > O.D. ≥ 0.5), ± (0.5 > O.D. ≥ 0.2), and – (O.D. < 0.2) a
From the results of this study, long missing information with respect to the loci and structural details of secreted glycoproteins carrying both human blood group A and B glycotopes is now provided. In particular, the identification of blood group A/A-Leb/y and B/B-Leb/y active glycotopes coexpressed on the cyst O-glycans is expected to further advance the field of structural immunohematology. Analysis of the glycosylation profiles of glycoconjugates from human blood AB-type erythrocytes is planned for the future.
3.6 Summary Pseudomucinous-type HOC is one of the pioneer sources of human blood active substances for studying blood group active glycoproteins and glycotopes. In this chapter, the most comprehensive analysis of the carbohydrate moieties of HOC with human blood group ABH, Lea, Leb, Lex, Ley, sLea, and sLex is described. The conclusions concerning blood group glycotopes present on the HOC and the common internal structures of O-glycans are based on the data obtained during the past six decades (Fig. 3.1, 3.3, 3.4 and Table 3.1).
Binding intensityb HOC 350 Native Asialo− 1+ − − ± 3+ − 3+ ± 3+ − 5+ − 5+ 3+ 5+ − 3+ 1+ 5+ 1+ 5+ − 4+ 5+ 5+ ± 5+ − 5+ − 5+ − 4+ − − − − − − − − − −
a
Carbohydrate specificity of lectins as expressed by lectin determinants: F, GalNAca1→3GalNAc; A, GalNAca1→3Gal; Ah, GalNAca1→3[lFuca1→2]Gal; Tn, GalNAca→Ser/Thr; T, Galb1→3GalNAc; I/II, Galb1→3/4GlcNAc; m, multivalent; B, Gala1→3Gal; E, Gala1→4Gal; M, the trimannosidic core structure in N-linked glycoprotein; C, GlcNAcb1→4GlcNAc (chitin disaccharide) b Results were interpreted according to the measured A405 after 4 h of incubation as follows: 5 + (OD ³ 2.5), 4 + (2.5 > OD ³ 2.0), 3 + (2.0 > OD ³ 1.5), 2 + (1.5 > OD ³ 1.0), 1 + (1.0 > OD ³ 0.5), ± (0.5 > OD ³ 0.2), and − (OD A(>Ah) ≧ Tn, T − − DBA F > Ah > A > Tn 20 − − 0.01 0.0 CFA F, Ta > Tn > Ah >> I/II 25 − 40.0 0.2 1.8 GSI-A4 A > Ah >> B 20 − 350.0 0.01 1.8 SBA A(>Ah), Tn, I/II 20 − 160.0 0.2 1.5 VVL-B4 mTn >> T 5 − 120.0 0.07 2.5 PNA T >> I/II 50 − 6.2 0.04 4.0 ACL T >> I/II 5 450.0 70.0 1.6 2.5 MPL T, Tn 5 − 140.0 0.1 1.5 Jacalin mT, mTn >> I/II 10 − 4.0 0.7 2.6 BPL T > I/II, Tn 5 − 5.0 0.8 2.6 APA T > I/II > E > B >> Tn 4 − 8.0 0.1 2.3 WGA mGlcNAcb1 → >mTn 5 200.0 22.0 3.7 4.3 RCA1 II > I > B > T >> Tn 5 − 30.0 0.3 3.7 Ricin T > I/II, Tn 5 − 300.0 0.01 3.9 ECL I/II > B > Tn 10 − 400.0 0.02 3.3 ECorL I/II > B > Tn 50 − 450.0 0.05 2.2 Abrin-a E, II 10 − − 0.0 0.0 GSI-B4 B > E > A 25 − − 0.0 0.01 M-II Mb1 → 4C 20 − − 0.1 0.1 PSA Mb1 → 4C 50 − − 0.01 0.01 50 0.01 0.01 Lentil − − Mb1 → 4C
3 Human Blood Group ABH/Ii, Lea,b,x,y, and Sialyl Lea,x Glycotopes 47
A.M. Wu
48
(i) Simple (short) type cyst carbohydrate chain in HOC 350
Branch 3 NeuAc 2 3/6Gal
1,3/4 sLea/x or Lea/x 4/3GlcNAc 1,3 Gal 1,4 GlcNAc NeuAc Fuc + 2,3/6 1,6 1,4/3 + Gal 1 3/4GlcNAc 1 3Gal 1 3GalNAc 1 Ser/Thr + 1,6 + sLea/x or Lea/x Fuc 1 3GlcNAc sLex or Lex 1,4 + NeuAc 2 3/6Gal Fuc 1
Branch 4 (ii) Internal structure of HOC 350
Branch 3 Gal
Gal 1
+
3/4GlcNAc 1 3Gal 1 + 1,6 I or II GlcNAc 1,4 Gal
1,3/4 I or II GlcNAc 1,3 Gal 1,4 II GlcNAc 1,6 3GalNAc 1 Ser/Thr II
Branch 4 Fig. 3.4 The proposed overall structures of a Lea, sLea, and sLex active glycoprotein isolated from pseudomucinous-type of human ovarian cyst fluid (HOC 350)
Acknowledgements This work was supported by grants from the Chang Gung Medical Research plan (CMRPD No. 180482 and 170443) Kwei-san, Tao-yuan, Taiwan, and the National Science Council (NSC 97-2628-B-182-002-MY3 and 97-2320-B-182-020-MY3) Taipei, Taiwan. The author also thanks Dr. Z. Yang and our co-workers for assistance of the work and arrangement of manuscript typing.
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References 1. Beauharnois ME, Lindquist KC, Marathe D, Vanderslice P, Xia J, Matta KL, Neelamegham S (2005) Affinity and kinetics of sialyl Lewis-X and core-2 based oligosaccharides binding to L- and P-selectin. Biochemistry 44:9507–9519 2. Buchanan DJ, Rapoport S (1951) Composition of meconium: serological study of blood group specific substances found in individual meconiums. Proc Soc Exp Biol Med 77:114–117 3. Cheese IAFL, Morgan WTJ (1961) Two serologically active trisaccharides isolated from human blood-group A substance. Nature 191:149–150 4. Clausen H, Hakomori S (1989) ABH and related histo-blood group antigens; immunochemical differences in carrier isotypes and their distribution. Vox Sang 56:1–20 5. Dall’Olio F, Chiricolo M (2001) Sialyltransferases in cancer. Glycoconj J 18:841–850 6. Del Villano BC, Brennan S, Brock P, Bucher C, Liu V, McClure M, Rake B, Space S, Westrick B, Schoemaker H, Zurawski VR (1983) Radioimmunometric assay for a monoclonal antibodydefined tumor marker, CA 19-9. Clin Chem 29:549–552 7. Donald ASR (1981) A-active trisaccharides isolated from A1 and A2 blood group specific glycoproteins. Eur J Biochem 120:243–249 8. Feizi T, Lloyd KO (2001) An appreciation of Elvin A. Kabat (1914-2000): scientist, educator and a founder of modern carbohydrate biology. Glycobiology 11:15G–18G 9. Fukuda M, Fukuda MN (1981) Changes in cell surface glycoproteins and carbohydrate structures during the development and differentiation of human erythroid cells. J Supramol Struct 17:313–324 10. Hakomori S (2001) Tumor-associated carbohydrate antigens defining tumor malignancy: basis for development of anti-cancer vaccines. In: Wu AM (ed) The molecular immunology of complex carbohydrates-2 in Adv Exp Med Biol. Plenum Press, New York and London 11. Hakomori S, Kannagi R (1986) Carbohydrate antigens in higher animals. In: Weir DM, Herzenberg LA, Blackwell CC (eds) Handbook of experimental immunology. Blackwell Scientific Publications, Oxford 12. Jain RK, Piskorz CF, Huang BG, Locke RD, Han HL, Koenig A, Varki A, Matta KL (1998) Inhibition of L- and P-selectin by a rationally synthesized novel core 2-like branched structure containing GalNAc-Lewisx and Neu5Acalpha2-3Galbeta1-3 GalNAc sequences. Glycobiology 8:707–717 13. Kabat EA (1956) Blood group substances: their chemistry and immunochemistry. Academic Press, New York 14. Kabat EA (1976) Antibody (and lectin) combining sites for elucidation of structures of antigenic determinant. In: Kabat EA(ed) Structural Concepts in Immunology and Immunochemistry, 2nd edn. In Holt, Rinehart and Winston, New York, pp. 167–200 15. Kabat EA (1982) Contributions of quantitative immunochemistry to knowledge of blood group A, B, H, Le, I and i antigens. Am J Clin Pathol 78:281–292 16. Kannagi R (2004) Molecular mechanism for cancer-associated induction of sialyl Lewis x and sialyl Lewis a expression- the Warburg effect revisited. Glycoconj J 20:353–364 17. Kannagi R, Kitahara A, Itai S, Zenita K, Shigeta K, Tachikawa T, Nuda A, Hirano H, Abe M, Shin S, Fukushi Y, Hakomori S, Imura H (1988) Quantitative and qualitative characterization of human cancer-associated serum glycoprotein antigens expressing epitopes consisting of sialyl or sialyl-fucosyl type 1 chain. Cancer Res 48:3856–3863 18. Kogan TP, Dupré B, Bui H, McAbee KL, Kassir JM, Scott IL, Hu X, Vanderslice P, Beck PJ, Dixon RA (1988) Novel synthetic inhibitors of selectin-mediated cell adhesion: synthesis of 1, 6-bis[3-(3-carboxymethylphenyl)-4-(2-alpha-D-mannopyranosyloxy) phenyl]hexane (TBC1269). J Med Chem 41:1099–1111 19. Landsteiner K, Wiener AS (1940) An agglutinable factor in human blood recognized by immune sera for rhesus blood. Proc Soc Exp Biol Med 43:223–224 20. Ley K, Kansas GS (2004) Selectins in T-cell recruitment to non-lymphoid tissues and sites of inflammation. Nat Rev Immunol 4:325–335
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21. Lloyd KO, Kabat EA (1968) Immunochemical studies on blood groups. XLI. Proposed structures for the carbohydrate portion of blood group A, B, H, Lewisa and Lewisb substances. Proc Natl Acad Sci USA 61:1470–1477 22. Lowe JB (2002) Glycosylation in the control of selectin counter-receptor structure and function. Immunol Rev 186:19–36 23. Lowe JB, Stoolman LM, Nair RP, Larsen RD, Berhend TL, Mark RM (1990) ELAM-1dependent cell adhesion to vascular endothelium determined by a transfected human fucosyltransferase cDNA. Cell 63:475–484 24. Magnani JL, Steplewski Z, Koprowski H, Ginsburg V (1983) Identification of the gastrointestinal and pancreatic cancer-associated antigen detected by monoclonal antibody 19-9 in the sera of patients as a mucin. Cancer Res 43:5489–5492 25. Morgan WTJ (1960) A contribution to human biochemical genetics; the chemical basis of blood-group specificity. Proc R Soc Lond B Biol Sci 151:308–347 26. Morgan WTJ, van Heyningen R (1944) The occurrence of A B and O blood group substances in pseudomucinous ovarian cyst fluids. Br J Exp Pathol 25:5–15 27. Morgan WTJ, Watkins WM (2000) Unravelling the biochemical basis of blood group ABO and Lewis antigenic specificity. Glycoconj J 17:501–530 28. Neelamegham S (2004) Transport features, reaction kinetics and receptor biomechanics controlling selectin and integrin mediated cell adhesion. Cell Commun Adhes 11:35–50 29. Oriol R, Le Pendu J, Mollicone R (1986) Genetics of ABO, H, Lewis, X and related antigens. Vox Sang 51:161–171 30. Pusztai A, Morgan WTJ (1961) Studies in immunochemistry. 18. The isolation and properties of a sialomucopolysaccharide possessing blood group Lea specificity and virus-receptor activity. Biochem J 78:135–146 31. Rao BN, Anderson MB, Musser JH, Gilbert JH, Schaefer ME, Foxall C, Brandley BK (1994) Sialyl Lewis x mimics derived from a pharmacophore search are selectin inhibitors with antiinflammatory activity. J Biol Chem 269:19663–19666 32. Rosen SD, Singer MS, Yednock TA, Stoolman LM (1985) Involvement of sialic acid on endothelial cells in organ-specific lymphocyte recirculation. Science 228:1005–1007 33. Vestweber D, Blanks JE (1999) Mechanisms that regulate the function of the selectins and their ligands. Physiol Rev 79:181–213 34. Watkins WM (1972) Blood group specific substance. In: Gottschalk A (ed) Glycoproteins. Elsevier, Amsterdam 35. Watkins WM (1981) Biochemistry and genetics of the ABO, Lewis, and P blood group systems. In: Harris H, Hirschhorn K (eds) Advances in human genetics. Plenum Press, New York 36. Watkins WM (1995) Molecular basis of antigenic specificity in the ABO, H and Lewis bloodgroup systems. In: Montreuil J, Vliegenthart JFG, Schachter H (eds) Glycoproteins. Elsevier, Amsterdam 37. Weston BW, Hiller KM, Mayben JP, Manousos G, Nelson CM, Klein MB, Goodman JL (1999) A cloned CD15s-negative variant of HL60 cells is deficient in expression of FUT7 and does not adhere to cytokine-stimulated endothelial cells. Eur J Haematol 63:42–49 38. Wu AM (1988) Structural concepts of the blood group A, B, H, Lea, Leb, I and i active glycoproteins purified from human ovarian cyst fluid. Adv Exp Med Biol 228:351–394 39. Wu AM, Kabat EA, Nilsson B, Zopf DA, Gruezo FG, Liao J (1984) Immunochemical studies on blood groups. Purification and characterization of radioactive 3H-reduced di- to hexasaccharides produced by alkaline beta-elimination-borohydride 3H-reduced of Smith degraded blood group A active glycoproteins. J Biol Chem 259:7178–7186 40. Wu AM, Kabat EA, Pereira MEA, Gruezo FG, Liao J (1982) Immunochemical studies on blood groups: the internal structure and immunological properties of water-soluble human blood group A substance studied by Smith degradation, liberation, and fractionation of oligosaccharides and reaction with lectins. Arch Biochem Biophys 215:390–404 41. Wu AM, Khoo KH, Yu SY, Yang Z, Kannagi R, Watkins WM (2007) Glycomic mapping of pseudomucinous human ovarian cyst glycoproteins: identification of Lewis and sialyl Lewis glycotopes. Proteomics 7:3699–3717
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42. Wu AM, Wu JH, Kuo HW, Herp A (2005) Further characterization of the binding properties of two monoclonal antibodies recognizing human Tn red blood cells. J Biomed Sci 12:153–166 43. Wu AM, Wu JH, Singh T, Liu JH, Tsai MS, Gilboa-Garber N (2006) Interactions of the fucose-specific Pseudomonas aeruginosa lectin, PA-IIL, with mammalian glycoconjugates bearing polyvalent Lewis and ABH blood group glycotopes. Biochimie 88:1479–1492 44. Wu AM, Wu JH, Tsai MS, Yang Z, Sharon N, Herp A (2007) Differential affinities of Erythrina cristagalli lectin (ECL) toward monosaccharides and polyvalent mammalian structural units. Glycoconj J 24:591–604 45. Yang Z, Tsai MS, Wu JH, Herp A, Wu AM (2008) Defining the carbohydrate specificities of Erythrina corallodendron lectin (ECorL) as polyvalent Galb1-4GlcNAc (II) > monomeric II > monomeric Gal and GalNAc. Chang Gung Med J 31:26–43 46. Yang Z, Wu AM, Kuo HW, Wu JH, Kannagi R (2009) Expression of sialyl Lex, sialyl Lea, Lex and Ley glycotopes insecreted human ovarian cyst glycoproteins. Biochimie 91:423–433 47. Yip SP (2002) Sequence variation at the human ABO locus. Ann Hum Genet 66:1–27 48. Zopf D, Hansson GC (1988) The chemical basis for expression of the sialyl-Lea antigen. In: Wu AM (ed) The molecular immunology of complex carbohydrates in Adv Exp Med Biol. Plenum Press, New York and London
Chapter 4
Lewis Glyco-Epitopes: Structure, Biosynthesis, and Functions Hui-Li Chen
Keywords Lewis antigen • Cancer metastasis • Cell biological behaviors The Lewis glyco-epitope, also called the Lewis antigen (Le), is a series of a1→3/a1→4 fucosylated oligosaccharide epitopes derived from the Galb1→3/1→4GlcNAcb1→R carbohydrate backbone and that have been identified on the surface of certain eukaryotic and prokaryotic cells. The a1→3 or a1→4 fucose in Lewis glyco-epitopes is attached on GlcNAc residue, and the Gal residue can be unsubstituted, a2→3 sialylated, a1→2 fucosylated, or sulfated. They are structurally related to the sugar determinants of the human ABH(O) blood group antigen system. The “Lewis” blood group antigen was named for a family of individuals suffering from a red blood cell incompatibility, which led to the discovery of this blood group [1]. Recently, the biological significance of Lewis epitopes has been widely studied. Some of them became recognized as tumor markers and are being used in the clinical diagnosis and prognosis of certain cancers, and others were found to be involved in the pathogenesis of stomach disorders and embryo development. More interestingly, Lewis epitopes have been implicated in the regulation of cell biological events, such as cell signaling, growth, apoptosis, adhesion, and migration. This review will introduce some of the major advances in the research of the structure and function of these Lewis epitopes.
4.1 Structures of Lewis Glyco-Epitopes Lewis epitopes are classified into type 1 a/b series and type 2 x/y series. The former is synthesized from Galb1→3GlcNAcb1→R (type 1 chain) with a1→4Fuc on the b1→3 linked GlcNAc residue, while the latter is from Galb1→4GlcNAcb1→R H.-L. Chen (*) Key Laboratory of Glycoconjugate Research, Ministry of Health, Department of Biochemistry, Shanghai Medical College, Fudan University, Shanghai 200032, China e-mail:
[email protected] A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_4, © Springer Science+Business Media, LLC 2011
53
54 Lea:
H.-L. Chen Galβ1®3GlcNAcb1®R
Lex:
4
3
� Fuca1 Leb:
Galβ1®4GlcNAcb1®R � Fuca1
Fuca1®2Galb1®3GlcNAcb1®R
Ley:
Fuc a1®2Galb1®4GlcNAcb1®R 3
4
� Fuca1
� Fuca1 SLea: NeuAca2®3Galb1®3GlcNAcb1®R
SLex:
NeuAca2®3Galb1®4GlcNAcb1®R
4
3
� Fuca1 a/b series
� Fuca1 x/y series
Fig. 4.1 Structure of six main Lewis antigens
(type 2 chain) with a1→3Fuc on the b1→4 linked GlcNAc residue. Lewis b (Leb) and Lewis y (Ley) contain another a1→2Fuc residue attached to the Gal residue, while Lewis a (Lea) and Lewis x (Lex) have only one Fuc linked to GlcNAc residue. The Gal on Lea and Lex can be sialylated with a2→3NeuAc to yield sialyl Lewis a (SLea) and sialyl Lewis x (SLex), respectively. The structures of the six main Lewis antigens mentioned above are shown in Fig. 4.1 [2–5]. In those sugar chains with [Galb1→4GlcNAcb1-3]n (n ³ 2) repeat sequence (also called type 2 poly-N-acetyllactosamine), the outer two or three b1→4GlcNAc residues may be all a1→3 fucosylated and produce sialyl difucosyl Lewis x (SDLex) and sialyl trifucosyl Lewis x (STLex). If the outermost b1→4GlcNAc residue in SDLex is not fucosylated, the oligosaccharide is called VIM-2 (Fig. 4.2) [2]. It has been found that the Gal residue in Lea and Lex can also link to the sulfo group (SO3−) at 3¢-position instead of the sialyl group as in 3¢-sulfo-Lewis a (3¢sulfo-Lea) and 3¢-sulfo-Lewis x (3¢sulfo-Lex). Moreover, sialyl Lewis x can be monosulfated at 6-position of GlcNAc or 6¢-position of Gal to produce 6-sulfo- or 6¢-sulfo-sialyl Lewis x (6sulfo-SLex, 6¢sulfo-SLex) (Fig. 4.3). Recently, disulfated Lewis x (6¢,6-bis-sulfo Lewis x) at both Gal (6¢-position) and GlcNAc (6-position) residues (Fig. 4.3) and disialylated Lewis a (DiSLea) at both Gal (NeuAca2→3) and GlcNAc (NeuAc a2→6) residues (Fig. 4.4) were discovered [2, 5]. In rare cases, GalNAc may be attached to GlcNAc residue instead of Gal and form GlaNAcLex (Fig. 4.4). If there are no a1→4 and a1→3 fucosyl substitutions at GlcNAc in Leb and Ley, the structures of Leb and Ley become the H antigen of blood group O, the simplest antigen in the ABH blood group system. If there is a a1→3GalNAc or a1→3Gal substitution at the Gal residue of the H antigen, the oligosaccharide will be modified to the A or B antigen of blood groups A and B, respectively (Fig. 4.5) [2]. Therefore, the structures of Lewis antigens are closely related to the ABH(O) blood group antigen system.
4 Lewis Glyco-Epitopes: Structure, Biosynthesis, and Functions SDLex:
55
Neua2®3Gal b 1®4GlcNAc b 1®3Gal b 1®4GlcNAc b 1®3Gal b 1®R 3 3 � � Fuca1 Fuca1
STLex: Neua2®3Gal b 1®4GlcNac b 1®3Gal b 1®4GlcNAc b 1®3Gal b 1®4GlcNAc b 1®3Gal b 1®R 3 3 3 ¯ � � Fuca1 Fuca1 Fuca1
VIM-2:
Neua2®3Gal b 1®4GlcNAc b 1®3Gal b 1®4GlcNAc b 1®3Gal b 1®R 3 � Fuca1
Fig. 4.2 Structures of long-chain Lewis antigens
3’sulfo-Lea:
3’sulfo-Lex:
SO3-® 3Galb1®3GlcNAcb1®R 4 � Fuca1
6sulfo-SLex:
SO3-® 3Galb1®4GlcNAcb1®R 3 � Fuca1
6’sulfo-SLex:
SO3¯ 6 NeuAca2 ® 3Galb1 ®4GlcNAcb1®R 3 � Fuca1
SO3¯ 6’ NeuAca2 ® 3Galb1 ®4GlcNAcb1®R 3 � Fuca1
6’6 bis-sulfo-SLex:
SO3SO3¯ ¯ 6 6 NeuAca2 ® 3Galb1 ®4GlcNAcb1®R 3 � Fuca1
Fig. 4.3 Structures of sulfated Lewis antigens
DiSLea:
NeuAca2 ¯ 6 NeuAca2 ® 3Galb1®3GlcNAcb1®R 4 � Fuca1
GalNAcLex:
Fig. 4.4 Structures of two uncommon Lewis antigens
GalNAcb1®4GlcNAcβ1®R 3 � Fuca1
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Type 1 (H type) H antigen:
A antigen:
B antigen:
Type 2 (H type) Galb1®4GlcNAcb1®R 2 � Fuca1
Galb1®3GlcNAcb1®R 2 � Fuca1 GalNAca1®3Galb1®3GlcNAcb1®R 2 � Fuca1
GalcNAca1®3Galb1®4GlcNAcb1®R 2 � Fuca1
Gala1®3Galb1®3GlcNAcb1®R 2 � Fuca1
Gala1®3Galb1®4GlcNAcb1®R 2 � Fuca1
Fig. 4.5 The antigen structures of the ABH(O) blood group
4.2 Localization of Lewis Glyco-Epitopes and Detection with Specific Antibodies Lewis antigens are located at the termini of Ser/Thr-linked oligosaccharides (O-glycans) and Asn-linked oligosaccharides (N-glycans) on glycoproteins, especially at O-glycans with b1→6 GlcNAc branching of the C2 or C4 core [6]. The neutral (unsialylated or unsulfated) Lewis antigens (Lea, Leb, Lex, Ley) are also present at the termini of some fucosylated glycosphingolipids [7]. Lewis antigens are predominately present on cell surface glycoconjugates and, to a lesser extent, in the cytoplasm, but not in the cell nuclei. They are also found in the capsular glycoconjugates of prokaryotic cells. However, some Lewis antigens can be detected in serum, and the mucin glycoproteins (rich in O-glycans) can be detected in the secretory fluids of humans, including the secretions of the salivary gland, gastrointestinal tract, and reproductive tract. Monoclonal antibodies have been raised for the detection of Lewis antigens. However, monoclonal antibodies used against the same Lewis antigen but produced by different companies usually have different names and numbers. Most of them are immunoglobulin G or M (IgG, IgM) [5, 8, 9]. Table 4.1 summarizes examples of some monoclonal antibodies that have been reported.
4.3 Biosynthesis of Lewis Glyco-Epitopes and Related Glycosyltransferases Lewis glyco-epitopes are biosynthesized by a series of glycotransferases. The trisaccharide Lea and Lex are usually considered minimum structures of Lewis epitopes. Their synthesis requires N-acetylglucosaminyltransferase (GnT),
4 Lewis Glyco-Epitopes: Structure, Biosynthesis, and Functions
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Table 4.1 The monoclonal antibodies used in the detection of Lewis antigens Lewis antigen Symbol Monoclonal antibody Lewis a Lea 7Le Lewis b Leb T128, 2.25Le Lewis x Lex CD15, FH-2, 6H3 (CB-10) Lewis y Ley AH-6, HpN35 (540), ABL364 Sialyl Lewis a SLea CA-19-9, N19-9, SPan-1.KMO1 Sialyl Lewis x SLex KM93, NCC-ST-439*, CSLEX-1 Sialyl difucosyl Lewis x SDLex FH-6 3¢-sulfo-Lewis a 3¢sulfo-Lea SU59# 3¢-sulfo-Lewis x 3¢sulfo-Lex SU59# x 6-sulfo-Lewis x 6sulfo-Le AG107 6¢-sulfo-sialyl Lewis x 6sulfo-SLex G159 FH-7 Disialyl Lewis a DiSLea * Recognizes Lewis x carried by O-glycans with C2/C4 core This monoclonal antibody cross-reacted with both 3¢sulfo-Lea and 3¢sulfo-Lex
#
galactosyltransferase (GalT), and fucosyltransferases (FucT), which are all enzyme families. In the synthesis of acidic Lewis antigens, sialyltransferases and, in some cases, sulfotransferases must be involved. In the cells, the glycosyltransferases are mainly located in the different portions (cis, medial, trans) of the Golgi apparatus.
4.3.1 N-Acetylglucosaminyltransferase GnT catalyzes the transference of GlcNAc residue from UDP-b-d-GlcNAc to an acceptor, usually a sugar chain. It can be divided into b1→2, b1→3, b1→4, and b1→6 subfamilies. Among them, b1→2 GnTs, including GnT-1 and GnT-2, participate in the synthesis of C2C2 biantennary N-glycans, and the transferred GlcNAc residue is attached to the a1→3 and a1→6 mannoside (a1→3 and a1→6 arm), respectively, on the pentasaccharide core of N-glycans [10] (Fig. 4.6). They are not directly involved in the synthesis of Lewis antigens. The b1→3GnT subfamily includes eight glycosyltransferases that add GlcNAc to the Gal residue in glycoproteins and glycolipids. To date, three b1→3GnTs (b3GnT-2, -4, -8) participate in the synthesis of poly-N-acetyllactosamine (also called i antigen), which is the precursor of long-chain Lewis antigens, such as sialyl difucosyl Lewis x, sialyl trifucosyl Lewis x, and VIM-2 (refer to Fig. 4.2). Among them, b1→3GnT-2 shows the strongest activity for poly-N-acetyllactosamine synthesis both in vitro and in vivo, while b1→3GnT-4 has very weak activity (the real substrate of b1→3GnT-4 in vivo is unclear at present) [11]. b1→3GnT-2 and -8 have been found to show synergistic activities in poly-N-acetyllactosamine synthesis and complement the function of each other in cells. In colon cancer, the synthesis of poly-N-acetyllactosamine is significantly enhanced. Interestingly, the transcript of b1→3GnT-8 is upregulated in colon cancer but that of b1→3GnT-2 is not.
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GnT-V
GlcNAcb1
GnT-VI
GlcNAcb1
GnT-II
GlcNAcb1
6 4 Mana1 2
6 GnT-III
GlcNAcb1
4Manb1®4GLcNAc b1®4GLcNAcβ1®Asn 3
GnT-IV
GnT-I
GlcNAcb1
GlcNAcb1
4 Mana1 2
Fig. 4.6 The positions of GlcNAc residue added by GnT-I to GnT-VI in the processing of N-glycans
In addition, b1→3GnT-3 that adds GlcNAc to b1→3 linked Gal in core 1 of O-glycans (Galb1→3GalNAca1−) is responsible for the elongation of core 1, and b1→3GnT-5 that adds GlcNAc to lactosylceramide participates in the synthesis of lactose (Lc)- and neolactose (nLc)-series of glycosphingolipids. b1→3GnT-6 adds the GlcNAc in core 3 of the O-glycans (GlcNAcb1→3GalNAca1−), while b1→3GnT-7 participates in the synthesis of keratan sulfate [11]. After the extension of N-, O-glycans, and glycosphingolipids by GalTs, these sugar chains may be further processed to produce Lewis antigens at their termini. b1→4GnTs consist of three GnTs (GnT-III, GnT-IV, and GnT-VI); however, GnT-VI is only found in birds and fish and not in mammals [12]. GnT-III is responsible for the synthesis of bisecting GlcNAc linked to the b1→4 mannoside in the N-glycan core. On the contrast, GnT-IV and GnT-VI are involved in the synthesis of multi-(3-, 4-, 5-) antennary N-glycans [10] (Fig. 4.6). They also do not directly participate in Lewis antigen synthesis but provide a structural basis for the elongation of the antenna (outer sugar chain) of N-glycans. The increased antennas, which are the products of GnT-IV and GnT-VI, may become the skeletal backbone for the synthesis of Lewis antigens. b1→6 GnT includes GnT-V in the processing of N-glycan, C2GnT in the branching of O-glycan core, and IGnT responsible for the branching of poly-Nacetyllactosamine (i antigen) in the formation of I antigen. GnT-V catalyzes the transfer of GlcNAc residue to the a1,6 mannoside of C2C2-biantennary or C2,4C2-triantennary N-glycans, to produce a GlcNAcb1→6Mana1→6 branching structure in the products, C2C2,6 tri- or C2,4C2,6 tetra-antennary N-glycans (Fig. 4.6). After a b1→6GlcNAc-branch is formed by GnT-V, poly-N-acetyllactosamine can be preferentially added to this antenna and its next antenna derived from the C2 position of a1→6 mannoside (product of GnT-II) [11], then poly-N-acetyllactosamine or i antigen becomes the preferred substrate for the synthesis of SLex, SDLex, or
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STLex. C2GnT (core 2 b1→6 GnT) is a family of glycosyltransferases that catalyze the synthesis of core 2 and core 4 of O-glycans. These enzymes transfer the GlcNAc group to the innermost GalNAc residue in the sequence of Galb1→3GalNAca1→Ser/Thr (core 1 of O-glycans, precursor of core 2) or GlcNAcb1→3GalNAca1→Ser/Thr (core 3 of O-glycans, precursor of core 4), producing a GlcNAcb1→6GalNAca1→Ser/Thr branch in the product core 2 and core 4. At least three C2GnT subtypes have been discovered [13, 14]. Among them, C2GnT-I and -III synthesize core 2 only, while C2GnT-II synthesizes both core 2 and core 4. C2GnT has been reported as the critical enzyme for the synthesis of the SLex precursor on O-glycans [6]. IGnT (I-branching b1→6GnT) is an N-acetylglucosaminyltransferase that transfers GlcNAc to b1→4 linked Gal residue in linear poly-N-acetyllactosamine, forming a GlcNAcb1→6Gal branch, such as Galb1→4GlcNAc b1→3 (GlcNAc b1→6) Galb1→4GlcNAc→R. The formation of this branch is usually followed by galactosylation by b1→4 GalT (generally b1→4GalT-1), resulting in the synthesis of I antigen [15]. The synthesis of I antigen on glycans is entirely dependent on the expression of IGnT [15], though C2GnT-II also shows an activity of IGnT [14]. It has been reported that at least two, possibly three, different IGnT are present that add GlcNAc to different Gal residues in poly-N-acetyllactosamine [15].
4.3.2 Galactosyltransferase GalT catalyzes the transference of Gal residue from UDP-b-d-Gal to GlcNAc on the N- or O-glycans. In the synthesis of glycosphingolipids, Gal can also be added to Glc, GalNAc, or another Gal residue in the sugar chains in addition to GlcNAc [16]. It can be divided into b1→3 and b1→4 subfamilies. At least six b1→3GalT [11, 17, 18] and seven b1→4GalT [19, 20] have been characterized in mammalian cells. b1→3GalT-1, -2, and -5 participate in the synthesis of a type 1 sugar chain or N-acetyllactosamine, Galb1→3GlcNAc→R (precursor of a/b series Lewis antigens), while b1→4GalT-1, -2, -3, -4, -5, and -6 are required for the synthesis of a type 2 sugar chain or N-acetylneolactosamine, Galb1→4GlcNAc→R (precursor of x/y series Lewis antigens). Among them, b1→4GalT-1 and -2 can synthesize lactose in the presence of a-lactalbumin. b1→4GalT-3 and -4 cannot synthesize lactose even in the presence of a-lactalbumin but can effectively utilize GlcNAcb1→3Galb1→4Glcb1→ceramide as their substrate. Eventually, b1→3GalT-3 was characterized instead as b1→3GalNAcT-1, which is involved in globoside synthesis. b1→3GalT-4 failed to galactosylate glycoprotein acceptors and was reported to be a ganglioside GM1 synthase [21] [GM1: Galb1→3GalNAcb1→4(NeuAca2→3) Galb1→4Glcb1→ceramide]. b1→4GalT-6 is also a synthase of lacto-ceramide (Galb1→4Glca1→ceramide) [21, 22]. b1→3GalT-6 and b1→4GalT-7 are predominately involved in the synthesis of the core of glycosaminoglycan (b1→3Galb1→4Xyb1→3Ser/Thr) but are not implicated in Lewis antigen synthesis [11, 20]. In addition, a core 1 b1→3GalT
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responsible for O-glycan core 1 synthesis was characterized by Ju et al. [23]. It was reported by Narimatsu’s laboratory that b1→3-galactosyltransferase 5 (b1→3GalT-5), which synthesizes the type 1 N-acetyllactosamine, is the rate- limiting enzyme in the synthesis of SLea. Its importance is greater than that of a1→3FucT-III, the only FucT for SLea synthesis. The expression of b1→3GalT-5 is in accordance with the expression of SLea in many cell lines [24]. In contrast, a1→3FucT-III is found to exhibit no difference between malignant and nonmalignant epithelial cells in digestive organs, even though the expression of SLea is highly increased in the malignant cells [25, 26]. It was reported that b1→4GalT-5 is the main enzyme for transferring Gal residue to GlcNAcb1→6Mama1→branch in N-glycans [26].
4.3.3 Fucosyltransferase Human FucT is a family of glycosyltransferases responsible for the synthesis of fucosyl-containing compounds. It catalyzes the transference of fucosyl residue (Fuc) from GDP-a-l-fucose to a sugar acceptor, usually galactose (Gal) or N-acetylglucosamine (GlcNAc or Gn), in the sugar chains of glycoconjugates. Human FucT is divided into three main subfamilies: a1→2FucT, a1→3FucT, and a1→6 FucT [27, 28]. a1→2FucT consists of FucT-I (H enzyme) and FucT-II (Se enzyme), participating in the synthesis of the ABH(O) blood group antigens (refer to Fig. 4.5) as well as the Lewis antigens b and y. a1→6 FucT (FucT-VIII) is responsible for the synthesis of core a1→6 fucose in N-glycans. The a1→3FucT subfamily is the main glycosyltransferase involved in the synthesis of Lewis antigens, and a1→3/a1→4 fucosylation is the last (in general) and most critical step in Lewis antigen synthesis. To date, six a1→3FucTs have been identified [28]. Each enzyme has a unique acceptor substrate binding pattern, and each generates a unique range of fucosylated products. Four of them (a1→3 FucT-III, -V, -VI, and -VII) efficiently fucosylate sialylated acceptors and produce sialyl Lewis antigens. As a rule, one glycosyltransferase can only produce one glycosidic linkage, but a1→3FucT-III is an exception; it is the only glycosyltransferase with two glycosidic linkage (a1→3 and a1→4) specificities that can synthesize both a1→3 fucosyl containing Lex, SLex, and Ley and a1→4 fucosyl containing Lea, SLea, and Leb (refer to Fig. 4.1). Therefore, FucT-III is also designated as a1→3/4 FucT and Lewis type FucT [28, 29]. Its a1→4 fucosylation activity is higher than its a1→3 fucosylation activity, being the only enzyme for the synthesis of a/b series Lewis antigens [29, 30]. The gene of a1,3FucT-V has been reported to be a silent gene that is not expressed in any tissues where other a1,3 FucTs are expressed. However, this enzyme can synthesize Lex and SLex effectively, as well as Lea and Leb with lower activity in vitro [28, 30]. a1→3FucT-VI is widely distributed in human tissues as a1→3FucT-III and -IV. Also, a1→3FucT-VI is an important FucT in the synthesis of sialylated and nonsialylated x/y series Lewis antigens (including SDLex) carried by plasma protein (therefore, it also called plasma type a1→3FucT)
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and epithelial cancer cells. However, a1→3FucT-VI cannot utilize a type 1 sugar chain to synthesize a/b series Lewis antigens [31]. In the fucosylation of sialylated substrate, only NeuAca2→3 substituted, but not NeuAca2→6 substituted lactosamine, serves as its substrate. Poly-N-acetyllactosamine having 6-sulfate modification at GlcNAc moiety can be utilized by FucT-VI, and 3¢-sulfate modification at the terminal Gal moiety are good substrates for both FucT-VI and FucT-III [31]. a1→3FucT-VII is mainly distributed in white blood cells, which catalyzes sialylated substrates only, and SLex is its main product. This FucT-VII is responsible for the increased SLex on leukemia cell surfaces, but it was reported also to be involved in the expression of SLex in cancer cells of epithelial origin [32]. On the other hand, a1→3FucT-IV and -IX prefer neutral acceptors and usually form nonsialyl Lewis antigens as their products. However, FucT-IV (myeloid type a1→3FucT) can also synthesize sialyl Lewis antigens under certain conditions. Its substrate specificity and its product are different from those of FucT-IX, which is mainly distributed in kidney, brain, and peripheral blood leukocyte (granulocyte, natural killer, and B lymphocyte) [33, 34]. For example, using neutral type 2 poly-N-acetyllactosamine as a substrate, FucT-IV preferentially transfers Fuc to the inner GlcNAc, while FucT-IX preferentially transfers Fuc to the distal GlcNAc of the substrate. When sialyl poly-N-acetyllactosamine is used instead of the neutral one, FucT-VII preferentially transfers Fuc to the distal GlcNAc, while FucT-IV preferentially transfers Fuc to the inner GlcNAc, and FucT-IX exhibits only weak activity for the transference of Fuc to the inner GlcNAc residue [33].
4.3.4 Sialyltransferase STs are divided into four subfamilies based on their substrate specificities and the positions of the newly formed a-glycosidic linkages in the products [35, 36]. a2→3 sialyltransferase (ST3Gal) transfers sialyl (NeuAc) group from CMP-Nacetylneuraminate (CMP-NeuAc) to the Gal residue of glycans, forming a2→3 linkage in its product, NeuAca 2→3Galb1→R. ST6Gal also transfers NeuAc group from CMP-NeuAc to Gal residue of glycans in glycoproteins but produces a a2→6 linkage as NeuAca 2→6Galb1→R, and its main substrate is N-glycans. The ST6GalNAc subfamily transfers the NeuAc group to the N-acetylgalactosaminyl (GalNAc) residue in O-glycans or glycosphingolipids and forms a a2→6 linkage, but it can also transfer NeuAc group to GlcNAc residue as in the synthesis of disialyl Lewis a (refer to Fig. 4.4). The ST8Sia subfamily transfers the NeuAc group to another NeuAc residue at the termini of glycans to create a a2→8 linkage as NeuAca2→8 NeuAca2→R. This ST8Sia subfamily does not participate in Lewis antigen synthesis. ST3Gal is the most popular sialyltransferase in the synthesis of sialylated Lewis antigens. It can be further divided into five subtypes. The substrates of type I and II are the Galb1→3GalNAca1→Ser/Thr sequence in O-glycans and Galb1→3GalNAcb1→R in glycosphingolipids, respectively. The preferred substrate of ST3Gal-III is Galb1→3GlcNAcb1→R (type 1 chain, precursor of
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SLea), while that of ST3Gal-IV is Galb1→4GlcNAcb1→R (type 2 chain, precursor of SLex). However, ST3Gal-III uses a type 2 chain and ST3Gal-IV uses a type 1 chain as their substrates but with lower activity. Both ST3Gal-III and ST3Gal-IV rarely utilize a glycolipid such as paragloboside (Galb1→4GlcNAcb1→3Galb1→ 4Glcb1→ceramide) [37, 38]. Type V was evidenced as a ganglioside GM3 synthase not involved in Lewis antigen synthesis [36].
4.3.5 Biosynthetic Pathway of Neutral and Sialylated Lewis Antigens Figure 4.7 summarizes the biosynthetic pathway of the main Lewis antigens. In the synthesis of neutral Lewis antigens, the precursor is substituted by different kinds of glycosyltransferases one by one with a sequence of GnT→GalT→FucT, while in the synthesis of sialyl Lewis antigens, the enzyme sequence is GnT→ GalT→ST→FucT. In other words, FucT is usually the last enzyme in the synthetic pathways. In the synthesis of Lewis b and Lewis y with double fucosyl substitutions, FucT-I or FucT-II always acts prior to other FucTs. After the action of FucT-I or -II, the 2-position of Gal in Leb or Ley is occupied, then sialyltransferase become unable to add sialyl residue to the 3-position of Gal residue; hence, there are no sialyl Leb and sialyl Ley Lewis antigens. It is noteworthy that the specific activity of FucT-III in most malignant and benign epithelial cells is usually one or two orders of magnitude higher than that
GlcNAcbb1,4GalT-I,II,III,VI,V,VI
b1,3GalT-I,II,III,V
Galb1-3GlcNAcb-, Type 1 chain
FucT-I,II
FucT-III
Galb1-4GlcNAcb-, Type 2 chain
ST3Gal-III,IV
FucT-I,II
Galb1-3GlcNAcb- Galb1-3GlcNAcb- SAa2-3Galb1-3GlcNAcb-
|a1-2 Fuc
H type 1
|a1-4 Fuc
|a1-2 Fuc
Lewis a
H type 2
FucT-III
FucT-III
Galb1-3GlcNAcb-
SAa2-3Galb1-3GlcNAcb-
|a1-2 Fuc
|a1-4 Fuc
Lewis b
Galb1-4GlcNAcb-
|a1-4 Fuc Sialyl Lewis a
FucT-III,IV,VI,IX
Galb1-4GlcNAcb-
|a1-2 |a1-3 Fuc Fuc Lewis y
Fig. 4.7 Biosynthetic pathway of main Lewis antigens
FucT-III,IV,VI,VII
ST3Gal-III,IV
Galb1-4GlcNAcb- SAa2-3Galb1-4GlcNAcb-
|a1-3 Fuc Lewis x FucT-III,IV,VI,VII
SAa2-3Galb1-4GlcNAcb-
|a1-3 Fuc Sialyl Lewis x
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of sialyltransferases [31]. Therefore, FucT-III is not the rate-limiting enzyme in the synthesis of SLea. As mentioned above, the rate-limiting enzyme is b1→3GalT-V. In the synthetic pathway of SLex, the rate-limiting step is speculated to be at the GnT level, such as b1→3 iGnT or b1→6C2GnT and IGnT, but FucT-VI, -VII, or sialyltransferase may also be the limiting enzyme in some cases. It is very possible that the rate-limiting enzyme in SLex synthesis varies from tissue to tissue. The contribution of different FucTs in SLex synthesis is also different from cell to cell. For instance, FucT-VII is the predominant FucT for SLex synthesis in white blood cells and leukemia cells, while FucT-VI and -III may be the major enzyme for SLex synthesis in epithelial cells (where FucT-VII is expressed in very low levels) [5]. 1. The underlined words are the names of the glycosyltransferases that catalyze the reactions. Among them, the bigger symbols are the major (more important) glycosyltransferases. FucT-V is not included in the figure because it is never expressed in tissues. 2. The products of the reactions in boxes are Lewis antigens or their precursors. H type 1 and H type 2 are the H epitopes in the ABH(O) blood group system. “SA” is sialyl residue equal to “NeuAc” in mammalian tissues/cells. If the 3-position of Galb1→3 in H type 1 and Galb1→4 in H type 2 epitopes is further substituted by aGalNAc or aGal, it will become the structure of A and B epitopes in the ABH(O) blood group, respectively (refer to Fig. 4.5). These two reactions are catalyzed by a1→3GalNAc transferase (A enzyme) and a1→3Gal transferase (B enzyme), respectively [29]. The biosynthesis of long-chain Lewis antigens is different from that indicated in Fig. 4.7. Their precursor is poly-N-acetyllactosamine (Ln–Ln–) with a type 1 or type 2 sugar chain structure. The biosynthesis of sialyl difucosyl Lewis x (SDLex) is taken as an example, and its synthetic pathway is shown in Fig. 4.8. There are alternative pathways for SDLex synthesis. The type 2 poly-N-acetyllactosamine can be first fucosylated and then sialylated to form VIM-2, or first sialylated and then fucosylated to produce VIM-2 or SLex structure. The sialylated poly-N-acetyllactosamine can also be first catalyzed by FucT-IV (add Fuc to inner GlcNAc, and the product is VIM-2) and then by FucT-VII (add Fuc to distal GlcNAc), or first by FucT-VII (the product is SLex) and then by FucT-IV. The final step is the second fucosylation, and the final product in all alternative pathways is SDLex. In addition, FucT-VI is an important enzyme for the synthesis of SDLex; it can fucosylate both distal and inner GlcNAc residues in the product SDLex [28, 39]. However, FucT-IX is not an important enzyme in SDLex synthesis since the direct precursor of SDLex, VIM-2, or SLex carries an acidic sialyl group. The biosynthesis of sialyl trifucosyl Lewis x (STLex) from poly-N-acetyllactosamine (Ln–Ln–Ln–) is similar to SDLex. The intermediate compound is also type 2 sialyl poly-N-acetyllactosamine, which can be fucosylated by the combination of FucT-VII (adding Fuc to the outmost GlcNAc residue) and FucT-IV (adding Fuc to the inner two GlcNAc residues) either by the enzyme sequence of FucTVII→Fuc-IV or FucT-IV→Fuc-VII [40].
64
H.-L. Chen Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-R Type 2 poly-N-acetyllactosamine
ST3Gal-III, IV
FucT-IV, -VI, -IX Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-R |a1-3 Fuc
ST3Gal-III, IV
NeuAca2-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-R
FucT-IV, VI, IX
FucT-IV, VI, IX
NeuAca2-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-R NeuAca2-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-R |a1-3 |a1-3 Fuc VIM-2 Fuc SLex
FucT-IV, VI, VII
FucT-IV, VI, IX
NeuAca2-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-R |a1-3 |a1-3 Fuc Fuc
Sialyl difucosyl Lewis x (SDLex)
Fig. 4.8 Biosynthetic pathway of sialyl difucosyl Lewis x (SDLex)
The underlined words are the names of the glycosyltransferases that catalyze the reactions. Among them, the bigger symbols are the major (more important) glycosyltransferases. FucT-V is not included in the figure because it never expressed in tissues.
4.3.6 Sulfotransferase and Its Role in the Synthesis of Lewis Antigens Sulfotransferase catalyzes the transfer of a sulfate group from the donor substrate 3¢-phosphoadenosine 5¢-phosphate (PAPS) to a sugar residue in glycans. Most sulfotransferases that participate in the biosynthesis of glycoaminoglycans, such as heparan sulfate and chondroitin sulfate, are not involved in Lewis antigen synthesis. However, some sulfotransferases have rather broad substrate specificity. For instance, chondroitin 6-sulfotransferase [41] and keratan sulfate Gal-6-sulfotranferase [42] can transfer a sulfate group to the 6¢-position of the Gal residue next to the terminal NeuAc in sialylated N-acetyllactosamine or sialylated poly-N-acetyllactosamine (usually type 2); hence, they are considered to be involved in the synthesis of 6¢-sulfo-sialyl Lewis x and 6¢,6-bis-sulfo Lewis x (refer to Fig. 4.3). The sulfate groups at 6-position of GlcNAc in 6-sulfo-sialyl Lewis x and 6¢,6-bis-sulfo Lewis x are added by N-acetylglucosamine 6-O-sulfotransferases [GlcNAc6STs (here the “ST” is
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sulfotransferase, not sialyltransferase)] [43]. The sulfation of 3¢-position of Gal in 3¢-sulfo-Lewis and 3¢-sulfo-Lewis x is catalyzed by the b-galactose-3-O-sulfotransferase (Gal3ST) family [44]. Gal3ST-2 acts on type 1 (Galb1→3GlcNAcb1→R) and type 2 (Galb1→4GlcNAcb1→R) N-acetyllactosamine as well as core 1 oligosaccharide of O-glycans (Galb1→3GalNAca1→R). Gal3ST-3 exclusively acts on type 2 chains in N- and O-glycans and also utilizes Galb1→4 (sulfo-6) GlcNAcb1→R as a substrate. Gal3ST-4 is an O-glycan-specific enzyme that recognizes core 1[Galb1→3GalNAca1→R] and core 2 [Galb1→3(GlcNAcb1→6)GalNAca1→R] as good substrates, but not Galb1→3/1→4 GlcNAcb1→R in N- or O-glycans [44]. In addition to the above-mentioned glycosyltransferases and sulfotransferases, some other proteins or enzymes are involved in the synthesis of Lewis antigens, such as the transporters of UDP-GlcNAc, UDP-Gal, GDP-Fuc, and CMP-NeuAc. They transfer the sugar groups required for Lewis antigen synthesis from cytosol to the Golgi apparatus [32].
4.4 Lewis Glyco-Epitopes and Cancer Metastasis It has been well documented that sialyl Lewis antigens are closely associated with the progression and metastasis of a variety of cancers, including lung, stomach, colon, pancreas, biliary tract, prostate, and bladder [3, 5]. The expression of SLex on cancer cells is positively correlated to the potency of hematogenous metastasis and negatively correlated to the survival of the patients [45–49]. In our studies on the expressions of Lex, SLex, and SDLex in clinical specimens of human nonsmall cell pulmonary cancer (NSCPC) and primary liver cancer (PLC), it was found that these Lewis antigens were predominately expressed on the surface of cancer cells [50]. The positive rates of these antigens in NSCPC were within the range of 75–86%, but the regions adjacent to the cancer tissues did not express any Lewis antigens. The expression intensities of all three antigens were significantly higher in samples with poor differentiation and metastasis as compared to those with well/ medium differentiation and no metastasis. SLex and SDLex were increased more significantly than nonsialylated Lex. The expressions of these antigens were also observed in the peripheral lymph nodes with metastasis but not in those without metastasis. On the other hand, the positive rates of Lex, SLex, and SDLex in human PLC were 83.3, 88.9, and 77.8%, respectively. In cases with cancer cell thrombosis (CCT) in portal vein (an index of metastasis), the expressions of all three antigens were stronger than those in the cases without CCT. SLex was the most abundant and most highly increased Lewis antigen on the surface of NSCPC and PLC cells, especially in the cases with poor differentiation and metastasis. The above results indicate that these three Lewis antigens are correlated with cancer metastasis [50]. Moreover, human fetal liver also expresses Lex, SLex, and SDLex, but adult liver does not express any of these antigens. Therefore, these Lewis antigens are considered to be onco-fetal antigens and tumor markers. Similarly, in the human hepatocellular carcinoma cell line H7721, the most expressed antigen was SLex, SDLex
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was expressed only a little, and the expressions of Lex and SLea were negligible. In H7721 cells, the messenger RNA (mRNA) of FucT-IV and -III were highly expressed, but the expressions of FucT-VI and -VII mRNA were low, and that of FucT-IX mRNA was nil. [51]. FucT-VI is supposed to be the main enzyme responsible for the synthesis SLex and SDLex (the efficiency of FucT-VI in the synthesis of SLex is 6.4 times and 1.5 times that of FucT-III and FucT-VII, respectively [52]), but FucT-VII and -III may also participate in SLex synthesis. Our results also indicate that SLex is the most important Lewis antigen in mediating the metastasis of NSCPC and PLC. However, Matsusaka et al. [53] reported that a Lewis antigen with a long sugar chain, such as SDLex, was more important than the short-chain Lewis antigens, Lex and SLex, in the metastasis of bladder cancer. They also reported that, in the liver metastatic foci of rectal cancer, the expression of SDLex was even higher than in the primary cancer tissue [54]. It is highly likely that the metastasis of different cancers mainly depends upon different sialyl Lewis antigens. Using H7721 cells as a model, our laboratory found that cell surface SLex, but not SLea and SDLex, was upregulated by the transfection of the metastasis-promoting gene c-erbB2/neu [55] and downregulated by the metastasis-suppressing gene nm23-H1 [56]. In addition, surface SLex was increased after the cells were treated with proliferation inducer, epidermal growth factor (EGF), or phorbol-12-myristate13-acetate (PMA) and was decreased after treatment with differentiation inducer, all-trans retinoic acid (ATRA), or 8-bromo-cyclic AMP [57]. Concomitantly, the expression of a1→3FucT-VII was increased and decreased in parallel with SLex, indicating that a1→3FucT-VII contributed in the increased synthesis of SLex in H7721 cells. Furthermore, the in vitro metastatic potential, including cell adhesion to human umbilical vein endothelial cells, cell migration, and invasion, was directly proportional to the expression of surface SLex and cell a1→3 FucT-VII. The metastatic potential was attenuated by using the SLex monoclonal antibody KM93 to block the surface SLex, but the antibodies to SLea and SDLex showed no or very little effect [55–57]. Therefore, SLex is considered to be the most important and main Lewis antigen responsible for the in vitro metastatic potential of H7721 cells. In further studies, it was revealed that insulin also enhanced the expression of SLex, FucT-VII, and the metastatic potential of H7721 cells, which was mediated by protein kinase B (PKB or Akt) [58]. On the other hand, nm23-H1 downregulated not only FucT-VII but also other FucTs involved in the synthesis of Lewis antigens, including FucT-III, -IV, and -VI [59]. It was very interesting to find that after the H7721 cells were transfected with the complementary DNA (cDNA) of the metastasis-related enzyme N-acetyl-glucosaminyltransferase V (GnT-V) [60] to increase the branching of N-glycan, the surface SLex was decreased and not increased as predicted [61]. The enzymatic mechanisms were proved to be the concomitantly decreased expression of a1→3FucT-III, -VI, and -VII and the branching enzyme of O-glycan cores, C2GnT-I, and -II. These two glycosyltransferase families have been suggested to be the key enzymes in the synthesis of SLex. Transfection of antisense GnT-V into H7721 cells showed entirely opposite effects on the expression of SLex and the above-mentioned glycosyltransferases.
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The underlying mechanisms for the appearance or overexpression of sialyl Lewis antigens in cancers include neosynthesis and incomplete synthesis of the Lewis antigens [5, 32, 62]. The former is based on the upregulation of some glycosyltransferases and transporters of nucleotide sugars in cancer cells resulting from the changes of oncogenes and/or anti-oncogenes. The latter is the downregulation or deletion of the key enzymes in the synthesis of disialyl Lewis a and 6-sulfo-sialyl Lewis x. These two sugar determinants are present in nonmalignant epithelial cells, such as normal colon mucosa. In the malignant transformation of colon epithelial cells, the impairment of GlcNAc 2→6 sialylation of SLea and GlcNAc 6-sulfation of SLex (possibly owing to the loss of a2→6GalNAc sialyl-transferase-VI or ST6GalNAc-VI [62] and N-acetylglucosamine 6-O-sulfotransferases, respectively) leads to the incomplete synthesis of disialyl Lewis a and 6-sulfo-sialyl Lewis x, and the synthetic pathways are blocked at the stages of SLea and SLex. In general, incomplete synthesis is the major mechanism in early stages of cancer, while the neosynthesis mechanism is more important in advanced stages of cancers [5]. It has been well known that sialyl Lewis antigens are the ligands of the selectin family, which includes E-, P-, and L-selectins. They are C-type (Ca++ dependent) lectins containing, from N terminus to C terminus, a carbohydrate recognize domain, EGF-like domain, complement regulatory repeats, transmembrane domain, and cytoplasmic domain in their protein structure [40]. The interaction between the sialylated and sulfated Lewis antigens on cancer cell surfaces and the E-/P-selectin expressed on microvascular endothelium is the prerequisite for cancer cells to adhere and penetrate through the vascular wall, which is the critical step in cancer metastasis. This is the reason why cancer metastasis is improved by the presence of sialylated and sulfated Lewis antigens on cell surfaces. Acidic Lewis antigens not only participate in the metastasis of cancer cells but are also involved in the infiltration of leukocytes to the inflammatory area, since leukocytes also express acidic Lewis antigens on their cell surface. The Lewis antigens allow leukocytes to roll on and adhere to the activated endothelium [40]. E-selectin can be induced on endothelial cells (such as those on blood vessels) by inflammatory stimulus, including tumor necrosis factor (TNF), interleukin (IL)-1a, and IL-1b, and recognizes mainly SLex, SDLex, SLea, and VIM-2, as well as 3¢-sulfo-SLea and 3¢-sulfo-SLex [3, 5, 40]. P-selectin is stored in Weibel–Palade granules or a-granules in endothelial cells or platelets and translocated at the cell surface by stimulation. Its binding specificity to a Lewis antigen is similar to E-selectin but with higher affinity toward sulfated determinants. [3]. However, P-selectin binding to sialylated or sulfated Lewis antigens requires a protein molecule as a mediator, called P-selectin glycoprotein ligand-1 (PSGL-1), carrying a specific tripartite arrangement of sialyl Lewis x-containing core 2-based O-glycan and three tyrosine sulfate determinants at its N-terminal [5, 40, 63]. L-selectin is constitutively expressed on white blood cells, and it requires the presence of a sulfate group on its ligands Lewis x and Lewis a, such as 3¢-sulfo-Lea, 3¢-sulfo-Lex, 6- or 6¢-sulfo-SLex, bis-sulfo-SLex, and sulfatide [3, 5, 40]. The L-selectin on lymphocytes can specifically bind to its ligands, such as GlyCAM-1 and CD34 (these molecules carry 6- and/or 6¢-sulfo-sialyl Lewis x) on high epithelial venules in lymph nodes, and this mechanism is implicated in the
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“homing” of lymphocytes. Hence, L-selectin is also designated as a “homing receptor” [40]. Neutral Lewis antigens without a sialyl or sulfo group are generally not recognized by selectins, but GalNacLex (refer to Fig. 4.4) can bind to E-selectin [40]. It is interesting to note that artificially substituted a2→6 sialyl Lewis x also cannot bind to E-selectin [40]. Sialyl- or sulfo-group-containing glycoconjugates other than Lewis antigens, like heparan sulfate proteoglycan, can also serve as a strong ligand of selectins [3]. On the other hand, not all sialyl Lewis antigens can bind to selectins. For example, disialyl Lewis a expressed on normal epithelial cells cannot bind to E- or P-selectin but can effectively serve as a ligand for immunosuppressive receptors such as siglec-7 and -9 (family members of sialic acid-recognizing lectin) expressed mainly on monocytes/macrophages and lymphocytes. The function of disialyl Lewis a, siglec-7, or siglec-9 interaction is to prevent excessive activation of monocytes/macrophages and maintain immunological homeostasis of mucosa membranes [62]. While SLea was found to be more important in the adhesion to vessel endothelial E-selectin of cancer cells derived from the colon, rectum, pancreas, and biliary tract, SLex was found to contribute more to the adhesion of cancer cells from the breasts, ovaries, and lungs [62]. SLea and SLex, originating mainly from malignant cells, are found in human serum. They can be used to diagnose and assess prognosis in cancers of the digestive system, including stomach, colon, rectum, pancreas, and biliary duct. In fact, the well-known CA 19-9 used in clinics as a tumor marker is SLea. However, the appearance of SLea or SLex in serum is not specific to cancer; patients with benign diseases such as inflammatory disorders sometimes show a slight or even medium elevation of CA 19-9 or SLex in their serum. Therefore, a false-positive or falsenegative result is a problem in the application of CA 19-9 or SLex. Kannagi suggested using the ratio of SLea to disialyl Lewis a in differential diagnoses of malignant and benign diseases of the digestive system. Since disialyl Lewis a is the normal counterpart of sialyl Lewis a from normal tissues, patients with cancer have a higher level of sialyl Lewis a than disialyl Lewis a, and patients with benign disorders tend to have a higher serum level of disialyl Lewis a relative to sialyl Lewis a [62].
4.5 Lewis B and the Infection of Helicobacter pylori Recently, several studies have reported that the Lewis antigen b (Leb) expressed on human gastric epithelial cells serves as the receptor to Helicobacter pylori (H. pylori), since H. pylori can adhere to Leb, at least in the limited number of strains that have been examined [2, 64]. H. pylori is a spiral, microaerophilic Gramnegative bacterium known as the major etiologic pathogen of chronic active gastritis and is generally accepted as a causative factor in the pathogenesis of gastritis, peptic ulcer, gastric adenocarcinoma, and gastrointestinal lymphoma [2, 65, 66]. It is estimated that more than 50% of the world’s human population is infected with H. pylori [67]. However, a clinical study found that there was no correlation
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between the H. pylori infection rate and the host Leb phenotype [2]. Further studies revealed that the lipopolysaccharides (LPS) of most H. pylori strains also contain Lewis antigens [8, 68]. Approximately 85% of H. pylori isolates express at least one of the Lewis antigens, while more than 80% of H. pylori strains were shown to express the type 2 chain Lex and Ley, and 5% express the type 1 chain Lea and Leb [68]. The presence or absence of Leb in H. pylori did not correlate to the adhesive rate of this bacterium to immobilized Leb, and blockage of H. pylori self Leb by preincubation with anti-Leb monoclonal antibody did not interfere with bacterial adhesion to Leb, suggesting that self Leb in H. pylori cannot serve as the bacterial self-receptor (or auto-receptor) [67]. Investigations on the genetic determinants involved in the biosynthesis of the Lewis antigen in H. pylori have led to the identification of nonmammalian a-FucTs, whose substrate specificities are different from those of the mammalian enzymes. In addition, H. pylori utilizes a different pathway compared to its host to synthesize the difucosylated Lewis antigens Leb and Ley [68]. In the synthesis of Leb, the a1→2FucT (equivalent to mammalian FucT-I, -II) of H. pylori utilizes Lea but not H type 1 as a precursor (refer to Fig. 4.7), while a1→4FucT of H. pylori (equivalent to mammalian FucT-III) cannot synthesize Leb from H type 1. Similarly, Ley is synthesized via Lex, but not H type 2, by a1→2FucT of H. pylori, and H type 2 is not the substrate of a1→3FucT in H. pylori [68]. In other words, the action of a1→2FucT in H. pylori is after the action of other FucTs in the synthetic pathways of Leb and Ley, whereas it is before other FucTs in mammalian cells. The biological role of Lewis antigens in H. pylori infection is unclear. It is hypothesized that its Lewis antigens mimic the host gastric epithelial Lewis antigens and thus enable H. pylori to escape detection by the host immune system. The observation that LPS of H. pylori has lower immunological activity compared to that of enterobacterial LPS supports this hypothesis. However, the Lewis antigens in H. pylori have been suggested as a contributor to the autoimmunity involved in the pathogenesis of chronic gastritis peptic ulcers [68].
4.6 Lewis Y and Embryo Implantation Lewis antigens are also implicated in reproductive physiology. For example, a1→3 fucosylated glycans are associated with the process of morula compaction and may contribute to sperm–egg interactions in some species [69]. Lex and Ley are both stage-specific embryonic antigens. Lex is first detected on the blastomeres of the eight-cell-stage embryo, which correlates with the onset of blastomere compaction. Ley is highly expressed on the surface of the blastocyst, which has been shown to be involved in blastocyst attachment in the mouse [70]. In rodents, several pieces of evidence support the hypothesis that an a1→2fucosylated glycan, such as Ley, helps blastocysts attach to the uterine epithelial wall during the initial stage of embryo implantation [69, 71]. Western blot analysis shows that Ley is carried on many uterine glycoproteins in both pregnant and nonpregnant females. The function
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of Ley in implantation was tested by injecting the monoclonal antibody against Ley directly into the uterine horn of a postcoital female mouse. Injection of purified anti-Ley (AH6) on the afternoon of Day 4 postcoitus significantly inhibited implantation. This effect was dose-dependent and was obtained during a narrow time window. Inhibition of implantation was not observed in contralateral uterine horns injected with saline or other monoclonal antibodies against Lex, Lea, and Leb [72]. In the blastocyte–uterine epithelial cell coculture system, the adhesion of blastocytes on the surface of uterine epithelial cells was obviously inhibited when monolayer epithelial cells or blastocytes were pre-incubated with AH6. The effect of AH6 was optimum at 2–4 h of co-incubation. There was no inhibition in the control group using other antibodies to oligosaccharides with structures closely related to that of Ley [73]. The adhesion is inferred to be dependent on a1→2 fucosylated glycans since adhesion is inhibited by the addition of a1→2 fucosylated glycoconjugates but not by control fucosylated glycans with no a1→2 fucosylation [69]. These results provide evidence that Ley is a specific determinant in the embryonic implantation of the blastocyte stage. When the FucT-I and FucT-IV involved in the synthesis of Ley were analyzed, it was found that these enzymes were expressed concomitantly with Ley in the embryos developed in the culture, suggesting that Ley is synthesized by the embryo itself but not transferred from the uterine epithelial cells [70]. In the uterine epithelial cells, the expression of Ley-synthesizing FucT is strictly controlled by hormonal changes that account for the physiology of the estrous cycle, at a time that correlates with endometrial receptivity for blastocyst implantation [69, 74]. The mRNA level of FucT-I and FucT-IV genes in mice were parallel to the expression of Ley and decreased during early pregnancy through Days 1–5, which was compatible with the increased level of progesterone. In ovariectomized mice, the mRNA levels of these two genes significantly decreased and increased after progesterone and estrogen treatment, respectively. These findings suggest that the expression of Ley in the endometrial epithelium of mice is regulated at the level of the transcription of FucT-I and FucT-IV genes and is suppressed by progesterone and stimulated by estrogen [75]. The presence of Ley on an embryo’s surface has been implicated in the growth and development of the embryo. This was supported by the observation that the gene expression and secretion of EGF significantly decreased after the embryo was pre-incubated with the Ley antibody AH6. The expression of the EGF receptor was also slightly downregulated after surface Ley was blocked by AH6 [76].
4.7 Function of Sialyl Lewis X in Cell Signaling and Growth SLex, the ligand of selectins, functions in leukocyte infiltration and cancer cell metastasis. The question as to what are the other functions of SLex has been addressed in our laboratory by transfecting the cDNA of FucT-VII into H7721 cells, which express only low levels of FucT-VII. Two transfectants with medium
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and high FucT-VII expression were established and designated as FucTVII-M and FucTVII-H, respectively. The SLex expression on the cell surface increased in accordance with the expression of FucT-VII mRNA in those cells [77, 78]. These two cell lines were used as the models of SLex overexpression, and their biological alterations were investigated. It was found that in FucT-VII-transfected cells, the protein expressions of insulin receptor (InR) a- and b-subunits and epidermal growth factor receptor (EGF-R) on the cell surface and in cells were unchanged when compared to cells mocktransfected with an empty vector. However, the SLex level on the InR a-subunit dramatically increased, while that on EGF-R was not altered. The reason why SLex did not increase on EGF-R might be as follows: (1) The SLex content on EGF-R is far more than that on the InR a-subunit, suggesting that the SLex on EGF-R is high enough and cannot be further upregulated by the overexpression of FucT-VII. (2) The sugar composition and structure of EGF-R glycans probably differ from those of the InR a-subunit, and the glycans of EGF-R are not suitable substrates for fucosylation by the exogenous FucT-VII. Interestingly, the tyrosine autophosphorylation of the InR b-subunit, but not that of EGF-R, was elevated concomitantly with the increased SLex on the InR a-subunit. The tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) was also upregulated. In the downstream signaling molecules, the Ser/Thr phosphorylation of PKB (Akt) as well as p42/44 mitogenactivated protein kinase (MAPK) and MAPK kinase (MEK) were significantly elevated. In addition, the proteins of some other signaling molecules, such as phosphoinositide-dependent kinase-1 (PDK-1), novel protein kinase (PKN), c-Raf-1, and b-catenin were also upregulated, and the activities of PKB and transcription factor TCF were obviously stimulated. The upregulation of InR-signaling molecules and their phosphorylation correlated with the level of SLex on the InR a-subunit and FucT-VII expression in cells. It was also observed that the phosphorylation intensity and difference in phosphorylation intensity among cells with different levels of FucT-VII expressions were attenuated significantly by the inhibitor of InR tyrosine kinase, HNMPA-(AM)3, and by the monoclonal antibody to SLex (KM93). Moreover, the insulin-induced signaling was facilitated in FucT-VII-transfected cells, particularly FucTVII-H. These findings provide strong evidence that FucT-VII may affect insulin signaling by upregulating the phosphorylation and expression of some signaling molecules involved in the InR signaling pathway. These effects are probably mediated by its product, SLex, on the glycans of surface InR. Therefore, transmembrane signaling of cells can be modified by the change in the SLex content in the glycans of surface receptors [77]. It is speculated that the increased density of SLex on the surface receptor will change the steric conformation of this receptor and promote binding to its ligand. Consequently, the changed conformation of a receptor may alter the recruitment of the adaptor proteins or downstream signaling molecules, resulting in the enhancement of receptor signaling. Another important finding is that the growth rate of FucT-VII cDNA-transfected H7721 cells was higher than that of control (mock-transfected) cells. When cell cycle was analyzed using flow cytometry, it was found that most of the control cells were at the G1 (including G0) stage. After transfection of FucT-VII cDNA, the
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percentage of G1 cells decreased, and the reduction was more obvious in FucTVII-H cells than in FucTVII-M cells. Conversely, S-stage cells were increased more in FucTVII-H than in FucTVII-M, but the percentages of G2/M cells were not significantly changed. These findings indicate that the transfection of FucT-VII cDNA accelerates the cell progression from the G1 to the S stage or stimulates the G1/S transition in the cell cycle [78]. It is well known that there is a “checkpoint” at the G1/S transition [79]. This checkpoint is controlled by three families of cell cyclerelated proteins, including cyclins [80], cyclin-dependent kinases (CDK 2, 4, 6), and cyclin-dependent kinase inhibitors (CDIs) [81, 82]. Cyclins are positively regulated by CDK activities via phosphorylation, and CDKs are negatively regulated by CKIs via protein–protein interactions. It was found that the expressions of cyclin D1, E, A, and CDK 4, 6, 2 were not significantly changed in FucT-VII-transfected cells as compared to the control cells [78]. The study was then focused on the CDIs. At least two subfamilies of CDI were reported. One is the INK4 (Inhibitor of CDK4) family, including p15INK4b, p16INK4a, p18INK4c, and p19INK4d, which inhibits the construction and activity of the cyclinD1–CDK4/6 complex. Another is the Cip/Kip (CDK inhibitory protein/Kinase inhibitory protein) family, containing mainly p21Waf1/Cip1, p27Kip1, and p57Kip2, which inhibits the construction and activity of cyclinE–CDK2 and cyclinA–CDK2 complexes [80–82]. Our results showed that the expression of p27Kip1, but not p16INK4 or p21waf1/Cip1, was decreased in the FucTVII-transfected cells. Using immuno-coprecipitation, it was discovered that not only did the total cell p27Kip1 decrease, but also the CDK2-combined p27Kip1 was reduced apparently. As a consequence of de-inhibition from p27Kip1, CDK2 activity was clearly elevated in FucT-VII-transfected cells. However, the mRNA level of p27Kip1 was not altered. This suggests that the reduction of p27Kip1 in FucT-VIItransfected cells is probably at the posttranscriptional level. One of the important target proteins of CDKs is retinoblastoma protein (Rb), known as a tumor suppressor and a dominant inhibitor of G1/S cell cycle progression. Rb is regulated by cyclinD1–CDK4/6, cyclinE–CDK2, and cyclinA–CDK2 complexes via phosphorylation at multiple sites [79]. The finding from Western blot experiment revealed that the phosphorylated Rb (p-Rb) was almost undetectable in control cells but was significantly elevated in FucT-VII-transfected cells. In contrast, the unphosphorylated Rb protein was correspondingly decreased. All of the above changes, including the decrease of p27Kip1, the activation of CDK2, and the increase of p-Rb were positively correlated to the content of SLex on cell surfaces and the expressions of FucT-VII in cells. To study the relationship between the expression of cell p27Kip1, p-Rb, and that of cell surface SLex, the expressions of p27Kip1 and p-Rb were determined after the cell surface SLex was blocked by KM93, the monoclonal antibody of SLex. It was found that p27Kip1 was significantly increased and p-Rb was definitely decreased by KM93 treatment in a dose-dependent manner in all three cell lines (control, FucTVII-M, and FucTVII-H), when compared with the cells without KM93 treatment. In addition, the intensity differences of both p27Kip1 and p-Rb among the three cell lines were greatly or totally abolished after the blockage of surface SLex by KM93 [78]. One of the molecular mechanisms for the accelerated cell proliferation induced by the transfection of FucT-VII is the decreased expression
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of CDI p27Kip1 protein, including its total amount and the amount that is bound to CDK2. The reduced p27Kip1 causes the de-inhibition of CDK2, which in turn stimulates the phosphorylation of Rb protein, resulting in the release of transcription factor E2F. E2F enters into nuclei and initiates the transcription of S-stage genes. Finally, the G1/S transition and growth rate of the cells are accelerated. These findings indicate that p27Kip1 expression is influenced by FucT-VII and its product, SLex. However, the reason why p27Kip1 is reduced in protein, but not in mRNA level, is unknown. It is possible that some regulatory factors that suppress the synthesis of p27Kip1 or accelerate the degradation of p27Kip1 protein were increased in FucTVII-transfected cells and thereby reduced the expression of p27Kip1 protein.
4.8 Lewis Antigens and Cell Apoptosis Very recently, our laboratory discovered that after H7721 cells were transfected with FucT-VII cDNA to increase surface SLex, cell signaling and growth were promoted as mentioned above. In addition, the expression of apoptotic protease, procaspase-3, was decreased, while the anti-apoptotic proteins, phospho-PKB (Akt) and phospho-Bad, were increased compared to those in mock-transfected control cells. This finding indicates that FucT-VII or SLex is a potential anti-apoptotic factor for H7721 cells. When mock- and FucT-VII-transfected cells were irradiated by ultraviolet (UV) light to induce apoptosis, the expressions of SLex on the surface and FucT-VII mRNA in cells were further significantly upregulated as compared with the unirradiated control cells [83]. Concomitantly, after irradiation, the percentages of apoptotic cells and active, cleaved caspase-3 were also elevated with the increased SLex. However, the increase of apoptotic cells and active caspase-3 was less in FucT-VII-transfected cells than that in mock-transfected cells. Phospho-p38 MAPK and Jun N-terminal kinase (JNK), the two apoptosis-related signaling molecules responsive to UV stress [84, 85], did not appear in unirradiated cells but increased promptly after UV irradiation. We have proved that these two signaling molecules are anti-apoptotic for H7721 cells [83]. The increase of phospho-p38 MAPK and JNK was more apparent in FucT-VII-transfected cells than in mock-transfected cells, further supporting that FucT-VII or SLex is an antiapoptotic factor, which can reduce the susceptibility of UV-induced apoptosis [83]. It was previously found by Russell et al. that at the late stage of apoptosis induced in thymocytes and P185 cells by dexamethasone, gliotoxin, or thapsigargin, the exposure of fucose residues on cell surfaces was increased [86]. Recently, Azuma et al. also reported [87] that the expression of FucT-IV and its products, Lex and Ley, was elevated after apoptosis of Jurket cells (a human T-cell line) was induced by the Fas antibody. The elevation of Lex and Ley could be blocked by the inhibition of caspase-3 and -8, two key proteases in the execution of cell apoptosis, suggesting that the enhancement of FucT-IV and its products was mediated by a molecule downstream of the caspases. It is considered that the mechanism of the upregulation of FucT-VII mRNA by UV irradiation in our study may be similar to the elevation
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of FucT-IV in Fas antibody-induced apoptosis, and the increase of mRNA was probably at the transcription level. It is reasonable to consider that the elevated expression of SLex on cell surfaces originates from the increased FucT-VII by UV. Therefore, the upregulation of fucosylated oligosaccharides, including Lewis antigens, on cell surfaces may be a common phenomenon and consequence of apoptosis induced by a number of chemical or physical agents. It is very interesting to find that apoptosis induced by UV upregulates the expression of FucT-VII and surface fucose-containing SLex, and the increase of FucT-VII and surface SLex reduces the susceptibility of UV-induced apoptosis. This may be a feedback mechanism for the protection of cells from apoptosis.
4.9 Function of Sialyl Lewis X on Cell Adhesion, Migration, and Integrin Expression To elucidate the mechanism through which SLex upregulates the metastasis of cancer cells, the effect of FucT-VII transfection on cell adhesion, migration, and integrin expression was studied since integrin is involved in the regulation of cell adhesion and migration [88, 89]. After transfection of FucT-VII cDNA into H7721 human hepatocarcinoma cells, cell adhesion to fibronectin (Fn) and chemotactic cell migration was obviously promoted. The cell adhesion could be blocked by the a5 integrin antibody, and cell migration was obviously attenuated by the antibodies to both a5 integrin and SLex [90]. These results suggest that the increased cell adhesion and migration are closely related to the expression of a5 integrin and SLex on a cell’s surface. In addition, the expression of a5 integrin, but not b1 integrin, was significantly upregulated in the FucT-VII-transfected cells. This was evidenced by the increase of a5 integrin on the cell surface as well as the increase of a5 mRNA and protein in the cells. However, the expressions of SLex on both a5 and b1 integrin subunits were unchanged. Concomitantly, the tyrosine autophosphorylated FAK and dephosphorylated Src (FAK and Src involved in the signal transduction of integrin a5b1 [88]) were upregulated, while the Tyr-527-phosphorylated Src was downregulated. The above-mentioned alterations were correlated to the expressions of FucT-VII in different FucT-VII-transfected H7721 cell lines [90]. These findings suggest that the overexpression of FucT-VII upregulates the mRNA of a5 integrin, leading to the increase of a5 integrin in the cells. The increased a5 integrin subunit combines with b1 integrin to produce more a5b1 dimer on the cell surface. Consequently, the a5b1 dimer promotes cell adhesion to Fn and cell migration. Moreover, Fn-induced signaling of the a5b1 integrin via Src/FAK was elevated, which also stimulated the cell adhesion to Fn as well as cell migration, as it was reported that cell adhesion and migration were also regulated by Src/FAK [88]. The upregulation of the surface SLex originating from the overexpression of FucT-VII also plays an important role in the stimulation of cell migration [90]. The above-mentioned functions of the Lewis antigen are summarized in Table 4.2.
4 Lewis Glyco-Epitopes: Structure, Biosynthesis, and Functions Table 4.2 Summary of biological functions of Lewis antigens Lewis antigen Proposed biological function Ley Embryo implantation and development Leb On gastric epithelium: H. pylori receptor On H. pylori LPS: Mimic host Lewis Ag form immune escape Trigger autoimmunity and cause gastritis Cancer metastasis: Colorectal, prostatic, nonsmall cell lung Sialyl Lex cancer, gastric, bladder, and hepatocellular carcinoma Insulin receptor signaling Promote cell proliferation: Transition from G1/S cell cycle stage ⋅CDI p27Kip1, ↑CDK2, ↑phophorylated RB Anti-apoptosis: procaspase-3, ↑Akt/P-Bad Promote cell adhesion and migration:⋅a5 integrin, a5b1 integrin signaling Metastasis of bladder cancer, rectal cancer Disialyl Lex Colorectal cancer Sialyl Lea
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References [71–73] [64] [68] [45–50] [77] [78] [83] [90] [53, 54] [62]
4.10 Future Perspectives It is generally accepted that glycobiology research is the third landmark in molecular biology after the research on proteins and nucleic acid (genes) since the carbohydrate chain is considered the third unique chain molecule in living organisms. The importance of glycoconjugates in the life sciences has attracted increased attention from biologists and chemists around the world. The complete nucleotide sequence of the human genome has been elucidated in recent years, but there remains a great gap between the genotype and the phenotype expressions in living systems. In the postgenomic era, proteomics became the hot topic in the field of molecular biology. However, more than 50% of proteins are glycoproteins containing N- and/or O-linked glycans, and Lewis antigens are common structures at the termini of many sugar chains. Furthermore, Lewis antigens are also found in the sugar chain of glycolipid, which is a necessary and sometimes crucial component of the cell membrane. Therefore, studies on Lewis antigens will be an important aspect of proteomics and glycomics research in order to elucidate the secrets of life, especially the secrets of cell–cell, cell–extracellular matrix recognition and adhesion. In the future, investigations will be focused on the search for novel structures of Lewis antigens, the structure–function relationship of the Lewis antigens and their receptors, the roles of these antigens in physiological events, and the molecular mechanisms of their actions. From a clinical point of view, it is worthwhile to study the expressions of Lewis antigens in different pathological conditions and the value of Lewis antigens as markers for disorder diagnosis and prognosis. It will be also possible to use the analogs or antagonists of Lewis antigens in family planning and in the treatment of inflammatory, infective, and malignant diseases.
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4.11 Summary The Lewis glyco-epitope or Lewis antigen is one of the most common and most important carbohydrate antigens in the glycoconjugates of most organisms. A fucosyl residue attached to an oligosaccharide by a a1→3 or a1→4 linkage is the essential structure of these antigens. They are synthesized from N-acetyllactosamine or poly-N-acetyllactosamine and their b1→6 branched variants by the modification of some Golgi apparatus resident glycosyltransferases and/or sulfotransferases in a tissue-specific manner. The Lewis antigen is not only structurally closely related to the ABH(O) blood group antigens but is also a glyco-determinant participating in a variety of biological events. They serve as the ligands of selectin, a receptor of bacteria, and as a bridge in an embryo’s uterine implantation. The novel results of the latest studies revealing that the Lewis antigen may function as a regulator of cell signaling, proliferation, and apoptosis, as well as cell adhesion and migration, are discussed in this chapter.
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Chapter 5
A Unique Endo-b-Galactosidase that Cleaves Both Blood Group A and B Glycotopes Su-Chen Li, Kimberly M. Anderson, and Yu-Teh Li
Keywords Endo-b-galactosidase • Glycosidase • Clostridium enzyme • Blood group A and B • Glycotopes Abbreviations A+-PGM B+-HOCG A-Tri A-Tetra A-Penta
Blood group A+ porcine gastric mucin Blood group B+ human ovarian cyst glycoprotein GalNAca1 → 3(Fuca1 → 2)Gal GalNAca1 → 3(Fuca1 → 2) Galb1 → 4Glc G a l N A c a 1 → 3 ( F u c a 1 → 2 ) G a l b 1 → 4(Fuca1 → 3)Glc A-Hexa GalNAca1 → 3-(Fuca1 → 2)Galb1 → 3GlcNA cb1 → 3Galb1 → 4Glc B-Tri Gala1 → 3(Fuca1 → 2)Gal B-Penta Gala1 → 3-(Fuca1 → 2)Galb1 → 4(Fuca1 → 3) Glc Endo-ABase Blood group A and B cleaving endo-bgalactosidase nEndo-ABase and rEndo-ABase The native and the recombinant Endo-ABase respectively FPLC Fast protein liquid chromatography TLC Thin-layer chromatography ConA Concanavalin A LB Luria–Bertani medium CNBr Cyanogen bromide DEAE Diethylaminoethyl
Su-Chen Li () Department of Biochemistry, Tulane University Health Sciences Center, School of Medicine, New Orleans, LA 70112, USA e-mail:
[email protected] A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_5, © Springer Science+Business Media, LLC 2011
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Sulfopropyl Ni-nitrilotri-acetic acid Column volume 17.5% Sodium chloride/8.8% sodium citrate, pH 7.0 0.58% Sodium chloride/0.20% magnesium sulfate/50 mM Tris–HCl, pH 7.5/0.01% gelatin Human red blood cells Fucolectin-related protein Fluorescene-5-isothiocyanate Fluorescene-activated cell sorter
Endo-b-galactosidases cleave the internal b-linked galactosyl residues of sugar chains in glycoconjugates and release oligosaccharides as their products. Based on the oligosaccharide products, the microbial endo-b-galactosidases [1] can be divided into four classes, of which the products are, respectively: (a) GlcNAcb1 → 3Gal, (b) Gala1 → 3Gal, (c) blood group A-trisaccharide and B-trisaccharide, and (d) GlcNAca1 → 4Gal. Endo-b-galactosidases, as well as other glycosidases, are often present as contaminants in commercial enzyme preparations. Their presence as contaminants is not easily recognized or clearly indicated. The use of an enzyme preparation with unknown contaminants can give unexplainable results or cause misinterpretation. However, the enzyme contaminants may sometimes provide opportunities for new discoveries. Endo-ABase was discovered through the use of an impure commercial clostridial sialidase preparation.
5.1 Initial Observation In 1994, we reported the isolation and characterization of a NeuAca2 → 3Galspecific sialidase, sialidase L, that produced 2,7-anhydro-NeuAc from the leech Macrobdella decora [2]. Sialidase L cleaves only the a2 → 3-linked NeuAc. This strict linkage specificity of sialidase L is distinct from the widely used commercial clostridial sialidase, which cleaves both the a2 → 3- and the a2 → 6-linked NeuAc [3, 4]. In 1996, we used clostridial sialidase and sialidase L to determine the ratio of a2 → 3-linked and a2 → 6-linked NeuAc in blood group A active human ovarian cyst glycoprotein (HOCG). Commercially available clostridial sialidase preparations have been widely used for studying the structure and function of sialoglycoconjugates [3]. This sialidase has been shown to also contain proteolytic [5–7], glycosidic [6], cytotoxic, and hemolytic [8–10] activities. We have also previously reported that the commercial sialidase prepared from Clostridium perfringens (ATCC 10543) was contaminated with an unusual endo-b-galactosidase [11] capable of releasing a specific disaccharide glycotope, GlcNAca1 → 4Gal, from blood group A+ porcine gastric mucin (A+-PGM). Upon our initial incubation of HOCG with the clostridial sialidase Type IX from Sigma, we found the production of an unknown sugar band in addition to the expected NeuAc band (Fig. 5.1). Those
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Fig. 5.1 Production of an unknown band from human ovarian cyst glycoprotein (HOCG) by clostridial sialidase. E, clostridial sialidase; S, A+-HOCG; NeuAc, N-acetylneuraminic acid
unknown bands released from the A+-HOCG and from the B +-HOCG were subsequently purified and identified to be the A+-trisaccharide (A-Tri) and the B+trisaccharide (B-Tri), respectively, by nuclear magnetic resonance (NMR) [12].
5.2 Enzyme Assay Since A+-HOCG was not easily available in large quantities, we searched for a convenient substrate and found that the commercially available A+-PGM was suitable as a substrate for studying this unknown saccharide-releasing enzyme. This enzyme was subsequently named Endo-ABase or E-Abase [12]. A+-PGM (Type II) from Sigma was exhaustively dialyzed against distilled water and used to assay Endo-ABase activity by thin-layer chromatography (TLC). A 10-mL reaction mixture containing 25 mg of A+-PGM and an appropriate amount of Endo-ABase in 25 mM sodium acetate buffer, pH 6.0, was incubated at 37°C for a predetermined time. The reaction was stopped by the addition of 2 mL of glacial acetic acid, and the entire reaction mixture was spotted onto a silica gel-coated TLC plate (EM Science). The plate was developed with 1-butanol:acetic acid:water (2:1:1, v/v/v), and the oligosaccharides were revealed by the diphenylamine-aniline-phosphoric acid reagent [13]. The intensities of the oligosaccharide bands were quantified by scanning the plates as described previously [11]. One unit of the enzyme activity was defined as the amount that releases 1 nmol of A-Tri from A+PGM/min at 37°C.
5.3 Purification of the Native Endo-ABase Unless otherwise indicated, all operations were performed at 0–5°C. Protein solutions were concentrated using an Amicon stirred cell with a PM-10 membrane. Centrifugations were routinely carried out at 8,000 × g for 20–30 min using a Sorvall RC5C refrigerated centrifuge.
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Step 1: Preparation of the crude enzyme C. perfringens, strain ATCC 10543, was cultured in 15 L of medium for 20 h at 37°C as previously described [14]. The culture supernatant was brought to 85% saturation with solid ammonium sulfate and left standing overnight. The precipitate thus formed was collected, dissolved in a small amount of water, and dialyzed exhaustively against water for 48 h. The dialyzed sample was centrifuged, and the supernatant was lyophilized to yield 11.2 g of crude enzyme powder. Step 2: Sephacryl S-200 gel filtration The crude enzyme powder from Step 1 was dissolved in 70 mL of 0.1 M ammonium acetate buffer, pH 5.8, and applied onto a Sephacryl S-200 column (5 × 90 cm) (Amersham Biosciences) equilibrated with the same buffer. The column was also eluted with the same buffer at 96 mL/h, and 20-mL fractions were collected. A 10-mL aliquot from every other fraction was assayed for the enzyme activity using A+-PGM as substrate. Fractions containing nEndo-ABase activity were pooled, concentrated, and dialyzed overnight against 10 mM Tris–HCl buffer, pH 7.5 (Buffer A). Step 3: Fractogel diethylaminoethyl (DEAE) chromatography The preparation from Step 2 was applied onto a Fractogel DEAE-650 (M) column (1.5 × 10.5 cm) (EM Science) equilibrated with Buffer A. After removing the unabsorbed proteins with Buffer A, nEndo-ABase was eluted with 10 mM sodium acetate buffer, pH 4.0, at 1 mL/min. Fractions of 2 mL were collected. The fractions containing nEndo-ABase were pooled, concentrated, and dialyzed overnight against 10 mM sodium acetate buffer, pH 5.5 (Buffer B). Step 4: Fractogel sulfopropyl (SP) chromatography The preparation from Step 3 was applied onto a Fractogel SP-650 (M) column (1.0 × 7.5 cm) (EM Science) equilibrated with Buffer B, and the column was eluted with the same buffer at 1 mL/min. Fractions of 1 mL were collected. The majority of nEndo-ABase activity was recovered in the unabsorbed fractions while the bulk of the contaminants were eluted by Buffer B containing 0.4 M NaCl. Fractions with the highest nEndo-ABase activity were pooled and dialyzed overnight against 25 mM sodium phosphate buffer, pH 7.0 (Buffer C). Step 5: Concanavalin A (ConA) Sepharose chromatography The preparation from Step 4 was concentrated to 0.3–0.5 mL and applied onto a ConA Sepharose column (1.0 × 48 cm) (Amersham Biosciences) equilibrated with Buffer C. nEndo-ABase was eluted in the early breakthrough fractions by Buffer C. Fractions containing nEndo-ABase activity were pooled, concentrated, and dialyzed against Buffer A. Step 6: Mono Q chromatography The enzyme preparation from Step 5 was applied onto a Mono Q HR 5/5 column (0.5 × 5.5 cm) equilibrated with Buffer A at 0.5 mL/min using an Amersham Biosciences ÄKTA fast protein liquid chromatography (FPLC). After washing with the same buffer, the column was eluted with Buffer A containing NaCl using the following gradient: 0–0.06 M NaCl for 5 column volumes (CV); 0.06 M NaCl for 5 CV; 0.06–0.1 M NaCl for 20 CV; 0.1 M NaCl for 20 CV; 0.1–0.12 M NaCl for 5 CV; 0.12 M NaCl for 5 CV; 0.12–0.3 M NaCl for 5 CV. Fractions of 1 mL were
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collected. Fractions containing the nEndo-ABase activity were eluted as a sharp peak at 0.1 M NaCl (Fig. 5.2). They were pooled, dialyzed against 5 mM sodium phosphate buffer, pH 6.0, and used in subsequent experiments. This final enzyme preparation was over 98% pure as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 5.3, lane 1). Table 5.1 summarizes the purification of nEndo-ABase from 15 L of culture medium.
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Fig. 5.3 SDS-PAGE of the nEndo-ABase and rEndo-ABase. Both the nEndo-ABase and rEndoABase were analyzed by SDS-PAGE using a 10% gel. Protein bands were visualized using Coomassie Brilliant Blue stain. Lanes: 1, nEndo-ABase (2 mg); 2, molecular weight markers; 3, rEndo-ABase (2 mg)
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Table 5.1 Purification of nEndo-ABase from C. perfringens culture supernatant (1.5 L) Purification Total protein Total Specific Steps (mg) activity (U) activity (U/mg) Yield (%) (-fold) 11,077 100 1. Crude enzyme 10,282 1.1 1 2. Sephacryl S-200 279.5 9,084 32.5 82 29.5 3. Fractogel DEAE 79.8 7,920 99.2 71.5 90.2 4. Fractogel SP 56.9 6,885 121.0 62.2 110 5. ConA Sepharose 5.9 4,638 786.1 41.9 714 2,630 6. Mono Q 0.7 2,026 2,894.0 18.3 By the above purification scheme, Sephacryl S-200 gel filtration removed over 90% of the contaminating proteins. In the final two steps, ConA Sepharose chromatography removed a 96-kDa contaminant, whereas Mono Q/FPLC resolved the nEndo-ABase from smaller contaminants. By these steps, the nEndo-ABase was purified over 2,600-fold with approximately 18% recovery. Using Azocoll as substrate, nEndo-ABase was found to be free of protease activity. It was also free of the following exoglycosidase activities using p-nitrophenyl glycosides as substrates: a-l-fucosidase, a- and b-glucosi-dases, a- and b-galactosidases, a- and b-mannosidases, a-N-acetylglucosaminidase, a-arabinosidase, b-xylosidase, and b-hexosaminidase
5.4 Cyanogen Bromide: Peptides from nEndo-ABase Cyanogen bromide (CNBr) cleavage products were generated from 200 mg of the purified nEndo-ABase according to Charbonneau [15]. Peptides were separated by 10% SDS-PAGE and blotted onto a polyvinylidene difluoride membrane using a Trans-Blot semidry transfer cell (Bio-Rad) at 20 V for 1.25 h. The most prominent bands were excised for sequencing. The amino acid sequences of CNBr peptides P1, P2, P3, and the N-terminal region of the native protein are shown below: N-Terminal: LEESRDVYLSDLDWLNATHGDDTK CNBr P1: MLNEAQSYVNPK CNBr P2: MRAKTKSLLYG CNBr P3: MSQSPAYTTGRYGNIPAV
5.5 Cloning of the Endo-ABase Gene Genomic DNA from C. perfringens (ATCC 10543) was prepared as described [16]. A sense primer, F1 (GAYYTIGAYTGGYTNAAYGC), and an antisense primer, R2 (YTTNGTYTT-NGCYCGCAT), were designed based on the N-terminal sequence and the CNBr peptide P2, respectively. Using these two primers and the C. perfringens genomic DNA as a template, a 1.5-kbp product was generated by polymerase chain reaction (PCR) under the following conditions: using Taq DNA polymerase (Gibco BRL) for 30 cycles consisting of 45 s denaturation at 94°C, 45 s annealing at 45°C, and 2 min extension at 72°C. This 1.5-kbp product (probe A) was sequenced and was found to contain the N-terminal sequence of the nEndo-ABase and the two CNBr peptides P1 and P2, indicating that this DNA fragment was part of the gene encoding Endo-ABase. Probe A was subcloned into the pGEM-T Easy vector (Promega).
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XL1-Blue MRF’ and SOLR cells (Stratagene) were grown in Luria–Bertani (LB) medium supplemented with 0.2% maltose and 10 mM MgSO4. Construction of the first genomic library in the Lambda ZAP II vector (Stratagene) and subsequent screenings were performed as previously described [17] using the two primers, EAB4.2F (AGTGGAAAG-AGGTTCCAGATG) and EAB4.2R (CTGCTGCGTTACTTTCTACA), complementing the sequence of Probe A. Of 10,000 plaques screened, 20 positives were identified, and one positive cloned phage was selected for in vivo excision using the ExAssist helper phage and SOLR strain. Single colonies of SOLR cells containing the excised phagemid (DNA insert in pBluescript SK (-) vector) were subsequently used in a third PCR screening under previous conditions. One clone, pEAB4E, was found to contain two thirds of the expected gene sequence from the 5¢-end. To identify the 3¢-end gene for the Endo-ABase, a second genomic library was constructed in Escherichia coli (E. coli) JM 109. Genomic DNA (15 mg) from C. perfringens was digested with 100 U of XbaI. Purified DNA fragments between 1.8 and 6 kbp were ligated into a pBluescript SK (-)/XbaI/calf intestinal alkaline phosphatasetreated vector and transformed into E. coli JM 109. One positive clone (pEABX1) was identified by PCR using the primers EAB4.1F (GCTACCCACGGAGATGATACAA) and EAB4.5R (ATCTG-ACTTCCAGATAAACGACTCTC). This clone was found to contain the full-length open reading frame of eabC; the DNA and the deduced amino acid sequences of this gene have been published [12]. The eabC gene consists of 2,400 bp that encodes 800 amino acid residues. Based on the PSORT program, the N terminus of this protein shows an extended region of hydrophobicity between the residues 10 and 35 as well as a predicted cleavage site after residue 35. All of these indicate a signal sequence for secretion into the culture medium. The mature protein consists of 765 amino acid residues with a calculated molecular mass of 87 kDa, which is very close to the value of 88 kDa estimated by SDS-PAGE for the nEndo-ABase (Fig. 5.3, lane 1). An expression plasmid, pEABHNB3, with a C-terminal 6 histidine tag (His tag), was constructed in the pET-15b vector. The eabC without the sequence for the N-terminal 35 amino acid signal peptide was amplified by PCR using Taq DNA polymerase with pEABX1 as template and EX1 (AACCATGGGATTGGAAGAAAGCAGA) and EX6 (AGCCGG- ATCCGTGATGATGATGATGATGCTTAATTACAATATC) as primers. The 2.3-kbp PCR product was purified, digested with NcoI and BamHI, ligated into a pET-15b vector, sequenced, and given the name pEABHNB3. The recombinant Endo-ABase (rEndo-ABase) was expressed in E. coli BL21(DE3) at 37°C in LB medium containing 100 mg/mL of ampicillin. rEndoABase was purified under nondenaturing conditions as described by Hoffmann and Roeder [18] but excluded dithiothreitol (DTT), glycerol, and NP40. Three 1-L portions of LB/ampicillin media were each inoculated with 20–30 mL of an overnight culture, and the cells were harvested 4 h after the optical density at 600 nm had reached 1.4. The cells were lysed in 60 mL of lysis buffer (10 mM Tris–HCl buffer, pH 7.9, 0.5 M NaCl, 0.5 mM phenylmethylsulfonyl fluoride, 5 mM imidazole) using a French press. The cell-free extract was heated at 60°C for 4 min and centrifuged. This heat treatment precipitated about 30% of nonenzymatic proteins but did not affect the rEndo-ABase activity. The supernatant was then
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applied onto a Ni2+-nitrilotri-acetic acid (Ni-NTA) column (1.5 × 12.5 cm) (Qiagen) equilibrated with the lysis buffer. After extensively washing the column with the same buffer, the bound proteins were eluted with an elution buffer (20 mM Tris– HCl buffer, pH 7.9, 100 mM KCl, 0.5 mM phenylmethylsulfonyl fluoride, 150 mM imidazole). Fractions containing the enzyme activity were concentrated and dialyzed for 18 h against 4 L of Buffer A (see purification of the native EndoABase, Step 2). Aliquots (9–12 mg) of protein were then applied onto a Mono Q HR (5/5) column previously equilibrated with Buffer A. The rEndo-ABase was eluted with Buffer A containing NaCl using the following gradient: 0–0.1 M NaCl for 5 CV; 0.1 M NaCl for 5 CV; 0.1 M–0.2 M NaCl for 40 CV. The rEndo-ABase was eluted as a sharp peak at around 0.13 M NaCl. The rEndo-ABase was expressed as a soluble protein at about 34 mg/L of culture without isopropyl-b-d-thiogalactopyranoside induction. We were able to purify 27 mg of the rEndo-ABase from 1 L of culture with a recovery of about 80%. Similarly to the nEndo-ABase, the purified rEndo-ABase moved as a sharp protein band of about 88 kDa by SDSPAGE with a very minor contaminant of 67 kDa (Fig. 5.3, lane 3). No exoglycosidase or protease activity was detected in the purified rEndo-ABase preparation. The specific activity of 2,667 U/mg of protein for the rEndo-ABase was very close to that of 2,894 U/mg of protein for the nEndo-ABase (Table 5.1), indicating that the rEndo-ABase was fully active.
5.6 General Properties of Endo-ABase Using A+-PGM or B+-HOCG as the substrate, the maximal activity of both rEndoABase and nEndo-ABase was found to be pH 5.5–6.0, and both enzymes maintained greater than 75% of their activity between pH 5.5 and 8.5. Furthermore, both enzymes were very stable with little or no loss of activity upon storage at −20°C for 2 years. Cations at 10 mM concentration, such as Ca2+, Co2+, Mg2+, and Mn2+, had little or no effect on Endo-ABase activity with either A+-PGM or B+-HOCG as the substrate. Also, b-mercaptoethanol (up to 100 mM) and various monosaccharides (Gal, Glc, Fuc, and GalNAc at 0.15 mM) had little or no effect on Endo-ABase activity.
5.7 Substrate Specificity of Endo-ABase nEndo-ABase and rEndo-ABase have identical substrate specificity. Endo-ABase was able to liberate the A-Tri from A+-PGM and the B-Tri from B+-HOCG (Fig. 5.4a, lanes 3 and 5). Endo-ABase also effectively liberated A-Tri or B-Tri from the following blood group-carrying oligosaccharides: A-Tetra, GalNAc-a1 → 3(Fuca1 → 2) Galb1 → 4Glc; A-Penta, GalNAca1 → 3(Fuca1 → 2)Galb1 → 4(Fuca1 → 3)Glc;
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Fig. 5.4 TLC analysis showing the hydrolysis of A+-PGM and B+ HOCG (a) and oligosaccharides containing blood group A and B glycotopes (b) by rEndo-ABase. (a) Lane 1, A+-PGM; lane 2, standard A-Tri; lane 3, A+-PGM + rEndo-ABase; lane 4, standard B-Tri; lane 5, B+HOCG + rEndo-ABase; lane 6, B+ HOCG; lane 7, rEndo-ABase only. Lanes 3 and 5 show the release of the A-Tri and B-Tri, respectively. A+-PGM or B+ HOCG (25 mg) was incubated with 25 mU of rEndo-ABase in 25 mM sodium acetate buffer, pH 6.0, at 37°C for 20 min. (b) Lane 1, standard A-Tri; lane 2, A-Tetra (GalNAc-a1 → 3(Fuca1 → 2)Galb1 → 4Glc); lane 3, A-Tetra + rEndo-ABase; lane 4, A-Penta (GalNAca1 → 3(Fuca1 → 2)Galb1 → 4(Fuca1 → 3)-Glc); lane 5, A-Penta + rEndo-ABase; lane 6, B-Penta (Gala1 → 3(Fuca1 → 2)Galb1 → 4(Fuca1 → 3) Glc); lane 7, B-Penta + rEndo-ABase; lane 8, A-Hexa (GalNAca1 → 3(Fuca1 → 2)Galb1 → 3Glc NAcb1 → 3Galb1 → 4Glc); lane 9, A-Hexa + rEndo-ABase; lane 10, A+-PGM; lane 11, A+PGM + rEndo-ABase; lane 12, rEndo-ABase only. Each oligosaccharide substrate (2 mg) was incubated with 2 U of rEndo-ABase in 25 mM sodium acetate buffer, pH 6.0, for 18 h at 37°C. Detailed conditions are described under experimental procedures
and B-Penta, Gala-1 → 3(Fuca1 → 2)Galb1 → 4(Fuca1 → 3)Glc (Fig. 5.4b). In these oligosaccharides, both blood group A and B glycotopes are linked through b1 → 4 to Glc. As shown in Fig. 5.5, Endo-Abase hydrolyzed A-Penta only slightly faster than B-Penta, indicating that the enzyme does not show a particular preference for blood group A or B glycotope. Unlike the above-mentioned blood group oligosaccharides, the A-Hexa, GalNAca1 → 3(Fuca-1 → 2) Galb1 → 3GlcNAcb1 → 3Ga lb1 → 4Glc, which carries the blood group A glycotope on a type 1 chain, was only slowly hydrolyzed (Fig. 5.4b, lane 9). These results suggest that while Endo-ABase does not have a strict preference for the blood group A or B glycotope, it does recognize the specific core chain by preferentially cleaving the endo-b-galactosyl linkage of type 2 core chain (–Galb1 → 4GlcNAc/Glc–) over type 1 core chain (–Galb1 → 3GlcNAc–) (Fig. 5.4). Endo-ABase also slowly released A-Tri from GalNAca1 → 3(Fuca1 → 2) Galb1 → 3GalNAca1 → Ser/Thr in the glycopeptides prepared from porcine submaxillary mucin. The Galili pentasaccharide, Gala1 → 3Galb1 → 4GlcNAcb1 → 3 Galb1 → 4Glc, which is devoid of an l-fucose linked a-1 → 2 to the penultimate b-galactosyl residue in the type 2 core structure, was not hydrolyzed. Thus, the presence of an l-fucose residue on the substrate is essential for Endo-ABase to carry out its action. Furthermore, Endo-ABase did not hydrolyze the Lea+- or the
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H+-HOCG blood group substances. Also, glycosaminoglycans, such as heparin, heparan sulfate, dermatan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, and keratan sulfate, were not susceptible to Endo-ABase.
5.8 Application of Endo-ABase The ability of Endo-ABase to modify the agglutination property of human red blood cells (RBCs) with the respective anti-blood group antibodies was examined by flow cytometry with a Becton Dickinson fluorescence-activated cell sorter (FACS) Calibur cytometer (San Jose, CA) at a low flow rate. Data from 30,000 events were collected and analyzed with the use of CELL Quest software (Becton Dickinson). To prepare the RBCs for enzyme treatment, 400 mL of type A or type B RBC (Immucor, Inc, Norcross, GA, 2–4% cell suspension) was centrifuged at 1,250 × g for 4 min. The packed RBCs were then washed with autoclaved phosphate-buffered saline (PBS) (pH 7.4), centrifuged, and resuspended in PBS to give a hematocrit of 8–16%. A 50-mL aliquot of each type of RBC was then treated with 50 mL (533 units) of rEndo-ABase, and the mixture was rotated end-over-end on a 5.5-cm diameter wheel at 8 rpm for 18 h at 23°C. After incubation, the RBCs were washed with 2.0 mL of PBS and resuspended in 20 mL of PBS. A 5-mL aliquot of packed RBC was then added to 100 mL of PBS Wash Buffer (PBS containing 1% fetal bovine serum). The indirect immunofluorescence method was used to label 1 × 107 RBC (each from the control and the enzyme-treated RBC samples) with anti-A or anti-B murine monoclonal antibodies (Immucor Inc., Norcross, GA) as the primary antibody at a dilution of 1:1,000 in PBS Wash Buffer, followed
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by the addition of fluorescein isothiocyanate (FITC)-labeled goat anti-mouse immunoglobulin G (IgG) (Fab specific) (Sigma) as the secondary antibody. The secondary antibody was used at a dilution of 1:200 in PBS Wash Buffer. All incubations were performed for 15–20 min at room temperature under rotation. The labeled RBC were washed with PBS Wash Buffer, resuspended in 1 mL of PBS, and then analyzed by FACS. Under the conditions described above, rEndo-ABase reduced the blood group A antigen expression of type A RBC by 92% (Fig. 5.6, left panel, bottom) while
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Fig. 5.6 FACS analysis showing the effect of Endo-ABase on blood group A and B antigenicity of human type A and type B RBC, respectively. Left panels, top and bottom, show the histograms of blood group A-FITC fluorescence of type A RBC stained in the absence (top) or presence (bottom) of rEndo-ABase. Right panels, top and bottom, show the histograms of blood group B-FITC fluorescence of type B RBC stained in the absence (top) or presence (bottom) of rEndo-ABase. The results are depicted as overlays in which the shaded histograms represent the negative control mock/RBC and the open histograms represent the FITC-labeled RBC. Mean fluorescence intensity in log scale is on the x-axis, and the relative number of cells is represented on the y-axis. Anti-A ab and Anti-B ab are anti-A and anti-B murine monoclonal antibodies. “2° ab” means FITC-labeled goat anti-mouse IgG
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completely removing the blood group B antigen from type B RBCs (Fig. 5.6, right panel, bottom). The incomplete destruction of blood group A antigenicity of type A RBC by rEndo-ABase is consistent with the heterogeneous nature of the blood group A immunodeterminants on type A RBC [19]. It has also been shown that various blood group A glycotopes on type A RBC differ in their susceptibility to a-N-acetylgalactosaminidase [20]. Since both A- and B-trisaccharide glycotopes are linked through an endo-b-galactosyl linkage, the ability of rEndo-ABase to abolish both blood group A and B antigenicity of RBC established this enzyme as a blood group A- and B-cleaving endo-b-galactosidase. rEndo-ABase was also used in a preliminary testing for the reduction of blood type A antigen expression by intravenous administration of the enzyme to a type A baboon [21]. Immunohistochemical analysis was carried out to compare the biopsy samples from the kidney and liver before and after 4 h of enzyme administration. The expression level in the glomeruli (kidney) was reduced to 9% and that of the sinusoids (liver) to 1% [21]. Although further research is necessary, Endo-ABase may be useful in developing an effective method for overcoming ABO incompatibility.
5.9 Comparison of Endo-ABase and Other Proteins A BLASTN search of the Endo-ABase gene against the genome of C. perfringens 13 deposited in the GeneBank database revealed a 100% match to the CPE0329 gene, whose gene product was labeled as a hypothetical protein [22]. This finding validated the results of our cloning work and also established the gene product of the CPE0329 gene of C. perfringens 13 as Endo-ABase. When FASTA and BLAST searches were performed, no significant sequence identity between Endo-ABase and any other nonglycosidase was revealed, except for a modest sequence identity (34% identity to 550 amino acid residues at the C terminus of Endo-ABase sequence) to a protein (NP 346573) identified as a “fucolectinrelated protein (FRP) of unknown function” of Streptococcus pneumoniae [23]. Since an enzyme of similar specificity to the Endo-ABase had been detected in Diplococcus pneumoniae [24], the cloning of the FRP gene was undertaken to verify the relationship between Endo-ABase and FRP. The gene encoding FRP (SP2159) [23] was isolated from the genomic DNA of S. pneumoniae (ATCC BAA-334), and the recombinant FRP was expressed in E. coli, BL21 (DE3). Although the purified recombinant FRP showed a strong affinity to an l-fucose-conjugated Agarose column, this protein was unable to cleave substrates containing the blood group A or blood group B determinant (data not shown). The ability of the FRP to bind to the l-fucose-conjugated Agarose column supports the initial identification of the gene product of SP2159 as an FRP by Tettelin et al. [23]. Endo-ABase was found to hydrolyze only the l-fucose-containing blood group A and B trisaccharide glycotopes, GalNAc/ Gala1 → 3(Fuca1 → 2)Gal–, but not substrates devoid of an l-fucose residue. The slight sequence identity of Endo-ABase to FRP indicates that the l-fucose recognition site for Endo-ABase may reside in the C-terminal half of the peptide sequence.
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5.10 New Glycoside Hydrolase Family We used the DNA and protein sequences of Endo-ABase as a query to search against the FASTA and BLAST databases to find other proteins that might be related to Endo-ABase. The results revealed that the amino acid sequence of EndoABase did not bear any detectable similarity to any of the established glycoside hydrolase (GH) families. There were 97 public families of carbohydrate-active enzymes (CAZy) when we completed our work (http://afmb.cnrs-mrs.fr/CAZY). Since the Endo-ABase sequence was distinct from the established glycoside hydrolases, it would define a novel CAZy family. In consultation with Dr. Bernard Henrissat of the Universités d’Aix-Marseille I and II, France, who had established a classification system for glycosidases, we had assigned Endo-ABase to a new glycoside hydrolase family designated as GH98. It is noteworthy that Endo-ABase does not share significant sequence homology with other endo-b-galactosidases cloned from Flavobacterium keratolyticus (AF083896) [25] and C. perfringens (AB038772 and AB059351) [17, 26]. The fact that the primary sequence of EndoABase does not contain an EXDX(X)E motif as found in other endo-b-galactosidases [17, 25, 26] indicates that Endo-ABase may have evolved along a separate evolutionary line.
5.11 Conclusion Glycoconjugates containing blood group antigens are known to be on the surface exposed to the external environment [19, 27]. The microorganism C. perfringens may utilize Endo-ABase to degrade the blood group antigens on cell surface glycoconjugates to enhance its infectivity and virulence. This unique endo-b-galactosidase should become useful for studying the structure and function of glycoconjugates as well as for identifying other glycosidases belonging to the new GH98 family.
5.12 Summary Human RBCs contain specific saccharide epitopes, glycotopes, which determine the human blood group A, B, and O (H) antigenicities. Blood group A determinant is a trisaccharide GalNAca1 → 3(Fuca1 → 2)-Gal, and the blood group B determinant is a different trisaccharide Gala1 → 3-(Fuca1 → 2)-Gal. These specific trisaccharides are called A-trisaccharide and B-trisaccharide. We have isolated an endo-b-galactosidase (Endo-ABase) from C. perfringens (ATCC 10543) capable of releasing both the A-trisaccharide and B-trisaccharide from glycoconjugates containing the blood group A and B glycotopes. We have purified the native EndoABase from C. perfringens using a series of chromatographic steps. From 15 L of the culture supernatant, we obtained 0.7 mg of homogeneous enzyme. The purified Endo-ABase migrated on SDS-PAGE as one band at ~88 kDa. We subsequently
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cloned the gene (eabC ) encoding Endo-ABase from this organism. The gene consists of 2,400 bp and encodes 800 amino acid residues. This gene was found to be identical to the gene CPE0329 of C. perfringens strain 13, whose product was labeled as a hypothetical protein. Since the amino acid sequence of Endo-ABase does not bear detectable similarity to any of the existing 97 families of GHs, this unusual enzyme was assigned to a new family, GH98. We have also expressed eabC in E. coli BL21(DE3) and obtained 27 mg of fully active recombinant EndoABase from 1 L of culture. The recombinant Endo-ABase not only destroyed the blood group A and B antigenicities of human type A and B erythrocytes, but also cleaved A and B trisaccharides from blood group A+ and B+ glycoconjugates. Acknowledgments We thank the late Professor Winifred M. Watkins for the generous gift of the HOCG samples used in this work. Thanks are also due to Dr. Bernard Henrissat for his invaluable advice on the classification of GHs. This work was supported by National Institutes of Health grant NS 09626.
References 1. Ashida H, Li SC, Li YT (2006) An unusual GlcNAca1-4Gal releasing endo-b-galactosidase. In: Endo M, Hasse S, Yamamoto K, Takagaki K (eds) Endoglycosidases, biochemistry, biotechnology, application. Kodansha/Springer, Tokyo, Japan 2. Chou MY, Li SC, Kiso M, Hasegawa A, Li YT (1994) Purification and characterization of sialidase L, NeuAca2 → 3Gal-specific sialidase. J Biol Chem 269:18821–18826 3. Cassidy JT, Jourdian GW, Roseman S (1965) The sialic acids IV. Purification and properties of sialidase from Clostridium perfringens. J Biol Chem 240:3501–3506 4. Corfield T (1992) Bacterial sialidases – roles in pathogenicity and nutrition. Glycobiology 2:509–521 5. Hatton MWC, Regoeczi E (1973) A simple method for the purification of commercial neuraminidase preparations free from proteases. Biochim Biophys Acta 327:114–120 6. Chien SF, Yevich SJ, Li SC, Li YT (1975) Presence of endo-b-N-acetylglucosaminidase and protease activities in the commercial neuraminidase preparations isolated from Clostridium perfringens. Biochem Biophys Res Commun 65:683–691 7. Boyle MDP, Ohanian SH, Borsos T (1976) Lysis of tumor cells by antibody and complement VI. Enhanced killing of enzyme pretreated tumor cells. J Immunol 116:661–668 8. Kraemer PM (1968) Cytotoxic, hemolytic and phospholipase contaminants of commercial neuraminidases. Biochim Biophys Acta 167:205–208 9. Den H, Malinzak DA, Rosenberg A (1975) Cytotoxic contaminants in commercial Clostridium perfringens neuraminidase preparations purified by affinity chromatography. J Chromatogr 111:217–222 10. Trams EG, Lauter CJ, Banfield WG (1976) On the activation of plasma membrane ectoenzymes by treatment with neuraminidase. J Neurochem 27:1035–1042 11. Ashida H, Anderson KM, Nakayama J, Maskos K, Chou CW, Cole R, Li SC, Li YT (2001) A novel endo-b-galactosidase from Clostridium perfringens that liberates the disacchride GlcNAca1 → 4Gal from glycans specifically expressed in the gastric gland mucous cell-type mucin. J Biol Chem 276:28226–28232 12. Anderson KM, Ashida H, Maskos K, Dell A, Li SC, Li YT (2005) A Clostridial endo-b-galactosidase that cleaves both blood group A and B glycotopes. J Biol Chem 280:7720–7728
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13. Anderson KM, Li SC, Li YT (2000) Diphenylamine-aniline-phosphoric acid reagent, a versatile spray reagent for revealing glycoconjugates on thin layer chromatography plates. Anal Biochem 287:337–339 14. Cassidy JT, Jourdian GW, Roseman S (1966) Sialidase from Clostridium perfringens. Methods Enzymol 8:680–685 15. Charbonneau H (1989) CNBr digest. In: Matsudaira PT (ed) A practical guide to protein and peptide purification for microsequencing. Academic, San Diego, CA 16. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1991) Current protocols in molecular biology. Wiley, New York 17. Ashida H, Maskos K, Li SC, Li YT (2002) Characterization of a novol endo-b-galactosidase specific for releasing the disaccharide GlcNAca1 → 4Gal from glycoconjugates. Biochemistry 41:2388–2395 18. Hoffmann A, Roeder RG (1991) Purification of His-tagged proteins in non-denaturing conditions suggests a convenient method for protein fraction studies. Nucleic Acids Res 19:6337–6338 19. Clausen H, Hakomori SI (1989) ABH and related histo-blood group antigens; immunochemical differences in carrier isotypes and their distribution. Vox Sang 56:1–20 20. Hoskins LC, Larson G, Naff GB (1995) Blood group A immunodeterminants on human red cells differ in biologic activity and sensitivity to a-N-acetylgalactosaminidase. Transfusion 35:813–821 21. Kobayashi T, Liu D, Ogawa H, Miwa Y, Nagasaka T, Maruyama S, Li YT, Onishi A, Kuzuya T, Kadomatsu K, Uchida K, Nakao A (2007) Alternative strategy for overcoming ABOincompatibility. Transplantation 83:1284–1286 22. Shimizu T, Ohtani K, Hirakawa H, Ohshima K, Yamashita A, Shiba T, Ogasawara N, Hattori M, Kuhara S, Hayashi H (2002) Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater. Proc Natl Acad Sci USA 99:996–1001 23. Tettelin H et al (2001) Complete genome sequence of a virulent isolate of Streptococcus pneumoniae. Science 293:498–506 24. Takasaki S, Kobata A (1976) Purification and characterization of an endo-b-galactosidase produced by Diplococcus pneumoniae. J Biol Chem 251:3603–3609 25. Leng L, Zhu A, Zhang Z, Hurst R, Goldstein J (1998) Cloning, functional expression and purification of endo-b-galactosidase from Flavobacterium keratolyticus. Gene 222:187–194 26. Ogawa H, Muramatsu H, Kobayashi T, Morozumi K, Yokoyama I, Kurosawa N, Nakao A, Muramatsu T (2000) Molecular cloning of endo-b-galactosidase C and its application in removing a-galactosyl xenoantigen from blood vessels in the pig kidney. J Biol Chem 275:19368–19374 27. Greenwell P (1997) Blood group antigens: molecules seeking a function? Glycoconj J 14:159–173
Part III
Lectins
Chapter 6
Recognition Roles of Mammalian Structural Units and Polyvalency in Lectin–Glycan Interactions Albert M. Wu, Jia-Haw Liu, Tanuja Singh, and Zhangung Yang
Keywords Lectins • Polyvalency • Recognition factor • Carbohydrate specificity • Carbohydrate combining sites • Glyco-recognition intensities Abbreviations C (GlcNAcb1→4)n NeuNAc or Neu5Ac NeuGly or NeuGc R m Ah
Chitin disaccharide (GlcNAcb1→4GlcNAc) Repeat unit of GlcNAcb1→4 N-Acetylneuraminic acid N-Glycolylneuraminic acid Carbohydrate residue multivalent GalNAca1→3(lFuca1→2)Gal
Lectins are an important class of proteins or glycoproteins of nonimmune origin that specifically or selectively bind carbohydrate moieties of complex carbohydrates. They play many critical roles in life processes, such as fertilization, embryogenesis, cell migration, organ formation, inflammation, immune defense, and microbial infection [8, 22, 40]. Specificity of lectins toward particular carbohydrate structures allows them to be used for characterization of unknown structures and identification and fractionation of glycoconjugates [23]. Moreover, this unique group of proteins has provided researchers with powerful tools to explore many biological processes in which lectins are involved. Considering the role of lectins in vivo, we have to realize that lectins in living organisms interact with multivalent macromolecules (glycoproteins) or clusters of oligosaccharide ligands on the cell surface. Many reports indicate that lectins show an altered reactivity (avidity) and, in some cases, even an altered specificity when the surface density of the carbohydrate ligand is changed [12, 25, 26, 47, 49, 50, 53]. Therefore, to provide a more valid and satisfactory depiction of the A.M. Wu (*) Glyco-Immunochemistry Research Laboratory, Institute of Molecular and Cellular Biology, College of Medicine, Chang Gung University, Kwei-san, Tao-yuan 333, Taiwan e-mail:
[email protected] A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_6, © Springer Science+Business Media, LLC 2011
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carbohydrate specificity of lectins, elucidate their functional roles, and optimize their biomedical applications, the following criteria should be taken into consideration [33, 40, 49]: (1) affinity for a monosaccharide and its derivatives; (2) expression of reactivities toward mammalian disaccharide structural units or their derivatives and finding the most active ligand; (3) simple oligovalent or cluster effect (e.g., multiantennary oligosaccharides or Tn glycopeptides); and (4) complex multivalent or cluster effects present in macromolecules with multiple known glycotopes. In earlier reports, the characterization of lectin specificity was limited to scales of (1) and (2). More recently, studies on the recognition profile of many lectins have expanded the scope to levels (3) and (4), on which this article is focused.
6.1 Grouping Lectins Based on Recognition of Monosaccharides and Oligosaccharides Lectins that can be used as tools to study the glycobiology system are defined as applied lectins. They must be easily obtainable, stable, and well-characterized with respect to specificity. Most lectins are inhibited by monosaccharides at relatively high concentrations and interact (usually more strongly) with oligosaccharides terminating with a given monosaccharide (Man/Glc, Gal, lFuc, GalNAc, GlcNAc, or sialic acid). Some lectins can recognize the specific monosaccharide (or oligosaccharide structure) substituted with one other residue. Lectins usually require an anomeric structure of the specific monosaccharide, but some lectins are not so restricted, i.e., they do not discriminate between a and b anomers. The contribution to lectin binding of monosaccharides (including their anomery and linkage positions) following the specific sugar differs from one lectin to another. For example, Griffonia simplicifolia isolectin B4 is specific for a-Gal and reacts with galactose and only four to ten times more strongly with Gala1→3Gal and several other Gala1→2/3/4-terminating disaccharides in which the second component is Gal, GalNAc, or GlcNAc [5, 45]. On the other hand, Amaranthus caudatus lectin (ACL) is specific for Galb1-3GalNAc (T antigen), reacts 320 times more actively than GalNAc, and shows no or negligible inhibition by Gal and Galb1→3/4GlcNAc at greater than 1,000 times higher concentration [65]. It should be mentioned that some lectins are not inhibited by any monosugar and recognize more complex larger structures. Examples are two Phaseolus vulgaris isolectins: leukoagglutinin (PHA-L), which reacts specifically with the tri- or tetraantennary complex N-glycans with b1→6-linked branches, and erythroagglutinin (PHA-E), which is specific for the biantennary N-glycans with bisecting GlcNAc [13]. Another example is the already forgotten Vicia graminea lectin, which requires in a receptor the presence of at least two clusters of Galb1→3GalNAc units linked to adjacent Ser/Thr residues. The activity is decreased by sialylation of the disaccharides and enhanced by a proximal hydrophobic residue [3]. This lectin reacts with human erythrocyte asialoglycophorins, which have clusters of O-glycans on adjacent amino acid residues, and does not react with the antifreeze glycoprotein, which has multiple Galb1→3GalNAc chains located on every third amino acid residue.
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In view of the interaction with mono- and oligosaccharides, the degree of lectin specificity is differentiated. Many lectins react with a series of structurally related oligosaccharides with more or less differentiated affinity. However, in the case of some lectins, the affinity may vary a great deal due to slight changes in the carbohydrate structure of the receptor. During the past decade, 11 disaccharide mammalian structural units have been used to characterize the binding properties of applied lectins (Table 6.1 and Fig. 6.1). Except Lb (Galb1→4Glcb1→ in glycosphingolipid) and P (GalNAcb1→3Gal in globoside), all of the units can be found in glycoproteins. Tn, which is an important marker for breast/colon cancer and vaccine development, exists only as O-glycans. Natural Tn glycoprotein, containing the simplest mammalian O-glycan, is exclusively expressed in the armadillo salivary gland [54]. Antifreeze glycoprotein is composed of repeating units of T [2]. Pneumococcus Type XIV capsular polysaccharide has uniform II disaccharide as carbohydrate side chains [18]. Asialo human a1-acid glycoprotein and asialofetuin provide multiantennary II structures. Human ovarian cyst glycoproteins, which belong to the complex type of glycoform, comprise most of the structural units [32, 67]. Selected applied lectins are classified into six groups according to their specificities to monosaccharides [35, 47]. They are further subgrouped by the affinities to (a) GalNAca1→O to Ser(Thr) of the peptide chain; (b) mammalian disaccharide structural units; (c) trisaccharides; (d) the number and location of lFuca1→linked to Galb1→3/4GlcNAc sequence; and (e) a2→3/6 linkages of sialic acid [44]. These structures are frequently found in soluble glycoproteins and as cell-surface glycoconjugates in mammals. A scheme of the classification based on monosaccharide Table 6.1 Carbohydrate structural units in mammalian glycoproteins and glycosphingolipids [34] Codes Structural units Sources Forssman pentasaccharide. Animal 1 F GalNAca1→3GalNAc tissue antigens and human FpentaGalNAca1→3GalNAcb1→ oncofetal glycotopes, mainly in 3Gala1→4Galb1→4Glc glycosphingolipids Fa In O-linked glycoproteins core GalNAca1→3GalNAca1→ Ser/Thr of protein core Fb Glycotope at the nonreducing end of GalNAca1→3GalNAcb1→ Fpenta2 A Human blood group A related diGalNAca1→3Gal saccharide Ah Human blood group A related triGalNAca1→3[lFuca1→ saccharide 2]Gal 3
Tn
GalNAca1→Ser/Thr of protein core
Tn antigen, only in O-linked glycoproteins
4
Ta
Galb1→3GalNAca1→ Ser/Thr of protein core
The mucin-type sugar sequence on the human erythrocyte membrane
Tb
Galb1→3GalNAcb1→…ceramide
Brain glycoconjugates and Gangliosides, GM1 (continued)
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Table 6.1 (continued) 5
Codes
Structural units
Sources
I
Galb1→3GlcNAc
Human blood group precursor type I and II carbohydrate sequences
Galb1→4GlcNAc
Branched or linear repeated II sequence is part of blood group I and i epitopes. I and II are precursors of ABH and Lea, Leb, Lex, Ley blood group active antigens. Most of the lectins reactive with II are also reactive with I. Lectin Tri-II and mII determinants are present at the nonreducing end of the carbohydrate chains derived from N- and O-glycans Human blood group B related disaccharide Human blood group B related trisaccharide Blood group Pk active and P1 disaccharide. Sheep hydatid cyst glycoproteins, salivary glycoproteins of the Chinese swiftlet, glycosphingolipids in human erythrocytes, and small intestine Constituent of mammalian milk
Ib 6
II IIb Tri-II
Galb1→3GlcNAcb1→ Galb1→4GlcNAcb1→
Triantennary Galb1→4GlcNAc
mII
Multivalent Galb1→4GlcNAc
B
Gala1→3Gal
Bh
Gala1→[lFuca1→2]Gal
8
E
Gala1→4Gal
9
L
Galb1→4Glc
7
Lb 10
P Pa
11
S Sb
Galb1→4Glcb1→ GalNAcb1→3Gal
GalNAcb1→3Gala1→
GalNAcb1→4Gal GalNAcb1→4Galb1→
Lactosyl ceramides in brain and part of carbohydrate structures in gangliosides Glycotope at the nonreducing end of globoside (antigen P) Brain and asialo-GM2 disaccharide; human blood group Sd(a+) related disaccharide in most human urine secretions, Tamm-Horsfall glycoprotein
a, b anomer of sugars, m multivalent, tri triantennary
specificity [10, 47] and the most active oligosaccharide structures (listed in Table 6.1) are shown as follows: 1. GalNAc-specific lectins (a) F/A, GalNAca1→3GalNAc (Forssman) and GalNAca1→3Gal (blood group A determinant disaccharide) – Dolichos biflorus (DBA), Helix pomatia (HPA), and Wisteria floribunda (WFA) (b) A, GalNAca1→3Gal – soybean (SBA), lima bean (LBA), and Psophocarpus tetragonolobus (PTA)
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I Group of GalNAcα1→structural units (Tn, Aβ, Fα and Fβ) Tn.
F. Tn CH2OH
CH2OH
O
OH
OH
OH O
(CH2)1 or 2
CO CH
OH
CO O (CH2)1 or 2 CH NH
O NHCOCH3
Tn, GalNAcα1→Ser/Thr (Tn, in O-linked glycoprotein)
A. CH2OH
CH2OH O
OH
OH
O
CH2OH O
O
OH
OH
OH
β
β
O
OH
NHCOCH3
NHCOCH3
Fa, GalNAcα1→3GalNAcα1→ (Core 5, GalNAcα1→ Tn at the reducing end of Oglycan)
CH2OH OH
O OH
O
NH
NHCOCH3
OH
CH2OH OH
O
NHCOCH3
Ab, GalNAcα1→3Galβ1→
NHCOCH3
Fb, GalNAcα1→3GalNAcβ1→ (Terminal disaccharide at the nonreducing end of Forssman glycotope)
II. Group of GalNAcβ1→structural units (Pα and Sβ) P.
S. CH2OH OH
CH2OH OH
CH2OH
O OH
O
α
O
OH
O
O
OH
OH
OH
CH2OH O
OH
β
OH OH
NHCOCH3
NHCOCH3
P a, GalNAcβ1→3Galα1→
S b, GalNAcβ1→4Galβ1→
III. Group of Galα1→structural units (Bβ and Eβ)
B.
E. CH2OH
OH
CH2OH OH
O
O
OH
β
O OH
OH
OH
Bb, Galα1→3Galβ1→
Fig. 6.1 (continued)
CH2OH OH
CH2OH OH
O O
OH
OH
O OH
OH
β
OH
Eb, Galα1→4Galβ1→
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IV. Group of Galβ1→structural units (Ta, Tb, Lb, Ib and IIb)
T.
I. Tn CH2OH
CH2OH OH
CH2OH OH
O
OH O
O
O (CH2)1 or 2
OH
CO CH
OH
O
CH2OH O
β O
OH
NH
NHCOCH3
NHCOCH3
OH
OH
Ib, Galβ1→3GlcNAcβ1→
Ta, Galβ1→3GalNAcα1→Ser/Thr (Ta, Galβ1→3Tn at the reducing end of O-glycan) CH2OH CH2OH OH
O
OH O
CH2OH
OH
OH
β
O
CH2OH O
OH
O O
OH
OH
β
OH NHCOCH3
OH
NHCOCH3
OH
Tb, Galβ1→3GalNAcβ1→ (Terminal disaccharide at the asialo GM1; GSL)
IIb, Galβ1→4GlcNAcβ1→
L. CH2OH OH
CH2OH O
O OH
O
OH
OH
OH
β
OH
Lb, Galβ1→4Glcβ1→(GSL)
Fig. 6.1 Mammalian glycoconjucates structural units used to express and classify the carbohydrate specificity of lectins (adopted and modified from Wang and Wu [31])
(c) Tn, GalNAca1→Ser/Thr – Vicia villosa B4 (VVA-B4) [36] and Salvia sclarea (SSA) [37] 2. Gal-specific lectins (a) T, Galb1→3GalNAc (Ta, or Tb) – peanut (PNA), Bauhinia purpurea alba (BPA) [52], Abrus precatorius (APA) [39, 51], Agaricus bisporus (ABA) [49], Sclerotium rolfsii (SRA) [62], Artocarpus integrifolia (AIA, jacalin) [50], Artocarpus lakoocha (ALA) [25], Maclura pomifera (MPA) [38], ricin [56], and Morus nigra (Morniga G) [27]
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(b) I/II, Galb1→3/4GlcNAcb1→ – Ricinus communis (RCA1) [57], Datura stramonium (thorn apple, TAA), wheat germ (WGA) [61], Erythrina cristagalli (ECL) [64], Erythrina corallodendron (ECorL) [66], and ricin [56] (c) B, Gala1 – G. (Bandeiraea) simplicifolia B4 (GSI-B4) [45] (d) E, Gala1→4Gal – abrin-a, mistletoe toxic lectin-I (ML-I) [43], and Aplysia gonad (AGA) [9, 42] 3. Man and/or Glc-specific lectins recognizing complex N-linked oligosaccharides Conavalia ensiformis (jack bean, ConA), Lens culinaris (LCA), Pisum sativum (PSA), Hippeastrum hybrid (HHA), Narcissus pseudonarcissus (NPL), and Morniga M [55] 4. GlcNAc, and/or Galb1→4GlcNAcb1→linked specific lectins Chitin oligosaccharide-specific agglutinins – wheat germ (WGA) [61], G. (Bandeiraea) simplicifolia II (GSA-II), Solanum tuberosum (STA), Ulex europaeus (UEA), and D. stramonium (DSA) 5. lFuc-specific lectins (subgroups based on the numbers and location of lFuca linkage) (a) Monofucosyl-specific agglutinins (blood group H, Lea, and/or Lex) – U. europaeus I (UEA-I), U. europaeus II (UEA-II), Pseudomonas aeruginosa (PA-II) [20, 59], and Anguilla anguilla (AAA) [58] (b) Difucosyl-specific agglutinins (Leb and Lex/y) – G. (Bandeiraea) simplicifolia IV (GSA-IV) and Lotus tetragonolobus (LTA) (c) Others requiring further characterization – Salmonella typhimurium (STA) and Ulva lactuca (Ulva-I) 6. Sialic acid-specific lectins (subgroups based on the recognized linkage of SA) (a) SAa2→6Gal(NAc) – Sambucus nigra (SNA-I) [24], Trichosanthes japonica (TJA1), and mistletoe toxic lectin-I (ML-I) [43] (b) SAa2→3Gal– Agrocybe cylindracea (ACA) and Maackia amurensis (MAA) [30] (c) Others requiring further characterization – Limax flavus (LFA) [14], Limulus polyphemus (LPA) [1], and Achatinin-H [21] Profiling the binding properties of lectins based on the affinity of decreasing order of mammalian glycotopes (determinants) is probably one of the best ways to express carbohydrate specificity and should facilitate the selection of lectins as structural probes for studying mammalian glycobiology. An example of such classification of Gal/GalNAc-reactive lectins is shown in Table 6.2. The lectins are divided into groups according to their highest affinity for one of the disaccharides or Tn residue, and it is completed with data showing the order of reactivity with other disaccharides [35]. These data demonstrate that lectins reacting most strongly with the same oligosaccharide may show differences in order of reactivity with other structures, which reflects subtle differences in lectin fine specificity.
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Table 6.2 Expression of binding properties of Gal/GalNAc reactive lectins by carbohydrate structural units Codes Lectins [11, 31, 33, 34, 44, 46, 47, 57] Carbohydrate specificity F/A
F/II A
Dolichos biflorus (DBA) Helix pomatia (HPA) Hog peanut (ABA, Amphicarpaea bracteata) Wistaria floribunda (WFA) Geodia cydonium (GCA) Griffonia (Bandeiraea) simplicifolia-A4 (GSI-A4) Caragana arborescens (CAA) Wistaria sinensis (WSA) Lima bean (LBA) Soybean (SBA, Glycine max) Vicia villosa (VVA, a mixture of A4, A2B2 and B4) V. villosa B4 (VVA-B4) Salvia sclarea (SSA)
Fpenta. > Aha > A > Tn >> P Fpenta. > A (>Ahb) ³ Tn, T >> P Fb > A >> L A (>Ahb), Fpenta. > F/P > Tn, I/II > L Fpenta., Ah > L > II, T > I >> E Fpenta. > Ah > GalNAc > E > B > I, T >> L, II Fpenta. > II > sialyl Tn Fpenta. > P > II, Tn, I and Ah ³ L/E Hexa-Aha > Ahb >> B A(>Ahb), Tn and I/II A (>Ahb) and Tn mainly
Two Tn >> one Tn >> one or two T Two Tn > single or three sequential Tn structures Glechoma hederacea (GHA) Tn cluster > Tn > A > Ah > F >> T T Peanut (PNA, Arachis hypogaea) T >> I/II >> Tn Amaranthus caudatus (ACL) T >> Tn >> I/II T/Tn Codium fragile subspecies tomentosoides (CFA) Fpenta. and Ta > Tn cluster > Ah > T Agaricus bisporus (ABA) Ta and Tn > I >> GalNAc >> II, L Maclura pomifera (MPA) T > Tn >> I/II and L Artocarpus integrifolia (jacalin, AIA) Ta > Pa > T, Tn, II > I >> Tb Artocarpus lakoocha (ALA) Ta, Tn cluster >> T, Tn >> I/II Bauhinia purpurea alba (BPA) Ta, Tn cluster > T, Tn > I/II Morus nigra galactose-specific lectin (Morniga G) Ta > Tn cluster >> T > Tn, P, Tri-IIc T/II Ricinus communis toxin (ricin, RCA2) T > I/II and Tn Abrus precatorius (APA) T > I/II > E > B > Tn Sophora japonica (SJA) T > I ³ II > L I/II R. communis (RCA1) Tri-IIc > II ³ I > E, B > T Datura stramonium (TAA, thorn apple) Biantennary I/II (penta-2,6) >> Cd Erythrina cristagalli (coral tree, ECA) Tri-II > II > L, I Erythrina corallodendron (ECorL) Tri-II > II > L > I Geodia cydonium (GCL) Tri-II > L > II, T > I Phaseolus vulgaris-L Tri-II > Penta-2,6 > Tri-2,6 > Hepta3,6 > IIb > GlcNAcb1→2Man B Griffonia (Bandeiraea) simplicifolia-B4 B > E > A (GSI-B4) A. precatorius toxin-a (Abrin-a) E, B > T, L, I/II E Mistletoe lectin-I (ML-I) E > II, L > T and I a Substitution of Fuca1→2 to subterminal Gal is important for binding b Substitution of Fuca1→2 to subterminal Gal blocks binding c Tri-II, triantennary II glycopeptides d C, chitin disaccharide Tn
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6.2 Effect of Polyvalency of Glycotopes on Lectin Binding The interaction between one component containing multiple receptors (cell surface, macromolecule) and another component containing multiple ligands shows significantly higher affinity than an interaction between a monomeric receptor and ligand. This phenomenon has great biological importance because polyvalent interactions between receptors on the surface of one cell type and ligands on the surface of another cell type are presumed to play critical roles in a wide variety of biochemical recognition processes. This effect can also be used to design polyvalent drugs with stronger therapeutic efficacy [29]. The effect of polyvalency also applies to lectins. Many studies have shown that an individual carbohydrate–lectin binding that is weak and hardly shows a specific interaction with a monomeric glycan may have a strong interaction with its polymeric forms [12, 17, 28, 49]. Therefore, it becomes pivotal to estimate the polyvalent effects while establishing the binding profile of a lectin. The most precise approach to this problem is to compare the lectin reactivity with a monomeric receptor (e.g., disaccharide) and a synthetic construct containing multiple receptors of defined orientation and density. Using this approach led to the following conclusions: (1) a polyvalent interaction may increase recognition intensities; (2) the binding of lectin initially increases as a function of ligand density and then decreases at higher densities; (3) the binding selectivity of a lectin may switch from one carbohydrate ligand to another when the surface density of a ligand increases [12]. The data used for this review were derived mainly from the results of lectin– glycan interactions analyzed by our sensitive enzyme-linked lectin sorbent assay (ELLSA) and inhibition of ELLSA [4, 19, 40]. ELLSA is a rapid and reagent-saving method in which the binding profile and recognition factors can be easily elucidated (Fig. 6.2) [4, 41, 48, 50]. This assay can provide insight into the specificities and size parameters of lectin-combining sites. The studies on the mode of interaction between lectins and various well-defined glycoproteins, glycosphingolipids, and polysaccharides should provide further information about actual requirements for binding, but they are hampered by the availability of various such glycoforms. This problem was basically solved by using natural glycoforms containing several or multiple/complex structural units. The data obtained provided us important concepts on the effect of polyvalency of glycotopes on lectin binding [40, 49, 53, 65]. Binding the lectins to plates coated with glycoproteins at various concentrations frequently shows an effect similar to that described by Horan et al. [12]: when the density of carbohydrate receptors on the well surface increases, the binding of a lectin initially increases and then decreases at higher coating densities [4, 40]. Therefore, it is important to select the proper coating concentration for analysis. The binding of lectins to immobilized polyvalent glycoconjugates does not allow for direct comparison to reactivity with monovalent and polyvalent receptors. This possibility is offered by inhibition of lectin binding (under selected conditions) with various monomeric, oligomeric, and polymeric inhibitors. Oligo-antennary N-glycans shown in Fig. 6.3 or Tn glycopeptides were used as oligomeric inhibitors.
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a
Fig. 6.2 Principle and an example [Ricinus communis agglutinin-1 (RCA1)] of (a) biotin/avidinbased microtiter plate enzyme-linked lectin sorbent assay (ELLSA) and (b) inhibition assays as tools for characterizing binding properties of soluble glycoproteins, their inhibitory potential, and/or determining carbohydrate specificities of lectins [40, 57, 64]. (a) The uncharacterized glycoprotein immobilized on a microtiter plate can bind an established biotinylated lectin, e.g., RCA1, which can recognize glycotopes (Galb1→4GlcNAcb) of unknown glycans. The overall activity of ExtrAvidinalkaline phosphatase complexed to the biotinylated lectin can be estimated. (b1) The biotinylated (known) lectin reacting with small haptens (M.W. 1.5 × 104), containing polyvalent glycotopes, can be used as an inhibitor to block the binding, and its inhibitory potential toward known lectins can also be determined
6 Recognition Roles of Mammalian Structural Units
a
b Monomeric I
Monomeric II Galβ1→4GlcNAc
c
Galβ1→3GlcNAc
d Lacto-N-neohexaose [LNnH; IIb1-6(Ib1-3)L]
Lacto-N-hexaose[LNH; Di-II or II b1-6(IIb1-3)L] Galβ1→4GlcNAc ↓β 1-6 Galβ1→4Glc ↑β1-3 Galβ1→4GlcNAc
e
109
Galβ1→4GlcNAc ↓β 1-6 Galβ1→4Glc ↑β1-3 Galβ1→3GlcNAc
Triantennary Galb1→4GlcNAc (Tri-II)
Galβ1→4GlcNAcβ1→ 2Man ↓ α 1-6 Galβ1→4GlcNAcβ1→2Manα1→3Manβ1→4GlcNAcβ1→4GlcNAcβ1-N-Asn ↑ β 1-4 Galβ1→4GlcNAc
f
Asialo human α1-acid glycoprotein
II β
LFuc
↓α1, 3 Galβ1→4GlcNAc ↓ β 1, 4 Thr Galβ1→4GlcNAcβ1→2Man ↓ α1, 3 Manβ1→4GlcNAcβ1→4GlcNAcβ1→Asn II β ↑ α1, 6 C Ala Galβ1→4GlcNAcβ1→2Man ↑ β 1, 6 Galβ1→4GlcNAc II β
CF
C
BF B A
II β
Fig. 6.3 Structures of monomeric, diantennary, triantennary, and multivalent Galb1→3/4GlcNAc (I/II). (a) Monomeric II; (b) Monomeric I; (c) Lacto-N-neohexaose [LNnH; IIb1→6(Ib1→3)L]; (d) Lacto-N-hexaose [LNH; Di-II or IIb1→6(IIb1→3)L]; (e) Triantennary Galb1→4GlcNAc (Tri-II); (f) Asialo human a1-acid glycoprotein. The primary structure of classes A, B, BF, C, and CF carbohydrate units of the glycosylation site in human plasma a1-acidic glycoprotein [6, 7] is indicated in the above structure for asialoorosomucoid. The carbohydrate units of this asialoglycoprotein can be grouped into compounds with biantennary (class A) and triantennary structures without a fucose residue (class B) or with a fucose residue (class BF or CF). C chitin disaccharide. Shaded areas (I/II) are proposed to be the reactive glycotopes for I/II-specific lectins
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Several examples of the effect of the polyvalency of glycotopes on lectin–carbohydrate interactions are given in Table 6.3. A lectin from the edible mushroom ABA [49] is an important example in which the disaccharide II monomer and triantennary Galb1→4GlcNAc (tri-II) glycopeptides were poor inhibitors, while their polyvalent carriers were very active, implying that other more complicated structural factors are involved in binding besides the multiantennary II sequences. The polyvalent glycotopes present on macromolecules generated a great enhancement in affinity with ABA of up to 106 times greater than that generated by GalNAc or Galb1→3GalNAc. A similar phenomenon was also seen in Morniga G [27] in which biantennary I/II and tri-II glycopeptides were weak or poor inhibitors compared to their polyvalent carrier. The V. villosa B4 (VVL-B4) [36] was inhibited by asialo OSM (which contains over 75% of Tn determinants) 1.6 × 103 times higher than by monomeric GalNAca1→Ser/Thr (Tn). A mixture of Tn-containing glycopeptides was the most active ligand among various oligomeric inhibitors; it was, on a nanogram basis, 52 times more active than monomeric Tn and about 3 × 102 times less active than asialo OSM. These results clearly suggest that when Tn is in high-density polyvalent form, it is the most potent ligand and plays an essential role in the binding of anti-Tn lectins. The strongest inhibitors of ACL and peanut agglutinin (PNA, Arachis hypogaea) [65] are polyvalent Ta-containing glycoproteins, but these lectins differ in inhibition by monosugars (subtopes): GalNAc (penultimate component of T). Its derivatives are inhibitors of ACL while Gal is inactive, and Gal (terminal component of T) is a stronger inhibitor of PNA than GalNAc, which is practically inactive. The polyvalent forms of Tn are weak inhibitors of ACL and do not react with PNA. Human blood group precursor disaccharides Galb1→3/4GlcNAcb1→ (Ib/IIb) gave negligible inhibition, but their polyvalent forms were active with both lectins. GalNAc was an active ligand for ECL [64], but its polyvalent structural units, in contrast to those containing oligosaccharides IIb, were poor inhibitors. Polyvalent II forms on macromolecules are a potent recognition force for ECL while II monomer and oligo-antennary II forms play only a limited role in binding. The examples discussed above provide a good demonstration of the structural importance of complex carbohydrates. However, the polyvalency effect of glycotopes on carbohydrate–protein binding does not always make such an important contribution. An interesting example is P. aeruginosa II lectin, which interacts more strongly with free Fuc than with lFuca1-containing oligosaccharides, and the reactivity with respective polyvalent glycoproteins is only slightly increased (Table 6.4). To explain these observations, the present concept of a glycoside cluster effect has to be further defined and can be classified into two groups: (1) the “oligo-antennary or simple glycoside cluster effect” as observed in the reaction of galactosides with hepatic lectin [15, 16] or triantennary II with a galectin from chicken liver (CG-16) [63]; (2) the “high-density polyvalent or complex glycoside cluster effect” as observed in macromolecular interactions of lectins with glycoproteins containing polyvalent glycotopes.
GalNAc >>> Gal, GalNAc > Gal GalNAc >>> Gal, inactive inactive Contribution of recognition factors Ta Polyvalent 4.7 × 106 4.0 × 103 2.5 × 103 Monomer 8.6 7.5 1.6 × 102 Tn Polyvalent 1.1 × 105 2.9 × 104 60 b Cluster – 4.0 × 102 9.7 Monomer 1.0d 4.8 1.0 IIb Polyvalency 1.5 × 104e 4.0 × 103e 6.7 × 103 e Cluster – 0.6 1.4 Monomer – – – a Mass RP scale from Wu et al. [49], Singh et al. [26], Wu et al. [65], Wu [36], Wu et al. [64] b Tn glycopeptides – means inactive c Triantennary IIb d Monomeric Tn was replaced by GalNAc e It has to be further confirmed
Monosaccharide specificity
Table 6.3 Summary of overall contribution of recognition factors of Gal/GalNAc-specific lectins Mass RP a Recognition effects ABA (T) Morniga G (T/Tn) ACL (T) GalNAc > Gal
– – 3.3 × 105 1.1 × 103 2.1 × 102 – – –
3.4 × 103 10 – – – 2.2 × 103 – 0.5
VVL-B4 (Tn)
Gal > GalNAc
PNA (T/II)
2.1 × 104 5.5 8.9
– 1.1 1.8
– 0.2
GalNAc » Gal
ECL (II)
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Table 6.4 Recognition intensity of the polyvalent and monomeric ligands in carbohydrate–lFuc specific lectins interaction Mass RP a, b Recognition effects RSL AAA PA-II Polyvalency effect Cyst 14 phenol insoluble (Ah) Cyst Mcdon (Ah) Cyst Tighe phenol insoluble (H, Leb) Cyst JS phenol insoluble (H) Cyst N-1 Lea 20% 2× (Lea) Hog gastric mucin # 9 (Ah, H) Hog gastric mucin # 4 (Ah, H) Cyst Beach phenol insoluble (Bh)
2.1 × 102 1.7 × 102 1.0 × 102 81 81 43 35 21
1.5 1.5 2.0 25 –c 1.0 × 102 1.5 × 102 2.5
2.2 2.2 0.8 1.5 1.0 0.8 1.0 2.1
Monomeric glycotope effect H-di (Fuca1→2Gal) 2.3 7.0 0.4 Bh (Gala1→3[Fuca1→2]Gal) 2.0 1.6 0.03 Ah (GalNAca1→3[Fuca1→2]Gal) 1.9 2.6 0.02 LDFT, gluco-analog of Ley epitope 1.1 0.1 – (Fuca1→2Galb1→4[Fuca1→3]Glc) lFuc 1.0 1.0 1.0 0.1 0.05 – Lex trisaccharide (Galb1→4[Fuca1→3]GlcNAc) LNDFH I, Leb hapten (Fuca1→2Galb1→3[Fuca1→4] 0.06 0.08 – GlcNAcb1→3Ga1b1→4Glc) 0.05 0.9 – LNFP II, Lea penta- (Galb1→3[Fuca1→4] GlcNAcb1→3Galb1→4Glc) a In mass RP scale from Tables 6.2–6.4 in Wu et al. [51, 52, 59] b The mass RP of ABH/Lewis active glycoprotein toward the binding of a fucolectin-related protein of unknown function (FRP) encoded by the SP2159 gene of S. pneumoniae ATCC BAA-334 is up to 5.0 × 105 (unpublished data) c –, inactive
6.3 Summary Lectins are an important class of proteins or glycoproteins that specifically or selectively bind to carbohydrates and play many critical roles in life processes. To characterize lectins, the following factors have been taken into account: (1) affinity to monosaccharides and (2) expression of reactivities toward oligosaccharides (mammalian structural units) and finding the most active ligand. However, it is not satisfactory because most lectins show an increased reactivity with high-molecular polyvalent ligands, and this polyvalency effect is differentiated. During the past decade, it has been found that lectins with the same mono- or oligosaccharide specificity may demonstrate different specificities in reaction with polyvalent forms. It has even shown a shift of binding specificity of lectin from one type of carbohydrate ligand to another when the density of the specific carbohydrate changed. Therefore, characterization of lectin specificity has been extended to (3) simple oligovalent or cluster effect and (4) complex multivalent or cluster effects. Simple oligovalent effect
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concerns the reactivity of lectins with oligomeric glycoconjugates (e.g., branched oligosaccharides carrying several active disaccharides, glycopeptides with several Ta or Tn glycotopes). A complex multivalent effect applies to interaction with highmolecular or aggregated molecules carrying multiple glycotopes recognized by a lectin. In this article, we are focusing on the contribution intensities of three basic recognition factors: essential mammalian structural units (2), their clusters (3), and polyvalency (4) in the recognition processes. Acknowledgments This work was supported by grants from the Chang Gung Medical Research plan (CMRPD no. 180482 and 170443) Kwei-san, Tao-yuan, Taiwan, and the National Science Council (NSC 97-2628-B-182-002-MY3 and 97-2320-B-182-020-MY3), Taipei, Taiwan.
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Chapter 7
Adhesion/Growth-Regulatory Galectins: Insights into Their Ligand Selectivity Using Natural Glycoproteins and Glycotopes Albert M. Wu, Tanuja Singh, Jia-Hau Liu, Sabine André, Martin Lensch, Hans-Christian Siebert, Mickael Krzeminski, Alexandre M.J.J. Bonvin, Herbert Kaltner, June H. Wu, and Hans-Joachim Gabius Keywords Agglutinin • Docking • Glycosylation • Lectin phylogeny • Sugar code “Biochemistry text books commonly make it appear that it is a foregone conclusion that the hardware of biological information storage and transfer is confined to nucleotides and amino acids, the letters of the genetic code. However, the remarkable talents of a third class of biomolecules are often overlooked” [1]. This statement from a recent review guides the readers to look at and fully appreciate the chemical/lectinochemical characteristics of carbohydrates that underlie the concept of the sugar code [2].
7.1 The Principles of the Sugar Code Beyond simple sequence permutations, as in oligonucleotides and peptides, there are four parameters that account for the unsurpassed coding capacity of glycans: (1) variability of linkage points; (2) possibility for two types of interglycosidic linkage by anomer; (3) alteration of ring size (furanose or pyranose); and (4) introduction of branches, both in the central and in the peripheral regions of glycans [3]. To define a disaccharide, such as the epitope of the histo-blood group H determinant, writing down the sequence, i.e. Fuc-Gal, is therefore not at all sufficient, as it would be for an oligonucleotide or peptide. Instead, considering the four parameters listed above, the structure of this histo-blood group antigen is only unequivocally defined when writing a-l-Fucp1-2-b-d-Galp (first dimension of the sugar code). As signified by the physiological importance of this structure, complex synthetic machinery is available to produce the indicated sequence diversity of glycans in nature, hereby realizing their theoretical coding capacity, e.g. facilitating a1-2, a1-3, A.M. Wu (*) Glyco-Immunochemistry Research Laboratory, Institute of Molecular and Cellular Biology, College of Medicine, Chang-Gung University, Kwei-Shan, Tao-Yuan, 333, Taiwan e-mail:
[email protected] A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_7, © Springer Science+Business Media, LLC 2011
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a1-4, and a1-6 linkages for fucose addition to glycan chains [4–8]. Each given linkage type forms a characteristic structural constellation as part of a branched glycan chain, often at terminal positions (the second dimension of the sugar code). Being spatially accessible at these sites is suggestive for being a target of proteins specific for distinct glycotopes. The way the structural assignment of the mentioned histo-blood group epitope had been initiated provides a telling example of the versatility of carbohydrates to engage in such intermolecular interactions. Their inherent specificity combined with hapten inhibition set the stage to track down the presence of the l-fucose moiety as part of the H-epitope. Hydrogen bonds, van der Waals interactions, and C–H/p-bonds with Trp as part of the contact sites are suitable means for yielding specific recognition and enabling, e.g. accurate discrimination against epimers [9–13]. When not tied to enzymatic processing, binding of a glycan defines a protein as a lectin (except for antibodies and transport proteins for free glycans), and the lectin of the European eel Anguilla anguilla, an agglutinin for erythrocytes of this type of blood group, was instrumental for identifying fucose as an essential part of the histo-blood group H epitope [14–17]. This molecular interplay, leading to hemagglutination, also underscores the mentioned spatial accessibility of glycans for intermolecular interactions. Thus, in the context of the cell surface, glycoproteins, such as integrins or laminin, can independently engage in protein–protein contacts and also in protein–carbohydrate interplay via their glycan chains. At this stage, another particular factor comes into play that predisposes carbohydrates as ideal hardware for biological information transfer. Whereas peptides are highly flexible, the bulky rings of the sugars limit the flexibility around glycosidic linkages so that oligosaccharides often adopt only a few energetically privileged conformations, the third dimension of the sugar code [13, 18]. Since lectins accommodate such conformers in their contact sites frequently without major structural changes by mutual adaptation (please see below for flexible ligand docking), the recognition of cell surface glycans as signals or code words does not require arresting a flexible oligomer into a particular conformation in the context of a polymer. This, in contrast, is the case for bioactive conformations of peptide motifs as part of proteins. Overall, glycans thus adorn protein and lipid scaffolds with bioactive signals. They are decoded by respective receptor proteins. We will next turn to a distinct class of these translators of the sugar code with b-sandwich folding, i.e. the galectins.
7.2 Galectins: A Class of Potent Translators of the Sugar Code A graphic example for this type of molecular complementarity, guiding decoding of glycan signals, is the illustration of the ribbon diagram (Fig. 7.1), which is emblematic for the fold of this family of adhesion/growth-regulatory lectins. Figure 7.2 shows the contact sites and profiles how the pentasaccharide of ganglioside GM1 makes contact to a galectin’s binding site as a distinct, energetically privileged conformer, representing a “valley” in the conformational “landscape” of
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Fig. 7.1 Ribbon diagram of the homodimeric human galectin-1 [19]. The b-strands in the fivestranded (F1–F5) and six-stranded (S1–S6a/S6b) b-sheets are indicated by letter–number code, and the contact site for the carbohydrate ligand is shown by the insertion of a lactose molecule
Fig. 7.2 Left panel: positioning of the contact site for the pentasaccharide chain of ganglioside GM1, shown for a galectin-1 subunit [19, 20]; right panel: the architecture of this site in a rat galectin-5 (rGal-5) [21]
the ligand [19–21]. Using the saturation transfer difference technique in nuclear magnetic resonance (NMR) spectroscopy, local vicinity between galectin-1 and the pentasaccharide have been mapped in detail [20]. Together with calculations of the binding energy of individual amino acids and sugar units, major contacts were ascribed to the Galb1-3GalNAcb-terminus (Tb antigen) and the a2-3-linked sialic acid of the branch [20]. Of note, this type of interaction between the lectin and the ganglioside’s glycan has significant biological activity, triggering growth inhibition in neuroblastoma cells in vitro and autoimmune suppression by efficiently facilitating communication between regulatory and effector T cell populations [22–24]. Beyond galectins, ganglioside GM1 is also the docking site for cholera toxin.
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Fig. 7.3 Illustration of two bioactive conformers of the pentasaccharide of ganglioside GM1. Whereas the upper structure is the ligand for human galectin-1, a shape change is required to let the pentasaccharide acquire affinity to cholera toxin (bottom) [20]
Intriguingly, the bacterial protein accommodates a different conformer [20]. The pentasaccharide, by adopting the two low-energy “key-like” conformations shown in Fig. 7.3, is thus suited to fit into “locks” of widely separate architecture. Interestingly, selection of a conformer from an equilibrium is not confined to an oligomer. It can already take place on the level of disaccharides, obvious proof for the enormous versatility of carbohydrates as ligands. NMR spectroscopy, especially the determination of interproton distances by picking up nuclear Overhauser effects, was essential for structurally defining bound-state conformations [12, 13, 18]. The two conformers of the disaccharide Galb1-2Gal, drawn in Fig. 7.4, together with the detectable interproton distances, represent the structures reactive either with a galectin or a plant agglutinin (toxin). The energy maps as part of Fig. 7.4 illustrate the meaning of a central low-energy valley with two further privileged positions. Overall, these results teach us the following lesson: animal/plant lectins are able to perform differential conformer selection, underscoring that conformers
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180 H2’
Φ: 0 Ψ: −63
120 60
H1
Ψ 0
H1’
−60 −120 −180 −180 −120 −60
0
Φ
60
120
180
max atom1 atom2 min distance [A] H1 - H1’ 2.0 - 3.3 H1 - H2’ 2.0 - 2.7
180 H3’
120
Φ: Ψ:
14 60
H1 60
Ψ 0
H2’
−60 −120 −180 −180 −120 −60
0
Φ
60
120
180
atom1 atom2 min max distance [A] H1 - H3’ 2.3 - 3.7 H1 - H2’ 2.0 - 3.4
Fig. 7.4 Illustration of two bioactive conformers of the disaccharide Galb1-2Gal, a potent ligand for galectins (upper panel) and the plant agglutinin viscumin (bottom panel). The two measured interproton contacts of the bound-state conformations of the disaccharide identify its position in the energy map defined by the dihedral angles Y/F of the glycosidic linkage, making the change in the Y-parameter obvious. A representation of the shape of the two conformers is given on the right side
can have distinct bioactivity profiles [8, 25, 26]. This finding of differential c onformer selection has notable ramifications for drug design. It uncovers a chemical means to reduce cross-reactivity at the molecular level by designing conformationally restrained lectin ligands. This approach documents the medical perspectives for turning insights of the third dimension of the sugar code into innovative therapeutic agents targeting bacterial and plant toxins without reacting with human lectins. Their synthesis will give a clear direction if a lectin’s fine specificity is characterized in detail, especially in comparison to related proteins. As the term “galectin” implies, a key contact involves the b-galactoside part of the ligand. The galectins form a family of homologous proteins, and the current status of analysis indicates that they have potential to serve as specific regulators of a variety of cellular activities by virtue of homing in on certain physiological glycan ligands [1, 27, 28]. Beyond the interaction with ganglioside GM1, binding of the glycoprotein CD7 on activated T cells, the fibronectin receptor (a5b1-integrin) on tumor suppressor-positive carcinoma cells, and the tissue plasminogen activator in pancreatic cancer by galectin-1 sets distinct biosignaling in motion to elicit the
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Fig. 7.5 Comparison of the sequences for prototype rat galectin-5 and the N-terminal domain of the rat tandem-repeat-type galectin-4 (G4-N or rGal-4N). Identical residues in both sequences are indicated as white letters on black background
particular cellular response [29–31]. Sequence diversification within this lectin family raises the question as to whether sequence divergence, which is apparent in the various family members (e.g. from the groups of prototype and tandem-repeattype galectins, please see Fig. 7.5), will have an impact on ligand binding. In this respect, binding assays with free glycans are a valuable tool for comparative specificity analysis, e.g. in calorimetry [32], mapping the interaction between a ligand and the lectin in solution. Considering the importance of spatial parameters in natural glycoconjugates as regulators in lectin affinity [1], testing surface-immobilized glycoproteins constitutes a major refinement of specificity analysis. What is more, the selection of such test compounds will establish a panel that covers the range of common natural glycotopes (please see Table 7.1 for a listing) to enable finespecificity determination as well as assessment of the impact of naturally clustered presentation [1, 33]. The next part of this chapter presents information on the experimental setup and answer the question as to whether the two mammalian lectins, whose sequences are presented in Fig. 7.5, have uniform or nonidentical binding properties to natural glycoproteins.
7.3 Natural Glycoproteins: Sensors to Map Functional Galectin Divergence? A reliable and sensitive method for determining a lectin’s binding specificity requiring minimum quantity of glycoprotein is the enzyme-linked lectin sorbent assay (ELLSA). A panel of glycoproteins well-characterized for the presentation of lectin-reactive glycotopes establishes the ligand side for the binding reaction. Their adsorption to the surface of a microtiter plate well leads to a ligand-presenting matrix. Keeping the lectin in solution precludes potentially harmful effects of adsorption on the receptor protein. The lectin will thus be confronted with different sets of glycotopes on the surface of the microtiter plate well, mimicking the interaction with cell surfaces. Quantitative monitoring of lectin binding requires labeling under activity-preserving conditions, conveniently using a fluorescent dye or by biotinylation, the extent of which is measured by two-dimensional gel electrophoresis and mass spectrometry [34, 35]. Extent of binding in the assay is routinely assessed to be dependent on carbohydrate and saturable. As shown in Fig. 7.6 for a galectin, the nature of the glycan chains, e.g. their status of sialylation, determines their reactivity. Typically for galectins, strong reactivity to terminal b-galactosides
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Table 7.1 Codes and abbreviations of structural units in glycans of mammalian glycoproteins (gps) and glycosphingolipids [33] Codesa Structural units Sources F 1 Forssman pentasaccharide; animal tissue GalNAca1-3GalNAc Fpentaantigens mainly in glycosphingolipids GalNAca1-3GalNAcb13Gala1-4Galb1-4Glc Fa GalNAca1-3GalNAca1In mucin-type O-glycosylation of glycoproteins Ser/Thr GalNAca1-3GalNAcb1Glycotope at the nonreducing end of FpentaFb 2 A GalNAca1-3Gal Human blood group A-active disaccharide Ah GalNAca1-3[lFuca1-2] Human blood group A-active trisaccharide Gal 3 Tn GalNAca1-Ser/Thr Tn antigen, only in mucin-type O-glycosylation of glycoproteins 4 Ta Galb1-3GalNAca1-Ser/ The mucin-type O-glycans sequence on the human Thr erythrocyte membrane Tb Galb1-3GalNAcb1-... Brain glycoconjugates and gangliosides such as ceramide asialo-GM1 tetrasaccharide 5 I Galb1-3GlcNAc Human blood group type I and II carbohydrate sequences. Branched or linear repeated 6 II Galb1-4GlcNAc II sequence is part of blood group I and i IIb Galb1-4GlcNAcb1epitopes. I and II are precursors of ABH Triantennary Galb1Tri-II and Lea, Leb, Lex, Ley blood group-active 4GlcNAcb1antigens. Most of the lectins reactive with II Multivalent Galb1mII are also reactive with I. Lectin Tri-II and mII 4GlcNAcb1determinants are present at the nonreducing end of carbohydrate chains in N- and O-glycans 7 B Gala1-3Gal Human blood group B-active disaccharide Bh Gala1-3[lFuca1-2]Gal Human blood group B-active trisaccharide 8 E Gala1-4Gal Blood group pk and P1 active disaccharide; sheep hydatid cyst glycoproteins, salivary glycoproteins of the Chinese swiftlet, glycosphingolipids in human erythrocytes and small intestine 9 L Galb1-4Glc Constituent of mammalian milk Lb Galb1-4Glcb1Lactosyl ceramides in brain and part of carbohydrate structures in gangliosides 10 P GalNAcb1-3Gal Blood group P-related disaccharide; glycotope at Pa GalNAcb1-3Gala1the nonreducing end of globoside Brain and asialo-GM2 disaccharide; human 11 S GalNAcb1-4Gal Sb blood group Sd(a+)-related disaccharide in GalNAcb1-4Galb1most human secretions, such as urine Tamm– Horsfall glycoprotein a a, b = anomer of sugars; m = multivalent
free of substitution is shared by the two mammalian family members (Table 7.2). Their sequences are listed in Fig. 7.5. Also, the common histo-blood group ABH substitutions are potent ligands [36–38]. This feature sets the galectins apart from the plant toxin ricin. The spatial architecture of this b-galactoside-specific lectin
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Fig. 7.6 Binding curves of rat galectin-5 to glycotopes in a solid-phase assay. The surface of 96-well microtiter plate wells was coated with glycoproteins dissolved in 0.05 M sodium carbonate buffer (0.05 M NaHCO3/0.05 M Na2CO3, pH 9.6) overnight at 4°C (for details, please see [36]). Biotinylated rat galectin-5 (5 mg/ml) was used to determine and compare the extent of binding to the following glycoproteins (gps): cyst MSS 1st Smith degraded (circle), human asialo a1-acid gp (diamond), human asialoglycophorin (+), human a1-acid gp (filled square), and asialo ovine submaxillary mucin (filled triangle). Total volume of the assay was 50 ml. A405 was recorded after a period of 24 h
will obviously not tolerate these extensions to the core structure [39]. Thus, galectins and the b-galactoside-binding plant protein differ in fine specificity despite sharing binding capacity to b-galactosides. Next, even a conspicuous difference between the two galectins tested is revealed in Table 7.2: the binding properties to mucin-type O-glycans, especially the Ta-disaccharide, differ significantly. Thus, despite the close sequence similarity, the two galectins deviate from each other markedly in this parameter. Our assay not only enables one to assess and compare the binding of lectins to surface-immobilized glycoproteins, but it also offers the attractive opportunity for fine-scale mapping by using free saccharides as inhibitors to glycotope binding. Basically, these inhibition assays are a control for specificity because they confirm the carbohydrate dependence of the binding [36–39]. Even more important, any difference in the relative potency identifies a disparity in the fine specificities between tested lectins, here especially the extension by a1-3-linked galactose and the effect of branching (Table 7.3). Moreover, the plant lectin proved relatively less sensitive to oligomer formation of the lactose core disaccharides (Table 7.3), as also summarized in Table 7.4. These results teach an important lesson that, despite sharing specificity to b-galactosides, the fine-specificity profiles can obviously be distinct between mammalian and plant lectins as well as among the members of this
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Table 7.2 Comparison of the reactivity of two mammalian galectins (rGal-4N and natural glycoproteins (gps) with that of a plant lectin (ricin)a Signal intensity for bindingc rGal-4N (ABH, I/II, T/Tn) rGal-5 (A/B, I/II) Glycoprotein (terminal epitope)b Terminal (I/II)-containing gps Cyst MSS 1st Smith degraded (I/II) +++++ +++++ Cyst Mcdon P-1 (I/II) +++++ +++++ Cyst Tighe P-1 (I/II) +++++ +++++ +++++ +++++ Human asialo a1-acid gp (mII) Asialofetuin (mII/I, Ta) ++++ ++++ iII/Lac Pneumococcus type 14 polysaccharide
+++
Ta-containing gps Human asialoglycophorin (Ta, Tn, mIIb/f) Antifreeze gp (Ta)
Ricin (II, T/Tn) +++ +++++ +++++ +++++ +++++
+++
–
++++ +++++
– +
+
–
+++++ +++++
– –
+++++ +++++
+++++
++++
+++++
+++++
±
–
Blood group ABH, Lea, Leb, Lex, and Leyactive gps Cyst Mcdon (Ah > Leb, Ley) +++++ Cyst Beach phenol insoluble (Bh > Leb, +++++ Ley) ++++ Cyst Tighe phenol insoluble (H, Lea, Leb, Lex, Ley) Ta/Tn-containing gps Asialo OSM (Tn, Ta, core 2 II) Asialo PSM (Tn, Ta, A, Ah, H)
rGal-5) for
Crypto II, Ta/Tn-containing gps Human a1-acid gp (a2-3/6 sialyl mII) + – – OSM (sialyl Tn, Ta, core 2 II) – – – – – – PSM (sialyl Tn, Ta, A, Ah, H) rGal-4N N-terminal domain of rat tandem-repeat-type galectin-4, rGal-5 rat galectin-5, ricin Ricinus communis toxin a For details, please see [36, 37, 39] b The symbols in parentheses indicate the terminal epitopes and are listed in Table 7.1. iII/ Lac = internal Galb1-4Glc(NAc); m = multiantennary; mIIb/f = biantennary N-glycan with core fucosylation and bisecting GlcNAc c The results were graded according to the spectrophotometric absorbance value at 405 nm (i.e. OD405) after 2 h (ricin), 4 h (rGal-4N), and 24 h (rGal-5) incubation as follows: +++++, (OD ³ 2.5); ++++, (2.5 > OD ³ 2.0); +++, (2.0 > OD ³ 1.5); ++, (1.5 > OD ³ 1.0); +, (1.0 > OD ³ 0.5); ±, (0.5 > OD ³ 0.2); –, (OD 10.0 (11% inhibition)c
30.0
9.0
Ricin (II, T/Tn) 0.9 0.9 1.5
Gala1-3Galb1-4GlcNAc (B active II) 1.2 50.0 0.4 Galb1-4Glc (L) 1.0 1.0 1.0 0.4 0.3 Gala1-3Gal (B di) 0.2d Galb1-3GlcNAc (I) 0.2 3.3 0.8 4.3 0.1 0.8 Galb1-4GlcNAc (II) a Lectin abbreviations are listed in the footnote of Table 7.2. Inhibitory potency of oligosaccharides on binding of rGal-4N (50 ng/50 ml) to a Galb1containing gp, cyst MSS 1st Smith degraded gp (1 ng/50 ml) [37]; of rGal-5 (125 ng/50 ml) to a Galb1-containing gp, cyst Beach P-1 (5 ng/50 ml) [36]; of ricin (2.5 ng/50 ml) to a Galb1-containing gp, asialofetuin (25 ng/50 ml) [39] b Relative potency of Galb1-4Glc (L) is given as 1.0, leading to a normalization c Could not reach 50% inhibition d Extrapolation
Gal 1-4GlcNAc 1-2Man α1-6 Gal 1-4GlcNAc 1-2Man 1-3Man 1-4GlcNA c 1-4GlcNAc 1-N-Asn 1-4 Gal 1-4GlcNAc Tri-antennary Gal 1-4GlcNAc (Tri-II)
1-6 Gal 1-4Glc 1-3 Gal 1-4GlcNAc
Table 7.3 Comparison of the relative potency of rGal-4N and rGal-5 for oligosaccharides with that of ricina Relative potencyb Type of saccharide rGal-4N (ABH, I/II, T/Tn) rGal-5 (A/B, I/II) 10.0 7.7 Galb1-3GlcNAcb1-3Galb1-4Glc (Type 1, Ib1-3L) Galb1-4GlcNAcb1-3Galb1-4Glc (Type 2, IIb1-3 ) 1.9 5.0 Gal 1-4GlcNAc 1.9 15.0
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Table 7.4 Comparison of the binding properties of rGal-4N and rGal-5 with ricina Type of lectin Carbohydrate rGal-4N (ABH, I/II, specificity T/Tn) rGal-5 (A/B, I/II) Ricin (II, T/Tn) Monosaccharide b-Anomer of Gal b-Anomer of Gal b-Anomer of Gal specificity and reduced by and enhanced by and slightly N-acetyl group in N-acetyl group in enhanced by GalNAc GalNAc N-acetyl group in GalNAc Galb1-4Glc (L) > Galb1Galb1-4GlcNAc Reactivity toward Galb1-4Glc 4GlcNAc (II) > Galb1-3GlcNAc (L) > Galb1disaccharide (II) ³ Galb1-3GlcNAc (I) > Galb1-3GalNAc 3GalNAc structural units (I) > Galb1-3GalNAc (T) > Galb1-4Glc (T) > Gala1-3Gal expressed in (T) > Gala1-3Gal (L) > GalNAcb1-3Gal (B) ³ Galb1decreasing (B) ³ Gala1-4Gal (P) > Gala1-3Gal 3GlcNAc order (based (E) ³ GalNAcb1(B) > GalNAcb1-4Gal (I) > Galb1on nanomoles 3Gal (P) > GalNAc (S) ³ GalNAca14GlcNAc (II); comparison) a1-Ser/Thr 3GalNAc Fuca1-2Gal (H) (Tn) ³ GalNAcb1(F) > GalNAca1-3Gal was inactive 4Gal (A); GalNAca1-Ser/ (S) > GalNAca1-3Gal Thr (Tn) and Gala1(A); GalNAca1-3 4Gal (E) were inactive GalNAc (F) was inactive Galb1-4GlcNAc core Gala1-3Galb1-4GlcNAc The most active [Fuca1-2]Galb1(B active II) and b-galactoside 3GlcNAcb1Gala1-3Galb13Galb1-4Glc 4GlcNAcb1-3Galb1(H active Ib1-3L) 4Glc (B active and Fuca1IIb1-3L) 2Galb1-4Glc (H active L) Triantennary glycopeptides Triantennary Ratio of glycotope Triantennary glycopeptides with mostly type II glycopeptides clusters with mostly type termini were seven with mostly type (simple II termini were 12 times more active than II termini were multivalent times more active monomeric II inactive form)/ than monomeric II monomeric II Histo-blood group Histo-blood group ABH Histo-blood group Substituted precursor precursor (equivalent) ABH precursor branch-end (equivalent) gps but gps and enhanced (equivalent) gps glycans hindered by ABH strongly by blood and enhanced histo-blood group group A, B determinant strongly by determinants sugar blood group A, B, and H determinant sugar Ratio of complex 8.8 × 105 times 3.2 × 102 times more active 4.6 × 102 times polyvalent I/ more active than than monomeric II more active than II glycotopes monomeric II monomeric II in natural glycoproteins/ monomeric II For detailed information on the binding properties of the lectins, please see [36, 37, 39]
a
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(formerly referred to as C-16/C-14) closely related to mammalian galectin-1 [40], had been subjected to crystallographic analysis so that their crystal structures were available [41, 42]. In addition, the DNA sequence traits beyond the proteins’ primary structures were known.
7.4 The Case Study of Two Prototype CGs Owing to complete genome sequencing, the intended comparison can thus include the promoter region in addition to the coding sequence. Consequently, insights into divergence of regulatory mechanisms of gene expression can be inferred, too. In other words, the question can be answered as to whether the two genes have acquired their own characteristic expression profiles. The ensuing computational processing using algorithms to spot putative sites for binding of transcription factors revealed a notable degree of sequence divergence (Table 7.5). The arising assumption for disparate expression profiles was tested immunohistochemically. The preparation of noncross-reactive antibodies against these two lectins facilitated mapping their tissue presence [40]. The divergence of the two gene sequences, originally stemming from a duplication event at around the separation of birds from mammals in the phylogenetic tree approximately 3 × 108 years ago [43], indeed translated into nonidentical localization patterns (Table 7.6). These findings, together with sequence variations noted around key residues relevant for lectin activity, such as the Trp moiety at positions 68 (CG-1A) or 70 (CG-1B) or the His moiety at equivalent positions 52/54 (Fig. 7.7), nourish the suggestion that the interaction profiles with glycans will also be different. This question was answered by running the galectin–glycan binding assays under identical conditions [44, 45]. As summarized in Table 7.7, although both galectins showed pronounced preference for I/II-containing human blood group precursor equivalent glycoproteins, reactivity differed significantly for multiantennary N-glycans and the AB blood group epitopes. The inhibitory potency of di- and oligosaccharides was rather comparable (Table 7.8), leading us to the overall comparison given in Table 7.9. A prominent feature is that the polyvalent nature of glycotopes could be sensed discriminatively by these two closely related galectins (Table 7.9). With sequences and crystal structures being available, this experimental analysis could be taken to the level of detailed structure–activity computations. As a means to move beyond common rigid-body docking, we implemented the high ambiguity-driven docking (HADDOCK) procedure for lectin interactions [45, 46]. It allowed lectins and ligands to maintain full flexibility, thereby yielding a detailed view on structural and energetic aspects. As stated above, in explaining the third dimension of the sugar code, the ligands were invariably accommodated by the lectin sites in their low-energy conformation, and a detailed calculation for energy of interaction disclosed the contribution of each amino acid to binding [45]. Files of movies showing the dynamic process of contact building are available at http://www.nmr.chem.uu.nl/haddock/movies/ [45]. A static view (snap-
cccTTGCAcaaata/ccCTTGCacaaata agTTTGCtgaaggt cgctatCCACAga cattttTGCAAgca acTTTGCtaagca tggaatGGAAGcgactaaagcctc catcagCATTGccacaaacagttg cccttcttgcctgCCCCCcacacc
−1291 (+) −1006 (+) −929 (−) −849 (−) −773 (+) −1482 (+) −556 (+) −134 (−)
n.f.
taacACGTGatt gaTTGATccc
CP2
c-Myc/Max CUTL-1
COMP1
−1454 (−) −1446 (+)
CATTCtg aATAATg gggtttcGCAAAgc
C/EBPa, b; CHOP-10
n.f.
−1892 (+) −1630 (+) −1592 (−)
tGTGGC tGTGGC tCTGGT GCCACa tGTGAT
−1518 (+) −1296 (+) −1226 (+) −546 (−) −95 (+)
Cdx-1
ACCAGa
−1742 (−)
Barbie-Box
AML-1a (Runx)
Position Motif for factor (orientation) Sequence
CG-1A
1.0 0.996
1.0/0.984 0.984 0.979 1.0/0.996 0.972 0.964 0.914 0.893
1.0 0.984 0.984
1.0 1.0 1.0 1.0 1.0
1.0
0.995 0.997
0.979/0.975 0.97 0.96 0.973/0.99 0.968 0.892 0.812 0.902
1.0 0.981 0.973
1.0 1.0 1.0 1.0 1.0
1.0
Ma Ma
M M M M; PM M PM Ma PM
PM M M
PM PM PM PM PM
PM
Core score Matrix score Program
−1139 (+) +132 (+)
−105 (−)
−1955 (−)
−1317 (−) −813 (−)
−1549 (−) −904 (+) −1658 (+)
−64 (+)
−1761 (+) −1288 (+) −1280 (−) −953 (−) −939 (+) −898 (−) −511 (−) −447 (−) −263 (−) −49 (+)
−1897 (+)
1.0 0.929 1.0
n.f. cATTGAtggg caTCGATccc
1.0
0.972 1.0
1.0 1.0 0.952
1.0
1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
1.0
0.925 0.999/1.0
1.0
0.84/0.936
0.962 0.973
1.0 1.0 0.953
1.0
1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
1.0
(continued)
Ma Ma; PM
PM
M a; PM
M M
PM PM M
PM
PM PM PM PM PM PM M, PM M, PM PM M, PM
PM
Core score Matrix score Program
CTGGGgttggg
gcccttcccatgcCAATCctgtcc
ccatgaGCAAAta ctttttCCAAAgg
gtCAAAT ATTGAag gaTGTGCtaacat
atttAAAGGcaaggg
aGTGGT cGTGGT ACCACt ATCACa tGTGAT GCCACa ACCACa ACCACa GCCACa tGTGGT
tGCGGT
Position (orientation) Sequence
CG-1B
Table 7.5 Compilation of putative binding sites for transcription factors in the proximal promoter regions of the genes for chicken galectins CG-1A/CG-1B
+78 (+)
−1248 (−)
−1703 (−) −1619 (−) −1284 (−) −1011 (+) −999 (+) −938 (+)
GKLF
GR
HOX A3
aaatGTGCA agcaCTGAA acaaATATG AGCACagtt TGAAGgttg CCTTCctgg
agaGGACAcatgggatatt
gaagggaggAAGGG
caCTATCtg
aagcaAACAAtc
−262 (−)
−1760 (−)
FoxD3
aTATTTcctctggta aGCTCAgctttggtt
−1234 (−) −1137 (−)
Evi-1
GATA-3
acAACAC GTTTTgc ccACTAC
−1671 (−) −1207 (+) −817 (−)
En-1
tctagagTCTGGaatg
n.f.
−673 (+)
Elk-1 (TCF-A)
E47 (Hand-1)
Position Motif for factor (orientation) Sequence
CG-1A
Table 7.5 (continued)
1.0 1.0 0.968 1.0 1.0 1.0
0.989
1.0
1.0
1.0
0.861 0.842
1.0 1.0 0.996
1.0
1.0 1.0 0.974 0.995 1.0 1.0
0.97
0.944
0.995
0.967
0.904 0.909
1.0 1.0 0.992
0.974
PM PM M PM PM PM
M
M
M
Ma
PM PM
PM PM M
M a
Core score Matrix score Program
−1990 (−) −1902 (+) −1801 (−) −1575 (−) −1463 (+) −1213 (−)
−467 (+)
−1850 (−) −1489 (−) −980 (−) −861 (−) −694 (−) −617 (−) +40 (+)
−1551 (−) −1456 (−) −1308 (−) +20(−)
−1494 (+) −986 (+) −825 (+) −825 (+) −612 (−) −333 (−) −302 (−)
−1128 (+)
aactGTCCT TTCAGtgcg acctTTAGG caccAAAGG ATGAGttag gcccAAAGG
tcagtcctctgTGTACttg
CCTTCctttccttg CCCTCtcctgttgt CCTCTtctatttct CCTCTtctttcagt CCCATccccttgtt CCTCCccatttttc agggaaggaATGGG
n.f.
n.f.
aGGTCAaatttcctt agTCTCTaatc aaTATCTagaa gCATCAtgtcttgtg
GTATTcc GTTTTgc GTAATtg GTAATtg ccATTTT ccATTTT agAATAC
ccaactGGAAGtaccc
n.f.
Position (orientation) Sequence
CG-1B
1.0 1.0 1.0 1.0 1.0 1.0
0.984
0.933 0.965 0.949 0.949 0.952 0.914 0.952
0.842 0.988 1.0 0.921
1.0 1.0 1.0 1.0 1.0 1.0 1.0
1.0
0.995 1.0 0.953/1.0 0.995 0.995 0.995
0.974
0.911 0.901 0.901 0.906 0.910 0.9 0.906
0.903 0.981 0.982 0.927
1.0 1.0 1.0 1.0 1.0 1.0 1.0
0.928
PM PM M; PM PM PM PM
M
M M M M M M M
PM PMa PMa PM
PM PM M PM PM PM PM
Ma
Core score Matrix score Program
−1458 (−) −1048 (+) −1009 (+) −979 (−) −48 (+) +35 (−)
Pax-2
tctctaacacGTGATtgat tagtGTCAGgagttggagt cacaGTTTGctgaaggttg gcagggcagcGTGTCtcct ctgaGTCACgttggctgtg ggggggtggcTTGTCttgt
ccCTTCCtgg
cGCTGAgtcac
−50 (+)
−939 (−)
NF-E2
NRF-2
atgctAGGGActg agtGGGGA
−1427 (+) −757 (+)
MZF-1
cAGTTAcct cAGTTAcag cACATAcag cACAAAcag cttTAAATg aagTGATTt
cacTATCTg
catgggacgaaaGCCAAcatacactgctg
−1316 (−) −1184 (−) −591 (−) −544 (−) −182 (+) −78 (+)
Msx-1
ctaaCTTAA gccaTCTCG acagGAAGG CCTACttta
Myogenin/NF-1 −338 (−)
−1760 (−)
Lmo2-complex
−580 (−) −362 (−) −289 (−) −186 (+)
Position Motif for factor (orientation) Sequence
CG-1A
0.902 0.98 0.992 1.0 0.992 0.98
1.0
1.0
1.0 1.0
1.0
0.988 0.988 0.928/1.0 1.0 0.996 1.0
1.0
1.0 1.0 1.0 1.0
0.862 0.971 0.989 0.967 0.982 0.968
1.0
0.986/1.0
0.989 1.0
0.779
0.927 0.991 0.95/1.0 1.0 0.958 1.0
1.0
0.995 1.0 1.0 0.995
M PM PM PM PM PM
PMa
Ma; PMa
PM M, PM
Ma
M M M; PM PM M PM
PM
PM PM PM PM
Core score Matrix score Program
−1947 (−) −1879 (−) −1819 (+) −1592 (+) −1418 (−) −1345 (+) −1312 (−) −1215 (−) −1101 (−) −919 (+) −653 (−) −601 (+) −564 (−)
−1432 (+) −690 (−) −619 (−) −482 (−)
−1802 (+)
−1708 (−) −940 (+) −827 (+) −827 (+) −768 (+)
−927 (−) −274 (+) −200 (−) −116 (+) +101 (+)
catgccaatcCTGTCctga agtgctgccaAGAACatgg taagGACATgtatcctcaa aacaGACAAgcatgggcca ttgggcatgcGTGACtccg aggaGACATgatttttaaa agcaaatatcTAGAAattt ctgcccaaagGCTGGaaca ggggttctgcATGACtggg gcaaGACAAggaaagattg gtgcaagagcCTGACactg agagGACAGgcttgctgtc atctacaagaGCTGGtggc
n.f.
n.f.
tcgGAGGA TCCCCttg ttcCTCCCcattt TCCCCaaa
aacctttaggttTTGGCatctctccatag
cAGGCAatg ttgTGATTt tagTAATTg tagTAATTg cacAAAGTg
n.f.
acaaATATG TTGACttcg catcCTGGA CCTATtggt AGCAGagtg
Position (orientation) Sequence
CG-1B
0.972 0.971 0.992 0.98 1.0/0.992 0.992 0.98 0.992 0.991/1.0 0.98 0.98 0.972 0.992
1.0 1.0 1.0 1.0
1.0
1.0 1.0 1.0 1.0 1.0
0.963 0.963 0.973 0.969 0.892/0.981 0.962 0.965 0.967 0.879/0.997 0.967 0.974 0.962 0.965
1.0 1.0 0.996 1.0
0.801
1.0 1.0 0.935 1.0 1.0
0.974 0.995 0.995 0.982 0.995
(continued)
PM PM PM PM M; PM PM PM PM M; PM PM PM PM PM
PM PM PM PM
Ma
PM PM M PM PM
M PM PM M PM
Core score Matrix score Program 0.968 1.0 1.0 0.985 1.0
taaCATCAt tagGATCAt aaaAATCAt
−1434(+) −275 (+) −241 (+)
−870 (+)
−1686 (+)
−1057 (+) −721 (−) −228 (−)
−1570 (+) −1566 (+) −965 (−) −685 (−)
Pbx-1
POU2F1/2 (Oct-1)
POU3F1 (Oct6, Tst-1)
Sp1/GC box
SRY
AAACAaa AAACAca ctCCTTT tcTCTTT
ggGGCAGggt accCAGCCct atcCTGCCca
gggGAATTggaatgg
ctctatGCAAAgctc
n.f.
Pax-6
ccaacataCACTGctgcgtagcagctaa
1.0 1.0 1.0 1.0
0.972 0.962 1.0
1.0
1.0
1.0 0.986 1.0
1.0
−325 (+)
1.0 1.0 1.0 1.0
0.978 0.954 0.994
0.935
0.901
0.989 0.982 0.996
0.913
M; PM PM PM PM
M M PM
M
Ma
PM PM PM
Ma
M
Pax-5 (BSAP)
0.786
0.95
−1420(−)
Pax-4a
ggactggggtaagcttaaggtggcaTTTCT
Core score Matrix score Program
Position Motif for factor (orientation) Sequence
CG-1A
Table 7.5 (continued)
−1995 (+) −1976 (−) −1818 (+) −1462 (−)
AATCTaa tgTCCTT AAGGAca tgAGTTA
aacCTGCCca
gtgTAATTacaattg
−849 (+) −1218 (−)
tcagtgtAATTAcaa
gtaGTAATtgggt
−828 (+) −852 (−)
cttcatGCAAAggaa
aTGATTttt aTGATTcct
tgcctagagtaGGTGCaagag
n.f.
ccttgggcatgCGTGActccg gGGGTGggttca aggcttggcctgctgcctgatatatTTTTT gGGGTGggatta
−1276 (+)
−1338 (−) −323 (−)
−665 (−)
−1420 (−) −1076 (−) −893 (−) −213 (−)
1.0 1.0 1.0 1.0
1.0
0.902
0.902
1.0
1.0
1.0 1.0
0.987
0.977 1.0 1.0 1.0
1.0 1.0 1.0 1.0
0.994
0.930
0.919
1.0
0.929
0.996 0.995
0.977
0.875 0.985 0.813 0.995
PM PM PM PM
PM
M
M
PM
Ma
PM PM
PM
Ma M M M
PM PM PM M PM
0.949 1.0 0.992 0.843 0.989
0.969 0.961 0.974 0.848 0.987
Core score Matrix score Program
−276 (−) −128 (+) +15 (−) +23 (+) +86 (−)
cattgacttcGTGGCcaca cttaTTCTCgatcctattg cagcagcatcATGTCttgt tcatGTCTTgtgtgagtag atgccttggcTTGACagca
Position (orientation) Sequence
CG-1B
atgcCACGTgaatg/atgccACGTGaatg gccaCGTGAa
−1096 (+/−) −1094 (−)
n.f.
n.f.
aaCAAACac agCAAACaa
v-Myb
YY1
ZEB (AREB6)
1.0 1.0
1.0 1.0
1.0
1.0
1.0
1.0 1.0 1.0
1.0 1.0
0.974 0.974
0.995
0.985
1.0
1.0 1.0 1.0
PM PM
M Ma
M
M
PMa
PM PM PM
Core score Matrix score Program
−617 (+)
−1927 (+)
−545 (−)
−329 (+/−)
−995 (−)
n.f.
cctcCCCATttttccaa
aatAACGGgc
aaggtctaggTCAGCagct
tttcCACGTgggat/ tttccACGTGggat
n.f.
n.f.
tgTGTTT
Position (orientation) Sequence
CG-1B
0.996
1.0
1.0
1.0
0.988
0.985
0.896
0.963
1.0
M
Ma
Ma
M
PM
Core score Matrix score Program 1.0
Putative transcription factor binding sites found in the proximal promoter regions (from −2,000 bp upstream of the transcription (in the case of CG-1A: translation) start site to +150 bp downstream) of chicken galectins CG-1A and CG-1B. Position (orientation): Position (relative to the transcription start site) and orientation of the putative binding site ((+): sense strand; (−): antisense strand). Sequence: DNA sequence of the putative binding site; core sequence is set in uppercase. Core score: score for the matrix core sequence; matrix score: score for the entire matrix sequence. Program: the algorithm yielding the displayed hit – M: Match™; PM: P-Match™Names of factors: AML1a acute myeloid leukemia gene 1a (Runx/runt homology domain), Barbie-Box barbiturate-inducible element, Cdx-1 chicken homeobox gene cdxA product, C/EBP CCAAT/enhancer binding protein, CHOP-10 C/EBP homologous protein 10, COMP1 cooperates with myogenic proteins 1, CP2 CCAAT-binding protein 2, c-Myc/Max c-Myc (cellular counterpart of viral myelocytomatosis oncogene): Max heterodimer, CUTL-1 cut-like homeodomain protein 1, E47 (Hand-1) E-box binding protein (heart- and neural crest derivatives-expressed 1), Elk-1 (TCF-A) ets-like factor 1 (transcription factor A), En-1 engrailed 1, Evi-1 ectopic viral integration site 1-encoded factor, FoxD3 fork head box D3, GATA-3 GATA-box binding factor 3, GKLF gut-enriched Krueppel-like factor, GR glucocorticoid receptor, HOX A3 homeobox cluster protein A3, Lmo2 LIM-only protein 2, Msx-1 msh-like homeobox protein 1, myogenin/NF-1 myogenin/ nuclear factor 1, MZF-1 myeloid zinc finger protein 1, NF-E2 nuclear factor erythroid 2, NRF-2 nuclear respiratory factor 2, Pax paired box gene product, Pbx-1 pre B-cell leukemia transcription factor 1, Sp1 stimulatory protein 1, SRY sex-determining region Y gene product, STAT signal transducer and activator of transcription 1, p91, USF upstream stimulatory factor, v-Maf avian musculoaponeurotic fibrosarcoma virus AS42 nuclear oncoprotein, v-Myb viral myoblastoma oncoprotein, YY1 Yin and Yang 1 (nuclear factor E1, delta factor), ZEB zinc finger E-box-binding protein, Atp1a1 regulatory element binding protein 6 (AREB6), n.f. no significant sequence hit found; these differences are highlighted in gray a Hits detected by “high-quality” matrices It is possible to obtain more than one hit for the same factor at the same or almost the same position. This is due to the use of two independent search algorithms and more than one factor-specific weight matrix. For the listed cases of such “multiple hits” at the same site, their scores and/or orientation differ significantly
n.f.
v-Maf
−1569 (−) −261 (−)
ctaaCACGTgattg/ctaacACGTGattg
−1455 (+/−)
USF
tgccctctTTTATat
−1203 (−)
ttacAGGAA
−291 (−)
TATA-Box
gaTGTTT TAACAaa AAAGAac
STAT1a/b, 2-6
−509 (−) −374 (+) −217 (+)
Position Motif for factor (orientation) Sequence
CG-1A
134
A.M. Wu et al. Table 7.6 Immunohistochemical profiling for the presence of the prototype chicken galectins CG-1A and CG-1B in various organs of adult animalsa Staining intensityb Type of organ CG-1A CG-1B Larynx – Respiratory epithelium ++c Lamina propria mucosae – + Trachea Respiratory epithelium Lamina propria mucosae
++c –
– ++
Lung Respiratory epithelium Connective tissue
– –
+++c +
Esophagus Lamina propria mucosae
–
++
Gut Epithelial lining of villi and intestinal glands Lamina propria mucosae
– –
– +
Liver Hepatocytes (parenchyma)
+++c
–
Kidney Epithelium Collecting ducts (medulla) Proximal/distal tubules (MTN I, MTN II, RTN)
– +++c
– –
Skin Epidermis Stratum corneum – – Stratum intermedium – +++d Stratum basalis – +c Dermis – ++ Subcutis – + MNT-I mammalian-type nephron I (juxtamedullar), MNT-II mammalian-type nephron II (mid-cortical), RTN reptilian-type nephron (superficial) a For details, please see [40] b The intensity of staining is grouped into categories: − no staining, + weak staining, ++ medium staining, +++ strong staining c Only cytoplasmic d Cytoplasmic and nuclear
Fig. 7.7 Comparison of the sequences for chicken galectins CG-1A (C-16) and CG-1B (C-14). Identical residues in both sequences are indicated as white letters on black background. Please note the sequence differences in the vicinity of residues indispensable for sugar binding, such as His 52/54 or Trp 68/70
7 Adhesion/Growth-Regulatory Galectins
135
Table 7.7 Comparison of reactivity of two prototype chicken galectins for natural glycoproteins (gps)a Signal intensity for bindingc Glycoprotein (terminal epitope)b CG-1A (I/II) CG-1B (AB, I/II) Terminal (I/II)-containing gps Cyst Beach P-1 (I/II) +++++ +++++ Cyst Mcdon P-1 (I/II) +++++ +++++ Cyst Tighe P-1 (I/II) +++++ ND Human asialo a1-acid gp (mII) ++++ + Asialofetuin (mII/I, Ta) +++ – iII/Lac Pneumococcus type 14 polysaccharide
++++
+
Blood group ABH, Lea, Leb, Lex, and Ley active gps Cyst Mcdon (Ah > Leb, Ley) Cyst Beach phenol insoluble (Bh > Leb, Ley) Cyst Tighe phenol insoluble (H, Lea, Leb, Lex, Ley)
+ + –
+++++ +++++ –
Ta/Tn-containing gps Asialo OSM (Tn, Ta, core 2 II) Asialo PSM (Tn, Ta, A, Ah, H)
– –
– ND
Ta-containing gps Human asialoglycophorin (Ta, Tn, mIIb/f) Antifreeze gp (Ta)
+ –
– –
Crypto II, Ta/Tn-containing gps Human a1-acid gp (a2-3/6 sialyl mII) – – OSM (sialyl Tn, Ta, core 2 II) – – – – PSM (sialyl Tn, Ta, A, Ah, H) CG-1A/B chicken galectin-1A/B, iII/Lac internal Galb1-4Glc(NAc), m multiantennary, mIIb/f biantennary N-glycan with core fucosylation and bisecting GlcNAc, ND not determined a For details, please see [44, 45] b The symbols in parentheses indicate the terminal epitopes and are listed in Table 7.1 c The results were graded according to the spectrophotometric absorbance value at 405 nm (i.e. OD405) after 4 h incubation as follows: +++++, (OD ³ 2.5); ++++, (2.5 > OD ³ 2.0); +++, (2.0 > OD ³ 1.5); ++, (1.5 > OD ³ 1.0); +, (1.0 > OD ³ 0.5); ±, (0.5 > OD ³ 0.2); –, (OD Galb1Galb1-4GlcNAc (II) > Galb14Glc (L) > GalNAcb1-3Gal 4Glc (L) > Galb1-3GlcNAc (P) > GalNAca1-3Gal (I) > Gala1-3Gal (B); Galb1(A) ³ GalNAca1-3GalNAc 3GalNAc (T) and Gala1(F) ³ Galb1-3GalNAc 4Gal (E) were inactive (T) ³ Galb1-3GlcNAc (I) ≫ Gala1-3Gal (B) > Fuca12Gal (H) ≫ Gala1-4Gal (E)
The most active b-galactoside
Galb1-4GlcNAc (II) mainly, extension to ABH epitopes reducing activity
Galb1-4GlcNAcb1-3Galb1-4Glc (IIb1-3L) and Galb1-4GlcNAc (II) and its H-type derivative
Ratio of glycotope clusters Triantennary glycopeptides with mostly type II termini (simple multivalent and 2,4,2-branching pattern form)/monomeric II from asialofetuin was three times more active than monomeric II
Triantennary glycopeptides with mostly type II termini and 2,4,2-branching pattern from asialofetuin was three times more active than monomeric II
Substituted branch-end glycans
Histo-blood group precursor (equivalent) gps but hindered by ABH histoblood group determinants
Histo-blood group ABH precursor (equivalent) gps and enhanced strongly by blood group A, B determinant sugar
Ratio of complex polyvalent glycotopes in natural glycoproteins/ monomeric II The most complementary chain length
Only 5.5 times more active than 77 Times more active than monomeric II monomeric II
Galb1-4GlcNAc (II) and Galb1- H active Ib1-3L and Galb14GlcNAcb1-3Galb1-4Glc 3GlcNAcb1-3Galb1-4Glc (IIb1-3L) (Ib1-3L) a For detailed information on the binding properties of the galectins, please see [45]
7 Adhesion/Growth-Regulatory Galectins
a
Cys51
His52
Glu123
137
b
His124
Ala53
Gal
Gal Arg48 His44
His46
Arg50 GlcNAc
GlcNAc
Trp68
c
His54
His52
Glu123
GalNAc
Trp70
Glu71
Cys51
Gal Fuc
d
His124
GalNAc
His54
GlcNAc
Trp68
Glu71
Ala53
Gal Fuc
Arg48
His44
Glu73
Arg50
His46
GlcNAc
Trp70
Glu73
Fig. 7.8 Graphical illustration of the carbohydrate recognition domains (CRDs) of CG-1A (C-16) (left panel) and CG-1B (C-14) (right panel) pinpointing the contact sites for Galb1-4GlcNAc (upper panel) and for histo-blood group A tetrasaccharide. The files for seeing the molecules in motion are available at http://www.nmr.chem.uu.nl/haddock/movies/. The positioning of the key contact sites was deliberately kept constant for direct comparison. Both sugars remained in lowenergy conformations, and the comparison identified the regions of each CRD responsible for contact to the a1-2/3 substitutions on the core galactose moiety [45]
shot) of the binding-site architecture and the major contacts is illustrated in Fig. 7.8. With this information, it is possible to gage the impact of amino acid substitutions between the proteins on ligand binding, e.g. that of the Ala53/Cys51 exchange (see Fig. 7.8) on the orientation of equivalent His54/His52 residues and the resulting consequences on energetic terms [45]. Clearly, the outlined data attest that even closely related members of the galectin family should not be considered as redundant modules. They prompt further analysis to tie the presence of amino acid substitutions to functional divergence. The strategic combination of the binding assays with the flexible ligand docking in silico and calculation of binding-energy terms is expected to be of pivotal importance for understanding the initial step of translating the sugar code in molecular terms [47, 48]. Also, with the emergence of insights into lectin involvement as endogenous effectors in
138
A.M. Wu et al.
d isease manifestation and progression, drug design on human galectins becomes a therapeutical perspective [49, 50], the concept and data presented herein serving as a proof-of-principle case study.
7.5 Summary The glycan part of cellular glycoconjugates harbors bioactive signals encoded in glycotopes. They are decoded and translated into signaling and cellular responses by lectins such as those of the family of galectins sharing the b-sandwich folding. As a physiologically relevant means for evaluating the binding properties of these medically important lectins to carbohydrate ligands in their natural structural context, we have introduced a panel of glycoproteins with a presentation of structurally well-defined glycotopes. Their application in a solid-phase assay reveals finespecificity differences between closely related lectins, even from the same subgroup, and also among mammalian and plant lectins sharing specificity to b-galactosides. These results support the notion for nonredundant assignments of different lectins from the same family in vivo, convincingly backed by promoter analysis and immunohistochemical expression profiling of two prototype CGs. By implementing flexible ligand docking to this research area in a proof-of-principle case study, we began to discern detailed structure–activity relationships, a promising approach for drug design when applied to human lectins. Acknowledgments This work was supported by grants from the Chang-Gung Medical Research Project (CMRP No. 33025 and 170441; Kwei-san, Tao-yuan, Taiwan), the National Science Council (NSC 97-2320-B-182-020-MY3 and 97-2628-B-182-002-MY3; Taipei, Taiwan), the Mizutani Foundation for Glycoscience (Tokyo, Japan), the research initiative LMUexcellent (Munich, Germany), the Verein zur Förderung des biologisch-technologischen Fortschritts in der Medizin e. V. (Heidelberg, Germany), and a European Community Marie Curie Research Training Network grant (contract no. MRTN-CT-2005-019561).
References 1. Gabius HJ, Wu AM (2006) The emerging functionality of endogenous lectins: a primer to the concept and a case study on galectins including medical implications. Chang Gung Med J 29:37–62 2. Gabius HJ (ed) (2009) The sugar code. Fundamentals of glycosciences. Wiley-VCH, Weinheim 3. Laine RA (1997) The information-storing potential of the sugar code. In: Gabius HJ, Gabius S (eds) Glycosciences: status and perspectives. Chapman & Hall, London, pp 1–14 4. Brockhausen I, Schachter H (1997) Glycosyltransferases involved in N- and O-glycan biosynthesis. In: Gabius HJ, Gabius S (eds) Glycosciences: status and perspectives. Chapman & Hall, London, pp 79–113 5. Reuter G, Gabius HJ (1999) Eukaryotic glycosylation: whim of nature or multipurpose tool? Cell Mol Life Sci 55:368–422
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6. Patsos G, Corfield A (2009) O-Glycosylation: structural diversity and functions. In: Gabius HJ (ed) The sugar code. Fundamentals of glycosciences. Wiley-VCH, Weinheim, pp 111–137 7. Wilson IBH, Paschinger K, Rendić D (2009) Glycosylation of model and ‘lower’ organisms. In: Gabius HJ (ed) The sugar code. Fundamentals of glycosciences. Wiley-VCH, Weinheim, pp 139–154 8. Zuber C, Roth J (2009) N-Glycosylation. In: Gabius HJ (ed) The sugar code. Fundamentals of glycosciences. Wiley-VCH, Weinheim, pp 87–110 9. Lemieux RU (1989) The origin of the specificity in the recognition of oligosaccharides by proteins. Chem Soc Rev 18:347–374 10. Lemieux RU (1996) How water provides the impetus for molecular recognition in aqueous solution. Acc Chem Res 29:373–380 11. Gabius HJ (1998) The how and why of protein–carbohydrate interaction: a primer to the theoretical concept and a guide to application in drug design. Pharm Res 15:23–30 12. Gabius HJ, Siebert HC, André S, Jiménez-Barbero J, Rüdiger H (2004) Chemical biology of the sugar code. Chembiochem 5:740–764 13. Solís D, Romero A, Menéndez M, Jiménez-Barbero J (2009) Protein–carbohydrate interactions: basic concepts and methods for analysis. In: Gabius HJ (ed) The sugar code. Fundamentals of glycosciences. Wiley-VCH, Weinheim, pp 233–245 14. Watkins WM, Morgan WTJ (1952) Neutralisation of the anti-H agglutinin in eel serum by simple sugars. Nature 169:825–826 15. Kilpatrick DC, Green C (1992) Lectins as blood typing reagents. Adv Lectin Res 5:51–94 16. Watkins WM (1999) A half century of blood-group antigen research: some personal recollections. Trends Glycosci Glycotechnol 11:391–411 17. Rüdiger H, Gabius HJ (2009) The history of lectinology. In: Gabius HJ (ed) The sugar code. Fundamentals of glycosciences. Wiley-VCH, Weinheim, pp 261–268 18. von der Lieth CW, Siebert HC, Kožár T, Burchert M, Frank M, Gilleron M, Kaltner H, Kayser G, Tajkhorshid E, Bovin NV, Vliegenthart JFG, Gabius HJ (1998) Lectin ligands: new insights into their conformations and their dynamic behavior and the discovery of conformer selection by lectins. Acta Anat 161:91–109 19. López-Lucendo MF, Solís D, André S, Hirabayashi J, Kasai KI, Kaltner H, Gabius HJ, Romero A (2004) Growth-regulatory human galectin-1: crystallographic characterisation of structural changes induced by single-site mutations and their impact on the thermodynamics of ligand binding. J Mol Biol 343:957–970 20. Siebert HC, André S, Lu SY, Frank M, Kaltner H, van Kuik JA, Korchagina EY, Bovin NV, Tajkhorshid E, Kaptein R, Vliegenthart JFG, von der Lieth CW, Jiménez-Barbero J, Kopitz J, Gabius HJ (2003) Unique conformer selection of human growth-regulatory lectin galectin-1 for ganglioside GM1 versus bacterial toxins. Biochemistry 42:14762–14773 21. André S, Kaltner H, Lensch M, Russwurm R, Siebert HC, Fallsehr C, Tajkhorshid E, Heck AJR, von Knebel DM, Gabius HJ, Kopitz J (2005) Determination of structural and functional overlap/ divergence of five proto-type galectins by analysis of the growth-regulatory interaction with ganglioside GM1 in silico and in vitro on human neuroblastoma cells. Int J Cancer 114:46–57 22. Kopitz J, von Reitzenstein C, Burchert M, Cantz M, Gabius HJ (1998) Galectin-1 is a major receptor for ganglioside GM1, a product of the growth-controlling activity of a cell surface ganglioside sialidase, on human neuroblastoma cells in culture. J Biol Chem 273:11205–11211 23. Kopitz J, von Reitzenstein C, André S, Kaltner H, Uhl J, Ehemann V, Cantz M, Gabius HJ (2001) Negative regulation of neuroblastoma cell growth by carbohydrate-dependent surface binding of galectin-1 and functional divergence from galectin-3. J Biol Chem 276:35917–35923 24. Wang J, Lu ZH, Gabius HJ, Rohowsky-Kochan C, Ledeen RW, Wu G (2009) Cross-linking of GM1 ganglioside by galectin-1 mediates regulatory T cell activity involving TRPC5 channel activation: possible role in suppressing experimental autoimmune encephalomyelitis. J Immunol 182:4036–4045
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25. Siebert HC, Gilleron M, Kaltner H, von der Lieth CW, Kožár T, Bovin NV, Korchagina EY, Vliegenthart JFG, Gabius HJ (1996) NMR-based, molecular dynamics- and random walk molecular mechanics-supported study of conformational aspects of a carbohydrate ligand (Galb1-2Galb1-R) for an animal galectin in the free and in the bound state. Biochem Biophys Res Commun 219:205–212 26. Gilleron M, Siebert HC, Kaltner H, von der Lieth CW, Kožár T, Halkes KM, Korchagina EY, Bovin NV, Gabius HJ, Vliegenthart JFG (1998) Conformer selection and differential restriction of ligand mobility by a plant lectin. Conformational behavior of Galb1-3GlcNAcb1-R, Galb1-3GalNAcb1-R and Galb1-2Galb1-R’ in the free state and complexed with mistletoe lectin as revealed by random walk and conformational clustering molecular mechanics calculations, molecular dynamics simulations and nuclear Overhauser experiments. Eur J Biochem 252:416–427 27. Kaltner H, Gabius HJ (2001) Animal lectins: from initial description to elaborated structural and functional classification. In: Wu AM (ed) The molecular immunology of complex carbohydrates. Adv Exp Med Biol, vol 491. Plenum Press, New York, pp 79–93 28. Cooper DNW (2002) Galectinomics: finding themes in complexity. Biochim Biophys Acta 1572:209–231 29. Rappl G, Abken H, Muche JM, Sterry W, Tilgen W, André S, Kaltner H, Ugurel S, Gabius HJ, Reinhold U (2002) CD4+CD7– leukemic T cells from patients with Sézary syndrome are protected from galectin-1-triggered T cell death. Leukemia 16:840–845 30. André S, Sanchez-Ruderisch H, Nakagawa H, Buchholz M, Kopitz J, Forberich P, Kemmner W, Böck C, Deguchi K, Detjen KM, Wiedenmann B, von Knebel DM, Gress TM, Nishimura SI, Rosewicz S, Gabius HJ (2007) Tumor suppressor p16INK4a: modulator of glycomic profile and galectin-1 expression to increase susceptibility to carbohydrate dependent induction of anoikis in pancreatic carcinoma cells. FEBS J 274:3233–3256 31. Roda O, Ortiz-Zapater E, Martínez-Bosch N, Gutiérrez-Gallego R, Vila-Perelló M, Ampurdanés C, Gabius HJ, André S, Andreu D, Real FX, Navarro P (2009) Galectin-1 is a novel functional receptor for tissue plasminogen activator in pancreatic cancer. Gastroenterology 136:1379–1390 32. Ahmad N, Gabius HJ, Kaltner H, André S, Kuwabara I, Liu FT, Oscarson S, Norberg T, Brewer CF (2002) Thermodynamic binding studies of cell surface carbohydrate epitopes to galectins-1, -3, and -7: evidence for differential binding specificities. Can J Chem 80:1096–1104 33. Wu AM (2003) Carbohydrate structural units in glycoproteins and polysaccharides as important ligands for Gal and GalNAc reactive lectins. J Biomed Sci 10:676–688 34. Purkrábková T, Smetana K Jr, Dvořánková B, Holíková Z, Böck C, Lensch M, André S, Pytlík R, Liu FT, Klíma J, Smetana K, Motlík J, Gabius HJ (2003) New aspects of galectin functionality in nuclei of cultured bone marrow stromal and epidermal cells: biotinylated galectins as tool to detect specific binding sites. Biol Cell 95:535–545 35. Kübler D, Hung CW, Dam TK, Kopitz J, André S, Kaltner H, Lohr M, Manning JC, He L, Wang H, Middelberg A, Brewer CF, Reed J, Lehmann WD, Gabius HJ (2008) Phosphorylated human galectin-3: facile large-scale preparation of active lectin and detection of structural changes by CD spectroscopy. Biochim Biophys Acta 1780:716–722 36. Wu AM, Singh T, Wu JH, Lensch M, André S, Gabius HJ (2006) Interaction profile of galectin-5 with free saccharides and mammalian glycoproteins: probing its fine specificity and the effect of naturally clustered ligand presentation. Glycobiology 16:524–537 37. Wu AM, Wu JH, Liu JH, Singh T, André S, Kaltner H, Gabius HJ (2004) Effects of polyvalency of glycotopes and natural modifications of human blood group ABH/Lewis sugars at the Galb1-terminated core saccharides on the binding of domain-I of recombinant tandem-repeattype galectin-4 from rat gastrointestinal tract (G4-N). Biochimie 86:317–326 38. Wu AM, Wu JH, Tsai MS, Liu JH, André S, Wasano K, Kaltner H, Gabius HJ (2002) Fine specificity of domain-I of recombinant tandem-repeat-type galectin-4 from rat gastrointestinal tract (G4-N). Biochem J 367:653–664
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Chapter 8
Glycotope Structures and Intramolecular Affinity Factors of Plant Lectins for Tn/T Antigens Pierre Rougé, Willy J. Peumans, Els J.M. Van Damme, Annick Barre, Tanuja Singh, June H. Wu, and Albert M. Wu
Keywords Tn glycotope • T glycotope • Glycosylation • Plant lectins O-glycosylation is a widely distributed posttranslational modification initiated by the addition of GalNAc to serine or threonine residues of polypeptide chains. The GalNAc may be further substituted to form linear/branched sugar chains. Some proteins, like the mucins of vertebrates, are extensively O-glycosylated and consist predominantly of carbohydrate. T antigens, which were originally designated as Thomsen–Friedenreich or TF antigens, are of particular interest in glycobiology since a high expression of the T antigen on the cell surface can be used as a glycomarker [1–3]. Due to the apparent loss of bGalactosyl-transferase, which normally converts the Tn antigen (GalNAca1-Thr/Ser) into the T antigen (Galb1,3GalNAca1Thr/Ser), the precursor Tn antigen accumulates in cancer cells (Fig. 8.1). It is well established that the Tn antigen is a specific marker for human tumor cells and an indicator of carcinoma aggressiveness [4]. The Forssman antigen (GalNAca1,3GalNAcb1,3Gala1,4Galb1,4Glcb1-1’ceramide), which shares a terminal GalNAc residue with the Tn antigen, is also expressed as a tumor-associated lipid antigen in different forms of cancers [5–9]. Hence, proteins capable of specifically recognizing Tn and T antigens, e.g., monoclonal antibodies [10], are valuable tools for both the diagnosis and prognosis of carcinomas. In this respect, a large number of plant lectins that interact with Tn and T antigens have been described. However, only a few of them bind with a sufficiently high selectivity and affinity to be considered useful probes for the detection of Tn and T antigens. Furthermore, the molecular basis for their selective binding to Tn/T antigens is still poorly understood. Therefore, to decipher in detail the molecular basis for the Tn and T specificity, we made a comprehensive analysis of the structural data provided by X-ray
P. Rougé () Surfaces Cellulaires et Signalisation chez les Végétaux, UMR UPS-CNRS 5546, Pôle de Biotechnologie végétale, 24 Chemin de Borde Rouge, 31326 Castanet Tolosan, France e-mail:
[email protected] A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_8, © Springer Science+Business Media, LLC 2011
143
144
P. Rougé et al. OH
OH
OH
O HO
b3GalT
OH
OH O
O O
HO
N
OH
A cHN O
N A cHN O O
O
Tn antigen
OH
T antigen
Fig. 8.1 Conversion of Tn antigen (GalNAca1O-Ser) into T antigen (Galb1,3GalNAca1O-Ser) by the bGalactosyl-transferase (b3GalT)
analysis of antigen–lectin complexes and complemented these data with novel insights generated by modeling and docking experiments.
8.1 Tn- and T-Specific Plant Lectins Plant lectins that were described as Tn or T specific are presented in Table 8.1 [11, 12]. Hitherto, Tn- or T-specific lectins have been found in the families of Amaranthaceae, Fabaceae, Lamiaceae, Moraceae, and Orchidaceae. These lectins belong to five families of structurally and evolutionarily related proteins (amaranthins, legume lectins, jacalin-related lectins, type 2 ribosome-inactivating proteins, and GNArelated lectins). The majority of them are in Fabaceae, Lamiaceae, and Moraceae species. Interestingly, all Lamiaceae lectins are Tn-specific lectins.
8.2 Two Plant Lectins Recently Identified as Tn and T Specific Two novel plant lectins with a preferential affinity for Tn and T antigens were recently identified and well characterized (Table 8.2). One of them was isolated from leaves of the ground ivy Glechoma hederacea (family Lamiaceae) [33] and accordingly was called Glechoma hederacea agglutinin (or Gleheda). Cloning of Gleheda revealed that this Lamiaceae lectin belongs to the legume lectin family and possesses the typical three-dimensional organization known as the jelly roll scaffold, which is in fact a b-sandwich structure similar to that found in legume lectins [33]. Moreover, a comparative analysis indicated that Gleheda and all other previously described Lamiaceae lectins represent a homogeneous subgroup of the legume lectin family. The second interesting Tn/T-specific lectin was purified from the bark of the black mulberry tree (Morus nigra, Moraceae) [31] and, based on its origin, called Morniga G (which stands for M. nigra agglutinin Gal-specific) to distinguish it from the mannose-specific homolog Morniga M (for M. nigra agglutinin Man-specific) [34]. The Morniga G protomer consists of two polypeptide chains
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Table 8.1 Tn- and/or T-specific plant lectins Family/Species Lectin family Amaranthaceae Amaranthus caudatus(amaranthin) Amaranthin Amaranthus leucocarpus Amaranthin
Structure
Specificity
References
Dimer Dimer
T Tn, T
[13] [14]
Fabaceae Abrus precatorius Arachis hypogaea (PNA) Griffonia simplicifolia (GSIA4) Vicia graminea Vicia villosa (VVLB4)
Type 2 RIP Legume lectin Legume lectin Legume lectin Legume lectin
Dimer Tetramer Tetramer Tetramer Tetramer
T T Tn T cluster Tn
[15] [16] [17] [18] [19, 20]
Lamiaceae Glechoma hederacea (Gleheda) Molucella laevis (MLL) Salvia sclarea Salvia bogotensis Salvia horminum
Legume lectin Legume lectin Legume lectin Legume lectin Legume lectin
? Dimer Dimer ? Tetramer
Tn, T Tn Tn Tn Tn
[21] [22] [23, 24] [25] [26]
Moraceae Artocarpus integrifolia (jacalin) Artocarpus lakoocha (ALA) Maclura pomifera (MPA) Morus nigra (Morniga G)
Jacalin Jacalin Jacalin Jacalin
Tetramer Tetramer Tetramer Tetramer
T Tn, T T Tn, T
[27, 28] [27, 28] [29, 30] [31]
Orchidaceae Laelia autumnalis
GNA-related?
Dimer
T
[32]
Table 8.2 Affinity of two plant lectins recognizing Tn and/or T glycotopes [37] Binding intensity a Relative potencyb Glycoprotein (lectin determinants; blood group Morniga Morniga G Gleheda Morniga Morniga G Gleheda specificity) M (mII) (T/Tn, II) (Tn, I/II) M (mII) (T/Tn, II) (Tn, I/II) Polyvalent T/Tn and I/II-containing O-linked gps +++++ +++++ −c 2.4 × 104 6.5 × 105 Native ASG-Tn (Tn) −c Asialo OSM (Tn) − +++++ +++++ − 1.2 × 104 6.5 × 105 Asialo PSM (Tn, Ta, A, Ah) − +++++ +++++ −c 7.2 × 103 1.8 × 105 c c 3 Tn-glycophorin (Tn) − +++++ +++++ − 2.9 × 10 1.3 × 104 c c 3 Cyst Mcdon P-1 (T/Tn, I/II) + +++++ +++++ − 6.5 × 10 4.1 × 104 Cyst Beach P-1 (T/Tn, I/II) ± +++++ +++++ −c 2.4 × 103 4.1 × 104 3.6 × 103 − − +++++ − −c Active antifreeze gp (Ta) Lectin abbreviations: Morniga M (mannose-specific M. nigra lectin) as control [34]; Morniga G (galactose-specific M. nigra lectin); Gleheda (Glechoma hederacea lectin) a The results were graded according to the spectrophotometric absorbance value at 405 nm (i.e. OD405) after 2 h incubation as follows: +++++, (OD ³ 2.5); ++++, (2.5 > OD ³ 2.0); +++, (2.0 > O.D ³ 1.5); ++, (1.5 > OD ³ 1.0); +, (1.0 > OD ³ 0.5); ±, (0.5 > OD ³ 0.2); −, (OD Fuc-binding) lectin R. solanacearum first (Fuc-binding lectin) Secretors Nonsecretors Surface plasmon resonance Soluble receptor-mimicking decoy glycans Tetraclita squamosa lectin Ulex europaeus agglutinin-I Ulva lactuca lectin Virulence factors
Among the ten different lectins discovered in the old biochemistry laboratory at Bar-Ilan University during the years 1972–2006 (Fig. 9.1), five were isolated from three soil bacteria: Pseudomonas aeruginosa (PA) [1–3], Ralstonia solanacearum (RS) [4, 5], and Chromobacterium violaceum (CV) [6]. The basic beneficial role of these three bacteria in nature is the vigorous decomposition of dead organic debris for carbon and nitrogen cycling. Accordingly, they are endowed with diverse adhesins, which enable their adhesion to target macromolecules and cells, including the lectins; a rich arsenal of extracellular toxins [12], which arrest protein synthesis in degenerating cells and kill them; and hydrolytic enzymes, which decompose the target cells and organic macromolecules, including proteins, lipids, glycoconjugates, and nucleoconjugates. Unfortunately, when these three bacteria encounter ill organisms, they regard them as targets for decomposition and change into opportunistic and very aggressive pathogens. Two of them (PA and CV) threaten animal and human life [13, 14], whereas RS causes lethal wilt in many crops, resulting in severe agricultural losses [15]. The lectins of these bacteria – PA first (LecA, galactophilic) lectin (PA-IL) and PA second (Lec B, fucose Manbinding) lectin (PA-IIL), CV (fucose-binding) lectin (CV-IIL), and RS first (Fuc-binding)
cells
cells
+
Mitogenicity
+
H(A,B),Le
(High/Normal)
Fuc>Ara>Man
11,732 x 4
Human pathogen
1983
+
HI
(Normal)
Gal+
30k x2
Coral tree
Erythrina corallodendron
N.D.
N.D .
(Low)
Chitin
Barnacle
Tetraclita squamosa
Animal ovary/ fertilized eggs
1981 Plant seeds
TSL
IV
ECorL
III
Sea lettuce 30k
Sea snail (hare)
N.D.
N .D.
saccharides
and N-acetyl
Sialic acid
Sea snail (hare)
1. GalUA/Gal
+
I,T
(High/Normal)
N.D.
H,Le
(Normal)
Fuc
10k x 3
Ulva lactuca
Aplysia various spp
Aplysia various spp
34k x 2
Plant pathogen
marine alga
Gonad & eggs
hemolymph
2002
N.D.
-
(High/Normal)
Fuc>Ara>Man
Ralstonia solanacearum
cells
Bacterial
Plant
1984
IX
X
Wimmerova et
H(A,B),Le N.D. N.D.
(High/Normal)
L-Gal>Fuc>Man
11,9k x 4
-
(High/Normal)
Man>Fru>Fuc
11,61k x 4
Animal (human) pathogen Plant pathogen
cells
Bacterial
2006
CV-IIL (PA-IIL-like)
Chromobacterium violaceum
al.
Ralstonia solanacearum
cells
Bacterial
2003
RS-IIL (PA-IIL-like)
Imberty,
B a c t e r i a l
1988
RSL
VIII
Animal
ULL
VII
1984,1985
AGL
VI
Animal
AHL
V
Fig. 9.1 The ten lectins, including the five bacterial ones [1–6], that were discovered, isolated, and studied in the old biochemistry laboratory at Bar-Ilan University during the years 1972–2006. The 3-D structures of these lectins, represented in this figure as ribbons with their calcium as spheres and their ligands as sticks, were unveiled thanks to a fruitful cooperation with Dr. Anne Imberty and Dr. Michaela Wimmerova, along with their collaborators and coworkers [4, 5, 7–11]
B,I,P
Gal+ (Normal)
12,763 x 4
Human pathogen
Pseudomonas Pseudomonas aeruginosa aeruginosa
Blood group specificity
(affinity)
specificity
Sugar
Mr (Da)
Producing organism
1977
Bacterial
1972
Bacterial
Year
(Lec B)
Source
(Lec A)
PA-IIL
Imberty, Wimmerova et al.
Bacterial
II
PA-IL
LECTIN
I
LECTINS DISCOVERED AND STUDIED IN PROF. NECHAMA GILBOA-GARBER’S LABORATORY (FACULTY OF LIFE SCIENCES)
9 The Five Bacterial Lectins 157
158
N. Gilboa-Garber et al.
lectin (RSL) and RS second (Man > Fuc-binding) lectin (RS-IIL) – are coproduced and c ofunction with the bacterial virulence factors (VIFs). They might be involved in bacterial sugar-specific homing and anchoring to target cells via glycosylated cell surface receptors, enabling the functions of the accompanying destructive agents [16]. These five lectins were discovered in the cell extracts of the three bacteria, where they exist in high concentrations with relatively low exposure on the bacterial cell surface [17]. This surface activity is probably enough to initiate bacterial biofilm formation and interact with heterologous target cells [17]. Heat-stress treatment of the bacteria increases the lectin surface activity [18]. The discovery of the first PA lectin, PA-IL, in 1972, was by serendipity [1], when crude PA cholinesterase preparation was added to papain-treated human erythrocytes [19]. This was the first description of a bacterial galactose (Gal)binding lectin and the first report on purification of a bacterial lectin by affinity chromatography using Sepharose [20]. At that time, lectins were still considered to be plant or animal products. Therefore, this finding was considered pioneer work in lectinology [21]. The second PA lectin, PA-IIL (L-Fuc > D-Man/Fru), was discovered 5 years later in the same PA cells grown in another medium [2]. This lectin was shown to display an outstandingly high affinity to fucose, at an order of mM (which is rare for lectins that generally exhibit mM affinity orders) [22]. It also displays Man-binding activity (at mM affinity order, like that of Conavalia ensiformis [ConA]) and, therefore, could be purified by affinity chromatography using Sepharose–mannose [2, 3]. Soon after, its reaction with the enzyme peroxidase was revealed, as well as its and PA-IL’s binding to other bacteria [23, 24], increasing their phagocytosis by human leukocytes [25]. The information on the sugar specificities of the PA lectins led to sugar application for abrogation of PA adhesion in the treatment of its infections [26–29]. The two PA lectins, like those of plants and animals, were further shown to possess mitogenic activities [30, 31]. During the following years, their properties, applications, interactions with diverse glycosylated molecules and cells, and their effects on them were investigated [32–34], always in parallel to plant and animal lectins that displayed similar specificities (the galactophilic Aplysia gonad lectin [AGL], Erythrina corallodendron lectin [ECorL], Maclura pomifera lectin [MPL], peanut agglutinin [PNA], the mannophilic ConA, and the fucophilic Ulex europaeus agglutinin-I [UEA-I]). The PA-IL-encoding gene was discovered in 1992, before the PA genomic project report in 2000, and expressed as a recombinant protein in Escherichia coli in 1994 [35, 36]. The gene sequence was the key for information on the lectin amino acid sequence, which had been hard to achieve before due to the high resistance of the PA lectins to proteases and chemicals. Winzer et al. [37], who received from us the lectin information, named the PA-IL gene lecA, intending to fit it to PA genomic nomenclature. That manipulation has introduced confusion as to the lectin names: calling PA-IL also Lec A and PA-IIL also Lec B, names which are not acceptable in lectinology. The detailed information on PA-IL carbohydrate specificities and affinities has further accumulated as a result of several additional studies using equilibrium dialysis [38], microtiter plate enzyme (alkaline phosphatase conjugated
9 The Five Bacterial Lectins
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Hemagglutination inhibition (Log2 dilution-1)
to avidin)-linked lectin (in biotinylated form) sorbent (glycan-binding) assay [ELL(S)A] [39], and X-ray crystallographic investigation of the 3-D structure of the PA-IL-galactose complex combined with microcalorimetric determinations [8]. The PA-IIL gene was discovered in 2000 [40], already enjoying concomitant PA genomic project information [41]. Its detailed carbohydrate specificities and affinities were studied during the following years, also using the above-described methods [9, 11, 22, 42–48]. The other three bacterial lectins (RSL, RS-IIL, and CV-IIL) were discovered about 30 years after the discovery of the PA lectins [4–7], following the publications of the genome projects of RS in 2002 [49] and CV in 2004 [50], that showed the presence of PA-IIL-like genes in them. The search for a PA-IIL-like lectin in RS cell extracts led first to the finding of RSL [4]. This lectin, which was not anticipated, is also Fuc-specific [4, 10] but is unrelated to PA-IIL in its structure and Ca2+ absence (Fig. 9.2). Alignment plus crystallographic X-ray studies showed its homology to the orange soil mushroom Aleuria aurantia lectin [10, 53]. The looked-for RS-IIL was discovered 2 years later in the same RS cell extracts using ethylenediaminetetraacetic acid (EDTA) for its differential release from the non-Ca2+-dependent RSL [5], which copurified with it by affinity chromatography. RS-IIL resembles PA-IIL in structure, including the double Ca2+-dependent, very high carbohydrate affinity (at mM order), but differs from it in highest preferential affinity to mannose instead of fucose, which is its secondary ligand. The RS-IIL Man > Fuc preference versus PA-IIL Fuc > Man preference is attributable to binding-site amino acid #22 variation: RS-IIL Ala versus PA-IIL Ser [11, 51] (Fig. 9.2). The other genetically anticipated putative CV lectin, CV-IIL, resembling PA-IIL in structure (including double Ca2+) and in preferential high Fuc affinity, was isolated
10
EDTA
8 6 4 2 0
Lectin:
PA-IL
PA-IIL
RSL
Ser22
Specificity:
Gal
Fuc>Man
RS-IIL Ala22
Fuc>Man
CV-IIL Ser22
Man>Fuc Fuc>Man
Fig. 9.2 The five bacterial lectin monosaccharide specificities, EDTA sensitivities, and Connolly surfaces with the calcium ions (1 in PA-IL; 2 in PA-IIL, RS-IIL, and CV-IIL; but none in RSL) as spheres and the ligands as sticks. The Connolly surfaces include the carbohydrate-binding site (22-23-24) amino-acid triads (Ser-Ser-Gly; Ala-Ala-Asn; Ser-Ala-Ala) of the homologous lectins – PA-IIL, RS-IIL, and CV-IIL, respectively – with arrows indicating the first amino acid, #22, in each triad [11, 51, 52]
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both in its native form from CV cell extracts [6] and in recombinant form from E. coli cells [7]. Its Fuc preference is like that of PA-IIL [52], albeit sharing only one (Ser #22) of the three (#22–24) binding-site amino acids (Ser-Ala-Ala versus Ser-Ser-Gly of PA-IIL) (Fig. 9.2). The differences in the other two amino acids probably contribute to the subtle differences in the homologous lectin interactions with fucose versus mannose and with diverse saccharide-bearing soluble molecules, various animal red blood cells (RBCs), and human RBCs of diverse blood types (as will be shown hereafter). They might contribute to the selective interactions with autologous microbial cells [32, 33, 54–56] for biofilm formation and with heterologous host cells [32, 54] for infection establishment.
9.1 Determinations of Pathogenic Bacterial Lectin Interactions with Diverse Glycoconjugates The studies on pathogenic bacterial lectin–carbohydrate interactions have introduced additional aspects associated with the possible involvement of these lectins in bacterial binding to target cells leading to subsequent infection. These aspects encompass both characterizations of the host cell surface receptors attracting these lectins and finding the best-fitting glycan derivatives that would most efficiently compete with those receptors as decoys for blocking the lectin-receptor binding according to the aims of the anti-adhesion strategy. The need for such a strategy, in addition to the antibiotic and bactericidal treatments, has evolved due to increasing antibiotic resistance of bacterial infections, including the above-described lectinproducing, human opportunistic pathogens PA and CV. The lectins of those bacteria, which are supposed to enable their homing to the patients’ cells, are regarded as important probes for the looked-for soluble receptor mimicking decoy glycans (SRMDGs). For preliminary estimation of bacterial–lectin inhibition by diverse glycans, the hemagglutination inhibition test is very convenient and efficient but is not accurate. Usage of equilibrium dialysis, ELL(S)A, X-ray crystallography, thermodynamic isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR) techniques profoundly upgrades the accuracy of the results.
9.1.1 PA-IL Interactions with Diverse Glycans PA-IL is a proper galactophilic lectin. Using equilibrium dialysis (with D-[6-3H] Gal) has demonstrated that it binds 1 Gal per subunit with a Ka of 3.4 × 104 M−1 and exhibits higher affinities for its hydrophobic (phenyl) derivatives, with highest affinities for the hydrophobic thio derivatives. Alpha-Me Gal is a stronger inhibitor than b Me Gal, but in the case of the hydrophobic derivatives, the b configuration is more potent than the respective a-galactosides [38]. This lectin is also inhibited
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by GalNAc. In this respect, it differs from the galactophilic lectin AGL (which prefers galacturonic acid over Gal but does not react with GalNAc). These two lectins also differ in their interaction with l-rhamnose: while PA-IL reacts with this sugar, AGL’s interactions with it, like those of ConA, are negligible (70:10:20 agglutination score units, respectively). Among the natural Gal derivatives, it prefers Gala1-6Glc/Gal derivatives, which are present in disaccharide melibiose (Gala1-6Glc), trisaccharide raffinose (Gala1-6Glcb1-2Fru), tetrasaccharide stachyose (Gala1-6Gala1-6Glcb1-2Fru), and in the branched galactans and galactomannans guar and locust gums (Tables 9.1 and 9.2; Fig. 9.3). PA-IL also very strongly reacts with Gala1-4 Gal and Gala1-3 Gal derivatives [39]. Using both ELL(S)A and inhibition of agglutinin–glycan interactions with sugar ligands, Chen et al. [39] showed that, among 36 glycans tested for binding, PA-IL reacted best with two pigeon glycoproteins (gps) containing Gala1-4Gal determinants and with a human blood group ABO precursor (Galb1-3/4GlcNAc)-equivalent gps. It also reacted strongly with many B-blood-group-active (Gala1-3Gal) gps, but weakly or not at all with A (GalNAca1-3Gal)- and H (Fuca1-2Gal)-active or sialylated gps. Among the mammalian disaccharides tested by the inhibition assay, human blood group P1- and Pk-active Gal a1-4 was the best, 1.8 times more active than Gal. However, it was 7.4-fold less active than melibiose (Gala1-6GLc). PA-IL was found to prefer the galactose a-anomer in the following decreasing order: Gala1-6 > Gala1-4 > Gala1-3. Of the monosaccharide derivatives studied, the Table 9.1 Relative inhibitory potencies of various saccharides based on 50% inhibition of PA-IL binding by hydatid cyst Gp [39] The saccharide examined Relative potency 57.1 PhenylbGal p-NO2-phenylbGal 20 Melibiose (Gala1-6Glc) 13.3 Stachyose (Gala1-6Gala1-6Glcb1-2Fru) 5.7 Gala1-3Gala1-methyl 4.7 Raffinose (Gala1-6Glcb1-2Fru) 4.4 p-NO2-phenylaGal 2.9 MethylaGal 2.7 MethylbGal 2.2 Gala1-4Gal (P1, Pk) 1.8 Gal 1.0 Gala1-3Gal (B) 0.8 GalNAc 0.5 Galb1-4Glc (Lac) 0.5 a Galb1-3GlcNAc a Galb1-4GlcNAc (LacNAc, present in Ii) Galb1-3Ara 0.1 a Galb1-3GalNAc (T) d-Fuc, l-rhamnose 0.02 a Weak inhibitors not determined at 50% inhibition but at lower level. Their location in the table is in accordance with the decreasing inhibitory potency order
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Table 9.2 The interactions of the five bacterial lectins (compared to AGL and ConA) with guar, locust, acacia (arabic) and xanthan gums, and with inulin and yeast mannan (all of them at 5 mg/ml concentration), examined by hemagglutination inhibition and represented as the inhibition titer log2 dilution−1 (Lerrer and Gilboa-Garber [57]). The results presented in this table are all part of the PhD thesis of K.D. Zinger-Yosovich, Bar-Ilan University Lectin AGL PA-IL ConA PA-IIL RSL RS-IIL CV-IIL Guar gum 1.0 ± 0.02 0.8 ± 0.07 0 16.5 ± 1.1 21.0 ± 1.5 0.25 ± 0.02 0 Locust gum 33 ± 3.6 35 ± 4.2 0.25 ± 0.03 0 1.8 ± 0.02 1.8 ± 0.1 0 Acacia gum 13 – 0.8 14 ± 0.8 0 2 ± 0.3 3.1 ± 0.6 4.1 ± 0.5 2.5 ± 0.4 Xanthan gum 6 ± 1.1 7.5 ± 1.2 1.8 ± 0.2 1.7 ± 0.3 2.2 ± 0.6 3.2 ± 0.35 2.1 ± 0.16 Yeast mannan 0 1.0 ± 0.2 20 ± 1.5 16 ± 1.5 2.1 ± 0 18.5 ± 1.3 1.0 ± 0.17 Inulin 0 1.5 ± 0.3 2.3 ± 0.4 0 0 3.9 ± 0.5 1.7 ± 0.3 (p < 0.01) Guar gum …..→4β-D-Man
6 ↑ 1 α-D-Gal
1→4β-D-Man 1→4β-D-Man 1→4β-D-Man 1→…. 6 ↑ 1 α-D-Gal
Fig. 9.3 Guar gum structure: galactomannan constructed of Gala1-6-linked branches and Man b1-4-linked backbone
p henyl b derivatives of Gal were much more inhibitory than methyl b derivatives. Only an insignificant difference was found between Gal a-anomer of methyl and p-NO2-phenyl aGal derivatives. Based on these results, it has been suggested that the PA-IL combining-site size better fits a disaccharide with terminal a nonreducing Gal anomer, Gal a1-6Glc being most complementary. The b Gal anomer does not fit it, and a hydrophobic interaction is important for the binding; phenyl hydrophobicity enables better binding than the methyl derivative [39] (Table 9.1). Among the gps and polysaccharides that bear human blood group epitopes – A (GalNAca1-3Gal); AH (GalNAca1-3(l-Fuca1-2)Gal); B (Gala1-3Gal); H (Fuca12Gal); P1 and Pk (Gala1-4Gal); T (Galb1-3GalNAc); and Ii (Galb1-4GlcNAc) – tested by the inhibition test, the human blood group Pk- and P1-active gps were the most inhibitory, while the A, H, or Leb blood group-active substances and sialic acid-containing gps were either very weak or inactive. Neither fetuin/human a1acid gps nor their asialo products reacted with PA-IL [39].
9.1.2 Interactions of PA-IL and the Other Four Bacterial Lectins with Plant and Microbial Polysaccharides Among the polysaccharides examined, PA-IL has been found to strongly interact with branched galactans [58] and with the galactomannans guar and locust gums [59] (Table 9.2). Guar (also called guaran), which is extracted from the leguminous
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shrub Cyamopsis tetragonoloba, contains about 10,000 residues, while locust, which is prepared from the locust bean (of the leguminous carob tree), is shorter. These two galactomannans, which act in the seeds as food and water stores, are used in the food industry as economical thickeners and stabilizers (E412 and E410, respectively). Structurally, guar and locust gums are constructed of Gala-1-6linked branches and Man b(1-4)-linked backbone. In guar, there are 1.5–2 mannose residues for every galactose residue (Fig. 9.3), and in locust gum, there are fewer galactose branches. PA-IL, which selectively reacts with the Gala1-6 branches, strongly adsorbs to these gums and also precipitates them. It also reacts with arabic gum, from the sap of African acacia trees, which is used in the food industry for similar purposes (E414). The chemical composition of this gum is less defined, but in contrast to guar and locust gums, which are neutral, it is anionic. It contains a complex mixture of saccharides and gps. Its arabic acid is a highly branched polymer of b-D-galactose, L-arabinose, D-gluconic acid, L-rhamnose, D-glucuronic acid, D-galacturonic acid, and L-guluronic acid. PA-IL also reacts with the microbial xanthan gum (E415) prepared for the food industry by aerobic submerged fermentation of Xanthomonas campestris. This gum is also an anionic polyelectrolyte with a cellulose-like backbone and side chains also containing – in addition to Gal – Manb1-4 (pyruvated) in ~40% of its terminal residues and D-glucuronic acid a1-2 Man (or Man 6 acetate) linked on alternating residues.
9.1.3 PA-IIL Interactions with Diverse Glycans The unusually high affinity (1–3 × 106 M−1) of PA-IIL to Fuc and its derivatives, which is unusual for lectins where the millimolar range is most common, was first shown using hemagglutination inhibition and equilibrium dialysis [22]. According to the carbohydrate concentration abolishing two hemagglutinating units and the equilibrium dialysis results, one binding site per subunit was defined. Its association constant for Fuc was estimated to be 1.5 × 106 M−1, and for d-mannose, 3.1 × 102 M−1. The equilibrium dialysis results indicated the following order of affinities: p-nitrophenyl-a-Fuc > Fuc > Fucamine > L-galactose > Ara > Man/Fru, while L-rhamnose was a relatively weak inhibitor [22], and xylose and ribose did not inhibit it. Examination of PA-IIL interactions with the above-described polysaccharides has shown that, like ConA, PA-IIL is not attracted by the unbranched mannan backbones of the galactomannans or by the unbranched inulin polyfructosan (which very weakly attracts ConA). It weakly binds to arabic and xanthan gums and strongly binds (although somewhat less than ConA) to yeast mannan, the highly branched polysaccharide with a1-2 and a1-3-linked mannose side chains attached to an a1-6-linked polymannosyl backbone [60]. This yeast polysaccharide also strongly attracts RS-IIL but does not bind CV-IIL or RSL (Table 9.2). ELL(S)A [44] and thermodynamic analyses of the lectin–carbohydrate binding energy using ITC according to Dam and Brewer [61] – together with very high resolution X-ray crystal structure studies (of the lectin in complex with the monosaccharides)
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independently performed in two laboratories [9, 42] and further upgraded [11, 43, 45, 52] accompanied by computational chemistry methods – have elucidated the enthalpydriven interactions that contribute to the high (mM order) PA-IIL Fuc-affinity. PA-IIL, which is a tetramer (like PA-IL), contains two calcium ions (PA-IL contains one) in each subunit binding site. These Ca2+ tweens attract the saccharide hydroxyl residues onto their coordination sphere, leading to extensive spatial delocalization of the binding-site charges, encompassing the negatively charged binding-site amino-acid side chains, the positively charged calcium ions, and the sugar hydroxyl groups. According to computational chemistry calculation rationalization, these delocalizations of charges are responsible for the high enthalpy of binding expressed as an unusually high affinity of the lectin to fucose (KD = 2.9 × 106 M−1) [43]. The PA-IIL complex with fucose shows that the sugar locks onto the pair of calcium ions, with three of its hydroxyls participating in the coordination of these cations. Such a lectin–carbohydrate binding mode has never been observed before [43]; generally, interactions between lectins and carbohydrates include hydrogen bonds and hydrophobic contacts. PA-IIL is rather unique in that its interaction with fucose is established mainly through coordination by calcium ions and hydrogen bonds, with a hydrophobic interaction only between the C5 methyl of fucose and the side chain of Thr 45. Changing the amino acid 22 from Ser to Ala aborts the Fuc > Man preference. The mutant S22A was the only one of three constructed mutant lectins (the other two directed to S23 and G24) that lost Fuc preference, due to greatest thermodynamic change [51]. Anti-adhesion studies have indicated that PA-IIL is most sensitive to inhibition by human milk [62], which protects newborns from microbial and viral infections [63, 64]. The major inhibiting saccharide in the human milk is Lewis (a) (Lea, Fuca1-4GlcNAc), to which PA-IIL displays the highest affinity (Fig. 9.4), higher
Fig. 9.4 Orthogonal view of PA-IIL crystal structure represented as ribbon in a complex, with the Lea trisaccharide ligand as sticks (a) ribbon representation of the lectin tetramer with stick-represented saccharide, (b) representation of the lectin active site detailed interactions with the two calcium ions (balls) and the Lea trisaccharide ligand (as sticks)
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than to Lex (Fuca1-3GlcNAc) or to the H epitope (Fuca1-2Gal) [44, 47, 65]. Among 40 glycans tested for PA-IIL binding by Wu et al. [44], using ELL(S)A and inhibition of lectin–glycan interaction, PA-IIL reacted well with all human blood groups ABH-, Lea-, and Leb-active gps, but weakly or not at all with their precursors. The Lea pentasaccharide lacto-N-fucopentaose (LNFP II, Fuca1-4(Galb1-3) GlcNAcb1-3Galb1-4Glc) was the most potent one, being fivefold more active than Fuc, tenfold and 38-fold more active than Lex pentasaccharide (LNFP III, Fuca13(Galb1-4)GlcNAcb1-3Galb1-4Glc) and sialyl Lex, and 125-fold more active than Man while inactive with Gal and GalNAc [44]. The crystal structure of PA-IIL with lacto-N-neofucopentaose-V (LNnFP-V) provided the first atomic-scale insights into the interactions of bacterial lectins with these natural anti-adhesive compounds [65]. These and Wu’s [44] results imply that the combining site of PA-IIL is a small cavity-type fitting to Fuca, with a specific shallow groove for the remaining Lea saccharides, and that polyvalent Lea glycotopes enhance the reactivity. Recently, Marotte et al. [46, 47] have described a study on binding of a series of Lea-related disaccharide derivatives to PA-IIL using X-ray structures, thermodynamics, and ELL(S)A assays. This study confirms that the Lea trisaccharide (Fuca1-4(Galb1-3)GlcNAc) is the best ligand, followed by Fuca1-4GlcNAc > Fuca1-2Galb1-4GlcNAc (H type 2) > mannosylated antennae containing five Man residues organized in two a3 and a6 forks > H type 3 > Man 8 = Fuc = H type 1 = Ley > sialyl Lea > Lex. That study’s conclusions are that Fuca14GlcNAc brings sufficient affinity to serve as the basis for designing glycomimetic anti-adhesion therapy. Other studies with a battery of synthetic receptor-mimeting compounds also yielded interesting results [48]. All of the above results, combined with reports on the adhesion specificity of the intact bacterium cells to Lex epitopebearing receptors [66], indicate that the Lewis epitopes might be useful SRMDGs for blocking the PA-IIL-dependent bacterial adhesion. The special specificity of PA-IIL to Lewis epitopes Lea > Ley > Lex also makes it an important tool for the detection of tumor cells that bear these epitopes in sialylated form [67, 68]. Similar sialylated epitopes are present on leukocytes. These epitopes are involved in the attraction of these two cell types by endothelial leukocyte adhesion molecule-1 (E-selectin, ELAM-1), which allows their adhesion to blood vessel walls and subsequent invasion adjacent to sites of inflammation [67, 68]. The interactions of PA-IIL with mannosylated compounds are also associated with its inhibition by quail egg white [57] and royal jelly (RJ) gps (as will be shown hereafter). The interactions of PA-IIL with mannosylated and fucosylated gps make it a good tool for purification of gps, including enzymes [69]. Among the enzymes that bind to PA-IIL-Sepharose are horseradish peroxidase, yeast invertase, and hyaluronidases from bovine and ovine testes [70], as well as calf intestinal mucus alkaline phosphatase [71]. Recently, Tielker et al. [72] have also described the usage of this lectin as a genetically constructed affinity tag for one-step purification of recombinant proteins.
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9.1.4 RSL Interactions with Diverse Glycans RSL, which is a trimeric Ca2+-devoid fucose-binding lectin composed of only 90 amino acid subunits [4], was found to display a strong sequence similarity to one third of the mushroom A. aurantia Fuc-binding lectin (AAL) [10]. The RSL monomer is composed of two consecutive similar four-stranded antiparallel b sheets, lying side by side in parallel and connected by one long loop. When assembled as a threefold trimer, RSL forms a six-bladed b propeller fold very similar in form to that generated by peptide repeats of one continuous monomeric chain of AAL. AAL is composed of two identical subunits of 312 amino acids [73], organized in six internal homologous regions displaying 26% homology with the Gram-negative bacterium Myxococcus xanthus hemagglutinin. Wimmerova et al. [53] described the crystal structure of that lectin with its six-bladed b-propeller fold and novel fucose recognition. Each RSL monomer has two binding sites for a total of six sites per trimer. The ligand-binding sites of the two lectins are very similar and are characterized by numerous hydrogen bonds to the side chains of polar amino acids and by strong hydrophobic interactions between aromatic residues, without calcium involvement [10]. In hemagglutination tests, the inhibitions of RSL by the saccharides are considerably weaker than those obtained with PA-IIL and also when examined by other methods, possibly due to stronger interaction of RSL with human RBCs. The order of its decreasing sugar affinities obtained by hemagglutination inhibition is Fuc (3.15 × 104 M−1) > L-Gal > Ara > Fru > Man (1 × 102 M−1) [4]. The results attained in studies of the interactions between RSL and fucosylated compounds by ITC and SPR [10], which are much more accurate, indicate much higher affinities of RSL to Fuca1-6GlcNAcMe, Fuca1-2Lac, MeFuc, and Fuc (KD = 3 × 107 M−1, 4 × 107 M−1, 7 × 107 M−1, and 1.4 × 106 M−1, respectively) (Table 9.3). RSL is not considerably inhibited by xylose and ribose but interacts with plant xyloglucan [10]. The ITC results demonstrate the presence of two binding sites per monomer and that RSL differs from PA-IIL in its better fit to B and A, followed by H blood group-specific oligosaccharides, than to fucose [10]. The very high affinity of RSL for a1-2 (H) and a1-6 (core) fucosylations markedly differs from PA-IIL preference of a1-4(Lea) fucosylations. Since a1,6 (core)-fucosylation of gps is widely distributed in mammalian tissues and is altered under pathological (inflammatory processes and cancer) conditions [74–77], a probe to specifically detect it is important for studying the role of this oligosaccharide structure and for diagnostic purposes. In particular, the serum a-fetoprotein, a well-established tumor marker produced by hepatocellular carcinomas, is rich in core Fuc [78]. The increased content of a1-6-fucosylated oligosaccharides in liver and serum gps during the development of malignant liver diseases (hepatocellular carcinoma and hepatoma) was ascribed to increased expression of fucosyltransferases (Fut8s), mainly Fut8 activity [79–81], to elevated GDP-Fuc, which is a common donor substrate for fucosylation [82], and to a high expression of transporter [77].
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Table 9.3 Inhibitory potencies (related to Fuc = 1) of RSL, as compared to PA-IIL, as attained with diverse fucosyl-bearing oligosaccharides measured by SPR with BIAcore at 25°C [10] Inhibitory potency The saccharide examined RSL PA-IIL 9.9 0.1 Fuca1-2(Galb1-3)Gal (BH) 8.2 0.1 Fuca1-2(GalNAcb1-3)Gal (AH) Fuca1-6GlcNAclb-O-Me 6.8 Fuca1-2Galb1-4Glc (2¢Fuc-Lac) 5.2 0.6 Fuca1-2Gal (H disaccharide) 3.4 0.9 Fuca-O-Me 3.2 2.0 Fuca1-2Galb1-4GlcNAc (H type 2) 2.5 p-NitrophenylaFuc 2.4 0.8 Fucose 1.0 1.0 Fuca1-4(Galb1-3)GlcNAc (Le a) 0.55 2.8 p-NitrophenylbFuc 0.24 0.15 l-Gal 0.23 0.4 b-d-Ara-O-Me 0.16 Fuca1-3(Galb1-4)GlcNAc (Lex) 0.14 0.2 Fuca1-3Galb1-4Glc (3¢Fuc-Lac) 0.14 1.4 0.11 0.13 Fuca1-3(Neu5Aca2-3Galb1-4) GlcNAc (Sialyl Lex)
There are only a few lectins exhibiting core fucose specificity. AAL [83] and Lens culinaris (LCA) have often been used as carbohydrate probes for core fucose in gps. AAL is even commercially available for that purpose [73]. Fractionation of Fuc-containing oligosaccharides on Sepharose-immobilized AAL by Yamashita et al. [84] indicated its preferential binding to core a-fucosyl residue linked to the proximal GlcNAc moiety. Their results indicated that AAL-Sepharose is useful for separating mixtures of complex-type asparagine-linked sugar chains. Oligosaccharides with Fuc-a1-2Galb1-4GlcNac and Galb1-4(Fuca1-3) GlcNAc also interact with this lectin, but less strongly than the complex-type sugar chains with fucosylated core. Weaker interactions of the lectin are obtained with Galb13(Fuca1-4)GlcNAc (of lacto-N-fucopentaitol II), and almost no binding is found with Fuca1-2Galb1-3GlcNAc [84]. Wimmerova et al. [53] have also shown that AAL binds to a1-2 Fuc, a1-3 Fuc, and a1-4-fucosylated oligosaccharides. Another fungal lectin, of Rhizopus stolonifer, was also reported to preferentially bind to a1-6-fucosylated oligosaccharides [85], and recently, Matsumura et al. [86] described a related Fuc-specific lectin, AOL, from Aspergillus oryzae (a filamentous fungus) displaying 26% homology with AAL and profound preference for core fucose. Using SPR analysis, they showed that AOL exhibits strongest preference for the a1-6 Fuc-pyridylaminated saccharides compared to a1-2, a1-3, and a1-4. Comparison of AOL to AAL and LCA, by SPR, showed that AOL activity toward a1-2 (H antigen) versus a1-6 core Fuc was weaker than theirs, and therefore, they suggested that it is a better novel probe for core fucose. Furthermore, they showed that staining by AOL was not observed in cultures of embryo fibroblasts of a1-6 Fut8 knock-out mice, but was observed with AAL.
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Although not yet compared to either AOL or AAL in differential core fucosyl detection and cancer diagnosis, RSL seems to be of interest for those aims. RSL might also be useful for study of and separation between high- and low-corefucosylated human antibodies of immunoglobulin 1 (IgG1) type used for cancer therapy. Since high fucosylation hampers the cytotoxic activity of these antibodies, a new strategy for generating fully nonfucosylated therapeutic antibodies with enhanced cellular cytotoxicity as “next-generation therapeutic antibodies” – based on double knock-down of a1,6-Fut8 and GDP-Man 4,6-dehydratase (GMD) in the antibody-producing cells [87, 88] – was offered.
9.1.5 RS-IIL Interactions with Diverse Glycans The yield of RS-IIL is generally lower than that of the other bacterial lectins, and its hemagglutinating activity is delayed (takes several hours), while that induced by RSL is immediate. The order of its saccharide affinities, determined by hemagglutination inhibition, differs from the others in displaying preferential, very high (mM) sensitivity to mannose and fructose while lower to fucose: Man/ Fru > Fuc > l-Gal/Ara [5]. Its crystal structure in a complex with mannose is similar to that of PA-IIL with fucose. It also possesses a pair of calcium ions at the bindingsite but differs from PA-IIL in the three binding site (22-23-24) amino acids (which are A-A-N) (Fig. 9.2) [5, 43, 52]. Adam et al. [51], as already described, have shown that switching PA-IIL amino acid (−22−) from Ser to Ala eliminates its preferential fucose affinity [51].
9.1.6 CV-IIL Interactions with Diverse Glycans Comparison of CV genomic ORFs to those of RS and PA has revealed 17.4 and 9.61% similarity, respectively. The CV-IIL sequence exhibits 60% similarity to PA-IIL and 62% to RS-IIL [6, 7]. The subunit size of CV-IIL is ~11.86 versus 11.73 kDa (PA-IIL) and 11.60 kDa (RS-IIL), and it also holds two Ca2+ ions. The lectin is tetrameric. Hemagglutination inhibition of the native CV-IIL has shown the following decreasing affinity order: L-Gal > Fuc > D-Ara > Fru > Man [6]. ELL(S)A tests using the recombinant CV-IIL (produced in E. coli) have shown [7] that it displays unusual entropy-driven affinity toward fucose and mannose, resembling PA-IIL in the Fuc > Man affinity order (Table 9.4). The saccharide-binding thermodynamic ITC analyses of aMeFuc and aMeMan gave dissociation constants of 1.7 and 19 mM, and their ELL(S)A potency ratios were 2.4 and 0.18 versus Fuc (=1), respectively. Pokorna et al. [7] results have indicated higher sensitivity of CV-IIL to inhibitions by D-Ara, Man, and Fru in comparison to PA-IIL (Table 9.4). The relatively higher sensitivity of CV-IIL to mannose as related to PA-IIL was not
9 The Five Bacterial Lectins Table 9.4 ELL(S) A-determined CV-IIL inhibitory potency of some saccharides in comparison to PA-IIL [7]
169 Inhibitory potency CV-IIL PA-IIL 2.4 2.6 a-Fuc Me l-Fucose 1 1 d-Arabinose 0.55 0.09 aMan Me 0.18 ND d-Mannose 0.16 0.0043 0.13 0.0036 d-Fructose ND not determined Lectin sugar
observed in the hemagglutination inhibition results [6], possibly due to the limitations of the accuracy of this method. In the X-ray structures of CV-IIL crystal complexes with FucaMe and ManaMe, the three hydroxyls (O2, O3, and O4) of the sugar have been shown to directly participate in coordination with the Ca2+ to establish many hydrogen bonds with the amino acids.
9.2 Usage of PA Lectin-Inhibiting Glycans for Anti-adhesion Treatment of PA Infections Prevention of PA infections by blocking its lectin-dependent adhesion to host cells might be achieved by antilectin antibodies [89], by preventing lectin production [90], and by competing glycans that mimic those of the target cell receptors. The latter goal has led to a considerable advancement in the studies of bacterial lectin inhibition by diverse glycans and related compounds. In any case, it should be remembered that PA lectins are part of the pathogen-adhesion system, which also includes sialophilic [91] and hydrophobic adhesins [92]. Upgrading the inhibiting compounds might be achieved by their coupling with hydrophobic components. Moreover, to abrogate infection, it might be better if the curative mixture included agents that can abrogate the production of “quorum sensing” signals that induce lectin and other VIF production [90]. Diverse in vitro models have already been used for checking sugar effects on PA adherence to host cells. In 1989, Marcus et al. [93], who studied the adherence of PA cells to tracheal epithelium of hamsters, guinea pigs, and mice using a perfusedtrachea model, reported that the highest adherence of mucoid PA cells to guinea pig cells was best inhibited by GlcNAc, Gal, and N-acetylneuraminic acid (NANA). Wentworth et al. [94] showed that adhesions of PA-IL- and PA-IIL-coated PA cells to rabbit corneal epithelial cell cultures were inhibited by Gal and Man, respectively. Since no such activity was observed with uncoated bacteria, it has been suggested that auto- or externally induced lysis of part of the bacterial population leads to lectin release as a salvage mechanism [95], contributing to survival of the other part of the bacteria (those coated by the released salvage lectins), enabling their
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adherence one to the other and to the host cells. In the same year, Ramphal et al. [96] showed the adhesion of mucoid and nonmucoid strains of PA to type 1 (Galb1-3 GlcNAc) and type 2 (Galb1-4 GlcNAc) disaccharides. Several years later, Stepínska and Trafny [97] showed highest inhibition of PA adherence to collagen type I by a Gal-Man-NANA (5:5:1) mixture and to collagen type II by a Glc-Gal (1:1) mixture. In 1999, Scharfman et al. [66] used flow cytometry for a panel of fluoresceinlabeled polyacrylamide-based glycoconjugates to determine the best carbohydrate receptors for PA. The neoglycoconjugates included neutral, sialylated, or sulfated chains analogous to carbohydrate determinants found at the periphery of the respiratory mucins, Lea, Lex, Ley, and sialyl Lex, as well as Gala1-2Galb and others. The interaction of these compounds was measured using a nonpiliated PA strain. Their results indicated highest affinity to the sialyl-Lex determinant. In 2000, King et al. [98], who determined the inhibition of PA adhesion to equine endometrial cells in vitro, found that mannose and GalNAc inhibited it while other sugars did not affect the bacterial adherence. They concluded that, in horses with uterine infections, the use of sugars to competitively displace bacteria from attachment sites on cells may provide an effective adjunct to antibiotic treatment.
9.3 Interactions of PA Lectins with Autologous and Heterologous Microorganisms The PA lectins were shown to interact with diverse microbial cells, including their own, as well as heterologous bacteria: E. coli strains, marine luminous bacteria, rhizobia, and archaea.
9.3.1 Interactions of PA Lectins with Autologous Bacterial Cells Interaction of the PA lectins PA-IL and PA-IIL with autologous bacterial cells under stress conditions was already described in 1989 by Sheffi et al. [18] and in 1991 by Wentworth et al. [94]. This phenomenon could be ascribed to either “in–out” shift or to the salvage mechanism suggested by Doyle and Koch [95], in which lectin release from lysed bacteria covers the other ones for their survival. During the past 4 years, dependence of PA biofilm formation on PA-IL and PA-IIL has also been shown by others in context with quorum sensing stress using advanced molecular biology and bioinformation techniques [55, 56]. Prevention of PA-IL and PA-IIL interactions with autologous cells might be the reason for their initial low distribution on the bacterial surface (despite their rich internal stock) and the increase of their surface levels only under stress conditions [18].
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9.3.2 Interactions of PA Lectins with Heterologous Bacteria Interactions of PA-IL and PA-IIL with various heterologous bacteria, including special strains of E. coli (O86B6 and O128B12, respectively) [23, 24] (Fig. 9.5a), marine luminous bacteria [99], and rhizobia [100], were described even before the autologous interactions. PA lectin coating increases E. coli phagocytosis by human leukocytes (Fig. 9.5b, c) [25] and augments Rhizobium adhesion to root hairs of legume plant Phaseolus lathyroides, leading to increased nodule formation [100]. The interactions of PA lectins with bacteria have been applied for their typing and for identification of enteropathogenic E. coli strains (E. coli O86 and O128) bearing epitopes that cross-react with human blood group (B and H) antigens [24]. Later on, PA-IL was used together with AGL, based on their increased interactions with malignant cells [34], for screening low-pathogenic E. coli strains bearing cancer-like antigens. The idea was that injection of such bacteria might increase patient resistance to cancer due to crossantigenic reactivity and that the antiserum against these bacteria might be helpful for cancer diagnosis, as suggested by Springer in 1997 [101]. The results obtained with the isolated bacteria indicated potential fulfillment of the second goal [102].
9.3.3 Interactions of PA Lectins with Archaea At the end of the twentieth century, for the first time in history, archaeal cell membranes were studied using lectins [103]. The PA lectins were included in the lectin battery used for the determination of the archaeal cell membrane saccharides and for their differentiation and typing (Fig. 9.6). Among the halophilic archaeal cells examined were Halobacterium salinarum strains (halobium and salinrium), Haloferax volcanii and mediterranei, and Haloarcula marismortui and vallismortis, from the collection of Professor A. Oren [103]. The reason why lectins were not previously used for the study of halophilic archaea was the dependence of these organisms on high salt concentration (30% NaCl) for their survival. At such high salinity, no interaction between cells and antibodies (for cell typing), conventional glycosidases (for glycomic characterization), or lectins (for both typing and saccharide detection) takes place. The trick used for overcoming this obstacle was very
Fig. 9.5 Electronic microscope photos of E. coli O86 B6 cell agglutination by PA-IL (a) [23, 24] and their phagocytosis by peripheral human leukocytes in the lectin presence (b) and absence (c) [25]
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ARCHAEA Fig. 9.6 Application of PA lectins for differentiation between and for typing of archaea, showing PA-IL preferential interaction with Haloferax mediterranei (IV) and PA-IIL preferential interaction with Haloarcula marismortui (V). The other halobacteria examined included two strains of Halobacterium salinarum, halobium (I), and salinrium (II), Haloferax volcanii (III), and H. vallismortis (VI). The interactions of the halobacteria with these and with additional lectins were described by Gilboa-Garber et al. [103]
short exposure of thrice-washed archaea (in their high salinity) to the lectins in a medium containing 16–20% NaCl concentration. Those nonoptimal conditions for both components enabled very short archaeal cell existence and a reliable (although suboptimal) lectin binding to their surface saccharides. Using this technique, it was shown that the H. salinarum cell envelope contains galacturonic acid (which reacts most strongly with AGL [103]). As seen in Fig. 9.6, PA-IL preferentially interacts with H. mediterranei and PA-IIL with H. marismortui.
9.3.4 PA Lectin Interactions with Diverse Free-Living Unicellular Organisms PA lectins agglutinated Euglena gracilis and Chlamydomonas reinhardi cells and stimulated their growth [104]. They were also shown to significantly increase the growth and phagocytic activity of Tetrahymena pyriformis, resembling ConA’s effect on it [105].
9.4 Interactions of the Five Bacterial Lectins with Diverse Animal Cells The five bacterial lectin interactions with RBCs from several mammals, with human RBCs of different blood types, human blood platelets, spermatozoa, and lymphocytes, as well as malignant cells from different sources, were followed by
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agglutination and adsorption assays. In addition, the effects of these lectins on human erythrocytes and lymphocytes were studied in vitro and on the malignant cells, both in vitro and in vivo.
9.4.1 Interactions of the Five Bacterial Lectins with Diverse Animal RBCs The five bacterial lectins have been shown to agglutinate most of the human (hundreds) and diverse animal RBCs examined. Generally, agglutination by all of them is much stronger following treatment of the RBCs by papain or by neuraminidase. Neuraminidase removes sialic acid with its repulsive negative charges, and papain abolishes the protein steric hindrance that disturbs lectin binding to glycolipid receptors. Comparison of the agglutination of human, cow, rabbit, rat, and sheep RBCs by the five bacterial lectins is presented in Fig. 9.7. This figure shows that
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all five lectins strongly agglutinate human, rat, and sheep erythrocytes, while varying in their interactions with cow and rabbit RBCs; PA-IL agglutinates both cell types, although somewhat less than the human and rat cells. PA-IIL does not agglutinate the cow RBCs but agglutinates the rabbit cells, in addition to the cells of the other three animals. RSL-IIL behavior is similar to that of PA-IIL, while RSL exhibits low activity toward the cow cells, and CV-IIL differs from the other four in not agglutinating the rabbit in addition to the cow RBCs (Fig. 9.7). PA-IL was shown to augment human RBC osmotic hemolysis [106].
9.4.2 PA Lectin Interaction with Human Spermatozoa Both PA-IL and PA-IIL agglutinate human sperm cells. Controls in the presence of the lectin-inhibiting sugars, where no agglutination is observed, prove that agglutination is sugar dependent. Agglutination of spermatozoa has been found to be selective for living motile cells and may be used for separation between vital and nonvital cells [107] (Fig. 9.8).
9.4.3 PA Lectin Binding to Human Blood Platelets PA lectins strongly agglutinate human blood platelets. Peroxidase-labeled lectins enable demonstration of the distribution of lectin-binding receptors on them by means of electron microscopy (Fig. 9.9).
Fig. 9.8 Human sperm cells in absence (a) and presence of PA lectins, leading to their agglutination (b)
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Fig. 9.9 Binding of PA lectins to human blood platelets (thrombocytes), detectable by their agglutination (a) and by peroxidase-labeled lectin binding (b), as seen in electron microscopy
9.4.4 PA Lectin Interactions with Human Peripheral Leukocytes Soon after PA-IL discovery, its interaction with human peripheral leukocytes was revealed. PA-IL at 50 mg/ml concentration induced lymphocyte vacuolization [108], but at 5 mg/ml, it induced mitogenic stimulation of human T lymphocytes [30] with disappearance of their microvilli [54]. PA-IL was also found to reduce phagocytosis of bacteria by neutrophiles that were pretreated with it [25]. PA-IIL at low concentrations was also found to be mitogenic for human peripheral lymphocytes and murine splenocytes [31]. The mitogenic stimulations by both PA lectins were inhibitable by their respective specific sugars. Use of the bacterial lectins for stimulation of lymphocytes in pretreated patients with cancer revealed significantly lower reactions than in those of healthy individuals. The difference between the healthy and patient groups was much more significant than that obtained using the standard plant lectin Phaseolus vulgaris (PHA) (Fig. 9.10). Hence, the PA lectins may be used as plant lectins to assay the stimulation of peripheral lymphocytes from patients with cancer for evaluation of their immunocompetence and for diagnostic and prognostic purposes [109].
9.4.5 PA Lectin Interactions with Malignant Tumor Cells In addition to the above-described efficient application of bacterial lectins for cancer diagnosis in patients based on their lymphocyte reduced response to lectin-induced mitogenic stimulation, the bacterial lectins have also been shown to be useful for direct experimental studies with transformed cells themselves. Since tumor cells
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Fig. 9.10 Interactions of PA lectins with human peripheral lymphocytes leading to their mitogenic stimulation, analyzed microscopically (left) and by 3H-thymidine incorporation (right). The latter test was used for lymphocytes from healthy people (H) and from patients with cancer (C) exposed to PA-IL (a) and PA-IIL (b)
often display surface saccharide variations, lectins might be useful tools for the detection of these and for the reduction of the transformed cell tumorigenicity. The effects of PA lectins were studied on cancer cells of murine AKR lymphoma and Lewis lung carcinoma (3LL). The AKR lymphoma included two variants that induce tumors in inbred AKR/Cu mice. One of them (TAU-39) is associated with large subcutaneous low malignant (LM) tumors, almost without metastases in the lymph nodes, and the other (TAU 38) is highly malignant (HM). The 3LL induces tumors in C57B1/6J mice. The PA lectin interactions with these malignant cells were compared to those with normal murine splenocytes and lung cells, respectively [110]. PA-IL and PA-IIL at 0.2–1 mg/ml concentration (like the plant lectins ConA and PHA) agglutinate tumor cells more strongly than the respective normal ones without discriminating between the LM and HM variants [110]. The agglutination is inhibitable by 0.15 M Gal and Man, respectively (Fig. 9.11). Examination of the PA lectin effects on in vitro 3LL cell viability showed that PA-IL exerted significant dose-dependent viability inhibition in 48-h cultured cells assayed by crystal violet vital staining. Fifty percent cytotoxic effect at 4 mg/0.2 ml concentration was observed with this lectin. PA-IIL cytotoxicity under the same conditions was very low. The in vitro examination of the lectin effects on tumor cell proliferation (assayed by 3H-thymidine incorporation test) revealed that the LM cells exhibited a very high sensitivity to ConA, PA-IL, and PHA, but low sensitivity to PA-IIL. The HM cells were also highly sensitive to ConA and somewhat less sensitive to PHA and the two PA lectins. Their sensitivity to PA-IIL was higher than that of the LM cells (Table 9.5). The 3LL cells were most sensitive to PA-IL and ConA. The PA-IL cytotoxicity was both dose and time dependent, and inhibitable by addition of 0.12 M methyl-a-D-Gal to the culture medium [34, 111] (Table 9.5). The lectin effects on cancer cells exhibited in vitro were also expressed in vivo, affecting primary tumor incidence and average size, as well as appearance of metastatic tumors, their size, and rate of development. Subcutaneous inoculation of
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Fig. 9.11 Agglutinations of LM murine TAU-39 and HM TAU-38 AKR lymphoma cell variants by PA-IL. 1 = nontreated; 2 = treated by PA-IL (20 mg/106 cells/0.2 ml); 3 = treated by PA-IL in the presence of 0.15 M galactose [34, 110] Table 9.5 Cytotoxicity (%) of the PA lectins for the AKR (LM and HM) lymphoma and 3LL cells exhibited by 3H-thymidine incorporation into AKR cells grown for 72 h and 3LL cells grown for 24 h [110–112] ConA Tumor type PA-IL PA-IIL PHA 95 99 LM AKR 98 29 HM AKR 60 63 81 98 9 55 3LL 95 12
untreated and lectin-pretreated (75 mg/0.2 ml for 90 min) AKR lymphoma cells into AKR mice revealed that pretreatments by PA lectins were less effective than by PHA and ConA for depressing the LM variant primary tumor incidence and size, and elongating the animal life span [110]. Similar pretreatment of the 3LL cells by PA-IL exhibited the most potent antitumoral effects (abolished by addition of Gal to the mixture), fully protecting the mice from mortality, followed by ConA with 20% and PHA with 80% mortality while PA-IIL was inactive (100% mortality). The in vivo PA-IL-induced reduced tumorigenicity was associated with the preservation of tumor cell immunogenicity [110–113]. Interestingly, PA-IIL, which did not display direct toxic effects on 3LL cells in vitro, nor did it provide any protection in vivo, was found to exert (at a concentration of 15 mg/ml) a profound in vivo inhibition of tumor cell growth in the presence of murine splenocytes [111, 112]. This cytolysis was completely inhibited by including 0.05 M Fuc in the culture medium. The same splenocytes, in the absence of PA-IIL, did not directly inhibit 3LL cell growth [111]. This phenomenon of indirect
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Fig. 9.12 Incubation for 24 h of human ovarian IGROV-1 cancer cell culture without (a) and with (b) PA-IL (10 mg/ml) led to increased cytoplasmic vacuolization and chromatin condensation, ending in necrotic cell morphology with organelle decomposition [34]
effects on cells – subduing them to cytotoxic effects of immune cells by either coating the target cells or by activation of the immune cells – resembles the PA-lectindependent (opsonic) increase of E. coli phagocytosis by human granulocytes. Examination of the binding of 125I-labeled PA lectins to human ovarian IGROV-1 and SKOV-3 carcinoma cells and to oral epidermoid KB and breast SKBr-3 carcinoma cells, grown in vitro in monolayers at 37°C for 24 h, revealed higher binding of PA-IL to the first three tumors and higher binding of PA-IIL to the last one (Table 9.6). Exposure of human ovarian IGROV-1 cancer cell culture to PA-IL (10 mg/ml) for 24 h led to increased cytoplasmic vacuolization and chromatin condensation, ending in necrotic cell morphology with organelle decomposition (Fig. 9.12).
9.4.6 PA Lectin Interactions with Diverse Animal Tissues In Vitro and In Vivo In 1991, Wentworth et al. [94] showed adhesions of PA-IL- and PA-IIL-coated PA cells to rabbit corneal epithelial culture cells and their inhibition by Gal and Man, respectively. In 1994, Grant et al. [114] demonstrated the in vitro effects of PA-IL on rat gut cell (enterocyte) structure, metabolism, and function, and also, later on, its in vivo
9 The Five Bacterial Lectins Table 9.7 Effects of orally administered growth and polyamine levels [115] Rat gut Control Small intestine weight 644 ± 48 Protein (mg) 405 ± 19 Putrescine (nmol) 403 ± 66 Cadaverine (nmol) 46 ± 12 Spermidine (nmol) 2,047 ± 67 Spermine (nmol) 1,607 ± 71
179 PA-IL and PHA on rat gut PA-IL 831 ± 33 489 ± 19 1,043 ± 86 196 ± 60 2,569 ± 52 1,814 ± 75
PHA 842 ± 34 488 ± 22 1,034 ± 66 307 ± 141 2,576 ± 26 1,799 ± 58
effects on the rat gut [115]. The results of these studies demonstrated that orally ingested PA-IL induced an increase in gut cell growth and polyamine accumulation, resembling the well-known effects of nonboiled leguminous seed lectins, represented in this study by that of PHA (Table 9.7). The findings presented in Table 9.7 show that purified PA-IL, like PHA, radically alters gut cellular metabolism, exhibited in increased polyamine levels, and induces gut hypertrophy. More recently, Laughlin et al. [116] also showed that PA-IL interacts with gut cells. They reported that it plays a key role in experimental gut-derived sepsis [116]. Concomitantly, PA-lectin effects on human endothelial and respiratory cells were studied by Plotkowski et al. [117] and by Bajolet-Laudinat et al. [118], who reported that PA-IL is cytotoxic for respiratory epithelium ciliated cells in primary cultures of nasal polyps in vitro, leading to a decrease in active ciliated cell surface and to the appearance of epithelial cell cytoplasmic vacuoles. Later on, Adam et al. [119] showed similar effects using PA-IIL. In 1997, Gilboa-Garber et al. [34] examined the in vivo differential binding of the two PA lectins to murine tissues following injection of 125I-labeled lectins. Examination of the labeled-lectin distribution in the mouse isolated tissues showed that, in general, PA-IIL binding levels were higher than those of PA-IL. The two lectins varied in regard to their preferential tissue affinity order: PA-IL showed highest binding to mouse lungs equal to that of PA-IIL to lungs and liver, but PA-IIL most strongly bound to the murine spleen and kidney (Fig. 9.13). In 1999, Kirkeby and Hoyer [120] showed selective PA-IL binding to murine skeletal muscle fibers, and several years later, they demonstrated the lectin binding to sections of wild-type and knock-out mice lacking a1,3-galactosyltransferase [121]. In that study, the wild-type mice lectin histochemistry showed a strong capillary reaction in the heart, kidney, and adrenal gland, but not in the pancreas. No such binding was found in the mutant mice. These studies were followed by other demonstrations of PA-IL- and PA-IIL-induced apoptosis in human endothelial cells [122] and immobilization of human airway ciliary beats, inhibitable by Gal and Fuc, respectively [123]. Concomitantly, Stoykova et al. [124] described PA-IIL binding to cystic fibrosis airway cells and its inhibition by fucosylated compounds, adding the suggestion to implicate these results for PA infection therapy [124]. Recently, Kirkeby et al. [125] used advanced, discriminating, histochemical techniques for the examination of PA-IL and PA-IIL bindings to sections from lungs and
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pancreas of minks, which are known to develop spontaneous PA respiratory infections. Their results indicated in situ binding of both lectins to the lung seromucinous glands that are located in the submucosa of the larger bronchi. In addition, PA-IL reacted with the capillaries in the alveolar walls and with the small blood vessels forming the vasa vasorum around the larger vessels while PA-IIL marked the goblet cells in the bronchial surface epithelium. In the pancreas, both lectins bound to the epithelium in the excretory ducts. In addition, PA-IL strongly stained the pancreatic capillaries while PA-IIL stained the apical part of acinar cells in the exocrine part of the gland, but no lectin reaction was recorded in the endocrine cells [125].
9.5 Specificity of the Five Bacterial Lectins to Human RBC Antigens One of the oldest and most common lectin applications is human blood group typing. Since the bacterial lectins bind to galactose (PA-IL) and fucose (the other four), their interactions with human RBC antigens B, I, T, P1, and Pk, all of them bearing terminal galactose, and H that bears fucose (Fig. 9.14), were examined [126–130].
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9.5.1 PA-IL Interactions with Human RBC Antigens PA-IL has been found to agglutinate adult human RBCs that bear B epitope (terminating in Gala1-3Gal) more strongly than A and O(H) type cells [126] (Fig. 9.15). It also strongly reacts with B-negative, A-, and O-type human adult RBCs (Figs. 9.15 and 9.16) due to its high affinities to P system antigens, especially the very common P1 and the rare P precursor Pk (that bear Gala1-4Gal) [126]. Its agglutination of P-type (lacking P1 and P antigens) RBCs of A and O types is very weak. Therefore, PA-IL may be regarded as a reliable reagent for differentiation between P-positive (including P1, Pk, and P antigens) versus P-negative (p) RBCs [127, 128, 131]. The strong binding of PA-IL to the B- and P-system antigens depends on their combination with the adult I antigen [132, 133] (Fig. 9.17), which, unlike the fetal-specific linear polyLacNAc (Gala1-4GlcNAc) I antigen, is a branched poly LacNAc (Fig. 9.14). It differentiates between I-positive adult and I-negative (fetal and very rare ii adult) erythrocytes [127, 132, 133]. The interactions of PA-IL with those antigens that are present not only on RBCs, but on all human cells, imply that it might contribute to the strong binding of PA to all human adult organs, cells, and tissues. PA-IL interaction with T antigen was found to be relatively weak, much weaker than those of PNA and AGL. Its much stronger agglutination of sialidase-treated (compared to untreated) RBCs is mainly due to the removal of sialic acid repulsing charges rather than the unmasking of the subterminal T-antigen Galb1-3GalNAc, as a ligand.
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9.5.2 PA-IL Interactions with Human H-Deficient Bombay- and Para-Bombay-Type RBCs As seen in Figs. 9.15 and 9.16, PA-IL (and also AGL) most strongly agglutinates the rare H-deficient Bombay- and para-Bombay-type RBCs [129, 130]. The H antigens are formed by b-galactoside a1-2 fucosyltransferases Fut1 and Fut2, which are encoded by the H and Se structural genes, respectively. The production of the
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Fig. 9.17 Agglutinations of human adult (I-positive) and fetal RBCs (cord erythrocytes, which are mainly I-negative [ii]) by the five bacterial lectins
H type-2 antigen on human RBCs, vascular endothelium, and other cells of mesodermal origin is mainly catalyzed by Fut1, while Fut2 is expressed in tissues of endodermal origin, including the exocrine system, contributing to the presence of type 1 and other H types in secretors (80% of the white population). The very rare Bombay and paraBombay phenotypes issue from mutations affecting either both Fut1 and Fut2 or production of their substrates or coenzymes. The Bombay phenotype is defined by total lack of H epitopes while in para-Bombay, there may be detectable heterogeneous traces of H epitopes on the RBCs due to very low transferase activities. UEA-I, which is specific for H type 2, is most widely used for the detection of the H epitope on human O(H)-type RBCs and in secretors’ secretions. The increased agglutinations of the Bombay and para-Bombay RBCs by PA-IL might be ascribed to its binding to the H-precursor terminal Galb1-4GlcNAc residue in combination with I antigen and to increased levels of the P-system and I antigens, instead of the missing H, by compensatory use of the unfucosylated H precursor for their crossing pathways [129].
9.5.3 Low-Temperature-Favored PA-IL Interaction with the Human RBC I Antigen Blood bank examinations of I-antigen interactions with human anti-I antibodies are performed at low temperatures. Checking for auto-anti-I antibodies is also carried on in a refrigerator while most other RBC–antigen interactions with their respective antibodies take place at room temperature or 37°C. Therefore, the temperature effect was also examined in regard to PA-IL interaction with the I antigen. Interestingly, PA-IL and AGL agglutinations of the I-positive RBCs, like those of the anti-I antibodies, were augmented at low temperatures, as opposed to the four other bacterial lectin interactions with their receptors [134, 135].
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9.5.4 Interactions of the Four Fucose- and Mannose-binding Bacterial Lectins (PA-IIL, RSL, RS-IIL, and CV-IIL) with Human RBC Antigens As may be seen in Figs. 9.15 and 9.16, the four fucose- and mannose-binding bacterial lectins vary in their relative preferential interactions with the ABH blood group antigens. PA-IIL and CV-IIL resemble UEA-I in sharply differentiating between H-positive (of O, A, B, and AB types) and H-deficient (Bombay and paraBombay) RBCs, differing from it in lower sensitivities to the H fucosyl-masking by adjacent galactose (of B) and GalNAc (of A). While UEA-I agglutinates O(H) RBCs much more strongly than the A and B type cells, their agglutination of the A and B cells is only mildly reduced (Fig. 9.15). RS-IIL exhibits much lower H preference, as shown in somewhat weaker Oh-cell agglutination, as could be anticipated regarding its Man > Fuc affinity. RSL behavior is totally different from those of the other three, being similar to PA-IL in Oh > O(H) preference; its hemagglutination profile does not show any H over Oh preference despite its fucophilicity [5] and Fuca1-2 affinity [10]. It seems to very strongly bind to another ligand, undetected by PA-IIL and CV-IIL, which is elevated in the H-deficient RBCs in compensation for the absence of the H antigen and the associated elevated level of available GDPFuc. The attractive ligand unique for RSL (being still an enigma for us) might be related to the a1-6 core fucosylation type, which is RSL’s most favored ligand. Figure 9.17 demonstrates that, unlike PA-IL, the Fuc/Man-binding bacterial lectins are not supported by the I-antigen presence for agglutination of human RBCs. The RS lectins even enjoy its absence as if its presence reduces the levels of their cell receptors or their contacts with them.
9.6 Blocking of the Five Bacterial Lectins by Human Body Fluid SRMDGs Following demonstration of the five bacterial lectin interactions with antigenic receptors of diverse animal and human cells, these lectins have been used as probes for discovering the presence – in the fluids surrounding the cells – of soluble protective glycoconjugates that might function as SRMDGs for blocking lectin-dependent bacterial adherence to their target cells [34, 62]. Animal body fluids protect tissues and sensitive immunologically immature embryos and newborns against microbial infections. This protection is provided by immune cells and their components, IgGs, and innate immunity systems encompassing glycoconjugates that act as pathogenattracting decoys blocking pathogen binding to the host cells. The best examples for the latter are human milk carbohydrates that have been shown to inhibit adherence of various bacteria and their toxins to target cells [63, 64, 136], including Streptococcus pneumoniae and Hemophilus influenzae [137], the heat-stable
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enterotoxin of E. coli [138, 139], enteropathogenic E. coli [140], and Campylobacter jejuni [141]. Human milk, saliva, and seminal fluid were chosen to represent protective human body fluids that interact with bacterial lectins as probes [142–145]: the first, due to its superb efficiency in providing highest infection-blocking service to newborns at a period of immature immunity subsequent to their detachment from the uterine (amniotic) immuno-sheltering; the second, due to its role in protecting the mouth, gums, teeth, pharynx, and larynx, and guarding the entrance through them to the respiratory and gastrointestinal systems; and the third, due to its role in protecting sperm cells, ensuring continuation of the organism. These fluids contain diverse glycans at different complexity levels: beginning in monosaccharides (e.g., seminal fluid fructose), followed by disaccharides (e.g., milk lactose) and oligosaccharides (e.g., the fucosylated H, Lea, Leb, and Lex epitopes present in most human body fluids), and ending in high MW gps that bear branched oligosaccharides (e.g., lactoferrin, which is present in milk, seminal fluid, saliva, and serum). The hemagglutination inhibitions by dialyzed body fluid preparations are lower than those without dialysis due to the removal of the low MW glycans, including the seminal fluid fructose (which inhibits PA-IIL, RSL, RS-IIL, and CV-IIL), milk lactose (which inhibits PA-IL), and fucosylated oligosaccharides of H and Lewis types (inhibiting the fucophilic lectins), but retaining the gps that are detectable in the Western blots. In addition to human body fluids, the five bacterial lectins were also used for the analyses of various kinds of mammal milk, avian egg whites (which are supposed to protect their embryos), and beehive products (royal jelly [RJ] and honey, which might similarly protect the new hatching bee larvae and queen). The interactions of the lectins with these glycans were examined using hemagglutination inhibition and Western blot analyses.
9.6.1 Interactions of the Galactophilic PA-IL with Human Milk, Saliva, and Seminal Fluid PA-IL interactions with human body fluids were examined (by hemagglutination inhibition and Western blotting) as compared to several other galactophilic lectins of plants (ECorL, MPL, and PNA) and AGL (Fig. 9.18). As may be seen in this figure, PA-IL interactions with the human body fluids are generally weaker than those of the wide spectrum galactophilic MPL and AGL lectins. It mainly interacts with the milk, like the limited spectrum PNA and ECorL. Saliva [142] and seminal fluid samples [144] inhibit it very weakly, excluding those of type AB secretors, which contain branched structures bearing both the A and B antigens [142]. The relevant Western blots show that PA-IL only binds to relatively few gps (Figs. 9.18 and 9.20); it does not stain saliva gps but binds to several milk and seminal fluid gps, which differ from those attracting PNA. The PA-IL-stained bands are included among those stained by ECorL, AGL, and MPL [143, 144] (Fig. 9.18).
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9.6.2 Interactions of the Fucophilic and Mannophilic Bacterial Lectins with Human Milk, Saliva, and Seminal Fluid The four fucophilic and mannophilic bacterial lectins PA-IIL, RSL, RS-IIL, and CV-IIL, as well as the plant lectins ConA and UEA-I, have been found to be strongly inhibited by human body fluids, differing in their preferential affinities to them (Figs. 9.19 and 9.20). PA-IIL, RSL, and ConA exhibit highest affinity to milk, followed by seminal fluid, and lowest affinity to saliva. ConA was already shown earlier to differ from them in highest inhibition by human serum, followed by milk [144]. As seen in Fig. 9.19, comparison of these three lectins shows that PA-IIL exhibits outstandingly high sensitivity to human milk [62], probably due to its richness in Lewis epitopes [9, 146] while RSL is more sensitive to it than human seminal fluid (almost equalling its own milk sensitivity) and saliva. RS-IIL, CV-IIL, and – even more so – UEA-I, differ from them in preference of seminal fluid over milk. UEA-I (which interacts only with samples obtained from secretors) and RS-IIL exhibit highest affinity to seminal fluid while CV-IIL is outstanding in its highest sensitivity to saliva. The latter is the only bacterial lectin from the examined series that is more sensitive to inhibition by saliva than by milk, and its saliva sensitivity matches its seminal fluid sensitivity (Figs. 9.19 and 9.20).
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Fig. 9.19 Inhibitions of the five bacterial lectins and two plant lectins (the mannophilic ConA and fucophilic UEA-I) by human (secretors’) milk, saliva, and seminal fluid [62, 143–145]
Fig. 9.20 Comparison of the Western blots of human milk (M), seminal fluid (SF), and saliva (Sa) from secretors (Se, +) and nonsecretors (sese, −) stained by the five bacterial and the two additional plant lectins (ConA and UEA-I) labeled by horseradish peroxidase [144] and the notyet-published results of K.D. Zinger-Yosovich’s PhD thesis at Bar-Ilan University
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9.6.3 The Ability of the Five Bacterial Lectins to Differentiate Between Secretors’ and Nonsecretors’ Body Fluids Secretors (individuals possessing Se structural gene [Se], encoding the enzyme GDP-Fuc-dependent Fut 2, that catalyzes the production of H blood group epitopes and their secretion to the body fluids, in contrast to nonsecretors [sese]) display the presence of H-type epitopes in their body fluids; their soluble H type-2 epitope is best detectable by UEA-I. This plant lectin is widely used in blood banks and research for differentiation between Se and sese based on its inhibition (or absence of inhibition) by their saliva. ConA – which is widely used for the detection of terminally mannosylated/glucosylated compounds and gps that bear N-glycans, containing a subterminal trimannosyl forking – does not differentiate between the two populations. These two lectins have been used for comparison along with the bacterial lectins in their investigation. Examination of the ability of the five bacterial lectins to differentiate between body fluids of Se and sese has revealed that PA-IL displays a light Se preference when AB saliva is examined [142]; otherwise, its interactions with saliva are negligible. PA-IIL does not differentiate between Se and sese saliva and seminal fluids, which contain Lewis epitopes [147], but is more strongly inhibited by sese than by Se milk [148], probably due to its preference of Lea over Leb [9, 44]. RSL is similarly inhibited by Se and sese milk, seminal fluid, and saliva. RS-IIL is also similarly inhibited by Se and sese saliva and seminal fluid, but somewhat more sensitive to Se than to sese milk. CV-IIL, in contrast to them, exhibits a significantly higher sensitivity to Se body fluids, being only weakly inhibited by the sese body fluids [145] (Fig. 9.20). The Western blots of the described lectin interactions with the Se and sese body fluids are shown in Fig. 9.20.
9.7 Blocking of the Five Bacterial Lectins by SRMDGs of Diverse Kinds of Mammalian Milk 9.7.1 Comparison of the Inhibitions of the Five Bacterial Lectins, ConA, and UEA-I by Human and Cow (Bovine) Milk Following demonstration of the very high efficacy of human milk in blocking the five bacterial lectins, headed by PA-IIL [62, 143, 144], this milk was compared to cow milk, which is used not only by human adults, but also for feeding human infants, as a human milk substituent. As can be seen in Fig. 9.21a, cow milk is not efficient in blocking the five bacterial lectins. Similar results were also obtained in analyses of baby-feeding formulas based on cow milk [146]. PA-IIL has been found to be a powerful probe for differentiation between human and cow milk [146], which also may be used for quality control and as an indicator for improving the
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Fig. 9.21 Comparison of the inhibitions of the five bacterial lectins, ConA, and UEA-I by human and cow milks (a) and by their lactoferrins (b) whose structures, as described by Eda et al. [149] and others, are presented in (c) [146, 149, 150]
milk formulas. UEA-IL reacts only with human milk but not with all of them, being selective for Se, and not reacting with cow milk. The respective lactoferrins at a high concentration (50 mg/mL) inhibit only the fucophilic and mannophilic bacterial lectins and ConA, but not PA-IL (Fig. 9.21b). The Fuc > Man PA-IIL, RSL, and CV-IIL are inhibited by human lactoferrin considerably more strongly than by bovine lactoferrin while the Man > Fuc RS-IIL is inhibited by bovine lactoferrins more strongly than by human ones, probably due to its very high sensitivity to their mannosylated N-glycans (Fig. 9.21c). ConA does not distinguish between them (Fig. 9.21b, c).
9.7.2 Interactions of the Five Bacterial Lectins and ConA with a Series of Diverse Kinds of Mammalian Milk Hemagglutination inhibition analyses of 11 different kinds of mammalian milk using ConA have shown that the different kinds of milk vary in their inhibitory activity and in the relative contribution of low MW (removable by dialysis) glycans versus high MW gps to the inhibition (Fig. 9.22). ConA is most strongly inhibited by alpaca and rabbit milk followed by cow > fallow deer > camel (human milk being a relatively weak ConA inhibitor). In human, alpaca, rabbit, mare, ewe (sheep), and dog milk, the relative part played by the low MW compounds in ConA inhibition is minute while in cow, fallow deer, camel, buffalo, and goat milk, it is high. Namely, free Man/Glc/Fru monosaccharides or oligosaccharides are present in the first group
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Fig. 9.22 Inhibition of ConA by 11 kinds of mammalian dialyzed and undialyzed milk (presented in an order of decreasing intensity) and the related Western blots stained by peroxidase-labeled ConA [150] Table 9.8 The mean concentrations of protein and lactose in diverse kinds of mammalian milk Milk Human Alpaca Rabbit Camel Cow Mare Buffalo Goat Dog Ewe Proteins (%) 1.23 6.7 9.5 3.7 3.11 2.2 4.3 3.11 6.8 5.4 Lactose (%) 6.94 4.8 1.12 4.1 4.7 6 4.9 4.26 4 6.7
in lower concentrations than in the second group. This state may be seen in the respective Western blots (Fig. 9.22), showing heaviest staining by peroxidase-labeled ConA of the alpaca and rabbit milk gps, and stronger staining of human than cow milk gps, despite higher inhibition of ConA by the undialyzed cow milk. This shows that the contribution of gps glycans in human milk is higher than in cow milk. The same phenomenon is also observed using the five bacterial lectins, but their interaction profiles are different. Human milk is their best inhibitor, displaying highest affinity to PA-IIL. PA-IL is most strongly inhibited by human milk, followed by milk of alpaca, mare, buffalo, and monkey, and moderately by milk of cow, ewe (sheep), goat, camel, and fallow deer. Only rabbit milk does not inhibit it. PA-IL inhibition by most milk is mainly due to low MW saccharides, mainly lactose (Table 9.8), that are removable by dialysis. As can be seen in Fig. 9.23, in most kinds of animal milk, including mare, buffalo, cow, ewe, goat, camel, and
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Fig. 9.23 Inhibition of the five bacterial lectins by 11 kinds of mammalian dialyzed and undialyzed milk (presented in an order of decreasing intensity) and related Western blots (of the most active ones stained by the peroxidase-labeled lectins) [150]
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f allow deer, dialysis removes most of the PA-IL-inhibiting activity. The respective Western blots show clear staining by PA-IL of human, alpaca, and mare milk (which are also stained by PNA) but not buffalo milk, which loses almost all its PA-IL-inhibiting activity by dialysis. Interesting is the finding that rabbit milk, which considerably inhibits ConA (Fig. 9.22) and the four other bacterial lectins, especially RS-IIL (Fig. 9.23), does not inhibit PA-IL. This finding is compatible with the reports on very low lactose concentration in this milk (Table 9.8). As seen in Fig. 9.23, examination of the inhibitory activities of diverse kinds of milk with the four fucophilic and mannophilic bacterial lectins shows that human, alpaca, and rabbit milk, followed by camel milk, are generally the strongest inhibitors while cow, buffalo, deer, and mare are the weakest. PA-IIL is outstandingly sensitive to human milk compared to both the other kinds of milk and the other lectins. It is also considerably sensitive to milk of alpaca, monkey, rabbit, and camel, less sensitive to milk of ewe, dog, and goat, and almost insensitive to milk of mare, buffalo, cow, and fallow deer. RSL differs from PA-IIL in being more sensitive to milk of goat, dog, and ewe than to that of alpaca and camel. RS-IIL (which displays Man > Fuc affinity), like ConA, is more strongly inhibited by rabbit than by human milk. CV-IIL, like PA-IIL, is most strongly inhibited by human milk, nicely blocked by camel, alpaca, rabbit, goat, ewe, monkey, dog, mare, fallow deer, and buffalo milk, but very weakly by cow milk. The camel inhibitory activity for it, as well as for most of the other lectins examined, is mainly associated with the low MW glycans (removable by dialysis) rather than the high MW gps. These results, which show the diverse bacterial lectin sensitivities to the different kinds of milk, support their usefulness for studies of milk glycans and their antibacterial protective value.
9.8 Interactions of the Five Bacterial Lectins and ConA with Avian Egg-White Glycans Avian egg whites also have a role in the nourishment of immature progeny and their protection from infections. Their embryo-surrounding fluids are rich in gps that might abrogate microbial infections [151]. Chicken, quail, and columbidae (dove/ pigeon) egg whites have been chosen to be examined using the five bacterial lectins compared to ConA, UEA-I, ECorL, and PNA. The chicken egg-white glycans are known to strongly react with wheat germ agglutinin and ConA, which are commercially used for their gps complex-type carbohydrate-chain purification [152–156]. Quail egg whites are rich in polymannosylated gps chains [157], and the pigeon egg whites are rich in gps that bear terminal aGal residues, which possess P1 blood group antigen activity [158–161]. The data presented in Figs. 9.24 and 9.25 show that chicken egg white considerably inhibits ConA and weakly inhibits RS-IIL, PA-IIL, and CV-IIL. RSL inhibition by it is negligible, and PA-IL (like PNA, ECorL, and UEA-IL) is not inhibited by it (Figs. 9.24 and 9.25) [57, 162–164]. Quail egg white is much more active than chicken egg white in inhibiting the
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Fig. 9.24 Decreasing orders of the chicken (C), quail (Q), and pigeon (P) egg-white inhibitory activities toward the five bacterial lectins and ConA, as analyzed by hemagglutination inhibition test [57, 162]. On the right are proposed representative structures of one major ovomucoid (OM, that constitutes around 55% of egg white proteins) glycan of each egg white: COM, according to Yamashita et al. [155]; QOM, according to Hase et al. [157]; and POM, according to Takahashi et al. [160]
uc- and Man-specific bacterial lectins due to the rich presence of mannose F residues on its gps. It inhibits ConA even more than the chicken egg white, but, in contrast to the latter, it also inhibits CV-IIL and RS-IIL and strongly inhibits PA-IIL. RSL inhibition by it is relatively weak while PA-IL (as well as ECorL, PNA, and UEA-IL) is not inhibited by it (Figs. 9.24 and 9.25) [57]. A totally different pattern is observed with the pigeon egg white, which, in contrast to the chicken and quail egg whites (which do not inhibit PA-IL), most strongly blocks PA-IL (but not ECorL, PNA, or UEA-IL) owing to its terminally galactosylated P1/Pk-type gps (Figs. 9.25 and 9.26) [57, 163]. The pigeon egg white also very strongly inhibits RS-IIL, PA-IIL, and CV-IIL (indicating the presence of additional gps that probably bear mannosylated ligands), more weakly ConA, and very weakly RSL (Figs. 9.24 and 9.25). The Western blots presented in Fig. 9.25 also show that the diverse avian egg whites differ in their bacterial lectin binding and that these lectins are most useful tools for their study. They disclose that PA-IL is unique in high specificity and very
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Fig. 9.25 Comparison of the interactions of the five bacterial lectins and ConA with chicken (C), quail (Q), and pigeon (P) egg whites, as analyzed by hemagglutination inhibition and Western blotting using the same lectins labeled by peroxidase [57, 162]
strong binding to the pigeon galactosylated gps, which display human blood group P1 antigenic activity [128], but not to the chicken and quail gps. They demonstrate that ConA’s strongest binding is to the quail, followed by the chicken gps, and the weakest binding is to the pigeon gps. The four Fuc- and Man-binding bacterial lectins bind to the quail and pigeon gps more strongly than to those of the chicken. The unique interaction of PA-IL with pigeon, but not with chicken or quail eggwhite gps, is nicely compatible with Suzuki et al.’s [167] results of a wide phylogenetic survey of many avian species for the gene encoding of the specific terminal a-Gal1-4 transferase activity, which showed the enzyme presence in pigeon but not in chicken.
9.9 Interactions of the Five Bacterial Lectins, ConA, and UEA-IL with Beehive Honey and RJ and with Flower Nectars Used by the Bees for Honey and RJ Production In the beehive, owing to the fascinating social behavior of the honeybees (Apis mellifera), the maternal contribution of feeding and protecting the new progeny is substituted by parental brood care. For up to 3 days, the larvae are supplied with RJ
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Fig. 9.26 Inhibition of the hemagglutinating activities of the five bacterial lectins and two plant lectins by dialyzed and undialyzed bee honey (from wild flowers) and RJ (from Kfar Habad, Israel). At the bottom of the figure, there is a structure of mannosylated N-glycan of major royal jelly proteins [MRJP]-I (apalbumin-I) according to Kimura et al. [165], representing the MRJP glycan structure, which is common to honey and RJ, but present in the latter in a much wider range than in the former (as seen in Fig. 9.27) [166]. Some of the results presented in this figure are part of K.D. Zinger-Yosovich’s PhD thesis at Bar-Ilan University
that is secreted by young worker bees (nurse bees) in the hive. Thereafter, only larvae designated to become queens receive RJ while a mixture of honey, pollen, and water is fed to larvae selected to become workers [168]. Honey is produced by worker honeybees from floral nectars, followed by water evaporation in the honeycomb. It is rich in carbohydrates (approximately 79%, including around 38% fructose [Fru], 31% glucose [Glc], 1% sucrose, and 9% other sugars), but its protein content is only around 0.7% [169–171]. RJ, which is produced by young nurse bees from honey and flower pollen mixture with
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s ecretions from their cephalic salivary glands [169], differs from honey in its higher water content (60–70%), 10–16% carbohydrates (some of honey origin), and much higher protein levels (around 14%), most of them gps [165, 172, 173], and 3–7% lipids as well as richer content of other vital components [174–177]. Beehive products have long been used for various therapeutic purposes. Over the past decade, there is a renewed usage of both honey and RJ for the treatment of infections due to the emergence of antibiotic-resistant bacteria. There have been many case reports and clinical trials proving their effectiveness against such bacteria, including PA [178], coagulase negative staphylococci [179], and others [180]. Honey’s antibacterial effects were mainly attributed to a combination of high osmolarity and bacteriocidal/static activities, including those of hydrogen peroxide, phenolic compounds, and antioxidants [178], as well as cytokine induction [171, 181, 182]. Recently, the blocking of the lectin adhesion was added to the list of the honey and RJ anti-infection effects [166]. PA-IL inhibitions by undialyzed honey and RJ are moderate, mostly exerted by low MW glycans that are almost fully removable by dialysis (Fig. 9.26) [166]. PA-IL’s very weak inhibition by dialyzed RJ and honey, and the staining of only several bands (between 40 and 55 kDa) in RJ Western blots and only the 55 kDa MRJP apalbumin-1 band in those of the honey (Fig. 9.27), show that there is very low galactosylation in these gps. That galactosylation might be of the b type (based on recent, not-yet-published results of K.D. Zinger-Yosovich’s PhD work using hemagglutination inhibition and Western blotting showing that PNA displays stronger and broader staining than PA-IL). PA-IIL inhibition by honey is the highest of all the lectins included in the study (resembling its inhibition by human milk), but like that of PA-IL, it is mainly associated with low MW components, being considerably reduced by dialysis of the honey and scant honey Western blot staining (the 55 kDa apalbumin-1 band) with additional gps around 60, 80, and 105 kDa gps (Fig. 9.27) [166]. RJ’s strong effect on PA-IIL is mainly associated with mannosylated gps [165], similar to those of the quail, which persist after dialysis [166]. The reason for the different effects of dialysis on the RJ and honey (Fig. 9.26) is the much higher concentration of gps in the RJ versus honey (14 versus 0.7%) combined with the honey’s much higher
Fig. 9.27 Comparative Western blotting of RJ (×200 dilution), honey (Hon; ×4 dilution), human milk (HM, undiluted, from O, Se+ volunteers), and bovine milk (BM, commercial cow milk) stained by the peroxidase-labeled five bacterial and two plant lectins, showing the royal jelly MRJP (apalbumins) distribution [166] and indicating (by an arrow) apalbumin-1 location in both RJ and honey. Some of these results are part of K.D. Zinger-Yosovich’s PhD thesis at Bar-Ilan University
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free-saccharide concentration (79%). This interpretation is confirmed in the Western blot results showing the relatively prosperous interactions of PA-IIL with the RJ gps (Fig. 9.27). ConA inhibition by the honey, which was close to that of PA-IIL, was less reduced by dialysis despite the fructose removal, owing to its interactions with several additional honey gps N-glycans, including the 55 kDa one [170, 173, 175]. Its tagging of additional gps between 36 and 105 kDa, observed in the more loaded Western blots, might be due to their terminal N-acetylglucosamine (GlcNAc) and internal Man residues. The concentration of such gps is about 11% of the RJ gps [165]. Only ConA binds to these gps [183], not PA-IIL. ConA Western blot results show relatively prosperous interactions of ConA with the RJ gps (Fig. 9.27). As seen in Fig. 9.27, ConA reacts not only with stronger avidity, but also with a much wider spectrum of gps than PA-IIL, which tags about 11 bands in the range of 20–105 kDa in the more highly loaded blot. Taking into account the dilutions used for the Western blotting (RJ × 200 versus honey × 4–6, and human milk, almost without dilution), the results indicate enormous affinities of PA-IIL and ConA to RJ gps. Kimura et al. [165], who have already described ConA’s reactions with RJ gps, characterized the structures of RJ N-glycans and showed that typical high-mannosetype (9Man) structure (Fig. 9.26, at the bottom) accounts for about 72% of them, followed by a biantennary structure (GlcNAc2 Man3 GlcNAc2) (about 8%), and a hybrid-type structure (GlcNAc1Man4GlcNAc2) (about 3%), all of them fitting to ConA specificity [183] and not containing fucosylated residues. Figures 9.26 and 9.27 show PA-IIL’s selective strong binding to and staining of part of the ConAstained MRJP bands. This binding is attributable to the highly mannosylated gps, resembling those of quail egg-white gps [57]. ConA also binds to around 11% additional gps that bear terminal GlcNAc to which PA-IIL does not bind. RSL is outstanding among the bacterial lectins; it is inhibited by undialyzed honey less than PA-IIL, but by dialyzed honey more than PA-IIL, like ConA. However, unlike both of them, it is inhibited by dialyzed honey gps considerably more strongly than by dialyzed RJ gps (Figs. 9.26 and 9.27). RS-IIL resembles ConA in its interactions with honey gps exhibited in both hemagglutination inhibition and Western blottings (staining around seven apalbumin bands between 35 and 80 kDa) (Fig. 9.27). The inhibitions of the mannophilic ConA and RS-IIL (Fig. 9.26) are associated with an interaction with a wider spectrum of MRJPs and additional RJ gps, at a range of 30–300 kDa (Fig. 9.27). CV-IIL’s behavior is between those of RS-IIL and PA-IIL. It is strongly inhibited by honey and RJ (Fig. 9.26), but displays a limited range of gps staining (resembling that of PA-IL, between 40 and 55 kDa), mainly staining the honey 55 kDa band and only three RJ bands (between 45 and 55 kDa), to which it probably binds more strongly than PA-IL does. UEA-I does not react at all with the honey or RJ components in either hemagglutination inhibition or Western blotting, even under overloading conditions, indicating that there are no a1-2 fucosylated oligosaccharides or gps in them. PA-IIL’s weak inhibition by honey gps also indicates that there are no relevant fucosylations in them.
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The interactions of the bacterial lectins with RJ show its relative richness in inhibitory gps glycans, which are retained in the dialyzed preparations. RSL deviates from the other bacterial lectins in being the only lectin that displays weaker reactions with RJ than honey gps. Taken together, it may be stated that RJ and honey are very efficient in blocking the bacterial lectins. Their anti-adhesion activity is mainly associated with honey high fructose and RJ mannosylated gps (mainly apalbumins), that act as decoys resembling the human milk glycans. The critical comparison of ConA, UAE-I, and the bacterial lectin interactions with honey and RJ to those with bovine and human milk using Western blotting (Fig. 9.27) shows that, in contrast to the strong inhibition of ConA by the beehive products, its inhibition by human milk is relatively weak, being equal to its inhibition by cow milk, which did not inhibit PA-IIL or UEA-I. The main difference between PA-IIL and UEA-I is that the latter (which is specific for human Fuca1-2 H type-2 epitope [184]) is only inhibited selectively by human Se milk. PA-IIL interactions with human milk are much stronger and involve more gps, with a significantly wider spectrum, extensively overlapping the combination of ConA and UEA-I. The high PA-IIL affinities to human milk gps is due to their fucosylation, which does not attract ConA, whereas its affinities to the RJ gps (which are at a 14-fold higher concentration than in the honey) are due to their mannosylation, which also attracts ConA. The use of PA-IIL as a probe shows that both honey and RJ provide excellent human-milk-like protection against PA-IILmediated PA adhesion. PA-IIL blocking by honey is mainly attributable to its high Fru content (which is removable by dialysis) and to low mannosylated gps contribution while RJ’s powerful PA-IIL inhibition is mainly associated with highly mannosylated MRJPs (retained after dialysis) together with lower Fru contribution. Examination of two floral nectars used by bees for the production of honey and RJ has shown that they also inhibit the bacterial lectins (Table 9.9) mainly due to low MW glycans.
Table 9.9 Inhibition of the bacterial lectins by floral nectars used for honey and RJ production, without and following dialysis (in parentheses), presented by simplified 0–10 grade scale RJ Preparation Tecomaria Jacaranda Honey ConA 6 (2) 6 (0) 8 (6) 7 (6) AGL 2 (1) 7 (2) 6 (2) 4 (1) PA-IL 2 (2) 7 (2) 4 (0) 3 (0) PA-IIL 8 (2) 6 (0) 10 (2) 8 (7) RSL 8 (2) 2 (1) 8 (4) 5 (3) RS-IIL 10 (5) 8 (3) 10 (4) 10 (9) CV-IIL 2 (0) 0 (0) 5 (2) 9 (7) Some of the results presented in this table are part of K.D. Zinger-Yosovich’s PhD thesis at Bar-Ilan University (not yet published elsewhere)
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9.10 Concluding Remarks The availability of the herein-described five bacterial lectins in purified, stable, and relatively high concentrations is a treasure trove for bacteriology, lectinology, and medicinal sciences, and we personally thank God for discovering them, their properties, interactions, and applications, enjoying a fruitful and fair collaboration with excellent experts in the field. The study of these special bacterial lectins by hemagglutination and its inhibition tests, advanced ELL(S)A, thermodynamic microcalorimetric titrations, SPR, and X-ray crystallographic assays, using both the native and recombinant lectins produced by E. coli, has introduced the following new scopes and ideas unique for bacterial lectins: 1. Regulation of lectin production, a subject which is not easy to explore in regard to plant, animal, or viral lectins. Knowledge of bacteria enables induction of higher lectin levels or repression of lectin production in relation to the pathological conditions associated with them (e.g., cystic fibrosis and choline induction of PA lectin production) and their therapeutic implications 2. Relationships between lectin production and the bacterial function in nature and in disease (e.g., saprophytic role and opportunistic pathogenicity of the three herein-described high-level lectin-producing soil bacteria) 3. Functional linkage between the lectins and coproduced VIFs, where the lectins select the targets and provide anchorage, enabling the VIF-concentrated destructive activities on the target host cell components (e.g., PA lectin functional linkage to pyocyanin effect and both proteolytic and hemolytic activities of this bacterium) [16] 4. Homologies versus diversities of structures and carbohydrate specificities and affinities between lectins of same and related bacteria (exemplified by the herein-described five bacterial lectins showing differences in structures, carbohydrate specificities, and binding intensities of the two PA [galactophilic and fucophilic] or RS [fucophilic and mannophilic] lectins, and structural homology of PA-IIL, RS-IIL, and CV-IIL) 5. Association between natural point variations in binding-site amino acids of structurally homologous lectins and their different sugar selectivities and preferential affinities (e.g., the contribution of serine–alanine variation in amino acid #22, of PA-IIL and RS-IIL, to Fuc > Man and Man > Fuc specificities of these two lectins, respectively) 6. Lectin roles in autologous biofilm production and in induction of agglutination and extermination of other bacteria (e.g., PA lectin contribution to PA’s own biofilm formation and to E. coli agglutination and opsonization) 7. Possible lectin contribution to the bacterial preferential host selection (e.g., PA-IL affinities to human P system, B and I antigens, and PA-IIL and CV-IIL avidities to the Lewis and H antigen might be involved in the human pathogenicity of PA and CV, and similarly, RS-IIL high affinities to mannosylated and fructosylated branches might determine RS plant pathogenicity)
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8. Possible lectin function in determining the infecting bacterium organ distribution (e.g., the possible involvement of PA lectins in PA-specific homing onto almost all human and murine organs and tissues with special organ preference) 9. Important lectin contribution to the bacterium’s crucial selective preferential binding to disease affected, altered, or transformed cells, as compared to normal host cells for infection establishment and subsequent serious damages (e.g., the increased interactions of PA lectins with abnormal and with sialidaseor protease-affected cells existing in cystic fibrosis and in secondary PA infections following other primary bacterial or viral infections) 10. Lectin usage for host protection against infections, using the isolated lectins as a vaccine and the antibodies that were produced against them (shown to specifically agglutinate those bacteria) in passive immunization (e.g., full and partial protection of mice against otherwise lethal PA injection by such treatments with PA lectins and rabbit antiserum produced against them, respectively) 11. Lectin application as probes for best fit (highest affinity) ligands that would block the lectin binding to their host cell receptors, acting as SRMDGs according to the antibacterial adhesion strategy for first step of anti-infection therapy (e.g., galactose and Lea derivatives for blocking PA-IL and PA-IIL-dependent PA binding to host cells) 12. Lectin usage for disclosing the natural anti-adhesion mechanisms existing in plants and animals, based on SRMDGs function for first-step innate protection against infections (e.g., the herein-described glycans of plant seeds and gums, of human body fluids and additional kinds of mammalian milk, of avian egg whites and beehive products, all of them supposed to provide protection to adult cells, reproductive elements, embryos, newborns, and newly hatched larvae against pathogen adhesion) 13. Abrogation of lectin production, using manipulations that would hinder bacterial formation of quorum sensing signals that induce the production of lectins and other VIFs (e.g., the treatment by subinhibitory erythromycin or other macrolide concentrations that reduce lectin and VIF levels) [90]. The combined usage of SRMDGs and lectin-formation abrogating treatments might help cure lectin-dependent pathogenic infections
9.11 Summary The soil-borne bacteria PA, CV (animal and human opportunistic pathogens), and RS (plant pathogen) possess homologous high-affinity fucose-/mannose-binding lectins: PA-IIL, CV-IIL, and RS-IIL, respectively. In addition, PA produces a unique galactophilic lectin, PA-IL, and RS forms another fucose- > mannose-binding lectin, RSL, which resembles AAL in structure and specificity. The properties and specific
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interactions of these five bacterial lectins with glycoconjugates, glycosylated macromolecules, and cells that bear them, their functions and effects on cells, as well as their implications in diverse systems, have been studied during the past 35 years and compared to plant (ConA, ECorl, MPL, PNA, and UEA-I) and animal (AGL) lectins, which display related sugar specificities. Important preliminary data on the sugar specificities and affinities of these special lectins were attained by hemagglutination inhibition. Further, on equilibrium dialysis, ELL(S)A, X-ray crystallography, ITC, and SPR analyses were used, owing to a fruitful cooperation between several investigators’ groups. These advanced methods have provided much more accurate, broad, and interesting multidisciplinary bioinformation on the five lectin–glycan interactions. The lectin-selective bindings to diverse cells, including microorganisms (bacteria and archaea), eukaryotic unicellulars, blood cells, spermatozoa, and cancer cells, were followed by agglutination and adsorption tests. These tests were also used for cell typing and identification of bacteria, archaea, and animal RBCs, with special focus on human A, B, O(H), Bombay (Oh), and para-Bombay types, as well as for differentiation between cord (mainly ii) and adult (mainly I-positive) RBCs, and between normal and transformed cancer cells. The lectin effects on the survival and function of these cells and also on rat gut cells (enterocytes) were examined both in vitro and in vivo (by rat feeding with PA lectins). PA-lectin preferential distribution in mouse organs was also examined in vivo, following 125I-labeled lectin injection. The five bacterial lectins were also used as probes in studies of glycans that prevail in plants, human body fluids, diverse kinds of mammalian milk, avian egg whites, and beehive products. Their binding to individual gps of these fluids was further analyzed by Western blotting using these lectins labeled with peroxidase instead of peroxidase-labeled antibodies. Comparison of the bacterial lectin interactions in all the examined systems has shown that they are no less valuable than plant and animal lectins as research and diagnostic tools that vary in their major and subtle specificities to saccharides, cells, and blood groups. Their bacterial origin is advantageous in their usage as probes for cell surface saccharides that act as pathogen receptors and for discovering soluble glycans that might act as SRMDGs for abrogation of lectin-dependent, pathogenic bacterial adhesion to target cells for infection establishment. Investigations of the bacterial lectin blocking by plant saccharides, human body fluids, diverse kinds of mammalian milk, avian egg whites, and beehive products allow insight into the highly skilled mechanism of natural SRMDG contribution to the innate protection of plant reproductive elements and embryos, adult human tissues, immunologically immature mammalian newborns, avian embryos, and newly hatched bee larvae against microbial infections. Acknowledgments The authors express their gratitude to Ms. Sharon Victor for her great helpwith editing and preparing this manuscript and to Ms. Ela Gindy for her great helpin the graphic presentation. This work is part of the PhD thesis of K.D. Zinger-Yosovich at Bar-Ilan University.
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Chapter 10
On the Differential Sialic Acid Specificity of Lectins from Different Parts of Saraca indica Bishnu P. Chatterjee and Mainak Majumder
Keywords Lectins • Sialic acid • Saraca indica • Seed integument • Gynaecium • Filament
10.1 Introduction Sialic acids are the most important sugar molecules in life processes since they play important regulatory and protective roles in cell biology, e.g., protection from proteolytic attack, stabilization of protein conformation, regulation of innate and acquired immune responses, involvement in fertilization, differentiation, aging, apoptosis, and masking of recognition sites, thus regulating molecular and cellular interactions and antioxidative effects [1–3]. In the pathogenesis of diseases, alteration of sialylation occurs in glycoproteins, which has significant implications in the physiological role of glycoproteins [4, 5]. Angata and Verki listed a large number of sialic acid-binding lectins that have been isolated from microorganisms, plants, and animals [2]. Their binding specificity is directed not only to different types of sialic acid, viz., N-acetylneuraminic acid or N-glycolylneuraminic acid and their derivatives, but also to oligosaccharides containing sialic acid at the nonreducing end and their specific linkages with the penultimate sugar. Several of them in immobilized form have been employed for the detection and separation of sialic acid-containing glycoconjugates [5]. They are also proved to be indispensable candidates in biochemical and immunological research and useful in detecting some physiological and pathological changes as different sialic acid derivatives serve as molecular markers in those developments. Compared to bacteria, viruses, fungi, and invertebrates, evidence of sialic acidbinding lectins is limited to the plant kingdom [2]. Wheat germ agglutinin (WGA)
B.P. Chatterjee (*) West Bengal University of Technology, Salt Lake, 700064 Kolkata, India A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_10, © Springer Science+Business Media, LLC 2011
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was reported to be the first plant lectin showing specificity toward Neu5Ac in addition to GlcNAc and its b-1,4-linked oligosaccharides [6]. The binding affinity of WGA to sialic acid was attributed to its structural similarity to GlcNAc, namely, the superimposable configuration of amino and hydroxyl groups at C-5 and C-4 of the pyranose ring of sialic acid with the C-2 and C-3 of GlcNAc [7]. Lectins that distinguish various linkages of sialic acid are elderberry bark lectin (Sambucus nigra agglutinin, SNA) [8], Polyporus squamosus lectin (PSA) [9], and Allomyrina dichotoma (beetle) lectin [10], which are specific for Neu5Aca2,6Gal/GalNAc, Neu5Aca2,6Galb1,4Glc/GlcNAc, and Neu5Aca2,6Galb1,4GlcNAc, respectively; Maackia amurensis leukoagglutinin (MAL), reacts with highest affinity to the trisaccharide sequence Neu5Ac/Neu5Gca2,3 Galb1,4GlcNAc/Glc, which is usually present in N-linked glycans [11]; and the hemagglutinin from the same source (MAH) that exhibits higher affinity for a disialylated tetrasaccharide Neu5Aca2,3Galb1,3(Neu5Aca2,6) GalNAca is found in the O-linked glycan [12]. There are a few mushroom lectins that react with Neu5Aca2,3Gal sequence, namely, Agrocybe cylindracea mushroom lectin, which has specificity for the trisaccharide Neu5Aca2,3Galb1,3GalNAc/GlcNAc [13], and Psathyrella velutina lectin (PVL) and Macrophomina phaseolina agglutinin (MPA), which exhibit the highest binding affinity toward neuraminyl trisaccharide Neu5Aca2,3 Galb1,4GlcNAc [14, 15]. Two other elderberry bark lectins of the Sambucus genus, S. canadensis (the North American species) and S. sieboldiana (the Japanese species), show carbohydrate binding specificity to the Neu5Aca2,6Gal/GalNAc sequence, which is identical to that of S. nigra [16]. The lectin saracin has been detected exclusively in the outer surface of developing seeds of Saraca indica (Ashok), and its activity has been found to be totally absent in matured and dried seeds [17]. Contrary to this, development of Griffonia simplicifolia, Lens culinaris, and Ricinus communis seeds revealed that lectin activity was absent in the immature seed but appeared in the ripe mature seeds [18]. After the discovery of the seed integument lectin saracin, an effort was made to search for the occurrence of any other high molecular weight lectins synthesized as precursors in the filament, which is a part of androecium, the male reproductive organ, and gynaecium, the female reproductive organ of the flower of S. indica. This chapter describes isolation, physiochemical characterization, and the subtle differences in sialic acid-binding specificity of the lectins in different parts of S. indica, viz., seed integument, filament, and gynaecium.
10.2 Materials and Methods The flowers and seeds from the green pod of the S. indica tree were collected in February and March from the garden of the Indian Association for the Cultivation of Science in Kolkata. Seed integuments were obtained by scraping the seeds. Filaments and gynaeciums were separated from the intact flower by fine forceps. All chemicals, biochemicals, sugars, and column matrices were from Sigma.
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Porcine thyroglobulin (PTG)-Sepharose was prepared by the conjugation of PTG with cyanogen-bromide-activated Sepharose-4B according to the method described in the Pharmacia booklet. Acetylated neuraminic acids were the kind gift of Professor Roland Schauer, Biochemisches Institute, Kiel, Germany. The homogeneity of the purified lectins was tested by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 7.5% nondenaturing gel at pH 8.9 [19]. The molecular weight of the lectins was determined by SDS-PAGE on 7.5% denaturing gel [20]. Dissociation and reduction of the proteins were performed as reported earlier [21]. Conjugation of HRP with saracin, filament lectin (FL), and gynoecium lectin (GL) was followed as described [22, 23]. With a view to understanding the detailed carbohydrate-binding specificity of saracin, FL, and GL, enzyme-linked lectin sorbent assay (ELLSA) was conducted in 96-well immunoassay plates [24, 25]. Prior to the addition of sugar inhibitors to the lectin–glycoprotein system, binding of enzyme-labeled lectins with glycoproteins was optimized [26]. Different concentrations of PTG (0.05–50 mg/100 ml/well) with saracin-HRP (5–25 ng/100 ml/well) and fetuin (0.05–15 mg/100 ml/well) with FL-HRP (2.5– 12.5 ng/100 ml/well) and GL-HRP (5–25 ng/100 ml/well) were incubated in an immunoassay plate at 4°C overnight. After washing with normal saline containing 0.05% Tween-20, different sugar inhibitors (50 ml) of varying concentrations were incubated separately with saracin-HRP (50 ml, 20 ng/100 ml), FL-HRP (50 ml, 20 ng/100 ml), and GL-HRP (50 ml, 40 ng/100 ml) for 3 h at 37°C. The mixtures were then added to the wells of the immunoassay plate precoated with PTG (5 mg/well) and fetuin (10 mg/well). The plates were further incubated for 1 h at 37°C followed by washing as before. The wells were incubated with 100 ml of O-phenylenediamine (1 mg/ml in 0.05 M citrate–phosphate buffer containing 0.01% H2O2, pH 5) for 15 min in the dark at 25°C. The absorbances were measured at 492 nm in an enzyme-linked immunosorbent assay (ELISA) reader after the addition of 3 N H2SO4. Protein content in the crude extracts and in different fractions was estimated by the method of Bradford [27].
10.3 Isolation of Lectins from S. indica 10.3.1 Seed Integument Lectin The lectin from S. indica seed integument extract (1 ml, 8 mg protein/ml) was purified successively by affinity chromatography on a PTG-Sepharose-4B column (1 cm × 5.2 cm) (Fig. 10.1a) and by gel permeation chromatography on a Sephadex G-50 column (0.5 cm × 36 cm) (Fig. 10.1b) as reported earlier [17]. The active protein (Fr.II) tested by hemagglutination activity was further purified on protein PAC 300 SW column (0.75 cm × 7.5 cm) with 50 mM sodium acetate (pH 5.0) containing 200 mM NaCl as eluent in high-performance liquid chromatography (HPLC) (Fig. 10.1c). The purification fold achieved was 272. The saracin was
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Fig. 10.1 Purification of the Saraca indica seed integument lectin, saracin. (a) Elution profile of seed integument extract of S. indica on PTG-Sepharose affinity column; (b) elution profile of PTG-Sepharose-bound protein on Sephadex G-50; (c) elution profile of the active protein fraction Fr.II from Sephadex G-50 on Protein-PAC 300 SW HPLC column; (d) SDS-PAGE (10%) of saracin, lane (i) with 2-mercaptoethanol, lane (ii) without 2-mercaptoethanol, and lane (iii) with protein markers (Ovalbumin [Mr 45 kDa], carbonic anhydrase [29 kDa], trypsinogen [24 kDa], a-lactalbumin [14.2 kDa], and aprotinin [6.5 kDa])
electrophoretically homogeneous and has an apparent molecular mass of 12 kDa, which was determined by SDS-PAGE in the presence and absence of 2-mercaptoethanol, proving it to be a monomer (Fig. 10.1d).
10.3.2 Filament Lectin The filament extract of S. indica flower (3 ml, 1.32 mg protein/ml) on addition to Sephadex G-200 column (106 cm × 0.95 cm) and subsequent elution with 10 mM TBS (pH 8.0) (Fig. 10.2a) yielded three fractions. The active fraction (Fr.III) by affinity chromatography on fetuin-agarose column (5 cm × 0.5 cm), followed by
Fig. 10.2 Purification scheme of filament lectin from S. indica flower. (a) Elution profile of S. indica filament extract on Sephadex G-200 column; (b) elution pattern of Fr.III from Sephadex G-200 column on fetuin agarose affinity column; (c) elution profile of the affinity purified fraction Fr.IIIb on Resource-Q anion-exchange column; (d) elution profile of the Resource Q-eluted active fraction, Fr.IIIbb on fetuin-affinity column; (e) SDS-PAGE (7.5%) of filamin, lane (ii) filamin, and lane (iii) protein markers (BSA [Mr 66 kDa], ovalbumin [45 kDa], and carbonic anhydrase [29 kDa])
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elution with citrate–phosphate buffer (pH 5.0) and subsequently by neutralization with Na2CO3, gave an active fraction (Fr.IIIb, Fig. 10.2b). Fr.IIIb was applied onto a Resource-Q anion-exchange column in FPLC and eluted with Tris–HCl–1 M NaCl (pH 8.0, Fig. 10.2c). Finally, the active fraction (Fr.IIIbb) after FPLC was subjected to reaffinity chromatography on fetuin-agarose column (Fig. 10.2d). The filament lectin (Fr.IIIbb [b]) achieved 421-fold purification and was designated as filamin. Filamin produced a single band in nondenaturing gel, indicating it to be homogeneous. The molecular mass of filamin was determined to be 65 kDa (Fig. 10.2e, ii) and it was also proved to be a monomer, which indicates the absence of any interpeptide disulphide linkage. Such a high molecular weight monomeric lectin was reported in Lycopersicon esculentum (tomato), which contains a single polypeptide chain (Mr 71 kDa [28], 40 kDa [29]). There are also reports of monomeric plant lectins, e.g., blood group A-specific lectin crotalarin from Crotalaria striata seeds and lectin from Ficus cunia (fig) seeds, both of which contain a single peptide chain of Mr » 4.5 kDa [30] and 3.5 kDa [31], respectively.
10.3.3 Gynaecium Lectin The gynoecium extract of S. indica flower (3 ml, 9.65 mg protein/ml) was loaded onto Sephacryl S-100 column (67 cm × 1.5 cm), and on subsequent elution with 10 mM TBS, it resolved into four fractions (Fig. 10.3a). Of them, the third fraction (Fr.gIII) having hemagglutination activity was applied to Resource-Q column in FPLC and eluted with 15–18% NaCl gradient resulting in two fractions, of which the second one (Fr.gIIIb, Fig. 10.3b) was active. By affinity chromatography on fetuin-Sepharose with citrate–phosphate buffer (pH 5.0) as eluent, this fraction yielded Fr.gIIIbb (Fig. 10.3c). The purification fold was 308. The purified gynaecium lectin, gynaecin, produced a single band in nondenaturating gel, suggesting it to be homogeneous and to have a molecular mass of 33 kDa (Fig. 10.2e, i).
10.4 Carbohydrate Specificity The carbohydrate specificity of saracin was determined by an inhibition study of the binding between saracin-HRP and PTG using different saccharides by solidphase assay under the established optimal conditions. The required concentration of the sugars for 50% inhibition was obtained from the inhibition curves (Fig. 10.4), and their relative inhibitory potency is given in Table 10.1. Compared to galactose (Gal), Me-a-Gal, Me-b-Gal, galactosamine, and N-acetylgalactosamine, Me-a-Nacetylgalactosamine showed no significant increase in binding. The same was true for lactose and a-methylglycoside of the T disaccharide, Galb1,3GalNAca-OMe. Uronic acids, where the CH2OH group at C-6 in the hexose ring replaced by the –COOH group reduced the binding. N-acetylglucosamine and its b1,4-linked
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Fig. 10.3 Purification of gynaecium lectin from S. indica flower. (a) Elution profile of S. indica gynoecium extract on Sephacryl S-100 column; (b) elution profile of Fr.gIII from Sephacryl S-100 on Resource-Q anion-exchange chromatography column; (c) affinity chromatography of Fr.gIIIb from Resource-Q anion-exchanger on fetuin-agarose column
dimer, chitobiose, showed equal binding, whereas lactosylamine was found to be three times more potent in binding than lactose indicating the importance of the acetamido group at C-2 of the reducing hexose; although with GalNAc and its a-glycoside enhancement was not significant. In this system, Neu5Ac was proved to be the most potent inhibitor, and that containing di- and trisaccharides inhibited the binding of saracin with PTG significantly. However, compared to Neu5Ac, Neu5Gc was 4.5 times less potent in inhibiting the binding. This suggests
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% Inhibition
100
50
0 0.01
0.1
1 Inhibitors (mM)
10
Fig. 10.4 Representative curves of neuraminic acid and different oligosaccharide inhibitors for the binding-inhibition of saracin with PTG. Filled horizontal rectangle, Galb1,3GalNAcaOMe; filled circle, lactose; filled triangle, chitobiose; filled inverted triangle, LacNAc; filled diamond, MeNeu5Aca2,6Galb1,4Glc; cross sign, Neu5Gc; plus sign, Neu5,7,9Ac3; asterisk, Neu5,9Ac2; horizontal dashed line, Neu5Aca2,3Galb1,4Glc; vertical dashed line, Neu5Aca2,3Galb1,4GlcNAc; unfilled horizontal rectangle, Melibiose; unfilled circle, MeNeu5Ac; unfilled triangle, Neu5Aca2,6Galb1,4GlcNAc; unfilled inverted triangle, Neu5Aca2,6Galb1,4Glc; unfilled diamond, Neu5Aca2,3Gal; filled vertical rectangle, Neu5Ac
that the acetamido group at C-5 of neuraminic acid is essential for binding. The –OH groups in the glyceryl chain of neuraminic acid also contribute to binding inhibition because di- and triacetylated Neu5Ac, viz., Neu5,9,Ac2, Neu5,7,9,Ac3 impaired their inhibitory power more than three and four times, respectively. The carboxyl group at C-1 of Neu5Ac is an important locus in binding with saracin since its methyl ester reduced the binding. Both Neu5Aca2,3Galb1,4Glc and Neu5Aca2,3Galb1,4GlcNAc inhibited with equal potency. Their relative affinity for binding was three times less than that of Neu5Ac, whereas the same for Neu5Aca2,6Galb1,4Glc and Neu5Aca2,6Galb1,4GlcNAc was 1.6 times less than Neu5Ac itself. Relatively better inhibition by these two oligosaccharides than the former two suggests the linkage specificity of Neu5Ac. Such linkage specificity was observed in lectins from different parts of the S. indica flower, viz., filament and gynaecium, as well as in SNA [8], PSA [9], A. dichotoma lectin [10], and Pertussis toxin (Pt) [32]. Thus, saracin preferably binds to oligosaccharides with Neu5Aca2,6-linkage and, in this regard, differs strikingly from MAL, which exclusively binds to Neu5Aca2,3-linkages [11]. With regard to the size of the combining site, it differs from SNA, PSA, and the A. dichotoma lectin. It is evident from the previous results that the combing site of saracin is best fitted to a monosaccharide, Neu5Ac, of which C-1, C-5, C-7, C-8, and C-9 are important loci for binding.
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Table 10.1 Relative binding affinity of different saccharides to saracin Inhibitors (mM) IC50a RA Glucuronic acid 9.647 0.74 Galacturonic acid 8.255 0.86 Galactose 7.118 1.00 Galb1,3GalNAcaOMe 5.678 1.25 Me-a-N-acetylgalactosamine 4.709 1.51 Lactose (Galb1,4Glc) 4.636 1.54 Me-b-Gal 4.494 1.58 Galactosamine 3.448 2.06 N-Acetylgalactosamine 3.239 2.20 Me-a-Gal 3.043 2.34 MeNeu5Aca2,6Galb1,4Glc 2.745 2.59 Chitobiose (GlcNAcb1,4GlcNAc) 2.335 3.03 N-Acetylglucosamine 2.093 3.40 LacNAc (Galb1,4GlcNAc) 1.633 4.36 Neu5Gc 1.440 4.94 Neu 5,7,9 Ac 3 1.406 5.06 Neu 5,9 Ac2 1.011 7.04 Neu5Aca2,3 Galb1,4Glc 0.998 7.13 Neu5Aca2,3 Galb1,4GlcNAc 0.998 7.13 Melibiose (Gala1,6Glc) 0.938 7.59 MeNeu5Ac 0.734 9.69 Neu5Aca2,6 Galb1,4Glc 0.518 13.72 Neu5Aca2,6 Galb1,4GlcNAc 0.506 14.05 Neu5Aca2,3 Gal 0.362 19.64 Neu5Ac 0.322 22.08 RA relative affinity a IC50 is the concentration of the inhibitors in mM required to give 50% inhibition
However, the importance of the anomeric nature at C-2 could not be ascertained from the above results. The number of sugars attached successively to Neu5Ac impaired its binding potency, as observed from the inhibition results of di- and trisaccharides having Neu5Ac at the terminal end. It may be explained that the attachment of one or more sugars at the a-anomeric position at C-2 restricts the binding due to steric hindrance of the bulky group. The carbohydrate specificity of filamin and gynaecin was assayed by an inhibition study of the binding between HRP-labeled filamin, gynaecin, and fetuin with a series of sugar inhibitors. The required concentration of the sugars for 50% inhibition was obtained from the inhibition curves (Fig. 10.5), and their relative inhibitory potency is given in Table 10.2, which shows that all simple monosaccharides (except Neu5Ac and Neu5Gc) were highly poor inhibitors. In the filamin–sugar inhibition assay, the Neu5Gc required for 50% binding inhibition was three times less than Neu5Ac, and that required for 50% binding inhibition in gynaecin–fetuin interaction was six times less than Neu5Ac, indicating that the N-acetyl group at
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Fig. 10.5 Representative curves of neuraminic acids and different oligosaccharide inhibitors for the binding-inhibition of filamin with fetuin. Filled circle, MeNeu5Aca2,6Galb1,4Glc; filled diamond, Neu5,7,9Ac3; filled inverted triangle, chitobiose; plus sign, Neu5,9Ac2; filled triangle, Neu5Gc; horizontal dashed line, melibiose; cross sign, lactose; asterisk, LacNac; vertical dashed line, Neu5Ac; unfilled horizontal rectangle, Galb1,3GalNAcaOMe; unfilled circle, Neu5Aca2,3Gal; unfilled triangle, Neu5Aca2,3Galb1,4Glc; unfilled inverted triangle, Neu5Aca2,3Galb1,4GlcNAc; unfilled diamond, Neu5Aca2,6Galb1,4Glc; circle with plus sign, Neu5Aca2,6Galb1,4GlcNAc; filled horizontal rectangle, MeNeu5Ac
C-5 is important for binding. Compared to Neu5Ac, its acetylated derivatives – Neu5, 9Ac2 and Neu5,7,9Ac3 – were found to have reduced their inhibitory potency in filamin by one-third and one-fifth, respectively. In gynaecin, both derivatives were one-fifth times less potent than Neu5Ac, suggesting that acetylation of 7- and 9-OHs in the glyceryl chain of Neu5Ac made the derivatives much more noninhibitory. Neu5Ac methyl ester in filamin and gynaecin glycoprotein interaction lost its inhibitory capacity more than 11 and 28 times, respectively, in comparison to its parent sugar, which clearly demonstrates that the –COOH group at C-1 is a highly indispensable locus for binding with lectins. A similar finding was observed with methyl ester of neuraminyllactose, MeNeu5Aca2,6Galb1,4Glc, which was 25 times and 8 times less potent in binding than Neu5Ac in filamin and gynaecin, respectively. Inhibition of the binding by disaccharides, viz., chitobiose, lactose, and melibiose, was also reduced in both lectins. However, the acetamido group at C-2 of the reducing sugar at the reducing end contributes more to binding as was observed in the competitive binding between lactose and lactosamine. Neu5Aca2,3Gal was six times more effective than Neu5Ac for 50% inhibition, suggesting that the binding site of filamin is more accommodative for neuraminyl disaccharide than Neu5Ac itself. This result corroborated with that for methyl glycosides, of Ta-disaccharide, Galb1,3GalNAcaOMe, which was 2.5 times more effective than Neu5Ac in inhibiting the binding of filamin to fetuin, which in turn is equally less potent than Neu5Aca2,3Gal. The enhanced inhibitory effect of the latter disaccharide is due to the presence of Neu5Ac at the terminal nonreducing end, which is absent in the T disaccharide. Thus, Neu5Ac may be considered to be
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Table 10.2 Relative binding affinity of different saccharides to filamin and gynaecin Filamin Gynaecin Inhibitors (mM) IC50a RA IC50a RA 4.743 0.02 3.717 0.01 Me-a-Galactose Galacturonic acid 4.356 0.03 3.140 0.01 Me-a-N-acetylgalactosamine 3.998 0.03 2.245 0.02 Glucuronic acid 3.998 0.03 3.998 0.01 Galactose 3.747 0.03 2.610 0.02 N-Acetylglucosamine 3.096 0.04 3.140 0.01 Me-b-galactose 2.423 0.05 1.497 0.03 N-Acetylgalactosamine 2.299 0.05 1.070 0.04 MeNeu5Ac 1.378 0.08 1.329 0.03 Galactosamine 1.359 0.09 1.470 0.03 MeNeu5Aca2,6Galb1,4Glc 3.120 0.04 0.377 0.12 Neu5,7,9 Ac 3 0.584 0.20 0.258 0.18 Chitobiose 0.455 0.26 0.290 0.16 Neu5,9 Ac2 0.399 0.30 0.210 0.22 Neu5Gc 0.317 0.37 0.293 0.16 Melibiose (Gala1,6Glc) 0.272 0.43 0.055 0.86 Lactose (Galb1,4Glc) 0.157 0.75 0.090 0.53 LacNAc (Galb1,4GlcNAc) 0.130 0.91 0.065 0.72 Neu5Ac 0.118 1.00 0.047 1.00 Galb1,3GalNAcaOMe 0.048 2.44 0.036 1.30 Neu5Aca2,3Gal 0.019 6.21 0.020 2.32 Neu5Aca2,3Galb1,4Glc 0.014 8.40 0.016 2.87 Neu5Aca2,3Galb1,4GlcNAc 0.011 10.72 0.013 3.65 Neu5Aca2,6Galb1,4Glc 0.004 26.02 0.004 9.90 0.003 32.60 0.003 13.32 Neu5Aca2,6Galb1,4GlcNAc RA relative affinity a IC50 is the concentration of the inhibitors in mM required to give 50% inhibition
a prime inhibitor in initiating the binding to the combining site, and substitution at the a-anomeric position at C-2 of Neu5Ac facilitated better inhibition compared to Neu5Ac alone. A similar trend of inhibition was observed in gynaecin. Considering the effect of trisaccharides, it was observed that Neu5Aca2,3Galb1,4Glc and Neu5Aca2,3Galb1,4GlcNAc were found to be 8 and 11 times more potent than Neu5Ac in the filamin–fetuin system, respectively, and three and four times more potent than Neu5Ac in the gynaecin–fetuin system, repsectively. Neu5Aca2,6Galb1,4GlcNAc and Neu5Aca2,6Galb1,4Glc were the most potent inhibitors, 33 and 26 times more effective than Neu5Ac in filamin, respectively. The two trisaccharides were 13 and 10 times more effective than Neu5Ac in gynaecin, respectively. Comparing the inhibitory potency of lactosylamine with lactose; N-acetyl-neuraminyl-2,3lactosylamine with N-acetyl-neuraminyl-2,3lactose; and N-acetyl-neuraminyl-2,6lactosylamine with N-acetyl-neuraminyl-2,6lactose in both the lectins, it can be clearly concluded that the presence of the acetamido
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group at C-2 of the reducing sugar at the reducing end facilitated binding and is considered to be an important factor in a binding-inhibition assay. Thus, the binding domain of Neu5Ac is confined around C-2, to which the –OH, –COOH, and the ring oxygen are linked (Fig. 10.6). In filamin and gynaecin, substitution at the a-anomeric position of C-2 of Neu5Ac showed better inhibition compared to Neu5Ac, as observed among the molecules carrying large bulky substituents, e.g., Neu5Aca2,3Gal; Neu5Aca2,3/2,6Galb1,4Glc; and Neu5Aca2,3/2,6Galb1,4GlcNAc. The bindinginhibition results demonstrated that filamin and gynaecin recognized the Neu5AcGal sequence for high-affinity binding, and attachment of a third sugar at the reducing end also contributed significantly. It can be concluded that there is an extended binding site for filamin and gynaecin that consists of three subsites for Neu5Ac, Gal, and GlcNAc residues (Fig. 10.7). It is evident from the foregoing results that the binding affinity of the trisaccharide toward filamin and gynaecin is also linkage-specific and depends on the type of sugars present. Neu5Ac was the prime determinant. Penultimate galactose strengthened the binding, though galactose itself was noninhibitory. The type of linkage and the number of sugar units following the nonreducing terminal Neu5Ac are the determinant factors. The interaction of filamin and gynaecin with fetuin as well as PTG, bovine thyroglobulin, and ceruloplasmin (not shown) is due to complex-type bi- and/or triantennary carbohydrate chains having Neu5Ac either 2,6 or 2,3-glycosidically linked to Gal unit. With regard to sialic acid linkage, the specificity of filamin and gynaecin differ greatly from that of MAL, which exclusively binds with high affinity to
Fig. 10.6 Representative curves of neuraminic acids and different oligosaccharide inhibitors for the binding-inhibition of gynaecin with fetuin. Vertical dashed line, MeNeu5Aca2,6Galb1,4Glc; filled circle, Neu5,7,9Ac3; filled triangle, chitobiose; filled inverted triangle, Neu5,9Ac2; filled diamond, Neu5Gc; cross sign, melibiose; asterisk, lactose; unfilled horizontal rectangle, LacNac; plus sign, Neu5Ac; horizontal dashed line, Galb1,3GalNAcaOMe; unfilled circle, Neu5Aca2,3Gal; unfilled triangle, Neu5Aca2,3Galb1,4Glc; unfilled inverted triangle, Neu5Aca2,3Galb1,4Glc NAc; unfilled diamond, Neu5Aca2,6Galb1,4Glc; circle with plus sign, Neu5Aca2,6Galb1,4GlcNAc; filled horizontal rectangle, MeNeu5Ac
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CH2OH CHOH CHOH
Me C
NH
O
COO− O
O
HO H2C
O O
HO OH A
B
NH Ac
HO CH2OH C
O
OH(H)
Fig. 10.7 A tentative model of the carbohydrate-binding site of filamin and gynaecin. Subsites A, B, and C fit to Neu5Ac, Gal, and GlcNAc, respectively
N-acetyl-neuraminyl-2,3lactose, while the isomeric structures N-acetyl-neuraminyl2,6lactose and N-acetyl-neuraminyl-2,6lactosamine are not bound. Filamin and gynaecin bound preferentially with Neu5Aca2,6 linkage and are identical in binding to SNA, PSA, the A. dichotoma lectin, and Pt, yet filamin and gynaecin differ greatly from them in regard to binding with Neu5Ac. The marked preference of filamin and gynaecin for the sequence Neu5Aca2,6Gal compared to Neu5Aca2,3Gal may be understood that substitution at C3–OH of the Gal residue by Neu5Ac restricted its attachment to the binding site of both lectins, resulting in decreased inhibition. Thus, the carbohydrate-binding specificity of filamin and gynaecin for Neu5Aca2,6Galb1,4Glc/GlcNAc is unique among many other sialic acid-binding lectins. Therefore, filamin and gynaecin would be very useful tools for studying glycoconjugates as well as host–pathogen interactions in bacteria, mycoplasma, and viruses because their receptors contain the Neu5AcGal sequence.
10.5 Summary A large number of sialic acid-binding lectins are found to be present in various microorganisms, plants, and animals. They bind specifically to N-acetylneuraminic acid or N-glycolylneuraminic acid and their derivatives or to their specific linkages in oligosaccharides present in N-glycans. Many microbial–host interactions have been observed depending on recognition and binding of sialylated ligands. The sialic acid-specific adhesion leads to infection processes, such as inflammation of gastric mucosa by Helicobacter pylori, by the adhesion of sialoglycoproteins of the cell surface, the binding of various pathogenic microbial toxins to mammalian cells, and the recognition of erythrocytes by Plasmodium falciparum merozoites. The wide expression of large quantities of soluble multivalent sialic acid-binding lectins in the body fluids of many lower organisms is thought to mediate host defense against microbes expressing sialic acid on their surfaces. For example, limulin in the hemolymph of the American horseshoe crab Limulus polyphemus has
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been shown to mediate foreign cell hemolysis. Sialic acid-binding lectins are proved to be tools for exploring the expression and biology of sialic acids. The agglutinins from diverse sources, e.g., wheat germ, Limax flavus, S. nigra, and M. amurensis, in their immobilized form are powerful tools for distinguishing among different types of sialic acid linkages on terminal lactosamines leading to separate sialoglycoconjugates. Saracin, a lectin from the seed integument of S. indica, and filamin and gynaecin from the filament and gynoecium of the S. indica flower have been isolated by different techniques of chromatography. Their molecular mass (saracin, 12 kDa; filamin, 65 kDa; and gynaecin, 33 kDa) has been determined by SDS-PAGE. Saracin is a thermostable glycoprotein, whereas filamin and gynaecin are not. The three lectins are most active at pH 7.6, and their activity is partially dependent on Ca2+. The carbohydrate specificity of saracin, filamin, and gynaecin has been determined by analyzing the binding inhibition between saracin-HRP and PTG and between filamin/gynaecin-HRP and fetuin, using series of saccharides in solid-phase assay. It has been revealed that filamin and gynaecin recognize Neu5Aca2,6Galb1,4GlcNAc, which shows specificity identical to that of SNA, PSA, and the A. dichotoma lectin when sialic acid linkage is concerned. Filamin and gynaecin differ greatly in their specificity from MAL, which is inhibited exclusively by Neu5Aca2,3Galb1,4Glc and is not inhibited by Neu5Aca2,6Galb1,4Glc. Although filamin and gynaecin show specificity toward Neu5Aca2,6Gal and Neu5Aca2,3Gal sequences, they preferably bind to Neu5Aca2,6Galb1,4GlcNAc. The specificity of saracin for Neu5Ac is unique for its maximum inhibition and in this respect differs greatly from SNA and MAL. The binding specificity of saracin is in the following order: Neu5Ac > Neu5Aca2 ,3Gal > Neu5Aca2,6Galb1,4GlcNAc > Neu5Aca2,6Galb1,4Glc > MeNeu5Ac. Acknowledgments The authors gratefully acknowledge Dr. U. Chatterjee and Mr. Gautam Mondal for their fruitful cooperation in preparing the manuscript. Dr. M. Majumder wishes to thank the Council of Scientific and Industrial Research, New Delhi, for the fellowship.
References 1. Schauer R (2000) Achievements and challenges of sialic acid research. Glycoconj J 17:485–499 2. Angata T, Varki A (2002) Chemical diversity in the sialic acids and related-keto acids: an evolutionary perspective. Chem Rev 102:439–470 3. Schauer R (2004) Sialic acids: fascinating sugars in higher animals and man. Zoology 107:49–64 4. Rutishauser U (1998) Polysialic acid at the cell surface: biophysics in service of cell interactions and tissue plasticity. J Cell Biochem 70:304–312 5. Varki A (1997) Sialic acids as ligands in recognition phenomena. FASEB J 11:248–255 6. Adair WL, Kornfeld S (1974) Isolation of the receptors for wheat germ agglutinin and Ricinus communis lectins from human erythrocytes using affinity chromatography. J Biol Chem 249:4696–4704
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7. Bhavanandan VP, Katlie AW (1979) The interaction of wheat germ agglutinin with sialoglycoproteins. J Biol Chem 254:4000–4008 8. Shibuya N, Goldstein IJ, Broekaert WF, Nsimba-Lubaki M, Peeters B, Peumans WJ (1987) The elderberry (Sambucus nigra L.) bark lectin recognizes the Neu5Ac (a 2-6) Gal/GalNAc sequence. J Biol Chem 62:1596–1601 9. Mo H, Winter HC, Goldstein IJ (2000) Purification and characterization of a Neu5Aca2– 6Galb1–4Glc/GlcNAc-specific lectin from the fruiting body of the polypore mushroom Polyporus squamosus. J Biol Chem 275:10623–10629 10. Yamashita K, Umetsu K, Suzuki T, Iwaki Y, Endo T, Kobata A (1988) Carbohydrate binding specificity of immobilized Allomyrina dichotoma lectin II. J Biol Chem 263:17482–17489 11. Knibbs RN, Goldstein IJ, Ratcliffe RM, Shibuya N (1991) Characterization of the carbohydrate binding specificity of the leukoagglutinating lectin from Maackia amurensis. Comparison with other sialic acid-specific lectins. J Biol Chem 266:83–88 12. Konami Y, Yamamoto K, Osawa T, Irimura T (1994) Strong affinity of Maackia amurensis hemagglutinin (MAH) for sialic acid-containing Ser/Thr-linked carbohydrate chains of N-terminal octapeptides from human glycophorin A. FEBS Lett 342:334–338 13. Yagi F, Miyamoto M, Abe T, Minami Y, Tadera K, Goldstein IJ (1997) Purification and carbohydrate-binding specificity of Agrocybe cylindracea lectin. Glycoconj J 14:281–288 14. Ueda H, Matsumoto H, Takahashi N, Ogawa H (2002) Psathyrella velutina mushroom lectin exhibits high affinity toward sialoglycoproteins possessing terminal N-acetylneuraminic acid a2, 3-linked to penultimate galactose residues of trisialyl N-glycans. J Biol Chem 277:24916–24925 15. Bhowal J, Guha AK, Chatterjee BP (2005) Purification and molecular characterization of a sialic acid specific lectin from the phytopathogenic fungus Macrophomina phaseolina. Carbohydr Res 340:1973–1982 16. Shibuya N, Tazaki K, Song Z, Tarr GE, Goldstein IJ, Peumans WJ (1989) A comparative study of bark lectins from three elderberry (Sambucus) species. J Biochem 106:1098–1103 17. Ray S, Chatterjee BP (1995) Saracin: a lectin from Saraca indica seed integument recognizes complex carbohydrates. Phytochemistry 40:643–649 18. Etzler ME (1986) Distribution and function of lectins. In: Liener IE, Sharon N, Goldstein IJ (eds) The lectins: properties, function and application in biology and medicine. Academic Press, New York, pp 371–445 19. Davis BJ (1964) Disc electrophoresis-II, method and application to human serum proteins. Ann NY Acad Sci 121:404–427 20. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277:680–685 21. Banerjee S, Chaki S, Bhowal J, Chatterjee BP (2003) Mucin binding mitogenic lectin from freshwater Indian gastropod Belamyia bengalensis: purification and molecular characterization. Arch Biochem Biophys 421:125–134 22. Avrameas S, Ternynck T (1971) Peroxidase labelled antibody and Fab conjugates with enhanced intracellular penetration. Immunochemistry 8:1175–1179 23. Engvall E, Perlman P (1972) Enzyme-linked immunosorbent assay ELISA: III. quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen-coated tubes. J Immunol 109:129–135 24. Singh T, Chatterjee U, Wu JH, Chatterjee BP, Wu AM (2005) Carbohydrate recognition factors of a Ta (Galb1-3GalNAca-Ser/Thr) and Tn (GalNAca1-Ser/Thr) specific lectin isolated from the seeds of Artocarpus lakoocha. Glycobiology 15:67–78 25. Singha B, Adhya M, Chatterjee BP (2007) Multivalent II [b-D-Galp-(1→4)-b-D-GlcpNAc] and Ta[b-D-Galp-(1→3)-a-D-GalpNAc] specific Moraceae family plant lectin from the seeds of Ficus bengalensis fruits. Carbohydr Res 342:1034–1043 26. Ahmed H, Fink NE, Pohl J, Vasta GR (1996) Galectin-1 from bovine spleen: biochemical characterization, carbohydrate specificity and tissue-specific isoforms profiles. J Biochem 20:1007–1019
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27. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 28. Nachbar MS, Oppenheim JD, Thomas JO (1980) Lectins in the US diet. Isolation and characterization of a lectin from the tomato (Lycopersicon esculentum). J Biol Chem 255:2056–2206 29. Kilpatrick DC (1980) Purification and some properties of a lectin from the fruit juice of the tomato (Lycopersicon esculentum). Biochem J 185:269–272 30. Sikdar S, Ahmed H, Chatterjee BP (1990) A pH-dependent low-molecular weight blood group A-specific lectin from Crotalaria striata seeds: purification and carbohydrate specificity. Biochem Arch 6:207–215 31. Ray S, Ahmed H, Basu S, Chatterjee BP (1992) Purification, characterization and carbohydrate specificity of Ficus cunia lectin. Carbohydr Res 242:247–263 32. Heerze LD, Armstrong GD (1990) Comparison of the lectin-like activity of Pertussis toxin with two plant lectins that have differential specificities for a(2–6) and a(2–3)-linked sialic acid. Biochem Biophys Res Commun 172:1224–1229
Chapter 11
Regulation of Lectin Production by the Human Pathogens Pseudomonas aeruginosa and Chromobacterium violaceum: Effects of Choline, Trehalose, and Ethanol Nachman C. Garber, Keren D. Zinger-Yosovich, Dvora Sudakevitz, Itschak Axelrad, and Nechama Gilboa-Garber Keywords Bacterial lectins • Choline • Chromobacterium violaceum • Cystic fibrosis • Erythromycin • Ethanol • Quorum sensing • Pseudomonas aeruginosa • Ralstonia solanacearum • Trehalase • Trehalose Abbreviations Ara CF Ch ChE C. violaceum CV-IIL (CV-lectin) ERM Et Fru Fuc Gal Glc HSLs Man PA PA lectins PA-IL PA-IIL PLC-H PLP
d-Arabinose Cystic fibrosis Choline Cholinesterase Chromobacterium violaceum C. violaceum (fucose > mannose-binding) lectin Erythromycin Ethanol d-Fructose l-Fucose d-Galactose d-Glucose N-acyl-l-homoserine lactones (autoinducers) d-Mannose Pseudomonas aeruginosa P. aeruginosa lectins P. aeruginosa first (LecA, galactophilic) lectin P. aeruginosa second (LecB, fucose > mannose-binding) lectin Hemolytic Phospholipase C Purified lectin preparations
N.C. Garber (*) The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel e-mail:
[email protected] A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_11, © Springer Science+Business Media, LLC 2011
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2-Heptyl-3-hydroxyl-4 quinolone Quorum sensing Ralstonia solanacearum Ralstonia solanacearum lectins R. solanacearum first (fucose > mannose-binding) lectin R. solanacearum second (mannose > fucose-binding) lectin Virulence factors
11.1 The Bacteria Pseudomonas aeruginosa, Ralstonia solanacearum, and Chromobacterium violaceum and the Discovery of Their Lectins The worldwide-distributed Pseudomonas aeruginosa (PA) and the geographically restricted (confined to tropical and subtropical zones) Ralstonia solanacearum and Chromobacterium violaceum are Gram-negative proteobacteria that dwell in soil and water. They are essentially beneficial saprophytes that vigorously decompose plant and animal remnants and organic debris, contributing to world carbon and nitrogen cycling (Fig. 11.1). In accordance with their distinguished role in nature, these bacteria are endowed with very prosperous arsenals of cell-binding adhesins, toxicating proteinaceous and nonproteinaceous factors, and hydrolytic enzymes as virulence factors (VIFs), enabling them to home in on dead or damaged cells and molecules and attack them.
Fig. 11.1 The beneficial contribution of PA, R. solanacearum, and C. violaceum (denoted by asterisks) to carbon and nitrogen cycling by toxicating dead cell synthetic pathways and decomposing them, with other organic debris by their hydrolytic enzymes
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The three bacteria display high versatility, environmental adaptability, and resistance to denaturing effects associated with highly sensitive regulatory factors. All of these properties and functions are encoded by rich genomes, which have been unveiled during the past decade [1–3]. Unfortunately, when PA and C. violaceum come in contact with impaired animal or human tissues displaying altered cell membranes or immunodeficiency, they attack them aggressively. While C. violaceum infections are relatively rare, PA is frequently involved in nosocomial human infections, endangering the lives of patients suffering from cystic fibrosis (CF), burns, or immunodeficiency (AIDS and therapy for cancer or tissue transplantation). R. solanacearum is mainly a phytopathogen responsible for the bacterial wilt of more than 200 plant species [4]. Plant infection takes place when the bacterium living saprophytically in the soil colonizes the root and subsequently multiplies in the apoplastic space, traversing the endoderm barrier and invading the vascular system. The development of wilting symptoms is probably a result of intensive bacterial multiplication within the xylem vessels and the concomitant production of copious amounts of exopolysaccharides that block water traffic, ultimately resulting in very heavy agricultural losses. The binding of the three bacteria to the host cells is mediated by their adhesins, including several related lectins whose production is closely regulated in association with additional VIFs. Historically, the PA first (galactophilic) lectin (PA-IL) and the PA second (fucose > mannose-binding) lectin (PA-IIL) were discovered first [5–8].
11.2 The PA Lectins PA-IL and PA-IIL 11.2.1 The PA Lectin Specificity and Function PA-IL was described in 1972 [5], and PA-IIL (displaying outstandingly high fucose > mannose-affinity) was described a few years later [6, 7, 9, 10]. Their definition as lectins was based on their hemagglutinating activities and their inhibition by specific carbohydrates [5–7] (Fig. 11.2), as well as equilibrium dialysis data that also confirmed PA-IIL’s very high affinity to fucose [9]. At that time, lectins were still mainly considered products of plants and animals, and we had to work hard to convince others that they are indeed lectins that behave as classical plant and animal hemagglutinating lectins and have properties, blood group specificities, effects on cells, and applications like those lectins [11, 12]. During that period, we very much enjoyed a fruitful cooperation with Dr. Albert M. Wu (Taiwan), who advanced the important aspect of the bacterial lectin interactions with blood group-specific glycoproteins and other glycotopes [13]. More detailed descriptions of the carbohydrate specificities of PA-IL are described in publications by Garber et al. [10], Chen et al. [13], and Cioci et al. [14], and those of PA-IIL are described in publications by Garber et al. [9], Mitchell et al. [15], and Wu et al. [16].
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Fig. 11.2 The basic simple carbohydrate specificities of PA-IL and PA-IIL to the monosaccharides: l-fucose (Fuc), l-galactose (l-Gal), d-arabinose (Ara), d-fructose (Fru), d-mannose (Man), d-glucose (Glc), d-galactose (Gal), and N-acetyl d-galactosamine (Gal-NAc)
Fig. 11.3 Adhesion of P. aeruginosa cells leading to biofilm formation (auto) and homing to target cells (hetero, auto and hetero) [17]
We had reported very early on that the major part of the PA-lectin molecule is internally located, and only a small fraction is exposed on the external bacterial cell surface, contributing to biofilm formation and adhesion to target cells [17] (Fig. 11.3).
11.2.2 Linkage Between the PA Lectin and VIF Production Our studies on the production of the two PA lectins showed that strains that produce high lectin levels are rich in proteolytic activities, pyocyanin, and hemolysins, while the lectin-deficient strains are poor VIF producers [11, 12] (Fig. 11.4). Based on these results, we suggested that the physiological role of lectin is to cofunction with lytic enzymes [18] (Fig. 11.5) and that the linkage between the production of PA lectins and the bacterium VIFs is due to a coregulatory mechanism. That was long before the description of the quorum sensing (QS) system and its central role in regulating the production of secondary metabolism VIFs. Today, partial knowledge of the QS mechanism controlling that coordination [19, 20] confirms our
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Fig. 11.5 Selective lectin-mediated PA adhesion to the host cell membrane, acting as an anchor, enabling coherent contact between the pathogen VIFs and their target substrates
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22-year-old claim [18] that lectin function is tightly linked to lytic activities, providing them with the ability to home in on and anchor onto the target cells, enabling them close contact with their substrates (Fig. 11.5). The accumulating molecular information on the QS multicomponential complex and the factors involved has shed light on some aspects of the lectin and VIF gene expression control. However, part of the whole picture puzzle, especially regarding the effects of additional active compounds existing in the microenvironment of the free-living and hosted bacterial ecosystems, is still missing.
11.2.3 PA Lectin Interactions with and Effects on Diverse Cells The PA target cell spectrum, ranging from microorganisms to human beings [8, 12, 22], implies possession of wide-range lectins. PA-IL and PA-IIL fulfill that requirement, displaying a wide spectrum of cell binding, representing that of the intact bacteria. Their binding to galactose and fucose apparently enables their binding to all human and many animal cells [23, 24]. Their binding to microorganisms may agglutinate them and expose them to the PA lytic enzymes as well as to the host circulatory complement and phagocytic peripheral leukocytes. In respect to the latter, the PA lectins function as selective opsonins, resulting in extinction of foreign bacteria that exist in their vicinity [25] (Fig. 11.6).
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Fig. 11.6 Specific opsonizations of Escherichia coli O86 B6 by PA-IL and E. coli O128 B12 by PA-IIL, selectively increasing their phagocytosis by human peripheral leukocytes (PLeu). The experimental controls included engulfment of untreated E. coli O86 B6 and E. coli O128 B12 cells (left column) and its inhibition in presence of NaF, exposure of the leukocytes (instead of bacteria) to the two lectins, and also cross-matching treatments of the bacteria by the lectins that do not react with them (E. coli O86 B6 by PA-IIL and E. coli O128 B12 by PA-IL) [25]
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%125I.D/g Tissue
PA lectin binding to free-living eukaryotic cells also agglutinates them and affects their metabolism and proliferation rate [26]. Their binding to animal cells induces membranal alterations [27], mitogenic stimulation of peripheral human lymphocytes [28, 29], interleukin secretion, and damages in many cell types, including corneal [30] and respiratory epithelial cells [31, 32], whose cilial movement and structure were shown to be demolished by them. PA-IL ingestion was shown to induce gut growth in rats [33] and changes leading to septicemia in stressed animals [34–36]. In vivo, the PA lectins were shown to resemble intact PA cells in target cell profile, binding to almost all the mammalian tissues examined and causing apparent transformations in them. The individual PA-IL and PA-IIL contributions to the pathogen distribution in different tissues and organs have been learned by injecting mice intraperitoneally with 125iodine-labeled PA-IL and PA-IIL and determining their distribution profile. The results of that experiment have shown a preferential organ affinity order similar to that of the bacterial infectivity [22] (Fig. 11.7). The data presented in Fig. 11.7 show the highest levels of lectin binding by murine spleen, kidney, lung, and liver, with PA-IIL being dominant. The high binding of PA-IL to murine organs was also shown later on by Kirkeby and Moe [37], using histochemical methods. They examined PA-IL’s binding to organ sections taken from wild-type and knock-out (lacking a13-galactosyltransferase) mice and found strong binding to the wild-type heart, kidney, and adrenal gland, but not to pancreatic sections. Their results are in accord with the a-Gal-specificity of that lectin [10, 13, 14] and the differential glycosylation of different organs. Recently, they have expanded the histochemical study to minks, showing the receptors for the two lectins in sections of mink pancreas and lungs [38].
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Fig. 11.7 The homing of intraperitoneally injected murine organs and cells [22]
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11.3 Blocking of PA Infection by Anti-PA-Lectin Antibodies The high affinity of both PA-IL and PA-IIL for lungs and their effects on them [22, 31, 32, 37, 38] are of great clinical importance since chronic lung infections caused by antibiotic-resistant PA are still a major cause of pulmonary damage, morbidity, and mortality in patients suffering from CF, diffuse panbronchiolitis [39], other forms of bronchiectasis, endotracheal intubation, AIDS, and additional chronic diseases and terminal nosocomial complications. Therefore, great efforts are being invested in anti-adhesive strategies aimed at preventing infections by abrogating the lectindependent adhesion [8]. One way to prevent lectin binding is by using antibodies or immune cells specifically produced against them in active or passive immunization. Such lectin vaccines have been shown to be very efficient. The antibodies obtained precipitated the purified lectins and agglutinated the bacteria, proving the crucial role of PA lectins in infection and their presence on the bacterial cell surface [40]. The injection of purified lectin preparations (PLP) as a vaccine fully protected all the treated mice against an otherwise lethal PA infection caused by PA live-cell inoculation (Fig. 11.8). As seen in this figure, passive protection by immune rabbit sera was also efficient (providing 80% protection, while normal rabbit serum protected only 10% of the mice). Passive cellular immunization by splenocytes and bone marrow cells withdrawn from immunized mice also provided considerable immune resistance (70% survival) [41].
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Fig. 11.8 Application of purified lectin preparations (PLP) of PA as a vaccine. PLP were used in active immunization – injecting them intraperitoneally [PLP(i.p.)] or giving them per os [PLP(p.o.)] – and in passive (i.p.) immunization, using PLP-immunized rabbit serum [Im(PLP) RS] versus normal rabbit serum (NRS) or PLP-immunized murine splenocytes [Im(PLP)Spl] or bone marrow cells [Im(PLP)BMC] versus normal splenocytes (NSpl), plant lectin (PHA)triggered splenocytes [Trig(PHA)Spl], and murine normal bone marrow cells (NBMC). The experimental controls also included mice injected i.p. with saline (0.85% NaCl solution) and mice subjected to i.p. injection of a very diluted live-PA cell suspension (live bacteria). The immunization efficacy was examined by an i.p.-injected lethal dose of live-PA cell suspension [41]
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11.4 Discovery of the PA-IL and PA-IIL Genes (lecA and lecB); the Unveiling of Their 3-D Crystal Structures; and the Revelation of RSL, RS-IIL, and CV-IIL The PA-IL gene (lecA) was unveiled first, in 1992 [42], and cloned in E. coli [43] before the release of PA’s whole-genome data (in 2000). The PA-IIL gene (lecB) was discovered 8 years later [44], immediately following the description of PA’s whole genome [1]. It was shown to differ from PA-IL in structure and to be far apart from it and other VIFs on the PA chromosome, rejecting the possibility that their observed coregulation is based on oligocistronic control (both are monocistronic). Following the report on R. solanacearum’s whole genome in 2002 [2], a PA-IILlike sequence was detected in it (Fig. 11.9). The search for the respective lectin in R. solanacearum cells yielded two lectins: RSL, which resembles PA-IIL in its fucose > mannose preference, but not structurally [45], and RS-IIL, which was the looked-for PA-IIL-structural analogue [46]. The latter lectin’s specificity differed from that of PA-IIL in displaying higher mannose than fucose avidity, but it kept the high affinity mode [46]. Two years later, following publication of C. violaceum’s whole genome [3], another PA-IIL-like lectin gene that of CV-IIL, was detected in it (Fig. 11.9) [47]. The native C. violaceum lectin CV-IIL was found in its cells [47], and a recombinant form was isolated from E. coli cells [48]. Further alignment studies indicated the presence of additional PA-IIL-like lectins in strains of Burkholderia cenocepacia [49], one of them, BClA, displaying mannose preference [50]. The simple sugar affinities of RSL, RS-IIL, and CV-IIL are represented in Fig. 11.10. The discovery of the five bacterial-lectin genes was a breakthrough that has opened new horizons for bacterial lectinology. Following the discovery of the PA-IL, PA-IIL, and RSL genes, we started a very fruitful cooperation with Dr. Anne Imberty and her collaborators in France and the Czech Republic. These experts
PA-IIL RS-IIL CV-IIL
PA-IIL RS-IIL CV-IIL
1 ATQGVFTLPANTRFGVTAFANSSGTQTVNVLVNNETAATFSGQSTNNAVIGTQVLNSGS ATQGVFTLPANTRFGVTAFANSSGTQTVNVLVNNETAATFSGQSTNNAVIGTQVLNSGSS : 60 1 AQQGVFTLPANTSFGVTAFANAANTQTIQVLVDNVVKATFTGSGTSDKLLGSQVLNSGSAQQGVFTLPANTSFGVTAFANAANTQTIQVLVDNVVKATFTGSGTSDKLLGSQVLNSGS : 59 1 AQQGVFTLPARINFGVTVLVNSAATQHVEIFVDNEPRAAFSGVGTGDNNLGTKVINSGSAQQGVFTLPARINFGVTVLVNSAATQHVEIFVDNEPRAAFSGVGTGDNNLGTKVINSGS : 59
61 GKVQVQVSVNGRPSDLVSAQVILTNELNFALVGSEDGTDNDYNDAVVVINWPLG : 114 60 GAIKIQVSVNGKPSDLVSNQTILANKLNFAMVGSEDGTDNDYNDGIAVLNWPLG : 113 60 GNVRVQITANGRQSDLVSSQLVLANKLNLAVVGSEDGTDMDYNDSIVILNWPLG : 113
22 23 24 PA-IIL Ser. Ser. Gly Saccharide-binding loop amino acids: RS-IIL Ala. Ala.Asn CV-IIL Ser. Ala.Ala
Fig. 11.9 Amino acid homology of PA-IIL, RS-IIL, and CV-IIL. Amino acids nos. 22, 23, 24, and 96 of the carbohydrate-binding loops are denoted by exclamation marks, and the Ca2+-binding ones are denoted by asterisks [45–49]
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Fig. 11.10 The basic simple sugar specificities of RSL, RS-IIL, and CV-IIL as compared to PA-IIL [47]
have very skillfully crystallized the lectins and demonstrated their 3-D structures, including the presence of Ca2+ and the crucial amino acids involved in its saccharide binding (both indicated in Fig. 11.9). They also studied the thermodynamic aspects of those interactions. The information derived from the gene data led to the following achievements: (1) exact molecular sequences of the five lectin genes and proteins were inferred; (2) recombinant lectins were produced in E. coli; (3) the number of subunits and their exact molecular weights could be determined (Fig. 11.11); (4) the amino-acid sequence homology between PA-IIL, RS-IIL, and CV-IIL [47, 48], and then the Burkholderia cenocepacia lectins, were established [49, 50]; (5) the 3-D structure of the PA-IIL crystal was revealed, showing the presence of unique Ca2+ tweens in each subunit and their direct involvement in the fucose/mannose binding [15], contributing to the exceptionally high carbohydrate affinity of that lectin and its homologues [9, 16, 49]; (6) the 3-D structure of the galactose-binding PA-IL crystal was revealed, showing the presence of one Ca2+ atom in each of its subunits [14], exhibiting conventional lectin-type affinity [10, 51]; (7) the 3-D structure of RSL was revealed and shown to be of b-propeller type, resembling that of the mushroom Aleuria aurantia lectin [52]; (8) the 3-D structure of RS-IIL was unveiled, showing its high similarity to PA-IIL, and the crucial difference in the carbohydrate-binding loop amino acids nos. 22, 23, and 24, being alanine–alanine–asparagine in RS-IIL instead of serine–serine–glycine in PA-IIL, which is supposed to contribute to the preferential affinity switch from fucose (of PA-IIL) to mannose (of RS-IIL) [46]; (9) the 3-D structure of CV-IIL was shown [47, 48], with its binding loop serine– alanine–alanine triad associated with dominant fucose preference; (10) the indication of the possible existence of several additional PA-IIL analogues in Burkholderia
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Fig. 11.11 Summary data on the five bacterial lectins, including their discovery year (y), subunit number, 3-D structure and its discovery year [14, 15], their subunit molecular weight, number of Ca2+ atoms, and carbohydrate-binding sites per subunit
cenocepacia strains [49] led to the isolation of recombinant BClA, possessing alanine–alanine–asparagine triad like RS-IIL [49] and resembling it in mannose preference, which was found to be useful for isolation and exploration of a-mannosylated copolymers [50]; and (11) deliberate in vitro mutations in the PA-IIL specificity loop serine 22 and serine 23 to alanines (resembling RS-IIL) led to preferential affinity switching from fucose to mannose [53].
11.5 Natural Protection Against the Binding of PA Lectins and CV-IIL to Cell Receptors by Their Surrounding Glycoconjugates In addition to the inhibition of lectins by antibodies produced against them, abrogation of lectin attachment to cells is also attainable by soluble receptor-mimicking glycotopes. This strategy is most widely encountered in nature, where embryos and newborns, as well as adult cells and tissues, are surrounded by soluble glycotopes that interact with and block the bacterial lectins, mimicking the lectin-binding cell receptors. These glycans function as decoys that competitively attract the lectins, hampering the bacterial adhesion to the cell receptors. Usage of purified bacterial lectins as probes is the best way of searching for such natural and synthetic glycans and evaluating their efficacy compared to that of the natural protecting glycoconjugates. Examples of natural sources of protecting glycoforms are the kinds of milk that protect mammalian newborns [54–56], avian egg whites that protect their
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embryos [57–60], and beehive products, royal jelly and honey, which protect newly hatched and developing larvae (respectively) [61]. Human milk glycans were found to effectively block PA-IL, PA-IIL [55], and CV-IIL. PA-IIL’s very high sensitivity to human milk [56] is due to the presence of the Lea epitope, which is its most favored ligand [15, 16, 62]. Both royal jelly and honey profoundly blocked PA-IIL and CV-IIL and weakly blocked PA-IL [61]. The very high sensitivity of PA-IIL and CV-IIL to the royal jelly glycoproteins is mainly due to their mannosylated glycans, and their inhibition by honey is due to its high fructose content. Cow’s milk cannot replace human milk, royal jelly, or honey in PA-IIL and CV-IIL inhibition (Figs. 11.12 and 11.13). In addition to usage of the natural anti-adhesive progeny-protecting compounds, there is a new quickly advancing research branch aimed at producing synthetic glycodendrimers designed to display higher affinity to the bacterial lectins and replace the natural ones [63–65]. 18 16 14 12 10 8 6 4 2 0 16 14 12 10 8 6 4 2 0 16 14 12 10 8 6 4 2 0
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Fig. 11.12 Inhibition of the bacterial lectins by honey, royal jelly, human milk, and cow’s milk, each of them nondialyzed and dialyzed (for removal of the low molecular weight saccharides)
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11.6 QS- and Choline-Dependent Regulation of PA Lectin Production The discovery of PA-IL [5] was not the intended result but the serendipitous by-product of a study on the usage of PA cholinesterase (ChE) [66] for replacing human erythrocyte enzyme. Three factors associated with that enzyme study resulted in the PA-IL discovery: (1) acetylcholine was added to the bacterial growth medium for induction of ChE production; (2) Ch was released from that acetylcholine by the induced ChE; and (3) the bacterial cell extracts were mixed with papain-treated human erythrocytes (for removal of their endogenous surface-associated acetylcholinesterase). The surprising experimental result was a very strong hemagglutination that was shown to be due to a lectin that was inhibited by galactose [5]. When PA-IIL was discovered, in the same PA cell extracts (under different growth conditions [7]), it was also shown to be similarly stimulated by Ch [6, 7]. Soon after, the lectin production was shown to correlate with that of additional VIFs [11, 12]. The suggested possible explanation for the coregulation was that it could be associated with either a close oligocistronic linkage or a common, not-yet-known central or global control of VIF production. The first possibility was rejected when the PA-IL gene was discovered [42, 43] and found to be monocistronic far apart from PA-IIL and the other VIF genes. The second possibility was confirmed to be true when, a decade later, the hierarchical QS cascade system of transcriptional regulating signal circuits associated with cell density and growth phase was unveiled [19, 67, 68]. Winzer et al. [19] showed that the transcriptional sensor regulatory protein RhlR and its cognate, N-acyl-l-homoserine lactones (HSL) C4-HSL activator (autoinducer synthesized by RhlI), induced expression of the PA-IL gene (lecA) by associating with a 20-bp luxI box-like sequence.
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Fig. 11.14 LuxI box-like 20-bp elements of PA-IL, PA-IIL, RSL, and CV-IIL. The inverted symmetry is dotted
That sequence was identified at 70 bp upstream of the lecA translational start codon (Fig. 11.14). They showed that the expression of lecA is induced by the RhlR/ C4-HSL complex [19], which controls the syntheses of many exoproducts in PA with homology to the autoinducer-responsive LUXR–LUXI family [69]. The LasR/3O-C12-HSL complex might support its expression, but it is not crucial for it since in LasR mutants, delayed PA-IL and PA-IIL production was observed. They further reported that RhlR/C4-HSL also activated the alternative stationary-phase sigma (sS) factor RpoS [70, 71], which is also involved in PA-IL induction. Winzer et al. [19] showed by transcript analyses of the PA-IL structural gene that both luxI box-like and RpoS consensus sequences were upstream of the transcriptional start site and that mutations in RpoS abolish lectin synthesis. Another compound found to modulate that system was a quinolone derivative shown to be required for production of rhl-dependent exoproducts at the onset of the stationary phase [72, 73]. Provision of 2-heptyl-3-hydroxy-4 quinolone (PQS) to the cultures significantly increased PA-IL, pyocyanin, and elastase production, overcoming transcriptional repression of lecA by the negative regulator MvaT and by the posttranscriptional modulator RsmA (two small RNA-binding regulatory proteins acting positively or negatively near the ribosome binding site [74]). Diggle et al. [72] attributed the augmented lecA expression in PQS presence to increases in RhlR, RpoS, and C4-HSL levels. Another global PA transcriptional regulator, the protein Alg R2 (Alg Q) – which was originally implicated in PA alginate upregulation and diverse VIF production versus rhamnolipid and proteases downregulation – was found to bind to lasR and rhlR promoters, affecting gene expression [75]. Taken together, the above-described works show that the QS circuits parallelly regulate numerous VIFs (up to 5% of PA genes) including PA elastase, alkaline protease, lasA protease, exotoxin A, pyocyanin, hemolysins, RpoS, rhamnolipids, and Xcp (secretion apparatus components). RhlR/C4-HSL controls expression of PA-IL both directly and indirectly via RpoS (the alternative sigma factor, which is considered to be responsible for activating the genes required for survival in stationary phase and general stress response). The switch-on of stationary-stage-associated genes enables bacterial multicellular behavior and survival under stress conditions.
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11.7 Stimulation of PA-IL, PA-IIL, RSL, RS-IIL, and CV-IIL Production by Osmoprotectants Our original reports that acetylcholine and Ch (0.2%) stimulate the production of PA lectins and ChE [5–7, 66] were followed by the reports of Lisa et al. [76] showing increased acid phosphatase, and Sage and Vasil’s findings on phospholipase C [77]. The latter have attributed the Ch effect to osmoprotection. The osmotic pressure in bacterial cytoplasm is normally higher than that of its surroundings, resulting in a profound positive turgor, which is regarded as the driving force for cell growth and expansion [78]. Hyperosmotic medium induces water efflux across the cytoplasmic membrane, leading to reduction of the turgor and dehydration. As a result of this process, various enzyme activities are decreased [79, 80], culminating in growth inhibition. To avoid such a situation, bacteria accumulate organic compounds that function as osmolytes, to protect macromolecules and preserve their function [79–83]. The compatible protecting solutes, which differ among species, are either synthesized in the cells or transported into them from the medium [80, 83]. Glycine betaine, which is one of the most useful natural osmolytes [79, 81], is produced from exogenous Ch that is transported into the cells by very efficient osmotically regulated transporters and is oxidized by two dehydrogenase-mediated steps [81, 84–86]. Bacteria that possess both the Ch-accumulating and dehydrogenating systems [87] exhibit high osmotic tolerance, while bacteria that lack the latter pathway display higher sensitivity to osmotic stress and to Ch. Bacillus subtilis mutants lacking the Ch-betaine pathway were found to differ from the osmotic stress-resistant wild type by being sensitive to osmotic stress and to Ch-rich medium [85, 86] because Ch accumulation in their cells, without its oxidation, inhibits their growth. The ability to survive in a Ch-rich hyperosmotic environment, owing to the ability to convert Ch to glycine betaine, is a better selective adaptation to human pathogenicity. PA enjoys this ability, which contributes to its dominant existence and pathogenicity in Ch-rich host microenvironments, such as those existing in the lungs of CF patients [88–90]. Increased Ch that stimulates PA lectin and VIF production (leading to sequential circles of additional Ch releases, by further increase of phospholipase and phosphatase hydrolytic activities) – together with increased levels of PA-IL and PA-IIL receptors (whose numbers are also increased due to their unmasking by the stimulated pathogen’s proteolytic activity) in CF patients’ lungs – might subdue them to its severe and lethal infections. This situation is more than compatible with Govan and Harris’s [88] definition: “Pseudomonas aeruginosa and cystic fibrosis: unusual bacterial adaptation and pathogenesis C. violaceum.” Examination of CV-IIL production by C. violaceum in Ch-containing culture medium that strongly stimulates PA-IL and PA-IIL production has revealed that, unlike PA, this purple violacein-producing bacterium [91] was not stimulated by Ch; its CV-IIL level was negligible (Fig. 11.15). R. solanacearum lectin production (RSL and RS-IIL) was also not stimulated by Ch, and the growth of the bacterium was even reduced in Ch-containing cultures, indicating damaging accumulation of Ch in its cells. These results accorded with the inability to convert Ch to glycine
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betaine in both C. violaceum and R. solanacearum. However, when another osmoprotectant, the nonreducing diglucoside (a-d-Glc 1-1a-d-Glc) trehalose, was used, a reciprocal pattern was obtained: considerable CV-IIL (and also RS lectin) production versus negligible PA-lectin activity (Fig. 11.15). These interesting findings indicate two important aspects that differentiate PA from C. violaceum and R. solanacearum: (1) only PA possesses the system that converts exogenous Ch to the endogenous osmoprotectant glycine betaine (Figs. 11.16 and 11.17) [84], and (2) only PA is not osmoprotected by trehalose. We proposed that the reason for the latter difference might be associated with decomposition of the trehalose by PA, but have not found in the literature any notion on the presence of trehalase activity in PA. However, in PA genomic data, we found that a report on the discovery of this enzyme putative gene in PA, PA7, was deposited on 5 July 2007 (by Dodson R.J., Harkins D., and Paulsen I.T. from the Institute for Genomic Research, 9712 Medical Center Dr, Rockville, MD 20850, USA), confirming our way of thinking. They described the enzyme 561 aa, linear structure, BCT (Locus [accession] ABR83668, GI: 150961643, DBSOURCE accession CP000744.1). This enzyme is considered as a hallmark of heat–shock response in yeast-protecting proteins and membranes against diverse stresses. As it may be seen from Fig. 11.16, 1 h after Ch addition to the PA cells, part of it was transported into the cells and completely converted to glycine betaine; 16 h later, all the Ch was absorbed by the cells, most of it converted to glycine betaine. At the same time, the negative strains adsorbed some Ch, but did not convert it to glycine betaine. Assay of glycine betaine dehydrogenase activity (Fig. 11.17) has also proved that PA possesses this activity. The above figures show how, in contrast to C. violaceum and R. solanacearum, PA is affected by Ch because of enzyme possession (missing in the C. violaceum and R. solanacearum) and is not affected by trehalose due to decomposition of this compound by its trehalase (E.C number = “3.2.1.28”), which is not produced by the other two bacteria, and therefore, they are affected by trehalose.
Glycine betaine
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Fig. 11.16 Conversion of exogenous radioactive (methyl- C) Ch to C glycine betaine. The residual 14C Ch and the newly formed labeled glycine betaine were detected following their separation by thin layer chromatography (TLC) according to Boch et al. [85]. The conversion was performed by 0.1 ml of an overnight bacterial culture inoculated into 1 ml of medium containing 38 mM [methyl-14C] Ch (5.4 mCi mmol−1). The culture was incubated with aeration at 37°C and 0.5-ml samples were taken after 1 and 16 h. The cells were pelleted from the two samples by centrifugation, and 0.2-ml samples of each supernatant were lyzed by treatment with a solution (50 ml) of 0.2 M NaOH and 1% sodium dodecyl sulfate (SDS). Following removal of cellular debris by centrifugation, 5-ml samples of the cell extracts and of the growth medium supernatants were spotted onto TLC plates (Silica Gel G) and subjected to running with methanol – 0.88 M ammonia (3:1). Glycine betaine and [methyl-14C] Ch were used as markers. The radiolabeled spots were visualized by Fujifilm phosphor imager. Lanes 1–4 are of PA, and lanes 5–8 are of bacteria that do not convert Ch to glycine betaine (representing C. violaceum and R. solanacearum). Each tetrad is composed of a cell lysate and culture supernatant (1 and 2) after 1 h, and the same (3 and 4) after 16 h. Lanes 9 and 10 contain standard radiolabeled Ch and glycine betaine, respectively. Ch itself does not migrate on the silica gel plates with the solvent used 14
a
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Fig. 11.17 (a) Ch oxidation by two dehydrogenase reactions and (b) trehalose hydrolysis by trehalase. PA’s sole possession of these two types (a and b) of activities differentiates it from C. violaceum and R. solanacearum (negative in both), determining the differential Ch-induced stimulation of PA-lectin production versus trehalose-induced stimulation of CV- and RS-lectin production
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Fig. 11.18 A schematic model of possible Ch involvement in stimulating PA (but not C. violaceum or R. solanacearum) virulence via QS activation. The induced PA-IL and PA-IIL contribute to biofilm formation and binding to host cell receptors that bear terminal galactose and fucose (mainly Lea) residues, respectively. That binding enables HSL to modulate the host immunity, proteolytic activity to unmask more glycolipid-associated galactosylated residues (sialidase also helps this process), PLC-H to degradate phospholipids with phosphoryl Ch release, and phosphatases to release Ch from the latter. The resulting Ch further amplifies QS and VIF formation and vice versa
The ability of our PA ATCC33347 to decompose trehalose was shown by our use of glucose oxidase as a reagent for detecting glucose release. No homologous sequence or similar activity was found in C. violaceum. The above-described findings – showing that trehalose, which is not associated with Ch in structure or metabolism, induces lectin production in C. violaceum and R. solanacearum, which cannot enjoy the Ch osmoprotecting potential – indicate that osmoprotection is the stimulating principle, as already claimed by Sage and Vasil in regard to PLC-H stimulation by Ch [77]. It therefore may be suggested that Ch-induced stimulation of PA-lectin production, owing to the ability of PA to convert it to the osmoprotectant glycine betaine, is the key for its special mode of pathogenicity, which is not shared with C. violaceum and R. solanacearum (which are stimulated by trehalose that is not hydrolyzed by them). The osmoprotectant stimulation might result from a positive modulating effect or stabilization of the QS circuit complexes, preventing their dissociation or decomposition, leading to augmented lectin and other VIF expression [92] (Fig. 11.18).
11.8 Ethanol Effects on the Levels of PA Lectins and Additional VIFs Addition of 1% ethanol (Et) to PA cultures, in absence or presence of Ch, considerably increases PA-lectin production, especially PA-IIL [92], as well as HSLs, pyocyanin, proteases, rhamnolipids (heat-stable hemolysins) [93], and alginate (PA mucopolysaccharide) [94] (Figs. 11.19 and 11.20), leading to a considerable virulence increase [92].
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Fig. 11.19 The effects of the addition of Et, subinhibitory erythromycin (ERM), and their combination to Ch-containing PA cultures on the production of PA-IL, PA-IIL, proteases (Pr), pyocyanin (Py), PLC-H, rhamnolipids (RhaL), and HSLs [107]
Fig. 11.20 The effects of the addition of Et, subinhibitory erythromycin (ERM), and their combination to Ch-containing PA cultures on the production of the short-chain HSL autoinducers. The HSLs obtained from concentrated supernatant extracts and separated on TLC plates were biostained by violacein produced by an HSL-dependent C. violaceum CVO26 mutant [107], according to McClean et al. [108]
Et consumption was reported to aggravate PA infections, especially in pulmonary diseases [94, 95] due to suppression of immune resistance [96, 97] and the concomitant increase in the levels of microbial VIFs [93, 94]. The combined Ch and Et effect varies between additivity in regard to pyocyanin and HSL and some antagonism in regard to the proteolytic, hemolytic, and acid phosphatase activities [92].
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11.9 Repression of PA Lectin Production The central role of the PA lectins in infection establishment, and their close linkage to the VIFs in function and production, pose the goal of finding a way to abolish their common production. This approach is more efficient than inhibition of the lectin activity since it is a root treatment and also includes the other VIF abolishment.
11.9.1 The Effects of Subinhibitory Erythromycin Concentration on PA Lectin and VIF Production One of the ways to achieve the purpose of PA lectin repression is to inhibit the QS system using either halogenated furanones as HSL-competing analogues or subinhibitory concentrations of antibiotics (mainly macrolides, e.g., erythromycin [ERM] and azithromycin) that repress lectin and other VIF production. The latter manipulation is based on encouraging reports that, despite global antibiotic resistance to PA, the condition of patients suffering from its pulmonary infections was profoundly improved after long-term (years) therapy using subinhibitory concentrations of ERM [98, 99] or azithromycin. Positive results were also reported in regard to patients with CF [100, 101]. These treatments increased the life expectancy of the treated patients and improved their respiratory function without eradicating the bacteria [99]. In vitro, subinhibitory ERM was shown to suppress PA production of exotoxin A and protease [102], PLC-H, and alginate [103], as well as biofilm formation and adherence to bronchial mucins of patients with CF. In 1999, subinhibitory ERM was also found to repress PA-IL and PA-IIL production together with its pyocyanin, hemolytic, and proteolytic activities, in association with HSL reduction [104]. Partial HSL reduction by azithromycin was also found in extracts of lung tissues of patients with CF infected with PA [105]. Recently, based on their experimental results on global transcription and protein expression, Nalca et al. [106] suggested that subinhibitory azithromycin interferes with QS and that this fact holds great promise for macrolide therapy of PA and other bacterial infections in patients with CF.
11.9.2 The Effects of Subinhibitory Concentration of ERM on Lectin and Other VIF Production in Presence of Ch with and Without Et The addition of ERM in subinhibitory concentration to Ch-containing PA cultures [107] was found to be less effective in reducing the PA-IIL [7], HSL [108], and PLC-H [109] levels than in cultures without Ch [107]. In contrast, its addition to 1% Et-containing cultures (displaying increased levels of PA lectins, rhamnolipids, pyocyanin, and HSLs) caused a profound reduction of these activities. Paradoxically,
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the addition of 1% Et to the Ch-containing cultures, which increased the VIF levels in the antibiotic absence, led to a dramatic annulment of the PA lectins and most of the other VIFs examined (Fig. 11.19), including the HSLs (Fig. 11.20) [107].
11.10 Summary PA and C. violaceum are soil-dwelling saprophytic Gram-negative bacteria that are convertible to opportunistic, life-threatening pathogens. Their pathogenicity depends on the possession of adhesins that bind to the target cells, including lectins, and a battery of additional VIFs that aggressively attack them. The PA lectins PA-IL (galactophilic) and PA-IIL (fucose > mannose-binding) were discovered first and proved to resemble classic plant and animal lectins in properties, activities, biological effects, and applications. The discovery of these bacterial lectins has introduced new aspects to lectinology, owing to their association with bacteria and pathogenicity: (1) their special sugar specificities that adapt them to mediate bacterial adhesion to target host cells; (2) their involvement in bacterial binding to target cells and molecules; (3) their contribution to PA biofilm formation; (4) their cofunction with the pathogen’s aggressive proteinaceous and nonproteinaceous VIFs; (5) their interactions with foreign bacteria in their vicinity, leading to their extinction; (6) their contribution to pathogen host selectivity and differential organ distribution; (7) their effects on the target cell membranes and metabolism; (8) their possible usage as a vaccine for PA infection prevention; (9) their usage as probes for natural and synthetic compounds (mainly glycoconjugates) that would block pathogen adhesion to the target cells; (10) the properties of the bacterial genes that encode their production; (11) existence of homologous lectins in related bacteria; (12) induction and regulation of their production; (13) interlinkage of their production to those of the VIFs that cofunction with them; (14) participation of hostderived compounds and nutrients in triggering their production; and (15) abrogation of the bacterial lectin production, preferentially together with that of the VIFs. Discovery of the PA-IL and PA-IIL genes (lecA and lecB) and their sequences enabled the unveiling of their 3-D crystal structures, including their Ca2+ atoms and the detailed interactions of the binding-site amino acids and Ca2+ with the specific carbohydrates. The PA-IIL gene information also led to the fascinating findings of additional fucose/mannose-binding bacterial lectins – RSL, RS-IIL and CV-IIL – in the aggressive plant pathogen R. solanacearum and in the human/animal pathogen C. violaceum. RSL is related to PA-IIL only in specificity but not in structure, while RS-IIL and CV-IIL are PA-IIL structural homologues. Their production in old (stationary phase) cultures is driven by QS signals and additional regulators. PA-lectin formation is strongly stimulated by exogenous Ch [(CH3)3–N+CH2CH2OH] (which is converted by PA to the endogenous osmoprotectant glycine betaine) but not by the nonreducing di-a-1-glucoside osmoprotectant trehalose (which is decomposed by PA trehalase). The levels of RSL, RS-IIL, and CV-IIL are only increased by trehalose since R. solanacearum and C. violaceum do not convert Ch to glycine
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betaine and do not hydrolyze trehalose. Hence, the combination of high Ch levels with many terminal PA-IL- and PA-IIL-attracting galactose and fucose (of Lea epitope) residues might underlie the increased PA-associated morbidity and mortality in patients with CF. The Ch stimulation of PA lectins and additional VIF production, e.g., PLC-H [109], hinders cure of the infection, even by the widely used, effective macrolide treatment. Our recent studies showed that the addition of 1% Et (which by itself stimulates PA-lectin production) paradoxically enforces the EMCrepressing effect in Ch presence, totally annulling the production of the lectins and most of the other VIFs examined, excluding PLC-H, which was partially reduced. These findings might be encouraging for local/external PA infection therapy. Acknowledgments The authors thank Ms. Sharon Victor and Ms. Ela Gindy for their skillful help in editing the manuscript, preparing it for publication, and assisting in the graphical presentations.
References 1. Stover CK, Pham XQ, Erwin AL et al (2000) Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406:959–964 2. Salanoubat M, Genin S, Artiguenave F et al (2002) Genome sequence of the plant pathogen Ralstonia solanacearum. Nature 415:497–502 3. Vasconcelos ATR et al and Brazilian National Genome Project Consortium (2003) The complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability. Proc Natl Acad Sci USA 100:11660–11665 4. Valls M, Genin S, Boucher C (2006) Integrated regulation of the type III secretion system and other virulence determinants in Ralstonia solanacearum. PLoS Pathog 2:e82 5. Gilboa-Garber N (1972) Inhibition of broad spectrum hemagglutinin from Pseudomonas aeruginosa by D-galactose and its derivatives. FEBS Lett 20:242–244 6. Gilboa-Garber N, Mizrahi L, Garber N (1977) Mannose-binding hemagglutinins in extracts of Pseudomonas aeruginosa. Can J Biochem 55:975–981 7. Gilboa-Garber N (1982) Pseudomonas aeruginosa lectins. Methods Enzymol 83:378–385 8. Gilboa-Garber N (1997) Multiple aspects of Pseudomonas aeruginosa lectins. Nova Acta Leopold 75:153–177 9. Garber N, Guempel U, Gilboa-Garber N, Doyle RJ (1987) Specificity of the fucose-binding lectin of Pseudomonas aeruginosa. FEMS Microbiol Lett 48:331–334 10. Garber N, Guempel U, Belz A, Gilboa-Garber N, Doyle RJ (1992) On the specificity of the D-galactose-binding lectin (PA-I) of Pseudomonas aeruginosa and its strong binding to hydrophobic derivatives of D -galactose and thiogalactose. Biochim Biophys Acta 1116:331–333 11. Gilboa-Garber N (1983) The biological functions of Pseudomonas aeruginosa lectins. In: Bog-Hansen TC, Spengler GA (eds) Lectins: biology, biochemistry, clinical biochemistry. Walter de Gruyter, Berlin, pp 495–502 12. Gilboa-Garber N (1986) Lectins of Pseudomonas aeruginosa: properties, biological effects and applications. In: Mirelman D (ed) Microbial lectins and agglutinins: properties and biological activity. Wiley, New York, pp 255–269 13. Chen CP, Song SC, Gilboa-Garber N, Chang KSS, Wu AM (1998) Studies on the binding site of the galactose-specific agglutinin PA-IL from Pseudomonas aeruginosa. Glycobiology 8:7–16 14. Cioci G, Mitchell EP, Gautier C, Wimmerova M, Sudakevitz D, Perez S, Gilboa-Garber N, Imberty A (2003) Structural basis of calcium and galactose recognition by the lectin PA-IL of Pseudomonas aeruginosa. FEBS Lett 555:297–301
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15. Mitchell E, Houles C, Sudakevitz D, Wimmerova M, Gautier C, Perez S, Wu AM, Gilboa-Garber N, Imberty A (2002) Structural basis for oligosaccharide-mediated adhesion of Pseudomonas aeruginosa in the lungs of cystic fibrosis patients. Nat Struct Biol 9:918–921 16. Wu AM, Wu JH, Singh T, Liu JH, Tsai MS, Gilboa-Garber N (2006) Interactions of the fucose-specific Pseudomonas aeruginosa lectin, PA-IIL, with mammalian glycoconjugates bearing polyvalent Lewis(a) and ABH blood group glycotopes. Biochimie 88:1479–1492 17. Glick J, Garber N (1983) The intracellular localization of Pseudomonas aeruginosa lectins. J Gen Microbiol 129:3085–3090 18. Gilboa-Garber N, Garber N (1989) Microbial lectin cofunction with lytic activities as a model for a general basic lectin role. FEMS Microbiol Rev 63:211–221 19. Winzer K, Falconer C, Garber NC, Diggle SP, Camara M, Williams P (2000) The Pseudomonas aeruginosa lectins PA-IL and PA-IIL are controlled by quorum sensing and by RpoS. J Bacteriol 182:6401–6411 20. Diggle SP, Winzer K, Lazdunski A, Williams P, Camara M (2002) Advancing the quorum in Pseudomonas aeruginosa: MvaT and the regulation of N-acylhomoserine lactone production and virulence gene expression. J Bacteriol 184:2576–2586 21. Liu PV, Hsieh HC (1969) Inhibition of protease production of various bacteria by ammonium salts: its effect on toxin production and virulence. J Bacteriol 99:406–413 22. Gilboa-Garber N, Avichezer D, Garber NC (1997) Bacterial lectins: properties, structure, effects, function and applications. In: Gabius HJ, Gabius S (eds) Glycosciences: status and perspectives. Chapman & Hall, Weinheim, pp 369–398 23. Gilboa-Garber N, Sudakevitz D, Sheffi M, Sela R, Levene C (1994) PA-I and PA-II lectin interactions with the ABO(H) and P blood group glycosphingolipid antigens may contribute to the broad spectrum adherence of Pseudomonas aeruginosa to human tissues in secondary infections. Glycoconj J 11:414–417 24. Lanne B, Ciopraga J, Bergstrom J, Motas C, Karlsson KA (1994) Binding of the galactosespecific Pseudomonas aeruginosa lectin, PA-I, to glycosphingolipids and other glycoconjugates. Glycoconj J 11:292–298 25. Sudakevitz D, Gilboa-Garber N (1982) Effect of Pseudomonas aeruginosa lectins on phagocytosis of Escherichia coli strains by human polymorphonuclear leucocytes. Microbios 34:159–166 26. Gilboa-Garber N, Sharabi Y (1980) Increase of growth-rate and phagocytic activity of Tetrahymena induced by Pseudomonas lectins. J Protozool 27:209–211 27. Gilboa-Garber N, Blonder E (1979) Augmented osmotic hemolysis of human erythrocytes exposed to the galactosephilic lectin of Pseudomonas aeruginosa. Isr J Med Sci 15:537–539 28. Sharabi Y, Gilboa-Garber N (1979) Mitogenic stimulation of human lymphocytes by Pseudomonas aeruginosa galactophilic lectin. FEMS Microbiol Lett 5:273–276 29. Avichezer D, Gilboa-Garber N (1987) PA-II, the L-fucose- and D-mannose-binding lectin of Pseudomonas aeruginosa stimulates human peripheral lymphocytes and murine splenocytes. FEBS Lett 216:62–66 30. Wentworth JS, Austin FE, Garber N, Gilboa-Garber N, Paterson CA, Doyle RJ (1991) Cytoplasmic lectins contribute to the adhesion of Pseudomonas aeruginosa. Biofouling 4:99–104 31. Bajolet-Laudinat O, Girod-de Bentzmann S, Tournier JM, Madoulet C, Plotkowski MC, Chippaux C, Puchelle E (1994) Cytotoxicity of Pseudomonas aeruginosa internal lectin PA-I to respiratory epithelial cells in primary culture. Infect Immun 62:4481–4487 32. Adam EC, Schumacher DU, Schumacher U (1997) Cilia from a cystic fibrosis patient react to the ciliotoxic Pseudomonas aeruginosa II lectin in a similar manner to normal control cilia – a case report. J Laryngol Otol 111:760–762 33. Grant G, Bardocz S, Ewen SWB, Brown DS, Duguid TJ, Pusztai A, Avichezer D, Sudakevitz D, Belz A, Garber NC, Gilboa-Garber N (1995) Purified Pseudomonas aeruginosa PA-I lectin induces gut growth when orally ingested by rats. FEMS Immunol Med Microbiol 11:191–195 34. Laughlin RS, Musch MW, Hollbrook CJ, Rocha FM, Chang EB, Alverdy JC (2000) The key role of Pseudomonas aeruginosa PA-I lectin on experimental gut-derived sepsis. Ann Surg 232:133–142
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35. Wu L, Holbrook C, Zaborina O, Ploplys E, Rocha F, Pelham D, Chang E, Musch M, Alverdy J (2003) Pseudomonas aeruginosa expresses a lethal virulence determinant, the PA-I lectin/ adhesin, in the intestinal tract of a stressed host: the role of epithelia cell contact and molecules of the Quorum Sensing Signaling System. Ann Surg 238:754–764 36. Wu L, Estrada O, Zaborina O, Bains M, Shen L, Kohler JE, Patel N, Musch MW, Chang EB, Fu YX, Jacobs MA, Nishimura MI, Hancock RE, Turner JR, Alverdy JC (2005) Recognition of host immune activation by Pseudomonas aeruginosa. Science 309:774–777 37. Kirkeby S, Moe D (2005) Analyses of Pseudomonas aeruginosa lectin binding to alphagalactosylated glycans. Curr Microbiol 50:309–313 38. Kirkeby S, Wimmerova M, Moe D, Hansen AK (2007) The mink as an animal model for Pseudomonas aeruginosa adhesion: binding of the bacterial lectins (PA-IL and PA-IIL) to neoglycoproteins and to sections of pancreas and lung tissues from healthy mink. Microbes Infect 9:566–573 39. Keicho N, Kudoh S (2002) Diffuse panbronchiolitis: role of macrolides in therapy. Am J Respir Med 1:119–131 40. Gilboa-Garber N, Sudakevitz D (1982) The use of Pseudomonas aeruginosa lectin preparations as a vaccine. In: Levy E (ed) Advances in pathology. Pergamon, Oxford, pp 31–33 41. Avichezer D, Gilboa-Garber N, Mumcuoglu M, Slavin S (1989) Adoptive transfer of resistance to Pseudomonas aeruginosa infection by splenocytes and bone marrow cells from BALB/c mice immunized by Pseudomonas aeruginosa lectin preparations. Infection 17:407–410 42. Avichezer D, Katcoff DJ, Garber NC, Gilboa-Garber N (1992) Analysis of the amino acid sequence of the Pseudomonas aeruginosa galactophilic PA-I lectin. J Biol Chem 267:23023–23027 43. Avichezer D, Gilboa-Garber N, Garber NC, Katcoff DJ (1994) Pseudomonas aeruginosa PA-I lectin gene molecular analysis and expression in Escherichia coli. Biochim Biophys Acta 1218:11–20 44. Gilboa-Garber N, Katcoff DJ, Garber NC (2000) Identification and characterization of Pseudomonas aeruginosa PA-IIL lectin gene and protein compared to PA-IL. FEMS Immunol Med Microbiol 29:53–57 45. Sudakevitz D, Imberty A, Gilboa-Garber N (2002) Production, properties and specificity of a new bacterial L-fucose and D-arabinose-binding lectin of the plant aggressive pathogen Ralstonia solanacearum, and its comparison to related plant and microbial lectins. J Biochem 132:353–358 46. Sudakevitz D, Kostlanova N, Blatman-Jan G, Mitchell EP, Lerrer B, Wimmerova M, Katcoff DJ, Imberty A, Gilboa-Garber N (2004) A new Ralstonia solanacearum high-affinity mannosebinding lectin RS-IIL structurally resembling the Pseudomonas aeruginosa fucose-specific lectin PA-IIL. Mol Microbiol 52:691–700 47. Zinger-Yosovich K, Sudakevitz D, Imberty A, Garber NC, Gilboa-Garber N (2006) Production and properties of the native Chromobacterium violaceum fucose-binding lectin (CV-IIL) compared to homologous lectins of Pseudomonas aeruginosa (PA-IIL) and Ralstonia solanacearum (RS-IIL). Microbiology 152(Pt 2):457–463 48. Pokorna M, Cioci G, Perret S, Rebuffet E, Kostlanova N, Adam J, Gilboa-Garber N, Mitchell EP, Imberty A, Wimmerova M (2006) Unusual entropy-driven affinity of Chromobacterium violaceum lectin CV-IIL toward fucose and mannose. Biochemistry 45:7501–7510 49. Mitchell EP, Sabin C, Snajdrova L, Pokorna M, Perret S, Gautier C, Hofr C, Gilboa-Garber N, Koca J, Wimmerova M, Imberty A (2005) High affinity fucose binding of Pseudomonas aeruginosa lectin PA-IIL: 1.0 Ǻ resolution crystal structure of the complex combined with thermodynamics and computational chemistry approaches. Proteins 58:735–746 50. Rieger J, Stoffelbach F, Cui D, Imberty A, Lameignere E, Putaux JL, Jerome R, Jerome C, Auzely VR (2007) Mannosylated poly(ethylene oxide)-b-poly(epsilon-caprolactone) diblock copolymers: synthesis, characterization, and interaction with a bacterial lectin. Biomacromolecules 8:2717–2725
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51. Imberty A, Wimmerova M, Mitchell EP, Gilboa-Garber N (2004) Structures of the lectins from Pseudomonas aeruginosa: insight into the molecular basis for host glycan recognition. Microbes Infect 6:221–228 52. Kostlanova N, Mitchell EP, Lortat-Jacob H, Oscarson S, Lahmann M, Gilboa-Garber N, Chambat G, Wimmerova M, Imberty A (2005) The fucose-binding lectin from Ralstonia solanacearum. A new type of beta-propeller architecture formed by oligomerization and interacting with fucoside, fucosyllactose, and plant xyloglucan. J Biol Chem 280: 27839–27849 53. Adam J, Pokorna M, Sabin C, Mitchell EP, Imberty A, Wimmerova M (2007) Engineering of PA-IIL lectin from Pseudomonas aeruginosa – unravelling the role of the specificity loop for sugar preference. BMC Struct Biol 7:36 54. Newburg DS, Ruiz-Palacios GM, Morrow AL (2005) Human milk glycans protect infants against enteric pathogens. Annu Rev Nutr 25:37–58 55. Lesman-Movshovich E, Lerrer B, Gilboa-Garber N (2003) Blocking of Pseudomonas aeruginosa lectins by human milk glycans. Can J Microbiol 49:230–235 56. Lesman-Movshovich E, Gilboa-Garber N (2003) Pseudomonas aeruginosa lectin PA-IIL as a powerful probe for human and bovine milk analysis. J Dairy Sci 86:2276–2282 57. Johnson JR, Berggren T (1994) Pigeon and dove eggwhite protect mice against renal infection due to P fimbriated Escherichia coli. Am J Med Sci 307:335–339 58. Suzuki N, Khoo KH, Chen HC, Johnson JR, Lee YC (2001) Isolation and characterization of major glycoproteins of pigeon egg white: ubiquitous presence of unique N-glycans containing Gala1-4Gal. J Biol Chem 276:23221–23229 59. Lerrer B, Gilboa-Garber N (2001) Interaction of Pseudomonas aeruginosa galactophilic lectin PA-IL with pigeon egg white glycoproteins. FEMS Immun Med Microbiol 32:33–36 60. Lerrer B, Gilboa-Garber N (2001) Interactions of Pseudomonas aeruginosa PA-IIL lectin with quail egg white glycoproteins. Can J Microbiol 47:1095–1100 61. Lerrer B, Zinger-Yosovich KD, Avrahami B, Gilboa-Garber N (2007) Honey and royal jelly, like human milk, abrogate lectin-dependent infection-preceding Pseudomonas aeruginosa adhesion. ISME J 1:149–155 62. Perret S, Sabin C, Dumon C, Pokorna M, Gautier C, Galanina O, Ilia S, Bovin N, Nicaise M, Desmadril M, Gilboa-Garber N, Wimmerova M, Mitchell EP, Imberty A (2005) Structural basis for the interaction between human milk oligosaccharides and the bacterial lectin PA-IIL of Pseudomonas aeruginosa. Biochem J 389:325–332 63. Deguise I, Lagnoux D, Roy R (2007) Synthesis of glycodendrimers containing both fucoside and galactoside residues and their binding properties to PA-IL and PA-IIL lectins from Pseudomonas aeruginosa. New J Chem 31:1321–1331 64. Johansson EMV, Kolomiets E, Rosenau F, Jaeger KE, Darbre T, Reymond JL (2007) Combinatorial variation of branching length and multivalency in a large (390 625 member) glycopeptide dendrimer library: ligands for fucose-specific lectins. New J Chem 31:1291–1299 65. Marotte K, Preville C, Sabin C, Moume-Pymbock M, Imberty A, Roy R (2007) Synthesis and binding properties of divalent and trivalent clusters of the Lewis a disaccharide moiety to Pseudomonas aeruginosa lectin PA-IIL. Org Biomol Chem 5:2953–2961 66. Gilboa-Garber N, Zakut V, Mizrahi L (1973) Production of cholinesterase by Pseudomonas aeruginosa, its regulation by glucose and cyclic AMP and inhibition by antiserum. Biochim Biophys Acta 297:120–124 67. Wagner VE, Frelinger JG, Barth RK, Iglewski BH (2006) Quorum sensing: dynamic response of Pseudomonas aeruginosa to external signals. Trends Microbiol 14:55–58 68. Schuster M, Greenberg EP (2006) A network of networks: quorum-sensing gene regulation in Pseudomonas aeruginosa. Int J Med Microbiol 296:73–81 69. Brint JM, Ohman DE (1995) Synthesis of multiple exoproducts in Pseudomonas aeruginosa is under the control of RHLR-RHLI, another set of regulators in strain PAO1 with homology to the autoinducer-responsive LUXR-LUXI family. J Bacteriol 177:7155–7163
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88. Govan JR, Harris GS (1986) Pseudomonas aeruginosa and cystic fibrosis: unusual bacterial adaptation and pathogenesis. Microbiol Sci 3:302–308 89. Govan JR, Nelson JW (1992) Microbiology of lung infection in cystic fibrosis. Br Med Bull 48:912–930 90. Chen AH, Innis SM, Davidson AG, James SJ (2005) Phosphatidylcholine and lysophosphatidylcholine excretion is increased in children with cystic fibrosis and is associated with plasma homo-cysteine, S-adenosylhomocysteine, and S-adenosylmethionine. Am J Clin Nutr 81:686–691 91. Antonio RV, Creczynski-Pasa TB (2004) Genetic analysis of violacein biosynthesis by Chromobacterium violaceum. Genet Mol Res 3:85–91 92. Katri N, Gilboa-Garber N (2007) Ethanol effects on Pseudomonas aeruginosa lectin, protease, hemolysin, pyocyanin, autoinducer and phosphatase levels depending on medium composition and choline presence. Curr Microbiol 54:296–301 93. Matsufuji M, Nakata K, Yoshimoto A (1997) High production of rhamnolipids by Pseudomonas aeruginosa growing on ethanol. Biotechnol Lett 19:1213–1215 94. DeVault JD, Kimbara K, Chakrabarty AM (1990) Pulmonary dehydration and infection in cystic fibrosis: evidence that ethanol activates alginate gene expression and induction of mucoidy in Pseudomonas aeruginosa. Mol Microbiol 4:737–745 95. de Roux A, Cavalcanti M, Marcos MA, Garcia E, Ewig S, Mensa J, Torres A (2006) Impact of alcohol abuse in the etiology and severity of community-acquired pneumonia. Chest 129:1219–1225 96. Greenberg SS, Zhao X, Hua L, Wang JF, Nelson S, Ouyang J (1999) Ethanol inhibits lung clearance of Pseudomonas aeruginosa by a neutrophil and nitric oxide-dependent mechanism, in vivo. Alcohol Clin Exp Res 23:735–744 97. Jerrells TR (1991) Immunodeficiency associated with ethanol abuse. Adv Exp Med Biol 288:229–236 98. Kita E, Sawaki M, Oku D, Hamuro A, Mikasa K, Konishi M, Emoto M, Takeuchi S, Narita N, Kashiba S (1991) Suppression of virulence factors of Pseudomonas aeruginosa by erythromycin. J Antimicrob Chemother 27:273–284 99. Fujii T, Kadota J, Kawakami K, Iida K, Shirai R, Kaseda M, Kawamoto S, Kohno S (1995) Long term effect of erythromycin therapy in patients with chronic Pseudomonas aeruginosa infection. Thorax 50:1246–1252 100. Jaffe A, Francis J, Rosenthal M, Bush A (1998) Long-term azithromycin may improve lung function in children with cystic fibrosis. Lancet 351:420 101. Carr RR, Nahata MC (2004) Azithromycin for improving pulmonary function in cystic fibrosis. Ann Pharmacother 38:1520–1524 102. Hirakata Y, Kaku M, Mizukane R, Ishida K, Furuya N, Matsumoto T, Tateda K, Yamaguchi K (1992) Potential effects of erythromycin on host defense systems and virulence of Pseudomonas aeruginosa. Antimicrob Agents Chemother 36:1922–1927 103. Majtan V, Hybenova D (1996) Inhibition of Pseudomonas aeruginosa alginate expression by subinhibitory concentrations of antibiotics. Folia Microbiol (Praha) 41:61–64 104. Sofer D, Gilboa-Garber N, Belz A, Garber NC (1999) ‘Subinhibitory’ erythromycin represses production of Pseudomonas aeruginosa lectins, autoinducer and virulence factors. Chemotherapy 45:335–341 105. Favre-Bonte S, Kohler T, Van Delden C (2003) Biofilm formation by Pseudomonas aeruginosa: role of the C4-HSL cell-to-cell signal and inhibition by azithromycin. J Antimicrob Chemother 52:598–604 106. Nalca Y, Jansch L, Bredenbruch F, Geffers R, Buer J, Hussler S (2006) Quorum-sensing antagonistic activities of azithromycin in Pseudomonas aeruginosa PAO1: a global approach. Antimicrob Agents Chemother 50:1680–1688 107. Katri N, Garber NC, Kilfin G, Gilboa-Garber N (2008) Abrogation of the resistance of choline-induced Pseudomonas aeruginosa virulence to sub-MIC erythromycin by ethanol. ISME J 2:1243–1246
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Chapter 12
Non-carbohydrate-Mediated Interaction of Lectins with Plant Proteins Jared Q. Gerlach, Michelle Kilcoyne, Seron Eaton, Veer Bhavanandan, and Lokesh Joshi
Keywords Plant protein • Lectins • Glycosylation • Carbohydrate • Non-carbohydratemediated binding • Non-mammalian • Glycoprotein
12.1 Introduction Glycosylation is the most common posttranslational modification of proteins and plays diverse roles in numerous biological processes, including fertilization, development, differentiation, inflammation, cancer metastasis, and host–pathogen/parasite interactions. A number of glycosylated proteins are bioactive molecules of medical/ therapeutic or other commercial interest and are currently produced by recombinantly transformed cells and organisms. Among non-animal expression systems, plant cells and transgenic plants are considered an attractive alternative system for recombinant human and animal glycoproteins. The advantages of using plants for the production of commercially important glycosylated proteins include lower manufacturing costs and a reduced risk of transmitting mammalian pathogens [11, 27]. However, a major roadblock in the use of plants for this purpose is the lack of available information on N- and O-linked glycans in plants and specifically those in the endogenous plant glycosylation pathways [9, 31]. Thus, gathering detailed structural information on plant-derived glycoproteins is of utmost importance. The traditional technologies used for the structural analysis of glycans, such as mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy, are highly sophisticated and precise in their analyses. These techniques are not only time and labor intensive but, also require expensive equipment, expert personnel,
J.Q. Gerlach (*) Glycoscience Group, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland e-mail:
[email protected] A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_12, © Springer Science+Business Media, LLC 2011
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and relatively pure and large sample quantities [16]. Furthermore, the emerging field of glycomics, i.e., the functional assignment of glycans and lectins [21, 29], requires rapid analysis of the glycosylation status of proteins and cell surfaces in a systematic manner. Therefore, for rapid and high-throughput structural analysis, carbohydrate-binding proteins, especially lectins, are thought to offer great potential [14, 19, 26]. Various lectins, which are capable of binding a diverse array of glycan structures, are a major means by which glycosignatures on cell surfaces and of various molecules are decoded and the dynamics of carbohydrate structural variations in diseases interpreted [4, 7]. Thus, for the past few decades, lectin-based assays such as agglutination, mitogen stimulation, histology, blotting, affinity chromatography, and flow cytometry have been popular tools for glycan detection and characterization [22, 24, 32]. More recent developments in lectin-based technologies have included lectin arrays and biosensors for uses such as differentiating terminal glycans [5, 10, 15]. The characteristics of lectin–carbohydrate interactions for mammalian glycoconjugates have been well established, and the information is utilized by researchers to obtain further details on animal glycan structure [1, 23]. In contrast, the specificity of lectins (many of which in common usage are plant-derived) for nonmammalian glycoconjugates, particularly those from plants, has not been critically evaluated, although this information is crucial for structure–function data interpretation [31]. To fully realize the potential of lectin-based rapid glycan analyses in the field of plant glycomics, a more complete understanding of lectin–plant glycoprotein specificity is necessary. We have been utilizing lectin-based technologies, in addition to techniques such as high-performance liquid chromatography (HPLC) and MS, to investigate glycosylation of proteins in plants such Arabidopsis thaliana, tobacco, and rice.
12.2 Investigation of the Glycoproteins in A. thaliana A. thaliana has been widely investigated and has several advantages as an experimental system. These include its small genome, which has been sequenced; an established suspension cell culture system with a rapid life cycle; and its popular and widespread use in molecular, genetic, and transfection studies. Previous research in our laboratory revealed a strong interaction of several lectins with cultured A. thaliana cells and their protoplasts, prepared by treatment with cell wallsolubilizing enzymes [Shah M (2005) Dissertation, Arizona State University]. Of special interest was the binding of Sambucus nigra-I (SNA-I) and Maackia amurensis (MAA) lectins with specificity for sialic acid and Vicia villosa agglutinin (VVA), Arachis hypogaea (peanut) agglutinin (PNA), and Artocarpus integrifolia (AIA, jacalin) with specificity for GalNAc-Ser/Thr-linked structures. This observation prompted us to investigate the structures of the potential glycoproteins in A. thaliana suspension-cultured cells, which may have interacted with the abovementioned lectins. The cells were grown in suspension at room temperature in a
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medium made from MS519 salts (Sigma), vitamins, sucrose, a-naphthalene acetic acid, and kinetin. After seven days, the cells were harvested and delipidated with acetone followed by a mixture of chloroform and methanol (2:1 and 1:2). The delipidated cells were suspended in phosphate-buffered saline (PBS) containing protease inhibitor cocktail (Sigma) and sodium azide (0.02%), disintegrated using a French press, and the suspension was stirred overnight at 4°C. The extract was centrifuged (10,000 RPM, 30 min), the supernatant saved, and the residue extracted again as above but with PBS containing 0.1% Triton X-100. The PBS and PBSTriton extracts were analyzed for protein content by the micro bicinchoninic acid (BCA) assay and the remainder was stored in aliquots at −20°C until required.
12.2.1 Enzyme-Linked Lectin Assay (ELLA) A common and readily available method to quantitate the interaction of lectins with macromolecular glycoconjugates is the microtiter plate ELLA [6, 17]. Accordingly, 96-well microtiter plates were coated with PBS and PBS-Triton extracts of A. thaliana cells, and the binding of various biotinylated lectins to the bound plant proteins was determined using avidin-alkaline phosphatase and p-nitrophenyl phosphate substrate (Sigma). The assays were performed in triplicate and the mean values of the results were plotted. The results of typical experiments, as illustrated in Fig. 12.1, show that several lectins bound strongly to both extracts. Concanavalin A (ConA), SNA-I, AIA (jacalin), and VVA showed high binding to components in the PBS extract, whereas VVA, Lotus tetragonolobus lectin (LTA), and AIA bound maximally to components in the PBS-Triton extract. Experiments were then done to
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determine the nature of the glycoconjugates present in these extracts that interacted with the lectins. Since the cells were delipidated prior to the extraction, it was considered unlikely that glycolipids and lipids were contributing to the binding. While proteins and glycoproteins are insoluble in perchloric acid, polysaccharides and glycoproteins with very high carbohydrate content (mucins) remain in solution [2]. Therefore, to distinguish between glycoproteins/proteins and mucins/polysaccharides, the interaction of jacalin with PBS extract, which was pretreated with perchloric acid to remove mucins and polysaccharides, was tested. The results illustrated in Fig. 12.2 demonstrate that jacalin binding was not significantly influenced. Furthermore, the binding of jacalin was abolished if the extract was exhaustively (overnight) treated with Pronase. These results suggest that the lectin-binding components in the PBS extract of A. thaliana are protein-based molecules and are likely to be glycoproteins. To determine the specific nature of the binding of the biotinylated lectins to the putative glycoproteins, a series of experiments were conducted to test the ability of saccharides to inhibit the binding. Surprisingly, the binding of biotinylated VVA to A. thaliana cell proteins was not inhibited by a variety of monosaccharides (d-GalNAc, d-Gal, d-ManNAc, l-Rha, l-Ara, d-Xyl, d-GalN, d-Man, d-Glc, and amethylmannoside) up to the highest concentration (100 mM) tested (Fig. 12.3). It should be noted that the saccharides tested included typical plant sugars such as l-Rha, l-Ara, d-Xyl, and d-Glc. In control experiments, the binding of VVA to asialo ovine submaxillary mucin was inhibited by 10 mM d-GalNAc and d-Gal, which are reported to be haptenic sugars for VVA [20, 28]. Similarly, the binding 0.7 0.6
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Fig. 12.2 Binding of biotinylated jacalin before (─●─) and after perchloric acid precipitation (∙∙∙○∙∙∙) or Pronase treatment (─■─) of PBS extract of delipidated Arabidopsis thaliana cells
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Fig. 12.3 Effect of monosaccharides on the binding of biotinylated VVA (left) and biotinylated SNA-I (right) to PBS extract of delipidated Arabidopsis thaliana cells. GalNAc (filled circle), ManNAc (open circle), Gal, (x), rhamnose (filled diamond), lactose (filled square), NeuNAc (open diamond). VVA binding was also not inhibited by d-arabinose, d-xylose, or d-galactosamine, and SNA-I binding was not inhibited by d-glucuronic acid, d-galacturonic acid, or KDO
of biotinylated SNA-I to A. thaliana cell proteins was also not inhibited by 100 mM of either typical animal sugars (NeuNAc, lactose, d-Gal, d-GalNAc, d-glucuronic acid) or plant sugars (ketodeoxyoctulosonic acid [KDO], d-galacturonic acid) (not illustrated). In control experiments, the binding of SNA-I to fetuin was inhibited by lactose, d-GalNAc, and d-Gal, which are reported to be haptenic sugars for SNA-I [3, 25]. In addition, VVA and jacalin conjugated to alkaline phosphatase gave results similar to those obtained with biotinylated VVA and jacalin, respectively, which eliminated the possibility that the observed phenomenon was a peculiarity of the biotin/avidin system.
12.2.2 Lectin Blotting After SDS-PAGE and Transfer to PVDF Membranes To gather more information on the nature of the components of the A. thaliana extract that interacted with the biotinylated lectins, blotting experiments were carried out. Lectins that interacted with either intact A. thaliana cells or cell extracts were also found to bind to several individual components, which were well resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The results of the binding of biotinylated VVA, SNA-I, and jacalin to components in PBS and PBSTriton extracts of A. thaliana cells are illustrated in Fig. 12.4.
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Fig. 12.4 Binding of biotinylated lectins to Arabidopsis thaliana proteins after 5–15% SDSPAGE and transfer to PVDF membranes. Lane 1, proteins extracted from delipidated A. thaliana cells; lane 2, proteins extracted by boiling A. thaliana cells in SDS sample buffer
12.2.3 Lectin Affinity Chromatography of the A. thaliana Extracts To isolate larger quantities of the lectin-binding components, PBS extract of the cells was subjected to affinity chromatography on sepharose-immobilized lectins (VVA, SNA-I, and jacalin). After extensive washing to remove unbound material, the columns were eluted first with a 100 mM solution of the appropriate haptenic sugar, followed by a 50 mM glycine buffer, pH 2.2, to elute any material that is bound to the lectin either specifically or non-specifically, respectively. Macromolecules eluted from the column were recovered by exhaustive dialysis against distilled water, followed by lyophilization.
12.2.4 Analysis of the Monosaccharide Composition of Lectin-Binding Material Portions of the isolated material were hydrolyzed with either 2 N trifluoroacetic acid at 100°C for 6 h to release neutral monosaccharide and hexosamines or with 0.1 N sulfuric acid at 80°C for 1 h to release sialic acids. In parallel, some major bands, shown in Fig. 12.4, were excised from preparative PVDF membrane blots and subjected to hydrolysis. The hydrolysates, after appropriate treatment to remove the acid, were analyzed by high-performance anion-exchange chromato graphy with pulsed amperometric detection (HPAEC-PAD). The results of the
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Fig. 12.5 Monosaccharide analysis by HPAEC-PAD of Arabidopsis thaliana glycoproteins isolated by affinity chromatography on VVA-Sepharose (top panel) as described in the text and the major jacalin reactive band excised from PVDF blots (bottom panel)
a nalysis of material eluted from VVA–sepharose with glycine buffer, which were typical of other preparations, or excised PVDF, are illustrated in Fig. 12.5. Some of the preparations contained significant quantities of plant sugars, specifically d-Glc, d-Man, and d-Xyl, and several unidentified peaks. It should be noted that the expected ligands of the lectins VVA and SNA-I (e.g., GalNAc and/or Gal for VVA or Jacalin and NeuNAc for SNA-I) were either absent from the monosaccharide analysis or present only in minute, insignificant quantities. In summary, microtiter plate assays and probing of blots revealed strong binding of several lectins to proteins extracted from A. thaliana cells. This binding was not inhibited by haptenic sugars. HPAEC-PAD analysis of bands excised from blots and lectin-affinity purified material revealed amounts of the expected monosaccharides, which were insufficient in quantity to explain the extent of the lectin– plant protein interaction.
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12.3 Interaction of Lectins with Glycoproteins in Tobacco Seeds and Seedlings Tobacco (Nicotiana spp) is another model system that is widely used for the investigation of plant biochemistry. Thus, it was of interest to examine the interactions of endogenous plant proteins with lectins to verify that the observed results were not unique to A. thaliana or, indeed, suspension cultured cells. Nicotiana sylvestris seeds were ground to a fine powder and then delipidated by treatment with acetone. The lipid-free powder was extracted with 50 mM Tris–HCl, pH 8.0, containing 200 mM NaCl and 0.1 mM phenylmethylsulfonyl fluoride (PMSF). The extract was centrifuged, the supernatant analyzed for protein, and aliquots were stored at −20°C, as in the case of the A. thaliana cell extracts.
12.3.1 Lectin Blotting After SDS-PAGE and Transfer to PVDF Membranes Aliquots of the extract were subjected to SDS-PAGE, transferred to PVDF membrane, and the blots probed with biotinylated lectins. The results of the binding of biotinylated VVA, SNA-I, MAA, and PNA to proteins extracted from tobacco seed and five-day-old seedlings, as well as their staining with Coomassie Blue, are illustrated in Fig. 12.6.
Fig. 12.6 Binding of biotinylated lectins to proteins extracted from Nicotiana sylvestris seeds (0) or five-day-old seedlings (5) subjected to 4–12% SDS-PAGE and transferred to PVDF membranes. For comparison, staining of proteins with Coomassie Blue (R250) is shown in the first panel
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12.3.2 Effect of Sialidase Treatment on the Binding of MAA and SNA-I Both MAA and SNA-I showed significant interactions with components in the tobacco extracts. Since these lectins are known to recognize ligands containing sialyl residues, we examined their binding after overnight treatment of the tobacco seed extracts with sialidases. The results illustrated in Fig. 12.7 show that exhaustive treatment of the extract with Arthrobacter ureafaciens or Clostridium perfringens sialidases had no effect in the binding of MAA to the tobacco proteins. The above treatments also had no effect on the binding pattern of SNA-I (not illustrated). Many sialidase preparations, particularly those from C. perfringens, are known to contain some protease activity. Therefore, the sialidase treatments were done in the presence of the protease inhibitor PMSF.
12.3.3 Effect of Various Pretreatments of VVA–Biotin on Its Interaction Ability The following experiments were carried out to obtain information on the specificity of the interaction of biotinylated VVA with tobacco proteins. VVA–biotin was first preincubated with 10 mM GalNAc, a haptenic sugar for the lectin, or with glycine buffer, pH 2.2, which should affect the secondary structure of the lectin and, therefore,
Fig. 12.7 Staining of Nicotiana sylvestris seed proteins with biotinylated MAA after overnight incubation with increasing concentrations of Clostridium perfringens sialidase (0, 10, 15, and 20 mU) in the presence of protease inhibitor (a). Staining of N. sylvestris seed proteins with biotinylated MAA before (1) and after (2) overnight incubation with Arthrobacter ureafaciens sialidase (10 mU) (b)
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Fig. 12.8 Staining of Nicotiana sylvestris seed protein (TSP) and ASGF with biotinylated VVA (lane A), biotinylated VVA in the presence of 10 mM GalNAc (B), heat-denatured biotinylated VVA–biotin (C), and biotinylated VVA in the presence of 50 mM glycine buffer, pH 2.2
eliminate its specific binding. These treatments did not influence the binding of VVA to tobacco proteins, while binding to asialoagalacto fetuin (ASGF) was abolished (Fig. 12.8). Similarly, treatment of VVA-biotin at 100 °C for 12 min (Fig. 12.8) and incubation with 1M sodium chloride or 1% Tween 20 also had no effect on its binding to the tobacco proteins, but did abolish binding of the lectin to the control (data not shown). These results demonstrate that the binding of VVA to tobacco proteins was non-carbohydrate mediated, since the haptenic sugar did not affect the binding. Furthermore, denaturation of the lectin by heat treatment or exposure to acidic pH also had no effect on its binding. Finally, the binding observed is not mediated by ionic or hydrophobic interaction, since neither salt nor detergent treatment had any influence on the binding. In summary, proteins extracted from tobacco seeds strongly interacted with various lectins. Pretreatment of the tobacco proteins with sialidases or preincubation of the lectins with haptenic sugar, heat, or acidic buffer, all of which would be expected to abolish specific binding, did not significantly affect binding. While these results suggest a non-carbohydrate-based binding between tobacco seed components (presumably protein-based, judging by SDS-PAGE and accompanying Coomassie Blue Staining) and the lectins, it is surprising that even reagents that disrupt ionic and hydrophobic interaction had no effect on the binding.
12.4 Interaction of Rice Prolamin with Jacalin SDS-PAGE analyses of alcohol-soluble (prolamin) rice (Oryza japonica) protein revealed bands in the molecular weight range of 14–16 kDa, which interacted strongly with VVA, PNA, and wheat germ agglutinin. These proteins were purified
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and analyzed by ELLA using biotinylated jacalin before and after treatment with endo-a-N-acetylgalactosaminidase (O-glycanase) [30]. While Galb1, 3GalNAc was released on digestion with the endoglycosidase, as confirmed by isolation and MS/ MS analysis, the binding of jacalin to the untreated and treated prolamin was not significantly different (Fig. 12.9). Furthermore, the total monosaccharide content of prolamin, consisting of galactose, galactosamine, and glucosamine, accounted for only about 0.01% by weight [12]. These findings demonstrated that the binding of the lectins to rice prolamins is primarily mediated by non-carbohydrate ligands, inferred to be protein-protein interaction. In summary, prolamin purified from rice still interacted with jacalin after treatment with O-glycanase to release the small amount of Galb1-3GalNAc associated with the protein.
12.5 Summary The characteristics of lectin–carbohydrate interactions with respect to animal glycoconjugates have been well established and have thus been reliably utilized by researchers. In contrast, the specificity of the interaction of lectins with plant glycoconjugates had not been critically evaluated previously. The above-discussed results, as well as other studies carried out in our laboratory, provide evidence that there is considerable non-carbohydrate-mediated interaction between lectins and
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as-yet-unidentified plant proteins. Similar results obtained with A. thaliana and Nicotiana spp, two widely used plant model systems, suggested that our observations are probably a general phenomenon involving lectins and components in the plant, tentatively identified as proteins. The interaction of lectins with various hydrophobic plant molecules, such as adenine derivatives, cytokinins, and porphyrins, has been previously reported [8, 13, 18]. These findings, in addition to our own, provide evidence of molecular mimicry in the context of lectin–plant protein interaction. Therefore, it is clear that lectins are not just simple probes, as has been proposed, and results from lectin microarrays, particularly in the case of plant material, should be interpreted with caution. The long-term goal of our research is to understand their nature and thereby minimize non-carbohydrate-mediated binding events that may lead to a false-positive identification of plant glycans, making improved and robust high-throughput analysis of the plant glycome possible. Acknowledgements Lokesh Joshi and Jared Q. Gerlach would like to thank Professors HansJoachim Gabius and Harold Rudiger for their helpful discussions. The authors would like to acknowledge the Wallace Research Foundation and the Biodesign Institute at Arizona State University for their financial support.
References 1. Angeloni S, Ridet JL, Kusy N, Gao H, Crevoisier F, Guinchard S, Kochhar S, Sigrest H, Sprenger N (2005) Glycoprofiling with microarrays of glycoconjugates and lectins. Glycobiology 15:31–41 2. Bolmer SD, Davidson EA (1981) Preparation and properties of a glycoprotein associated with malignancy. Biochemistry 20:1047–1053 3. Broekaert W, Nsimba-Lubaki M, Peeters B, Peumans WJ (1984) A lectin from elder (Sambucus nigra L.) bark. Biochem J 221:163–169 4. Dabelsteen E (1996) Cell surface carbohydrates as prognostic indicators in human carcinomas. J Pathol 179:358–369 5. Dai Z, Kawade AN, Xiang Y, La Belle JT, Gerlach J, Bhavanandan VP, Joshi L, Wang J (2006) Nano-particle-based sensing of glycan-lectin interactions. J Am Chem Soc 128:10018–10019 6. Duk M, Lisowska E, Wu JH, Wu AM (1994) The biotin/avidin-mediated microtiter plate lectin assay with the use of chemically modified glycoprotein ligand. Anal Biochem 221:266–272 7. Durand G, Seta N (2000) Protein glycosylation and diseases: blood and urinary oligosaccharides as markers for diagnosis and therapeutic monitoring. Clin Chem 46:795–805 8. Goel M, Jain D, Kaur KJ, Kenoth R, Maiya BG, Swamy MJ, Salunke DM (2001) Functional equality in the absence of structural similarity: an added dimension to molecular mimicry. J Biol Chem 276:39277–39281 9. Gomord V, Faye L (2004) Posttranslational modification of therapeutic proteins in plants. Curr Opin Plant Biol 7:171–181 10. Jelinek R, Kolusheva S (2004) Carbohydrate sensors. Chem Rev 104:5987–6015 11. Joshi L, Lopez LC (2005) Bioprocessing in plants for engineered proteins. Curr Opin Plant Biol 8:223–226 12. Kilcoyne M, Shah M, Gerlach JQ, Bhavanandan V, Nagaraj V, Smith AD, Fujiyama K, Sommer U, Costello CE, Olszewski N, Joshi L (2009) O-glycosylation of protein subpopulations in alcohol-extracted rice proteins. J Plant Physiol 166:219–232
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13. Komath S, Kavitha M, Swamy MJ (2006) Beyond carbohydrate binding: new directions in plant lectin research. Org Biomol Chem 4:973–988 14. Kuno A, Uchiyama N, Koseki-Kuno S, Ebe Y, Takashima S, Yamada M, Hirabayashi J (2005) Evanescent-field fluorescence-assisted lectin microarray: a new strategy for glycan profiling. Nat Methods 2:851–855 15. La Belle JT, Gerlach JQ, Svarovsky S, Joshi L (2007) Label-free impedimetric detection of glycan-lectin interactions. Anal Chem 79:6959–6964 16. Lee KB, Loganathan D, Merchant ZM, Linhardt RJ (1990) Carbohydrate analysis of glycoproteins: a review. Appl Biochem Biotechnol 23:53–80 17. Leriche V, Sibille P, Carpentier B (2000) Use of an enzyme-linked lectinsorbent assay to monitor the shift in polysaccharide composition in bacterial biofilms. Appl Environ Microbiol 66:1851–1856 18. Montalto MC, Collard CD, Buras JA, Reenstra WR, McClaine R, Gies DR, Rother RP, Stahl GL (2001) A keratin peptide inhibits mannose-binding lectin. J Immunol 15:4148–4153 19. Pilobello KT, Krishnamoorthy L, Slawek D, Mahal LK (2005) Development of a lectin microarray for the rapid analysis of protein glycopatterns. Chembiochem 6:1–4 20. Puri KD, Gopalkrishnan B, Surolia A (1992) Carbohydrate binding specificity of the Tn antigen binding lectin from Vicia villosa seeds. FEBS Lett 312:208–212 21. Raman R, Venkataraman M, Ramakrishnan S, Lang W, Ragurm S, Sasisekharan R (2006) Advancing glycomics: implementation strategies at the consortium for functional glycomics. Glycobiology 16:82R–90R 22. Rudiger H, Gabius HJ (2001) Plant lectins: occurrence, biochemistry, functions and applications. Glycoconj J 18:589–613 23. Sharon N (2007) Lectins: carbohydrate-specific reagents and biological recognition molecules. J Biol Chem 282:2753–2764 24. Sharon N, Lis H (2004) History of lectins: from hemagglutinins to biological recognition molecules. Glycobiology 14:53R–62R 25. Shibuya N, Goldstein IJ, Broekaert WF, Nismba-Lubaki M, Peeters B, Peumans WJ (1987) The elderberry (Sambucus nigra L.) bark lectin recognizes the NeuNAc(alpha 2-6)Gal/ GalNAc sequence. J Biol Chem 262:1596–1601 26. Shin I, Park S, Lee M (2005) Carbohydrate microarrays: an advanced technology for functional studies of glycans. Chem Eur J 11:2894–2901 27. Tekoah Y, Ko K, Koprowski H, Harvey DJ, Wormald MR, Dwek RA, Rudd PM (2004) Controlled glycosylation of therapeutic antibodies in plants. Arch Biochem Biophys 426:266–278 28. Tollefsen S, Kornfeld S (1983) The B4 lectin from Vicia villosa seeds interacts with N-acetylgalactosamine residues alpha-linked to serine or threonine residues in cell surface glycoproteins. J Biol Chem 258:5165–5171 29. Turnbull JE, Field RA (2007) Emerging glycomics technologies. Nat Chem Biol 3:74–77 30. Umemoto J, Bhavanandan VP, Davidson EA (1977) Purification and properties of an endoalpha-N-acetyl-d-galactosaminidase from Diplococcus pneumoniae. J Biol Chem 252:8609–8614 31. Wilson IBH (2002) Glycosylation of proteins in plants and invertebrates. Curr Opin Struct Biol 12:569–577 32. Yamamoto K, Ito S, Yasukawa F, Konami Y, Matsumoto N (2004) Measurement of the carbohydrate-binding specificity of lectins by a multiplexed bead-based flow cytometric assay. Anal Biochem 336:28–38
Chapter 13
Novel Concepts About the Role of Lectins in the Plant Cell Els J.M. Van Dammes, Elke Fouquaert, Nausicaä Lannoo, Gianni Vandenborre, Dieter Schouppe, and Willy J. Peumans
Keywords Classification • Nucleocytoplasmic protein • Plant lectin • Physiological role • Specificity The history of plant lectins dates back to 1888 when Stillmark published his dissertation Über Ricin ein giftiges Ferment aus den Samen von Ricinus communis L. und einigen anderen Euphorbiaceen, in which he linked the toxicity of castor beans to the presence of a proteinaceous hemagglutinating factor called ricin [1]. Only in 1952, it was shown that the agglutination properties of lectins are based on a specific sugar-binding activity [2]. Since then, a lot of carbohydrate-binding proteins have been reported in plants. For a long time, research was concentrated on those plant tissues that contain readily detectable amounts of lectin by agglutination assays. As such, a selection was made for plant lectins occurring in high concentrations, mostly in seeds and vegetative tissues. In recent years, evidence has accumulated of a different class of plant lectins occurring in the nucleus and the cytoplasm of the cell in low concentrations. This chapter aims to give an overview of the most recent findings and the impact thereof on our understanding of the physiological role of lectins in plants.
13.1 Plant Lectins: A Group of Bioactive Plant Proteins Many plants, including important food plants such as wheat, potato, tomato, and bean, contain carbohydrate-binding proteins commonly referred to as lectins, agglutinins, or hemagglutinins. This group of proteins comprises all plant proteins possessing at least one noncatalytic domain that binds reversibly to specific
E.J.M. Van Dammes (*) Laboratory of Biochemistry and Glycobiology, Department of Molecular Biotechnology, Ghent University, Coupure Links 653, 9000 Gent, Belgium e-mail:
[email protected] A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_13, © Springer Science+Business Media, LLC 2011
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ono- or oligosaccharides [3, 4]. Hitherto, about 500 different plant lectins have m been isolated and (partially) characterized [5]. At first glance, all these lectins form a heterogeneous group of proteins because of the obvious differences in structure, specificity, and biological activities. However, structural and molecular studies have revealed the existence of a limited number of plant lectin families. Hitherto, different sugar-binding domains/motifs have been identified with certainty in plants (Table 13.1). Using these domains as basic structural units, a relatively simple system was elaborated that allows classifying virtually all known plant lectins into families of structurally and evolutionarily related proteins. Most plant lectins belong to one of the seven common families, which are (in alphabetical order) the amaranthins, the Cucurbitaceae phloem lectins, the Galanthus nivalis agglutinin family, the lectins with hevein domain(s), the jacalin-related lectins, the legume lectins, and the ricin-B family [3, 4]. In addition, two families with a narrow taxonomic distribution have been identified only recently, namely, orthologs of the fungal Agaricus bisporus agglutinin [6] and catalytically inactive homologs of class-V chitinases [7]. Furthermore, recent studies have provided information on the existence of a new motif with lectin activity, referred to as the Euonymus lectin (EUL) domain. Although there are still a lot of unanswered questions regarding the physiological role of plant carbohydrate-binding proteins, during the past decade, some substantial progress has been made in our general understanding of the role of those plant lectins that are constitutively expressed in reasonable quantities. Biochemical and molecular studies of numerous lectins eventually demonstrated that only a Table 13.1 Overview of different lectin families occurring in the vacuole and/or the nucleus and cytoplasm of plant cells Lectin family Vacuolar lectins Nucleocytoplasmic lectins Amaranthins No examples known Documented in Amaranthus spp and Prunus spp No examples known Wide taxonomic Cucurbitaceae phloem distribution lectins/lectins with Nictaba domain Galanthus nivalis Wide taxonomic distribution Found in diverse taxa agglutinin family Lectins with hevein Wide taxonomic distribution No examples known domains Jacalin-related lectins Only documented in a few Moraceae Ubiquitous spp Legume lectins Common in Fabaceae and Lamiaceae No examples known Ricin-B family Wide taxonomic distribution No examples known Orthologs of Agaricus No examples known Documented in Marchantia bisporus agglutinin polymorpha and Tortula ruralis Homologs of class-V Only documented in a few legumes No examples known chitinases Ubiquitous Lectins with Euonymus No examples known lectin domain
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limited number of carbohydrate-binding motifs evolved in plants [8]. Since the specificity of these binding motifs is primarily directed against foreign glycans, it is generally accepted now that many plant lectins are involved in the recognition and binding of glycans from foreign organisms and accordingly play a role in plant defense. Animal and insect feeding studies with purified lectins and experiments with transgenic plants confirmed that at least some lectins enhance the plant’s resistance against herbivorous higher animals or phytophagous invertebrates. To reconcile the presumed defensive role with the high concentration, the concept was developed that many plant lectins are storage proteins that can be used as nonspecific defense proteins in case the plant is challenged by a predator [8, 9]. Evidently, such a defense/storage role applies only to lectins that are present in relatively high concentrations. This role of plant lectins for defense against foreign attack is in marked contrast with the role of animal lectins; most of these lectins are believed to recognize and bind endogenous receptors and, accordingly, are involved in recognition mechanisms within the organism itself [10, 11]. Furthermore, there is increasing evidence that protein–carbohydrate interactions are very important for the normal development and functioning of animal organisms. In addition, it was shown that different types of lectins are the mediators of these protein–carbohydrate interactions. Therefore, the question arises whether protein–carbohydrate interactions are also important in signal transduction in plants.
13.2 Classical and Inducible Plant Lectins During the past 5 years, evidence has accumulated that plants synthesize well-defined carbohydrate-binding proteins upon exposure to stressful situations such as drought, high salt, wounding, treatment with some plant hormones, or pathogen attack. Localization studies demonstrated that, in contrast to the “classical” plant lectins, which are typically found in vacuoles, the “inducible” lectins are exclusively located in the cytoplasm and the nucleus. Based on these observations, the concept that lectin-mediated protein–carbohydrate interactions in the cytoplasm and the nucleus play an important role in the stress physiology of the plant cell was developed [12, 13]. The development of this novel concept was founded primarily on the results obtained with the inducible lectins discovered in rice [14] and tobacco [15]. Since then, firm evidence has been obtained that plants express several families of nucleocytoplasmic lectins that, according to the available sequence information, are definitely unrelated to each other (Table 13.1). Since most of these inducible lectins are synthesized only as a response to specific physical, chemical, and biotic stress factors and occur in low but physiologically relevant concentrations, it can be assumed that they play a specific role in the plant. Taking into consideration that any physiological role of plant lectins most likely relies on their specific carbohydrate-binding activity and specificity, the discovery of the novel stress-related lectins provides strong evidence for the importance of protein– carbohydrate interactions in plants.
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Hitherto, five families of nucleocytoplasmic lectins have been identified (Table 13.1). Some of these lectin families have been known for several years but have only now been shown to reside in the cytoplasm and/or nucleus of the plant cells (e.g., amaranthin-like sequences and Nictaba-related sequences). Other lectin families, such as the mannose-binding lectins related to Galanthus nivalis agglutinin (GNA) and the jacalin-related lectins, comprise both vacuolar and nucleocytoplasmic homologs. Finally, a new lectin family of nucleocytoplasmic lectins has been discovered for which no vacuolar homologs have been reported until now. The latter family is characterized by the presence of a so-called EUL domain. Each of these nucleocytoplasmic lectin families is discussed in more detail below.
13.2.1 Jacalin-Related Lectins The family of jacalin-related lectins groups all proteins with one or more domains that are structurally equivalent to jacalin, a galactoside-binding lectin from jack fruit (Artocarpus integrifolia) seeds [16]. In recent years, several lectins related to jacalin but with specificity toward mannose have been discovered and characterized in detail [17, 18]. Therefore, the family of jacalin-related lectins is now divided into two subfamilies with a distinct specificity and molecular structure. The galactosespecific jacalin-related lectins are built up of four cleaved protomers comprising a small (b) (20 amino acid residues) and a large (a) (133 amino acid residues) subunit and exhibit a clear preference for galactose over mannose [19]. In contrast, mannose-specific jacalin-related lectins are built up of either uncleaved protomers of approximately 150 amino acids (Fig. 13.1a) or protomers comprising two to seven tandemly arrayed (uncleaved) jacalin domains that exhibit an exclusive specificity toward mannose. The first unambiguous evidence for the cytoplasmic location of a mannose-specific jacalin-related lectin came from localization studies in rhizomes of Calystegia sepium [20]. It was shown that the localization pattern for the mannose-specific Calystegia lectin definitely differed from the vacuolar location of the galactose-specific jacalin from Artocarpus integrifolia. Based on these observations, the hypothesis was put forward that the galactose-specific jacalinrelated lectins evolved from their mannose-specific homologs through the acquisition of vacuolar targeting sequences [12, 13, 20]. The first inducible jacalin-related lectin was purified and characterized from rice. This protein, called Oryza sativa agglutinin or Orysata, was already described in 1990 as SalT (a salt-inducible protein) [21] but was identified as a lectin belonging to the family of “mannose-specific jacalin-related lectins” in 2000 [14, 22]. Orysata cannot be detected in untreated plants but is rapidly expressed in roots and sheaths after exposing the whole plant to salt or drought stress, or jasmonic acid and abscisic acid treatment [21, 23, 24] (Table 13.2). The lectin is also expressed in excised leaves after infection with an incompatible Magnaporthe grisea race [25, 26] and during senescence [27]. Irrespective of the inducing agent, the lectin level remains very low. Orysata is synthesized without signal peptide on free
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Fig. 13.1 Schematic representation of the structural relationships between nucleocytoplasmic lectins and their vacuolar homologs. Comparison of sequences for (a) vacuolar galactose-binding and cytoplasmic mannose-binding jacalin-related lectins from jack fruit (jacalin) and Calystegia sepium (Calsepa), respectively; (b) Nictaba and Cucurbita phloem lectin PP2; and (c) vacuolar GNA and cytoplasmic GNA-related sequence from maize (GNAmaize)
r ibosomes and, based on the analogy of other mannose-specific jacalin-related lectins with a known location, probably remains in the cytoplasmic/nuclear compartment. We have recently confirmed the nucleocytoplasmic location of Orysata using confocal microscopy of tobacco BY-2 cells expressing a lectin sequence fused to enhanced green fluorescent protein (EGFP) (Fig. 13.2a). Fluorescence was seen in the nucleus and cytoplasm of the tobacco cells, whereas the vacuole was completely devoid of any signal. A search for proteins and genes comprising domain(s) equivalent to Orysata revealed that these sequences are widespread. Jasmonate-inducible orthologs of Orysata have been identified in several other Gramineae species, as well as in Helianthus tuberosus [28] and Ipomoea batatas [29]. Moreover, according to transcriptome analysis, all higher plants (Tracheophyta) studied thus far apparently express (low levels of) one or more of these lectins. An extended family of genes encoding proteins with single or multiple jacalin domains was found in Arabidopsis. One of these proteins (RTM1) is involved in the restriction of long-distance movement of tobacco etch virus, whereas others correspond to wounding/jasmonateinduced myrosinase-binding proteins [30, 31]. Within the Gramineae family, several inducible stress/defense-related proteins containing a jacalin domain fused
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Table 13.2 Overview of stress conditions inducing lectin activity Lectin or Lectin family Plant species Biotic or abiotic factor Rice Abscisic acid Mannose-specific jacalin-related Rice Salt stress lectins Rice Drought stress Rice Jasmonate Rice Wounding Rice Magnaporthe grisea infection Rice Leaf senescence Rice Gibberellins Wheat, Rice Vernalization, jasmonate Barley Light Barley Jasmonate Barley Salt stress Oilseed rape Jasmonate Sweet potato Jasmonate Jerusalem artichoke Jasmonate Nictaba Tobacco Jasmonate Insect herbivory Proteins with EUL Rice Abscisic acid domain Rice Salt stress Maize Drought stress Banana Desiccation
References [21] [21] [21] [23] [24] [25, 26] [27] [33] [32, 33] [116] [117] [118] [119] [29] [120] [15] [39] [121] [62] [122] [123]
to an unrelated domain have been found. One of these proteins (called VER2) is specifically expressed during vernalization [32, 33]. The VER2 protein in rice and wheat was shown to be jasmonate inducible. Expression of wheat VER2 is also inducible by gibberellins [33]. Characterization of the recombinant rice protein (expressed in Escherichia coli) revealed inhibition of the agglutination activity by mannose [34], confirming that VER2 is indeed a lectin. Recent studies have also showed that overexpression of the gene in rice suppresses coleoptile and stem elongation, indicating that this lectin plays an important role in rice growth and development [35]. Another structurally similar protein containing a jacalin domain occurs in maize, where it is known as a b-glucosidase-aggregating factor [36–38]. Judging from the available sequence information, we can conclude that the jacalin domain is widespread in plants.
13.2.2 Proteins with a Nictaba Domain In 2002, Chen et al. reported that jasmonic acid methyl ester induces lectin activity in leaves of Nicotiana tabacum (var. Samsun NN) [15]. This lectin (called Nicotiana tabacum agglutinin or Nictaba) cannot be detected in untreated tobacco plants but is specifically induced in leaves after treatment with jasmonic acid and other
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Fig. 13.2 Confocal images of living, transiently transformed BY-2 cells expressing EGFP-Orysata (Genbank Accession No. CB632549) (a); EGFP-Nictaba 24 h after transformation (AF389848) (b); EGFP-Nictaba 48 h after transformation (c); EGFP-Nictaba mutated in NLS (d); EGFPGNAmaize (GNA-related sequence from maize, BM351398) (e); EGFP-EULArabidopsis (Arabidopsis sequence related to EUL, AF411801; [114, 115]) (f); AmaranthinPrunus-EGFP (Amaranthin-like sequence from Prunus, DN554056) (g). Free EGFP (h) was used as a control for nuclecytoplasmic localization, whereas the expression of sporamin-EGFP (U12436) [95, 96] was a control for vacuolar targeting (i). Scale bars represent 25 nm
jasmonates (Fig. 13.3). It was shown that jasmonate treatment of a single leaf of a tobacco plant results in lectin expression not only in the treated leaf but also in the leaves below and above the treated leaf, indicating that some signal is transported between different leaves and triggers lectin expression. In addition, Nictaba was recently also shown to be induced by insect herbivory [39] (Table 13.2). Nictaba and its corresponding gene have been isolated and characterized. In its native form, Nictaba is a homodimer consisting of two identical unglycosylated subunits of approximately 19 kDa. The deduced amino acid sequence of the complementary
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Fig. 13.3 Induction of Nictaba expression in tobacco plants after treatment with jasmonates. (a) Using semiquantitative agglutination assays, Nictaba expression was determined at the protein level in different leaves of a tobacco plant, of which only one leaf was treated with methyl jasmonate. Lectin activity was analyzed 72 h after treatment and calculated as ug lectin per gram fresh weight (FW). (b) The treated leaf was assigned as leaf 0; the two leaves above the treated leaf are referred to as leaf 1 and 2, whereas the two lower leaves are referred to as leaf 1 and 2. (c) Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis on different tobacco leaves for lectin messenger RNA (mRNA). After 12 h of treatment of leaf 0, mRNA for Nictaba could already be detected in the treated leaf (a). To clearly show mRNA for Nictaba in the systemic leaves, a nested PCR was performed (b). The quality of the RNA samples was assured by an internal control for ribosomal RNA (rRNA) (c). Samples were analyzed after 12 h of treatment
DNA (cDNA) clone encoding Nictaba revealed a 165-amino acid sequence containing a putative nuclear localization signal (NLS) sequence (102KKKK105). Immunocytochemical localization studies using polyclonal Nictaba-specific antibodies revealed that the lectin is located in the nucleus and cytoplasm of leaf cells. No labeling could be detected in vacuoles or chloroplasts. All immunolabeling could be detected in the leaf parenchyma cells and not in vascular tissues [15]. More recently, the nucleocytoplasmic location of Nictaba was confirmed using confocal microscopy of tobacco BY-2 cells expressing a Nictaba sequence fused to EGFP (Fig. 13.2b, c) [40]. Furthermore, it became evident that the lectin is not uniformly distributed over the nucleus or the cytoplasm of BY-2 cells. Confocal microscopy of transiently transformed BY-2 cells revealed that 24 h after biolistic delivery, the expressed EGFP-Nictaba is predominantly located in the nucleus and, to a lesser extent, in the cytoplasm surrounding the central vacuole and the strands of cytoplasm transversing the vacuole (Fig. 13.2b). However, 48 h after DNA delivery, the ×48 nucleus still contained most of the EGFP-Nictaba but showed a rimlike staining pattern, suggesting that the fusion protein was now concentrated at the periphery of the nucleus (Fig. 13.2c). Very similar staining patterns were observed in BY-2 cells stably transformed with the EGFP-Nictaba construct. Similar experiments with a fusion protein of EGFP and a Nictaba mutant, in which the presumed NLS (102KKKK105) was changed into (102KTAK105), provided evidence for the involvement of an NLS-dependent transport mechanism. Confocal microscopy of the transiently transformed BY-2 cells clearly demonstrated that the mutant protein is exclusively located in the cytoplasm and cannot be detected in the nucleus (Fig. 13.2d). The distribution pattern seen 24 h after biolistic delivery of the DNA
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did not change during the subsequent 36 h, indicating that the NLS is required and sufficient for transport of Nictaba from the cytoplasm into the nucleus. Based on the results of hapten inhibition assays, the lectin was originally classified as a chitin-binding protein. However, more detailed specificity studies with glycan arrays revealed that Nictaba preferentially recognizes high-mannose as well as complex N-glycans and strongly interacts with glycoproteins carrying such glycans [40]. A detailed analysis of the binding studies suggests that the binding site of Nictaba is most complementary to (Man)3b1-4GlcNAcb1-4GlcNAcb-N-Asn. The tobacco lectin was originally discovered in Nicotiana tabacum L. cv. Samsun NN leaves. To check whether and, if so, to what extent the specific induction of this lectin applies to related tobacco species, a collection of 19 Nicotiana species, covering 12 Nicotiana sections and eight Nicotiana tabacum cultivars, was screened for their capability to synthesize the jasmonate-inducible lectin. Protein analyses by agglutination assays and Western blot confirmed that only nine out of the 19 species examined synthesize lectin after jasmonate treatment. Remarkably, all cultivars tested of the allotetraploid species N. tabacum L. express the lectin after jasmonate treatment. Polymerase chain reaction (PCR) analyses demonstrated that all responsive species possess one or more lectin genes, whereas no lectin gene(s) could be traced in the nonresponding tobacco species. These findings provide the first firm evidence for a striking intragenus difference with respect to the activation of a well-defined jasmonate-inducible gene that can be correlated with the presence/ absence of orthologous genes in the genomes of closely related species [41]. Searches in the databases revealed that many flowering plants contain sequences encoding putative homologs of the tobacco lectin, suggesting that Nictaba is the prototype of a widespread or possibly ubiquitous family of lectins with a specific endogenous role. These database searches also revealed sequence homology with the family of Cucurbitaceae phloem lectins, a small group of chitin-binding agglutinins found in the phloem exudates of a number of Cucurbitaceae species [42]. These lectins, also called the phloem proteins 2 (PP2 proteins), are homodimers consisting of unglycosylated subunits of 17–25 kDa that show a high affinity toward oligomers of GlcNAc [43]. Nictaba shares 33% and 51% sequence identity and similarity, respectively, with PP2. However, alignment of the sequences of Nictaba and the Cucurbita maxima PP2 revealed several striking differences. First, the Nictaba sequence is 53 amino acid residues shorter than the sequence of PP2 (Fig. 13.1b). A more detailed sequence analysis shows that Nictaba lacks the C-terminal cysteine-rich pentapeptide of PP2 and 65 amino acid residues at the N-terminus of PP2. Second, the NLS found in the Nictaba sequence is missing in the PP2 sequence. Another important difference concerns the localization of both lectins in the plant cell. As already mentioned above, Nictaba could be detected in all leaf cells except in the vasculature. In contrast, the Cucurbitaceae phloem lectins are typically present in phloem exudates of cucurbit species [44]. By virtue of their cysteine-rich pentapeptide, the Cucurbitaceae phloem lectins can establish intermolecular disulfide bridges with the phloem protein PP1. Finally, it should also be mentioned that, in contrast to Nictaba, the Cucurbitaceae phloem lectins are constitutively expressed.
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In view of the sequence similarity and sugar specificity, it is believed that Nictaba and the Cucurbitaceae phloem lectins are closely related to each other, but taking into account the differences mentioned above, both types of lectin probably do not fulfill the same role. Though it is possible that the extra N-terminal sequence determines ligand specificity, it is more likely that the sugar-binding module is not the equivalent of PP2 but rather of Nictaba. Since Nictaba is a potent hemagglutinin, it must comprise a complete carbohydrate-binding domain. Accordingly, the extra sequences found in the PP2 proteins can be considered accessory domains with an unrelated function. Other Nictaba homologs could be found in many flowering plants, among them Solanum tuberosum (potato), S. esculentum (tomato), Glycine max (soybean), Lotus japonicus, Hordeum vulgare (barley), and Oryza sativa (rice). Arabidopsis thaliana contains the largest number of Nictaba homologs [45]. Most of these Arabidopsis proteins are chimeric proteins consisting of a C-terminal Nictaba domain and an unrelated N-terminal domain. Amongst these N-terminal domains F-box, TIR (Tollinterleukin 1-resistance) domains and AIG1 (avirulence-induced gene) domains could be identified, which are believed to function in protein degradation and defense signaling, respectively [46–48]. Taking into account the widespread occurrence of proteins with Nictaba domains, we propose that Nictaba is the prototype of a lectin family much larger than the Cucurbitaceae phloem lectins. Since, in addition, Nictaba can by no means be considered a phloem lectin, we refer to these Nictaba-like proteins as the “superfamily of proteins with a Nictaba domain.”
13.2.3 Cytoplasmic Galanthus nivalis Agglutinin (GNA)-Related Lectins In 1987, a lectin with exclusive specificity toward mannose was isolated and characterized in snowdrop (Galanthus nivalis) bulbs [49]. GNA is a homotetramer of noncovalently linked 12-kDa monomers. Originally, this group of lectins was referred to as the “monocot mannose-binding lectins” [5], since similar mannosebinding lectins were found in numerous monocot plant families (e.g., Alliaceae, Liliaceae, Orchidaceae, Araceae, Bromeliaceae, Ruscaceae, and Iridaceae) [3, 5, 50, 51]. However, in recent years, very similar lectins have been identified in plants other than Liliopsida (e.g., in the liverwort, Marchantia polymorpha) [52]. Therefore, this group of lectins is now referred to as “GNA-related lectins” after the first identified member. All GNA-related plant lectins are synthesized as preproproteins with an N-terminal signal peptide and a C-terminal propeptide [3, 53] and accordingly are thought to be located in the vacuolar compartment. However, recent findings revealed that some plants (Triticum aestivum, Zea mays, and Medicago truncatula) express proteins that closely resemble the vacuolar GNA-related lectins but lack the signal peptide and C-terminal propeptide [12] (Fig. 13.1c). Analysis of the sequences of the GNA-related proteins from Zea mays indicated that they lack
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specific sorting sequences such as a signal peptide. Transient expression of the fusion construct of the coding sequence of the GNA orthologs with EGFP in tobacco BY-2 cells revealed that the GNA homolog from maize is located in the nucleus and cytoplasm of the cell (Fig. 13.2e). Until now, no information is available on the carbohydrate-binding specificity of the cytoplasmic GNA-related lectins, since none of these lectins have been purified. However, molecular modeling studies using the three-dimensional structure of GNA as a model have shown that all amino acids known to be involved in the carbohydrate-binding site of GNA are conserved in most of the cytoplasmic GNArelated lectins. Hence, it can be envisaged that these cytoplasmic lectins will have very similar binding properties as their vacuolar homologs. During the past few years, evidence has accumulated for the occurrence of GNA-like proteins outside the plant kingdom. A first nonplant protein was identified in Dictyostelium discoideum [54] as comitin, a bifunctional actin-binding protein with a mannose-binding GNA domain. More recently, a lectin-like bacteriocin with high sequence similarity to the GNA domain has been isolated from the bacterium Pseudomonas putida [55]. Furthermore, it was demonstrated that the skin and intestine mucus of the Japanese puffer fish (Fugu rubripes) produce mannose-binding lectins that share striking sequence similarity with GNA [56, 57]. Finally, genome and transcriptome sequencing programs revealed the occurrence of expressed GNA-like proteins in several fungi [58] and in the freshwater sponge Lubomirskia baicalensis [59]. These observations leave no doubt that the GNA-like plant lectins represent only a subgroup of a more extended family of proteins. Biochemical analyses of the so-called comitin from Dictyostelium discoideum [54] and puffer fish from Fugu rubripes [56, 57] provided firm evidence that these proteins contain a functional mannose-binding domain. Importantly, cloning of the corresponding genes revealed that comitin and puffer fish lack a signal peptide and accordingly do not follow the secretory pathway but are synthesized on free ribosomes in the cytoplasm. Expression analysis of fusion proteins with EGFP revealed very similar location patterns for GNA orthologs from the fish and the fungus as was observed for maize [60]. The identification of these nonvacuolar GNA-like plant proteins sheds a new light on the molecular and functional evolution of plant lectins. It is suggested that the newly identified cytoplasmic GNA homologs are regulatory/signaling plant proteins functionally well different from the vacuolar lectins that are thought to play a role in plant defense and storage. In addition, it was proposed that the cytoplasmic lectins may have served as templates for the development of their vacuolar homologs through the insertion of a signal peptide and a C-terminal propeptide [12].
13.2.4 Cytoplasmic Lectins with an EUL Domain For many years, it has been known that the Euonymus europaeus (spindle tree) contains an agglutinin, referred to as Euonymus europaeus agglutinin [61].
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Since no sequence information was available, this lectin could not be classified yet in one of the currently known families of plant lectins. Recently, molecular cloning of EUL and detailed analysis of the sequence indicated that this lectin shares a marked sequence identity (46%) and similarity (62%) with a family of rice proteins that were found to be induced in roots by abscisic acid and salt stress, and presumably play a role in the adaptation of the roots to a hyperosmotic environment [62]. This family of so-called OSR40 proteins comprises four different proteins. Two of them consist of a single OSR40 domain of approximately 150 amino acid residues preceded by a short glycine-rich (osr40g3) or a long histidine-rich N-terminal peptide (unnamed osr40g3homolog). The two other proteins are built up of two OSR40 domains separated by a short linker and preceded by a short histidine-rich N-terminal peptide (osr40g2 and osr40c1, respectively). Hitherto, no specific biological activity could be attributed to this OSR40 domain. However, taking into consideration the marked sequence similarity with the EUL, it seems likely that this sequence represents a new carbohydrate-binding domain and can be referred to as the EUL domain. According to the available sequence data, all rice proteins with OSR40 domain(s) are synthesized on free polysomes and are presumed to be located in the cytoplasm (and possibly also in the nucleus). This implies that the rice OSR40 proteins represent a family of cytoplasmic proteins, the expression of which is abscisic acid and salt-stress responsive. Confocal microscopy of a fusion construct of an Arabidopsis thaliana protein containing an EUL domain linked to EGFP confirmed its localization in the nucleus and cytoplasm (Fig. 13.2f). A preliminary screening of the databases further revealed that proteins comprising one or two EUL domains occur not only in flowering plants but also in gymnosperms and mosses [63], suggesting that the EUL domain probably plays a universal role in stress-related physiological processes. At present, one can only speculate about the working mechanism of proteins containing EUL domain(s). Taking into consideration (1) that all proteins with EUL domain(s) are synthesized in the cytoplasm and (2) that the EUL domain apparently possesses lectin activity, it seems reasonable to expect that the activity of these proteins relies on their binding to cytoplasmic and/or nuclear glycoconjugates.
13.2.5 Amaranthin-Like Sequences The Amaranthin family is a rather small family of closely related lectins found in different Amaranthus species. This group is called after the first lectin of this family isolated from Amaranthus caudatus seeds. All known amaranthins are homodimers built up of 33-kDa subunits. Detailed specificity studies have shown that amaranthin preferentially recognizes the T-antigen disaccharide Galb(1,3)GalNAc [64]. Recently, amaranthin-like sequences have also been reported outside the family Amaranthaceae [65, 66]. Until now, no data have been reported on the biosynthesis, processing, or subcellular location of amaranthin. However, some predictions can be made on the basis
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of the sequence of the mature amaranthin and the deduced sequence of the presumed Amaranthus hypochondriacus lectin [67]. Both sequences are almost identical except that the deduced sequence of the Amaranthus hypochondriacus lectin has four extra residues at its C-terminus, indicating that a short C-terminal propeptide may be cleaved from the primary translation product of the amaranthins. Analysis of the amino acid sequence data further revealed the absence of a signal peptide, suggesting that the lectin is synthesized on free polysomes and possibly located in the nucleus or the cytoplasm. To analyze the location of this group of lectins, a cDNA clone encoding an amaranthin-like sequence from Prunus persica, found in the EST database prepared from leaves infected with the plum pox virus, was used to make a construct encoding a fusion protein of the amaranthin-like sequence with EGFP and expressed in tobacco BY-2 cells. Confocal images indicated that the amaranthin-like lectin is expressed mainly in the nucleus and partially in the cytoplasm (Fig. 13.2g). The fluorescence pattern did not change over time. These findings meet the evidence that the amaranthin-like lectin is synthesized without signal peptide.
13.3 Physiological Role of Inducible Plant Lectins As indicated above, evidence has recently accumulated that plants synthesize welldefined carbohydrate-binding proteins upon exposure to stressful situations. In contrast to the classical plant lectins, which are typically found in vacuoles, the inducible lectins are exclusively located in the cytoplasm and nucleus. Therefore, it can be envisaged that this new class of lectins might play a specific role within the plant cell. In addition, the observation that many of these inducible lectins are apparently widespread in the plant kingdom makes them interesting tools to study the importance of protein–carbohydrate interactions in the plant cell. Although there is good evidence for the carbohydrate-binding properties of at least some of the inducible lectins, there are at present few indications for the possible receptors for these lectins in the plant cell. In the case of Orysata, specificity studies indicated that this cytoplasmic jacalin-related lectin has a relatively poor affinity for mannose but binds strongly to oligomannosides and high-mannose N-glycans [14]. For Nictaba, it was shown that the lectin reacts well with GlcNAc oligomers but exhibits a higher affinity for high-mannose N-glycans [40]. The preference of both Orysata and Nictaba for high-mannose N-glycans indicates that N-glycosylated glycoproteins are the most likely glycan-receptors for these lectins. At present, the possible occurrence in the cytoplasmic/nuclear compartment of glycoproteins with N-linked glycans is still controversial. However, several research groups have already reported the presence of nuclear and cytoplasmic N-glycosylated glycoproteins in animal systems [68–73]. Irrespective of the exact nature of the receptor glycans, conclusive evidence was obtained for both in vitro and in situ interactions between Nictaba and nuclear/ cytoplasmic tobacco proteins. Far Western blots clearly demonstrated that Nictaba
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reacts in a GlcNAc oligomer-inhibitable manner with many proteins present in a crude extract from purified nuclei [40]. PNGase treatment of the proteins almost completely abolished the interaction with Nictaba, suggesting that Nictaba reacts with N-glycans. Therefore, one can reasonably assume that Nictaba interacts through its carbohydrate-binding activity with endogenous glycoprotein receptors in the cytoplasmic/nuclear compartment. This, taken together with the nucleocytoplasmic location and the induction by jasmonate, strongly argues for a specific role of Nictaba in jasmonate-inducible or jasmonate-dependent physiological processes [12, 15]. To get a better insight into the physiological role of these lectins in plants, it is important to understand what the possible receptors for these lectins are.
13.3.1 Role of Lectins in Nucleocytoplasmic Transport It has been shown that transport of proteins as well as RNA molecules between the cytoplasm and the nucleus plays an important role in animal and plant cells [74]. Several studies unambiguously demonstrated that glycoproteins substituted with O-glycans play an important role in nuclear transport. It has been shown, for example, that the import of proteins into the nucleus of animal cells is inhibited by antibodies directed against O-GlcNAc-modified proteins of the nuclear pore complex (NPC) as well as by an intracellular application of wheat germ agglutinin, a GlcNAc-specific lectin that binds to the O-GlcNAc-residues of glycoproteins in the NPC [75]. Microscopical analysis of EGFP-Nictaba expression in tobacco cells revealed a strong staining of the nuclear rim, indicating that Nictaba may interact with proteins in the NPC. Taking into account the carbohydrate-binding specificity of Nictaba for GlcNAc oligomers and N-glycans, it can be envisaged that Nictaba could interact with O-GlcNAc-modified NPC proteins. Previous reports have also shown that some NPC proteins are glycosylated [76–78]. Based on all these observations and the presence of Nictaba both in the cytoplasm and the nucleus, the hypothesis that Nictaba could be a shuttle protein between the nucleus and the cytoplasm was put forward [12, 13, 15].
13.3.2 Role of Inducible Lectins in Plant Defense Plants possess several constitutive as well as inducible defense mechanisms to protect themselves against insect attack and other threats. Over the last few years, it has become clear that insect herbivory also influences the expression of several lectins or proteins containing lectin domains. As already indicated above, a jasmonate-inducible lectin was identified in tobacco leaves. It was shown that insect herbivory (e.g., by cotton leaf worm Spodoptera littoralis) induces lectin expression in tobacco plants, most probably
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through activation of the jasmonate pathway. When larvae were allowed to feed on a single leaf, systemic induction of lectin activity was observed in all leaves of the plant. In addition, preliminary experiments showed that the tobacco lectin exerts a repellent effect on chewing insects [79]. Evidence for the expression of lectin-like sequences was also obtained from molecular studies showing that wheat plants respond to insects by the expression of lectin genes [80]. These wheat plants react to the initiation of first-instar larval feeding of specific biotypes of Hessian fly (Mayetiola destructor) with rapid changes in the expression of several mRNA transcripts at the feeding site, among which was an mRNA encoding Hfr-1, a protein containing a C-terminal domain with sequence similarity to jacalin-related lectins [80, 81]. More recently, two other Hessian flyresponsive wheat genes, called Hfr-2 and Hfr-3, containing a lectin domain similar to amaranthin and hevein, respectively [65, 82], have been reported. Sequence analysis of Hfr-2 revealed an N-terminal lectin domain similar to amaranthin fused to a region similar to hemolytic lectins and channel-forming toxins. The Hfr-3 sequence contains four predicted chitin-binding hevein domains. All these data suggest the involvement of a set of lectins in the resistance of wheat to Hessian fly. The fact that insect herbivory stimulates the expression of lectins or proteins with lectin domains suggests that plants respond to insect herbivory through the synthesis of proteins with carbohydrate-binding activity. Certainly in the case of the tobacco lectin, it has been shown that the lectin is fully active and is able to recognize and bind carbohydrates. Future experiments will have to show whether these lectins induced by insects are expressed in sufficient amounts to exert a toxic effect on the insect.
13.4 Biomedical Applications At present, only a limited amount of information is available with regard to the carbohydrate-binding properties and specificity of this novel class of nucleocytoplasmic lectins. However, despite the relatively short history, some of these new lectins have already proven to be useful tools. Nictaba was shown to be highly inhibitory to human immunodeficiency virus, similar to the chitin-binding lectin from stinging nettle (Urtica dioica, [83]) (Balzarini J, unpublished results). These results are in good agreement with the strikingly similar carbohydrate-binding properties of both lectins. Amaranthin and EUL have intensively been studied for a long time but have only recently been recognized as nucleocytoplasmic lectins. It has been shown previously that amaranthin is a valuable, highly specific tool for the detection of T- and cryptic T-antigens [64] and accordingly is very useful in cancer diagnosis [84–86]. EUL was proven useful to detect M-cells [87] but was reported unsuitable for delivering vaccines to M-cells [88]. The jacalin-related lectin from Morus nigra, called Morniga M, was shown to exhibit a strong interaction with high-mannose N-glycans [89]. It was suggested
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that the capability of Morniga M to recognize the N-linked structure makes this lectin a powerful tool for the purification of oligomannosyl residues containing N-glycans and glycoproteins, and as a selective marker to identify N-glycans. Recently, it has been shown that Morniga M binds glycotopes of mammalian retinal neurons [90]. Biomedical applications of many other nucleocytoplasmic lectins are hampered at the moment because the proteins have not been purified yet. It can be envisaged that once sufficient amounts of the (recombinant) lectins have been made available, detailed studies of the carbohydrate-binding properties of the new lectins will reveal more new interesting specificities that possibly can be exploited, e.g., for purification of particular glycoproteins or for the detection of changes in the glycosylation pattern on cell surfaces.
13.5 Nucleocytoplasmic Lectins Outside the Plant Kingdom Extensive studies with animal lectins have shown that most of them recognize endogenous glycoconjugates and accordingly are involved in specific recognition processes within the organism itself. Depending on their location (extracellular, surface exposed, or intracellular), animal lectins mediate either cell–cell interactions or intracellular protein–glycoconjugate interactions. In the past, there have also been several reports of nucleocytoplasmic lectins in animal cells [91]. In particular, the b-galactoside-binding lectins, referred to as galectins, have been studied in detail, and at least eight galectins have been documented in the nucleus and cytoplasm. It should be mentioned that, for some galectins, a dual localization was reported in that these lectins were found both intracellularly (in nucleus and cytoplasm) as well as in the extracellular compartment (cell surface). Each individual galectin is expressed in a tissue-specific or developmentally regulated fashion. Galectins are involved in many biological processes, such as morphogenesis, control of cell death, immunological responses, and cancer. Galectin-1 and galectin-3 have been identified as pre-mRNA splicing factors in the nucleus and interact with Gemin4 [92]. Some reports suggest that the splicing activity of galectins depends on their carbohydrate-recognition domain [93, 94]. However, other reports indicate that the binding of galectin-3 to DNA and RNA is carbohydrate independent [95, 96]. Detailed analyses have further provided evidence to show that galectin-3 serves as a shuttle between the nucleus and the cytoplasm [97–100]. The expression of at least one of the galectins was shown to be inducible. Ovga11 is a lectin in the cytoplasm and nucleus of the upper epithelial cells of the gastrointestinal tract, the mRNA of which is greatly upregulated in tissues infected with the nematode parasite Haemonchus contortus [101]. The lectin is believed to play an immunomodulatory role. Until now, no galectin-like sequences have been reported in plants. Certain members of the annexin family may interact with carbohydrates and like the galectins are predominantly intracellular proteins [91]. Another glycosylated nuclear lectin, called CBP70, was reported as a GlcNAc-binding lectin interacting
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with an 82-kDa nuclear glycoprotein [69, 102]. In addition, it was shown that CBP70 can interact with laminin, suggesting a role in the interaction with the cytoskeleton [103]. In addition, several intracellular animal lectins have been reported in the endoplasmic reticulum (ER) where they play an important role in folding of glycoproteins (calnexin, calreticulin), intracellular routing of glycoproteins (ERGIC-53, VIP-36), and targeting of misfolded glycoproteins to ER-associated degradation (ERAD) [104–107]. Once misfolded glycoproteins have been targeted into the cytoplasm, they are recognized by sugar-binding F-box proteins and tagged with ubiquitin to be degraded by the proteasome [108, 109]. Functional homologs for at least some of these lectins have also been found in plants [110]. Comitin is a cytoplasmic actin-binding protein that until now has exclusively been found in the slime mold Dictyostelium discoideum. The N-terminal core domain of comitin has lectin-like activity [54] and has been modeled from the X-ray coordinates of the mannose-binding lectin from snowdrop (Galanthus nivalis). Docking experiments performed on the three-dimensional model showed that two of the three mannose-binding sites of the comitin monomer are functional. They are located at both ends of the comitin dimer, whereas the actin-interacting region occurs in the central hinge region where both monomers are noncovalently associated. This distribution is fully consistent with the bifunctional character of comitin, which is believed to link the Golgi vesicles exhibiting mannosylated membrane glycans to the actin cytoskeleton in the cell [111, 112]. Analyses with mutant Dictyostelium discoideum lines lacking comitin revealed some defects in phagocytosis and altered response to hyperosmotic shock [113].
13.6 General Remarks Recently, evidence has accumulated that nucleocytoplasmic plant lectins also occur in plants and are far more abundant throughout the plant kingdom than was believed until a couple of years ago. As such, carbohydrate-binding motifs seem to be important molecules in the animal as well as the plant kingdom. Thanks to novel technologies, such as confocal fluorescence microscopy and life-time imaging, localization of lectins fused to fluorescent marker proteins such as the green fluorescent protein could be studied. It was unambiguously shown that these fusion proteins are expressed in the nucleocytoplasmic compartment. In addition, analyses of the new sequence data resulting from whole-genome sequencing projects and transcriptome analyses predict that more lectins or proteins with lectin domains might be present in the cytoplasm and nucleus of plant cells. Interestingly, a detailed study of a few lectin families for which vacuolar as well as nucleocytoplasmic lectins have been reported revealed that both forms seem to be related evolutionarily. At present, a lot of questions still exist regarding the possible function of these nucleocytoplasmic lectins. To get a better insight into the physiological role of
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nucleocytoplasmic lectins, it is important to have detailed information on their carbohydrate-binding properties and the proteins they could interact with in the cell. Since these nucleocytoplasmic plant lectins are present in very low concentrations, the challenge in plant lectin research is put nowadays on recombinant protein technology. As soon as a small amount of recombinant protein is available, the new glycan array technology will allow one to study or refine the carbohydrate-binding properties of these lectins. In addition, interaction studies of the recombinant protein with putative receptors for the lectin in the cell will help to identify the binding partners for the nucleocytoplasmic lectins. Acknowledgments This work was supported in part by grants from the Research Council of Ghent University and the Fund for Scientific Research-Flanders (FWO grants G.0201.04 and 3G.0163.06). Gianni Vandenborre acknowledges the receipt of a scholarship from the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen).
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Part IV
Structures and Functions of Glycolipids
Chapter 14
Role of Gangliosides and Plasma MembraneAssociated Sialidase in the Process of Cell Membrane Organization Sandro Sonnino, Vanna Chigorno, Massimo Aureli, Anie Priscilla Masilamani, Manuela Valsecchi, Nicoletta Loberto, Simona Prioni, Laura Mauri, and Alessandro Prinetti Keywords Sialidase • Plasma membranes • Ganglioside Glycosphingolipids are amphiphilic membrane lipids characterized by the presence of a long-chain (C18 or C20) amino alcohol, which has the trivial name “sphingosine.” Glycosphingolipids are components of all eukaryotic cell membranes, and gangliosides (glycosphingolipids containing sialic acid residues in their oligosaccharide chains) are particularly abundant in the plasma membranes of neurons. As sphingolipids are concentrated at the subcellular level in the plasma membrane, where they reside asymmetrically in the extracellular leaflet, they are relatively abundant in this district. Keeping in mind that sphingolipids are not homogeneously distributed throughout the membrane plane but rather are concentrated in restricted membrane areas [1] due to their spontaneous segregation with respect to glycerophospholipids, it can be predicted that their local concentration in specific “lipid membrane domains” would be very high.
14.1 Regulation of Plasma Membrane Glycosphingolipid Composition by Biosynthesis and Degradation The regulation of plasma membrane glycosphingolipid composition is of crucial importance for cell biology and is mainly dependent on the biosynthetic and catabolic processes occurring within the cells. Both biosynthesis and degradation take place in intracellular districts, thus the turnover of plasma membrane sphingolipids is intimately connected with the bidirectional flow of molecules from and to the plasma membrane that mainly occurs via vesicular traffic, even if nonvesicular S. Sonnino (*) Department of Medical Chemistry, Biochemistry and Biotechnology, Center of Excellence on Neurodegenerative Diseases, University of Milan, Via Fratelli Cervi 93, Segrate, Milano 20090, Italy e-mail:
[email protected] A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_14, © Springer Science+Business Media, LLC 2011
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Fig. 14.1 Different metabolic pathways possibly involved in changing plasma membrane glycosphingolipids. The following pathways can be altered by modulating the enzyme activities/ expressions or process rates. (1) Plasma membrane uptake of extracellular glycolipids shed by different cells; (2) shedding of glycolipid monomers (some directly reenter the membrane, while others interact with the extracellular proteins or lipoproteins and are subsequently taken up by the cells and catabolized into lysosomes); (3) release of glycolipid-containing vesicles from the plasma membrane; (4) membrane endocytosis followed by sorting to lysosomes and lysosomal catabolism; (5) biosynthetic modifications by plasma membrane-associated glycosyltransferases and glycosidases
transport via sphingolipid binding proteins plays an important role in specific steps (Fig. 14.1) [2–4]. The de novo biosynthetic pathway of sphingolipids starts at the cytosolic face of the endoplasmic reticulum, where enzyme activities responsible for the reaction sequence leading to the formation of ceramide are localized. The neosynthesized ceramide reaches the Golgi apparatus by a not-yet-known mechanism, where it is used as the common precursor of glycosphingolipids. Different membrane-bound glycosyltransferases are responsible for the sequential addition of sugar residues to the ceramide, leading to the growth of the oligosaccharide chain. Glucosylceramide is the first glycosylated product, formed by a ceramide glucosyltransferase activity localized at the cytosolic side of the early Golgi membrane. Glucosylceramide can either directly reach the plasma membrane [5], presumably transported in a nonvesicular way, or be translocated to the luminal side of the Golgi, where it is further glycosylated by other glycosyltransferases located in this cellular district to generate more complex glycosphingolipids. Neosynthesized glycosphingolipids move through the Golgi apparatus to the plasma membrane following the mainstream exocytotic vesicular traffic.
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The enzymology and the intracellular topology of the de novo biosynthesis of glycosphingolipid have been unveiled in their details, whereas very little is known about its regulation, which has been regarded for a long time as the main mechanism responsible for the formation of a specific glycosphingolipid pattern. It is generally assumed that glycosphingolipid synthesis is mainly regulated at the transcriptional level through the control of the expression levels of all the enzymes involved in the synthesis (glycosyltransferases) or the trafficking among the different intracellular districts (transporter proteins). Indeed, changes in the expression of glycosyltransferases have been observed in several phenomena characterized by changes in cellular glycosphingolipid patterns, such as those occurring during neuronal development, oncogenic transformation, or acquisition of drug resistance in tumor cells. However, the possibility that differential intracellular flows of different glycosphingolipids could influence the resulting glycosphingolipid patterns (independently from the expression levels of relevant glycosyltransferases) should not be neglected [6]. In other words, the regulation of intracellular sphingolipid traffic might be as important as the control of synthetic enzyme expression in determining the final glycosphingolipid composition of the plasma membrane. Another important point about the regulation of plasma membrane glycosphingolipid composition is the degradation that takes place in the lysosomes, where glycosphingolipids are transported by the endocytic vesicular flow through the early and late endosomal compartment to be catabolized. During this retrograde transport from plasma membrane to lysosomes, some glycosphingolipids, originally residents in the plasma membrane, can be diverted to different intracellular sites (presumably the Golgi apparatus), where they undergo direct glycosylation with the formation of more complex products and thus are able to return to the plasma membrane. It has been suggested that this process might be quantitatively relevant for certain cell types, including neurons [7], thus being another potential mechanism for regulating plasma membrane ganglioside composition at the level of intracellular traffic. Analogously, intermediate or final degradation products can escape the lysosomes and be recycled along the biosynthetic pathway. The salvage pathways for gangliosides in neurons should not be neglected from the quantitative point of view [8], but very little is known about the mechanisms of escape from the lysosome, the transfer of these intermediates to the Golgi or other cellular districts, or the regulation of these processes. The presence of soluble ganglioside–protein complexes in the cytosol, as reported by some authors [9–12], might reflect the intracellular traffic linked to the recycling of these intermediates.
14.2 Biological Functions of Glycosphingolipids and the Importance of Their Local Concentration Glycosphingolipids are essential for the survival, proliferation, and differentiation of eukaryotic cells within complex multicellular systems (i.e., tissues). This becomes particularly evident when cellular or animal models, lacking the activity of some of the enzymes involved in glycosphingolipid metabolism, are used.
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Important observations about the vital importance of glycosphingolipids in the “social life” of cells, i.e., in cells that are dealing with a multifaceted extracellular reality, have been made in comparing cellular models lacking ceramide glucosyltransferase activity with the corresponding animal model. In the GM-95 mutant melanoma cell line [13], ceramide glucosyltransferase activity is absent. This enzyme catalyzes the synthesis of glucosylceramide, a common glycosylation step in the biosynthetic pathway of all glucosylceramide-based complex glycosphingolipids. In the same way, embryonic stem cells derived from ceramide glucosyltransferase knockout mice [14] become glycolipid- deficient cells. Like the GM-95 cells, they are able to survive, grow, and undergo in vitro differentiation as well as their counterparts expressing the enzyme activity. However, ceramide glucosyltransferase knockout mice are embryonically lethal and showed no cellular differentiation beyond the primitive germ layers [15]. As already mentioned, glycosphingolipids are not randomly distributed along the membrane surface, but they are rather highly segregated with cholesterol in lipid domains with specialized signaling functions [1], typically referred to as “lipid rafts.” In these lipid domains, glycosphingolipids modulate the functional features of several membrane proteins through direct specific lipid–protein interactions or through the maintenance of a dynamic membrane organization. Thus, these complex membrane lipids participate in the modulation of several processes, such as cell proliferation, survival, adhesion, and cell differentiation. A high local concentration of glycosphingolipids in the plasma membrane has important implications with regard to their ability to engage both trans and cis functional interactions with other cellular components. In the first case, the recognition of lipid-bound oligosaccharides by soluble ligands (such as antibodies or toxins) or by complementary carbohydrates and carbohydrate-binding proteins (such as selectins, siglecs, and other lectins) belonging to the interfacing membrane of adjacent cells is strongly affected by their degree of dispersion (or segregation) [16]. In the same way, sphingolipid-enriched membrane domains could favor cis interactions, i.e., direct lateral interactions with plasma membrane proteins or short-range alterations of the lipid microenvironment of plasma membrane proteins [16]. During the development of the nervous system and along differentiation in cultured neurons, the glycosphingolipid patterns undergo deep qualitative and quantitative modifications [17–25], and this reflects the crucial role played by gangliosides in controlling various aspects of neural cell function [26, 27], as suggested by several experimental observations. The study of glycosphingolipid biological functions has been pursued for a long time by several experimental approaches; one of the widely used experimental models is the exogenous administration of gangliosides dissolved in culture medium to intact cells or membrane preparations. The binding, uptake, and metabolic fate of exogenous gangliosides under different experimental conditions have been well characterized [4, 28], and it has been shown that, after removing the amount of administered ganglioside loosely bound to the membrane, a portion of the stably associated ganglioside was inserted into the membrane. As a consequence,
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the subsequent cellular events can be ascribed to the resulting modifications of membrane composition and organization [29–32]. The addition of exogenous gangliosides resulted in the modulation of the biological activity of several proteins as tyrosine kinase receptors, protein kinases and phosphatases, ion channels, and pumps. Moreover, this exogenous addition is able to exert neuritogenic, neurotrophic, and neuroprotective effects on cultured neurons and neurotumor cell lines [20, 26, 30]. As supported by many study results, the differentiation and function of neurons in culture is strongly dependent on sphingolipid biosynthesis. In neuroblastoma cell lines, for example, the ability to extend neurites in response to various stimuli was correlated with the cellular gangliotetraose content [33], and treating neuroblastoma cells with Clostridium perfringens sialidase increased surface expression of GM1 and potentiated PGE1-induced neurite formation [33, 34]. Several pharmacological approaches could influence neuronal function and normal differentiation, causing either inhibition or upregulation of specific enzymes involved in glycosphingolipid metabolism. The inhibition of glycosphingolipid biosynthesis by synthetic inhibitors of glucosylceramide synthase (d-threo-1- phenyl-2-decanoylamino-3-morpholino-1-propanol [d-PDMP] and analogs) [35] or by inhibitors of sphinganine N-acyltransferase (the enzyme that catalyzes the synthesis of dihydroceramide, the biosynthetic precursor of ceramide and of all complex sphingolipids) [36], such as Fusarium moniliforme mycotoxins (fumonisins), caused a reduction in axonal elongation and branching in cultured hippocampal and neocortical neurons [37–39], and nerve growth factor (NGF)-induced neurite outgrowth in human neuroblastoma and PC12 cells [40, 41]. Conversely, upregulation of glycosphingolipid biosynthesis by L-PDMP stimulated neurite outgrowth in cultured cortical neurons [39, 42]. In the same cellular model, d- and l-PDMP exerted opposite effects on the formation of functional synapses and synaptic activity [42]. Induced expression of GD3 synthase was able to switch neuroblastoma cells to a differentiated phenotype [43]. NGF- and forskolin-induced differentiation in PC12 was accompanied by the upregulation of several glycosyltransferase activities (GalGb3-, GM3-, GD1a-, and GM2 synthases) [44], and basic fibroblast growth factor (bFGF)-stimulated axonal growth in cultured hippocampal neurons resulted in the activation of ceramide glucosyltransferase [45]. As mentioned above, the role of glycosphingolipids in the maintenance of neuronal structure and function can be explained, at least in part, by their ability to laterally interact with specific proteins (including growth factor receptors and neuronal adhesion molecules) at the level of the plasma membrane and to modulate their activity (cis interactions). Possible functionally significant interactions between gangliosides and plasma membrane proteins have been intensively studied in the past [27, 46, 47], and they usually resulted in being highly specific. Well-studied examples are represented by the interactions of epidermal growth factor receptor (EGFr) or insulin receptor with tyrosine kinases. In the first case, the phosphorylation on tyrosine residues and the dimerization of EGFr are inhibited by GM3 but uninfluenced by GM1 [48]; the insulin receptor is inhibited by GM3 but not by GD1a [49]. In the nervous system, it has been reported that GM1 is able
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to potentiate the neuritogenic effect of NGF in PC12 cells, i.e., it is able to induce neuronal differentiation in the presence of an NGF concentration that is ineffective by itself [50–52]. Within sphingolipid- and cholesterol-enriched membrane domains, glycosphingolipids and signaling proteins colocalize, and many papers have indicated that this could be sufficient for the realization of functional links, even in the absence of direct, strong, and specific glycosphingolipid–protein interactions. Thus, it is clear how the overall lipid raft dynamics, as determined by the peculiar (and possibly regulated) lipid composition of these domains, might be rather responsible for the functional modulation of raft-associated signaling proteins [1, 53–57]. Within lipid membrane domains isolated from cultured neural cells (neurons, oligodendrocytes, astrocytes, and neurotumor cell lines), brain tissues, myelin, and synaptic plasma membranes, it has been shown that sphingolipids (glycosphingolipids, sphingomyelin, and ceramide) and cholesterol segregate together with many classes of proteins involved in mechanisms of signal transduction that are relevant for neural cell biology (including receptor tyrosine kinases, G protein-coupled receptors, nonreceptor tyrosine kinases of the Src family, adapter and regulatory molecules of tyrosine kinase signaling, heterotrimeric and small guanosine triphosphate (GTP)binding proteins, protein kinase C isoenzymes, cell adhesion molecules, ion channels, proteins involved in neurotransmitter release, and postsynaptic density complex proteins) [55, 58–66]. This specific protein enrichment of lipid membrane domains is in accordance with the functional role played by these domains in several aspects of nervous system development and functional specialization. Many pieces of evidence demonstrated that sphingolipid- and cholesterol-enriched membrane domains have been involved in neurotrophic factor signaling [55, 64–66], cell adhesion and migration [55, 67, 68], axon guidance, synaptic transmission [55, 69], neuron–glia interactions [56, 70], and myelin genesis [71]. In some cases, it has been shown that signal initiation and propagation in neural cells involve receptors and effectors that permanently reside in lipid membrane domains [55, 64–66, 72, 73]. Alternatively, the activation of membrane receptors is followed by the translocation of the receptors themselves or effector signaling proteins to or from the domain to other cellular districts [55, 64, 66, 68]. In both cases, these events imply changes in the reciprocal interactions among lipid membrane domain components. Sphingolipids play an active role in the regulation of these interactions, as has been reported in several papers. In rat cerebellar granule cells, an increase in the surface occupied by the sphingolipid- and cholesterol-enriched membrane domains during the different stages of development in culture has been observed [25]. In particular, during axonal sprouting and neurite extension, the sphingolipid–glycerophospholipid molar ratio more than doubled, and the maximum ganglioside density was reached in fully differentiated neurons. On the contrary, a high content of ceramide was found in the domains of aging neurons. By different experimental approaches, some interactions between gangliosides and proteins have been identified within the lipid membrane domains. Ganglioside GM3 has been found to be closely associated with c-Src and Csk in neuroblastoma Neuro2a cells [74], and in rat brain and cerebellar granule cells, GD3 was associated with the Src-family kinase Lyn and
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the neural cell adhesion molecule TAG-1 [60, 75]. In these cells, a complex lipid environment seems to be essential for the interaction on the domain of c-Src, Lyn, Fyn, TAG-1, and prion protein [25, 59, 63, 76]. In differentiated rat cerebellar neurons, the membrane environment of PrPC has been studied by immunoprecipitation experiments [76]. In the separated PrPC-rich membrane domains, about 50% of the sphingolipids, cholesterol, and phosphatidylcholine present in the detergent-resistant sphingolipid-enriched membrane fraction have been found. The enrichments of all main sphingolipids in the PrPC-rich membrane domains, including sphingomyelin, neutral glycosphingolipids, and gangliosides, were very similar to those in the detergent-resistant sphingolipid-enriched membrane fraction. Moreover, a complex pattern of proteins was associated with the PrPC-enriched membrane domains, in a way depending on the existence of lipid-mediated interactions. Thus, the prion protein plasma membrane environment in differentiated neurons resulted in being a complex entity, with its integrity requiring a network of lipid-mediated noncovalent interactions rather than (or as well as) specific direct molecular interactions. Further supporting the notion that glycosphingolipids are essential in lipid domain-dependent cellular events was the multifaceted evidence that experimental manipulations able to change the concentration or pattern of glycosphingolipids in the plasma membrane profoundly affect – together with the organization of lipid membrane domain – the association of protein components with the domain itself and lipid domain-dependent signal transduction. Administration of exogenous GM1 and GM3 induced dissociation of Csk (the physiological inhibitor of Src kinases) from the lipid domain in neuroblastoma cells, followed by c-Src activation and neuritogenesis [74]. Treatment with fumonisin B1 or with ceramide glucosyltransferase inhibitors was able to deplete a detergent-insoluble lipid membrane domain from the glycosphingolipids and GPI-anchored proteins (e.g., Thy-1 in hippocampal neurons) [77–82] and to impair lipid domain-mediated biological functions [83–90]. Selective depletion of cell-surface sphingolipids, achieved by treating living cells with bacterial sphingomyelinases [84, 91] or with endoglycoceramidase (which are able to remove the oligosaccharide chain from cell-surface glycosphingolipids) [72] reduced the amount of sphingomyelin in detergent-insoluble membrane fractions in neuroblastoma cells [84] and inhibited TAG-1 signaling in cerebellar neurons, respectively [72].
14.3 Local Plasma Membrane Events and Their Role in the Modulation of Glycosphingolipid Composition The reduced molecular heterogeneity of the sphingolipid simple breakdown products – ceramide, sphingosine, and sphingosine-1-phosphate – greatly clarified the biological roles of this class of lipids, particularly when compared with the more complex glycosphingolipids. Moreover, the regulation of cellular sphingoid
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levels in response to physiological stimuli is relatively simple for this kind of molecule and relies on the activity of a limited number of metabolic enzymes. For a long time, the production of bioactive ceramide was regarded as being caused exclusively by sphingomyelin hydrolysis by sphingomyelinases [92]. It soon became clear that “signaling” sphingomyelinases are residents in the plasma membrane (as is the case for the Mg2+-dependent neutral SMase) or translocated to it from intracellular sites upon stimulus (as happens for the acid SMase, usually described as the lysosomal enzyme involved in the catabolic degradation of sphingomyelin) and are active on plasma membrane sphingomyelin pool(s) [93, 94]. More recently, a sphingomyelin synthase enzyme activity (SMS2), encoded by a different gene than that of the Golgi enzyme, was also shown to be present at the plasma membrane [95]. Thus, ceramide and sphingomyelin levels within the plasma membrane are regulated by two different enzyme activities acting – in opposite directions – directly on the plasma membrane in response to changes in cellular physiology, without needing to sort any of the substrates to intracellular sites of metabolism. The sphingomyelin–ceramide interconversion on the plasma membrane leads to changes in membrane curvature, as schematically reported in Fig. 14.2. Sphingomyelin, as a component of the external leaflet of the membrane, participates with its large and hydrophilic head group to confer a positive curvature
SM synthase
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Flip-flop Negative curvature
Positive curvature
Sphingomyelin
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Fig. 14.2 Changes of membrane geometry and organization following plasma membrane- associated sphingomyelin–ceramide interconversion
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to the cell surface. Ceramide, due to its much stronger hydrophobic character, is likely associated with a less positive, or negative, membrane curvature. Accordingly, ceramide formed enzymatically at the plasma membrane very rapidly should determine the formation of membrane areas with a negative curvature (or move to preexisting areas with these features). A further possibility is that a flip-flop process is thermodynamically favored, allowing ceramide to move from a positive to a negative membrane curvature. The information on sphingomyelin synthase is very scant, and no information is available on the membrane topology of this enzyme. In any case, due to geometrical considerations, sphingomyelin formed from ceramide belonging to a membrane area with a negative curvature will return to being a component of the extracellular leaflet of the membrane with positive curvature. Similar observations have been made for other enzymes responsible for regulating bioactive sphingoid levels. Plasma membrane-associated ceramidases and sphingosine kinases have been described as putatively responsible for the generation of sphingosine and/or sphingosine-1-phosphate at the cell surface [96–98]. Thus, in the case of simple sphingoids, the role of the plasma membrane as the site for those metabolic events responsible for locally regulating sphingoid levels in response to specific biological events is well established, even if not fully unveiled. In the case of glycosphingolipids, the number of enzymes responsible for their metabolism that have been shown to be associated with the plasma membrane is growing very rapidly, as is the information on their features, allowing for a precise characterization of some of them. A long time ago, it was shown that synaptosomal membranes, a subset of neuronal membranes highly enriched in gangliosides, carry both sialidase [99–102] and sialyltransferase [103] activity. However, the existence of a plasma membrane-associated sialidase distinct from the lysosomal enzyme was suggested by enzymatic and immunological studies [104–109], as well as by metabolic studies of intact cells; cultured rat cerebellar granule and human neuroblastoma cells possessed the capability to desialylate exogenously added GM3, GD1a, and GD1b under experimental conditions, preventing ganglioside internalization and lysosomal function [110, 111]. In human neuroblastoma SK-N-MC cells, the desialylation of GM3 and polysialogangliosides, but not of GM1, was strongly inhibited by a cell-impermeable sialidase inhibitor [112]. The membrane-bound sialidase was purified from human brain gray matter [113] and from bovine brain [114] and further characterized [115]. In 1999, the existence of a specific membrane-linked sialidase, distinct from other known sialidases, was unambiguously proven by Miyagi’s group, who cloned the complementary DNA (cDNA) sequence for human [116], bovine [117], and mouse [118] plasma membrane-associated sialidase, subsequently termed Neu3 [119]. Following studies elucidated the role of this enzyme in modifying the cell-surface ganglioside composition, causing a shift from polysialylated species to GM1, a decrease of GM3, and a parallel increase in lactosylceramide, with deep consequences on very important cellular events such as neuronal differentiation and apoptosis in colon cancer. In mouse and human neuroblastoma cells, Neu3 expression increased during pharmacologically induced neuronal differentiation [120],
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and Neu3 gene transfection accompanied by a corresponding increase in the enzyme activity enhanced the extension or branching of neurites induced by 5-bromodeoxyuridine [118] or by dibutyryl cAMP treatment, and was sufficient by itself to induce neurite outgrowth [120]. Conversely, inhibition of plasma membrane sialidase activity resulted in the loss of neuronal differentiation markers [111, 121]. In cultured hippocampal neurons, the activity of the plasma membraneassociated ganglioside sialidase locally regulated GM1 surface levels and was essential for axonal growth and regeneration after axotomy [122]. In these cells, Neu3 activity was asymmetrically concentrated at the end of one single neurite and determined the neurite’s axonal fate by a local increase in TrkA activity [123]. In colon and renal cancer, this sialidase seemed to be responsible for maintaining high cellular levels of lactosylceramide that would exert a Bcl-2-dependent antiapoptotic effect, contributing to the survival of cancer cells and consequent tumor progression [124, 125]. The nonrandom distribution of Neu3 at the cellular surface was confirmed by the observation that this ganglioside sialidase associated with Triton X-100 insoluble glycosphingolipid-enriched membranes [126] and closely associated with caveolin-1 in Neu3-transfected COS-1 cells [127]. The colocalization of Neu3 and its putative substrates at the cell surface is probably not surprising; on the contrary, it raises the possibility that the biological effects of this enzyme are due to the local reorganization of glycosphingolipid-based signaling units (Fig. 14.3). Remarkably, the ability of Neu3 to modulate the cell-surface glycolipid composition was not restricted to cis interactions. In fact, mouse Neu3 overexpressed in COS-7 cells was able to hydrolyze ganglioside substrate belonging to the surface of neighboring cells [128]. Subsequently, it has been shown that Neu3 was able to modulate the production of bioactive ceramide at the cell surface when overexpressed in cultured skin fibroblasts, providing the first direct evidence on a link between glycosphingolipid metabolism and ceramide-mediated signaling [129]. Neu3-assisted cell-surface ceramide generation from ganglioside GM3 indirectly demonstrates the presence of the other two active glycosyl hydrolases, b-glucosidase and b-galactosidase, in the same plasma membrane district. The presence of active b-hexosaminidase A in the external leaflet of plasma membrane has been also demonstrated in cultured fibroblasts [130]. Different from the sialidase, the immunological and biochemical characterization of the membrane-associated b-hexosaminidase suggested that this enzyme has the same structure as the lysosomal enzyme. Since it has been shown that the regulated fusion of lysosomes with the plasma membrane might be a general mechanism of repair for the plasma membrane [131], these observations open the possibility that other lysosomal glycolipid-metabolizing enzymes could reach the cell surface and play an active role in remodeling its glycolipid composition. However, the existence of specific membrane-associated isoenzymes for glycosyl hydrolases, other than sialidases, cannot be excluded. A lot of further experimental work will be needed to fully understand the real significance of these events, but it is indubitable that cell-surface hydrolysis of complex glycosphingolipids does occur. Some information is also available about the in situ sialylation of gangliosides at the cell surface. The original report on the existence of a synaptosomal membrane
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H+ sialidase
OH− sialyltransferase
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Fig. 14.3 Changes of membrane geometry and organization following plasma membrane- associated GD1b–GM1, or GD1b–GD1b lactone interconversion
sialyltransferase in calf brain [103] has been confirmed by metabolic studies in chicken embryos [132] and rat brain [133, 134]. More recently, it has been shown that dexamethasone treatment markedly increased GM3 synthesis due to enhanced gene expression and increased enzyme activity of GM3 synthase. Radiolabeling metabolic studies indicated that this event was localized at the plasma membrane [135], thus confirming that glycolipid sialylation might occur outside the Golgi compartment, contributing to the local modulation of cell-surface glycolipid patterns. Glycosylation and deglycosylation pathways are not the only chance to modify the plasma membrane glycosphingolipid composition. In this sense, a very intriguing (even if very poorly understood) mechanism is the possible lactonization of gangliosides containing a disialosyl residue, such as GD1b. Ganglioside lactones are present as minor components in vertebrate brains [136, 137]. GD1b monolactone
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formation in the presence of catalytic proton concentrations has been studied in vitro [138], and it has been shown that the lactonization process profoundly influenced the conformational, aggregational [139], and biological properties of GD1b [140]. GD1b is able to directly interact with several cellular proteins [59] and to modulate several plasma membrane-associated protein kinase activities [140]. But when gangliosides were lactonized, these properties were strongly reduced or lost [140, 141]. This suggests that lactonization/delactonization might be a localized event that is able to trigger specific ganglioside-mediated cellular events. Unfortunately, no information is available about the possible mechanism responsible for this conversion in vivo. Metabolic remodeling is not the only local event that could contribute to the surface composition and organization of cell glycosphingolipids. It is known that glycosphingolipids and sphingolipids can be released from the cell surface to the extracellular milieu in the form of monomers or aggregates, including shedding vesicles [142–145]. Glycolipid-containing shedding vesicles seemed to originate from caveolin- and glycolipid-enriched membrane areas, thus their release could be used by the cell to modify the lipid membrane domain composition and organization. On the contrary, it has been suggested that shed gangliosides could be taken up by neighboring cells, modifying their lipid composition [146]. However, the metabolic fate of shed glycolipids after reuptake seems oriented toward degradation, thus the contribution of this event to the determination of cell lipid composition remains unclear [144].
14.4 Summary A long time ago, it was shown that synaptosomal membranes, a subset of neuronal membranes highly enriched in glycosphingolipids, particularly gangliosides, carry both a sialidase and a sialyltransferase activity on sialoglycolipids. The existence of a plasma membrane-associated ganglioside sialidase distinct from the lysosomal enzyme has been suggested by enzymatic, immunological, and metabolic studies, and then unambiguously proven by cloning the cDNA sequence for the human, bovine, and mouse enzyme, subsequently termed Neu3. In neuroblastoma cells, Neu3 expression increased during pharmacologically induced neuronal differentiation, and Neu3 gene transfection, accompanied by a corresponding increase in the enzyme activity, enhanced the extension or branching of neurites induced by 5-bromodeoxyuridine and was sufficient by itself to induce neurite outgrowth. Conversely, inhibition of plasma membrane sialidase activity resulted in the loss of neuronal differentiation markers. In cultured hippocampal neurons, the activity of the plasma membrane-associated ganglioside sialidase locally regulated GM1 surface levels and was essential for axonal growth and regeneration after axotomy. In colon and renal cancer, this sialidase seemed to be responsible for maintaining high cellular levels of lactosylceramide, which would exert a Bcl-2dependent antiapoptotic effect, contributing to the survival of cancer cells and
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consequent tumor progression. Remarkably, the ability of Neu3 to modulate the cell-surface glycolipid composition was not restricted to cis interactions. In fact, mouse Neu3 overexpressed in COS-7 cells was able to hydrolyze ganglioside substrate belonging to the surface of neighboring cells. More recently, it has been shown that Neu3 was able to modulate the production of bioactive ceramide at the cell surface when overexpressed in cultured skin fibroblasts, providing the first direct evidence of a link between glycosphingolipid metabolism and ceramidemediated signaling. Neu3-assisted cell-surface ceramide generation from gangliosides indirectly implies the presence of other glycosyl hydrolases (b-glucosidase and b-galactosidase) in the same plasma membrane district. In addition to this, the presence of active b-hexosaminidase A in the external leaflet of plasma membrane has been demonstrated in cultured fibroblasts. Much less information is available about the possible in situ sialylation of gangliosides at the cell surface. Nevertheless, the original report on the existence of a synaptosomal membrane sialyltransferase has been confirmed by several metabolic studies. Acknowledgments This work was supported by the Mitzutani Foundation for Glycoscience Grant 070002, which was given to Alessandro Prinetti, and by the CARIPLO Foundation Grant 2006, which was given to Sandro Sonnino.
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associated mature beta-hexo-saminidase A, active towards GM2 ganglioside, in human fibroblasts. FEBS Lett 579(25):5501–5506 131. Reddy A, Caler EV, Andrews NW (2001) Plasma membrane repair is mediated by Ca(2+)regulated exocytosis of lysosomes. Cell 106(2):157–169 132. Matsui Y, Lombard D, Massarelli R, Mandel P, Dreyfus H (1986) Surface glycosyltransferase activities during development of neuronal cell cultures. J Neurochem 46(1):144–150 133. Durrie R, Saito M, Rosenberg A (1988) Endogenous glycosphingolipid acceptor specificity of sialosyl-transferase systems in intact Golgi membranes, synaptosomes, and synaptic plasma membranes from rat brain. Biochemistry 27(10):3759–3764 134. Durrie R, Rosenberg A (1989) Anabolic sialosylation of gangliosides in situ in rat brain cortical slices. J Lipid Res 30(8):1259–1266 135. Iwamori M, Iwamori Y (2005) Changes in the glycolipid composition and characteristic activation of GM3 synthase in the thymus of mouse after administration of dexamethasone. Glycoconj J 22(3):119–126 136. Sonnino S, Ghidoni R, Chigorno V, Masserini M, Tettamanti G (1983) Recognition by twodimensional thin-layer chromatography and densitometric quantification of alkali-labile gangliosides from the brain of different animals. Anal Biochem 128(1):104–114 137. Riboni L, Sonnino S, Acquotti D, Malesci A, Ghidoni R, Egge H, Mingrino S, Tettamanti G (1986) Natural occurrence of ganglioside lactones. Isolation and characterization of GD1b inner ester from adult human brain. J Biol Chem 261(18):8514–8519 138. Bassi R, Riboni L, Sonnino S, Tettamanti G (1989) Lactonization of GD1b ganglioside under acidic conditions. Carbohydr Res 193:141–146 139. Acquotti D, Fronza G, Riboni L, Sonnino S, Tettamanti G (1987) Ganglioside lactones: 1H-NMR determination of the inner ester position of GD1b-ganglioside lactone naturally occurring in human brain or produced by chemical synthesis. Glycoconj J V4(2):119–127 140. Bassi R, Chigorno V, Fiorilli A, Sonnino S, Tettamanti G (1991) Exogenous gangliosides GD1b and GD1b-lactone, stably associated to rat brain P2 subcellular fraction, modulate differently the process of protein phosphorylation. J Neurochem 57(4):1207–1211 141. Sonnino S, Chigorno V, Valsecchi M, Bassi R, Acquotti D, Cantu L, Corti M, Tettamanti G (1990) Relationship between the regulation of membrane enzyme activities by gangliosides and a possible ganglioside segregation in membrane microdomains. Indian J Biochem Biophys 27(6):353–358 142. Kong Y, Li R, Ladisch S (1998) Natural forms of shed tumor gangliosides. Biochim Biophys Acta 1394(1):43–56 143. Deng W, Li R, Ladisch S (2000) Influence of cellular ganglioside depletion on tumor formation. J Natl Cancer Inst 92(11):912–917 144. Chigorno V, Giannotta C, Ottico E, Sciannamblo M, Mikulak J, Prinetti A, Sonnino S (2005) Sphingolipid uptake by cultured cells: complex aggregates of cell sphingolipids with serum proteins and lipoproteins are rapidly catabolized. J Biol Chem 280(4):2668–2675 145. Dolo V, Li R, Dillinger M, Flati S, Manela J, Taylor BJ, Pavan A, Ladisch S (2000) Enrichment and localization of ganglioside G(D3) and caveolin-1 in shed tumor cell membrane vesicles. Biochim Biophys Acta 1486(2–3):265–274 146. McKallip R, Li R, Ladisch S (1999) Tumor gangliosides inhibit the tumor-specific immune response. J Immunol 163(7):3718–3726
Chapter 15
9-O-Acetyl GD3 in Lymphoid and Erythroid Cells Kankana Mukherjee, Suchandra Chowdhury, Susmita Mondal, Chandan Mandal, Sarmila Chandra, and Chitra Mandal
Keywords Apoptosis • Childhood acute lymphoblastic leukemia • Erythrocytes • Erythropoiesis • Lymphoblasts • GD3 • 9-O-acetyl-GD3 • 9-O-acetylated sialoglycoprotein Sialic acids are electronegatively charged sugars that contribute to the enormous structural diversity of complex carbohydrates, which are major constituents of mostly proteins and lipids of cell membranes and secreted macromolecules. They are usually positioned at the outer end of these molecules and thus are well suited for interacting with other cells, pathogens, or molecules in the cell environment. Sialic acids are 9-carbon-containing monosaccharides, and the structural diversity of glycan chains is further increased by the various modifications of sialic acids [1]. Amongst 50 known derivatives of sialic acids, 7-, 8-, and 9-O-acetylated derivatives (O-AcSA) are important constituents of the cell membrane and are known to influence many physiological and pathological processes [1, 2], including cell–cell adhesion, signaling, differentiation, and metastasis [3–6]. However, as O-acetyl esters from positions C-7 and C-8 spontaneously migrate to C-9, even under physiologic conditions, O-acetylation at C-9 is considered the most common biologically occurring modification [7]. The appearance of O-acetylated sialic acids on glycoproteins or glycolipids is cell-type specific and developmentally regulated, their synthesis and turnover being a highly orchestrated phenomenon. O-acetylation can have a significant role in cell physiology and can alter the functional effects of important molecular determinants in various disease conditions. In this chapter, we deal with the O-acetylation of glycosphingolipids (GSLs), specifically GD3 in both erythroid and lymphoid cells.
C. Mandal (*) Infectious Disease and Immunology Division, Indian Institute of Chemical Biology, A Unit of Council of Scientific and Industrial Research (CSIR), 4, Raja S. C. Mullick Road, Kolkata 700032, India e-mail:
[email protected] A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_15, © Springer Science+Business Media, LLC 2011
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Structure 15.1. Structure of ganglioside GD3 (a) and 9-O-acetyl GD3. Ceramide (Cer), galactose (Gal), glucose(Glc), sialic acid (Neu5Ac), and 9-O-acetyl sialic acid (Neu5,9Ac)
15.1 GD3 and 9-O-Acetyl GD3 Play Antagonistic Roles in Apoptosis GSLs are amphipathic molecules with a polar glycan chain as the head group and a hydrophobic sphingosine-containing ceramide tail, which is typically embedded in the outer leaflet of the plasma membrane. The mono- or multisialosylated GSLs are named gangliosides. GD3 is a ganglioside with two sialic acids linked to a lactosylceramide core common to many GSLs (Fig. 15.1). It is a minor ganglioside in most normal tissues, except placenta and thymus [8], and is specifically expressed on the surface of a small subset of normal human peripheral blood T cells [9]. GD3 ganglioside is highly expressed only during development and is present at high levels in the embryonic brain, with marked decrease during postnatal development [10]. This ganglioside is also expressed in certain pathological conditions as a tumor-associated ganglioside: melanomas, medulloblastomas, neuroblastomas, meningiomas, gliomas, soft tissue sarcomas, leukemias, colorectal and pancreatic carcinomas, and metastases of breast and lung cancer, as well as adenocarcinoma obtained by excision biopsy [11–18]. Increased GD3 expression has been found in brain tissue from patients
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Fig. 15.1 General scheme for ganglioside biosynthesis. The formation of O-, a-, b-, and c-series gangliosides is catalyzed by glycosyltransferases of Golgi membranes. All enzymatic steps (possibly except formation of LacCer) take place at the luminal surfaces of the Golgi membranes
with various neurodegenerative disorders, such as Creutzfeldt–Jakob disease, and subacute sclerosis panencephalitis [19, 20]. De Maria first proposed that GD3 could act as an apoptosis-inducing ganglioside in human hemopoietic cells [21]. In human tumor lymphoid and myeloid cell lines, GD3 rapidly accumulates upon CD95 triggering or ceramide exposure and directly induces apoptosis [22, 23]. Fas-mediated activation of membrane-associated acidic sphingomyelinase (ASMase) or its involvement in tumor necrosis factor (TNF) signaling generates free ceramides that are believed to return to Golgi-like compartments to be converted to GD3 [24, 25]. Addition of GD3 to intact cells induced apoptosis, and addition to isolated mitochondria gave a loss of mitochondrial transmembrane potential, along with release of apoptogenic factors such as cytochrome c and caspase-9. Antisense RNA against GD3 synthase prevents apoptosis, implying the need for newly synthesized GD3. On the other hand, enforced expression of GD3 synthase was sufficient to trigger apoptosis. The dissipation of mitochondrial membrane potential and membrane permeabilization is followed by generation of reactive oxygen species and release of apoptogenic factors, such as cytochrome c, adenosine triphosphate (ATP), apoptosis-inducing factor (AIF) [26], and mitochondrial caspases [27]. Once released in the cytosol, cytochrome c and ATP serve as cofactors for the Apaf-1-mediated activation of caspase-9 [28], while AIF is able to drive nuclear apoptosis [29]. 9-O-acetylated GD3 (9-O-AcGD3) is an acetylated modification of GD3 in which the outer sialic acids get 9-O-acetylated. Like GD3, 9-O-AcGD3 is expressed during neuronal development, and its expression becomes very limited
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Fig. 15.2 Role of GD3 and 9-O-AcGD3. The interaction of GD3 with mitochondria elicits the mitochondrial apoptosome resulting in activation of executioner caspase-3. 9-O-AcGD3 fails to induce these changes and fails to cause apoptosis. (⊥) symbolizes the failure of 9-O-AcGD3 to activate the mentioned pathway. The route of trafficking of GD3 and 9-O-AcGD3 to mitochondria is not fully understood
in adult tissues [18, 30–32]. The presence of 9-O-AcGD3 was observed in some tumors, such as melanoma [14, 33] and breast cancer [34, 35], as well as in tumor cell lines like MOLT-4 [36] and SKMel28 [37]. In many tumors, it appears only intracellularly and is not simultaneously exposed on the cell membrane [38]. Unlike GD3, 9-O-AcGD3 is a selective marker for germinal cells of the central nervous system [39] and shows a dorsoventral gradient across the developing retina. The CD60 subset of human T-lymphocyte markers includes GD3 and 9-O-AcGD3 [40]. Malisan et al. postulated that 9-O-acetylation could rescue the cell from GD3-induced apoptosis [41]. The study shows that 9-O-AcGD3 can counteract the proapoptotic effects of GD3. The authors conclude that by turning part of proapoptotic GD3 into “harmless” 9-O-AcGD3, 9-O-acetylation acts as an effective antiapoptotic mechanism. Subsequently, leukemic cell lines (MOLT-4 and Jurkat) were involved to show the antiapoptotic potential of 9-O-AcGD3 [42] (Fig. 15.2). Although 9-O-AcGD3 is reported to be an antiapoptotic molecule in cells of lymphoid origin, so far there are no reports on its status or role in erythroid lineage. This chapter focuses on the status and role of 9-O-AcGD3 in the erythroid progenitors during the process of erythropoiesis as well as in the mature erythrocytes.
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15.2 9-O-AcGD3 in Erythroid Cells Acute lymphoblastic leukemia (ALL) is a malignant transformation of lymphoblasts and represents the single most common type of cancer in the pediatric population. With the advent of modern chemotherapy, virtually all patients achieve remission, and approximately 80% are cured. The risk of relapse remains at 20% as patients in remission may harbor residual leukemic blasts referred to as minimal residual disease, and prediction of this still remains a major challenge in leukemia research [43]. Our group aims towards a comprehensive study to reveal the role of 9-O-acetylation of sialic acids in the disease biology of childhood ALL [44–47]. In previous studies, we have detected 9-O-acetylated sialoglycoproteins (Neu5,9Ac2-GPs) on peripheral blood mononuclear cells of ALL patients but not in patients with other hematological disorders [44–46]. The high level of Neu5,9Ac2-GPs and antibodies against Neu5,9Ac2-GPs helps these lymphoblasts to evade apoptosis [47–49]. The circulating immune-complexed Neu5,9Ac2-GPs, Neu5,9Ac2-GPs, and anti-Neu5, 9Ac2GPs have been used for monitoring disease status [50–52]. Neu5,9Ac2-GPs have been shown to be actual signal molecules and to promote survival of leukemic blasts. We have also observed enhanced expression of 9-O-AcGD3 on these lymphoblasts (unpublished data); it could be an interesting molecule as ALL is a disease caused by maturation arrest. Here we have highlighted the status of 9-O-AcGD3 in the erythroid progenitors during the process of erythropoiesis as well as in the mature erythrocytes and a possible biological role.
15.3 Identification of Different Erythroid Progenitor Populations Depending on the light-scattering property, four distinct regions were designated as B, L, R and G in the mononuclear cell (MNC) [52]. Based on the morphology, the least mature erythroid precursors was identified which reside in the region B. Immature cells of other lineages were also found in this region based on their colony-forming ability. Population L contained less mature nucleated erythroid precursors (normoblasts) and lymphoid cells. However, population R enriched with mature, non-nucleated erythrocytes and reticulocytes. No erythroid cells were observed in the region G which predominantly contained maturing cells of the granulocytic lineage [69]. The forward and side light-scattering contour plot of MNCs present in BM is shown in Fig. 15.3a. The Glycophorin A and CD45 were used as markers for erythroid progenitor cells and nucleated cells respectively. The status of 9-O-AcGD3 on these cells was investigated subsequently [69].
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Fig. 15.3 Status of 9-O-AcGD3 in erythroid maturation. MNCs separated from BM were stained with anti-glycophorin A and anti-9-O-AcGD3 monoclonal antibody and acquired in a FACSCalibur. (a) Forward scatter and side scatter of the MNCs in linear scale. The regions are gated as follows: B = the least mature erythroid precursors along with immature cells of all other lineages; L = less-mature nucleated erythroid precursors (normoblasts) along with lymphoid cells; R = mature nonnucleated erythrocytes and reticulocytes; G = granulocytes. (b) Status of 9-O-AcGD3 and glycophorin A in the B region. (c) Status of 9-O-AcGD3 and glycophorin A in the L region. (d) Status of 9-O-AcGD3 and glycophorin A in the R region [69]
15.4 The Expression of 9-O-AcGD3 in Different Maturational Stages of Erythropoiesis In erythroid progenitor and mature erythrocytes, Glycophorin A is expressed differentially as a cell surface antigen. Therefore, to determine the expression level of 9-O-AcGD3 during erythroid maturation we explored only the Glycophorin A+ cells in B, L and R windows. Quantitatively, on the surface of MNCs, the Glycophorin A expression was enhanced from B to R region. In contrast, the surface expression of CD45 was maximum in the B region and insignificant in the R region. In the total MNC population, 9-O-AcGD3+Glycophorin A+ cells in the B region was 13.45 ± 6.24% while that in the L and R region were 4.21 ± 3.68% and 1.54 ± 1.08% respectively (Fig. 15.3b–d, Table 15.1) [69]. This result clearly demonstrated that in BM, during the maturation of erythrocytes to non-nucleated erythrocytes the expression of 9-O-AcGD3 decreases significantly. The gradual decrease of the mean fluorescence
15 9-O-Acetyl GD3 in Lymphoid and Erythroid Cells Table 15.1 Status of 9-O-Acetyl GD3 in erythroid maturation stages in BM Percentage of cells in total MNCsb Erythroid populations in different regions of BMa BM1 BM2 BM3 BM4
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B 25.17 49.55 26.0 85.49 1,809 ± 91 9-O-AcGD3+ glycophorin A+ cells in B 11.3 20.16 7.02 17.2 L 6.98 13.86 6.85 7.33 1,115 ± 85 9-O-AcGD3+ glycophorin A+ cells in L 4.11 9.42 2.16 1.16 R 1.37 4.76 11.25 2.08 715 ± 53 1.07 3.01 1.62 0.47 9-O-AcGD3+ glycophorin A+ cells in R MFI mean fluorescence intensity a Regions B, L, and R contained cells as described in the text b BM1, BM2, and BM3 = BMs of three representative samples collected from individuals who were declared normal by the clinicians; BM4 = a representative patient with ALL at the onset of disease, i.e., before any chemotherapy [69]
Fig. 15.4 Status of glycophorin A, CD45, and 9-O-AcGD3 through erythropoiesis. MNCs separated from normal BM were stained with anti-glycophorin A, anti-CD 45, and anti-9-O-AcGD3 mAb and acquired in a FACSCalibur. The y-axis represents the mean MFI, and the x-axis represents the stages in erythroid maturation
intensity (MFI) being 1809 ± 91, 1115 ± 85 and 715 ± 53 in B, L and R regions respectively (Table 15.1) further support the previous findings. It is well known that BM of ALL patients contain immature lymphoblasts due to maturational defect of the lymphoid progenitors. Hence, the bulk of the B region was enriched with leukocytes (80 ± 10%) and less erythroid progenitors (10 ± 7%). Interestingly, even in the disease condition, the expression of 9-O-AcGD3 on these erythroid progenitors showed comparable decrease as compared to normal BM. In the non-nucleated mature erythrocytes population with negligible CD45 antigen expression, the MFI of 9-O-AcGD3 was found to be 675 ± 48 while that of glycophorin A was 13,220 ± 177 (Fig. 15.4) [69].
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15.5 Anti-9-O-AcGD3 Monoclonal Antibody Induces Alteration of Mature Erythrocyte Membrane 15.5.1 Membrane Osmotic Fragility Osmotic fragility has been found to be altered in various pathological conditions [53–56]. Erythrocytes sensitized with anti-9-O-AcGD3 monoclonal antibodies (mAb) were more fragile than untreated cells. Optimum osmotic fragility of the membrane of freshly isolated erythrocyte was observed with 0.5% sodium chloride exposure at 37°C for 1 h (Fig. 15.5a).
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Fig. 15.5 Membrane of mature erythrocytes sensitized with 9-O-AcGD3 mAb becomes more osmofragile. Erythrocytes (1 × 106) were incubated at 37°C for 0–8 h with 9-O-AcGD3 mAb (1:25) or with Ca2+ ionophore (0.5 mM) in the presence of Ca2+ (2.5 mM) or only phosphatebuffered saline (PBS) (pH 7.4). These sensitized cells were washed with PBS and again incubated at 37°C for 1 h with 1.0 ml of 0.5% NaCl or distilled water. The degree of hemolysis in the supernatant was measured spectrophotometrically at 412 nm. Lysis obtained with cells incubated with distilled water was considered 100% (a) Degree of hemolysis of 9-O-AcGD3-sensitized erythrocytes or treatment with Ca2+ ionophore in the presence of Ca2+ for 1 h. (b) Degree of hemolysis after stimulating erythrocytes with 9-O-AcGD3 mAb or with Ca2+ ionophore in the presence of Ca2+, for 1 h. Data are mean ± SD of three independent experiments (c) [69]
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These erythrocytes undergo rapid lysis when preexposed with 9-O-AcGD3 mAb and then incubated in 0.5% saline at 37°C for 1 h (Fig. 15.5b, c). The rapid lysis of 9-O-AcGD3-sensitized erythrocytes hints towards a probable role of these cell surface gangliosides in inducing cell death. Calcium ionophore in the presence of extracellular Ca2+ is known to induce death of mature human erythrocytes [57–59]. Ca2+ ionophore in the presence of Ca2+ induced maximum hemolysis of the erythrocytes at 37°C for 1 h and was treated as a positive control for the assay. The anti-9-O-AcGD3 mAb (clone Jones, Sigma) is however known to crossreact with a number of other gangliosides. The involvement of some of these lipids in response to stimulation with this antibody cannot be ruled out. Moreover, the presence and role of many gangliosides in downstream signaling in mature erythrocytes have been reported [60, 61].
15.5.2 9-O-AcGD3 Sensitized Matured Erythrocytes Showed Enhanced Membrane Hydrophobicity After sensitization of 9-O-AcGD3, erythrocytes showed enhanced intensity of 8-anilino-1-napthalenesulfonic acid (ANS) binding in arbitrary fluorescence units as compared to that of untreated erythrocytes. This result indicated that sensitized erythrocytes become more hydrophobic by nature (Fig. 15.6) [69]. Besides that, sensitized erythrocytes also showed a significant shift of the emission maxima from 520 nm to 480 nm suggesting distinct alteration of the erythrocyte membrane on sensitization of 9-O-AcGD3 with anti-9-O-AcGD3 mAb.
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Fig. 15.6 Membrane of erythrocytes sensitized with anti-9-O-AcGD3 mAb became more hydrophobic. Erythrocytes (1 × 106) were incubated at 37°C for 1 h with 9-O-AcGD3 mAb (1:25) or only PBS (pH 7.4). These sensitized cells were washed with PBS, loaded with ANS (5.0 ml, 1.0 mM), and further incubated at 37°C for 1 h. The binding of ANS to hydrophobic sites on erythrocyte membrane was measured using a fluorometer (EXmax = 365 and EMmax = 490); fluorescence spectra were scanned from 420 to 600 nm. The excitation and emission band passes were 5.0 nm in width. The graph represents results from one of three independent experiments [69]
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Fig. 15.7 Morphological changes of erythrocytes sensitized with anti-9-O-AcGD3 mAb. SEM analysis was carried out using erythrocytes treated with (a) only PBS and (b) sensitized with anti9-O-AcGD3 mAb at 37°C for 1 h. Micrographs are taken at a magnification of 18,000, and about 200 cells were counted to calculate the percentage of deformed cells [69]
15.5.3 Morphological Changes of 9-O-AcGD3 Sensitized Mature Erythrocytes Membrane The role of 9-O-AcGD3 in the maintenance of the shape of erythrocytes was further evaluated by scanning electron microscopy (SEM). Sensitization of 9-O-AcGD3 induces ultra structural and morphological changes, whereas the unsensitized cells maintain the normal discoid shape (Fig. 15.7). These qualitative results were confirmed by morphometric analyses, indicating about a 50% increase in the number of shrunken erythrocytes.
15.6 Programmed Cell Death Induced by Stimulating Mature Erythrocytes via 9-O-AcGD3 Human mature erythrocytes are terminally differentiated cells of the erythroid lineage that are devoid of mitochondria, as well as nucleus and other organelles. They have a normal life span of 120 days, which ends by a process of senescence leading to their clearance from the peripheral blood by reticuloendothelial cells. Programmed cell death (PCD) or apoptosis is a physiological process that contributes to the homeostasis of multicellular organisms and maintains the balance between cell proliferation and cell death. Erythrocytes were, until recently, considered to lack pathways that have been traditionally linked to activation of the apoptotic machinery in eukaryotic, nucleated cells. However, recent studies have pointed in a direction indicating that part of the machinery that is associated with execution
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of apoptosis in nucleated cells also exists in mature human erythrocytes. Erythrocyte senescence shares features common to apoptosis in nucleated cells, such as cell membrane blebbing and breakdown of cell membrane asymmetry with phosphatidylserine (PS) exposure at the cell surface [62–65].
15.6.1 Sensitized Mature Erythrocytes Showed Enhanced Phosphatidyl Serine Externalisation
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Fig. 15.8 Death induced by anti-9-O-AcGD3 mAb in mature erythrocytes. (a) Erythrocytes (1 × 106) untreated, sensitized with anti-9-O-AcGD3 mAb (antibody treated) or Ca2+ ionophore in the presence of Ca2+ (calcium ionophore) for 30 min at 37°C and stained with FITC-annexin V. Dot plots are representative of three independent experiments. (b) Erythrocytes (5 × 106) untreated, sensitized with anti-9-O-AcGD3 mAb (antibody treated) or Ca2+ ionophore in the presence of Ca2+ for 1 h at 37°C, and tested for the activation of caspase-3 using Caspase-3/CPP32 Fluorometric Assay Kit according to the manufacturer’s instructions. For antibody treatment in the presence of caspase-3 inhibitor, cells were pretreated with 100 mM DEVD-CHO at 37°C for 30 min. The values are mean ± SD of three independent experiments [69]
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incubated at 37°C for 30 min compared to the cells incubated with only buffer (14.5 ± 6.8%). The 9-O-AcGD3-sensitized erythrocytes showed annexin-V positivity (49.3 ± 13.1%) [70]. Hence, the anti-9-O-AcGD3 mAb induced cell death that shares its characteristics with apoptosis.
15.6.2 A Caspase-3 Dependent Death Induced by Sensitization of the 9-O-AcGD3 on Erythrocyte Membrane PCD mainly rely on two major pathways, one is intrinsic pathway that involves mitochondria and another is extrinsic or death receptor mediated pathway which usually function together and amplify each other. In mitochondrial pathway, depolarization of mitochondria initiates the release of mitochondrial pro-apoptotic proteins in cytosol that either induces caspase activation, such as cytochrome c, or trigger caspase-independent effectors pathways such as apoptosis-inducing factor AIF, Endo G etc. Pro-apoptotic stimuli, in general, require mitochondrion-dependent step in addition to the death receptor mediated direct activation of caspase 8 [70–73]. In the process of programmed senescence, caspases, the aspartate-directed cysteine proteases are the crucial mediator [74]. Caspases either function as initiator (e.g. caspase-8 and -9) or effector (e.g. caspase-3,6,7) caspase in response to the apoptotic signals, which then cleave different important proteins to manifest apoptotic phenotype [74–76]. We evaluate the role of the main effector caspase i.e. caspase-3, considering that erythrocytes lack mitochondria [69]. A 49-fold increase in active caspase-3 was observed after sensitization of 9-O-AcGD3 of freshly isolated mature erythrocytes as compared to the unsensitized cells under similar conditions. The level of active caspase-3 decreased to 23 fold in the presence of caspase-3 inhibitor DEVD-CHO (Fig. 15.8b). Caspase-3 activation was maximum in cells exposed to Ca2+ ionophore in the presence of Ca2+ used as control. During environmental signals like Ca2+ exposure, Ca2+ dependent cystein protease like calpain are activated during erythrocyte death and in turn are responsible for degradation of membrane proteins that finally leads to senescence [58]. These observations suggested a caspase-3 dependent death induced by the extrinsic signal, i.e., sensitization of the 9-O-AcGD3 on erythrocyte membrane (Fig. 15.9). However, the involvement of other caspases or calpain should not be ignored. Until recently, 9-O-AcGD3 has been reported to have an antiapoptotic role and to help in tumor progression in lymphoid cells [42]. We reported for the first time that signaling through 9-O-AcGD3 triggers PCD of mature erythrocytes, suggesting a cell-specific role for this acetylated ganglioside [62]. So far, our observation is based on normal mature erythrocytes, both from normal healthy volunteers as well as from patients with ALL under maintenance therapy and declared normal by the clinicians. It would be interesting to explore this phenomenon in erythrocytes from patients with ALL at the presentation of the disease.
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15.7 Summary Sialic acids are important constituents of the cell membrane and are known to influence many physiological and pathological processes. O-acetylation of sialic acids at C-9 is considered the most common biologically occurring modification. The appearance of O-acetylated sialic acids on glycoproteins or glycolipids is cell-type specific and developmentally regulated, their synthesis and turnover being a highly orchestrated phenomenon. Childhood ALL is a malignant transformation of lymphoblasts and represents the single most common type of cancer in the pediatric population. In the search for better biomarkers, we have demonstrated an enhanced presence of disease-associated 9-O-acetylated sialoglycoproteins (Neu5,9Ac2-GPs) on lymphoblasts and erythrocytes of these children, indicative of defective sialylation associated with ALL. The importance of these Neu5,9Ac2-GPs was demonstrated by their gradual decline with treatment and their reappearance with clinical relapse. To understand the functional implications of Neu5,9Ac2-GPs, lymphoblasts have been activated through Neu5,9Ac2a2-6GalNAc glycotopes, which led to the release of high amounts of interferon (IFN)-g. Exposure of lymphoblasts to IFN-g led to the production of nitric oxide, promoting their survival by evading apoptosis. Thus, Neu5,9Ac2-GPs may be considered as the actual signal molecules. Even though the increase of O-acetylated sialoglycoproteins is an important determinant in lymphocytes and erythrocytes, the status and role of O-acetylated sialic acid on GSLs in the disease biology of ALL remain unexplored. Although GD3 is a minor ganglioside in most normal tissues and lymphocytes, it is highly expressed in a variety of tumors. An acetylated modification of GD3, i.e., 9-O-acetyl GD3 (9-O-AcGD3), is also expressed in basal cell carcinomas, melanomas, and leukemias. GD3 acts as an apoptosis inducer in many cells. Addition of GD3 or enforced expression of GD3 synthase was sufficient to trigger apoptosis. In contrast,
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9-O-AcGD3 protects cells from apoptosis. It did not induce apoptosis when added to intact cells and could rescue cells from GD3-induced apoptosis. The acetylation– deacetylation cycle is suggested as a subtle means of regulating the ratio of GD3 and 9-O-AcGD3, thereby regulating the apoptotic effect. Interestingly, GD3 itself can induce its 9-O-acetylation by initiating new transcription and protein synthesis. In this context, the elevated level of 9-O-AcGD3 on lymphoblasts of childhood ALL may have a significant contribution in survival strategies in cancer cells and hence emphasizes the potential of these molecules in therapy. Interestingly, the level of 9-O-AcGD3 is downregulated during normal erythropoiesis with maturation. Also, at presentation of ALL, the erythroid progenitors show a decrease in their 9-O-AcGD3 levels with maturation. Mature erythrocytes on stimulation with anti-9-O-AcGD3 mAb are driven towards a cell death program that shares its features with apoptosis. Therefore, 9-O-AcGD3 triggers death signals in erythroid cells contrary to their role in lymphoblasts, suggesting a cell-specific role for this molecule. Acknowledgments Ms. Kankana, Mr. Chandan Mandal, Ms. Suchandra Chowdhury, and Ms. Susmita Mondal are senior and junior research fellows of the Council of Scientific and Industrial Research (CSIR) and the University Grant Commission for the Government of India. This work received financial support from the Department of Science and Technology, Indian Council of Medical Research, and CSIR, New Delhi, Government of India. We are thankful to all previous coworkers for their contributions. Figures 15.3, 15.5–15.8) and Table 15.1 have been reprinted from Biochemical and Biophysical Research Communications 2007;362:651–657 with permission from Elsevier.
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Chapter 16
GM3 Upregulation of Matrix Metalloproteinase-9 Possibly Through PI3K, AKT, RICTOR, RHOGDI-2, and TNF-A Pathways in Mouse Melanoma B16 Cells Pu Wang, Xiaodong Wang, Peixing Wu, Jinghai Zhang, Toshinori Sato, Sadako Yamagata, and Tatsuya Yamagata Keywords GM3 • Matrix metalloproteinase-9 • Tumor necrosis factor-alpha • Cell motility • GM6001 The murine melanoma B16 cell line is characterized by its highly invasive and metastatic capacity. Growth factors, adhesion molecules, proteases, and other components are involved in the process of metastasis [8]. MMP family members have been clearly shown to play an important role in this process [7, 19]. Among the MMPs thus far studied, MMP-9 (gelatinase B) appears to have an important role in a wide array of physiological and pathophysiological processes, including placental development, wound healing, angiogenesis, inflammation, tumor invasion, and metastasis [20]. Thus, studies of the mechanism(s) regulating the expression of MMP-9 are also important to the understanding of mechanisms underlying tumor metastasis. MMP-9 secretion can be stimulated by interleukin-1b [12], TNF-a [13], hepatocyte growth factor [26], and epithelial growth factor [17]. MMP-9 is stimulated in several cell lines via the PI3K-AKT signaling pathway [18]. Hyperactivated PI3K results in the activation of several transcriptional factors, such as nuclear factor (NF)-kB and activator protein (AP)-1, further leading to promotion of MMP-9 gene expression [1]. Restoration of phosphatase and tensin homolog to hyperactivated PI3K cell lines reversibly suppresses MMP-9 expression. S6K located downstream of PI3K is involved in the regulation of MMP-9 expression following stimulation with hepatocyte growth factor [26]. These lines of evidence clearly show that the PI3K signaling pathway plays an important role in MMP-9 regulation.
T. Yamagata (*) Laboratory of Tumor Biology and Glycobiology, Shenyang Pharmaceutical University, Shenyang, 110016, People’s Republic of China e-mail:
[email protected] A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_16, © Springer Science+Business Media, LLC 2011
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Reports from several laboratories have concluded that MMP-9 expression is modulated not only by cytokines, but also by gangliosides [9, 16, 25]. GM1, present in the glycolipid-enriched microdomain, is one of the crucial factors regulating cancer metastatic potential via the modulation of MMP-9 localization and secretion, as well as suppression of tumor invasion potential [25]. Overexpression of the GD3 synthase gene suppresses MMP-9 expression by inhibiting the combination between the MMP-9 promoter and transcription factors (NF-kB and AP-1) in vascular smooth muscle cells [16]. In murine FBJ cells, GD1a is found to suppress MMP-9 expression at the transcriptional level [9]. On the other hand, overexpression of plasma membrane-expressed sialidase Neu3 inhibits MMP-9 expression in vascular smooth muscle cells implying gangliosides promote MMP-9 [15]. Thus, there is no definite concept as to whether gangliosides positively or negatively regulate MMP-9 expression. Among tumor-associated glycolipids, ganglioside GM3 is the simplest ganglioside in structure that resides in the membrane of murine melanoma B16 cells [10]. GM3 has been shown to regulate TNF-a both at the transcriptional and translational levels in murine melanoma B16 cells [22, 23]. TNF-a expression was increased by the addition of GM3 to the B16 transfectants and decreased after the treatment with D-PDMP, an inhibitor of glucosylceramide synthesis. These results clearly indicate that GM3 positively regulates TNF-a expression in B16 cells. Phosphoinositide 3-kinase inhibitors, wortmannin and LY294002, suppressed the TNF-a expression that is stimulated by GM3 in B16 cells, suggesting that the GM3 signal is located upstream of the PI3K-AKT pathway. GM3 was shown to increase phosphorylation of AKT. Treatment of B16 cells with small interfering RNA (siRNA) targeted to AKT1/2 resulted in TNF-a suppression, indicating that AKT plays an important role in regulation of TNF-a expression. Suppression of AKT1/2 rendered cells insensitive to GM3, suggesting that the GM3 signal may be transduced via AKT [22]. Rapamycin suppressed TNF-a expression, indicating mammalian target of rapamycin (mTOR) to be involved in the pathway. Either siRNA Raptor or siRNA rapamycin-insensitive companion of mTOR (RICTOR) suppressed TNF-a expression, but the latter suppressed the effects of GM3 on TNF-a expression and AKT phosphorylation at Ser473, indicating the GM3 signal to be transduced via mTOR-RICTOR and AKT (Ser473), leading to TNF-a stimulation. Finally, Rho-GDP dissociation inhibitor (RhoGDI)-2, the tumor suppressor gene, whose expression is associated with GM3, was shown to be upstream of TNF-a [23]. Thus, the GM3 signal is transduced in B16 cells through a PI3K, mTOR-RICTOR, AKT, RhoGDI-2 pathway, leading to stimulated expression of TNF-a. Since TNF-a is known to stimulate MMP-9 synthesis, which is highly involved in tumor cell metastasis, we investigated the possibility that MMP-9 is regulated by GM3. In the present study, MMP-9, but not MMP-2, messenger RNA (mRNA) expression was found to be consistent with GM3 levels in every B16-derived cell variant. GM3 has been suggested to stimulate the PI3K/AKT signaling pathway in previous investigations [3, 5]. GM3 signals are thus transduced via the PI3K/AKT pathway, leading to the regulation of MMP-9 expression.
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16.1 Materials and Methods 16.1.1 Cell Lines and Culture Murine melanoma B16 cells were kindly provided by Dr. Kiyoshi Furukawa of Nagaoka University of Technology, Japan. The cells were maintained in medium containing Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Invitrogen Corporation, NY, USA) supplemented with 10% fetal bovine serum (TBD; Tianjin Hao Yang Biological Company, Tianjin, China), 100 U/mL penicillin, and 100 mg/ mL streptomycin and incubated in a humidified (37°C, 5% CO2 and 95% air) incubator (Sanyo, Tokyo, Japan). The cells were usually grown in a 60-mm culture dish (BD Falcon, CA, USA) and passaged once they reached 75% confluence. To observe the effects of gangliosides on MMP-9 expression, the cells were incubated with 25 mM GM3 in the absence of serum for 24 h. In some experiments, the cells were starved for 6 h, followed by incubation with 10 mM GM3 under serum-free conditions for 10 min. To observe the effects of TNF-a on MMP-9 expression or B16 cell motility, cells were treated with TNF-a (10 ng/mL) for 24 h before analyzing MMP-9 expression by RT-PCR.
16.1.2 Chemicals and Antibodies Ganglioside GM3 from bovine brain was purchased from Sigma (USA). LY294002, LY303511, and recombinant TNF-a were purchased from Sigma, and D-PDMP was purchased from Matreya (USA). Rabbit anti-AKT, anti-phospho-AKT (Ser473), anti-phospho-AKT (Thr308), and horseradish peroxidase-linked anti-rabbit secondary antibody were from Cell Signaling (MA, USA). The RNeasy mini kit to extract total RNA was obtained from Qiagen (Hilden, Germany). The RT-PCR kit was from Takara Biotechnology Corporation (Dalian, China).
16.1.3 RNA Extraction and RT-PCR RNA extraction and analysis of amplified DNA were detailed in our previous work [21]. The primers used in this study were designed with Primer 3 software and synthesized by Shanghai Genebase Biotechnology Corporation (China). Primer sequences used for the PCR in this study were as follows: for Eukaryotic elongation factor (Eef1a1), Sense 5¢-CGCTGCTGGAAGCTTTGGAT-3¢ and Antisense 5¢-GGGGCCATC-TTCCAGCTTCT-3¢; for MMP-9, Sense 5¢-CTGACTACGATAAGGACGGCAA-3¢ and Antisense 5¢-ATACTGGATGCCGTCTATGTCG-3¢; for ST3GAL5, Sense5¢-GCTCAAGGACCTCCCTGCAA-3¢ and Antisense 5¢-CGGGCAGCATATCC-AAGAGG-3¢. The mRNA levels of the genes under
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consideration, using Eef1a1 mRNA as a control, were determined by RT-PCR semiquantitatively, as described previously [21]. TNF-a mRNA values are expressed as a ratio of TNF-a to Eef1a1 mRNA and are usually expressed as unity for control experiments.
16.1.4 Ganglioside Extraction and HPTLC B16 cells were grown to ~90% confluency in 10-cm dishes, harvested, and washed three times with phosphate-buffered saline (PBS)(-). GM3 was extracted once with 1 mL of chloroform/methanol (2:1, v/v) and twice with chloroform/isopropanol/ methanol (7:11:2, v/v/v) with sonication for 1 h. The supernatants were evaporated at 60°C, and lipid fractions were dissolved in chloroform/methanol (2:1, v/v), developed in chloroform/methanol/0.25% KCl (5:4:1, v/v/v), and stained with orcinol/ sulfuric acid reagent followed by incubation at 120°C for 5 min.
16.1.5 siRNA The target sequence of ST3GAL5 was selected using the Protein Lounge program. The sequences were made to constitute a retroviral vector with neomycin resistance at Takara Biotechnology Corporation. Plasmids were transfected into B16 cells in the presence of Fugene (Roche, USA) as specified by the manufacturer. Three days after transfection, RNA was extracted and assayed for expression of corresponding gene and MMP-9 expression. The most effective target sequences were ST3GAL5, 5¢-AGACGGCTATGGC-TCTGTTAT-3¢. The control scrambled siRNA contained the sequence 5¢-CGAAGTTCG-TTGCACTATGGT-3¢. Stable transfections were carried out with Fugene reagent, essentially following the instructions of the manufacturer. Briefly, cells were seeded at a density of 20% confluency in a 60-mm dish, transfected with siRNA plasmid for three days, and selected by G418 to obtain stable monoclonal transfectants. The expression of targeted mRNA was analyzed by RT-PCR.
16.1.6 Gelatin Zymography Gelatinase activity was determined according to the method previously described [24]. In brief, an 8% polyacrylamide gel of 1-mm thickness containing 0.3 mg/mL gelatin was used, and the proteins were separated by Laemmli’s buffer system. The cells were inoculated into a 100-mm culture dish with 4 mL of culture medium. After overnight culture, the medium was discarded, and the cells were washed with DMEM containing no serum and further incubated for the time indicated in the
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medium without serum. At the indicated time, conditioned medium was obtained to use for the measurement of gelatinase activity. An aliquot of conditioned medium was mixed with the equal amount of Laemmli’s sample buffer with no reducing reagent and without heating applied to the electrophoresis. The gel following electrophoresis was rinsed with 2.5% Triton X-100 for 1 h at room temperature followed by incubation in the reaction buffer (10 mM CaCl2, 50 mM Tris–HCl, pH 7.4, and 0.02% NaN3) for 24 h at 37°C, fixed with 50% methanol–10% acetic acid for 30 min, stained with 0.02% Coomassie Brilliant Blue in 50% methanol–10% acetic acid for 1 h, and destained with 20% methanol–10% acetic acid until clear white bands appeared on the blue background. Gelatin zymography depicts MMPs as negatively stained bands that were scanned, and the optical density was determined using the Bio-profile Bio, ID image analyzer.
16.1.7 Transwell Experiments Motility was assayed using 24-well chambers with 8-mm pores, which were obtained from Costar (USA). For this, 0.6 mL of 0.5% fetal bovine serum medium was placed in the lower wells of the chamber. The upper chamber, which contains an 8-mm porous membrane, was placed above the lower wells. Cells were quickly trypsinized and washed twice with PBS, after which cells were resuspended in serum-free medium, and 1 × 105 cells in 0.1 mL of serum-free medium were placed in each of the upper wells of the chamber above the membrane. The cells were allowed to migrate for 24 h at 37°C, and the numbers of cells that moved to the lower chamber were counted under the microscope or with a WST kit (DOJIN Co., Japan).
16.2 Results 16.2.1 GM3 Is Associated with Cell Motility Cell migration is of fundamental importance in tumor metastasis [2, 11]. We determined cell migration by Transwell experiments with the following: B4GALT6 sense cDNA transfectant, CSSH-1; its mock transfectant cells, SM-1; B4GALT6 antisense cDNA transfectant, CAH-3; and its mock transfectant cells, CM-1. CSSH-1 cells expressed GM3 twice as much as SM-1, and GM3 expression was suppressed in CAH-3 cells to half the level of that in CM-1 (control). As shown in Fig. 16.1a, the number of GM3-enriched CSSH-1 cells migrating into the lower chamber in the Transwell experiment was five times as many as the number of control SM-1 cells. On the other hand, the number of GM3-deficient CAH-3 cells translocating to the lower chamber was one fourth compared to the control CM-1 cells. These results suggest that GM3 positively regulates cell motility. In an effort to confirm the above findings, B16 cells were transfected with an siRNA targeting the ST3GAL5 gene
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(GM3 synthase) or with a scrambled sequence siRNA as a control. Stable clones were selected by adding G418 to the culture medium at a concentration of 1.5 mg/mL. Expression of ST3GAL5 mRNA by the transfectants thus obtained was suppressed roughly 0.6-fold to that of the scrambled control (Fig. 16.1b), and the GM3 levels of the transfectants were accordingly less than those of the control (Fig. 16.1c). The motility of one of the transfectants, B11, was suppressed to one tenth of the scrambled control (Fig. 16.1a). These results further support the possibility that GM3 is responsible for cell motility.
Fig. 16.1 Cell migration capability is positively related to the amount of GM3 of the cell. In (a), cell migration was determined using a Transwell plate. As such, 24 h later, the number of SM-1 (a mock transfectant) cells that moved into the lower chamber was taken as unity to that of CSSH-1, and that of CM-1 was taken as unity to that of CAH-3. Likewise, cell motility of B11 cells is expressed as a ratio to the control B16 scrambled. In (b), St3gal5 expression in A7 and B11 cells was determined by RT-PCR and compared with a scrambled siRNA sequence (A7 and B11 cell lines are the stable monoclonal transfectants obtained by St3gal5 siRNA). In (c), GM3 contents were analyzed by high-performance thin-layer chromatography (HPTLC). Independent experiments were performed twice, and mean values are given. Typical results are shown in the lefthand panels
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16.2.2 Endogenous GM3 Positively Regulates MMP-9 Synthesis Since MMPs are molecules associated with tumor cell motility and invasion, expression of MMP-9 in these cells was determined by RT-PCR. As shown in Fig. 16.2a, MMP-9 expression in GM3-enriched CSSH-1 was twice as much as that in the control SM-1, whereas GM3-deficient CAH-3 cells decreased MMP-9 expression by half. MMP-9 mRNA of B11 cells, whose expression of ST3GAL5 and GM3 was suppressed as mentioned above, was 70% of that of the scrambled cells (Fig. 16.2b). Gelatin zymography revealed that MMP-9 of B11 cells was onefifth-fold of the scrambled control while MMP-2 was not affected (Fig. 16.2c).
Fig. 16.2 MMP-9 expression is positively associated with GM3 levels in the cell. MMP-9 expression in SM-1, CSSH-1, CM-1, and CAH-3 cells was determined by RT-PCR along with Eef as the control and shown in (a) and that of B11 cells is shown in (b). The activity of MMP-9 and MMP2 in B11 cells compared with that of the scrambled sequence transfected cells was determined by gelatin zymography in (c). Independent experiments were performed twice, and mean values are given. Typical results are shown in the lefthand panels
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These results clearly suggest that GM3 positively regulates expression of MMP-9, but not MMP-2, in B16 cells.
16.2.3 Exogenous GM3 Addition or GM3 Depletion Modulates MMP-9 Expression To further confirm that GM3 positively regulates MMP-9 expression and secretion, B16 and B11 cells were incubated with 25 mM GM3 for 24 h in serum-free medium, for which GM3 induced MMP-9 mRNA expression in B16 and B11 cells (Fig. 16.3a). Treatment of cells with D-PDMP, an inhibitor of glycosphingolipid synthesis, for three or six days suppressed GM3 levels in B16 cells (Fig. 16.3b, left panel), leading to the suppression of MMP-9 synthesis (Fig. 16.3b, right panel). These data in combination with the above-mentioned results clearly suggest that GM3 positively regulates MMP-9 expression in murine melanoma B16 cells at the transcriptional level.
Fig. 16.3 MMP-9 expression is regulated by GM3. In (a), B16 or B11 cells were incubated with 25 mM GM3 in serum-free medium for 24 h, and MMP-9 expression was determined by RT-PCR, where Eef served as an internal control. In (b), B16 cells were incubated with 12.5 mM D-PDMP for three or six days, and MMP-9 was determined by RT-PCR. Independent experiments were performed twice, and mean values are given. Typical results are shown in the lefthand panels. In both experiments, GM3 contents as revealed by HPTLC are shown at the lower panels
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16.2.4 The PI3K/AKT Pathway Is Involved in MMP-9 Regulation During the previous investigation of the GM3 signaling pathway, GM3 was found to stimulate the PI3K/AKT pathway [23]. We thus examined whether the PI3K/ AKT signal pathway was involved in GM3 regulation of MMP-9 expression. For this, 25 mM LY294002, an inhibitor of PI3K, was found to inhibit MMP-9 expression in B16 and CSSH-1 cells (Fig. 16.4a). Another inhibitor of PI3K, LY303511, also inhibited MMP-9 expression at a concentration of 100 mM (Fig. 16.4b). These results imply that the PI3K/AKT pathway is involved in MMP-9 regulation in B16 cells. Our previous study has shown that GM3 is capable of activating the PI3K/AKT pathway [22]. The GM3-induced enhancement of MMP-9 expression was completely blocked by the presence of the PI3K inhibitor LY294002 or LY303511 (Fig. 16.5a). In addition, GM3-induced AKT phosphorylation [23] was also inhibited by 25 mM LY294002 or 100 mM LY303511 (Fig. 16.5b). These results indicate that GM3 stimulates MMP-9 expression through a PI3Kdependent pathway.
Fig. 16.4 The effects of PI3K inhibitors on MMP-9 expression. B16 and CSSH-1 cells were incubated with LY294002 (25 mM) for 24 h, and MMP-9 expression was determined by RT-PCR (a). B16 cells were incubated with LY303511 (25 mM) for 24 h, and MMP-9 expression was determined by RT-PCR (b). Independent experiments were performed twice, and mean values are given. Typical results are given in the upper panels
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Fig. 16.5 GM3 is located upstream of the PI3K/AKT pathway through which MMP-9 expression is regulated. B16 cells were incubated with 25 mM GM3 and/or 25 mM PI3K inhibitors in serumfree medium for 24 h, and MMP-9 expression was determined by RT-PCR in (a). Cell lysates were prepared from the above-mentioned cells, and AKT phosphorylation was determined by Western blots (b). Similar results were obtained in two independent experiments
Fig. 16.6 The motility of B16 cells was consistent with MMP-9 expression. (a) B16 cells were incubated with 10 ng/mL TNF-a or 25 mM GM6001 for 24 h, and MMP-9 expression was determined by RT-PCR. (b) The secretion of MMP-9 in B16 cells incubated with 10 ng/mL TNF-a in the presence or absence of 25 mM GM6001 for 24 h was analyzed by gelatin zymography. (c) The motility of B16 cells treated with 10 ng/mL TNF-a or 25 mM GM6001 for 24 h was determined by a Transwell plate, and the number of cells invading the lower chamber was compared to that of the control migration. Similar results were obtained in two independent experiments, and the typical results are shown on the lefthand side
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16.2.5 MMP-9 Plays an Important Role in B16 Cell Motility In order to determine the role of MMP-9 in cell motility, we examined the effects of an MMP-9 agonist and antagonist on cell motility, as stimulation of MMP-9 expression by TNF-a has been reported in several studies [4, 6, 14]. We therefore used TNF-a as a positive control against the MMP-9 inhibitor GM6001. MMP-9 expression as assessed by RT-PCR showed that TNF-a was increased to 3.5 times the levels in controls while GM6001 reversibly suppressed MMP-9 expression (Fig. 16.6a). Gelatin zymography revealed that TNF-a enhanced MMP-9 expression five times that of the levels in controls, but the presence of GM6001 lowered MMP-9 expression and activity (Fig. 16.6b). Cell migration tested by Transwell experiments showed that the numbers of cells migrating were consistent with MMP-9 expression (Fig. 16.6c). These data strongly suggest that the capacity of cell migration in B16 cells is proportional to MMP-9 expression, which is under the positive control of GM3.
16.3 Discussion The development of metastasis is a major cause of death in many patients with cancer. Mechanisms for the acquisition of metastatic potential are, however, not well understood. In recent decades, a number of systems and molecules involved in metastasis have been identified. Among these, protease-mediated migration and invasion may play a major role in cell migration. Above all, MMP-2 and MMP-9, known as gelatinases, have been considered to be major proteolytic enzymes in the degradation of the extracellular matrix during cancer cell progression and invasion. Recently, a number of gangliosides have also been identified as upstream molecules affecting MMP production. In this study, the B16 cell’s capability to migrate has been attributed to expression of ganglioside GM3 through MMP-9, but not MMP-2. MMP-9 was clearly induced by GM3, whose amount was increased either by B4GALT6 sense cDNA transfection or exogenous addition of GM3. Inversely, GM3 suppression by ST3GAL5 siRNA, B4GALT6 antisense cDNA transfection, or D-PDMP treatment suppressed MMP-9 expression. All of these results support the notion that MMP-9 is positively regulated by GM3 in B16 cells. However, in the case of mouse osteosarcoma FBJ cells, we have found that ganglioside GD1a is shown to negatively regulate MMP-9 [9]. Exogenous addition of GD1a leads to increased MMP-9 expression at the transcriptional level in mouse B16 cells (X. Wang, unpublished data). Thus, the effects of gangliosides are different among ganglioside species and cell lines. In support of this, experiments are being performed to further explain the different effects of gangliosides. A PI3K inhibitor, LY294002, suppressed MMP-9 expression, suggesting that MMP-9 expression is controlled via the PI3K/AKT pathway. It was previously
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observed that GM3 activates AKT phosphorylation via the PI3K pathway [22]. This implies that GM3 is located upstream of the PI3K/AKT pathway. To further investigate this, we treated B16 cells with the PI3K inhibitor in the presence of GM3. The results showed that GM3 failed to upregulate MMP-9 expression when the PI3K inhibitor was present in the medium. This result strongly suggests that GM3 regulates MMP-9 expression through the PI3K/AKT pathway. However, it has not been elucidated as to how GM3 signals are transduced into the cell, leading to enhanced expression of MMP-9 via PI3K. The investigation regarding the GM3 signal is ongoing in our laboratory. As one of the MMP inhibitors, GM6001, inhibited cell motility as well as MMP9 expression, it is thus clear from this study that GM3 of B16 cells activates cell motility via increased MMP-9 expression. Yet, MMP-9 expression in murine B16 cells has never been reported except for in this investigation, possibly because of a scant amount of MMP-9 in B16 cells, which, even when MMP-9 production is upregulated by GM3 addition or TNF-a administration, cannot migrate in the Matrigel-coated Transwell. Thus, the function of MMP-9 on B16 cell metastasis remains unclear. We have reported elsewhere that GM3 regulates RhoGDI-2, which suppresses anchorage-independent cell growth of B16 cells (P. Wang, in preparation). Anchorage-independent cell growth is a tumor hallmark that is suppressed by the presence of GM3. Therefore, GM3 has an ambivalent effect on B16 cells. We are puzzled by the ambivalence of GM3 in that it suppresses anchorage-independent cell growth, a distinct tumor feature, through RhoGDI-2, while GM3 helps B16 cells to produce MMP-9, as shown here, as well as TNF-a [22, 23], which are believed to be associated with tumor malignancy. It is noteworthy that both murine B16 and GM3-enriched CSSH-1 cells were equally highly malignant while the latter was less malignant compared with the former in terms of number of nodules in the lung (P. Wang, unpublished data). GM3 was found to regulate TNF-a in our previous work, and TNF-a was shown to promote MMP-9 in this study. It is therefore highly possible that MMP-9 of B16 cells is upregulated by GM3 via TNF-a upregulation. Acknowledgments This work was supported in part by funding from The Mizutani Glycoscience Foundation given to Tatsuya Yamagata.
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Chapter 17
Pathological Roles of Ganglioside Mimicry in Guillain–Barré Syndrome and Related Neuropathies Robert K. Yu, Toshio Ariga, Seigo Usuki, and Ken-ichi Kaida
Keywords Antiganglioside antibodies • Autoantibodies against ganglioside c omplex • Campylobacter jejuni • Guillain–Barré syndrome • Lipopolysaccharides • Molecular mimicry
17.1 Introduction Gangliosides, sialic acid-containing glycosphingolipids (GSLs), are a family of diverse, highly complex molecules localized primarily on the plasma membrane and particularly abundant in the nervous tissues of vertebrates. Research interest in gangliosides is not limited to their normal biological functions, such as neurotrophicity, cell–cell recognition and adhesion, cellular differentiation and growth, intercellular signaling, and trafficking and/or sorting [17, 19, 61, 62], or on the important constituents of cell surface microdomains or lipid rafts [18, 25, 44]. Research is also focused on the role of gangliosides in the pathogenic mechanisms of many immune-mediated neurological disorders, such as Guillain–Barré syndrome (GBS) [5, 57, 63]. For the putative pathogenic roles of gangliosides, accumulating evidence indicates that (a) gangliosides are localized in peripheral nerve system (PNS) myelin and axolemma, and degeneration of myelin and axons accounts for the loss of sensory and motor functions; (b) animal models of peripheral neuropathies can be established using certain pure gangliosides as the immunogens; and (c) the pathophysiological effects of the antibodies could be due to one or more of the following mechanisms: an antibody-mediated, complementdependent process; a cell-mediated degenerating process; and a conduction block at the node of Ranvier.
R.K. Yu (*) Institute of Molecular Medicine and Genetics and Institute of Neuroscience, Medical College of Georgia, Augusta, GA 30912, USA e-mail:
[email protected] A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_17, © Springer Science+Business Media, LLC 2011
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17.2 GBS and Related Neuropathies GBS is the most frequent cause of acute flaccid paralysis in humans, occurring with an annual incidence of one to two cases per 100,000 people. GBS is slightly more common in men than in women, occurring at a ratio of 1.3:1. GBS is primarily an immune-mediated disorder of the PNS. The immune system attacks spinal nerve roots, peripheral nerves, and cranial nerves, resulting in focal inflammation with variable damage to myelin sheaths and axon fibers. In recent years, studies have shed new light on a number of disease aspects that have enhanced the understanding of the pathogenic mechanisms of GBS. As an acute inflammatory polyradiculoneuropathy, GBS frequently develops following a gastrointestinal infection. Clinical symptoms often occur 1–3 weeks after a bacterial or viral infection. The most commonly identified triggering agents are Campylobacter jejuni (in 13–39% of cases), followed by cytomegalovirus (5–22%), Epstein–Barr virus (1–13%), and Mycoplasma pneumoniae (5%) [14, 42]. All of these pathogens have carbohydrate sequences (antigens) in common with glycoconjugates of peripheral nerve tissue. GBS is recognized as several disorders characterized by an immune-mediated attack on the peripheral nerves, particularly in the myelin sheath or Schwann cells of sensory and motor nerves. Several subtypes of GBS have been characterized based on their clinical manifestations. The most common form is a multifocal demyelinating disorder caused by damage to the myelin sheath of the peripheral nerves and is called acute inflammatory demyelinating polyneuropathy (AIDP). Clinically, AIDP is characterized by progressive areflexic weakness and mild sensory changes. Sensory symptoms often precede motor weakness. About 20% of patients with AIDP eventually have respiratory failure. The chronic variant is called chronic inflammatory demyelinating polyneuropathy (CIDP). It is characterized by progressive weakness and impaired sensory function in the legs and arms. Several variants of CIDP are known, including one form that has no sensory involvement, i.e., no numbness or tingling in the hands or feet. This pure motor chronic acquired demyelinating neuropathy variant is also called multifocal motor neuropathy (MMN). Patients with MMN frequently demonstrate signs of conduction block in the peripheral nerves. On the other hand, sensory nerve conduction evaluations are normal in patients with MMN. Other cases of GBS are associated primarily with axonal processes with axonal degeneration and sparing of the myelin; these cases are called acute motor axonal neuropathy (AMAN). Yet another form of GBS appears to involve both sensory and motor axons and is termed acute motor and sensory axonal neuropathy (AMSAN) [23]. More than 90% of patients with GBS in Europe and North America are classified as having AIDP. The most distinct immunological marker that distinguishes MMN from CIDP is the presence in the serum of anti-GM1 ganglioside antibodies, which occur in approximately 50% of patients with MMN. Some motor neuropathies have been classified as amyotrophic lateral sclerosis (ALS) variants, with predominantly LMN signs and axonal changes based on electrodiagnostic studies. AMAN occurs in less than 10% of persons with GBS in the Western hemisphere but in more than 40% of those affected in China
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and Japan. The incidence of AMSAN is very low, less than 10% of that of AMAN. Miller–Fisher syndrome (MFS) is another GBS variant that occurs in about 5% of people affected by GBS. MFS is characterized by ophthalmoplegia, areflexia, ataxia, and, in some cases, facial and bulbar palsy. The incidence of the pharyngeal–cervical–brachial variant, which is characterized by proximal descending weakness, is very low. When immunoglobulin M (IgM) proteins react with myelinassociated glycoprotein or sulfated glucuronyl glycolipids, there is a correlation with specific syndromes called IgM paraproteinemia neuropathy [10].
17.3 Anti-GSL Antibodies in GBS and Related Neuropathies Much of the research into GBS over the past decade has focused on the forms that are mediated by anti-GSL antibodies [4, 54]. It has been found that the sera of 60% of patients with GBS contain one or more anti-GSL antibodies. Measurements of these antibody titers are therefore very important for diagnosing GBS and evaluating the effectiveness of treatment strategies in clinical trials [5, 57, 63, 64]. Early studies performed on the basis of elevated anti-GSL antibody titers had identified patients with chronic demyelinating polyneuropathies, ALS, and MMNs [39]. More than 200 papers have reported antibodies to a wide range of GSLs, including GM1, GM1(NeuGc), GM1b, GalNAc-GM1b, GD1a, GalNAc-GD1a, GD1b, 9-O-acetyl GD1b, GD3, GT1a, GT1b, GQ1b, GQ1b, LM1, galactocerebroside, and SGPG [57]. For example, up to 75% of patients with AMAN have positive C. jejuni serology and at the same time have anti-GM1, anti-GD1a [22, 59], anti-GM1b, and anti-GalNAc-GD1a antibodies [65]; and 10–15% of patients with CIDP and AIDP develop anti-GM1 [34, 49]. In certain cases, anti-GD3 and anti-GT3 antibodies may participate in the pathogenesis of CIDP and AIDP [51]; 96% of patients with MFS present elevated anti-GQ1b antibody titers [7]; and patients with the pharyngeal–cervical–brachial variant often develop associated immunoglobulin G (IgG) anti-GT1a antibodies [32] (Table 17.1; [5]). The target antigens for antibodies in GBS are frequently identified in clinical analysis by a solid-phase immunoassay, such as thin-layer chromatography (TLC) immunostaining and/or enzyme-linked immunosorbent assay (ELISA), employing purified gangliosides as the test antigens. Interestingly, Kaida et al. [29] have reported that 8 of 100 patients with GBS who tested as having little or no antibody tested positive by the TLC immuno-overlay technique employing a crude mixture of whole-brain gangliosides. They identified a strong immunoreactive band migrating between GD1a and GD1b in the “antibody negative” sera, suggesting that the sera contained an antibody species that reacted with GD1a and GD1b in a complex form, but not with either purified ganglioside alone (see Fig. 17.1). This result indicates that “antibody-negative” GBS sera may also react with gangliosides that are present in the form of a GD1a/GD1b ganglioside complex. These authors observed similar results for GD1a/GM1, GM1/GT1b, and GD1b/ GT1b. Kaida et al. [30] recently examined sera from 234 patients with GBS.
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Table 17.1 Relationship between anti-GSL antibodies, putative infectious pathogens, and clinical symptoms Anti-glycosphingolipid antibodies Infectious pathogens Clinical symptoms AMAN, injury to motor axon’s axonal GM1 Campylobacter degeneration, occasional persistent jejuni (antecedent motor weakness, distal-dominant gastrointestinal weakness of the extremities, rare infection) cranial nerve palsy, ALS, MN (cross-reactivity with GA1), AIDP/ CIDP GM1 and GD1a C. jejuni AMAN, injury to motor axons GA1 Mycoplasma pneumonia MND, MN, ALS (cross-reactivity with GM1), motor variant of CIDP C. jejuni MN, ALS (cross-reactivities with GM1 GD1b and other and GA1), chronic sensory ataxia, b-series motor and sensory disturbance gangliosides IgM paraproteinemia with chronic polyneuropathy AMAN, injury to motor axons distalGD1a-GalNAc C. jejuni (antecedent dominant, weakness, no sensory signs, gastrointestinal axonal dysfunction. Pure motor variant infection) of GBS, cranial nerve involvement (facial palsy) IgG, axonal degeneration of the motor GM1b-GalNAc C. jejuni (antecedent nerves (cross-reactivity with GD1agastrointestinal GalNAc) infection) GM2 CMV (antecedent Sensory dysfunction, frequent facial palsy respiratory infection) (cross-reactivity with GD1a-GalNAc or GM1b-GalNAc) GBS with oropharyngeal involvement GT1a and GM1b Dysphasia (aspiration pharyngeal–cervical–brachial pneumonia, etc.), weakness, bulbar palsy, tendon botulinum toxin reflexes Miller–Fisher syndrome. Bickerstaff’s GQ1b C. jejuni (antecedent brain stem encephalitis, muscle respiratory tract paralysis, ataxia, hyporeflexia, infection) ophthalmoplegia GM1b, GM1a C. jejuni (gastrointestinal Acute motor neuropathy, AMAN limb weakness, flaccid quadriparesis infection) M. pneumonia GQ1ba C. jejuni GBS with sensory ataxia, IgM GD3, GT3, C. jejuni Miller–Fisher syndrome, cranial nerve and OAc-GT3 oculomotor nerve involvement GM3, GD3, GT3 C. jejuni AIDP, CIDP LM1 C. jejuni IgG, AMSAN/AMAN, motor and sensory fiber involvement (cross-reactivities with GM1, GM1b, GD1a, GD1aGalNAc, GD1b, and GQ1b) (continued)
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Clinical symptoms
F-GM1, F-GD1b, B-GD1b
ALS, cerebral infarction (cross-reactivity with GM1) GBS with sensory ataxic neuropathy GalCer M. pneumonia (antecedent Broncho pneumonia, muscle pain, febrile seizures, GBS with encephalitis and sore throat pulmonary meningitis infection) IgM paraproteinemia demyelinating SGPG CMV neuropathies, CIDP Antibodies against ganglioside complexes in GBS and its variants
Fig. 17.1 TLC immunostaining of serum of a patient with GBS. (a) TLC results made visible by orcinol–sulfuric acid reagent. (b) TLC-immunostaining using serum from a patient with GBS shows that the serum is anti-GD1a/GD1b-positive. Bovine brain gangliosides obtained by extraction with 0.1 M ammonium acetate were applied to lane 1, GD1a (3 mg); lane 2, GD1b (3 mg); lane 3, GD1a; and lane 4, GD1a and GD1b (3 mg each). The arrowhead indicates the immunostaining on the overlapping portion of GD1a and GD1b. The developing solvent consisted of chloroform:methanol:0.2% aqueous CaCl2⋅2H2O (50:45:10, v/v)
ELISA showed that 39 (17%) of the patients had IgG antibodies against at least one ganglioside complex, such as GD1a/GD1b, GM1/GD1a, GD1b/GT1b, GM1/GT1b, or GM1/GD1b. Among the 39 patients who were antiganglioside complex-positive, all had IgG anti-GM1/GD1a antibodies, and 27 had anti-GM1/GT1b antibodies. Sixteen patients had anti-GD1a/GD1b antibodies, 13 had anti-GD1b/GT1b antibodies, and 6 had anti-GM1/GD1b antibodies. In addition, ten patients had both anti-GD1a/GD1b and anti-GD1b/GT1b antibody activities. The distribution of antiganglioside complex-positive groups of patients was described in a Venn diagram (Fig. 17.2). Antibodies against ganglioside complexes can be used as markers of severe GBS and may provide a clue to elucidate its pathogenetic mechanisms. Because MFS is
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considered a variant of GBS, the presence of antiganglioside complex antibodies in MFS was therefore investigated using seven ganglioside antigens: GM1, GM2, GD1a, GD1b, GT1a, GT1b, and GQ1b. ELISA results showed that 58% of the patients had serum antibodies to ganglioside complexes such as GQ1b/GM1 and GQ1b/GD1a. Sensory signs were infrequent in MFS patients with antibodies to GQ1b/GM1 [27, 31]. Following these observations, there is also an intriguing possibility that certain bacterial lipooligosaccharides (LOSs) may present an epitope that mimics a mixed GM1 and GD1a antigenic determinant, resulting in the generation of an autoantibody that recognizes the new epitope rather than the individual ganglioside alone [64]. Thus, in sera that are antibody negative, it may be necessary to examine the antibody activity by using appropriate ganglioside complexes and suitable methods, such as liposome-incorporated GSLs [55]. Nonetheless, measurements of these GSL antibody titers remain the most effective and reliable way for diagnosing GBS and evaluating the effectiveness of treatments in clinical trials [5, 54, 63].
17.4 Ganglioside Molecular Mimicry With respect to the etiology of GBS, it has generally been accepted that it is an autoimmune disease in which the immune system mistakenly attacks myelin or axons, the nerve conduits for signals transmitting to and from the brain [16]. Emerging evidence suggests that infectious agents trigger the disease because clinical symptoms occur frequently after an antecedent infection. The most well-studied microbial agent is the gram-negative bacterium C. jejuni, which contains an LOS coat that also shares putative antigenic epitopes with those found in human nerve tissues. This resemblance has been termed “molecular mimicry,” which is defined as the dual recognition, by a single B- or T-cell receptor, of a microbe’s structure and an antigen of the host. This recognition is the mechanism by which infectious agents trigger cross-reactive antibodies or T cells that can lead to autoimmune diseases [1]. According to the molecular-mimicry hypothesis, a cross-reactive immune response is originally directed to bacterial lipopolysaccharides (LPSs), and autoantibodies and/or autoreactive T cells induced by infection are initially directed against the microbial antigens [1]. Subsequently, immune responses against nerve tissue gangliosides are expected as a result of molecular mimicry between gangliosides of the nerve axolemmal membrane and the LOSs of C. jejuni ([42, 57]; see Fig. 17.2). This concept has gained strong support from Moran and Prendergast [36] and Moran et al. [35], who reported that sera of rabbits immunized with ganglioside-mimicking C. jejuni LOSs revealed high titers of anti-LOS antibodies that were cross-reactive with a panel of gangliosides. In addition, sensitization of Lewis rats with a C. jejuni LOS bearing the GD3 epitope induced anti-GD3 antibody [52]. These findings strongly support the idea that molecular mimicry between microbial antigens and host tissues represents an attractive mechanism for triggering autoimmune responses in hosts. Initially, Yuki et al. [66, 67] reported that the structural mimicry of gangliosides by core oligosaccharides in LOSs from C. jejuni had been implicated
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Fig. 17.2 The Venn diagram shows distribution of antiganglioside complex-positive (+) groups of patients. All the antiganglioside complex-positive (+) groups of patients had IgG anti-GM1/ GD1a antibodies. Anti-GD1a/GD1b-positive patients also had IgG anti-GM1/GD1a and GM1/ GT1b antibodies. Ten antiganglioside complex-positive (+) patients had IgG anti-GD1a/GD1b and anti-GD1b/GT1b antibodies
in inducing cross-reactive antiganglioside antibodies. Detailed chemical studies of LOSs from GBS-associated C. jejuni isolates, HS:41, using GLC-MS and NMR, identified a tetrasaccharide structure consistent with GM1 mimicry (Fig. 17.3). Several serotypes of C. jejuni LOSs and ganglioside mimicry have also been reported, including GM1 and GD2 (HS:1, HS:2, HS:4, HS:10, HS:19, HS:23, HS:36, and HS:41); asialo-GM1 (HS:41); GD1a (HS:1, HS:2, HS:4, HS:10, HS:19, HS:23, HS:36, HS41, and HS:19); GD2 (HS:1, HS:2, HS:4, HS:10, HS:23, HS:36, and HS:41); GD3 (HS:1, HS:2, HS:4, HS:10, HS:19, HS:19, HS:23, HS:36, and HS:41); GM3 and GD1b (HS:23, HS:36); and GQ1b and GT1a (HS:2, HS:19, and HS:23) [63]. Ganglioside-like LOS synthesis is catalyzed by sialyltransferase Cst-I, a2, 3-sialyltransferase-I; cgtA, b1, 4-N-acetylgalacto-saminyltransferase Cst-II; N-acetylgalactosaminyltransferase CgtA; and galactosyl-transferase CgtB, all of which have been characterized in C. jejuni. In fact, the cst-II, cgtA, and cgtB genes that encode these enzymes have also been cloned [11], facilitating further studies of the generation of various ganglioside-like epitopes.
17.5 Ganglioside Molecular Mimicry at the Neuromuscular Junction and Node of Ranvier The presynaptic neuromuscular junction (NMJ) is considered to be a potential target for autoimmune attack in GBS and peripheral neuropathies for the following reasons [38, 57]: (1) NMJ is rich in gangliosides, including GQ1b, GM1, and
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Fig. 17.3 Molecular mimicry of gangliosides and Campylobacter jejuni LOSs. Gangliosides are highly expressed in nerve cell membranes and consist of a ceramide portion and a polar head group that contains glucose (Glc), galactose (Gal), N-acetylgalactosamine (GalNAc), and N-acetyl-neuraminic acid (NeuAc). LOS-containing ganglioside mimics are located in the outer part of the cell wall of C. jejuni. The specific structure of gangliosides is crucial in understanding the pathogenesis of GBS
GD1a; (2) NMJ lacks the blood–nerve barrier, readily allowing access to circulating autoantibodies; and (3) the NMJ is a vulnerable site for antibody-mediated paralytic diseases, including myasthenia gravis and Lambert–Eaton myasthenic syndrome. Antiganglioside antibodies may facilitate studies on the functional roles of gangliosides by inducing specific structural and functional changes in the NMJ. For example, Santafe et al. [40] reported a monoclonal IgM antibody from a patient with a pure motor chronic demyelinating polyneuropathy that binds specifically to the gangliosides GM2, GalNAc-GD1a, and GalNAc-GM1b, all of which have a common epitope of [GalNAcb1-4Gal(3-2aNeuAc)b1]. Thus, it is possible to (1) localize these gangliosides in specific cellular components of the NMJ; (2) describe the antiganglioside antibody-induced structural and functional changes in NMJs to gain insights into the role of gangliosides in the synaptic function; and (3) elucidate how these gangliosides are involved in Schwann cell–nerve terminal interactions, including structural stability and neurotransmission. With respect to the neuronal components of the nodes of Ranvier, GM1 is present on the cytoplasmic surface of motor neurons and has been specifically identified on paranodal and internodal axolemma, as well as on distal motor nerve terminals [8, 9, 41, 48]. Similarly, disialogangliosides have been shown to be expressed on internodal axolemma and/or on adaxonal Schwann cell cytoplasm [56]. Antibodies to GD1a
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and GalNAc-GD1a associated with pure motor axonal neuropathy preferentially immunostain ventral root axons (VRs) rather than dorsal root axons (DRs) [12, 60]. Interestingly, these disialogangliosides are located only in the presynaptic component of the motor end plates. Taguchi et al. [45] also reported that the epitopes recognized by anti-GalNAc-GD1a antibodies were observed in the soma of large neurons in the anterior horn of the adult rat spinal cord and its motor axons, as well as in the VRs and NMJs. Kaida et al. [28] showed that anti-GalNAcGD1a antibody immunostained an inner surface of compact myelin and a periaxonal axolemma-related portion in the VR, small-diameter DR fibers, and intramuscular (IM) nerves. These studies suggest that anti-GalNAc-GD1a antibodies in patients’ sera may bind to those regions in the VR and IM nerves where GalNAc-GD1a is localized, and the antibodies may function in the pathogenesis of pure motor-type GBS. An immune attack directed at antigenic determinants located at the paranodal Schwann cell surface may lead to paranodal demyelination, whereas antigens targeted on the exposed axolemma may result in axonal degeneration, both of which would result in conduction failure. Ligand-binding studies have suggested that GM1, GD1b, and polysialosylated gangliosides are enriched in the paranodal myelin loops of the peripheral nerve [57]. In addition, GQ1b is particularly enriched at the nodes of Ranvier [7]. In MFS, the serum antibody specifically binds to the disialosyl epitope on gangliosides, such as GQ1b, GT1a, and GD3. Since these gangliosides are enriched in synaptic membranes, antiganglioside antibodies may target the NMJ, contributing to the disease symptoms [20]. Ex vivo studies of mouse NMJs revealed that anti-GQ1b-positive MFS serum and human anti-GQ1b monoclonal antibodies induced a dramatic increase in spontaneous quantal acetylcholine (ACh) release, measured as miniature endplate potential frequency, and subsequent blockade of neuromuscular synaptic transmission, due to a failure of ACh release upon nerve impulses [13, 26]. The binding of anti-GSL antibodies to nerve terminals also may result in concomitant immunohistological and ultrastructural damage of the terminals [38, 57]. Taguchi et al. [45] also reported that anti-GalNAc-GD1a antibodies blocked neuromuscular transmission in muscle–spinal cord cocultured cells. The ACh-induced potential was not reduced by the addition of antibodies, suggesting that the blockade is presynaptic, probably affecting the ion channels in presynaptic motor axons. As the anti-GA1 antibodies inhibited the voltage-gated Ca2+ channel (VGCC) [46], antiGalNAc-GD1a antibodies may block neurotransmission by suppressing VGCC on the axonal terminals of motor nerves. Although it is not clearly understood how anti-GM1 antibodies cause nerve dysfunction and injury, sodium and/or potassium ion channel dysfunction at the nodes of Ranvier has been implicated [43]. Voltage-gated sodium channels (VGSCs) exist in the nodes of Ranvier [21], in which GM1 is localized [2]. Several studies have described the relationship between sodium channel block and the development of GBS. Anti-GM1 antibodies induced reduction of the nerve impulse amplitude at common entrapment sites, resulting in frequent Wallerian degeneration or physiological conduction block at the nodes of Ranvier [33]. Autopsy studies of patients with AMAN have indicated that immunoglobulins and complement deposits are frequently located at the nodes of Ranvier, where sodium
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channels are clustered [15]. In GBS, blocking factors of sodium channels are p resent in the cerebrospinal fluid, impairing neuron impulse conduction, thereby causing muscle weakness and sensory disturbances in affected patients [6, 58]. Patch-clamp studies revealed direct inhibition of the ion-conducting pores of VGSCs by exposure to serum from patients with GBS [53], resulting in muscle weakness and sensory disturbances in patients with GBS. These studies suggest that inhibition of voltage-gated ion channels might be a mechanism by which nerve conduction is impaired in patients with GBS [37]. Illa et al. [24] reported that purified anti-GM1 antibodies from patients who exhibited AMAN after immunization with a ganglioside preparation recognized epitopes at the nodes of Ranvier and at the presynaptic nerve terminals of motor end plates from human nerve biopsies. Accumulation of these antibodies at the nodes of Ranvier can cause disruption of Na+ and K+ channels and, thus, interference of nerve conduction. Therefore, a causal link between C. jejuni infection, the presence of antiganglioside antibodies, and development of GBS is considered likely [54]. Anti-GM1 antibodies have been shown to mediate complement-dependent destruction of sodium channel clusters in peripheral motor nerves [47]. Thus, anti-GSL antibodies, such as anti-GM1, may cause nerve dysfunction and injury by interfering with ion-channel function at the nodes of Ranvier and may contribute to the pathogenic mechanisms of certain neuropathies [3, 47]. The possible pathogenesis of AMAN subsequent to C. jejuni enteritis can be envisaged as follows: infection by C. jejuni bearing GM1-like LOSs induces production of the anti-GM1 IgG antibody, the autoantibody binds to GM1 at the nodes of Ranvier in spinal anterior roots, and activated complements are recruited by the anti-GM1 antibodies, which then form a membrane-attack complex. Autoimmune attack disrupts sodium channels, producing muscle weakness in the early phase of illness, and, in severe cases, Wallerian-like degeneration ensues. Moran et al. [35] reported that rabbits immunized with ganglioside-mimicking C. jejuni LOSs presented high titers of anti-LOS antibodies in sera that were cross-reactive with a panel of gangliosides. However, nonganglioside-mimicking C. jejuni LOSs induced a strong anti-LOS response but no antiganglioside antibodies. This result suggests that immunization with ganglioside-mimicking C. jejuni LOSs triggers the production of cross-reactive antiganglioside antibodies that recognize epitopes at the nodes of Ranvier. Usuki et al. [51] reported two cases of AIDP or CIDP showing elevated titers of anti-GD3 antibodies, which occur rarely in GBS. The antibodies showed an inhibitory effect on the spontaneous muscle action potential of the NMJ. To examine the correlation between the anti-GD3 antibody titers and C. jejuni infection, Usuki et al. [52] sensitized female Lewis rats with LOSs from serotype HS:19 of C. jejuni and examined changes in nerve conduction velocity and nerve conduction block. After 16 weeks of sensitization, the animals revealed transient decreases in nerve conduction velocity and conduction blocks and high titers of anti-GD3 antibodies. These anti-GD3 antibodies also blocked the spontaneous muscle action potential of NMJs in spinal cord–muscle cell cocultures. To determine the target epitope for GD3 antibodies in causing nerve dysfunction, the LOS fraction containing the GD3 epitope was purified from the total LOSs using an anti-GD3 affinity column.
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Fig. 17.4 TLC immuno-overlay of LOS with chicken and human sera. Samples of 100 ng of GM1 and an LOS fraction (strain HS19 of Campylobacter jejuni) were developed on a highperformance TLC plate using n-propanol:H2O:25% NH3 (6:3:1, v/v/v) as the developing solvent, and the plate was examined by TLC immuno-overlay at 1:100 dilution of antisera in phosphatebuffered saline. (1) Chicken sera from the infection experiment with C. jejuni (A1.1, B1.1, or C1.1). Parentheses indicate strain HS6, HS21, or VLA2/18 for the inoculation. (2) Human sera from a laboratory worker with campylobacteriosis (H1.1 day 1, day 1 postisolation of the strain VLA2/18; H1.1 day 22, day 22 postisolation of the strain VLA2/18) (data from [50])
Subsequently, chemical analysis of the oligosaccharide portion was performed and confirmed the presence of a GD3-like epitope with the following tetrasaccharide structure: NeuAc2-8NeuAc2-3Galß1-3Hep. The data thus support the possibility of GD3 mimicry as a potential pathogenic mechanism in peripheral nerve dysfunction. These in vitro electrophysiological studies provide strong evidence that ganglioside molecular mimicry is likely responsible for muscle weaknesses, possibly via their action on the NMJ. The data also support the possibility of GD3 mimicry contributing to a potential pathogenic mechanism of peripheral nerve dysfunction. In addition, campylobacteriosis is often associated with GBS. Poultry are frequently highly colonized with C. jejuni as a major foodborne vehicle for campylobacteriosis. Usuki et al. [50] demonstrated the high titer of anti-GM1 antibodies in the serum of a laboratory worker who developed campylobacteriosis. The microbiologically confirmed strain VLA2/18 (nonserotyped) was isolated from the worker and subsequently inoculated into chickens, resulting in high titers of serum antibodies to GM1. Interestingly, hightiter anti-GM1 antibodies in chicken and human sera strongly inhibited spontaneous muscle action potential in an in vitro system of spinal cord–muscle cell coculture. Further studies indicate that the chicken sera contained a high titer of anti-lipid A antibody together with antiganglioside antibodies for GM1 and GM3 (Fig. 17.4). Production of antiganglioside antibodies is considered to be due to molecular mimicry of the carbohydrate structure between the bacterial LOSs and human peripheral nervous gangliosides. Anti-lipid antibody is the result of an immune response to the hydrophobic antigen of the LOS molecule. We studied the effect of the chicken sera on voltage-gated Na currents by whole-cell patch-clamped NSC34 cells, a motor neuron-like cell line, in culture. A decrease of sodium current was observed, which was dependent on anti-lipid A antibody activity (Fig. 17.5).
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To better understand the mechanism of the inhibitory effect, we treated NSC-34 cells with tunicamycin, an inhibitor for N-glycosylation, and found that the decrease of sodium currents did not depend on the channel glycosylation. This finding suggests that anti-lipid A antibody could instead induce a functional inhibition of sodium channels at the protein portion. Next, we compared the inhibition of nine sodium channels (Nav1.1–1.9) in NSC-34 cells; only the Nav1.4 that was robust in this cell line was affected (Fig. 17.6). Since the LOSs of C. jejuni are thought to play an important role in the pathogenesis of GBS, our finding suggests that serum antibody against the lipid A portion of LPS was generated in the infected chicken and that the antibody depressed the function of the sodium channel to account for the neurophysiological changes in GBS.
17.6 Summary GBS and its variants are autoimmune neuropathies that frequently occur as a result of enteritis resulting from an infection with agents such as C. jejuni. These neuropathies are recognized as several disorders characterized by an immune-mediated attack on the peripheral nerve, particularly in the myelin sheath of sensory and motor nerves.
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Fig. 17.6 Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for Nav1.1–1.9. N18TG2, NSC-34, and NSC-19 cells were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum for 3 days. Total RNA was extracted, and the expression of messenger RNA a subunit of Nav1.1–1.9 was examined by RT-PCR, separated by 1.5% agarose gel electrophoresis, and visualized by ethidium bromide staining. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control for loading and efficiency of RT-PCR (lane 1 from left). The size markers of 100 bp to 1,000 kb were used as reference (lane 2 from left) (data from [50])
Increased antibody titers in GBS and variants are thought to be a result of the production of antibodies to bacterial carbohydrate-containing surface antigen(s) that cross-react with the GSLs, including gangliosides, of the myelin sheath and the axons of nerve cells. For this reason, the most common diagnostic test of GBS is detection of circulating anti-GSL antibodies in patients thought to have GBS. Recently, it has been shown that it is not sufficient to rely on pure GSLs as the test antigens because some GBS-associated autoantibodies failed to bind to pure gangliosides alone, but bound instead to ganglioside-associated neoepitopes consisting of ganglioside mixtures. This finding may represent a new strategy to address potential immune targets for underlying pathogenic autoantibodies in GBS. Pathogenesis is believed to involve molecular mimicry between epitopes on bacterial (e.g., C. jejuni) LPSs and neural gangliosides, resulting in demyelination and axonal degeneration. Antibody- and/or cell-mediated immune responses are believed to produce pathological lesions of the nerve [4] and interruption of neurotransmission, such as the sodium ion channel dysfunction at the nodes of Ranvier [45, 46, 50]. We have shown that anti-GD3 antibodies block the spontaneous muscle action potential of the NMJs in spinal cord–muscle cell cocultures and that serum antibodies against the lipid A portion of LPS depress the function of the sodium channel that may be involved in neurophysiological changes in GBS [50]. We compared the inhibition of nine sodium channels (Nav1.1–1.9) in NSC-34 cells; only Nav1.4 that was robust in
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this cell line was affected. Accumulating in vitro electrophysiological evidence suggests that ganglioside molecular mimicry may be responsible for muscle weakness, possibly via the action of antiganglioside antibodies on the NMJ. Acknowledgments This study was supported by National Institutes of Health grants to Robert K. Yu. We thank Ms. Diana Westbrook for her skillful editorial assistance.
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56. Willison HJ, O’Hanlon GM, Paterson G, Veitch J, Wilson G, Roberts M et al (1996) A somatically mutated human antiganglioside IgM antibody that induces experimental neuropathy in mice is encoded by the variable region heavy chain gene, V1-18. J Clin Invest 97:1155–1164 57. Willison HJ, Yuki N (2002) Peripheral neuropathies and anti-glycolipid antibodies. Brain 125:2591–2625 58. Wutrz A, Brinkmeier H, Wollinsky KH, Mehrkens HH, Kornhuber HH, Rudel R (1995) Cerebrospinal fluid and serum from patients with polyradiculoneuropathy have opposite effects on sodium channels. Muscle Nerve 18:772–781 59. Yoshino H, Harukawa H, Asano A (2000) IgG antiganglioside antibodies in Guillain-Barré syndrome with bulbar palsy. J Neuroimmunol 105:195–201 60. Yoshino H, Utsunomiya I, Taguchi K, Ariga T, Nagaoka T, Aoyagi H, Asano A, Yamada M, Miyatake T (2005) GalNAc-GD1a is localized specifically in ventral spinal roots, but not in dorsal roots. Brain Res 1057:177–180 61. Yu RK (1994) Developmental regulation of ganglioside metabolism. Prog Brain Res 101:31–44 62. Yu RK, Ariga T, Yanagisawa M, Zeng G (2008) Biosynthesis and degradation of gangliosides in the nervous system. In: Fraser-Reid B, Tatsuka K, Thiem J (eds) Glycoscience. Springer, Berlin, pp 1671–1695 63. Yu RK, Usuki S, Ariga T (2006) Ganglioside molecular mimicry and its pathological roles in Guillain-Barré syndrome and related diseases. Infect Immun 74:6517–6527 64. Yuki N (2007) Ganglioside mimicry and peripheral nerve disease. Muscle Nerve 35:691–711 65. Yuki N, Ho TW, Tagawa Y, Koga M, Li CY, Hirata K, Griffin JW (1999) Autoantibodies to GM1b and GalNAc-GD1a: relationship to Campylobacter jejuni infection and acute motor axonal neuropathy in China. J Neurol Sci 164:134–138 66. Yuki N, Taki T, Inagaki F, Kasama T, Takahashi M, Saito K, Tai T, Handa S, Miyatake T (1993) A bacterium lipopolysaccharide that elicits Guillain-Barré syndrome has a GM1 ganglioside-like structure. J Exp Med 178:1771–1775 67. Yuki N, Taki T, Takahashi M, Saito K, Tai T, Miyatake T, Handa S (1994) Penner’s serotype 4 of Campylobacter jejuni has a lipopolysaccharide that bears a GM1 ganglioside epitope as well as one that bears a GD1a epitope. Infect Immun 62:2101–2103
Chapter 18
Structure and Function of Glycolipids in Thermophilic Bacteria Feng-Ling Yang, Yu-Liang Yang, and Shih-Hsiung Wu
Keywords Thermal adaptation • Membrane fluidity • Polar lipids • Immunomodulator The bilayer concept was established by Singer and Nicolson in 1972 with the fluid mosaic membrane model. The outline structure of biological membranes is a lipid bilayer with integral proteins inside [1]. The membrane can be thought of as a barrier to the free entry and exit of cellular molecules and as a matrix in which (or on which) biochemical reactions take place. It was found that the structures of membranes are dynamic and contain areas of heterogeneity that are vital for their formation [2, 3]. In membranes, both proteins and lipids have lateral and rotational freedom. The functional roles of glycolipids (GLs) include maintenance of membrane structure and fluidity, lipid–protein interactions, and cell surface recognition [4]. Microorganisms constantly face environmental changes. However, the lipid composition of a given membrane remains constant. How does the composition of a membrane help it function efficiently, adapt to temperature changes, and process its own physiology by regulating chaperone proteins? For temperature adaptation, yeast cells have two independent heat-stress response pathways, including the heat-shock response (HSR) and the general stress response (GSR) [5]. Genes in the GSR system contain a different stress-response promoter element (STRE) [6]. When STRE is activated, some events – including increased permeability, decreased membrane potential, and elevated membrane fluidity – occur in the membrane [5]. Heat stress is suggested to be detected by a membrane-linked thermostat whose activation is a consequence not only of the elevated temperature but also of the specific composition and physical state of the membrane lipids. In bacteria, the HSR is regulated by heat-shock proteins controlled by alternative sigma factors; s32 in gram-negative bacteria Escherichia [7], Xanthomonas [8], Rhodobacter [9], Agrobacterium [10], and Vibrio [11]; and HrcA and sB in the grampositive bacterium Bacillus subtilis [12, 13]. The thermotolerance of the thermophilic F.-L. Yang (*) Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan e-mail:
[email protected] A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_18, © Springer Science+Business Media, LLC 2011
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bacterium Bacillus caldolyticus, the archaeon Sulfolobus shibatae and Pyrococcus furiosus, and the eukaryote Thermomyces lanuginosus has been studied. The synthesis of heat-shock proteins was found to be related to temperature adaptation [14–16]. Usually, the total lipid content amounts to 6–8% of the dry weight of bacteria, and 80–90% of the total lipids belong to polar lipids [17]. Major lipids of bacteria include phospholipids and GLs. When the temperature changes, it may cause a variation of membrane components to prevent cell damage. Unbalanced membrane phospholipid composition was shown to affect the expression of several regulatory genes in Escherichia coli [18]. Overproduction of the membrane-bound sn-glycerol- acyltransferase in E. coli triggered an HSR [19]. The role of the lipid phase of membranes is to sense and signal high-temperature stress, like “cellular thermometers.” This early response was followed by a rapid adaptation (priming) of the cells to otherwise lethally elevated temperatures, which strongly correlated with an observed remodeling of the composition and alkyl chain desaturation of membrane lipids. Both in E. coli and Salmonella typhi, whenever a high-temperature signal is transduced, in part, the CpxA–CpxR is activated. CpxA is a histidine kinase that contains two transmembrane regions, and CpxR is a response regulator that functions as a transcription factor to regulate the expression of heat-inducible genes [20]. Since the activity of CpxA is greatly influenced by the composition of membrane lipids [21], it is tempting to suggest that CpxA senses change in the physical state of the membrane lipids of E. coli and Salmonella cells exposed to high- temperature stress [22]. In the mesophilic bacterium E. coli and in many other bacteria that were studied, the fatty acid composition of membrane lipids varied as a function of growth temperatures [23]. When the culture temperature is higher, there is an increasing tendency to incorporate longer and more saturated fatty acids into phospholipids [24]. The alteration of membrane lipid composition plays an important role in bacterial response to heat stress, the so-called “homeoviscous adaptation” [25, 26]. At higher growth temperatures, the mechanism of regulation theoretically occurs via the incorporation of more saturated fatty acids, such as palmitic acid, into membrane lipids due to their higher melting points [27, 28]. Membrane fluidity is important for cells because it affects membrane functions, such as biochemical reactions, transport systems, and protein secretion. The ratio of saturated to unsaturated fatty acids affects membrane fluidity [29]. An increase in the ratio represents decreased membrane fluidity.
18.1 GLs of Thermophilic Bacteria: Structure, Thermodynamics, and Biosynthesis Thermophilic organisms require, for growth, temperatures substantially higher than the upper limit of the atmospheric temperature on earth (~50°C). They include a wide variety of aerobic, anaerobic, spore-forming, gram-positive, and gram-negative microorganisms capable of growing at a wide range of pH [2–9] values [30].
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Thermophilic bacteria such as Aquifex pyrophilus, Thermodesulfotobacterium commune, Thermus scotoductus, Thermomicrobium roseum, and Thermode sulfatator indicus contain unique polar lipids as major membrane components [31–35]. The polar lipids from thermophilic eubacteria are based on either ester or ether derivatives of glycerol with 1,2-sn configuration [36]. Those lipids are essential for the thermal stability and biological functions of the bacteria in extreme environments [37–39]. The polar lipids are mostly phospholipids and GLs [39, 40]. One of the main structures of GLs is composed of three hexoses (glucose and galactose mainly), one hexosamine (glucosamine or galactosamine), and one glycerol [39, 40] (Fig. 18.1). When the temperature is elevated, the proportion of branchedchain fatty acids increases, and the proportions of monoenoic and heptanoic fatty acids decrease in E. coli, Thermus aquaticus, Candida species, thermophilic Bacillus species, and Staphylococcus aureus [41]. Thermus and Meiothermus species are gram-negative thermophilic rods isolated from thermal hot springs, industrial and domestic water traps, and hydrothermal vents with neutral-to-alkaline pH [42]. Thermus bacteria possess a thick murein layer [43, 44] like gram-positive bacteria, bearing an outer membrane that, as in gram-negative bacteria, appears to be central in adapting to high temperatures [29, 45]. Several kinds of polar GLs have been isolated from the membranes of Thermus [34, 38, 46, 47] and Meiothermus [39, 48] bacteria, which share some common structural features, mainly concerning the kind and sequence of monosaccharides present and the nature of the lipid component [47]. The lipids from the genus Thermus and Meiothermus are based on 1,2-sn-diacyl glycerol. The abundant acyl residues are iso-C15 and iso-C17, anteiso-C15 and anteiso-C17 [46–48]. The relative abundance of the different fatty acids varies with the cultural medium, temperature, age, and bacterial strains [30]. In Bacillus caldotenax, B. caldovelox, and B. caldolyticus, iso-C15, iso-C16, and iso-C17 amount to about 80% of the total fatty acids. A shifting from iso-C15 to iso-C17 and from iso-C16 to n-C16 is observed as the temperature of growth is raised from 45°C to 80°C [49]. The fatty acid compositions of the total lipids in Thermus and Meiothermus – including major GLs and phospholipids – have been reported in the following references: Wait et al. [34]; Donato et al. [40]; Nobre et al. [50]; Ferreira et al. [39]; and Yang et al. 2004 [48]. CH2OCOR CHOCOR Hex
Hex
HexNH
Hex
O
CH2
COR Fig. 18.1 Structure of GL. Hex hexose, HexNH hexosamine; R alkyl chain
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The other major polar GLs are phosphoglycolipids. Phosphoglycolipids of Deinococcus radiodurans and Thermus/Meiothermus spp. are 2¢-O-(1,2-diacyl-snglycero-3-phospho)-3¢-O-(a-galactosyl)-N-glyceroyl alkylamine [51], 2¢-O-(1,2diacyl-sn-glycero-3-phospho)-3¢-O-(a-N-acetylglucosaminyl)-glyceroyl alkylamine [52], 2¢-O-(1,2-diacyl-sn-glycero-3-phospho)-3¢-O-(a-N-acetylglucosaminyl)-N-glyceroyl alkylamine (PGL1), and the novel structure 2¢-O-(2acylalkyldio-1-O-phospho)-3¢-O-(a-N-acetylglucosaminyl)-N-glyceroyl alkylamine (PGL2) [53] (Fig. 18.2). Thermophilic and hyperthermophilic organisms contain phospho-sugar-related solutes, such as cyclic-2,3-bisphosphoglycerate [54], di-myo-inositol phosphate [55, 56], mannosylglycerate and mannosylglyceramide [57, 58], di-myo-inositol phosphate [55], diglycerol phosphate (1,1¢-diglyceryl-phosphate) [59, 60], and galactosyl-5-hydroxylysine [61]. These solutes might be adaptive features of these organisms to high temperatures [62] and might protect enzymes against heat inactivation [31, 63, 64]. The genetics, biosynthesis, and metabolism of GLs and phosphoglycolipids of thermophiles remain unclear and are still under investigation. Some studies in mesophiles and universal biochemical pathways help us to further understand the related metabolic pathways of thermophilic microorganisms. For example, in GL synthesis in Acholeplasma laidlawii, the monogalactosyldiacylglycerol synthase transfers one glucose residue from UDP-Glc onto diacylglycerol to produce a-GlcD, which serves as a substrate for the second glycosylation step catalyzed by a UDP-Gal- or UDP-Glc-specific glycosyltransferase. The mono- and diglycosyldiacylglycerol synthases from A. laidlawii and Streptococcus pneumoniae were shown to be responsible for the formation of a-GlcD, a-Glc(1→2)-a-GlcD, and a-Gal(1→2)-a-GlcD [65, 66]. A similar enzymatic reaction might occur in thermophiles. The presence of a-GlcD and diglucosyldiacylglycerol in Acholeplasma is crucial for the adjustment of the bilayer-to-nonbilayer-forming lipid ratio in the membranes [67]. The characterization of the glucosyltransferases showed that they are responsible for the synthesis of mono-, di-, and oligoglycolipids with DAG as
Fig. 18.2 Structures of phosphoglycolipids PGL1 and PGL2. R alkyl chain
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the primary acceptor and with a head group configuration identical to the one found in native bacterial GLs [67–70]. The carbohydrate part of GLs may be made of different UDP-sugar, sugar transferase, and sugar-glycerol synthase. The membrane lipid composition in bacteria generally contains dialkyl glycerol diester lipids, 1,2-di-O-alkyl-sn-glycerol. It is known that G-3-P-dehydrogenase plays the role [71]. The establishment of the sn-glycerol-3-phosphate backbone is out of dihydroxyacetone phosphate by G-3-P-dehydrogenase. In step, two isopentadecane moieties are connected via an ether bond at the sn-1 and sn-2 positions of the glycerophosphate backbone, creating a diether [33, 72]. The enzymes carrying out the ether bond formation in step are still unknown [73]. Branched-chain fatty acids with a methyl group at the penultimate or antepenultimate carbon atom are synthesized by both gram-positive and gram-negative bacteria [74]. In hydroxy fatty acids, either 2-hydroxy or 3-hydroxy, the steric configuration (S, L) should be by separate biosynthetic origins. Generally, the biosynthesis of phospholipids occurs via the Kennedy pathway (fatty acid synthesis pathway) [75]. The fatty acids of GLs and phosphoglycolipids in Thermus/Meiothermus are C15 and C17 branched-chain mainly. However, in Deinococcus spp., they are rich in straight-chained fatty acids [50, 52, 76]. Once the cytoplasmic steps of biosynthesis are complete, newly synthesized phospholipids are quickly transported across the inner membrane bilayer through the flip–flop mechanism into the biological membranes (seconds to tens of seconds) [77]. In prokaryotes, only the protein MsbA (putative flippase) has been identified as having a role in lipid transport [78, 79]. Deinococcus radiodurans and the spirochaetes Borrelia burgdorferi and Treponema pallidum organisms possess outer membranes but do not make lipid A lipopolysaccharide (LPS) [80]. It is suggested that the role of LPSs in membranes may be replaced by GLs in thermophilic bacteria.
18.2 Roles, Biological Activities, and Perspectives of GLs The fatty acids in GLs can be used as a biochemical marker in taxonomy because many of the lipids of thermophilic bacteria isolated from microbial mats in hot springs have unique diol, plasmalogen, monoether, and diether structures [31]. They are also involved in the adaptation to high temperatures since their hydrocarbon chains are fully saturated, and the corresponding fatty acids have relatively high-melting temperatures (iso, anteiso, w-cyclic, long-chain) [36]. It was proposed that during abrupt temperature fluctuations, the membranes represent the most thermally sensitive macromolecular structures [81, 82]. A major function of bacterial diglucosyldiacylglycerol is to serve as the membrane anchor for lipoteichoic acids [70]. Although Thermus and Meiothermus belong to gram-negative bacteria, they have been found to be deficient in LPSs (Leone et al. [47]; unpublished data by our team), like Sphingomonas capsulata [83], Treponema denticola [84], Fibrobacter succinogenes [85], Borrelia burgdorferi [86], and Chloroflexus aurantiacus [87]. This suggests that the chemicophysical role played by LPSs may be
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accomplished by these molecules that are provided with a lipid portion, which forms the main part of the outer leaflet of the membrane itself, and with a saccharide part directed toward the surrounding environment. The occurrence of long-chain alkyl diols within GL membrane structure can be seen as an adaptive response by Thermus to environmental stressors due to the greater chemical strength of the alkyl chain compared with the more labile ester bonds of acyl glycerol. Other thermophilic species also express uncommon structural features, as with long-chain ethers in Aquifex pyrophilus [88] or a, w-dicarboxylic fatty acids in Thermotoga maritima [89], which may result in an increased resistance to heat, regarding either chemical stability or preservation of the physical properties of the whole membrane. Glycoglycerolipids are required for membrane bilayer stability in some bacteria, they serve as precursors for the formation of complex membrane components, or they are crucial for supporting oxygenic photosynthesis or growth during phosphate deficiency. In general, bacterial glycoglycerolipids mostly contain one or two sugars or sugar derivatives bound to diacylglycerol. The head group diversity of these glycoglycerolipids is further determined by the variety of different glycosidic linkages. The sugars occur in alpha- or beta-anomeric configuration and are bound in (1→2), (1→3), (1→4), or (1→6) linkage. The lipid composition of the extremely thermophilic species Alicyclobacillus acidocaldarius is composed of a high proportion of GLs (64% of the total lipids) with very unusual compositions of the head group and of the lipophilic part [90]. One of the possible biological roles of phosphoglycolipids in thermophilic bacteria is to ensure the thermal stability of the cellular membrane. Lipid composition was affected by growth temperature [91]. Bulky head groups would enhance steric protection [92], possibly by stabilizing the membrane against environmental stress (e.g., osmotic stress and temperature changes) through hydrogen bonding via glycosyl head groups [93]. This study identified bulky head groups in the phosphoglycolipids of Thermus and Meiothermus strains. The compatible solute diglycerol phosphate protects proteins of Desulfovibrio gigas and Clostridium pasteurianum; for example, the half-life of rubredoxin is increased fourfold [59]. In A. laidlawii, the amount of phosphatidylglycerol, the only phosphatide present, relative to the amount of neutral GLs, reflects the relative balance of uncharged and charged lipids in the membrane [94]. GLs play important roles in augmenting and regulating innate and adaptive immune systems in humans [95–97], for example, some gangliosides interacting with CD1d molecules and releasing proinflammatory and immunomodulatory cytokines [98–100]. Furthermore, monogalactosyldiacylglycerol of the thermophilic blue-green alga ETS-05 had in vivo antiinflammatory activity [101]. The phosphoglycolipids of thermophilic bacteria have immunomodulatory functions, which were reported recently [102]. PGL1/PGL2 mixture (PGL1:PGL2 = 10: 1–10:2) from Meiothermus taiwanensis and Thermus oshimai, but not Thermus thermophilus and Meiothermus ruber, upregulated interleukin-1b (IL-1b) production in human THP-1 monocytes and blood-isolated primary monocytes. PGL2 did not induce proIL-1 production, even partially (35–40%) inhibited PGL1-mediated proIL-1 production, showing that PGL1 is the main inducer of proIL-1 production
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in PGL1/PGL2 mixture. The production of proIL-1 stimulated by phosphoglycolipids was strongly inhibited by specific PKC-a, MEK1/2, and JNK inhibitors, but not by p38-specific inhibitor. The intracellular calcium influx was involved in phosphoglycolipids-mediated proIL-1 production. Using blocking antibody and Toll-like receptor (TLR)-linked NF-kB luciferase assays, we found that the cellular receptor(s) for phosphoglycolipids on proIL-1 production was TLR-independent. Especially, the fatty acid composition of phosphoglycolipids from both T. thermophilus and M. ruber consists of a low percentage of C15 (75%). This suggests that the C15 percentage of PGL plays a critical role in PGL-mediated proIL-1 induction. Glycoglycerolipids from A. laidlawii (Mycoplasma): 3-O-[2¢-O-(a-dglucopyranosyl)-6¢-O-acyl-a-glucopyranosyl]-1,2-di-O-acyl-sn-glycerol (GAGDGs) can bind to lymphoid cells and HIV-1 and facilitate the entry of HIV-1 into cells [103]. The branching forms of the acyl chains with C14 or C16 were required for the ability. Both glucose and the acyl chains binding to glucose were also highly critical [104]. However, GAGDGs failed to enhance the production of proinflammatory cytokines and LTR promoter activity [105]. GAGDGs are found not only in the cell membranes of mycoplasmas but also in those of gram-positive and gram-negative bacteria and their l-forms [106]. The chain lengths of the fatty acid constituents of PGL might play a significant role in their biological activity. Alkyl chain length is known to be the factor controlling the loading of PGL into the hydrophobic cavities of lipid-binding receptors [107]. One kind of synthetic monogalactosyl diacylglycerol, BbGL-IIc (from Borrelia burgdorferi), of which the species of fatty acid seem to play an important role in biological activity since C18:1 was in the sn-1 position and C16:0 was in the sn-2 position of glycerol, was the most potent antigen in most mouse and human natural killer T (NKT) cells [99]. Studies have shown that glycosphingolipids from the nonpathogenic gramnegative bacteria Sphingomonas have a-linked sugars similar to a-GalCer [108]. a-GalCer and glycosphingolipids both can stimulate iNKT, especially in Va14i and Va24i NKT cells [98]. Based on chemical structure, the a-configuration is important for their biological activities. There are several scientific methods used to study the dynamics of membranes, the aggregation of membranes, and lipid–lipid and membrane–protein interactions, including Fourier transform infrared spectroscopy, synchrotron radiation small angle X-ray diffraction, and fluorescence resonance energy transfer technique. The related references can be seen in Vigh et al.’s paper [2]. Membranes are the initial targets for stress. Subtle membrane alterations are critically involved in the conversion of signals from the environment into the transcriptional activation of stress genes (e.g., heat-shock protein genes) [109]. The structures of PGL1 and PGL2 of Thermus/Meiothermus are partially similar to that of lipid A. Lipid A is a bacterial cell wall phospholipid composed of a diglucosamine with several ester-linked and amide-linked long-chain fatty acids. It is the principal component of gram-negative bacteria that activates the innate immune system [109]. Some studies have shown that lipid A-like compounds, such as lipid
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X and lipid IVa, form inactive complexes with TLR4 or its accessory proteins and result in the inhibition of acute lethal toxicity induced by LPS [2, 110, 111]. They act as agonists of proinflammatory responses in the mouse and as an antagonist in humans [2, 110–112], probably due to the similar structures of lipid A and PGL. In medical therapy, the pro- and anti-inflammatory responses following infection are complex and involve many mediators with unique actions. These mediators, however, have multiple interrelationships. Tumor necrosis factor and IL-1 are considered to be central in the sepsis responses; therefore, blocking their actions appears to be an attractive strategy in combating the infection.
18.3 Summary The functional roles of GLs are to maintain biological membrane structure and fluidity, lipid–protein interactions, surface recognition, and environmental adaptation. Thermophilic bacteria possess high percentages of polar GLs, including glycoglycerolipids and phosphoglycolipids instead of LPSs in gram-negative bacteria, to adapt to a thermal environment. The biochemical diversity of GLs is generated by numerous fatty acids, including chain lengths and degree of saturation and hydroxylation. The phosphoglycolipids of thermophilic bacteria have potential in medical applications since they have immunomodulatory bioactivities. In the near future, designing synthetic analogs of GLs could become a highly active research field for the development of a simple target compound with potent biological activity. Acknowledgments This work was financially supported by Academia Sinica and the National Science Council, Taiwan.
References 1. Singer SJ, Nicolson GL (1972) The fluid mosaic model of the structure of cell membranes. Science 175:720–731 2. Vigh L, Escribá PV, Sonnleitner A, Sonnleitner M, Piotto S, Maresca B, Horváth I, Harwood JL (2005) The significance of lipid composition for membrane activity: new concepts and ways of assessing function. Prog Lipid Res 44:303–344 3. Bony J, Lopez A, Gilleron M, Welby M, Lancelle G, Rousseau B, Beaucourt JP, Tocanne JF (1989) Transverse and lateral distribution of phospholipids and glycolipids in the membrane of the bacterium Micrococcus luteus. Biochemistry 28:3728–3737 4. Tillman TS, Cascio M (2003) Effects of membrane lipids on ion channel structure and function. Cell Biochem Biophys 38:161–190 5. Carratù L, Franceschelli S, Pardini CL, Kobayashi GS, Horvath I, Vigh L, Maresca B (1996) Membrane lipid perturbation modifies the set point of the temperature of heat shock response in yeast. Proc Natl Acad Sci USA 93:3870–3875 6. Chatterjee MT, Khalawan SA, Curran BPG (2000) Cellular lipid composition influences stress activation of the yeast general stress response element (STRE). Microbiology 146:877–884
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Chapter 19
Lipooligosaccharides of Neisseria Species: Similarity Between N. polysaccharea and N. meningitidis LOSs Chao-Ming Tsai†
Keywords Neisseria polysaccharea • Lipooligosaccharides • lgt genes • Serological cross-reactivities The Neisseria genus consists of 20 species, and humans host ten of the species. N. meningitidis (meningococcus) and N. gonorrhoeae (gonococcus) are the two pathogenic species that can cause meningitis and gonorrhea in humans, respectively. The eight other (nonpathogenic) Neisseria species found in humans are N. lactamica, N. polysaccharea, N. cinerea, N. elongata, N. flavescens, N. mucosa, N. sicca, and N. subflava. Ten other species are found in animals [10]. This chapter deals with the lipooligosaccharides (LOSs) of Neisseria. The LOSs in the two pathogens are surface antigens and also play important roles in their pathogenesis [19]. The biosynthesis and chemistry of N. meningitidis LOSs have been studied extensively and reviewed [13, 23]. The focus here is on the LOSs of commensal N. polysaccharea, which is closely related to N. meningitidis. Meningococcal disease caused by N. meningitidis is life-threatening and remains a serious health problem worldwide. Of adults, 10–20% carry N. meningitidis in their nasopharynxes without causing any symptoms or problems. However, people who acquire N. meningitidis and have no preexisting serum bactericidal antibodies to meningococci or a deficiency in complements may develop serious meningococcal disease. The pathogenesis of meningococcal disease is the subject of many investigations but has not been fully understood yet. The incidence of the disease is approximately 1 in 100,000 in developed countries, with a 10% mortality rate. The incidence can be 100 times higher in underdeveloped countries, particularly those in Africa. Twenty percent of patients who have recovered from the disease may develop neurological or other sequelae, such as hearing impairments and serious sepsis necessitating limb amputation.
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Carriage of N. meningitidis and commensal Neisseria such as N. lactamica in humans reduces the risk of meningococcal disease through the induction of antibodies that react or cross-react with N. meningitidis [7, 15, 18]. N. polysaccharea was historically grouped with nonencapsulated N. meningitidis due to the similarity in their morphology and many biochemical properties, such as utilization of glucose and maltose. N. polysaccharea was differentiated from N. meningitidis in 1983 as a new species based on its production of extracellular amylopectin and lack of g-glutamyl-transferase, which is a characteristic for N. meningitidis [20, 21]. In studies on the carriage of commensal N. polysaccharea in children, as high as one third of nonencapsulated N. meningitidis were identified as N. polysaccharea [1, 4, 5]. In a study of 50 isolates of N. polysaccharea, a high percentage of strains showed cross-reactivity with antimeningococcal antisera, especially with sera against Group B, by the antiserum agar method [4]. Since N. polysaccharea has no capsule, LOS and outer membrane proteins (OMPs) are most likely the crossreacting antigens.
19.1 Analysis of LOS or Lipopolysaccharide in Neisseria Species by SDS-PAGE [26] The lipopolysaccharide (LPS) in N. meningitidis or N. gonorrhoeae does not possess a polysaccharide chain of O-repeating unit but an oligosaccharide chain linked to lipid A; therefore, many investigators have been using LOSs to describe the LPSs of these two Neisseria species [6, 22]. In-depth reviews on genetics, phenotypic expression, and structures of N. meningitidis and N. gonorrhoeae LOSs are available [13, 14, 17, 19]. However, relatively fewer studies on the LOSs in other Neisseria species have been reported [2]. We have characterized the LOSs or LPSs in 19 Neisseria strains from ten species in humans and four species in animals by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as shown in Fig. 19.1, using one representative strain from each species, except for N. lactamica (four strains) and N. subflava (three strains). Crude LOSs or LPSs were extracted from cells with 2% SDS and 10% glycerol followed by a phenol/chloroform treatment of the extracts [32]. Figure 19.1 shows the profiles of LOSs or the region of R-LPSs from these strains. SDS-PAGE analysis revealed that the LOSs in most of the Neisseria strains were heterogeneous, having two or more LOS species in them and at least four LOS species in the strain of N. cinerea. A few Neisseria strains, however, appeared as single LOS species (Fig. 19.1, panel a, lanes 5 and 9; panel b, lane 10). The molecular sizes of LOSs were estimated to be in the range of 3.6–4.5 kDa using N. meningitidis M986-NCV LOS (L7) as a size marker, which had two 3.6- and 4.0-kDa LOS species [25]. All four N. lactamica strains (Fig. 19.1, panel a, lanes 2–5) have five lgtABCDH genes [33], but their LOS expressions are quite different, suggesting that certain genes are inactive in the strains (lanes 4 and 5); and consequently, their LOS sizes are relatively smaller.
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Fig. 19.1 SDS-PAGE patterns of LOSs or R-LPS regions in 19 Neisseria strains from 14 species. Lane 1 in both panels (a) and (b) is N. meningitidis M986-NCV LOS containing 3.6- and 4.0-kDa components. Panel (a), lanes 2–5: N. lactamica 81186, 89375, 89421, and 93302; lane 6: N. polysaccharea 85323; lane 7: N. canis 81172 (R-LPS region); lane 8: N. cinerea 81176; lane 9: N. caviae 81173; lane 10: N. elongate N9; lane 11: N. flavescens 81173. Panel (b), lane 2: N. flavescens (repeating of panel (a)); lane 3: N. mucosa 81170 (R-LPS region); lane 4: N. ovis 81184; lane 5: N. sicca 96291; lanes 6–8: N. subflava 81187, 81201, and 85071; lane 9: N. weaverii SB 356; lane 10: Moraxella catarrhalis 81191; and lane 11: N. gonorrhoeae strain F62
At fivefold higher sample loading of the cell extracts or outer-membrane vesicles [9], the strains of N. canis and N. mucosa, but not the rest of the 17 Neisseria stains, had small amounts of ladder-like components at much higher molecular weight regions on SDS-PAGE gel as shown in Fig. 19.2. The ladder-like components indicated the presence of O-polysaccharide repeating units of LPSs in N. canis and N. mucosa; therefore, these two strains produced LPS rather than LOS. Since only one strain was examined from each of the nine Neisseria species, further studies are needed to see whether these species produce LOS or LPS.
19.2 LOS Profiles from 15 N. polysaccharea Strains on SDS-PAGE Gel Phenotypic LOS expression in N. meningitidis on SDS-PAGE was presented previously [23]. Our genetic studies on the LOSs in Neisseria species and 15 N. polysaccharea strains have revealed that most N. polysaccharea and N. meningitidis have three common LOS genes, lgtABH, and that N. lactamica has two additional genes, lgtCD at the lgt-1 locus [33, 34], which are presented in the following section. Figure 19.3 shows the phenotypic LOS expression in 13 N. polysaccharea strains
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Fig. 19.2 SDS-PAGE profiles of LPS in N. canis 81172 and N. mucosa 81170 from proteinase K-treated outer-membrane vesicles. Lanes 1 and 8: N. meningitidis M986-NCV and its 3.6- and 4.0-kDa LOS species, relative sample loads 5 and 1, respectively; lane 2: proteinase K control sample; lane 3: E. coli O55 LPS (Sigma Co.), not proteinase K treated; lanes 4 and 5: N. canis 81172, relative sample loads 5 and 1; and lanes 6 and 7: N. mucosa 81170, relative sample loads 1 and 5. Crude LPS preparations from cells showed similar profiles
Fig. 19.3 SDS-PAGE analysis of LOSs in 13 N. polysacchareae stains on 16% gel. Lane 1 is stain 85323, as in lane 6 of Fig. 19.1a, but underloaded. Lanes 2–14: 2, 85321; 3, 87042; 4, 87188; 5 89354; 6, 89357; 7, 90400; 8, 85322; 9, 87043; 10, 87190; 11, 89353; 12, 89355; 13, 89357 (duplicate of 6); and 14, 91275. Lane 15 is N. meningitis 93246, an L1 LOS (3.8 kDa). The arrows on the left are the locations of the 3.6- and 4.0-kDa LOS species in N. meningitis M986-NCV
on an SDS-PAGE gel. The arrows on the edges of the gel indicate the locations of the 3.6- and 4.0-kDa LOS species of N. meningitidis M986-NCV and 3.8-kDa of L1 LOS. The results show that the majority of N. polysaccharea LOSs were heterogeneous and contained two to four LOS species in a strain, with estimated molecular
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sizes in the range from 3.6 to 4.1 kDa. Three strains, however, showed single LOSs with a size of 3.7 kDa for strains 89353 and 89355 (lanes 11 and 12) and 3.6 kDa for strain 89357 (lanes 6 and 13, duplicate). The LOS profiles of two additional strains, 86376 and 89356 (not shown), were similar to that of 87042 (lane 3). The heterogeneity and size variation of N. polysaccharea LOSs resemble those of N. meningitidis LOSs observed in previous studies [6, 27, 28].
19.3 Genetic Studies of LOS Glycosyltransferase Genes in Neisseria Species For LOS biosynthesis in N. meningitidis and N. gonorrhoeae, the genes that encode glycosyltransferases for the assembly of oligosaccharides in their LOSs have been identified and reviewed in many publications [13, 19]. The genes responsible for the biosynthesis of oligosaccharide of LOSs in these two species are shown in Fig. 19.4. There are five LOS glycosyltransferase (lgt) genes, lgtABCDE, in
lgt-1
alternative a - chain
Gal
a-chain GalNAc
b
Gal
b
GlcNAc
b
lgtA
lgtB
lgt-2
a
lgtC
Gal
b
lgtE or lgtH
b
Glc
lgtF
lgt-3 lgtG
b-chain
Gal (Ng)
b
a
rfaC
Glc (PEA)
II Kdo
II Hep
a
a
GlcNAc
I Kdo
a
Lipid A
coo−
a
rfaF a
lgtD (Ng)
I Hep
coo−
rfaK
g-chain
Ac
Fig. 19.4 A general structure of Neisseria LOS and functional relationship of the lgt genes. The a-chain of L8-LOS immunotype is limited to Galb1-4Glc attached to the Heptose I (Hep I) of the inner core. The a-chain of L3, 7-LOS immunotype contains a lacto-N-neotetraose structure of Galb1-4GlcNAcb1-3Galb1-4Glc [16]. The alternative a- and b-chain of oligosaccharides are indicated with dotted lines. PEA and Ac refer to phosphoethanolamine and acetyl residue, respectively. PEA replaces the b chain at the 3¢ position of Hep II for some N. meningitidis LOSs. The terminal sugar residues present in some of N. gonorrhoeae are indicated with (Ng). The lgt genes at the three chromosomal loci are indicated as lgt-1 (lgtA, lgtB, lgtC, lgtD, and lgtE), lgt-2 (lgtF and rfaK ), and lgt-3 (lgtG). Modified from Zhu et al. [33], Fig. 1, with permission
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N. gonorrhoeae at the lgt-1 locus [8]. The three genes, lgtA, B, and E (or H ), encode the three glycosyltransferases required for the assembly of the trisaccharide of lacto-N-neotetraose (LNnT, Galb1-4GlcNAcb1-3Galb1-4Glc) at the nonreducing end of the long a-chain in these two species [8, 11, 30, 33]. The lgtC and lgtD, two genes of the lgt-1 locus, are present in almost all N. gonorrhoeae but only infrequently found in N. meningitidis [33]. The lgtF and lgtG genes at two other loci, lgt-2 and lgt-3, encode two different b-glucosyltransferases for adding glucose to the heptose-I (Hep-I) in the a-chain and Hep-II to form a short b-chain, respectively [3, 14]. Using nine lgt primers designed from published DNA sequences of N. meningitidis and N. gonorrhoeae, we analyzed these LOS genes in 26 strains of N. meningitidis, including all 12 LOS immunotypes [35, 36], 51 strains of N. gonorrhoeae, and 18 strains of commensal Neisseria from 12 species; the distribution of the lgt genes at three loci are shown in Table 19.1 [33]. All N. gonorrhoeae tested had five, lgtA, B, C, D, E, genes at the lgt-1 locus, lgtF at the lgt-2 locus, and lgtG at the lgt-3 locus. However, the lgt genes were variable in N. meningitidis. At the lgt-1 locus, most strains had lgtA, B, E or lgtA, B, H genes, and the lgtE and lgtH genes were mutually exclusive. The lgtC and lgtD genes were found only in three and one strains, respectively. The lgtF gene at the lgt-2 locus was present in all N. meningitidis. The lgtG gene at the lgt-3 locus was found in less than half of the 12 LOS immunotypes. For commensal Neisseria species, four of the N. lactamica strains and two of the N. polysaccharea strains tested, all contained lgtA, B, H genes. N. lactamica strains also contained lgtC and lgtD genes in addition to the three genes, as in N. gonorrhoeae. The lgt genes were also found in N. cinerea, N. elongata, and N. subflava but were not detected in seven other Neisseria species tested, one strain each. Commensal N. polysaccharea is most closely related to N. meningitidis, while N. lactamica is most closely related to N. gonorrhoeae at the lgt-1 locus among Neisseria species, as shown in Table 19.1. A further analysis on 13 additional strains by polymerase chain reaction (PCR) method revealed that all 15 N. polysaccharea strains had the lgtA, lgtB, and lgtH genes, and two of them also had the lgtC and lgtD genes, as shown in Table 19.2 [34]. All N. polysaccharea strains also possess a characteristic amS gene, which encodes amylosucrase for the production of amylopectin from sucrose, as anticipated. The presence of the lgtF gene, a gene at the lgt-2 locus in N. meningitidis, was also detected in most of the N. polysaccharea strains [24]. Thus, N. polysaccharea resembles N. meningitidis in having the same LOS genes in the lgt-1 and lgt-2 loci. In addition, an lst gene at another locus in N. meningitidis that encodes sialyltransferase for LOS sialylation was also found in N. polysaccharea, in which the LOS is nonsialylated due to the lack of an endogenous substrate, CMP-NeuNAc, in this organism [29]. Both the lgtB and lgtE genes encode a b-galactosyltransferase, but these enzymes transfer galactose from the substrate, UDP-Gal, to two different acceptors as shown in Fig. 19.4; LgtB and LgtE add Gal to GlcNAc and Glc moieties in the a-chain, respectively [30]. DNA sequence and phylogenetic analyses suggest that the lgtH gene found in four Neisseria species is also a b-galactosyltransferase.
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Table 19.1 Distribution of nine lgt genes in 95 strains of Neisseria lgt-1 Major Species N. meningitidisc
Straina 126E 35E 6275 M986 BB-305 44/76 MC58 89I M981 M992 6155 M978 A1 M120 Z2491 7880 7889 7897
N. gonorrhoeae d
LOS L1 L2 L3 L3 L3 L3 L3 L4 L5 L6 L7 L8 L8 L9 L9 L10 L11 L12
lgt-2 lgt-3
lgtAb − + + + + + + + + + + + + + + + + +
lgtB − + + + + + + + + + + + − + + + − +
lgtC + − − − + − − − − − − + − − − − − −
lgtD + − − − − − − − − − − − − − − − − −
lgtE + + − − − + + + + + + + − − − − − −
lgtZ + − − − + − − − − − − + − − − − − −
lgtH − − + + + − − − − − − − + + + + + +
lgtF + + + + + + + + + + + + + + + + + +
lgtG − + + + + + + + + + − − + − − − − −
880250 FA1090 F62
+ + +
+ + +
+ + +
+ + +
+ + +
− − −
− − −
+ + +
+ + +
N. lactamica
81186 89375 89421 93302
+ + + +
+ + + +
+ + + +
+ + + +
− − − −
− − − −
+ + + +
+ − − −
+ + + +
N. subflava
81187 81201 85071
− − +
− − +
− − +
− − +
− − −
− − −
− − +
− − −
− − +
N. polysaccharea
85323 87043 81172 81176 81173 N9 81189 81170 81184 96291 SB356
+ + − + − (+) − − − − −
+ + − − − − − − − − −
− − − (+) − − − − − − −
− − − − − − − − − − −
− − − − − − − − − − −
− − − − − − − − − − −
+ + − + − (+) − − − − −
− + − + − − − − − − −
− − − − − (+) − − − − −
N. canis N. cinerea N. caviae N. elongata N. flavescens N. mucosa N. ovis N. sicca N. weaverii
Modified from Zhu et al. [33], Table 2, with permission Genes at the locus lgt-1 in strains 126E, A1, M978, MC58, Z2491, FA1090, and F62 were previously reported [8, 11, 12, 32] b Genes were observed by dot blot DNA hybridization and PCR. +, positive by both hybridization and PCR; (+), positive by hybridization but negative by PCR; −, negative by both hybridization and PCR c Eight N. meningitidis strains (2001083, 2001130, 2001113, 2001117, 2001110, 2001111, 2001147, and 2001148) with L2 immunotype have the same lgt pattern as strain 6275 d Forty-eight N. gonorrhoeae strains examined in this study have the same lgt pattern as strain FA1090 a
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Table 19.2 The genetic characteristics of 15 N. polysaccharea strains lgt-1 siaD/ Species N. meningitidis
Strain Z2491 MC58 M986 126E S4383 6304Y
orf 1 +A +B +B +C +W135 +Y
amS − − − − − −
lgtA + + + − + +
lgtB + + + − + +
lgtC − − − + − −
lgtD − − − + − −
lgtE − + − + − +
lgtZ − − − + − −
lgtH + − + − + −
N. polysaccharea
85321 85322 85323 86376 87042 87043 87188 87190 89353 89354 89355 89356 89357 90400 91275
− − − − − − − − − − − − − − −
+ + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + +
− − − − − − + + − − − − − − −
− − − − − − + + − − − − − − −
− − − − − − − − − − − − − − −
− − − − − − − − − − − − − − −
+ + + + + + + + + + + + + + +
N. gonorrhoeae
FA1090 F62
− −
− −
+ +
+ +
+ +
+ +
+ +
− −
− −
A, B, C, W135, and Y indicate PCR-based prediction for the serogroups of N. meningitidis [Taha (2000) J Clin Microbiol 38:855–857]. The results are consistent with the serogroup classification of N. meningitidis strains Adapted from Zhu et al. [34], Table 3
Although the lgtH gene had a higher DNA homology to the lgtB gene, the lgtE and lgtH genes were mutually exclusive [33]. Thus, we generated an lgtH mutant of N. meningitidis 6275 (L3 LOS with lgtABH genes) by mutagenesis and characterized the function of the lgtH gene [31]. Figure 19.5 shows that the size of lgtH mutant LOS on SDS-PAGE (I) decreased to 3.6 kDa and that the LOS had no reactivity with an L3 monoclonal antibody in immunoblot analysis (II-a) as compared to the parent LOS. The monosaccharide analysis (III) showed that the oligosaccharide of the mutant LOS contained no galactose. MALDI-TOF mass spectrometric analysis further revealed that the mutant LOS lost a mass of Gal. GlcNAc.Gal trisaccharide plus sialic acid if the parent LOS was sialylated (Table 1 in [31]). Therefore, the LgtH is a b-galactosyltransferase, and it uses the Glc moiety in the a-chain as acceptor; the mutually exclusive lgtE and lgtH genes serve the same function. The function of the lgtH gene was reconfirmed by a mutant of N. polysaccharea, in which all strains tested had an lgtH gene instead of lgtE gene.
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Fig. 19.5 Characterization of the LOSs of N. meningitidis 6275 and its lgtH mutant by (I) SDSPAGE (16%), (II) immunoblot, and (III) monosaccharide analysis of their oligosaccharides. Samples are the LOSs or oligosaccharides (OS) from A, N. meningitidis 6275 (L3); B, its lgtH mutant; 1 and 2, N. meningitidis M986 (L3,7) and its OP− variant with a truncated LOS having only Glc but no Gal. Monoclonal antibodies used in immunoblot are (a) 9-2-L3,7 and (b) OP− G5593. Adapted from Zhu et al. [31], supplementary Fig. S2
19.4 Serological Cross-Reactivities of N. polysaccharea and N. meningitidis LOSs A high percentage of N. polysaccharea strains were reported to cross-react with antimeningococcal antisera [4]. Their LOSs and OMPs are most likely the crossreacting antigens. Since most N. polysaccharea contained two or more LOS species
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Fig. 19.6 Cross-reactivity of 12 N. polysacchareae LOSs with a rabbit antiserum to N. meningitis M986-NCV (L7): panel (a), SDS-PAGE analysis of remaining LOSs on 16% gel after electrophoretically transferring LOSs to a nitrocellulose membrane; and panel (b), immunoblot analysis as described [25]. Lane 1 is N. meningitis M986-NCV LOS (a homologous antigen with a major 4.0-kDa LOS species), fast LOS mobility due to a leaky lane on the edge of the gel. Lanes 2–14: 2, 85323; 3, 85321; 4, 87042; 5, 87188; 6, 89354; 7 and 8, 90400 (duplicate); 9, 87043; 10, 87190; 11, 89353; 12, 89355; 13, 89357; and 14, 91275. The stars on the left of lane 4 are the position of the 4.1-kDa LOS species in N. polysacchareae
in a strain, separation of LOS species by SDS-PAGE followed by immunoblot was used to analyze the cross-reactive species in N. polysaccharea LOSs with antisera to N. meningitidis LOSs. Figure 19.6 shows that most N. polysaccharea LOSs cross-reacted with a rabbit antiserum to N. meningitidis M986-NCV LOS (L7), of which the a-chain of oligosaccharide is depicted in Fig. 19.4 but without GalNAc at the end [16]. All 12 N. polysaccharea LOSs, except two from strains 87188 and 87190 in lanes 5 and 10, respectively, showed cross-reactivity with the antiserum. It is intriguing that these two strains contain two additional genes, lgtC and lgtD, in addition to three lgtABH genes at the lgt-1 locus among 15 N. polysaccharea as shown in Table 19.2. The 4.1-kDa LOS species in N. polysaccharea LOSs was the main reactive species (indicated by a star in lane 4). A weak reactivity with the 4.0-kDa species was observed in a few N. polysaccharea LOSs (lanes 4 and 6). The antiserum also had reactivity with the 3.6- or 3.7-kDa component in three LOSs (lanes 11, 12, and 13). The 4.1-kDa and 4.0-kDa LOS species in three additional N. polysaccharea LOSs (strains 85322, 86376, and 89356) also reacted with the antiserum (not shown).
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Several N. polysaccharea LOSs also cross-reacted with a rabbit antiserum to N. meningitidis 6275 LOS (L3). N. polysaccharea 89353 LOS, which showed a single 3.7-kDa species on SDS-PAGE, was found to cross-react with a rabbit antiserum specific for L5. The above results demonstrate that N. polysaccharea LOSs are very cross-reactive with rabbit antisera to N. meningitidis LOSs. Similar to many N. meningitidis LOSs, the majority of 4.1-kDa species in N. polysaccharea LOSs showed reactivity with a monoclonal antibody that binds LNnT, which is the product of lgtABH and lgtF genes, indicating that this epitope is present as part of the structure in N. polysaccharea LOSs [24]. To examine the cross-reactivity of N. meningitidis LOSs with antibodies to N. polysaccharea LOSs, we immunized rabbits against N. polysaccharea LOSs from strains 85322 and 89357, two rabbits for each LOS. The former contained 4.1- and 4.0-kDa species in its LOSs, and the latter contained 3.6-kDa species and cross-reacted with a meningococcal L8 antibody. Both pairs of LOS antisera showed strong cross-reactivities with the majority of 12 N. meningitidis LOS types but weak cross-reactivity with types 9, 10, and 12 LOSs in enzyme-linked immunosorbent assay (ELISA). The degree of cross-reactivity was higher for the antisera to N. polysaccharea 89357 LOS than the other pair of antisera. The serological cross-reactivities of N. polysaccharea LOSs with antibodies to N. meningitidis LOSs and vice versa suggest that their LOS structures are similar. Comparative structural analysis of N. polysaccharea and N. meningitidis LOSs is needed to substantiate this proposal.
19.5 Summary The Neisseria genus consists of 20 species, and humans host ten of the species. N. meningitidis and N. gonorrhoeae are the two pathogenic species that can cause meningitis and gonorrhea in humans, respectively. Carriage of nonpathogenic commensal Neisseria has been reported to reduce the risk of meningococcal disease through the induction of antibodies in the host that cross-react with N. meningitidis. Among commensal Neisseria, N. lactamica and N. polysaccharea are frequently found in humans. LOS is a cell-surface antigen and also a virulent factor in N. meningitidis and N. gonorrhoeae. The two organisms contain common genes in their LOS biosynthesis. Five possible genes exist in their lgt-1 locus: lgtA, lgtB, lgtC, lgtD, and mutual exclusive lgtE or lgtH. Genetic analysis of commensal Neisseria revealed that 15 N. polysaccharea all had three genes, lgtABH, a gene organization in most N. meningitidis. Two of the 15 N. polysaccharea and all four of the N. lactamica examined had two additional genes, lgtC and lgtD, which are only occasionally found in N. meningitidis. Thus, commensal N. polysaccharea closely resembles N. meningitidis in the lgt-1 locus. Phenotypic LOS expression in N. polysaccharea was examined by SDS-PAGE and by Western blot for cross-reactivity with N. meningitidis LOS antibodies.
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SDS-PAGE showed that the majority of 15 N. polysaccharea LOSs were heterogeneous, having 4.1-kDa and other minor smaller components, but three of them had single 3.6- or 3.7-kDa LOSs. The LOS heterogeneity and size variation were also observed in N. meningitidis previously. Serological analysis showed that N. polysaccharea LOSs cross-reacted with rabbit antisera to N. meningitidis LOSs and vice versa. In conclusion, genetic, SDS-PAGE, and serological analyses show that N. polysaccharea and N. meningitidis LOSs are very similar. Acknowledgments The genetic studies on the Neisseria species done in the author’s laboratory were conducted by Dr. Peixuan Zhu, who is now at Creatv MicroTech, Potomac, MD 20854, USA. The author thanks Dr. Raymond Tsang of National Microbiology Laboratory, Health Canada, for providing the N. polysaccharea strains. He also thanks Mr. George Kao and Ms. Theresa Wang for their technical assistance.
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14. Kahler CM, Carlson RW, Rahman MM, Martin LE, Stephens DS (1996) Two glycosyltransferase genes, lgtF and rfaK, constitute the lipooligosaccharide ice (inner core extension) biosynthesis operon of Neisseria meningitidis. J Bacteriol 178:6677–6684 15. Kim JJ, Mandrell RE, Griffis JM (1989) Neisseria lactamica and Neisseria meningitidis share lipooligo-saccharide epitopes but lack common capsular and class 1, 2, and 3 protein epitopes. Infect Immun 57:602–608 16. Kogan G, Uhrin D, Brisson JR, Jennings HJ (1997) Structural basis of Neisseria meningitidis immunotypes including the L4 and L7 immunotypes. Carbohydr Res 298:191–199 17. Mandrell RE, Griffiss JM, Macher BA (1988) Lipooligosaccharides (LOS) of Neisseria gonorrhoeae and Neisseria meningitidis have components that are immunochemically similar to precursors of human blood group antigens. J Exp Med 168:107–126 18. Oliver KJ, Reddin KM, Bracegirdle P, Hudson MJ, Borrow R, Feavers IM, Robinson A, Cartwright K, Gorringe AR (2002) Neisseria lactamica protects against experimental meningococcal infection. Infect Immun 70:3621–3626 19. Preston A, Mandrell RE, Gibson BW, Apicella MA (1996) The lipooligosaccharides of pathogenic gram-negative bacteria. Crit Rev Microbiol 22:139–180 20. Riou JY, Guibourdenche M (1987) Neisseria polysaccharea sp. nov. Int J Syst Bateriol 37:163–165 21. Riou JY, Guibourdenche M, Popoff MY (1983) A new taxon in the genus Neisseria. Ann Microbiol (Paris) 134B:257–267 22. Schneider H, Hale TL, Zollinger WD, Seid RC, Hammack CA, Griffiss JM (1984) Heterogeneity of molecular size and antigenic expression within lipooligosaccharides individual strains of Neisseria gonorrhoeae and Neisseria meningitidis. Infect Immun 45:544–549 23. Tsai CM (2001) Molecular mimicry of host structures by lipooligosaccharides in Neisseria meningitidis. In: Wu AM (ed) The molecular immunology of complex carbohydrates. Kluwer Academic/Plenum, New York, pp 525–542 24. Tsai CM, Zhu P, Kao G, Tsang RSW (2006) Similarity between Neisseria meningitidis and Neisseria poly-saccharea lipooligosaccharides (rough-type lipopolysaccharides). In: Joint meeting of Society of Leukocyte Biology and International Endotoxin and Innate Immunity Society, San Antonio, TX, 9–11 November 2006 25. Tsai CM, Civin CI (1991) Eight lipooligosaccharides of Neisseria meningitidis react with monoclonal antibody which binds lacto-N-neotetraose (Galb1-4GlcNAcb1-3Galb1-4Glc). Infect Immun 59:3604–3609 26. Tsai CM, Frasch CE (1982) A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem 119:115–119 27. Tsai CM, Chen W, Balakonis PA (1998) Characterization of terminal NeuNAca2-3Galb14GlcNAc sequence in lipooligosaccharides of Neisseria meningitidis. Glycobiology 8:359–365 28. Tsai CM, Mocca LF, Frasch CE (1987) Immunotype epitopes of Neisseria meningitidis lipooligosaccharide type 1 through 8. Infect Immun 55:1652–1656 29. Tsang RSW, Law DKS, Tsai CM, Ng LK (2001) Detection of the lst gene different serogroups and LOS immunotypes of Neisseria meningitidis. FEMS Microbiol Lett 199:203–206 30. Wakarchuk W, Martin A, Jennings MP, Moxon ER, Richards JC (1996) Functional relationships of the genetic locus encoding the glycosyltransferase enzymes involved in expression of the lacto-N-neotetraose terminal lipopolysaccharide structure in Neisseria meningitidis. J Biol Chem 271:19166–19173 31. Zhu P, Boykins RA, Tsai CM (2006) Genetic and functional analyses of the lgtH gene, a member of the b-1,4-galactosyltransferase gene family in the genus Neisseria. Microbiology 152:123–134 32. Zhu P, Klutch MJ, Tsai CM (2001) Genetic analysis of conservation and variation of lipooligosaccharide expression in two L8-immunotype strains of Neisseria meningitidis. FEMS Microbiol Lett 203:173–177 33. Zhu P, Klutch MJ, Bash MJ, Tsang RSW, Ng LK, Tsai CM (2002) Genetic diversity of three lgt loci for biosynthesis of lipooligosaccharide (LOS) in Neisseria species. Microbiology 148:1833–1844
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34. Zhu P, Tsang RSW, Tsai CM (2003) A noncapsulated Neisseria meningitidis produces amylopectin from sucrose: altering the concept for differentiation between N. meningitidis and N. polysaccharea. J Clin Microbiol 41:273–278 35. Zollinger WD, Mandrell RE (1977) Outer-membrane protein and lipopolysaccharide serotyping of Neisseria meningitidis by inhibition of a solid-phase radioimmunoassay. Infect Immun 18:424–433 36. Zollinger WD, Mandrell RE (1980) Type-specific antigens of group A Neisseria meningitidis: lipopolysaccharide and heat-modifiable outer membrane proteins. Infect Immun 28:451–458
Part V
Structures and Functions of Complex Carbohydrates
Chapter 20
Roles for N- and O-Glycans in Early Mouse Development Suzannah A. Williams and Pamela Stanley
Keywords Conditional gene targeting • N-glycans • O-glycans • Fertilization • Stage-specific embryonic antigens Glycosylation is the most abundant posttranslational protein modification. Specific glycans covalently attached to glycoproteins contribute to their functions, ensuring appropriate folding, secretion, half-life, and receptor–ligand interactions [1]. Many different classes of glycans exist, but those discussed herein are the complex and hybrid N-glycans, core 1-derived O-glycans, and O-linked fucose glycans. The synthesis of each class of glycan is initiated by the addition of a single sugar, or group of sugars, to certain amino acids or amino acid sequons by specific glycosyltransferases via a particular linkage. The subsequent sugars are added individually in a carefully orchestrated pathway by specific glycosyltransferases that reside in the secretory compartments of the cell. Thus, the glycans ultimately synthesized by a cell depend on the cohort of glycosyltransferases, nucleotide sugar synthases, and transporters expressed by that cell, which will be influenced by metabolic state and stage of development. To determine roles for complex and hybrid N-glycans, core 1-derived O-glycans, and O-fucose glycans (Fig. 20.1) in oogenesis, fertilization, blastogenesis, implantation, and embryonic development, we used a maternal and zygotic gene-targeting approach.
20.1 Targeting Complex and Hybrid N-Glycans N-glycans are linked to the asparagine residue in Asn–X–Ser/Thr sequons. Complex and hybrid N-glycans are generated from oligomannosyl N-glycans by the addition of an N-acetylglucosamine (GlcNAc) catalyzed by N-acetylglucosaminyltransferase P. Stanley () Department of Cell Biology, Albert Einstein College of Medicine, New York, NY 1046, USA e-mail:
[email protected] A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_20, © Springer Science+Business Media, LLC 2011
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Fig. 20.1 Mammalian glycans targeted. (a) The generation of complex and hybrid N-glycans at N-X-S/T sequons is initiated by the action of GlcNAcT-1 encoded by the Mgat1 gene. (b) The generation of mucin O-glycans is initiated by the transfer of GalNAc to Ser or Thr. Core 1 is synthesized by the action of T-synthase encoded by the C1galt1 gene, and core-2 O-glycans are generated by the addition of a b1,6GlcNAc to a core-1 O-glycan. (c) O-fucose glycans are generated by the action of the protein O-FucT-1 encoded by the Pofut1 gene, which transfers fucose to EGF repeats with a particular consensus sequence. Fringe GlcNAc transferases encoded by the Lfng, Mfng, or Rfng gene add GlcNAc to the O-fucose; b1,4GalT-1 subsequently adds a Gal, and an a2,3 sialylT adds a sialic acid. A dashed line signifies that several reactions must occur before obtaining the product shown. A solid line designates a single reaction. Maternal and zygotic mutants of Mgat1, C1galt1, and Pofut1 are discussed in the text
I (GlcNAcT-I) (Fig. 20.1a). GlcNAcT-I is encoded by the Mgat1 gene, and deletion of Mgat1 prevents the generation of complex and hybrid N-glycans leaving oligomannosyl N-glycans at all complex or hybrid N-glycan sites [2, 3]. Since oligomannosyl N-glycans of the endoplasmic reticulum are generated normally in this model, N-glycan processing and glycoprotein trafficking from the endoplasmic reticulum to the mid-Golgi theoretically remain unaffected. Mgat1-null embryos generated from crosses of Mgat1neo/+ mice were identified using fluorescein isothiocyanate-labeled Phaseolus vulgaris leucoagglutinin (FITCL-PHA) [2]. L-PHA specifically binds complex N-glycans, and therefore mutant embryos lacking Mgat1 were identified by the lack of L-PHA binding compared
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with controls that fluoresce brightly. L-PHA-negative embryos developed normally until E9.5, indicating that complex N-glycans were not required for all stages of development prior to E9.5 [2]. However, all blastocysts from crosses of Mgat1neo/+ mice stained positive for L-PHA, and further analysis revealed that maternal transcripts were responsible for the generation of complex N-glycans in Mgat1neo/neo blastocysts [4]. Therefore, an alternate approach was needed to determine if maternal Mgat1 transcripts rescue preimplantation development of Mgat1neo/neo blastocysts or if complex N-glycans are simply superfluous before mid-gestation. Cre–loxP recombination technology [5] enabled us to delete the Mgat1 gene specifically in the oocyte early in oogenesis ensuring that, at the time of ovulation ~3 weeks later, maternal transcripts no longer exist in the oocyte and cannot contribute to the mutant blastocyst phenotype. Oocyte-specific deletion was achieved by targeted expression of Cre recombinase using the zona pellucida protein 3 (ZP3) promoter, which is expressed exclusively in the oocyte [6] at the primary follicle stage [7] (Fig. 20.2). Eggs ovulated by Mgat1-mutant females did not bind FITCL-PHA, revealing the absence of complex and hybrid N-glycans. An increase in the binding of Rhodamine-conjugated concanavalin A (ConA) was also observed, consistent with an increased proportion of oligomannosyl N-glycans on Mgat1−/− eggs compared to control eggs. Therefore, efficient deletion of both floxed Mgat1 alleles in Mgat1F/F:ZP3Cre females was achieved [8]. Interestingly, a proportion of maternal
Fig. 20.2 Folliculogenesis and ovulation. (a) Diagram showing different stages of folliculogenesis, including ZP3 expression at the primary stage. Adapted from Zhao and Dean [49]. (b) Mgat1−/− eggs with a thinner zona and retained cumulus cells. Mgat1-mutant eggs lack FITCL-PHA binding and have enhanced Rhodamine–ConA binding. (c) C1galt1−/− eggs lack peanut agglutinin lectin (PNA) binding and have enhanced expression of the Tn antigen
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and zygotic mutant embryos generated from Mgat1−/− eggs and Mgat1− sperm was developed to E9.5 with an indistinguishable phenotype to blastocysts containing maternal Mgat1 transcripts. Therefore, complex and hybrid N-glycans are not required for ovulation, fertilization, blastogenesis, or embryogenesis up to midgestation [8].
20.2 Oogenesis in the Absence of Complex and Hybrid N-Glycans Germ cells are generated in the ovary during embryonic development so that at birth, the entire complement of oocytes available to the female is already established. Each of these primordial germ cells is enclosed within a primordial follicle and is meiotically quiescent. Throughout postnatal life, a steady trickle of primordial germ cells leaves the quiescent pool. Each germ cell is contained within a follicle, and successful development of each oocyte is dependent on bidirectional signaling between the oocyte and the cells of the follicle [9]. The follicles develop from primordial germ cells into primary follicles when the expression of zona pellucida proteins is initiated, followed by secondary, preantral, antral, and finally ovulatory follicles [10] (Fig. 20.2). Once the follicle and oocyte have begun to grow, the only options are ovulation or, more likely, apoptosis of the follicle at some point prior to ovulation. During follicle development, the oocyte is surrounded by granulosa cells that in the last stage of development differentiate to cumulus cells in response to the preovulatory luteinizing hormone surge. The cumulus cell mass surrounding the ovulated egg is required for transfer of the egg into the oviduct and also for normal subsequent embryonic development. Cumulus cells dissociate from the zona surface after fertilization. The zona glycoproteins ZP1, ZP2, and ZP3 from Mgat1−/− oocytes that lack complex N-glycans generate a thinner zona pellucida, which contains less of all three zona proteins than does the control zona [8]. A zona lacking complex and hybrid N-glycans also retains cumulus cells after hyaluronidase treatment as opposed to the control zona, where all cumulus cells are removed by hyaluronidase [8, 11]. However, despite the modified form of the zona pellucida around mutant eggs, it is functional with respect to sperm binding and in preventing polyspermy [8]. These data revealed that complex or hybrid N-glycans are not essential for sperm–zona binding, although fewer sperm bind to a zona pellucida devoid of complex and hybrid N-glycans than to a wild-type zona [12]. While complex and hybrid N-glycans are clearly not essential for mouse female fertility (because Mgat1−/−-mutant females ovulate functional eggs that are fertilized and develop), the lack of these glycans does modify fertility since Mgat1mutant females generate smaller litters than do the control mice [8]. To determine if the decrease in fertility is due to a decrease in follicle number, follicles were counted in ovaries stimulated with pregnant mare’s serum gonadotrophin (PMSG) to synchronize follicle development. These Mgat1-mutant ovaries contained
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approximately equal numbers of large antral follicles compared to controls, indicating that preantral and antral follicle development is normal in Mgat1-mutant females. However, ovulation induction after PMSG produced significantly fewer eggs from Mgat1 females than from the controls, indicating a difference in follicle function. Furthermore, ~50% of eggs naturally ovulated by Mgat1F/F:ZP3Cre females were developmentally compromised, resulting in aberrant blastogenesis regardless of whether the mutant egg was fertilized by a Mgat1+ or Mgat1− sperm. However, ~50% of abnormal blastocysts resumed normal development upon implantation [8]. To understand the physiological basis behind the decrease in the number of Mgat1-null eggs ovulated despite the normal numbers of antral follicles just 9 h earlier, ovaries containing preovulatory follicles were examined [11]. These analyses revealed a significant decrease in the number of preovulatory follicles in Mgat1mutant females. Furthermore, ovaries with oocytes lacking Mgat1 contained follicles undergoing premature luteinization, a phenomenon not found in control ovaries. The luteinizing follicles contributed to the decrease in preovulatory follicle number in mutant females, but did not account for the overall decrease [11]. Mgat1-mutant ovaries have ~3.5 times more grossly abnormal preovulatory follicles than control ovaries do [11]. These abnormalities include oocytes with a separate zona pellucida, cumulus cells beneath the zona, and blebbing oocytes. Of the preovulatory follicles that are not grossly abnormal, more subtle abnormalities are detected, such as the presence of large PAS-positive vesicles within the oocyte. The perivitelline space around Mgat1−/− oocytes is more often absent than present, unlike in controls. The cumulus mass surrounding mutant eggs undergoes less expansion and is smaller, containing less proliferating cells. However, expansion is not defective due to altered hyaluronan content. The cumulus mass remains smaller around ovulated Mgat1-null eggs and cumulus cells adjacent to the zona are resistant to removal by hyaluronidase and sperm. Therefore, aberrant development of preimplantation embryos lacking complex and hybrid N-glycans most likely results from defective oogenesis in preovulatory follicles of Mgat1−/− oocytes [11].
20.3 Targeting Core 1-Derived Mucin O-Glycans The initial N-acetylgalactosamine (GalNAc) of core 1-derived mucin O-glycans is linked to Ser/Thr residues and is termed the Tn antigen (Fig. 20.1b). The Tn antigen can be extended by the addition of a galactose (Gal) residue to generate the T antigen, otherwise known as a core-1 O-glycan. T-synthase encoded by the C1galt1 gene catalyzes the addition of the Gal [13], which can be extended further by the action of additional glycosyltransferases. Furthermore, a GlcNAc residue may be added to the core-1 O-glycan to generate a core-2 O-glycan, which may be further extended by the action of galactosyltransferases, sialyltransferases, and fucosyltransferases [1]. Deletion of the C1galt1 gene removes T-synthase and prevents the generation of both core-1 and core-2 (core 1-derived) O-glycans, leaving only GalNAc (the Tn antigen) at O-GalNAc glycan sites.
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Core 1-derived O-glycans are required for embryonic development beyond E12 since C1galt1−/− embryos begin to hemorrhage at E12 and die by E14 [14], indicating that preimplantation development does not require core 1-derived O-glycans. However, as observed with Mgat1, maternal C1galt1 transcripts may persist in embryos generated from crossing C1galt1+/− mice and allow early development to progress normally. To investigate this question, females carrying two floxed C1galt1 alleles and the ZP3Cre recombinase transgene were used to generate oocytes lacking the C1galt1 gene from the primary follicle stage onwards [15]. The lack of core 1-derived O-glycans was confirmed using FITC-conjugated peanut agglutinin lectin (PNA), which binds the T antigen. Ovulated wild-type eggs bind FITC–PNA well, whereas eggs ovulated by C1galt1 mutants do not bind FITC– PNA. The Tn antigen detected by antibodies is present in increased amounts on C1galt1−/− eggs compared to its low levels on wild-type eggs [15]. This is consistent with the loss of Tn-antigen extension in C1galt1 mutants. Maternal and zygotic C1galt1−/− embryos generated from C1galt1−/− eggs survive and develop beyond mid-gestation. However, at E11.5, hemorrhaging and defective angiogenesis becomes evident, and embryos die by ~E13.5. Therefore, core 1-derived O-glycans are not required for oogenesis, fertilization, blastogenesis, implantation, or early embryonic development [15]. On the other hand, oogenesis is affected in females carrying C1galt1−/− oocytes [16]. C1galt1−/− oocytes generate a zona pellucida that is ~25% thinner than that in wild type, but contains all three zona glycoproteins. Surprisingly, despite evidence that core 1-derived O-glycans are required for sperm to bind to the zona pellucida [17, 18], sperm bind well to a zona lacking core 1-derived O-glycans [15]. In addition, C1galt1F/F:ZP3Cre mutant females give birth to larger litters than the controls do [15, 16]. The increase in litter size is due to an increase in the number of eggs ovulated by C1galt1-mutant females. Mutant ovaries are heavier and contain more follicles, predominantly at the preantral stage. Considering that the number of follicles leaving the quiescent pool of primordial follicles and enter the growing pool should be unaffected since targeted gene deletion occurs after this event, changes in follicle number were expected to be due either to a decrease in follicle apoptosis or modified follicle growth rate. Follicle apoptosis was assessed using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and a reduction in follicle apoptosis was eliminated as the reason for the increase in follicle number. Therefore, a model of slowed follicle growth is proposed, resulting in an overall accumulation of ovarian follicles [16]. These experiments revealed a role for oocyte O-glycans as regulators of female fertility. Ovaries containing developing oocytes lacking core 1-derived O-glycans also contain follicles with multiple oocytes at a much higher frequency than the control ovaries do. These multiple-oocyte follicles (MOFs) are most prevalent at the later stages of follicle development, indicating that they are being formed by the joining of adjacent follicles. However, these MOFs do not contribute to the increase in fertility of C1galt1F/F:ZP3Cre mutant females because the number of corpora lutea, each of which develops from a single ovulated follicle, is equal to the number of ovulated eggs. These experiments reveal that the oocyte has an active role in
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maintaining follicle integrity and that glycoproteins carrying core 1-derived O-glycans are involved, either directly or indirectly, in this process [16].
20.3.1 N-Glycans and O-Glycans as Sperm Receptors The first step in fertilization is sperm binding to the extracellular matrix of the zona pellucida that surrounds the ovulated egg. Terminal Gal residues on the O-glycans of zona glycoproteins have long been implicated as receptors for sperm binding in the mouse [17, 18] (Fig. 20.3a). The zona pellucida surrounding the egg is generated exclusively by the egg, and therefore the zona generated by C1galt1-mutant females lacks terminal Gal on core 1-derived O-glycans (Fig. 20.3c). This lack of O-glycans on C1glat1−/− eggs was confirmed by lectin binding and was also demonstrated by a decrease in molecular weight determined by Western analysis of O-glycosylated ZP1 and ZP3 [15]. Against all expectations, C1galt1-mutant females are not just fertile, but fertility is enhanced as described above, clearly demonstrating that terminal Gal residues on core 1-derived O-glycans are not essential for sperm–zona binding. Other experiments implicate a terminal GlcNAc residue on complex N- or O-glycans in sperm binding to the mouse zona pellucida [19, 20] (Fig. 20.3b). However, females generating eggs with a zona that lacks terminal GlcNAc on N-linked glycans are also fertile [8, 11] (Fig. 20.3d). This provides strong evidence that GlcNAc on complex N-glycans is not required for sperm binding. However, it is possible that terminal Gal on complex N-glycans of C1galt1-mutant eggs or terminal GlcNAc on core 1-derived O-glycans of Mgat1-mutant eggs [21] may compensate for the respective loss of a single class of glycan. To investigate this, the generation of eggs lacking both complex and hybrid N-glycans and core 1-derived O-glycans was undertaken. Double-mutant females carrying floxed Mgat1 and C1galt1 and the ZP3Cre transgene produce eggs lacking both core 1-derived O-glycans and complex and hybrid N-glycans [15] (Fig. 20.3e). Doublemutant eggs lack FITC-PNA and FITC-L-PHA binding and have enhanced Rhodamine–Con A binding, confirming their glycosylation-defective phenotype. Most surprisingly, double-mutant females are fertile, demonstrating conclusively that terminal Gal or terminal GlcNAc on O- or N-glycans is not required for sperm binding. The double mutants produce litters approximately half the size of the controls’ litters, although the time to first litter is no different from that in the controls. In addition, no double-mutant female had a second litter, and therefore fertility is clearly markedly affected by the lack of both complex and hybrid N- and core 1-derived O-glycans on oocyte glycoproteins. It is intriguing that the mild reduction in fertility observed in Mgat1F/F:ZP3Cre females [8] is greatly exacerbated by the combined removal of complex and hybrid N-glycans and O-GalNAc glycans in the double mutant [15], while the latter removed alone actually enhances fertility [15, 16].
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Fig. 20.3 N- and O-glycans in sperm binding. (a) a1,3galactose on O- and N-glycans (circled in red) has been proposed to be required for sperm binding to the zona pellucida (ZP) [17, 18]. (b) Terminal GlcNAc on N- and O-glycans on the zona has been proposed to be recognized by a galactosyltransferase (GalT) on the sperm head [19, 20]. (c) A zona lacking core 1-derived O-glycans and thus terminal Gal on O-glycans tested the hypothesis in (a). (d) ZP lacking terminal GlcNAc on N-glycans tested the hypothesis in (b). (e) The ZP on eggs generated by mice lacking both C1galt1 and Mgat1 and therefore all terminal Gal on O-glycans and all terminal Gal and GlcNAc on N-glycans tested if there was rescue for the lack of either class of glycans
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20.4 N -Glycans and O-Glycans as Carriers of Stage-Specific Embryonic Antigens Embryonic antigens are expressed on ovulated eggs and preimplantation embryos at specific stages of development, and many of these are glycan structures [22, 23]. Eggs, and most likely oocytes, express stage-specific embryonic antigen 3 (SSEA-3; Galb1,3GalNacb1,-3Gala1,4Galb1,4Glcb1-R), and SSEA-4 (sialylated SSEA3) until the eight-cell stage [24, 25]. SSEA-1, also known as the Lewis X antigen (LeX; Galb1,4[Fuca1,3]GlcNAcb1,3Galb1-R), is expressed from the eight-cell stage through the early blastocyst stage, but is not detectable on late blastocysts [26–28]. The Lewis Y antigen (LeY; Fuca1,2Galb1,4[Fuca1,3]GlcNAcb1,3Gal-R) is expressed at the morula and blastocyst stages [28]. Due to the specific temporal expression of these antigens, in particular SSEA-1 and LeY, it is proposed that they have roles in preimplantation embryonic development. Incubation of SSEA-1 glycans with four-to-eight-cell embryos causes inhibition of compaction, indicating that SSEA-1 plays a role in compaction [29, 30]. Furthermore, implantation is prevented in the presence of monoclonal antibodies to LeY [31, 32]. While these antigens appear to have physiological roles in embryogenesis, it is unknown if they are attached to N- or O-glycans on glycoproteins or on glycolipids of preimplantation embryos. To determine if these glycans are expressed on N- or O-glycans during blastogenesis, SSEA-1, SSEA-3, SSEA-4, and Le Y were examined on embryos lacking either complex N-glycans or core 1-derived O-glycans using indirect immunofluorescence [33]. Embryos lacking core 1-derived O-glycans were distinguished based on their lack of FITC-PNA binding. Interestingly, SSEA-3 and SSEA-4 were detected equally on mutant and control four-to-eight-cell embryos. Analysis of compacted embryos lacking C1galt1 revealed equivalent staining of SSEA-1 to that of controls. Finally, the LeY detected on C1galt1−/− embryos was equivalent to the controls. Therefore, SSEA-1, SSEA-3, SSEA-4, and LeY are not carried on core 1-derived O-glycans of glycoproteins expressed on developing embryos. Ovulated eggs and preimplantation embryos lacking complex N-glycans were identified by their lack of FITC-L-PHA binding. Mgat1−/− eggs were used to assess the presence of SSEA-3 and SSEA-4 antigens because, despite the production of small litters by Mgat1F/F:ZP3Cre females, prepubertal females ovulate a considerable numbers of eggs when stimulated with exogenous gonadotrophins [8]. SSEA-3 and SSEA-4 were detected on Mgat1−/− zona-free eggs at equivalent levels to those of the controls. Four-to-eight-cell Mgat1−/− embryos assessed for the presence of SSEA-1 also expressed this antigen equivalently to the controls. Blastocysts lacking Mgat1 also expressed LeY at levels equivalent to the controls’. Therefore, SSEA-1, SSEA-3, SSEA-4, and LeY are not carried exclusively on complex or hybrid N-glycans. While it is possible that an oocyte unable to synthesize complex N-glycans adds the stage-specific sugar epitopes to core-2 O-glycans, and conversely,
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an oocyte unable to synthesize the latter adds the SSEA epitopes to N-glycans, this is unlikely. Rather, the combined data suggest that the SSEA glycan antigens examined are presented solely on glycolipids.
20.5 Targeting O-Fucose Glycans Notch receptors are transmembrane glycoproteins that are activated in mammals by binding of the Notch ligands Jagged or Delta. Ligand binding induces proteolytic cleavages that result in the release of Notch intracellular domain, which is transferred to the nucleus and, in complex with other factors, modifies the transcription of target genes [34, 35]. The extracellular domain contains 29–36 epidermal growth factor (EGF)-like repeats, 21 of which have the consensus sequence for receiving an O-linked fucose [36]. The fucose is added to Ser/Thr in EGF repeats by the enzyme protein O-fucosyltransferase 1 encoded by the Pofut1 gene. O-fucose is the substrate for Fringe b1,3-N-acetylglucosaminyltransferases, which generate an O-Fucb1,3GlcNAc disaccharide that can be extended further by the addition of a Gal by b4GalT1 (b4galt1) and sialic acid by an a2,3 sialyltransferase [37] (Fig. 20.1c). Pofut1−/− embryos have defects in embryological development consistent with a lack of Notch signaling through all four Notch receptors [38]. Pofut−/− embryos at E8.5 have a kinked neuroepithelium, and the somites are misshapen and fused. At E9.5, vascularization of the yolk sac is absent, and the embryos lack major branched vessels. By E10, mutant Pofut−/− embryos are very developmentally retarded and dying; the undeveloped heart tube is obvious, and there is often a grossly extended pericardial sac. Lfng−/− mice lack the Lunatic Fringe enzyme, and therefore Notch receptors are modified at O-fucose sites solely by O-fucose (Fig. 20.1c). The presence of O-fucose results in embryo survival to birth unlike embryos lacking O-fucose. However, Lfng−/− mice have severe skeletal defects with compressed spinal columns, and they usually die by about 3–4 months of age [39–41]. A role for b4galt1 in Notch signaling was identified in a coculture Notch reporter assay [42]. Mice lacking b4galt1 are developmentally compromised, but are born and live for varying lengths of time, depending on the genetic background [43, 44]. Examination of b4galt1 −/−-mutant embryos revealed reduced expression of four Notch pathway genes and was consistent with a subtle defect in Notch signaling that might affect somitogenesis [45]. An effect on the number of lumbar vertebrae was observed in embryos just prior to birth [45]. To date, no examination of a2,3-sialyltransferase-deficient embryos has been reported. Notch pathway genes are well expressed in oocytes, the ovary, and embryos [41, 46, 47]. Therefore, to determine the role of O-fucose on Notch in oogenesis and embryogenesis, the Pofut1 gene was deleted in the developing oocyte. Most surprisingly, Pofut1F/F:ZP3Cre females ovulate eggs that are fertilized, pass normally through all stages of blastogenesis, implant, and develop through gastrulation,
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demonstrating that Notch signaling through the canonical pathway is not required for oogenesis, preimplantation development, implantation, or the formation of the three germ layers [38]. However, by E9.5, embryo growth is severely retarded, and embryos develop an enlarged pericardial sac as previously observed in Pofut1−/− embryos generated from heterozygous parents [48].
20.6 Conclusions By characterizing maternal and zygotic null oocytes and embryos, roles for complex N- and O-glycans in oogenesis and embryonic development have been revealed (Fig. 20.4). Surprisingly, zygotes that lack complex and hybrid N-glycans, core 1-derived O-glycans, both these types of glycan, or O-fucose glycans are formed and develop though blastogenesis to approximately mid-gestation. Therefore, none of these glycans is essential for preimplantation development or gastrulation. However, oocyte function is impaired when oocytes lack complex and hybrid N-glycans, resulting in smaller litters of maternal and zygotic null pups. Conversely, a lack of core 1-derived O-glycans on oocyte glycoproteins leads to an increase in the number of ovulated eggs and sustained elevated fertility. Furthermore, neither complex O-glycans nor complex and hybrid N-glycans, previously implicated in sperm binding, are essential for fertilization. Finally, none of the major classes of complex or hybrid N-glycans, core 1-derived O-glycans, or O-fucose glycans is required for ovulation, fertilization, blastogenesis, the expression of several stage-specific developmental antigens, implantation, or gastrulation. However, all these classes of glycans are necessary for embryonic development beyond approximately mid-gestation.
Fig. 20.4 Summary of roles for N- and O-glycans in oogenesis, fertilization, and early embryonic development. Adapted from a diagram prepared by Dr. Shaolin Shi
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References 1. Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler ME Essentials of glycobiology, 2nd edn. Cold Spring Harbor Laboratory Press, Plainview (in press) 2. Ioffe E, Stanley P (1994) Mice lacking N-acetylglucosaminyltransferase I activity die at midgestation, revealing an essential role for complex or hybrid N-linked carbohydrates. Proc Natl Acad Sci USA 91:728 3. Metzler M, Gertz A, Sarkar M, Schachter H, Schrader JW, Marth JD (1994) Complex asparaginelinked oligosaccharides are required for morphogenic events during post-implantation development. EMBO J 13:2056 4. Ioffe E, Liu Y, Stanley P (1997) Complex N-glycans in Mgat1 null preimplantation embryos arise from maternal Mgat1 RNA. Glycobiology 7:913 5. Orban PC, Chui D, Marth JD (1992) Tissue- and site-specific DNA recombination in transgenic mice. Proc Natl Acad Sci USA 89:6861 6. Bleil JD, Wassarman PM (1980) Synthesis of zona pellucida proteins by denuded and follicleenclosed mouse oocytes during culture in vitro. Proc Natl Acad Sci USA 77:1029 7. Philpott CC, Ringuette MJ, Dean J (1987) Oocyte-specific expression and developmental regulation of ZP3, the sperm receptor of the mouse zona pellucida. Dev Biol 121:568 8. Shi S, Williams SA, Seppo A, Kurniawan H, Chen W, Ye Z, Marth JD, Stanley P (2004) Inactivation of the Mgat1 gene in oocytes impairs oogenesis, but embryos lacking complex and hybrid N-glycans develop and implant. Mol Cell Biol 24:9920 9. Dong J, Albertini DF, Nishimori K, Kumar TR, Lu N, Matzuk MM (1996) Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature 383:531 10. Pedersen T, Peters H (1968) Proposal for a classification of oocytes and follicles in the mouse ovary. J Reprod Fertil 17:555 11. Williams SA, Stanley P (2009) Oocyte-specific deletion of complex and hybrid N-glycans leads to defects in preovulatory follicle and cumulus mass development. Reproduction 137:321–331 12. Hoodbhoy T, Joshi S, Boja ES, Williams SA, Stanley P, Dean J (2005) Human sperm do not bind to rat zonae pellucidae despite the presence of four homologous glycoproteins. J Biol Chem 280:12721 13. Ju T, Brewer K, D’Souza A, Cummings RD, Canfield WM (2002) Cloning and expression of human core 1 beta1, 3-galactosyltransferase. J Biol Chem 277:178 14. Xia L, Ju T, Westmuckett A, An G, Ivanciu L, McDaniel JM, Lupu F, Cummings RD, McEver RP (2004) Defective angiogenesis and fatal embryonic hemorrhage in mice lacking core 1-derived O-glycans. J Cell Biol 164:451 15. Williams SA, Xia L, McEver R, Cummings R, Stanley P (2007) Fertilization in mouse does not require terminal galactose or N-acetylglucosamine on the zona pellucida glycans. J Cell Sci 120:1341 16. Williams SA, Stanley P (2008) Mouse fertility is enhanced by oocyte-specific loss of core 1-derived O-glycans. FASEB J 22:2273 17. Wassarman PM (2005) Contribution of mouse egg zona pellucida glycoproteins to gamete recognition during fertilization. J Cell Physiol 204:388 18. Wassarman PM, Jovine L, Qi H, Williams Z, Darie C, Litscher ES (2005) Recent aspects of mammalian fertilization research. Mol Cell Endocrinol 234:95 19. Talbot P, Shur BD, Myles DG (2003) Cell adhesion and fertilization: steps in oocyte transport, sperm-zona pellucida interactions, and sperm-egg fusion. Biol Reprod 68:1 20. Clark GF, Dell A (2006) Molecular models for murine sperm-egg binding. J Biol Chem 281:13853 21. Dell A, Chalabi S, Easton RL, Haslam SM, Sutton-Smith M, Patankar MS, Lattanzio F, Panico M, Morris HR, Clark GF (2003) Murine and human zona pellucida 3 derived from mouse eggs express identical O-glycans. Proc Natl Acad Sci USA 100:15631
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22. Fenderson BA, Eddy EM, Hakomori S (1990) Glycoconjugate expression during embryogenesis and its biological significance. Bioessays 12:173 23. Kimber SJ (1990) Glycoconjugates and cell surface interactions in pre- and peri-implantation mammalian embryonic development. Int Rev Cytol 120:53 24. Kannagi R, Cochran NA, Ishigami F, Hakomori S, Andrews PW, Knowles BB, Solter D (1983) Stage-specific embryonic antigens (SSEA-3 and -4) are epitopes of a unique globo-series ganglioside isolated from human teratocarcinoma cells. EMBO J 2:2355 25. Kimber SJ, Brown DG, Pahlsson P, Nilsson B (1993) Carbohydrate antigen expression in murine embryonic stem cells and embryos. II. Sialylated antigens and glycolipid analysis. Histochem J 25:628 26. Solter D, Knowles BB (1978) Monoclonal antibody defining a stage specific mouse embryonic antigen (SSEA-1). Proc Natl Acad Sci USA 75:5565 27. Fenderson BA, O’Brien DA, Millette CF, Eddy E (1984) Stage-specific expression of three cell surface carbohydrate antigens during murine spermatogenesis detected with monoclonal antibodies. Dev Biol 103:117 28. Brown DG, Warren VN, Pahlsson P, Kimber SJ (1993) Carbohydrate antigen expression in murine embryonic stem cells and embryos. I. Lacto and neo-lacto determinants. Histochem J 25:452 29. Bird JM, Kimber SJ (1984) Oligosaccharides containing fucose linked alpha(1-3) and alpha (1-4) to N-acetylglucosamine cause decompaction of mouse morulae. Dev Biol 104:449 30. Fenderson BA, Zehavi U, Hakomori S (1984) A multivalent lacto-N-fucopentaose III-lysyllysine conjugate decompacts preimplantation mouse embryos, while the free oligosaccharide is ineffective. J Exp Med 160:1591 31. Zhu ZM, Kojima N, Stroud MR, Hakomori SI, Fenderson BA (1995) Monoclonal antibody directed to LeY oligosaccharide inhibits implantation in the mouse. Biol Reprod 52:903 32. Wang XQ, Zhu ZM, Fenderson BA, Zeng GQ, Cao YJ, Jiang GT (1998) Effects of monoclonal antibody directed to LeY on implantation in the mouse. Mol Hum Reprod 4:295 33. Williams SA, Stanley P (2008) Complex N-glycans or core 1-derived O-glycans are not required for the expression of stage-specific antigens SSEA-1, SSEA-3, SSEA-4, or LeY in the preimplantation mouse embryo. Glycoconj J 26:335–347 34. Lai EC (2004) Notch signaling: control of cell communication and cell fate. Development 131:965 35. Schweisguth F (2004) Notch signaling activity. Curr Biol 14:R129 36. Panin VM, Shao L, Lei L, Moloney DJ, Irvine KD, Haltiwanger RS (2002) Notch ligands are substrates for protein O-fucosyltransferase-1 and Fringe. J Biol Chem 277:29945 37. Moloney DJ, Shair LH, Lu FM, Xia J, Locke R, Matta KL, Haltiwanger RS (2000) Mammalian Notch1 is modified with two unusual forms of O-linked glycosylation found on epidermal growth factor-like modules. J Biol Chem 275:9604 38. Shi S, Stanley P (2003) Protein O-fucosyltransferase 1 is an essential component of Notch signaling pathways. Proc Natl Acad Sci USA 100:5234 39. Evrard YA, Lun Y, Aulehla A, Gan L, Johnson RL (1998) Lunatic fringe is an essential mediator of somite segmentation and patterning. Nature 394:377 40. Zhang N, Gridley T (1998) Defects in somite formation in lunatic fringe-deficient mice. Nature 394:374 41. Hahn KL, Johnson J, Beres BJ, Howard S, Wilson-Rawls J (2005) Lunatic fringe null female mice are infertile due to defects in meiotic maturation. Development 132:817 42. Chen J, Moloney DJ, Stanley P (2001) Fringe modulation of Jagged1-induced Notch signaling requires the action of beta 4galactosyltransferase-1. Proc Natl Acad Sci USA 98:13716 43. Asano M, Furukawa K, Kido M, Matsumoto S, Umesaki Y, Kochibe N, Iwakura Y (1997) Growth retardation and early death of beta-1, 4-galactosyltransferase knockout mice with augmented proliferation and abnormal differentiation of epithelial cells. EMBO J 16:1850 44. Lu Q, Hasty P, Shur BD (1997) Targeted mutation in beta1,4-galactosyltransferase leads to pituitary insufficiency and neonatal lethality. Dev Biol 181:257
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45. Chen J, Lu L, Shi S, Stanley P (2006) Expression of Notch signaling pathway genes in mouse embryos lacking b4galactosyltransferase-1. Gene Expr Patterns 6:376 46. Cormier S, Vandormael-Pournin S, Babinet C, Cohen-Tannoudji M (2004) Developmental expression of the Notch signaling pathway genes during mouse preimplantation development. Gene Expr Patterns 4:713 47. Wang QT, Piotrowska K, Ciemerych MA, Milenkovic L, Scott MP, Davis RW, Zernicka-Goetz M (2004) A genome-wide study of gene activity reveals developmental signaling pathways in the preimplantation mouse embryo. Dev Cell 6:133 48. Shi S, Stahl M, Lu L, Stanley P (2005) Canonical notch signaling is dispensable for early cell fate specifications in mammals. Mol Cell Biol 25:9503 49. Zhao M, Dean J (2002) The zona pellucida in folliculogenesis, fertilization and early development. Rev Endocr Metab Disord 3:19
Chapter 21
Glycobiology in the Field of Gerontology (Glycogerontology) Akira Kobata
Keywords Alzheimer’s disease • b-Actin • b-Amyloid peptide • Cathepsin D • IgG • Lens culinaris agglutinin • Maackia amurensis lectin • N-linked sugar chain • P0 • Psathyrella velutina lectin • Rheumatoid arthritis • Sambucus sieboldiana agglutinin • Sialylation • Tau Nucleic acids, proteins, and sugar chains are three major macromolecules that are widely distributed in the bodies of multicellular organisms. Through the study of molecular biology in the latter half of the past century, the roles of nucleic acids and proteins as information molecules have been elucidated. The information codes of nucleic acids are formed by the linear arrangement of nucleotides, while those of proteins are constructed not only by the linear arrangement of amino acids but also by the three-dimensional arrangement of amino acids, which are mutually remote in the thread of a polypeptide chain. The structures of sugar chains are formed only by three-dimensional arrangements of monosaccharides that include branching also. Through the application of many techniques devised in the field of molecular biology, various phenomena in living organisms have been clarified on a molecular basis, and revolutionary developments have started to arise in the field of medical treatment through the elucidation of the human genome. The main purpose of gerontology is to understand the mechanisms of aging in living organisms and thus improve quality of life for elderly people. Therefore, the development of molecular biology is expected to highly contribute to the field of gerontology. Actually, many important genes related to the aging processes of various organs have been found and are expected to be useful in the future development of geriatric medicine. For example, illumination of the longevity gene of Caenorhabditis elegans, followed by the finding that its structure is similar to that of the human insulin receptor gene, is expected to become an important clue to understanding the biochemical basis of an interesting and well-established A. Kobata (*) The Noguchi Institute, 1-8-1 Kaga, Itabashi-ku, Tokyo 173-0003, Japan e-mail:
[email protected] A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_21, © Springer Science+Business Media, LLC 2011
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phenomenon that the life spans of animals are extended significantly by restricting food intake [1]. Finding the various genes involved in Alzheimer’s disease – as discussed in more detail in the last section of this review – will be useful in developing effective therapies for this disgusting disease, which is one of the most important targets in geriatric medicine [2–4]. Finding CBFA-1, a gene related to bone formation, may open a way to taking preventive measures against osteoporosis, which seriously affects many aged women [5]. As discussed so far, the contribution of molecular biology to the development of geriatric medicine is already enormous. However, many proteins produced by the human body contain covalently linked sugar chains and are known as glycoproteins [6]. The sugar chains of various glycoproteins are not simply decorations, but play important roles as biosignals in multicellular organisms. Accumulated data, obtained through studies of the sugar chains of glycoconjugates, indicate that revealing the information included in the sugar chains together with that in nucleic acids and proteins is essential for the future development of biology. Based on such a trend in the bioscience world, a new life-science field called glycobiology was launched in the mid-1980s [7]. Since the information networks constructed by nucleic acids and proteins are very strict and are essential for maintaining the functions of living organisms, most of the defects induced in these biomolecules will be fatal to living organisms. On the contrary, the biosynthesis of sugar chains is performed by the concerted action of glycosyltransferases, which are coded by their respective structural genes [8]. Since no template is included in this biosynthetic machinery, the structures of the sugar chains are not formed as strictly as those of proteins. Accordingly, much of the information included in the sugar chains is not essential for maintaining life itself, but is necessary for maintaining the ordered social life of cells to construct multicellular organisms. Hence, investigating the structural changes of sugar chains, which are drawn by aging, is expected to afford very useful information about diseases induced by aging. However, the number of papers reporting studies of glycoconjugates in relation to the aging of animals is still quite limited. In this review article, I would like to introduce the importance of glycobiology research in the field of gerontology, mainly based on the data of N-linked sugar chains obtained by our own research and by studies performed by Tamao Endo’s group in the Tokyo Metropolitan Institute of Gerontology.
21.1 Changes in the Sugar Chain Structures of Immunoglobulin G Through Aging and Their Physiological Significance The first reliable data of altered N-glycosylation of glycoproteins through aging was obtained by the studies of the N-linked sugar chains of human serum immunoglobulin G (IgG). IgG is a glycoprotein composed of two types of polypeptide chains called H and L chains, with a stoichiometry of H2L2. Of the approximately
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2.8 mol of the N-linked sugar chains detected in one molecule of IgG samples p urified from human sera, 2 mol is linked to the Asn297 residues of the two H chains, and the remainder is linked to the potential N-glycosylation sites in the variable regions of both the H and L chains [9]. Establishment of hydrazinolysis opened a way for obtaining the complete oligosaccharide pattern of a glycoprotein [10]. A structural study of whole sugar chains of IgG samples, purified from sera of healthy individuals, revealed that several unique characteristics are included in them [11]. Only 24% of the sugar chains are sialylated. This is very unusual because most of the N-linked sugar chains of other serum glycoproteins are sialylated. Another characteristic feature of the sugar chains of IgG is the occurrence of extremely high microheterogeneity. The largest neutral portion of the N-linked sugar chains, isolated from serum IgG by hydrazinolysis followed by reduction with NaB3H4, is shown in Fig. 21.1. The microheterogeneity is produced by the presence or absence of the two galactose residues, the bisecting GlcNAc, and the fucose residue as underlined in the figure. The addition of sialic acid residues to these neutral sugar chains would produce more than 30 different sugar chains. Despite this extremely high multiplicity, the molar ratio of each oligosaccharide included in the IgG samples obtained from the sera of healthy individuals is quite constant. Therefore, the desialylated oligosaccharide fractions obtained from IgG samples always gave the fractionation pattern shown in Fig. 21.2a upon Bio-Gel P-4 column chromatography. While investigating the sugar chains of many IgG samples, we met with an IgG sample, the desialylated oligosaccharide fraction that gave the fractionation pattern as shown in Fig. 21.2b. The search for the physiological background of the donor of the serum revealed that he was a patient with rheumatoid arthritis (RA). Further investigation of the desialylated sugar chains of IgG samples, obtained from the sera of many patients with RA, revealed that all of them gave more or less similar abnormal elution patterns enriched in the oligosaccharides with smaller effective sizes. Structural studies of the oligosaccharides from these samples revealed that the IgG samples from patients with RA are prominently devoid of galactose residues [12]. No difference in the ratio of bisected oligosaccharides and fucosylated oligosaccharides to total oligosaccharides was detected as compared to IgG samples obtained from healthy individuals. Although many sugar chains of human IgG contain galactose residues at their nonreducing termini, the galactose residues could not be removed by digestion with jack bean or diplococcal b-galactosidase. By investigating b-galactosidases from
Fig. 21.1 The largest desialylated N-linked sugar chain obtained from human serum IgG by hydrazinolysis followed by NaB3H4 reduction. GlcNAcOT represents [1-3H]N-acetylglucosaminitol
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Fig. 21.2 Elution profiles from a Bio-Gel P-4 column of the desialylated radioactive oligosaccharide fraction obtained from IgG samples from the sera of a healthy individual (a) and a patient with RA (b). Arrows at the top of the figure indicate the elution positions of glucose oligomers used as internal standards, and the numbers indicate the glucose units. Cited in an altered form from Glycobiology (1990) 1:5–8
other sources, we found that the enzyme from Streptococcus 6646K can completely remove the galactose residues from desialylated human IgG [13]. This opened the way for comparing the function of this glycoprotein before and after removal of its galactose residues. Binding to C1q was significantly impaired in agalacto IgG compared with the control IgG (Fig. 21.3a) [13]. To estimate the effect of reduced C1q binding in actual immunological reactions, the complement activation of agalacto IgG and control IgG were comparatively investigated. Each IgG was heat aggregated, added to normal human serum at varying concentrations, and the degree of the complement consumption was assayed by determining the hemolytic activity to sheep erythrocytes. As shown in Fig. 21.3b, complement consumption was decreased significantly when the serum was incubated with heat-aggregated agalacto IgG at a concentration of 180 mg/ml, indicating that complement activation was reduced by degalactosylation [13]. Rheumatoid factor (RF) is an autoantibody directed at denatured IgG. No difference between the binding capacities of control and agalacto IgG to solid-phase IgM-RF was observed, although the antigenic determinants for IgM-RF are considered to reside in the CH2 and CH3 domains of the IgG molecule (data not shown). We finally investigated the binding affinity of the two IgG samples to protein A,
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Fig. 21.3 (a): Binding of 125I-control IgG (open circle) and 125I-agalacto IgG (closed circle) to C1q. Each IgG solution at the indicated concentration was applied to microtiter plates coated with C1q. After incubation for 5 h, the wells were cut out, and the radioactivity in the well was counted. The counts per minute were divided by the specific activity of each IgG, and the results are expressed as ng of IgG bound to each well. (b): Complement activation by heat-aggregated IgG. Control (open circle) or agalacto (closed circle) IgG was heat-aggregated and mixed with the normal human serum at the indicated final concentration. Untreated sample (0 mg/ml) was prepared by adding the same volume of phophate-buffered saline instead of aggregated IgG. After incubation for 1 h at 37°C, 50% complement dependent hemolytic activity of each sample was measured using sheep erythrocytes coated with IgM hemolysin. The experiment was repeated twice with similar results. (c): Binding of 125I-control IgG (open circle) and 125I-agalacto IgG (closed circle) to U937 cells. U937 cells (1 × 106 cells) were incubated with varying concentrations of each IgG at 0°C for 3 h. To determine nonspecific binding, 100-fold excess of unlabeled ligand was preincubated at each concentration of the labeled ligand. The amounts of bound ligands, total minus nonspecific binding, are expressed as ng IgG bound/106 cells. The experiment was repeated twice with similar results. Revised from Ref. [13]
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since the protein is also known to react with the CH2 and CH3 domains of IgG. No difference was detected between the binding capacities of the control and agalacto IgG (data not shown) [13]. U937, a human macrophage cell line, has been known to possess Fc receptors for human IgG [14]. One of these Fc receptors (FcRI) binds monomeric IgG more efficiently than aggregated IgG [15]. We investigated the effect of degalactosylation on the affinity of human IgG for the Fc receptors on the surface of U937 cells by using radiolabeled IgG samples. As shown in Fig. 21.3c, significant reduction in the binding of agalacto IgG was observed as compared with control IgG [13]. Although the structural analysis described so far affords the most accurate determination of the galactose content of IgG, it involves a series of sophisticated but tedious procedures. For further clinical studies, including a family study of the interesting phenomenon, a more simple analytical method was required. At the beginning, we expected that an immobilized RCA-I column might be useful for such purposes because the column would retain the glycoproteins with galactosylated sugar chains and pass through those with nongalactosylated sugar chains. However, all serum IgG molecules passed through the column without any interaction, although many of them definitely contained galactosylated sugar chains. By investigating the crystal of the Fc portion obtained from rabbit IgG by X-ray analysis, Sutton and Phillips found that the galactose residues of its sugar chains are buried within the pocket of its polypeptide portion [16]. Probably because of this situation, RCA-I cannot interact with the galactose residues of the Fc portion. This situation also explains the already described phenomenon that the galactose residues of IgG are hard to remove by digestion with various b-galactosidases. We considered that if the terminal galactose residue of IgG is hard to detect by an immobilized RCA-I column, the N-acetylglucosamine residues in the nongalactosylated IgG could be targets in developing an assay method using a lectin column. Among several GlcNAc-binding lectins, we chose the Psathyrella velutina lectin (PVL) isolated from the fungus by Kochibe and Matta [17]. An immobilized PVL column interacts preferentially with the sugar chains containing the GlcNAcb1-2Man group. Therefore, nongalactosylated biantennary complex-type sugar chains bound to the column and eluted with the buffer containing 1 mM N-acetylglucosamine, while the digalactosylated biantennary sugar chains passed through the column without interaction. Monogalactosylated biantennary complex-type sugar chains were slightly retarded but did not bind to the column [17, 18]. Furthermore, biotinylated PVL was found to react strongly in Western blotting with IgG samples from patients with RA. Based on this preliminary evidence, an enzyme-linked immunosorbent assay (ELISA) was developed for the detection of agalacto IgG [19]. As shown in Fig. 21.4, sera from patients with RA showed significantly higher PVL binding than those from healthy individuals. By using the PVL binding assay, we investigated the values of IgG in the sera of 112 healthy individuals and classified them by age. An interesting observation is that the values increase in aged males and females, confirming the data obtained by using a conventional method [20]. Hence, the alteration induced in the sugar chains of IgG can partly explain the phenomenon of immunodeficiency observed in aged individuals.
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Fig. 21.4 PVL binding of sera IgGs of patients with RA and healthy individuals [19]
IgG mediates anti-inflammatory activities as well as proinflammatory activities by the interaction of its Fc fragment with distinct Fcg receptors. Kanemoto et al. have recently reported that these distinct properties of IgG result from different sialylations of its N-linked sugar chains of the Fc core. It was found that sialylation affords IgG anti-inflammatory properties. It was also found that the extent of sialylation is reduced in the IgG molecule by the induction of an antigen-specific immune response [21]. Accordingly, reduction of galactosylation in the circulating IgG in aged individuals may lead to reduced anti-inflammatory activities.
21.2 Alteration of the N-linked Sugar Chains of the Glycoproteins in the Brain Through Aging To find out how aging alters the N-linked sugar chains of glycoproteins in the brain, Sato et al. comparatively analyzed the glycoproteins of various portions of the brain and spinal cord, which were obtained from 9-week-old and 29-months-old Fisher rats [22]. The tissues were homogenized and separated into soluble fraction and membrane fraction by centrifugation at 100,000 × g. The two fractions from each tissue were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then, the proteins were transferred on a polyvinylidene difluoride (PVDF) membrane,
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and the glycoproteins on the membrane were reacted with biotin-conjugated lectins, which were selected to detect representative groups of N-linked sugar chains. The lectins bound to the glycoproteins were reacted with avidin-conjugated horseradish peroxidase. Detection of the bound peroxidase, by using 3,3¢-diamino-benzamide tetrahydrochloride as a substrate, revealed that many age-related alterations occur in the glycoproteins of various portions of the central nervous system [22]. The most prominent alteration was found by using Lens culinaris agglutinin (LCA). Several age-related alterations were detected in various portions of the brain, but the most prominent difference was detected in the spinal cord. A band, which moved to the front of the gel, was detected in the membrane preparation of spinal cord obtained from aged rats, but not in that from young adult rats. The exact molecular weight of the LCA-positive glycoprotein, as measured by SDS-PAGE using a higher concentration of gel, was 30 kDa. Accordingly, this glycoprotein was named gp30 [23]. By investigating a large number of rats, it was confirmed that gp30 is detected in the membrane fractions of the spinal cord from all the aged rats, but not from the young adult rats. To determine the structure of gp30, it was fractionated by reverse-phase highperformance liquid chromatography (HPLC) after digestion with lysyl endopeptidase. The line in Fig. 21.5 shows the elution pattern of the peptides detected by
Fig. 21.5 Detection of a glycopeptide fragment from gp30. The peptide mixture obtained from gp30 by digestion with lysyl endopeptidase was fractionated by HPLC using a Wacopak column. A solid line indicates the elution profile of peptides detected by absorbance at 215 nm. A dotted line indicates the concentration of acetonitrile. Gray bars indicate the reactivity of each fraction with biotin-conjugated LCA. Bound lectin was detected by ELISA using avidin-conjugated horseradish peroxidase and O-phenylenediamine dihydrochloride. The color was measured by absorbance at 492 nm [23]
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absorption at 215 nm. The gray bars indicate the reactivity of each fraction with LCA. A glycopeptide fraction, which eluted at 35 min and strongly reacted with LCA, was collected and its amino-acid sequence was analyzed. The N-terminal amino acid could not be identified, but the sequence of GSIVIHNLD was obtained after the second amino acid of the N-terminal position of the glycopeptide. A database search of the amino-acid sequence revealed that it was completely identical to the 110(G)-118(D) of rat P0 [24]. As rat P0 contains a single N-glycosylation site at Asn122, recovery of 109(D)-130(K) was expected as the glycosylated product of lysyl endopeptidase digestion. Based on this reasoning, gp30 was identified as P0. P0 is a member of an immunoglobulin superfamily and has been known as a major structural component of mammalian peripheral nerve myelin. Targeted disruption of the structural gene of P0 in mice induced hypomyelination, abnormal expression of recognition molecules, and degeneration of myelin and axons [25]. Furthermore, it was reported that several mutations in the gene of human P0 cause genetic neural disorders [26, 27]. Therefore, a crucial role of P0 in the function of the peripheral nerve was expected [28]. As it had long been believed that P0 is not present in the central nervous system [29–31], detection of P0 in the rat spinal cord became a target of dispute. One possible explanation was that a search for P0 had never been performed in the brain nervous system of aged rats. For the absence of P0 in the spinal cord of young adult rats, two possibilities were considered: One is that the sugar chains of young adult rat P0 cannot interact with LCA because their structures are different from those of aged rats’ P0. The other possibility is that the P0 molecule is expressed only in the spinal cord of aged animals. To find out which possibility is the correct one, a polyclonal antibody that specifically recognizes the C-terminal sequence of rat P0 was prepared. Western blot analyses of the membrane proteins of spinal cords and sciatic nerves revealed that P0 was detected in the spinal cords as well as the sciatic nerves of both aged and young adult rats. Therefore, P0 does exist in the spinal cord membrane of young adult rats [23]. A glycoprotein detection kit (Amersham Pharmacia Biotech Co.) facilitates the detection of glycoproteins on a PVDF membrane. It was revealed by using this kit that the P0 in the spinal cord and sciatic nerve of aged rats contain sugar chains, while the P0 of the spinal cord of young adult rats does not [23]. Therefore, the absence of sugar chains in the P0 in the spinal cords of young adult rats caused its negative reaction with LCA. A cell line that shows strong homophilic adhesion was obtained by P0 complementary DNA transfection into C6 glioma cells. Addition of glycopeptide fragment (residues 91–95) obtained from P0 strongly inhibited the homophilic adhesion of this cell. In contrast, the corresponding peptide without sugar chain showed much lower inhibition [32], indicating that the sugar moiety of P0 plays a very important role in cell-to-cell adhesion by homophilic binding. It was also found that a nonglycosylated P0, obtained by site-directed mutagenesis, did not show homophilic adhesion [33]. Therefore, the biological meaning of the occurrence of nonglycosylated P0 in the spinal cord of young adult rats is an interesting target for future study. Elucidation of the mechanism that starts the glycosylation of spinal cord P0 through aging is also
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an interesting target for future study. In view of the presence of other glycoproteins containing N-linked sugar chains in the spinal cords of young adult rats, the mechanism should be working only for P0 or for some limited proteins including P0. In any event, clarification of the background of this interesting phenomenon will surely provide useful data not only for finding out the functional roles of the sugar chains but also for understanding the molecular events that occur during aging.
21.3 Altered Sialylation of Glycoconjugates in Various Portions of the Brain Through Aging Among various lectins, Maackia amurensis lectin (MAL) and Sambucus sieboldiana agglutinin (SSA) specifically bind to the Siaa2-3Gal group and the Siaa2-6Gal group, respectively. Accordingly, a histochemical method using MAL and SSA is useful for investigating the distribution of glycoconjugates containing the Siaa23Gal group and the Siaa2-6Gal group in various tissues. By using this technique, Sasaki et al. investigated the distribution of sialoglycoconjugates in the cerebellum of 9-week-old rats and 30-month-old rats [34]. It was found that MAL strongly stained the granular layer but weakly stained the molecular layer and the medullary lamina, while SSA stained more strongly the medullary lamina than the molecular and granular layers in young adult rats. In contrast, intense SSA staining of the granular layer and intense MAL staining of the medullary lamina were observed in the cerebellum from the aged rats. These results indicate that the Siaa2-3Gal group and the Siaa2-6Gal group were expressed in distinct regions of rat cerebellum, and their expression patterns changed in the aged rats. By using the same technique, Sato et al. analyzed the distribution of sialoglycoconjugates in the hippocampus from young adult rats and aged rats [35]. It was found that both the Siaa2-3Gal group and the Siaa2-6Gal group were expressed in the plasma membranes of pyramidal cells and the synapses in the stratum lacunosum, and the expression of these groups decreased in the aged hippocampus. The age-related alterations of the hippocampal synaptosome proteins were further investigated by using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) [36]. Approximately 1,000 protein spots were detected in the samples from the two groups. Among them, at least 24 protein spots were expressed differently in the hippocampal synaptosomes of young adult rats and aged rats. Nineteen of these 24 spots were identified by peptide mass fingerprinting. These proteins included various synaptic components such as chaperone proteins and proteins related to cytoskeleton, neurotransmission, signal transduction, and energy supply. The cytoskeleton-related proteins included b-actin and T-complex 1, which is thought to play a role in b-actin folding. b-Actin was upregulated, but T-complex 1 was downregulated in the synapses of aged rat. Among these changes, altered expression of b-actin and its related proteins through aging is of particular interest because b-actin is a major cytoskeletal protein composed of actin filaments that are abundant in synaptic areas, such as presynaptic nerve endings and postsynaptic dendrites. In these areas, b-actin and its
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related proteins form a submembranous cytoskeleton and are involved in neurite growth, cell adhesion, synapse formation, and exocytosis of neurotransmitters. Therefore, age-related alterations in b-actin and b-actin-associated molecules may play a role in the neuronal dysfunction of aging.
21.4 Increase of Cytosolic Cathepsin D Through Aging Most glycoproteins are found in the membrane fractions, but not in the cytoplasm. However, as described in the next section, a cytosolic protein tau, isolated from the brain of a person with Alzheimer’s disease (AD), was found to be N-glycosylated [37]. By analyzing the proteins in the cytosolic fraction of rat cerebral cortex by 2D-PAGE followed by Concanavalin A (ConA) staining, Sato et al. found that several spots increase in the aged rats [38]. To elucidate the molecular basis of this interesting phenomenon, cytosolic glycoproteins were collected by using a ConA–agarose column. Analysis of the ConA-bound fraction by 2D-PAGE followed by ConA blotting revealed that several spots were detected in the samples of both aged and young adult rats. At least 14 glycoproteins were enriched in the samples from aged rats compared to those from young adult rats. Among them, a glycoprotein that is most prominently increased in the samples from aged rats was identified as cathepsin D, by combination of tryptic digestion and nano liquid chromatography electrospray ionization quadrupole time of flight mass spectrometric analysis [38]. Analysis by 1D-PAGE, followed by staining with anti-rat cathepsin D antibody, revealed that cathepsin D was detected in the microsomal fractions of the cerebral cortex of both aged and young adult rats. In contrast, the glycoprotein was also detected in the cytosolic fractions of aged rats, but not in those of young adult rats. That all cathepsin D spots detected so far contain N-linked sugar chains was confirmed by their molecular shift to smaller sizes by digestion with Endo H. That the increase of cytosolic cathepsin D was not due to disruption of lysosomal membranes was confirmed by the analysis of lysosomal enzymes in the cytosol by age. It was confirmed that the amount of cathepsin D in the cytosolic fraction of rat cerebral cortex increases linearly through aging and reaches, at the age of 34 months, a level 13 times that of the level detected at 2 months of age. No change in the amount of microsomal cathepsin D was observed through aging. In contrast to cathepsin D, no significant increase in the cytosolic fraction was observed in other lysosomal enzymes, such as b-glucuronidase and b-N-acetylhexosaminidase [38]. The level of cathepsin D transcripts in the aged rats was approximately 1.6 times higher than that in the young adult rats, indicating that the increase of cathepsin D messenger RNA caused the increase of this glycoprotein through aging. The cytosolic cathepsin D also increased through aging in the cerebellum, hippocampus, spleen, liver, and kidney, but not in the lung. These results indicated that cathepsin D might play a role as a new aging marker. Histochemical analysis of rat brain by using anti-rat cathepsin D antibody revealed that the glycoprotein was distributed mainly in the neuronal cells and
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increased through aging. The increase of cytosolic cathepsin D through aging was also observed in mouse cerebral cortex, although processing of the single chain of cathepsin D into a light and a heavy chain was observed as reported already [39, 40]. Therefore, this interesting phenomenon could widely occur in various mammals. It has already been found by several laboratories that the activity of cathepsin D in the brain is altered through aging [41–43]. Cathepsin D is known to digest neurofilaments and tau [44, 45], which are components of the cytoskeleton of neuronal cells. Therefore, cytosolic cathepsin D may gradually digest cytoskeletal components in the neurons and may finally induce morphological changes and functional loss of neurons in the aged brain [46].
21.5 Future Prospects Dementia is one of the most important targets of aging research. Through the microhistological study of the brains of patients with AD, the occurrence of extracellular senile plaques and intracellular neurofibrillary tangles, together with extensive loss of neural cells, was found to be highly correlated with this disease [47]. It was found that b-amyloid peptide (Ab) is a major component of senile plaques [48]. Structural studies of Ab and subsequent cloning of the gene, which includes the code of the amino-acid sequence of Ab, revealed that the sequence is included in a membrane glycoprotein called amyloid precursor protein (APP), the structure of which is schematically shown in Fig. 21.6 [49]. Since a large amount of APP is produced in the brains of healthy individuals at almost the same level as it is in the brains of patients with AD, elucidating the mechanism for inducing an abnormal cleavage of APP, which leads to the production of Ab, was considered key for this line of study. Although AD is predominantly a disease of late life, there are families in which AD is inherited as an autosomal dominant disorder of midlife [50]. By searching the genes of familial AD, presenilin 1 gene and presenilin 2 gene were picked up, in addition to the gene of APP, as causative genes of early onset AD [51–53]. Both presenilins are
Fig. 21.6 The structure of APP buried within the plasma membrane. CHOs represent N-linked sugar chains. b-Site and g-site to produce Ab are shown by arrows
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multi-transmembrane-spanning proteins [54]. Currently, it is proposed as a scenario that APP is first cleaved at its b-site by b-secretase [55–58], forms a complex with presenilins, and is cleaved at the g-site by the protease postulated to reside in presenilins [58]. The g-site cleavage generates a variety of Ab species: predominantly a 40-amino-acid peptide, Ab1-40, with a smaller amount of a 42-amino-acid peptide Ab1–42. The latter peptide is more prone to form amyloid deposits. Mutations in all three genes, which were detected in studies of familial AD, alter the processing of APP to produce more Ab1–42. Two potential N-glycosylation sites were detected in the extracellular portion of APP, which is close to the N-terminal portion of the Ab fragment, as shown in Fig. 21.6. Study of the sugar chains of recombinant human APP produced by Chinese hamster ovary cells revealed that sialylated bi- and triantennary N-linked sugar chains with fucosylated and nonfucosylated trimannosyl core and sialylated O-linked sugar chains with core I structure are included in this glycoprotein [59]. In view of P0, studies of age-related alterations of the structures of the APP sugar chains may introduce another new aspect of producing Ab1–42 from this glycoprotein. As b- and g-secretases play critical roles in Ab production, inhibitors of these proteases were expected to be effective drugs for the treatment of AD [60, 61]. However, Kitazume et al. found that one of the major b-secretases, named BACE1, also acts on the Golgi-resident sialyltransferase, ST6Gal1, which forms the Siaa26Gal group in glycoconjugates and secretes enzymes out of cells [62]. Accordingly, BACE1 deficiency may cause abnormality in a2,6-sialylation of glycoconjugates and induce various side effects. Actually, mice deficient in the polysialyltransferase show impaired long-term potentiation and long-term depression of hippocampal neurons with increasing age [63]. Furthermore, BACE1 could be responsible for the secretion of other glycosyltransferases. Therefore, treatment of AD by inhibitors of secretases might induce unexpected side effects. It has recently been found that enhanced a2,6-sialylation of APP by transgenic overexpression of ST6Gal1, together with the APP gene in various cell lines, increased the extracellular level of Ab. Furthermore, a much lower level of Ab was produced by Lec-2 cells, a sialylation-deficient mutant of CHO cells, than by wild-type cells by the same transgenic treatment. Accordingly, production of Ab from APP is enhanced by a2,6-sialylation. Secretion of Ab was not enhanced upon ST6Gal1 overexpression, when the mutant APP gene, in which Asn residues of the two potential N-glycosylation sites were replaced by Ala, was cotransfected in the cell lines [64]. The major fibrous components of neurofibrillary tangles are paired helical filaments (PHFs). PHFs are composed of a microtubule-associated protein, called tau, in an abnormally hyperphosphorylated state [65, 66]. The majority of the PHF-tau polymerization property is induced by the abnormal phosphorylation at serine 262 in the human protein [66]. To find out that hyperphosphorylation of tau is actually the main cause of AD, tau kinase was thoroughly investigated [67]. However, the enhancement of tau kinase was not positively correlated with AD. Gerry Hart’s group found that the tau in the brains of normal animals is extensively modified by O-N-acetyl-glucosaminylation (O-GlcNAc) instead of phosphorylation [68]. Therefore, it is possible that the hyperphosphorylation of PHF-tau is the direct result of defective regulation resulting from either increased activity of cytosolic b-Nacetylglucosaminidase [69] or decreased activity of O-GlcNAc transferase [70].
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It was found that tau occurs in normal neurons (normal tau). In a brain affected by AD, however, there is a cytosolic pool of abnormally hyperphosphorylated tau (AD P-tau) as amorphous aggregates, in addition to tau in PHF (PHF-tau) [71]. No such cytosolic tau was detected in normal neurons. Wang et al. [72] found that PHF-tau and AD P-tau were stained by lectins, while normal tau was not. Comparative analysis of the sugar chains of PHF-tau and AD P-tau revealed that they have different N-glycosylation patterns. High mannose-type and complex-type sugar chains were found in both tau samples. More truncated N-glycans were detected in PHF-tau than in AD P-tau (Table 21.1), indicating that AD P-tau might be a precursor form of PHF-tau [37]. Table 21.1 Proposed structures and their percent molar ratio of N-linked sugar chains of tau samples [37] Structures AD P-tau PHF-tau 5.9 –
5.7
–
10.7
–
15.1
16.6
26.4
18.0
–
13.2
–
15.9
–
20.8
18.0
14.0
6.9
0.4
1.9
1.1
0.9
–
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The data described in this section are just a beginning in the glycobiology of the glycoproteins related to AD. As already discussed, sugar chains can be altered by the physiological conditions of cells. Accordingly age-related alteration of various glycoconjugates is likely to be an important target for solving various pathological problems found in aged individuals. Therefore, gerontology would be a gold mine for glycobiological studies.
21.6 Summary The main purpose of gerontology is to elucidate the mechanisms of age-related deterioration occurring in various parts of the human body and to use this knowledge to improve quality of life for the elderly. In the genomic age, a big development has begun in the field of gerontology by investigating the various genes related to the aging processes of various organs. However, most proteins produced by animal bodies contain sugar chains. The recent development of glycobiology indicated that these sugar chains play roles as various kinds of biosignals within multicellular organisms. As sugar chains are formed as secondary gene products by the concerted actions of glycosyltransferases, the structures of sugar chains are less strictly regulated than those of proteins. Therefore, most biosignals associated with sugar chains are not essential for the maintenance of life itself but are necessary for maintaining the ordered social life of cells constructing multicellular organisms. Therefore, investigating the structural changes of sugar chains that are induced by aging is expected to afford very useful information for elucidating the disorders of the elderly. This review summarizes our current knowledge of the changes detected in the sugar chains of glycoconjugates through the aging process.
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Chapter 22
Galectins in Regulation of Apoptosis Fu-Tong Liu, Ri-Yao Yang, Jun Saegusa, Huan-Yuan Chen, and Daniel K. Hsu
Keywords Galectins • Apoptosis • T cells • Glycans • Signal transduction
22.1 Introduction The term “galectins” was given in 1994 to a family of animal lectins that bind to b-galactosides and contain comparable sequences of amino acids in their genes and proteins [1]. At that time, there were just four identified members, but now 15 members have been described in mammals, with similar proteins found in various animal species, as well as in insects and plants. All galectins have, as part of their structure, regions of about 130 amino acids that are responsible for carbohydrate binding; these areas are called carbohydraterecognition domains (CRDs). Some galectins have one CRD (galectin-1, -2, -3, -5, -7, -10, -11, -13, -14, and -15), while others contain two CRDs in a single polypeptide chain; these are separated by a “linker” area of up to 70 amino acids (galectin-4, -6, -8, -9, and -12). Galectin-3 is the only member of this family that contains a long N-terminal region (about 120 amino acids) connected to a CRD. A great deal of progress has been made in understanding this multifaceted protein family, including structure, expression, and functions. This review focuses on one of the most extensively studied functions of the family members: regulation of programmed cell death (apoptosis).
Fu-Tong Liu () Department of Dermatology, University of California, 3301 C Street, Sacramento, CA 95816, USA e-mail:
[email protected] A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_22, © Springer Science+Business Media, LLC 2011
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22.2 General Characteristics of Galectins Many members of this family have a wide tissue distribution, while others are found only in specific tissues. While different members will bind the same carbohydrate, galactose, each may bind to specific oligosaccharides [2]. None of the family members contains a classical signal sequence, but they are secreted (although the secretary pathway is not well understood) [3]. Galectins, in fact, possess other features that are characteristic of intracellular proteins, and consistent with this, they are found in the cytosol, as well as in the nucleus, and are associated with intracellular structures (reviewed in [4]).
22.2.1 Oligovalency and Galectin Lattice Some galectins with one CRD have been shown to exist as dimers (where two galectin proteins function together). Those with two CRDs are capable of binding two carbohydrate molecules and can be described as being bivalent. Galectin-3 forms pentamers in the presence of multivalent carbohydrate ligands [5]. The concept of a galectin lattice – meaning the meshwork formed by a bivalent or oligovalent galectin and glycoproteins with multiple carbohydrate side chains recognizable by the galectin – is getting increased attention. Dennis’s group demonstrated the formation of lattices between galectin-3 and cell surface glycoproteins, in particular, glycoproteins modified by the N-acetylglucosaminyltransferase, Mgat5. One example of such a glycoprotein is the T-cell receptor (TCR). They showed that such lattice formation restricted TCR motility and reduced the T-cell response [6]. This group subsequently showed that other glycoproteins, such as epidermal growth factor receptor and transforming growth factor-b receptor, combine with galectin-3 on the surfaces of cells, resulting in the lack of movement of receptors into the cell, termed endocytosis [7]. Similarly, another study by Ohtsubo et al. [8] showed that a glucose transporter forms aggregates with galectin-9, resulting in reduced endocytosis of this transporter. In the absence of appropriate glycosylation, these receptors form weaker complexes with galectins and are more readily internalized.
22.2.2 Extracellular Functions The current view is that the secreted galectins can act either on the cell that secreted it or on neighboring cells (i.e., in an autocrine or paracrine manner) by binding to and cross-linking selected glycoproteins or glycolipids present on the cell surfaces to form a lattice. Unusually, galectins do not appear to have specific individual
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Fig. 22.1 Many galectins are either bivalent or multivalent with regard to their carbohydratebinding activities. They can cross-link cell surface glycoconjugates and trigger transmembrane signaling events, leading to responses such as apoptosis
receptors; rather, each galectin binds to a number of different glycoproteins (and glycolipids) that are on the cell surface and carry suitable galactose-containing oligosaccharides. As such, galectins can be regarded as pattern-recognition molecules, with different galectins recognizing distinct but similar patterns. Because they are capable of binding two or more carbohydrate molecules (i.e., bivalency or oligovalency, as mentioned above), galectins are able to cross-link cell surface glycans [9], which can result in the initiation of cascade signaling events across the cell membrane (Fig. 22.1).
22.2.3 Intracellular Functions A large number of studies have demonstrated that galectins can function inside the cell and, interestingly, in a fashion that is independent of their carbohydrate-binding activities. Moreover, inside the cell, galectins can translocate and combine with the membranes of subcellular structures. Evidence is accumulating that galectins regulate cellular functions by binding to intracellular ligands and participating in intracellular signaling pathways (reviewed in [4]). Thus, galectins have dual extracellular and intracellular functions and may regulate some cellular processes through both pathways (Fig. 22.2). Whether galectins are able to bind intracellular glycans is an intriguing question. An interesting recent finding showed that galectin-3 is localized in vesicles released from an intracellular organelle (the Golgi apparatus), which contain glycoproteins
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Fig. 22.2 Galectins do not have specific receptors but bind to a group of cell surface glycoproteins containing suitable oligosaccharides. Galectins delivered exogenously can exert effects on cells by binding to these glycoproteins. Endogenous galectins can also regulate these cellular responses through intracellular actions
destined to be exported out of the cell on the apical side [10]. Inactivating galectin-3 with small interfering RNA (which reduces the levels of galectin-3 messenger RNA) resulted in the apical glycoproteins being misdirected to the basolateral cell surfaces. These observations suggest that galectin-3 may help in controlling the expression of selected cell surface glycoproteins.
22.3 Galectin-1 22.3.1 In Vitro Studies Many studies have demonstrated that galectin-1 induces apoptosis in activated human T cells and T-cell leukemia cell lines, and this topic has been comprehensively reviewed [11,12]. Most recently, Toscano et al. [13] showed that galectin-1 binds preferentially to specific T cells, namely Th1 and Th17, over Th2 cells, and induces apoptosis in Th1 and Th17 cells, but not in Th2 cells. In addition, splenocytes from genetically modified mice that lack the gene for galectin-1 (galectin-1deficient mice) produce more of the chemicals, interferon-g (IFN-g, a Th1 cytokine) and interleukin-17 (IL-17) [13], which is consistent with Th1 and Th17 cells being more sensitive to apoptosis induced by galectin-1. Galectin-1 induces apoptosis of cells by binding to selected cell surface glycoproteins and triggering signaling pathways within the cell that result in apoptosis (reviewed in [12]). These signaling pathways have been investigated. Some studies showed that galectin-1 does not induce cytochrome c release from mitochondria and caspase activation, which are common features associated with apoptosis [14]. Another study found that galectin-1 induced the exposure of phosphatidylserine on the cell surface of a T-cell line, MOLT-4, which is an early event of apoptosis, but there was no detectable DNA fragmentation, which is also commonly associated
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with apoptosis [15]. Therefore, it appears that, depending on the type of cells and culture conditions, galectin-1 may lead to a different end point in the apoptotic pathways and may or may not cause apoptosis. Most studies on the apoptosis-inducing functions of galectin-1 were performed by the addition of synthesized (recombinant) protein, although native galectin-1 is also capable of inducing apoptosis. It has been shown that galectin-1 secreted and presented by thymic epithelial cells is effective in inducing apoptosis in T-cell lines that are cultured together with the former cells [16]. Also, dendritic cells genetically modified to overexpress galectin-1 induce apoptosis when cocultured with activated T cells, and galectin-1 is released by these modified dendritic cells and binds to T cells. In addition, by using an antisense strategy (whereby antisense RNA is added to a cell where it binds to a specific messenger RNA molecule and inactivates it), it was demonstrated that galectin-1 secreted by mouse melanoma cells can induce apoptosis in activated antitumor T cells [17]. The apoptosis-inducing activity of galectin-1 has also been reported in other biological systems. By the method of proteomic analysis, Plachta et al. [18] identified galectin-1 as one of the proteins with significantly increased activity in degenerating neurons. They then demonstrated that exogenously applied recombinant galectin-1 caused degeneration of neuronal processes. The apoptosis-inducing function of natural galectin-1 was confirmed when it was shown that inhibitors of galectin-1, including lactose and anti-galectin-1 antibody, could prevent the neuronal degeneration.
22.3.2 In Vivo Studies The above experimental studies suggest that galectin-1 may suppress the Th1 immune response by inducing T-cell apoptosis, and this is supported by animal studies. Administration of recombinant galectin-1 was shown to suppress the immune response in a number of animal models of inflammatory and autoimmune responses, including collagen-induced arthritis, concanavalin A-induced hepatitis, experimental colitis, graft-versus-host disease, and experimental autoimmune uveitis (reviewed in [19]). In addition, inoculation of galectin-1 knockout (genetically inhibited/removed) B16 cells to mice resulted in a Th1-polarized response, unlike the unmodified cells, which do not elicit a substantial T-cell response. This is consistent with the Th1-suppressing effect of galectin-1 [17]. The immunosuppressive role of endogenous galectin-1 has also been convincingly demonstrated in galectin-1-deficient mice; these mice exhibit more pronounced disease in a model of experimental autoimmune encephalitis [13]. The apoptosis-inducing activity of galectin-1 on neurons has also been demonstrated in vivo. First, degeneration of neurons induced by the injection of the neurotoxin ibotenic acid into the medial septum of mouse brain could be inhibited by simultaneously injecting with either lactose or anti-galectin-1 antibody. In addition, the elimination of nerve endings following the cutting of axons (axotomy) was significantly delayed in galectin-1-deficient mice compared to normal (wild-type) mice [18].
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22.4 Galectin-3 22.4.1 Studies with Cells Expressing Transferred DNA (Transfectants) By using the method of adding foreign genes to cells (gene transfection) and antisense approaches to inhibit the activity of certain genes, galectin-3 has been found to have antiapoptotic activity in a number of cell types, including immune and inflammatory cells, against many different stimuli that are known to induce apoptosis. This topic has been extensively reviewed [20,21]. The mechanisms by which intracellular galectin-3 confers resistance to apoptosis have also been extensively investigated, and current information suggests that this may involve interactions with other intracellular regulators of apoptosis, including those operating in mitochondria [4]. Many studies of the antiapoptotic functions of galectin-3 were demonstrated with tumor cells exposed to known inducers of apoptosis [22,23]. However, additional insights into the mechanisms of this antiapoptotic effect are worthy of mention. Takenaka et al. [24] described the association of antiapoptotic activity of overexpressed galectin-3 in human breast carcinoma cells with an upregulation of phosphorylated ERK. Cecchinelli et al. [25] showed that apoptosis induced by the tumor suppressor p53 involves repression of galectin-3.
22.4.2 Studies with Galectin-3-Deficient Mice The antiapoptotic functions of galectin-3 have been supported by studying cells from galectin-3-deficient (gal3−/−) mice. Peritoneal macrophages from gal3−/− mice are more sensitive to apoptosis that has been induced by lipopolysaccharide plus IFN-g, compared to those from wild-type mice [26]. More recently, we found that keratinocytes from gal3−/− mice were significantly more sensitive to apoptosis induced by UVB light in addition to various other stimuli, both in vitro and in vivo. As to possible mechanisms, galectin-3 deficiency is associated with increased activation of the signaling molecules JNK and ERK, and decreased activation of AKT [27].
22.4.3 Studies with Soluble Recombinant Galectin-3 Like galectin-1, galectin-3 has been shown to induce apoptosis in T cells, including human T-cell leukemia cell lines, human peripheral blood mononuclear cells, and activated mouse T cells [28,29]. One study found that the cell surface glycoproteins, CD7 and CD29, mediate galectin-3’s effect [28], but another study showed that CD45 and CD71, but not CD29 and CD43, are involved [29] and that galectin-3 is more potent than galectin-1. Galectin-3 has also been shown to induce apoptosis in neutrophils [30].
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22.5 Galectin-7 In a study by Bernerd et al. [31], it was found that UVB irradiation of keratinocytes resulted in the rapid upregulation of galectin-7 expression, and this was correlated with the induction of apoptosis. They also showed that overexpression of galectin-7 in keratinocytes caused increased apoptosis. Kuwabara and colleagues [32] confirmed and extended this finding by demonstrating that HeLa cells overexpressing galectin-7 were more sensitive to apoptosis induced by a number of different apoptotic stimuli. Additional studies have revealed that galectin-7 promotes apoptosis by potentiating JNK activation and mitochondrial cytochrome c release [32]. Ueda et al. [33] showed that human colon carcinoma DLD-1 cells transfected with galectin-7 were also more sensitive to apoptosis. In addition, these cells failed to grow and formed fewer colonies when compared with control cultures. Tumor formation from these cells was greatly diminished compared to control cells when they were injected subcutaneously into immunodeficient mice [33]. Whether this is related to increased apoptosis affected by galectin-7 is unknown, although the existing data suggest that galectin-7 is able to both induce apoptosis and inhibit cell growth.
22.6 Galectin-9 Wada et al. [34] found that recombinant galectin-9 was able to induce apoptosis in thymocytes. Kashio et al. [35] showed that galectin-9 induces apoptosis in cell cultures of B cells, monocytic cells, and promyelocytic cells, and it does so via the caspase-1 pathway, but caspase-8, -9, and -10 are not involved. More recently, Zhu et al. [36] demonstrated that galectin-9 induced apoptosis in specific immune cells (Th1 cells) and related this finding to binding of the lectin to a Th1-specific cell surface molecule, Tim-3. Like galectin-1, galectin-9 can also suppress the immune response in vivo. For example, galectin-9 decreases the severity of the disease in an animal model of multiple sclerosis (experimental autoimmune encephalomyelitis) [36].
22.7 Other Galectins Galectin-2 can also induce apoptosis in immune cells (T cells) [37]. This effect appears to involve activation of caspase-3 and -9, cytochrome c release, disruption of the mitochondrial membrane potential, and DNA fragmentation. In addition, galectin-2 causes an increase in the ratio of protein Bax, which is proapoptotic, to another protein, Bcl-2, which is antiapoptotic [37]. Galectin-8 has been shown to induce apoptosis in a human carcinoma cell line in a fashion that is dependent on inhibition of cell adhesion [38]. A more recent
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study showed that this galectin can either inhibit cell growth or apoptosis, depending on the level of cyclin-dependent kinase inhibitor p21 [39]. Galectin-12 has also been shown to promote apoptosis when overexpressed in a fibroblast cell line [40].
22.8 Conclusions Galectins are regulators of cellular homeostasis; some galectins promote while others inhibit apoptosis, and some function through binding to cell surface glycans, while others function intracellularly. Galectin-1, -2, -8, and -9 induce apoptosis through extracellular pathways; galectin-3 is antiapoptotic through an intracellular mechanism but induces apoptosis through an extracellular mechanism; galectin-7 promotes apoptosis through an intracellular mechanism (Fig. 22.3).
Fig. 22.3 Depending on cell types, binding of galectin (Gal)-9 to cell surface glycoproteins, such as Tim-3, can cause calcium influx and induce apoptosis through the calcium-calpain-caspase-1 pathway or the classical pathway mediated by cytochrome c (Cyt c) and apoptosis-inducing factor (AIF) released from the mitochondria with disrupted transmembrane potential. Galectin-1 causes translocation of endonuclease G (EndoG) from the mitochondrial intermembrane space to the nucleus without the release of Cyt c or AIF and induces DNA fragmentation and apoptosis in the absence of caspase activation. Galectin-7 appears to involve an intrinsic apoptotic pathway featuring activation of JNK. Galectin-3 is translocated to the mitochondria after associating with synexin, where it may interact with Bcl-2 and inhibit apoptosis
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For those galectins demonstrated to have apoptosis-inducing activities in studies where exogenous recombinant galectins have been used, a challenging question exists: does the endogenous protein exert the same activity? In particular, the concentrations required for extracellular induction are generally high, thus would the concentrations of extracellular galectins demonstrated for in vitro responses be adequately present in vivo? There are an increasing number of studies supporting this concept. Moreover, galectins may be more effective in inducing extracellular effects when presented by extracellular matrices, as described for galectin-1 [16]. In vivo data have also emerged to support the apoptosis-inducing activity of some galectins. However, it should be noted that galectin-3, when added exogenously, can induce cell death, but endogenous galectin-3 is antiapoptotic. Thus, activities demonstrated with recombinant proteins added in vivo may not represent the functions of the endogenous protein. Additional studies are needed to definitively establish the apoptosis-inducing functions of endogenous galectins in physiological and pathological processes. The cell surface receptors responsible for galectins’ actions also remain to be clarified. As mentioned earlier, galectins do not have specific individual receptors. Indeed, galectin-1 and -3 have been shown to bind to a large number of different glycoproteins [29,41]. They may bind to different cell surface glycoproteins in different cell types. It will continue to be a challenge to establish which glycoprotein(s) recognized by the lectin is (are) responsible for its function exerted on a specific cell type. The intracellular apoptosis-regulating functions of galectins are unexpected activities for proteins with lectin properties but are consistent with the protein being located intracellularly. These functions were revealed from studies involving gene transfection and antisense nucleotides to influence expression and, more recently, confirmed by the use of cells from mice deficient in galectin-3. In a number of cases, however, the intracellular site of action is also supported by the fact that the activities in question are not affected by lactose added to the culture medium, which would inhibit galectin-3’s carbohydrate-dependent extracellular action. In addition, in the case of galectin-3, a number of intracellular proteins with which this galectin interacts and which are conceivably responsible for its function have been identified. The functions of galectins when acting intracellularly do not depend on their interactions with intracellular carbohydrates. However, as described earlier in this review, galectin-3 may be involved in the transport of selected glycoproteins out of the cell [10]. Whether galectins bind to intracellular glycans in other subcellular compartments and whether such associations contribute to their intracellular functions, including apoptosis regulation, remain to be determined. Although there are many additional studies that remain to be conducted, the current information suggests that galectin family members are important regulators of apoptosis. Because apoptosis is involved in a variety of physiological and pathological processes, these members play important roles in regulating these processes.
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19. Rabinovich GA, Liu FT, Hirashima M, Anderson A (2007) An emerging role for galectins in tuning the immune response: lessons from experimental models of inflammatory disease, autoimmunity and cancer. Scand J Immunol 66(2–3):143–158 20. Liu FT (2005) Regulatory roles of galectins in the immune response. Int Arch Allergy Immunol 136(4):385–400 21. Hsu DK, Liu FT (2004) Regulation of cellular homeostasis by galectins. Glycoconj J 19(7–9):507–515 22. Liu FT, Rabinovich GA (2005) Galectins as modulators of tumour progression. Nat Rev Cancer 5(1):29–41 23. Nakahara S, Oka N, Raz A (2005) On the role of galectin-3 in cancer apoptosis. Apoptosis 10(2):267–275 24. Takenaka Y, Fukumori T, Yoshii T, Oka N, Inohara H, Kim HR, Bresalier RS, Raz A (2004) Nuclear export of phosphorylated galectin-3 regulates its antiapoptotic activity in response to chemotherapeutic drugs. Mol Cell Biol 24(10):4395–4406 25. Cecchinelli B, Lavra L, Rinaldo C, Iacovelli S, Gurtner A, Gasbarri A, Ulivieri A, Del Prete F, Trovato M, Piaggio G, Bartolazzi A, Soddu S, Sciacchitano S (2006) Repression of the antiapoptotic molecule galectin-3 by homeodomain-interacting protein kinase 2-activated p53 is required for p53-induced apoptosis. Mol Cell Biol 26(12):4746–4757 26. Hsu DK, Yang RY, Yu L, Pan Z, Salomon DR, Fung-Leung WP, Liu FT (2000) Targeted disruption of the galectin-3 gene results in attenuated peritoneal inflammatory responses. Am J Pathol 156:1073–1083 27. Saegusa J, Hsu DK, Liu W, Kuwabara I, Kuwabara Y, Yu L, Liu FT (2008) Galectin-3 protects keratinocytes from UVB-induced apoptosis by enhancing AKT activation and suppressing ERK activation. J Invest Dermatol 128:2403–2411 28. Fukumori T, Takenaka Y, Yoshii T, Kim HR, Hogan V, Inohara H, Kagawa S, Raz A (2003) CD29 and CD7 mediate galectin-3-induced type II T-cell apoptosis. Cancer Res 63(23):8302–8311 29. Stillman BN, Hsu DK, Pang M, Brewer CF, Johnson P, Liu FT, Baum LG (2006) Galectin-3 and galectin-1 bind distinct cell surface glycoprotein receptors to induce T cell death. J Immunol 176(2):778–789 30. Fernandez GC, Ilarregui JM, Rubel CJ, Toscano MA, Gomez SA, Beigier Bompadre M, Isturiz MA, Rabinovich GA, Palermo MS (2005) Galectin-3 and soluble fibrinogen act in concert to modulate neutrophil activation and survival: involvement of alternative MAPK pathways. Glycobiology 15(5):519–527 31. Bernerd F, Sarasin A, Magnaldo T (1999) Galectin-7 overexpression is associated with the apoptotic process in UVB-induced sunburn keratinocytes. Proc Natl Acad Sci USA 96:11329–11334 32. Kuwabara I, Kuwabara Y, Yang RY, Schuler M, Green DR, Zuraw BL, Hsu DK, Liu FT (2002) Galectin-7 (PIG1) exhibits pro-apoptotic function through JNK activation and mitochondrial cytochrome c release. J Biol Chem 277(5):3487–3497 33. Ueda S, Kuwabara I, Liu FT (2004) Suppression of tumor growth by galectin-7 gene transfer. Cancer Res 64(16):5672–5676 34. Wada J, Ota K, Kumar A, Wallner EI, Kanwar YS (1997) Developmental regulation, expression, and apoptotic potential of galectin-9, a b-galactoside binding lectin. J Clin Invest 99:2452–2461 35. Kashio Y, Nakamura K, Abedin MJ, Seki M, Nishi N, Yoshida N, Nakamura T, Hirashima M (2003) Galectin-9 induces apoptosis through the calcium-calpain-caspase-1 pathway. J Immunol 170(7):3631–3636 36. Zhu C, Anderson AC, Schubart A, Xiong H, Imitola J, Khoury SJ, Zheng XX, Strom TB, Kuchroo VK (2005) The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity. Nat Immunol 6(12):1245–1252 37. Sturm A, Lensch M, Andre S, Kaltner H, Wiedenmann B, Rosewicz S, Dignass AU, Gabius HJ (2004) Human galectin-2: novel inducer of T cell apoptosis with distinct profile of caspase activation. J Immunol 173(6):3825–3837
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Chapter 23
Avian and Human Influenza Virus Receptors and Their Distribution Yasuo Suzuki
Keywords Influenza virus • Hemagglutinin • H5N1 • Pandemic • Receptor
23.1 Introduction Influenza is one of the most widely distributed zoonotic infectious diseases in the world, and its pathogen, the influenza virus, is extremely mutable. Currently, the highly pathogenic avian influenza subtype H5N1, which is continuing to spread across Asia, Europe, and Africa, has already been transmitted into humans and has been generating mutation for possible propagation from human to human [1–8]. When this new variant of the virus becomes an epidemic among humans, it will spread throughout the world in a short time, resulting in a pandemic because humans do not have immunity to the virus. The avian influenza virus is rarely directly transmitted to humans in nature. Conversely, human influenza virus could not successfully infect ducks experimentally [1, 3, 4]. In other words, the host range of influenza viruses is changeable, wherein each virus is primarily prescribed with receptor binding specificity. In nature, receptor binding specificity changes due to mutations of the viral spike hemagglutinin molecule, resulting in transmission to other animal species across the host range.
23.2 Host Range of the Influenza Virus Influenza viruses (types A and B) recognize sialo-sugar chains on the host cell membranes as their receptors. With the exception of insect cells, the sialo-sugar chains exist universally in all mammal, bird, reptile, amphibian, and fish cells, in Y. Suzuki (*) Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, 1200 Matsumoto-cho, Kasugai-shi 487-8501, Aichi, Japan e-mail:
[email protected] A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_23, © Springer Science+Business Media, LLC 2011
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Raccoon Gray hound
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Equine
Chicken Quail
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H1-7, 9-11 N1-4, 6-8 H5N1 WHO phase 3 H7N7
Quail Domestic water fowl
Low Pathogenic
H: Hemagglutinin N: Neuraminidase
Direct Transmission
1997~ Highly Pathogenic Human H1N1, H2N2 H3N2 H5N1, H9N2, H5N1 H9N2 H7N7 Pandemic H1N1 2009
Host range Restriction
Native Host
Ferret H10N4 H5N1 H1N1
Wild water fowl Duck, etc
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H1-16 N1-9 Swine
Whale H1N3, H13N2 H13N9
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H1N1, H1N2, H2N3, H3N1, H3N2, H3N3, H3N8, H4N6, H5N1, H5N2, H9N2
Fig. 23.1 Host range of influenza A viruses and direct transmission of avian influenza viruses (H5N1, H9N2, H7N7) into humans and animals
addition to specific bacteria and protozoa. Therefore, it can be considered that influenza viruses have evolved and expanded their host range on the earth by using sialo-sugar chains, which are widely distributed in the animal world. Nevertheless, influenza does not necessarily spread to all animal species that have sialo-sugar chains; influenza circulates among a fairly limited range of animal species in nature. Various composite factors in addition to virus receptors are related to influenza virus epidemics. All influenza A viruses are pooled in wild waterfowl, but they can be isolated from other animals, including land birds, swine, horses, humans, whales, and seals. Furthermore, H5N1 viruses have been found recently in tigers [9, 10], cats [11–13], and dogs [14], indicating that monitoring of domestic animals during H5N1 outbreaks is needed (Fig. 23.1). The host range of influenza A viruses extends across a variety of animal species in addition to humans. On the other hand, type B viruses are circulating solely in the human world.
23.3 Receptor Binding Specificity of Influenza Viruses Influenza A viruses spread quickly within the same animal species; however, transmission to another type of host (e.g., bird → human) does not occur easily in nature. To cross over the host barrier, a host–range mutation must occur. The most important
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factor in crossing the host barrier is mutation of the host receptor recognition specificity of viral hemagglutinin [3, 4, 15, 16]. We found that the host cell membrane receptors for the influenza viruses are based on the carbohydrate structures of sialyl lactosamine chains (sialic acid [Sia] a2-3 [6] galactose [Gal] b1-4 [3] N-acetylglucosamine [GlcNAc] b1-). The hemagglutinin of avian and human influenza viruses recognize fine structures of Sia-Gal linkage (a2-3 or a2-6) of the terminus of the sialyl lactosamine structures and molecular species of nonreducing terminal sialic acid, such as 5-N-acetylneuramic acid (Neu5Ac) and 5-N-glycoly-lneuraminic acid (Neu5Gc) [3, 4, 15, 16]. The sialyl lactosamine sugar chains are carried on glycoproteins and glycosphingolipids in the host cell membranes. In the case of humans, these sugar chains universally exist in the somatic cells of a variety of tissues, rather than in the central nervous system. However, distribution of the Siaa2-3Gal (2-3), Siaa2-6Gal (2-6) linkage in glycoconjugates and the molecular species of terminal sialic acid (Neu5Ac, Neu5Gc) clearly differ depending on the tissues and animal species. This difference defines the host range of the influenza viruses. Actually, influenza A viruses isolated from duck preferentially bind to Siaa2-3Gal-, and the clinical isolates from humans preferentially bind to Siaa2-6Gal-linkages [3, 4, 15, 16] (Fig. 23.2). Only one or two amino acid substitutions in the viral hemagglutinin molecule can alter the binding specificity to terminal sialyl-Gal linkage (2-3, 2-6) and molecular species of sialic acid (Neu5Ac, Neu5Gc) [3, 4, 15, 16]. We also found that the subtype H9N2 from quail in China can bind to 2-6 human-type receptor, even though it was isolated from a bird [17]. Furthermore,
Fig. 23.2 Structure of terminal sialo-sugar chains (Neu5Aca2-3Gal- and Neu5Aca2-6Gal-) in mammalian tissues, which serve as receptors of human and avian influenza viruses
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H5N1 isolated from a boy in Fujian, China, had already mutated to bind to both a2-3 and 2-6 [18], and the hemagglutinin of H5N1, isolated from a girl infected by her elder brother in Vietnam, demonstrated affinity to human receptors [19].
23.4 Molecular Basis for Selecting the Receptor Binding Specificity of Influenza Viruses The hemagglutinin glycoprotein (homotrimeric) spike of influenza A viruses is responsible for binding to host cell receptors (Fig. 23.3). The structure and amino acids that make up the receptor binding pocket of the hemagglutinin subtypes H3 and H5 have already been reported [20–22]. In the case of H3 hemagglutinin, which is currently circulating in the human world, the amino acid no. 226 is closely associated with the sialic acid–Gal linkage (2-3, 2-6) determinants, and the amino acid no. 228 is similarly associated with the recognition of the sialic acid molecular species (Neu5Ac, Neu5Gc) [3, 4, 15, 16]. Substitution of Gln226Leu in the H3 hemagglutinin molecule brings about a dramatic change in the binding to Neu5Aca23Gal → Neu5Aca2-6Gal in sialo-sugar chain receptors, which consequently makes
Fig. 23.3 Influenza A virus, function of hemagglutinin and neuraminidase spikes, and the receptor sialo-sugar chains in host cell membranes. Illustration of the virus was drawn by Dr. Osamu Kanie, Riken, Japan
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it possible for the virus to cross the host range. Similarly, substitution of Ser228Gly brings about a Neu5Aca2-3Gal → Neu5Gca2-3Gal mutation for recognition of the sialic acid molecular species. Distribution of the sialic acid molecular species is clearly animal-species and tissue specific. For example, normal human and chicken tissues contain Neu5Ac, and swine and horse tissues have both Neu5Ac and Neu5Gc, which are factors in determining the host range of the influenza viruses for the sialic acid molecular species as well. Furthermore, we recently reported that the human-type 2-6 receptor exists in the respiratory tracts and intestines of domestic birds, such as quail and chicken [23]. This shows that quail and chickens have the molecular characterization to be potential intermediary hosts for avian influenza virus transmission to humans and could generate new influenza viruses with pandemic potential (Fig. 23.4). The 1918 Spanish influenza virus (subtype H1N1) was originally derived from birds. It was recently reported [24–26] that substitution of the no.190 and no. 225 amino acids in the hemagglutinin molecule of the 1918 Spanish influenza virus, A/South Carolina/1/18 (H1N1) (Glu190Asp, Gly225Asp), and A/New York/1/18 (H1N1) (Glu190Asp, Gly225Gly), resulted in a mutation for recognizing the 2-6 human-type receptor expressed in the human respiratory tract or both the bird- and human-type receptor 2-3/2-6, respectively (Fig. 23.5).
Fig. 23.4 Possible mutation of the highly pathogenic avian H5N1 virus to a new virus that binds to human receptor (2-6)
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Highly Pathogenic Avian Influenza virus (HPAV) H5N1 Asia, Europe, Africa Bird
H5N1
2-3
Current situation
High Conc. Virus; Direct contact
1918 Spanish Influenza Avian flu H1N1
a2-3 Mutation
*A/South Carolina/1/18 (H1N1) (binds to Sia2-6Gal receptor) Glu190Asp, Gly225Asp *A/New York/1/18 (H1N1) (binds to Sia2-3, 2-6 receptor) Glu190Asp, Gly225Gly Kobasa et al Nature, 431, 703707 (2004); T.M. Tumpey et al Science 2007; L. Glaser et al J. Virol. (2005)
a2-6, a2-6/a2-3 1918 Spanish influenza Human to Human transmission Pandemic
Humans (322 dead/552 case) World 15 countries (Apr. 21, 2011) If ……
Mutation of Receptor Binding Specificity:
Siaa2-3Galb1- (Avian type) Æ Siaa2-6Galb1- (Human type)
a2-6, a2-6/a2-3 (New human Influenza which spread human to human
Pandemic
New Vaccine, New Drug should be developed.
Fig. 23.5 Mutations of the receptor binding specificity of the highly pathogenic avian influenza virus (H1N1) caused the 1918 influenza pandemic. If the receptor binding specificity of the highly pathogenic H5N1 influenza virus changes from Siaa2-3Galb1- (avian type) to Siaa2-6Galb1(human type), a new human-type influenza virus may spread among humans
23.5 Receptor Sialo-Sugar Chains for the Highly Pathogenic Avian Influenza Viruses in Humans Since the highly pathogenic avian influenza virus H5N1 was directly transmitted from chickens to humans in Hong Kong in 1997, this virus has spread to more than 63 countries and infected 532 people in 15 countries around the world; 322 of the infected people died (according to the World Health Organization [WHO], as of 21 April 2011). The fatality rate was 63.2%, which is very high. By what mechanism did the highly pathogenic avian influenza virus spread from chickens to humans? It has been reported that the sialic acid a2-6Gal is predominant in the tracheal epithelial cells of humans [27]. A variety of viruses infect humans via the respiratory organs, where the parainfluenza virus uses the sialo-sugar chains as receptors. Blood-group sugar chains containing Neu5Aca2-3Gal have been identified as receptors for parainfluenza virus type 1, whereas 2-6 is not [28]. It has been assumed that Neu5Aca2-3Gal exists in the human respiratory tract and lungs because parainfluenza virus type 1, which binds to Neu5Aca2-3Gal, can be infected into the human respiratory tract, and H5N1, which binds to Neu5Aca23Gal, can also be infected into humans when they are directly exposed to the virus in high concentrations. Recently, the existence of Neu5Aca2-3Gal and Neu5Aca2-6Gal
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in the lower part of the human respiratory tract and lungs has been identified by a staining technique using lectins that differentiate sialyl a2-3 or 2-6 linkages, respectively. Matrosovich et al. [29] reported that influenza viruses enter the human airway epithelium through specific nonciliated cells, whereas avian viruses, as well as an egg-adapted human virus variant with an avian-virus-like receptor specificity, mainly infected ciliated cells; this pattern correlated with the predominant localization of receptors for human viruses (2-6-linked sialic acids) on nonciliated cells and of receptors for avian viruses (2-3-linked sialic acids) on ciliated cells. We also reported [30] that Neu5Aca2-3Gal exists in addition to Neu5Aca2-6Gal in primary culture of human tracheal epithelial cells. Recently, it was reported that Neu5Aca23Gal, which is believed to be a receptor for H5N1, is comparatively abundant in the lower part of the respiratory tract and lungs, indicating that 2-6 exists mainly in the upper respiratory tract [31, 32] and that H5N1 viruses preferentially recognizing Siaa2-3Gal can be transmitted from birds to humans, replicating efficiently in cells in the lower region of the respiratory tract, where the avian virus receptor is prevalent. This restriction may contribute to the inefficient human-to-human transmission of H5N1 viruses reported so far [31]. Tropism of avian influenza A (H5N1) in the upper and lower respiratory tract has also been reported [33]. These findings suggest that direct exposure of human trachea epithelial cells to high concentrations of H5N1 can mediate direct infection of the avian virus in humans through Siaa23Gal receptors in the trachea. Uiprasertkul et al. [34] reported the possibility of H5N1 infection in the human intestinal tract since viral genes were detected in the intestinal tract of a patient who died. Yamada et al. [35] determined the amino acids (no. 182 and no. 192) in H5N1 virus hemagglutinin, which is responsible for the binding to human-type receptors (Siaa2-6Gal). Mutations at positions 182 and 192 independently convert the hemagglutinins of H5N1 viruses known to recognize the avian receptor to those that recognize the human receptor. Auewarakul et al. [36] found that a simultaneous two-amino-acid substitution, Leu129Val, Ala134Val in H5N1 virus hemagglutinin, makes it possible for the virus to bind not only to avian receptor (Siaa2-3Gal), but also to human-type receptor (Siaa2-6Gal).
23.6 Conclusion Highly pathogenic avian influenza virus requires the following events to cross the host barriers and spread from human to human: (1) a mutation occurs so that the highly pathogenic avian virus can bind to viral receptors in the human respiratory tract and lungs; (2) a mutation occurs whereby an avian virus that replicates within the body of birds is able to replicate more efficiently in human bodies (as reported in the case of mice; Glu627Lys mutation in PB2 viral polymerase) [37, 38]; and (3) a mutation occurs causing more rapid transmission from human to human while the virus maintains high virulence. These mutations may occur by only one or two amino acid substitutions of any of the virus proteins. Currently, surveillance of the global scale of the highly pathogenic avian influenza virus by WHO and by countries,
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where the virus has spread is focusing on viral antigenic and genetic analyses. However, it is difficult to determine the phenotypic mutations, such as receptor binding specificity, by these antigenic and genotype surveillances. Therefore, monitoring of virus phenotype mutations, especially mutation of the receptor binding specificity from avian type to human type, should be started globally as soon as possible.
References 1. Webster RG, Peiris M, Chen H, Guan Y (2006) H5N1 outbreaks and enzootic influenza. Emerg Infect Dis 12:3–8 2. Enserink M (2006) Avian influenza. H5N1 moves into Africa, European Union, deepening global crisis. Science 311:932 3. Suzuki Y (2006) Sialobiology of influenza – molecular mechanism of host range variation of influenza viruses (review). Biol Pharm Bull 28:399–408 4. Suzuki Y (2007) The highly pathogenic avian flu viruses and the molecular mechanism of the transmission of the viruses into humans (Review). In: Comprehensive Glycoscience from Chemistry to Systems Biology, Editor-in-Chief, Johannis P. Kamerlig, Vol. 4, Cell Glycobiology and Development; Health and Disease in Glycomedicine, Elsevier, Amsterdam, pp 465–471, ISBN:978-0-444-51967-2 5. Kuiken T, Holmes EC, McCauley J, Rimmelzwaan GF, Williams CS, Grenfell BT (2006) Host species barriers to influenza virus infections. Science 312:394–397 6. Olsen B, Munster VJ, Wallensten A, Waldenstrom J, Osterhaus ADME, Fouchier RAM (2006) Global patterns of influenza A virus in wild birds. Science 312:384–388 7. Horimoto T, Kawaoka Y (2005) Influenza: lessons from past pandemics, warnings from current incidents. Nat Rev Microbiol 3:591–600 8. Duan L, Campitelli L, Fan XH, Leung YHC, Vijaykrishna D, Zang JX, Donatelli I, Delogu M, Li KS, Foni E, Chiapponi C, Wu WL, Kai H, Webster RG, Shortridge KF, Peiris JSM, Smith GJD, Chen H, Guan Y (2007) Characterization of low pathogenic H5 subtype influenza viruses from Eurasia: implications for the origin of highly pathogenic H5N1 viruses. J Virol 81:7529–7539 9. Keawcharoen J, Oraveerakul K, Kuiken T, Fouchier RAM, Amonsin A, Payungporn S, Noppornpanth S, Wattanodorn S, Theamboonlers A, Tantilertcharoen R, Ratanakorn P, Osterhaus ADME, Poovorawan Y (2004) Avian influenza H5N1 in tigers and leopards. Emerg Infect Dis 10:2189–2191 10. Amonsin A, Payungporn S, Theamboonlers A (2006) Genetic characterization of H5N1 influenza A viruses isolated from zoo tigers in Thailand. Virology 344:480–491 11. Kuiken T, Rimmelzwaan G, van Riel D, van Amerongen G, Baars M, Fouchier R, Osterhous A (2004) Avian H5N1 influenza in cats. Science 306:241 12. Songserm T, Amonsin A, Jam-on R, Sae-Heng N, Meemak N, Pariyothorn N, Payungporn S, Theamboonlers A, Poovorawant Y (2006) Avian influenza H5N1 in naturally infected domestic cat. Emerg Infect Dis 12:681–683 13. Leschnik M, Weikel J, Mostl K, Revilla-Fernandez S, Wodak E, Bago Z, Vanek E, Benetka V, Hess M, Thalhammer JG (2007) Subclinical infection with avian influenza A (H5N1) virus in cats. Emerg Infect Dis 13:243–246 14. Songserm T, Amonsin A, Jam-on R, Sae-Heng N, Pariyothorn N, Payungporn S, Theamboonlers A, Chutinimitkul S, Thanawongnuwech R, Poovorawan Y (2006) Fatal avian influenza A H5N1 in a dog. Emerg Infect Dis 12:1744–1747 15. Ito T, Suzuki Y, Suzuki T, Tanaka A, Horimoto T, Wells K, Kida H, Otsuki K, Kiso M, Ishida H, Kawaoka Y (2000) Recognition of N-glycolylneuraminic acid linked to galactose by a2-3
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linkage is associated with the intestinal replication of influenza A virus in ducks. J Virol 73:6743–6745 16. Suzuki Y, Ito T, Suzuki T, Holland RE, Chambers TM, Kiso M, Ishida H, Kawaoka Y (2000) Sialic acid species as a determinant of the host range of influenza A viruses. J Virol 74:11825–11831 17. Saito T, Nakaya Y, Suzuki T, Ito R, Saito T, Saito H, Takao S, Sahara K, Odagiri T, Murata T, Usui T, Suzuki Y, Tashiro M (2004) Antigenic alteration of influenza B virus associated with loss of a glycosylation site due to host-cell adaptation. J Med Virol 74:336–343 18. Shinya K, Hatta M, Yamada S, Takada A, Watanabe S, Halfmann P, Horimoto T, Neumann G, Kim JH, Lim W, Guan Y, Peiris M, Kiso M, Suzuki T, Suzuki Y, Kawaoka Y (2005) Characterization of a Human H5N1 Influenza A virus isolated in 2003. J Virol 79: 9926–9932 19. Le QM, Kiso M, Someya K, Sakai YT, Nguyen TH, Nguyen KHL, Pham HN, Ngyen DH, Yamada S, Muramoto Y, Horimoto T, Takada A, Goto H, Suzuki T, Suzuki Y, Kawaoka Y (2005) Isolation of drug-resistant H5N1 virus. Nature 437:1108 20. Wilson IA, Skehel JJ, Wiley DC (1981) Sructure of the haemagglutinin membrane glycoprotein of influenza virus at 3A resolution. Nature 289:366–373 21. Sauter NK, Glick GD, Crowther RL, Park SJ, Eisen MB, Skehel JJ, Knowles JR, Wiley DC (1982) Crystallographic detection of a second ligand binding site in influenza virus hemagglutinin. Proc Natl Acad Sci USA 89:324–328 22. Ha Y, Stevens DJ, Skehel JJ, Wiley DC (2001) X-ray structures of H5 avian and H9 swine influenza virus hemagglutinins bound to avian and human receptor analogs. Proc Natl Acad Sci USA 20:11181–11186 23. Guo CT, Takahashi N, Yagi H, Kato K, Takahashi T, Yi S-Q, Chen Y, Ito T, Otsuki K, Kida H, Kawaoka Y, Hidari KI-PJ, Miyamoto D, Suzuki T, Suzuki Y (2007) The quail and chicken have sialyl-Gal sugar chains responsible for the binding of influenza A viruses to human type receptors. Glycobiology 17:713–724 24. Tumpey TM, Maines TR, Hoeven NV, Glase L, Solorzano A, Pappas C, Cox NJ, Swayne D, Palese P, Katz KJM, Garcia-Sastre A (2007) A two-amino acid change in the hemagglutinin of the 1918 influenza virus abolishes transmission. Science 315:655–659 25. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, Basler CF, Taubenberger JK, Palese P (2005) A single amino acid substitution in 1918 influenza virus hemagglutinin change receptor binding specificity. J Virol 79:11533–11536 26. Kobasa D, Takada A, Shinya K, Halfman P, Hatta M, Theriault S, Suzuki H, Nishimura H, Mitamura K, Sugaya N, Usui T, Murata T, Suzuki T, Suzuki Y, Feldman H, Kawaoka Y (2004) Enhanced pathogenicity of influenza A viruses possessing the haemagglutinin of the 1918 pandemic. Nature 431:703–707 27. Baum LG, Paulson JC (1990) Sialyloligosaccharides of the respiratory epithelium in the selection of human influenza virus receptor specificity. Acta Histochem 40(Suppl):35–38 28. Suzuki T, Portner A, Scroggs RA, Uchikawa M, Koyama N, Matsuo K, Suzuki Y, Takimoto T (2001) Receptor specificities of human respiroviruses. J Virol 75:4604–4613 29. Matrosovich MN, Matrosovitch TY, Gray T, Roberts NA, Klenk HD (2004) Human and avian influenza viruses target different cell types in cultures of human airway epithelium. Proc Natl Acad Sci USA 101:4620–4624 30. Kogure T, Suzuki T, Takahashi T, Miyamoto D, Hidari KIPJ, Guo CT, Ito T, Kawaoka Y, Suzuki Y (2006) Human trachea primary epithelial cells express both sialyl (a2-3) Gal receptor for human parainfluenza virus type 1 and avian influenza viruses, and sialyl (a2-6) Gal receptor for human influenza viruses. Glycoconj J 23:101–106 31. Shinya K, Ebina M, Yamada M, Ono M, Kasai N, Kawaoka Y (2006) Influenza virus receptors in the human airway. Nature 440:435–436 32. van Riel D, Munster VJ, de Wit E, Rimmelzwaan GF, Fouchier RAM, Osterhaus ADME, Kuiken T (2006) H5N1 virus attachment to lower respiratory tract. Science 312:399 33. Nicholls JM, Chan MCW, Chan WY, Wong HK, Cheung CY, Kwong DLW, Wong MP, Chui WH, Poon LLM, Tsao SW, Guan Y, Peiris JSM (2007) Tropism of avian influenza A (H5N1) in the upper and lower respiratory tract. Nat Med 13:147–149
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Chapter 24
Importance of a Factor VIIIc-Like Glycoprotein Expressed in Capillary Endothelial Cells (eFactor VIIIc) in Angiogenesis Dipak K. Banerjee, Caroline M. Oliveira, José J. Tavárez, Viswa N. Katiyar, Subiman Saha, Juan A. Martínez, Aditi Banerjee, Aurymar Sánchez, and Krishna Baksi
Keywords Angiogenesis • Mannosylphospho dolichol synthase • Unfolded protein response • ER stress • Apoptosis • Cell cycle • N-linked glycoproteins • Tunicamycin • Lipid-linked oligosaccharide
24.1 Introduction Factor VIII is a large, 2,332-residue plasma glycoprotein that acts as a regulatory cofactor in the process of blood coagulation [1–3]. It binds to activated factor IX (factor IXa) in the presence of calcium and negatively charged phospholipids at the surface of activated platelets to form a membrane-associated, proteolytically active complex. Upon complex formation, the Vmax of factor IXa is increased by approximately 200,000-fold, promoting the rapid activation of its substrate, the serine protease factor X. The proteolytic conversion of factor X to its active form, factor Xa, is a central control point in the coagulation cascade, leading to activation of thrombin, formation of a fibrin mesh, and establishment of a stable blood clot. The binding of factor VIIIc and other activated proteins to these membrane surfaces allows for localization of the procoagulation process to sites of vascular damage. The factor VIII sequence contains six sequential domains arranged in the order of A1–A2–B–A3–C1–C2 (Fig. 24.1) [4–6]. The A domains are homologous to one another and display sequence similarity to the copper-binding protein ceruloplasmin. They are flanked by short spacer sequences that are highly acidic. The C domains are also homologous to each other and have a weak homology to the discoidin protein-fold family (e.g. the lipid-binding domain of galactose oxidase) [7, 8]. The circulating form of factor VIII protein is a metal-bridged heterodimer consisting of a heavy chain (A1–A2–B) and a light chain (A3–C1–C2). This form of factor D.K. Banerjee (*) Department of Biochemistry, School of Medicine, University of Puerto Rico, Medical Sciences Campus, San Juan 00936-5067, Puerto Rico e-mail:
[email protected] A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_24, © Springer Science+Business Media, LLC 2011
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Fig. 24.1 Domain structure of factor VIII. Arg 372, Arg 740, and Arg 1648 are proteolytic cleavage sites
VIII is bound tightly to von Willebrand factor (vWF). Factor VIII is processed further by specific thrombin cleavages into a heterotrimeric form. This active form, factor VIIIc, dissociates from vWF and binds to negatively charged phospholipids on activated platelet surfaces. The carboxyl terminal C2 domain of factor VIII contains binding sites for vWF and for negatively charged phospholipids. The binding of factor VIIIc to membranes involves stereoselection for O-phospho-l-serine, the negatively charged head group of phosphatidylserine (PS) [9]. The binding of factor VIII or VIIIc to vWF or PS is mutually exclusive, even though the activation of factor VIII involves cleavages outside the C2 domain [10]. Factor VIIIc deficiency has been documented in a congenital bleeding disorder, hemophilia A. The current estimate suggests one case of hemophilia A occurs in every 5,000 live births in the USA. We have seen expression of a factor VIIIc-like molecule in capillary endothelial cells, i.e. eFactor VIIIc, but its role in the capillary endothelial cell function, e.g. angiogenesis, has not yet been established. In this article, we present evidence that (a) an extracellular signal stimulates eFactor VIIIc N-glycosylation, which consequently enhances angiogenesis, and (b) eFactor VIIIc supports capillary endothelial cell invasion in Matrigel™.
24.2 Establishment of a Capillary Endothelial Cell Line to Study Angiogenesis In Vitro A nontransformed capillary endothelial cell line has been established from the microvasculature of bovine adrenal medulla [11]. Dissociated cells, when cultured in Eagle’s minimal essential medium (EMEM) with Earl’s salt containing fetal bovine serum and antibiotics, attached to one another in an end-to-end and side-toside fashion. On successive days, they became more flattened, elongated, and established more contact, developing extensive and ordered networks of cells that generated macroscopic capillary-like structures. Ultrastructural studies indicated extension of thin processes; cytoplasm filled with rough endoplasmic reticulum; and numerous surface cisterns of smooth, coated, and small vesicles exhibiting both exo and endocytosis. Dispersed mitochondria of the long tubular type parallel to the cellular axis were frequently seen. Both
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tight and gap junctions, as well as intercellular filaments and intercellular spaces reminiscent of capillary lumen, were also present [11].
24.3 Influence of Microenvironment and Extracellular Signaling on Angiogenesis The capillary endothelial cell population doubled in 56 h when cultured in the presence of 10% fetal bovine serum (G1 = 24 h, S = 8 h, G2 + M = 24 h [12]) in an atmosphere of 95% air and 5% CO2. A dramatic shift in cell morphology and a considerable amount of cell death occurred when they were cultured in the absence of CO2. In the absence of CO2, the cells failed to attach and died within 24 h. Supplementation with 10 mM Hepes–NaHCO3, pH 7.4, improved cell attachment, but proliferation and cell-to-cell contact remained low [13]. Cells cultured in a media containing 2% fetal bovine serum increased the cell doubling to 68 h due to an extension of the G1 phase to 36 h. On the other hand, culturing the cells in the presence of 2 mM 8Br-cAMP in a 2% fetal bovine serumcontaining media increased cell proliferation by ~70% and exhibited cell cycle shortening. During this treatment, almost one half of the cell population entered in the S phase, and the cellular morphology indicated increased mitosis. Bcl-2 expression and caspase-3, -8, and -9 activity remained unchanged. Western blot results indicated that the expression of the cytosolic chaperones HSP-70 and HSP-90 was enhanced by 1.4- to 1.6-fold in 8Br-cAMP-treated cells with a significant reduction in the expression of endoplasmic reticulum (ER) chaperones GRP-78/Bip and GRP-94 [14]. Metabolic labeling with 35S-methionine as a function of time followed by immunoprecipitation and autoradiography indicated no change in the GRP-78/ Bip level between 3 and 32 h in control cells (Fig. 24.2).
24.4 Expression of Factor VIIIc-Like Protein in Capillary Endothelial Cells Upon culturing, the cells were fixed and treated with a mouse monoclonal antibody to human factor VIIIc, followed by a fluorescently labeled secondary antibody and analyzed microscopically. The result indicated perinuclear localization of factor VIIIc 0h
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Fig. 24.2 Expression of GRP-78 in capillary endothelial cells as a function of time. Cells were labeled with [35S]methionine, and GRP-78 was immunoprecipitated from the cell lysate with antiGRP-78 monoclonal antibody and analyzed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography
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Fig. 24.3 Expression of factor VIIIc in capillary endothelial cells. (a) Cellular localization of factor VIIIc by immunofluorescence microscopy; (b) autoradiography of actively synthesized factor VIIIc; and (c) bioactivity of immunoprecipiated factor VIIIc: 1 = cell lysate; 2 = conditioned media; 3 = bovine plasma; 4 = human plasma; 5 = EMEM with 10% fetal bovine serum; 6 = cell lysate with a protease inhibitor (aprotinine)
(Fig. 24.3a). To provide further support for factor VIIIc expression, capillary endothelial cells were labeled with 35S-methionine (40 mCi/mL; Sp. Act. 1,400 Ci/mmol) for 2 h at 37°C. Factor VIIIc from the cell lysate and the conditioned media was immunoprecipitated with an antifactor VIIIc monoclonal antibody and analyzed by 7.5% SDSPAGE followed by autoradiography. The newly synthesized factor VIIIc consisted of a heavy chain (Mr 200,000 Da) and a light chain (Mr 46,000 Da) in the cell lysate. The values for the heavy and light chains for the secretory factor VIIIc were Mr 210,000 and Mr 40,000 Da, respectively (Fig. 24.3b). Secretory factor VIIIc, on the other hand, has a molecular mass of Mr 270,000 under nonreducing conditions, suggesting that the heavy and light chains are held together by S–S bonds [15]. To analyze the functional status of factor VIIIc, the immune complexes were subjected to a chromogenic assay (COATEST, Kabivitrum, Stockholm, Sweden), where the conversion of Factor X to Factor Xa was quantified by spectrophotometrically measuring the proteolytic release of p-nitroaniline bound to a synthetic substrate (S-2222). Both cell lysate and the conditioned media contained factor VIIIc-like activity (Fig. 24.3c).
24.5 Endothelial Factor VIIIc Is an Asparagine-Linked Glycoprotein Cells were metabolically labeled with [35S]methionine, and factor VIIIc in the conditioned media was immunoprecipitated. The glycosylation status of factor VIIIc was analyzed by SDS-PAGE followed by autoradiography after digesting the
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Fig. 24.4 Factor VIIIc is an asparagine-linked glycoprotein. Cells were labeled with [35S]methionine, and immunoprecipitated factor VIIIc was digested with N- and O-glycanases and analyzed by SDS-PAGE followed by autoradiography. Lane 1 = control; lane 2 = after digestion with N-glycanase; lane 3 = after digestion with O-glycanase
immunoprecipitate with N-glycanase (PNGase F). Reduction in the molecular mass of the light chain by ~8,000 Da (i.e. from Mr 46,000 to Mr 38,000 Da) supported that the light chain contains ~17% asparagine-linked (N-linked) glycans. Digestion with O-glycanase did not exhibit any appreciable change in the molecular mass, indicating that factor VIIIc may not contain covalently attached O-linked glycans (Fig. 24.4).
24.6 Regulation of Factor VIIIc N-Glycosylation by Transmembrane Signaling Anatomically, endothelial cells interface blood and the interstitium and are in constant contact with various growth factors, cytokines, chemokines, and other bioactive molecules present in the circulation. Therefore, we have tested the effect of the two circulatory hormones insulin and isoproterenol (a b-agonist and a synthetic catecholamine) on factor VIIIc N-glycosylation.
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24.6.1 Regulation by Insulin Insulin, a vital hormone of metabolism and a growth factor, stimulated growth of the liver, spleen, and heart during mammalian embryogenesis, as well as the growth of Reuber H35 hepatoma cells [16–18]. On the other hand, insulin downregulated the proliferation of capillary endothelial cells [19, 21]. This raised a question about the insulin receptors on the capillary endothelial cell surface. Examination of insulin receptors on the capillary endothelial cell membrane indicated that (1) 125I-insulin binding plus internalization reached to a steady state in 20 min; (2) acid-washable fraction accounted for nearly half of the total specifically bound insulin; and (3) the dissociation constants (Kd) for insulin receptor in acid-washable fraction were 4.0 × 10−11 M (high affinity) and 4.7 × 10−9 M (low affinity), with a total number of 210,000 receptors per cell [20]. To evaluate the effect of insulin on factor VIIIc biosynthesis and glycosylation, cells were metabolically labeled with [35S]methionine in the presence or absence of different concentrations of insulin (0.01, 1.0, and 10.0 mg/mL) and as a function of time (30 min to 24 h). Factor VIIIc in the cell lysate as well as in the conditioned media was immunoprecipitated with a mouse monoclonal antihuman factor VIIIc antibody and analyzed by SDS-PAGE followed by autoradiography. [35S]-Methionine incorporation in the heavy chain (Mr 210,000 Da) of cellular factor VIIIc was maximum at 1.0 mg/mL insulin, followed by 0.01 and 10.0 mg/mL in 2 h compared to the untreated controls. This was different from the light chain (Mr 46,000 Da). Maximum incorporation of radioactivity was observed at 10.00 mg/mL insulin, followed by 0.01 and 1.0 mg/mL. Secreted heavy and light chains, however, behaved differently. Maximum radioactivity was incorporated in the heavy and light chains at 1.0 mg/mL insulin concentration, followed by 0.01 and 10.0 mg/mL. When the cellular and the secretory components were combined for analysis for each insulin concentration, the incorporation of radioactivity into the heavy and light chains was always higher in each insulin concentration. To emphasize further, a higher level of radioactivity was present in secretory factor VIIIc when the cells were labeled for 2 h in the presence of 1 mg/mL insulin [22]. To address whether insulin increases the glycosylation of factor VIIIc, cells were double labeled with [3H]mannose and [35S]methionine in the presence or absence of insulin (1.0 mg/mL). Factor VIIIc in the cell lysate and in the media was immunoprecipitated with antifactor VIIIc antibody and subjected to SDS-PAGE. Bands corresponding to the heavy chain (Mr 210,000 Da) and the light chain (Mr 46,000 Da) were excised from the gel, and the radioactivity was quantified in a liquid scintillation spectrometer. The ratio of [3H]mannose to [35S]methionine in factor VIIIc was increased by 83% after insulin treatment (Table 24.1). The rate of Glc3Man9GlcNAc2-PP-Dol, lipid-linked oligosaccharide (LLO) biosynthesis and its turnover in cells were also increased. Mannosylphospho dolichol synthase (DPMS) activity was increased by 1.5- to 2-fold. Inability of actinomycin D to inhibit the DPMS activity suggested against increased transcription. Intracellular transport of 2-dexyglucose was increased by ~30% under this condition [23]. Epinephrine is a “fright, flight, or fight” hormone and prepares the cell for either combat or escape upon binding with b-adrenoreceptors. Therefore, the presence of
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Table 24.1 Effect of insulin on the ratio of [3H]mannose to [35S]methionine incorporation in factor VIIIc Total (cellular ± Cellular Media media) % increase % increase % increase Mean ± SEM over control Mean ± SEM over control over control Sample Control 1.00 ± 0.14 – 1.00 ± 0.27 – – Insulin 1.82 ± 0.15 82 1.00 ± 0.13 0 82 (1.0 mg/mL) Regulation by a b-agonist isoproterenol
b-adrenoreceptors on capillary endothelial cells was evaluated. Examination of [3H] dihydroalprenolol ([3H]DHA) binding indicated the presence of b-adrenoreceptors with two different affinities on the capillary endothelial cell plasma membrane. The dissociation constants (Kd) were 2.7 ± 0.9 × 10−10 M and 2.96 ± 0.31 × 10−9 M with the corresponding Bmax of 5.1 ± 0.05 and 70.0 ± 0.2 pmol/mg protein, respectively. Inhibition of [3H]DHA binding with atenolol (a b1-antagonist) and ICI 118,551 (a b2-antagonist) has suggested that the IC50cor (=Ki) for atenolol and ICI 118,551 for high-affinity site were 8.0 ± 3.0 × 10−14 M and 25.0 ± 8.0 × 10−14 M, respectively. This indicated that both atenolol and ICI 118,551 were able to displace the bound ligand effectively, but the b1-selective antagonist atenolol was three times more potent than its b2 counterpart, ICI 118,551 [23]. To evaluate whether the b-adrenergic response of isoproterenol is mediated via intracellular 3¢,5¢-cyclic adenosine monophosphate (cAMP), cells were exposed to isoproterenol (1 × 10−7 M) for 30 min in a serum-free media containing 5 × 10−7 M isobutylmethylxanthine (a phosphodiesterase inhibitor). Extracted cAMP was quantified by radiobinding assay [22]. A nearly 1.5-fold increase in cAMP concentration supported that capillary endothelial cells have functional b-receptors. Prior to studying the influence of b-adrenoreceptor activation on factor VIIIc glycosylation, cellular glycoproteins were analyzed in the presence or absence of the b-blockers atenolol and ICI 118,551. The cells were labeled with [3H]mannose (10 mCi/mL) and [14C]leucine (1.25 mCi/mL) in a continuous pulse for 1 h at 37°C in the presence of isoproterenol (1 × 10−9 M) with or without atenolol or ICI 118,551 (5 × 10−8 M). The ratio of [3H]mannose to [14C]leucine incorporation in proteins was quantified in a liquid scintillation spectrometer after precipitating the proteins with 10% trichloroacetic acid. The results indicated that the ratio of [3H]mannose to [14C]leucine incorporation into proteins was reduced by ~45% in cells pretreated with either atenolol or ICI 118,551 [24], thus establishing activation of protein N-glycosylation in capillary endothelial cells by isoproterenol. To address whether isoproterenol specifically increases the glycosylation of factor VIIIc, cells were labeled with 35S-methionine in the presence of increasing concentration of isoproterenol (i.e. 1 × 10−9, 1 × 10−7, 1 × 10−5, and 1 × 10−3 M) and analyzed as before. The incorporation of 35S-methionine was highest at 1 × 10−7 M isoproterenol in both cell lysate and in the conditioned media. In the cell lysate, the order was 10−7 > 10−5 > 10−3 > 10−9 M > control, whereas in the conditioned media, the order was 10−7 > 10−9 > 10−5 > 10−3 M > control (Fig. 24.5).
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Fig. 24.5 Effect of isoproterenol on factor VIIIc biosynthesis. The cells were labeled with [35S] methionine for 1 h at 37°C in the presence of isoproterenol, and factor VIIIc was analyzed in the cell lysate and in the conditioned media. Con control; a = 1 × 10−3 M isoproterenol; b = 1 × 10−5 M isoproterenol; c = 1 × 10−7 M isoprotrenol; d = 1 × 10−9 M isoproterenol Table 24.2 Effect of isoproterenol on the ratio of [3H]mannose to [35S]methionine incorporation in factor VIIIc Total (cellular + Cellular Media media) % increase % increase % increase Mean ± SEM over control Mean ± SEM over control over control Sample Control 1.00 ± 0.14 – 1.00 ± 0.27 – – 1.73 ± 0.08 73 1.45 ± 0.08 45 118 Isoproterenol (1 × 10−7M)
To analyze the glycosylation status, cells were dually labeled with 3H-mannose (10 mCi/mL) and 35S-methionine (40 mCi/mL) for 1 h at 37°C in a low-glucose serum-free/methionine-free DMEM (BioFluids, Inc.) in the presence or absence of isoproterenol (1 × 10−7 M). Factor VIIIc in cell extract and in the conditioned media was immunoprecipitated with the antifactor VIIIc monoclonal antibody and separated on a 10% SDS-PAGE. The protein bands of Mr 200,000 (cellular)/Mr 210,000 (media) and Mr 46,000 (cellular)/Mr 40,000 (media) Da were excised, and the radioactivity was quantified in a liquid scintillation spectrometer. The results indicated that the ratio of 3H-mannose to 35S-methionine in cellular factor VIIIc was increased by 73% after isoproterenol treatment, whereas that of the secretory factor VIIIc was increased by 45% (Table 24.2).
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24.7 Factor VIIIc Expression and Angiogenesis Are Coupled Angiogenesis, i.e. neovascularization, is key to tumor growth and invasion [24, 25]. Significant components of angiogenesis are endothelial cell migration, capillary budding, establishment of capillary loops, and neovascular remodeling. We have seen above that cAMP-related stimuli increase factor VIIIc N-glycosylation, and we have evaluated further whether the same stimulus would accelerate the capillary endothelial cell proliferation and the lumen formation. To examine the cellular proliferation, synchronized culture of capillary endothelial cells was treated with 2 mM 8Br-cAMP in 2% fetal bovine serum. The cells were collected after every 8 h and counted. There was a time-dependent increase in cellular proliferation (Fig. 24.6a). Photomicrographs indicated a lumen-like structure formation after 4 days of 8Br-cAMP treatment (Fig. 24.6b, c). To establish such a coupling more precisely, we used the protein N-glycosylation inhibitor tunicamycin. Tunicamycin is a glucosamine-containing pyrimidine nucleoside and is a competitive inhibitor of N-acetylglucosaminyl-1 phosphate transferase of the dolichol pathway in the ER. We have observed that tunicamycin (1 mg/mL) treatment reduced the LLO formation in capillary endothelial cells. This resulted in decreased factor VIIIc expression and cellular proliferation (Fig. 24.7).
24.8 Role of Factor VIIIc in Capillary Invasion Factor VIIIc is a cofactor in the blood coagulation cascade, but its role in capillary endothelial cell physiology needs to be established. One of our hypotheses is that eFactor VIIIc activates matrix metalloproteinases (MMPs) during capillary invasion a Control
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and supports tumor growth. To test this hypothesis, we have cultured capillary endothelial cells on plates coated with growth-factor-reduced Matrigel™ in the presence and absence of antifactor VIIIc monoclonal antibody as well as tunicamycin. The chemoattractant used in this study was conditioned media from human breast cancer cells MCF7. Capillary endothelial cells were cultured for 24 h, the inserts were processed, and the number of cells passed through the membrane was counted under a microscope after staining with hematoxylin. The control inserts were without Matrigel™ coating. The results in Table 24.3 indicated that antifactor VIIIc monoclonal antibody blocked Matrigel™ invasion of capillary endothelial cells. Tunicamycin at 1 mg/mL showed an increased invasion, but it was reduced to almost one-third at 10 mg/mL. One plausible explanation is that tunicamycin needs more time to act and that it needs higher concentrations to neutralize the effect of a high concentration of the growth factor(s) present in the tumor cell-conditioned media.
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24.9 Summary The physiological role of factor VIIIc in the blood coagulation cascade is established. But very little is known about the biosynthetic regulator(s) of this asparagine-linked (N-linked) glycoprotein. In addition, precise determination of cell-type specificity of factor VIIIc biosynthesis has also not been elucidated. Earlier, we demonstrated the expression of active factor VIIIc protein in a capillary endothelial cell line. This endothelial cell factor VIIIc, i.e. eFactor VIIIc, is a 270-kDa N-linked glycoprotein in which the heavy chain (Mr 210,000 Da) and the light chain (Mr 46,000 Da) are joined together by disulfide bridge(s). In addition, we have also demonstrated that factor VIIIc expression precedes the endothelial cell proliferation. In this article, we have presented evidence that factor VIIIc N-glycosylation is upregulated by cAMP signaling. Upregulation of factor VIIIc N-glycosylation consequently accelerates the capillary endothelial cell proliferation, i.e. angiogenesis. This has been supported by the use of a protein N-glycosylation inhibitor, tunicamycin. Capillary endothelial cells treated with tunicamycin exhibit reduced factor VIIIc level and inhibition of cellular proliferation. In addition, we have also provided evidence that factor VIIIc is responsible for tissue invasion during tumor progression. We used Matrigel™ for this study. Cells cultured in the presence of antifactor VIIIc monoclonal antibody failed to invade the Matrigel™ matrix. This suggested that eFactor VIIIc activates MMPs during tumor invasion, a function analogous to what has been observed in factor VIIIc-dependent activation of factor X in blood coagulation. Our study has also suggested that N-glycosylation of factor VIIIc is very much required for its tissue invasion because cells cultured in the presence of tunicamycin have exhibited considerable inhibition of Matrigel™ invasion. Acknowledgments The authors greatly appreciate the editorial assistance of Ms. Laura M. Bretaña. The authors also acknowledge the technical help provided by Mr. Jonathan Caldera Colón and Miss Maria Teresa Milán Mello. The authors are also indebted to Dr. Amitava Banerjee for his critical reading of the manuscript and for enhancing the quality of the images. The work has been supported in part by grants from the Department of Defense DAMD17-03-1-0754, the NIHU54-CA096297, and the Susan G. Komen Breast Cancer Foundation BCTR58206 (to Dipak K. Banerjee) and NIH/NCRR/RCMI grant G12-RR03035 (to Krishna Baksi).
References 1. Fay PJ (1999) Regulation of factor VIIIa in the intrinsic factor Xase. Thromb Haemost 82:193–200 2. Kane WH, Davie EW (1988) Blood coagulation factors V and VIII: structural and functional similarities and their relationship to hemorrhagic and thrombotic disorders. Blood 71:539–555 3. Lenting PJ, van Mourik JA, Mertens K (1998) The life cycle of coagulation factor VIII in view of its structure and function. Blood 92:3983–3996
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4. Saenko EL, Scandella D (1995) A mechanism for inhibition of factor VIII binding to phospholipid by von Willebrand factor. J Biol Chem 270:13826–13833 5. Toole JJ, Knopf JL, Wozney JM, Sultzman LA, Buecker JL, Pittman DD, Kaufman RJ, Brown E, Shoemaker C, Orr EC, Amphlett GW, Foster WB, Coe ML, Knutson GJ, Fass DN, Hewick RM (1984) Molecular cloning of a cDNA encoding human antihaemophilic factor. Nature 312:342–347 6. Vehar GA, Keyt B, Eaton D, Rodriguez H, O’Brien DP, Rotblat F, Oppermann H, Kock R, Wood WI, Harkins RN, Tuddenhan GD, Lauen RM, Capon DJ (1984) Structure of human factor VIII. Nature 312:337–342 7. Baumgartner S, Hofmann K, Chiquest-Ehrismann R, Bucher P (1998) The discoidin domain family revisited: new members from prokaryotes and a homology-based fold prediction. Protein Sci 7:1626–1631 8. Pellequer JI, Gale AJ, Griffin JH, Getzoff ED (1998) Homology models of the C domains of blood coagulation factors V and VIII: a proposed membrane binding mode for FV and FVIII C2 domains. Blood Cells Mol Dis 24:448–461 9. Gilbert GE, Drinkwater D (1993) Specific membrane binding of factor VIII is mediated by O-phospho-L-serine, a moiety of phosphatidylserine. Biochemistry 32:9577–9585 10. Spiegel PC Jr, Jacquemin M, Saint-Remy JM, Stoddard BL, Pratt KP (2001) Plenary paper: structure of a factor VIII C2-domain–immunoglobulin G4? Fab complex: identification of an inhibitory antibody epitope on the surface of factor VIII. Blood 98:13–19 11. Banerjee DK, Ornberg RL, Youdim MB, Heldman E, Pollard HB (1985) Endothelial cells from bovine adrenal medulla develop capillary-like growth patterns in culture. Proc Natl Acad Sci USA 82(14):4702–4706 12. Banerjee DK (1988) Microenvironment of endothelial cell growth and regulation of protein N-glycosylation. Indian J Biochem Biophys 25:8–13 13. Martinez JA, Torres-Negrón I, Amígo LA, Banerjee DK (1999) Expression of Glc3Man9GlcNAc2PP-Dol is a prerequisite for capillary endothelial cell proliferation. Cell Mol Biol 45:137–152 14. Martínez JA, Tavárez JJ, Oliveira CM, Banerjee DK (2006) Potentiation of angiogenic switch in capillary endothelial cells by cAMP: a cross-talk between up-regulated LLO biosynthesis and the HSP-70 expression. Glycoconj J 23:209–220 15. Banerjee DK, Tavárez JJ, Oliveira CM (1992) Expression of blood clotting factor VIII: C gene in capillary endothelial cells. FEBS Lett 306:33–37 16. Eriksson UJ, Lewis NJ, Freinkel N (1984) Growth retardation during early organogenesis in embryos of experimentally diabetic rats. Diabetes 33:281–284 17. Susa JB, Neave C, Sehgal P, Singer DB, Zeller WP, Schwartz R (1984) Chronic hyperinsulinemia in the fetal rhesus monkey. Effects of physiologic hyperinsulinemia on fetal growth and composition. Diabetes 33:656–660 18. Taub R, Roy A, Diater R, Koontz J (1987) Insulin as a growth factor in rat hepatoma cells stimulation of proto-oncogene expression. J Biol Chem 262:10893–10897 19. Oliveira CM, Banerjee DK (1990) Role of extracellular signaling on endothelial cell proliferation and protein N-glycosylation. J Cell Physiol 144:467–472 20. Brush JS, Tavárez-Pagán JJ, Banerjee DK (1991) Insulin and IGF-1 manifest differential effects in a clonal capillary endothelial cell line. Biochem Int 25:537–545 21. Tavarez-Pagan JJ, Oliveira CM, Banerjee DK (2004) Insulin up-regulates a Glc3Man9GlcNAc2PP-Dol pool in capillary endothelial cells not essential for angiogenesis. Glycoconj J 20:179–188 22. Banerjee DK, Vendrell-Ramos M (1993) Is asparagine-linked protein glycosylation an obligatory requirement for angiogenesis? Indian J Biochem Biophys 30:389–394 23. Das SK, Mukherjee S, Banerjee DK (1994) Beta-adrenoreceptors of multiple affinities in a clonal capillary endothelial cell line and its functional implication. Mol Cell Biochem 140:49–54 24. Uhr JW, Scheuermann RH, Street NE, Vitetta ES (1997) Cancer dormancy: opportunities for new therapeutic approaches. Nat Med 3:505–509 25. Gastl G, Hermann T, Steurer M, Zmija J, Gunsilius E, Unger C, Kraft A (1997) Angiogenesis as a target for tumor treatment. Oncology 54:177–184
Chapter 25
Mucin O-Glycan Branching Enzymes: Structure, Function, and Gene Regulation Pi-Wan Cheng and Prakash Radhakrishnan
Keywords Mucin O-glycan • Branching enzymes • C2GnT • b6N-acetylglucosa minyltransferase
25.1 Introduction Mucin O-glycans are conjugated carbohydrate chains that contain at the reducing terminus N-acetylgalactosamine linked covalently to serine/threonine in the peptide backbone. They are found in mucins and many nonmucin glycoproteins. The presence of a tandem repeat peptide heavily glycosylated with mucin O-glycans distinguishes mucins from other glycoproteins that contain mucin O-glycans. Muc14, -15, and -18, which do not contain a tandem repeat peptide, are the exceptions. To date, about six secreted and 14 membrane-tethered mucins have been reported based on cloned complementary DNA (cDNA) sequences [1, 2]. The functions of mucin O-glycans vary according to where they reside. For secreted mucins, O-glycans can retain water, maintain the viscoelastic properties of mucus secretion, and bind and clear inhaled and ingested pathogens, such as mycoplasma [3, 4], viruses [5], and bacteria [6]. This function depends primarily on heterogeneous carbohydrates, while the other two functions are determined by high carbohydrate content. High carbohydrate content and very heterogeneous carbohydrate structures found in secreted mucins enable them to perform the first line of innate immune defense at the epithelial surface of many mucus-secretory tissues, such as airways and the gastrointestinal tract. Under pathological conditions, overproduction of secreted mucins coupled with poor clearance of mucus causes obstructive lung diseases [7]. On the contrary, loss of secreted mucins as shown in Muc2-gene-knockout mice can result in the loss of mucus protective Pi-Wan Cheng (*) Department of Biochemistry and Molecular Biology, College of Medicine and Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198-5870, USA e-mail:
[email protected] A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_25, © Springer Science+Business Media, LLC 2011
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function and lead to the development of colitis [8] and colorectal cancer [9]. Mucin-type O-glycans in membrane-tethered mucins protect the epithelium by trapping water at the cell surface, transmitting signals from the extracellular environment to the cells, and serving as selectin ligands [10, 11]. Expression or elevated expression of these mucins tends to be associated with malignancy, despite the fact that the significance is not fully understood [12]. The best characterized function of mucin-type glycans comes from membrane-bound nonmucin glycoproteins, including the P-selectin glycoprotein ligand (PSGL)-1 and peripheral node addressin. These glycoproteins contain sialyl Lewis X (sLex) or 6-sulfo-sLex located at the nonreducing termini of mucin O-glycans. These glycotopes can guide the migration of leukocytes to the site of injury and lymphoid organs [13]. These two processes depend primarily on interactions of sLeX, sLeX plus sulfated tyrosine, and sulfated sLeX with E-, P-, and L-selectins, respectively [14]. Such interactions may also play a key role in cancer metastasis [15]. The above-mentioned functions of mucin O-glycans are controlled by b6GlcNAc branch structures, which include core 2, Galb1-3(GlcNAcb1-6) GalNAcaSer/Thr; core 4, GlcNAcb1-3(GlcNAcb1-6)GalNAcaSer/Thr; and blood group I antigen, GlcNAcb1-3(GlcNAcb1-6)Gal. Core 2 is the branch structure present in membrane-tethered mucins and other nonmucin glycoproteins, while all three branch structures are found in secreted mucins. The differences in these mucin glycan branch structures contribute to the differences in the function of these glycoproteins. The enzyme activity responsible for the synthesis of the core-2 structure was first reported in 1980 [16]. The enzyme activity responsible for the core-4 structure was identified in 1985 [17] and that for the blood group I structure in 1986 [18]. In 1991, our group employed a purified enzyme to demonstrate that this enzyme can make all three b6GlcNAc structures found in secreted mucins [19]. cDNA coding C2GnT-1/L was first cloned in 1992 [20], IGnT in 1993 [21], C2GnT-2/M in 1999 [22, 23], and C2GnT-3/T in 2000 [24]. Enzymatic characterization of the recombinant protein of C2GnT-M [22, 23] has confirmed its multiple substrate specificity as previously reported [19]. These b6GlcNAc transferases contain nine conserved cysteines [20–24]. Recently, the disulfide bond distribution in C2GnT-L [25] and C2GnT-M [26] has been reported. Also, the genomic structures of the IGnT [27], C2GnT-L [28], and C2GnT-M [29] genes have been elucidated. Furthermore, the 3D structure of C2GnT-L has been determined [30]. Since many biological functions of mucins reside in the carbohydrates extended from the b6GlcNAc branch structures, alterations of the activities of these enzymes and their gene expressions can have a profound effect on mucin functions. The current review is an update of an article published previously [31].
25.2 Role of B6GlcNAc Transferases in Mucin O-Glycan Biosynthesis The three mucin O-glycan b6GlcNAc structures [31, 32], including core 2, core 4, and I antigen, are shown in Fig. 25.1. Mucin O-glycan synthesis is initiated by the formation of the GalNAcaSer/Thr structure as catalyzed by peptidyl GalNAc transferases (ppGalNAc-T) [33]. At least 20 ppGalNAc-Ts have been identified to date.
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Fig. 25.1 Biosynthesis of mucin O-glycan core 2, core 4, and blood group I antigen as catalyzed by b6GlcNAc transferases. Core 2 can be synthesized by C2GnT-1/L, C2GnT-2/M, and C2GnT3/T; core 4 can be synthesized only by C2GnT-M; blood group I antigen can be synthesized by IGnT and C2GnT-M
These enzymes can be grouped into three categories based on acceptor specificities: one works on naked peptides, one works on partially glycosylated peptides, and one works on both types of acceptors [34–36]. To extend the glycan structure, GalNAc can be decorated with either b3Gal or b3GlcNAc to form core-1 and core-3 structures, respectively. Each of these two enzymes is encoded by a single gene. Core-1 (or T) synthase requires a chaperone protein for its function [37]. Loss of this chaperone protein can result in the loss of core-1 synthase activity [37]. The core-1 structure can be extended by either b3GlcNAcT-3 or C2GnTs, which produce linear or b1-6 branch structures, respectively. There are three types of C2GnTs: C2GnT-1/L, C2GnT-2/M, and C2GnT-3/T. These enzymes can catalyze the transfer of GlcNAc from UDPGlcNAc to GalNAc in the core-1 acceptor. The core-1 structure (or T antigen) is required for C2GnT activity. To generate L-selectin ligand, C-6 of GlcNAc is sulfated by ST6GlcNAcT-1/2 [38, 39] before galactosylation by b4GalT [40], as shown in Fig. 25.2. To generate P- and E-selectin ligands, GlcNAc on core 2 and extended core 1 is galactosylated without sulfation. If a polylactosamine structure is to be generated, the terminal b4Gal will be extended by N-acetyllactosamine unit(s), as catalyzed sequentially by b3GlcNAcT-3 [41] and b4GalT. To generate an sLex structure, the terminal Gal will be further decorated with a2-3 sialic acid as catalyzed by ST3Gal-III, -IV, or -VI [42] followed by fucosylation as catalyzed by FUT-III, -IV, or -VII [42]. Core 4 is the second b6GlcNAc branch structure found in secreted mucins. It is synthesized by C2GnT-M after the core-3 structure has been generated by core-3 synthase [43]. A core-3 structure is required for C4GnT activity. There is only one gene that can produce core-3 synthase. Furthermore, C2GnT-M is the only enzyme that exhibits C4GnT activity. Core-3 and core-4 structures can be further extended by galactosyltransferases, N-acetylglucosaminyltransferases, sialyltransferases, and fucosyltransferases to generate biologically active glycotopes, such as sLex, as described above.
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Fig. 25.2 Structure and biosynthesis of core 1- and core 2-associated sLex and key enzymes involved in the synthesis. (1) ppGalNAcT; (2) b3Gal-T1 (T synthase); (3) C2GnT-1/2/3;(4 and 9) GlcNAc6ST1/3; (5) b4GalT-4; (6 and 11) FUT-III/IV/VII; (7 and 12) ST3Gal-III/VI; (8) b3GlcNAcT-3; and (10) b4GalT
The blood group I antigen is the third b6GlcNAc branch structure found in polylactosamine chains of the glycans in secreted mucins and N-linked glycans. The structures can be generated by two different enzymes, IGnT [21] and C2GnT-M [22, 23], which act centrally and distally, respectively. C2GnT-M transfers GlcNAc from UDP-GlcNAc to Gal in GlcNAcb1-3Gal, while IGnT transfers GlcNAc to Gal at the reducing end of Galb1-3/4GlcNAcb1-3Gal. It is clear that carbohydrate content and complexity of mucin O-glycan structures are controlled to a large extent by these mucin O-glycan branching enzymes. Therefore, modulation of the activities of these branching enzymes is crucial for regulating mucin functions. The next two sections focus on the protein and genomic structures of these mucin O-glycan branching enzymes.
25.3 Protein Structures of b6GlcNAc Transferases As described above, mucin core-2 structure can be synthesized by three different b6GlcNAc transferases, the core-4 structure by C2GnT-M, and I antigen by two different b6GlcNAc transferases. cDNAs of these b6GlcNAc transferases have been cloned from several different species, including bovine, bovine herpesvirus, dog, chimpanzee, human, mouse, rat, and zebra fish. The amino acid sequences deduced from these cDNAs show that these branching enzymes are single-pass type II membrane proteins. They are similar in size (399–454 amino acids) and exhibit domain structures typical of most glycosyltransferases. They contain a short cytoplasmic tail at the N-terminus followed by a short transmembrane region, a short stem, and a long catalytic domain. The transmembrane domain plus cytoplasmic tail is the primary signal for targeting C2GnT-L to the cis-medial Golgi compartment [44].
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The alignment of the amino acid sequences deduced from the cDNAs of these b6GlcNAc branching enzymes shows several distinct features (Fig. 25.3). First, all b6GlcNAc transferases contain nine conserved cysteines located beyond the stem region. C2GnT-T contains an additional cysteine located at the cytoplasmic tail. C2GnT-M contains four additional cysteines located at the N-terminal region,
Fig. 25.3 Alignment of deduced amino acid sequences of four b6GlcNAc transferases from human (Homo sapiens) (h), chimpanzee (Pan troglodytes) (c), rat (Rattus norvegicus) (r), mouse (Mus musculus) (m), bovine (Bos taurus) (b), bovine herpesvirus (BHV), dog (Canis familiaris) (dog), and zebra fish (Danio rerio) (d). Multiple sequence alignment was performed with Invitrogen Vector NTI 10 software. The amino acid sequences of dog C2GnT-M and C2GnT-T, and mC2GnT-T used for alignment, include only those that are comparable to those of other b6GlcNAc transferases. The N-terminal amino acid sequences that are excluded are 96 amino acid residues of dog C2GnT-M, 137 residues of dog C2GnT-T, and 133 residues of mC2GnT-T. The N-glycosylation sites (N) are highlighted dark, and the nine cysteines conserved among all b6GlcNAc transferases, the four cysteines conserved among C2GnT-Ms, and the one cysteine conserved among C2GnT-Ts are highlighted light. The potential N-glycosyltaion sites in each sequence are indicated by arrow heads. The accession numbers for these proteins are C2GnT2/M: bovine, NP_991378.1; bovine herpesvirus, Q80RC7; dog, XP_544703; chimpanzee, XP_510451.2; human, NP_004742.1; mouse, NP_082363.2; rat, NP_775434.1. C2GnT-3/T: chimpanzee, XP_517702.2; human, NP_057675.1; dog, XP_546063.2; mouse, XP_980153.1; zebra fish, NP_963877.1. IGnT: human, Q06430-1; mouse, NP_032131.
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including the cytoplasmic tail, transmembrane region, and stem. These four cysteines are not required for enzyme activity. The designation of the various regions of these branching enzymes, such as cytoplasmic tail, transmembrane domain, stem, and catalytic domain, is to indicate the general domain structures. The exact lengths of the cytoplasmic tail and the transmembrane domain of each branching enzyme as predicted by different software may vary and need to be determined individually because cDNA devoid of this region still retain all three activities [26]. The function of these four cysteines is not known, although they may be involved in disulfide bond formation to help retain C2GnT-M in specific Golgi compartments according to the oligomerization/kin recognition model of the Golgi retention theory [45]. Second, all b6GlcNAc transferases, except h- and mIGnT, contain one conserved N-glycosylation site: b- and bhvC2GnT-M (N-72); dogC2GnT-M (N-71); c-, h-, m-, and rC2GnT-M (N-69); c- and hC2GnT-T (N-72); dogC2GnT-T (N-73); dC2GnT-T (N-59); and all C2GnT-L (N-58). The corresponding N-glycosylation site for h- and mIGnT is located at N-37 instead. While there is no additional N-glycosylation site for zebra fish C2GnT-T, the number of additional N-glycosylation sites found in other b6GlcNAc transferases ranges from one to four. The locations of these N-glycosylation sites are b- and bhvC2GnT-M (N-108); dogC2GnT-M (N-107); c- and hC2GnT-M (N-289); m- and rC2GnT-M (N-288); c- and hC2GnT-T (N-286, N-317, and N-448); dog- (N-318) and mC2GnTT (N-318 and N-381); hIGnT (N-212, N-255, N-314, and N-388); mIGnT (N-212, N-255, N-315, and N-389); and all C2GnT-L (N-95). Third, amino acid sequence identity analysis of these b6GlcNAc transferases reveals that human is closest to chimpanzee, mouse to rat, and bovine to bovine herpesvirus (Fig. 25.4). The pattern of distribution of the disulfide bonds derived from these nine cysteines conserved among all b6GlcNAc transferases has been elucidated for mC2GnT-L [25] and bC2GnT-M [26]. For both isozymes, eight cysteines are engaged in the formation of disulfide bonds, although different cysteines are involved. In mC2GnT-L [25], the sulfhydryl group of the sixth cysteine (Cys217) is not conjugated, and the cysteine pairs involved in the formation of disulfide bonds are first and ninth (Cys59-Cys413), second and fourth (Cys100-Cys172), third and fifth (Cys151-Cys199), and seventh and eighth (Cys372-Cys381). Enzyme activity is retained when Cys217 is at the reduced state [25]. In addition to these nine conserved cysteines, mC2GnT-L contains an extra cysteine (Cys235), which is responsible for
Fig. 25.3 (continued) C2GnT-1/L: bovine, NP_803476.1; mouse, NP_034395.1; rat, NP_071612.1; chimpanzee, XP_001145936.1; human, NP_001091103.1; dog, XP_541274.2. The accession numbers of the corresponding cDNAs are C2GnT-2/M: bovine, NM_205809.1; bovine herpesvirus, NC_002665; dog, XM_544703;chimpanzee, XM_510451.2; human, NM_004751.1; mouse, NM_028087.2; rat, NM_173312.1. C2GnT-3/T: human, NM_016591.1; chimpanzee, XM_517702.2; dog, XM_546063.2; mouse, XM_975059.1; zebra fish, NM_201583.1. IGnT: human, Z19550; mouse, NM_008105. C2GnT-1/L: bovine, NM_177510.2; mouse, NM_010265.2; rat, NM_022276.1; chimpanzee, XM_001145936.1; human, NM_001097634.1; dog, XM_541274.2
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Fig. 25.4 Cladogram of four b6GlcNAc transferases from different species. Amino acid sequences of the same isozyme are closest between human and chimpanzee, mouse and rat, and bovine and bovine herpesvirus. Multiple sequence alignment was performed with European Molecular Biology Laboratory–European Bioinformatics Institute (EMBL–EBI) clustalW. The number in parenthesis represents the degree of divergence in a pair of sequences
the formation of a dimer through the formation of an intermolecular disulfide bond. In the case of bC2GnT-M, the sulfhydryl group of the second conserved cysteine (Cys113) is not conjugated. The disulfide bonds are formed between the following cysteine pairs: first and ninth (Cys73-Cys425), third and seventh (Cys164-Cys381), fourth and fifth (Cys185-Cys212), and sixth and eighth (Cys230-Cys393). Except for the disulfide bond formed between the first and ninth cysteines, bC2GnT-M and mC2GnT-L do not share the same pattern of distribution of free cysteine and disulfide bonds among the remaining seven conserved cysteines, which may contribute to the difference in substrate specificity between these two enzymes [26]. Recently, Pak et al. have elucidated the 3D structure of mC2GnT-L by X-ray crystallography [30]. This enzyme exhibits a GT-A fold but lacks the characteristics of a metal ion-binding DXD motif. The amino acids involved in the binding to UDP-GlcNAc include Arg-378, Lys-401, Asp-155, Val-128, Val-354, Val-380, Glu320, and Cys-217. Arg-378 and Lys-401, which are conserved among all GT-14 family members and take the place of a metal ion in all other GT-A structures, stabilize the b-phosphate of UDP-GlcNAc. The use of basic amino acid side chains in this way is strikingly similar to that observed in a number of metal ion-independent GT-B-fold glycosyltransferases and suggests a convergence of catalytic mechanisms shared by both GT-A- and GT-B-fold glycosyltransferases [30]. The existence of Lys-401 in the cis-peptide conformation suggests an important functional role. Asp-155 is an amino acid well-conserved among many GT-A-fold glycosyltransferases. It forms a key hydrogen bond with N-3 of the uracil moiety. The backbone carbonyl group accepts a hydrogen bond from O-3¢ of the ribose moiety. Val-354 and Val-380 form an apolar pocket that interacts with the GlcNAc N-acetyl methyl group. Furthermore, Glu-320 is closely aligned in the three-dimensional space in the catalytic site. It is positioned for an in-line attack on the C-1 of GlcNAc
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in UDP-GlcNAc. It activates the C-6 hydroxyl group of GalNAc in the core-1 acceptor to make it more nucleophilic to attack C-1 of the donor substrate. Cys-217 is located in the UDP-GlcNAc binding pocket near the ribose moiety and involved in catalysis. C2GnT-L activity is activated by b-mercaptoethanol and dithiothreitol, indicating requirement of maintaining the sulfhydryl group in the reduced state for enzyme activity [46]. The enzyme activity is partially, i.e., 40%, retained when Cys217 is converted to serine, indicating that the serine OH group can partially substitute the SH group in this cysteine [46]. Treatment of this enzyme with iodoacetamide, 5,5¢-dithiobis-(2-nitrobenzoic acid) (DTNB), or N-ethylmaleimide inactivates the enzyme by covalent blocking of the sulfhydryl group in Cys-217. The enzyme activity is protected from inactivation by iodoacetamide or DTNB in the presence of UDP-GlcNAc, but not the disaccharide acceptor. The result supports the X-ray crystallography result that this cysteine is in close contact with UDP-GlcNAc and not the acceptor. The amino acids involved in binding to the disaccharide acceptor through formation of hydrogen bonds include Glu-320, Arg-254, Glu-243, Tyr-358, Lys-251, and Try-356. Glu-320, which is a critical amino acid for the catalytic activity of GT-A-fold glycosyltransferases [47, 48] and conserved among all b6GlcNAc transferases, forms a bidentate with O-4 and O-6 of GalNAc by accepting hydrogen bonds from O-4 and the nucleophilic O-6. Arg-254 donates a hydrogen bond to O-4 of GalNAc in the acceptor. Glu-243 forms a bidentate with O-4 and O-6 of Gal by accepting hydrogen bonds from both oxygens. Tyr-358 bridges the two monosaccharides in the acceptor by simultaneously accepting a hydrogen bond from GalNAc NH and donating a hydrogen bond to Gal O-2. Lys-251 forms a hydrogen bond with the glycosidic oxygen of the acceptor disaccharide. The acceptor binding is further stabilized by a stacking interaction between Try-356 and both Gal and GalNAc moieties. It is of interest to note that the amino acid Y358, which was identified to be the amino acid involved in the binding of mC2GnT-L with core-1 disaccharide acceptor, was proposed to be unique to C2GnT-L because a different amino acid was found in the same location of both human (G458) and bovine (G460) C2GnT-M. Since tyrosine (Y460) instead of glycine is found at the same location in bhvC2GnT-M, tyrosine cannot be the amino acid that is unique to C2GnT-L. Therefore, the difference in this amino acid between mC2GnT-L and h-/ bC2GnT-M cannot explain the difference in acceptor specificity between these two isozymes, suggesting that amino acids other than Y460 are involved in determining the multiacceptor specificity of C2GnT-M.
25.4 Genomic Organization of C2GnT Genes To date, the complete genomic structures of C2GnT1/-L [49], C2GnT2/-M [50], and IGnT [21] and a partial genomic structure of C2GnT3/-T [47] have been reported (Fig. 25.5). It is worth noting that the open reading frame (ORF) of IGnT is distributed over three exons [23], while the entire ORF of the three C2GnT genes is located in a single exon [24, 31]. As shown in Table 25.1, these four human b6GnT genes
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Fig. 25.5 Genomic structures and expression of human (a) C2GnT-1/L (GCNT1), (b) C2GnT2/M (GCNT3), and (c) C2GnT-3/T (GCNT4) genes. ORF of all the three C2GnT isozymes is located in one exon
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Table 25.1 Chromosomal localization of human b6GlcNAc transferase genes and tissue distribution Chromosomal Enzyme location Tissue specificity C2GnT-L 9q13 Ubiquitously expressed in all tissues and highly expressed in activated T lymphocytes and myeloid cells C2GnT-M 15q21.3 Primarily expressed in mucus-secreting tissues, including colon, testis, stomach, small intestine, kidney, trachea, adrenal gland, thyroid gland, uterus, ovary, and pancreas C2GnT-T 5q12 Predominantly expressed in the thymus. Weakly expressed in pancreas, peripheral blood leukocytes, placenta, small intestine, and stomach. Barely detectable in liver, spleen, lung, and lymph node IGnT 9q21 Erythroid cells, lymphocytes, monocytes, granulocytes, platelets, lens epithelium, and other tissues. Differential expression of specific transcripts in different tissues
are located at different chromosomes. Since the hIGnT and mC2GnT-1/L gene structures have been reviewed previously [31], the current chapter concentrates on the structures of the hC2GnT-1/L; b-, m-, and hC2GnT-2/M; and hC2GnT-3/T genes. Mouse [51–53] and human [49] C2GnT-L genes contain six exons distributed over 60 and 48 kb, respectively. Human C2GnT-L gene (48.2 kb) is made of six exons – A (650 bp), B (89 bp), C (118 bp), D (190 bp), E (426 bp), and F (2,022 bp) – and five introns – I1 (16,902 bp), I2 (755 bp), I3 (18,164 bp), I4 (22,143 bp), and I5 (1,178 bp) (Fig. 25.5a). Like the mC2GnT-L gene, the hC2GnT-L gene is expressed in a tissue-specific manner, employing multiple transcription initiation and alternative splicing mechanisms. The four promoters identified are located at the regions upstream of exons A, B, D, and E. The major promoter is promoter 2, which lacks a TATA box and is very GC rich. In addition, exons B, C, D, and part of exon E are alternately spaced out [54]. The structures of bovine [29], mouse [55], and human [50] C2GnT-M genes have recently been characterized. The C2GnT-M genes from these three species are made of three exons and two introns. The bovine gene exhibits a tissue-specific expression not observed in the other two species [50, 55]. The human C2GnT-M gene is located on chromosome 15 in the region of q21.3, oriented from centromere to telomere. The human C2GnT-M gene (~8.26 kb) is made of 3 exons – E1 (69– 198 bp), E2 (333–401 bp), and E3 (1,864 bp) – and 2 introns – I1 (4.5 kb) and I2 (1.3 kb). It produces three transcripts of 2.3–2.5 kb, 3.6–3.8 kb, and 6.8–7.0 kb (Fig. 25.5b). The shortest transcript (~2.3–2.5 kb) is made of three exons: 69–198 bp (exon 1); 333–401 bp (exon 2); and 1,864 bp (exon 3). This exon includes 56-bp 5¢-UTR, 1,317-bp ORF, and 491-bp 3¢-UTR. It does not contain any intron and is the most abundant transcript. The intermediate size transcript (3.6– 3.8 kb) contains intron 2, and the longest transcript (6.8–7.0 kb) contains intron 1 in addition to all three exons. The bovine C2GnT-M gene (5.3 kb) is made of a 128-bp exon 1, a 1,427-bp intron 1, a 186-bp exon 2, a 1,771-bp intron 2, and a 1,796-bp exon 3. Exon 3 contains 51-bp 5’-UTR, 1,323-bp ORF, and 424-bp 3¢-UTR.
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The smaller-size transcripts are the most abundant transcripts. There are two northern transcript bands, one at 2.1 kb and the other one at 3.7 kb. Each transcript band contains one transcript with and one transcript without exon 1. The transcripts that contain exon 1 are detected only in trachea and testis, indicating tissue-specific expression of these exon-1–containing transcripts. Mouse C2GnT-M gene (6.6 kb) is made of a 116-bp exon 1, a 998-bp intron 1, a 342-bp exon 2, a 1,294-bp intron 2, and a 1,794/3,843-bp exon 3. The mC2GnT-M transcripts do not contain any intron, and the difference in the transcript size, i.e., 2.25 kb versus 4.3 kb, lies in a difference in the length of the 3¢-UTR of exon 3; the short transcript is 561 bp and that of the long transcript is 2.6-kb. In addition, both transcripts contain 54-bp 5¢UTR and 1,311-bp ORF. The human C2GnT-T gene characterized to date is 3,435 kb. It contains 861-bp 5¢-UTR, 1,359-bp ORF, and 1,215-bp 3¢-UTR (Fig. 25.5c). The expression of this enzyme is highly restricted to the thymus tissue [24]. Human IGnT belongs to the b6GlcNAc transferase gene family and forms branched poly-N-acetyllactosamine from linear poly-N-acetyllactosamine [56]. This enzyme is mainly involved in red-cell differentiation and maturation during embryonic development. It has three transcripts: IGnTA, IGnTB, and IGnTC. The last transcript is mainly involved in red-cell development. Expression of IGnTC is regulated by transcription factor C/EBP alpha [57]. Also, a nonsense mutation in this gene is associated with autosomal recessive congenital cataracts in four distantly related Arab families from Israel [58]. During tumor development, this IGnT expression is upregulated. The structure and function of the IGnT enzyme have previously been described in detail [31].
25.5 Role of Human C2GnTs in Health and Disease Core-2 branching is an important step in the biosynthesis of mucin O-glycans. Core-2-associated glycotopes, such as sLex, can serve as ligands for selectin- and galectin-mediated cell–cell interactions involved in T-cell development [31, 54, 59–64] and cancer metastasis [15, 65–68]. The roles of C2GnT in cell development [59, 60], differentiation [69–71], and diseases, such as Wiskott–Aldrich syndrome [72–78], acquired immune deficiency syndrome (AIDS) [79], multiple sclerosis [80], and diabetes [81, 82], have been reviewed previously [31]. Therefore, they will not be dealt with in the current review. Instead, we focus on the role of C2GnT in immune functions, cancer progression, and the estrous cycle.
25.5.1 Role of C2GnT in Immune Function As many important functions of the immune cells reside in core-2-associated glycotopes, changes in C2GnT activity can greatly affect the functions of these cells.
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For example, a dramatic increase of C2GnT enzyme activity has been reported in activated T cells [83–85]. Stimulation of T lymphocytes with anti-CD3 antibodies or interleukin (IL)–2 increases the apparent size, as shown by decreased electrophoretic mobility, of CD43 expressed on the cell surface from 115 kDa to 130 kDa [86, 87]. The mobility shift of CD43 is due to changes in the CD43 mucin O-glycan structures, which include conversion of these glycans from core 1- to core 2-based structures and decoration of core 2 with new glycotopes, such as sLex. This transition resulted from a tenfold increase of C2GnT enzyme activity after T-cell activation coupled with a moderate decrease of a2-3sialyltransferase activity. This enzyme competes with C2GnT for core 1. Since C2GnT-L is localized at cis- and medial-Golgi [88] and a2-3sialyltransferase is localized at medial- and trans-Golgi [88], increase of C2GnT-L activity ensures the synthesis of predominantly core-2associated sLex, which helps recruit circulating neutrophils to the inflamed peritoneum [59, 64, 89]. a2-3Sialyltransferase I is highly expressed during Th2, but not Th1, differentiation [90]. Lack of expression of this glycosyltransferase in Th1 allows the formation of more core-2 glycans to help Th1 cells bind to selectins on the endothelial cells. Similarly, transfection of the EL-4 T-lymphoma cell line with C2GnT-L cDNA upregulates not only the highly glycosylated form of CD43 (130 kDa) but also the sizes of RPTPa, CD44, and CD45 by 3–5 kDa [91]. Since CD44 and CD45 along with CD43 are involved in cell–cell interactions, changes in their glycoforms after activation of T cells can have a significant impact on T-cell functions. Furthermore, Kikuchi et al. [92] found that C2GnT-L is essential for differentiation of human precursor B cells. The connection between C2GnT activity and polylactosamine came from the finding that mucin glycans from granulocytic cells [93] contain core-2-based polylactosamine structure. Such core-2-based polylactosamines along with sLex and sLea epitopes were detected in HL-60 cells, which expressed C2GnT, but not in K562 cells, which did not express C2GnT [94]. The polylactosamine chains are often terminated with sLea, sLex, and 6-sulfo sLex, which serve as selectin ligands. These selectin–ligand interactions are involved in directing leukocyte trafficking under inflamed conditions. P-selectin is found on the surface of activated endothelial cells and platelets, E-selectin on the endothelial cells, and L-selectin on the surface of leukocytes [95–98]. Circulating leukocytes also provide carbohydrate ligands for P- and E-selectins, and endothelial cells at the high endothelial venues provide sulfated carbohydrate ligands for L-selectin. Activated endothelial cells at the injured site mobilize prestored P-selectin to the cell surface to capture circulating leukocytes, which contain PSGL-1 [99], a molecule containing sulfated tyrosine and sLex on mucin in the core 2 chain at the N-terminus. The synthesis of selectin ligands on PSGL-1 in CD4+ T cells is enhanced by IL-12, which upregulated C2GnT gene expression via the STAT4 pathway [100]. E-selectin recognizes sLex located at either mucin-type or N-linked glycan, while L-selectin recognizes sulfated sLex on mucin-type glycan [101, 102]. These lectin–glycan interactions facilitate initial rolling of leukocytes on endothelial cells and subsequent firm binding prior to extravasation. In this case, endothelial cells at the high endothelial venues provide sulfated sLex located on the mucin core-2 branch of membrane-bound
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g lycoproteins [103], such as GlyCAM-1, CD34, podocalyxin-like protein, sgp200, endomucin, and MAdCAM-1. These results strongly demonstrate the involvement of mucin-type core-2 glycans in leukocyte trafficking during inflammation. It is of interest to note that eosinophil and neutrophil, which express core 2 glycans, are mobilized to the peritoneum and not to the lungs [104]. Dimerization of PSGL-1 coupled with formation of core-2 and associated glycans increases tethering rate under flow, while C2GnT-L levels influence tether bond strength [105]. Such selectin–ligand interactions were verified by binding assay between immobilized selectins and recombinant Chinese hamster ovary (CHO) cells that had expressed various combinations of PSGL-1, C2GnT, and a1-3fucosyltransferase, the key players involved in the formation of PSGL-1-associated sLex, which is absent in wild-type CHO cells. P- and E-selectins were found to bind recombinant CHO cells that expressed PSGL-1 only when both C2GnT and a1-3fucosyltransferase were coexpressed [106, 107]. Furthermore, L-selectin bound to the CHO cells that coexpressed C2GnT-L, FUT-VII, and L-selectin ligand sulfotransferase [108]. Although core-2-associated glycan is the preferred ligand for L-selectin, L-selectin also can bind to similar structures located on the core-1 chain but with lesser efficiency [62]. In oral cavity carcinomas, both C2GnT and a1-3fucosyltransferase contribute to the formation of sLex and binding to E-selectin, but not to other selectins [66]. These reports indicate that cell-specific expression of these two enzymes exhibits different specificity for different selectins. The L-selectin ligands also include sulfated core 2-based O-glycans, unlike the glycan ligands for P- and E-selectins. The selectin ligands on mucin O-glycans are contributed mainly by C2GnT-L and FUTVII, although C2GnT-L may play a more significant role than FUT-VII [109]. This report is consistent with the finding that transfection of antisense oligonucleotides against C2GnT-L suppresses the formation of selectin ligands [110]. C2GnT activity is also implicated in T-cell maturation [59]. The immature thymocytes express higher level of core-2-based O-glycans than matured thymocytes. Increased core-2-associated glycan is correlated with increased binding of galectin-1 containing epithelial cells to T cells. Galectin 1 is known to induce cell death in immature thymocytes and activated T cells by binding to the cell surface glycoproteins CD45, CD43, and CD7. Cells that lack C2GnT are resistant to galectin-1-induced cell death [111]. This concept has been confirmed by Nguyen et al. [112], who showed that transfection of CD45+ BW5147 T cells with C2GnT cDNA rendered these cells susceptible to galectin-1-induced cell death. The results suggest that core-2-based O-glycans play an important role in T-cell development. Furthermore, this concept was supported by decreased core-2 O-glycans (130 kDa) on CD43 in thymic-positive selection. These results clearly demonstrate the biological significance of C2GnT in immune system development. Mucin core-2 structure can be generated by three isozymes: C2GnT1, C2GnT2, and C2GnT3. C2GnT1 and C2GnT3 are involved in the synthesis of core 2 found in the mucin-type glycans of membrane-bound glycoproteins, while C2GnT2 is involved in the synthesis of core 2 found in secreted mucins. C2GnT1-deficient mice exhibited compromised functions of not only innate but also acquired immunities [59]. C2GnT3-deficient mice did not show any noticeable abnormal phenotype
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in thymus or immune functions, although a slight neutrophilia was observed. However, it is rather intriguing that these mice exhibited a significant increase in social dominance behavior, which was linked to a reduced level of circulating thyroxin T4 [113]. C2GnT2-deficient mice displayed reduced levels of immunoglobulins, including IgG1, IgG2a, IgG2b, and mucosal IgA. Although the mechanism for this phenomenon is not clear, it may explain the increased susceptibility of these animals to developing colitis [113] and possibly colorectal cancer [114]. Furthermore, mice deficient in multiple combinations of these three C2GnTs did not show any apparent abnormalities in viability, development, and fertility [113].
25.5.2 Role of Mucin Glycan Branch Structure in Cancer Development, Progression, and Metastasis It has been well documented that the development and progression of cancer are accompanied by alterations in glycoconjugates. However, the significance of these changes has only begun to emerge recently. It has been noted that most of the reported tumor-associated antigens belong to mucin-type glycans. Since the functions of mucin-type glycans vary according to where they reside, i.e., membranebound glycoproteins or secreted mucins, the functions of these two tumor-associated mucin-type glycans are reviewed accordingly. 25.5.2.1 Membrane-Associated Mucin-Type O-Glycans Generation of core 2 and its associated glycotopes on membrane-bound glycoproteins in advanced cancer can alter tumor-associated antigens and help promote tumor metastasis. We have previously shown that the tandem repeat peptide of MUC1 ectopically expressed in pancreatic cancer (Panc 1) cells was easily detected with an antibody specific for this peptide [67]. However, this peptide epitope was no longer recognized by the same antibody after forced expression of C2GnT-L cDNA [67]. Removal of sialic acid did not uncover the antigen, thus excluding the role of sialic acid in masking the antigen. In addition, ectopic expression of the core-2 branch generated sLex on the core-2 branch at the expense of sialyl-T antigen, sialic acida2-3Galb1-3GalNAcaSer/Thr, in this cell. This example illustrates how mucin glycan branching enzyme can alter tumor-associated antigens and enhance tumorigenicity. As described above, sLex is a key glycotope that can help target circulating leukocytes to distant sites, including lymphoid tissues [15]. Expression of sLex in advanced cancer is a common finding, which could provide a rational explanation for how these tumors metastasize [115, 116]. Several studies have confirmed the involvement of mucin-associated sLea and sLex glycotopes [117, 118] in lymphatic and venous invasion [15]. In mucin-type glycans, the core-2 branch is frequently decorated with sLex and sLea [119] (Fig. 25.2). Core-2 branches can be synthesized
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by C2GnTs, and expression of C2GnT directly correlates with the formation of those epitopes on mucin O-glycan. A recent study has shown enhanced adhesion and aggressive tumor formation [120] after transfection of human prostate cancer cells (LNCaP) with C2GnT-L cDNA. Therefore, C2GnT-L can be considered an oncogene and function as a progression marker for prostate cancer. Expression of C2GnT-L also correlates with tumor progression of other types of cancers, including colorectal and lung cancers [30, 65, 121]. These observations suggest that the expressions of C2GnT and resultant core-2-associated glycans play an important role in the progression of numerous tumors. Changes in C2GnT activity have also been documented in cells from leukemia patients, in particular, leukocytes of two patients, one with chronic myelogenous leukemia and one with acute myelogenous leukemia. A four- and 18-fold increase of C2GnT activity was found in the leukocytes of these two patients, respectively, as compared to normal donors [65]. Similar to that described above (D-1), expression of the core 2 branch can be regulated by relative expression of C2GnT-L and a2-3sialyltransferase I genes. Compared to normal breast tissues, breast cancer cells have reduced C2GnT-L gene expression by up to 50% but increased expression of a2-3sialyltransferase I gene, which culminates in the production of sialyl T antigen [122]. The involvement of P-, E-, and L-selectins in promoting cancer metastasis also has been demonstrated. P-selectin deficiency attenuates tumor growth and metastasis. Tumors are significantly smaller in size when mice are treated with receptor antagonists [123]. Lung colonization of B-16 melanoma cells that express sLex is significantly reduced in E- and P-selectin-deficient mice [123]. Furthermore, expression of L-selectin on tumor cells can enhance cancer metastasis to lymph nodes [124]. It is clear that there is a strong link between selectin–ligand interactions in blood-borne metastasis of various cancers. Figure 25.6 shows how the selectin–ligand interactions help promote cancer metastasis [123].
Fig. 25.6 Proposed mechanisms of cancer metastasis. Following detachment from the primary site, the cancer cells in circulation utilize three possible routes to metastasize: (a) direct binding to selectins on activated endothelium at distant sites using sLex and/or selectin expressed on the surface of cancer cells; (b) enlisting the help of circulating leukocytes via interaction of sLex with L-selectin present on the surface of leukocytes; (c) utilizing the help of platelets and leukocytes [123]
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It is important to point out that upregulation of C2GnT activity is not always associated with poor prognosis of cancer. In certain cancers, such as cutaneous T-cell lymphoma, downregulation/deletion of C2GnT-L renders these cells resistant to galectin-1–induced cell death [125]. Although upregulation of C2GnT-L is associated with poor prognosis for a large number of cancers, downregulation of C2GnT-L can benefit the progression of some types of cancer. Therefore, understanding the function of core 2-associated glycans in each cancer is the key to understanding the mechanism of tumorigenesis. 25.5.2.2 Branch Structures in Secreted Mucins As described above, the primary function of secreted mucins is to protect the underlying mucus-secretory tissues. Some of these tissues are constantly exposed to very harsh conditions, such as noxious gas in the lungs from polluted air, strong acid in the stomach, proteases throughout the gastrointestinal tract, and heavy loads of microorganisms in the colon. To protect the peptide backbones of these mucins from being degraded, the tandem repeat peptides rich in ser and thr are decorated with clusters of mucin-type O-glycans. To accomplish this goal, secreted mucins are equipped with high carbohydrate content, which can be up to 90% of the molecule by weight, and an extremely heterogeneous carbohydrate structure, which can be >300–400 oligosaccharide structures [126]. In addition, secreted mucins contain core 3 and core 4, which are unique to these mucins. As described above, core 3 is synthesized by core-3 synthase and core 4 by C2GnT-M. Downregulation of core-3 synthase or the C2GnT-M enzyme has been reported in colorectal cancers [114, 127]. Loss of core-3 or core-4 structure can lead to loss of integrity in these mucins, resulting in prolonged residency of bacteria, irritation of the epithelium, induction of chronic inflammation, and development of pathological conditions, such as colitis and cancer. This condition is similar to that found in Muc2 (−/−) mice. In this case, loss of secreted mucins resulted in loss of mucus protection, which led to the development of colorectal cancer [9] and colitis [8]. Core 3 is the obligatory precursor for core 4 [24, 128] in a reaction catalyzed by C2GnT-M, the only enzyme that can synthesize core 4 [22, 23]. Therefore, loss of the core-3 enzyme would result in the loss of not only core 3 but also core 4. Interestingly, when core-3 synthase cDNA was introduced to a colonic cancer cell line devoid of core-3 synthase gene, in vitro migration and in vivo lung metastasis were suppressed in an athymic mouse tumor xenograft model [127]. This result was confirmed in core3-synthase-gene knockout mice [129]. In this mouse model, both core-3 and core-4 O-glycans in secreted mucins were missing, which resulted in increased susceptibility to development of colitis and colorectal tumors [129]. This report also showed that mice devoid of this gene had increased degradation of Muc2, intestinal permeability, and retention of bacteria. Furthermore, this animal showed increased colonic infiltration of T lymphocytes and monocytes/macrophages after dextran sulfate sodium challenge. These results show that animals devoid of the core-3 synthase gene lose the integrity of colorectal epithelium, as well as the ability to efficiently clear
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p athogens, and have increased inflammation and incidence of colorectal cancer. Since core 3 is the precursor of core 4, these results suggest that core-3- and core-4-derived glycans are important for the epithelium-protective function of secreted mucins. Loss of all three mucin glycan branch structures as a result of downregulation of the C2GnT-M gene has also been observed in human colorectal cancers [114]. Loss of more than 50% of C2GnT-M gene expression was found in about 61–66% of human colorectal tumors. Forced expression of C2GnT-M cDNA in a colonic cancer cell line devoid of this enzyme suppresses cell spreading, attachment to extracellular matrix, colony formation, and invasion. In addition, introduction of C2GnT-M to this cell line induces apoptosis and suppresses cell growth in vitro and in vivo. These results support the idea that core-4 glycans are important for inhibiting the above-mentioned tumorigenic properties. As described previously, ablation of C2GnT2 gene leads to reduced levels of immunoglobulins and development of colitis [113], a likely prelude to development of colorectal cancer. In summary, except in rare cases as described above, elevated expression of C2GnT activity is associated with poor prognosis of cancer. Enhanced metastasis through formation of sLex on the core-2 branch in membrane-associated glycoproteins is the most likely explanation. However, loss of C4GnT or core-3 synthase activity in mucus-secretory tissues results in loss of the protective function of secreted mucins, which can lead to development of colorectal cancer. In this regard, the C2GnT-L gene functions like an oncogene, and core-3 synthase and C2GnT-M function like tumor suppressors.
25.6 Role of C2GnT in the Estrous Cycle C2GnT enzymes are also implicated in the reproductive system of golden hamster (Mesocricetus auratus) [130]. Glycosylation of hamster oviductin, a member of the mucin glycoprotein family, is regulated during the estrous cycle. The glycosylation process of oviductal glycoproteins is mainly involved in the synthesis of mucin O-glycans in the hamster oviduct. Hamster oviduct has high activities of glycosyltransferases that synthesize O-glycans that contain core-1, -2, -3, and -4 branched structures. During the estrous cycle, C2GnT-M enzyme activity is elevated at the stages of proestrus and estrus, but reduced at diestrus 1. Furthermore, regulation of the activities of these enzymes is correlated with messenger RNA levels of C2GnT-M in the estrous cycle stages. Increase of C2GnT-M activity in the hamster oviduct at the time of ovulation suggests that glycosylation of oviductal glycoproteins may be essential for the function of these proteins during fertilization.
25.7 Regulation of C2GnT Gene Expression Given the important role C2GnTs play in health and disease as described above, changes in the activities of these enzymes would have a significant impact on the overall health of an individual. To date, C2GnT-L and C2GnT-M are the only two
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mucin glycan branching enzymes of which detailed genomic structure and some characterization of gene regulation have been reported. C2GnT-L gene expression has been shown to be activated by IL-12 in T cells [100], butyrate in CHO cells [70], and Th2 cytokines (IL-4 and IL-13) in NCIH292 lung carcinoma cells [131]; moderately downregulated by epidermal growth factor (EGF) [132]; and not affected by retinoic acid [131]. SP1 is the transcription factor involved in the activation of this gene [49]. It remains to be established whether this transcription factor plays a role in the activation of this gene in metastatic cancer. IL-12 induces STAT4 expression in CD4+ T cells. STAT4 acts either directly or indirectly through the transcription factor T-bet to influence the expression of C2GnT-L and thus the function of PSGL-1 [133]. Sp1 transcription factor is essential for transcription of this gene in Jurkat cells, which are T cells derived from mesoderm, and NCI-H292 cells, which are derived from lung carcinomas [49]. Dose dependency of Sp1 is found in Jurkat cells, and only the Sp1s located at the proximal region are required for the expression of this gene. C2GnT-M gene expression can be upregulated by retinoic acid and Th2 cytokines [50, 131] and tumor necrosis factor (TNF)a [134], but downregulated by EGF in H292 cells [132]. EGF-mediated inhibition is through suppression of the EGF receptor (EGFR)-Ras-MEK-ERK signaling pathway. The EGFRphosphatidylinositol-phospholipase C pathway is involved in TNFa-mediated activation of the C2GnT-M gene. Retinoic acid acts through retinoic acid receptora, while Th2 cytokines act through the JAK3-mediated signaling pathway. The promoter of the C2GnT-M gene is responsive to retinoic acid and Th2 cytokine [50]. However, the transcription factors involved in the activation of this gene remain to be identified. In addition, introduction of N-glycan branching N-acetylglucosaminyltransferase V (GnTV) cDNA into H7721 hepatocellular carcinoma cells results in decreased expression of the sLex epitope. This is attributed to decreased expression of O-glycan branching enzymes such as C2GnT-L/-M and FUT-III, -VI, and -VII [135]. The mechanism remains unclear. Table 25.2 summarizes the regulation of C2GnTs under different growth conditions.
Table 25.2 Regulation of human C2GnT gene expression under various conditions hC2GnT Agents Effects Cells Mechanism References C2GnT-L butyrate, Th2 Increased Pancreas, Transcription [131, 136, cytokines, LPS, airway enzyme activity 137, 132] EGF Decreased Airway C2GnT-M Butyrate, all-trans Increased Pancreas, Transcription [131, 136, airway enzyme activity 137] retinoic acid, Th2 cytokines, LPS Airway EGF Decreased [132]
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25.8 Summary and Future Directions b6GlcNAc branching through formation of core 2, core 4, and I antigen is an important event in the synthesis of mucin O-glycans. These branch structures allow further elaboration of mucin-type glycans to increase not only content but also heterogeneous structure of mucin-type glycans. This is particularly significant for secreted mucins in view of their essential role in protecting the mucus-secretory epithelium. As many biologically important glycotopes are built on these branch structures, regulation of the synthesis of these branch structures is critically important for the regulation of many biological events, such as inflammation and cancer metastasis. With rare exceptions, increased expression of the C2GnT gene leads to decoration of the core-2 branch with selectin ligands, which correlates with poor prognosis of cancer. On the contrary, downregulation of the C2GnT-M gene could result in loss of epithelium-protective function of secreted mucins, which can lead to development of colitis and colorectal cancers. Thus, understanding the regulation of the expression of these genes could help elucidate the pathogenesis of cancer. Hence, future emphasis should be placed on understanding the mechanisms of coordinated gene expression of mucins, mucin glycan branching enzymes, and other glycosyltransferases involved in the synthesis of biologically important glycotopes, such as selectin ligands. Acknowledgements The authors wish to acknowledge the support of the following funding agencies: NIH RO1 HL48282, R21 HL097238, Cystic Fibrosis Foundation, and the State of Nebraska-NRI Cancer Glycobiology Program, LB 595 and LB506.
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76. Remold-O’Donnell E, Kenney DM, Parkman R, Cairns L, Savage B, Rosen FS (1984) Characterization of a human lymphocyte surface sialoglycoprotein that is defective in Wiskott-Aldrich syndrome. J Exp Med 159:1705–1723 77. Park JK, Rosenstein YJ, Remold-O’Donnell E, Bierer BE, Rosen FS, Burakoff SJ (1991) Enhancement of T-cell activation by the CD43 molecule whose expression is defective in Wiskott-Aldrich syndrome. Nature 350:706–709 78. Manjunath N, Johnson RS, Staunton DE, Pasqualini R, Ardman B (1993) Targeted disruption of CD43 gene enhances T lymphocyte adhesion. J Immunol 151:1528–1534 79. Lefebvre JC, Giordanengo V, Limouse M, Doglio A, Cucchiarini M, Monpoux F, Mariani R, Peyron JF (1994) Altered glycosylation of leukosialin, CD43, in HIV-1-infected cells of the CEM line. J Exp Med 180:1609–1617 80. Orlacchio A, Sarchielli P, Gallai V, Datti A, Saccardi C, Palmerini CA (1997) Activity levels of a beta1, 6N-acetylglucosaminyltransferase in lymphomonocytes from multiple sclerosis patients. J Neurol Sci 151:177–183 81. Nishio Y, Warren CE, Buczek-Thomas JA, Rulfs J, Koya D, Aiello LP, Feener EP, Miller TB Jr, Dennis JW, King GL (1995) Identification and characterization of a gene regulating enzymatic glycosylation which is induced by diabetes and hyperglycemia specifically in rat cardiac tissue. J Clin Invest 96:1759–1767 82. Panicot L, Mas E, Thivolet C, Lombardo D (1999) Circulating antibodies against an exocrine pancreatic enzyme in type 1 diabetes. Diabetes 48:2316–2323 83. Piller F, Piller V, Fox RI, Fukuda M (1988) Human T-lymphocyte activation is associated with changes in O-glycan biosynthesis. J Biol Chem 263:15146–15150 84. Fukuda M (2006) Roles of mucin-type O-glycans synthesized by core2beta1, 6-N-acetylglucosaminyltransferase. Methods Enzymol 416:332–346 85. Sperandio M, Thatte A, Foy D, Ellies LG, Marth JD, Ley K (2001) Severe impairment of leukocyte rolling in venules of core 2 glucosaminyltransferase-deficient mice. Blood 97:3812–3819 86. Kimura AK, Wigzell H (1978) Cell surface glycoproteins of murine cytotoxic T lymphocytes. I. T 145, a new cell surface glycoprotein selectively expressed on Ly 1−2+ cytotoxic T lymphocytes. J Exp Med 147:1418–1434 87. Andersson LC, Gahmberg CG, Kimura AK, Wigzell H (1978) Activated human T lymphocytes display new surface glycoproteins. Proc Natl Acad Sci USA 75:3455–3458 88. Dalziel M, Whitehouse C, McFarlane I, Brockhausen I, Gschmeissner S, Schwientek T, Clausen H, Burchell JM, Taylor-Papadimitriou J (2001) The relative activities of the C2GnT1 and ST3Gal-I glycosyltransferases determine O-glycan structure and expression of a tumor-associated epitope on MUC1. J Biol Chem 276:11007–11015 89. Carlow DA, Ziltener HJ (2006) CD43 deficiency has no impact in competitive in vivo assays of neutrophil or activated T cell recruitment efficiency. J Immunol 177:6450–6459 90. Grabie N, Delfs MW, Lim YC, Westrich JR, Luscinskas FW, Lichtman AH (2002) Betagalactoside alpha2, 3-sialyltransferase-I gene expression during Th2 but not Th1 differentiation: implications for core2-glycan formation on cell surface proteins. Eur J Immunol 32:2766–2772 91. Barran P, Fellinger W, Warren CE, Dennis JW, Ziltener HJ (1997) Modification of CD43 and other lymphocyte O-glycoproteins by core 2N-acetylglucosaminyltransferase. Glycobiology 7:129–136 92. Kikuchi J, Shinohara H, Nonomura C, Ando H, Takaku S, Nojiri H, Nakamura M (2005) Not core 2 beta 1, 6-N-acetylglucosaminyltransferase-2 or -3 but-1 regulates sialyl-Lewis x expression in human precursor B cells. Glycobiology 15:271–280 93. Fukuda M, Carlsson SR, Klock JC, Dell A (1986) Structures of O-linked oligosaccharides isolated from normal granulocytes, chronic myelogenous leukemia cells, and acute myelogenous leukemia cells. J Biol Chem 261:12796–12806 94. Maemura K, Fukuda M (1992) Poly-N-acetyllactosaminyl O-glycans attached to leukosialin. The presence of sialyl Le(x) structures in O-glycans. J Biol Chem 267: 24379–24386
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95. Yang J, Hirata T, Croce K, Merrill-Skoloff G, Tchernychev B, Williams E, Flaumenhaft R, Furie BC, Furie B (1999) Targeted gene disruption demonstrates that P-selectin glycoprotein ligand 1 (PSGL-1) is required for P-selectin-mediated but not E-selectin-mediated neutrophil rolling and migration. J Exp Med 190:1769–1782 96. Shimizu Y, Shaw S, Graber N, Gopal TV, Horgan KJ, Van Seventer GA, Newman W (1991) Activation-independent binding of human memory T cells to adhesion molecule ELAM-1. Nature 349:799–802 97. Von Andrian UH, Hansell P, Chambers JD, Berger EM, Torres Filho I, Butcher EC, Arfors KE (1992) L-selectin function is required for beta 2-integrin-mediated neutrophil adhesion at physiological shear rates in vivo. Am J Physiol 263:H1034–1044 98. Lowe JB (2003) Glycan-dependent leukocyte adhesion and recruitment in inflammation. Curr Opin Cell Biol 15:531–538 99. Lenter M, Levinovitz A, Isenmann S, Vestweber D (1994) Monospecific and common glycoprotein ligands for E- and P-selectin on myeloid cells. J Cell Biol 125:471–481 100. Lim YC, Xie H, Come CE, Alexander SI, Grusby MJ, Lichtman AH, Luscinskas FW (2001) IL-12, STAT4-dependent up-regulation of CD4(+) T cell core 2 beta-1, 6-n-acetylglucosaminyltransferase, an enzyme essential for biosynthesis of P-selectin ligands. J Immunol 167:4476–4484 101. Honn KV, Tang DG, Crissman JD (1992) Platelets and cancer metastasis: a causal relationship? Cancer Metastasis Rev 11:325–351 102. Karpatkin S, Pearlstein E (1981) Role of platelets in tumor cell metastases. Ann Intern Med 95:636–641 103. Hiraoka N, Kawashima H, Petryniak B, Nakayama J, Mitoma J, Marth JD, Lowe JB, Fukuda M (2004) Core 2 branching beta1, 6-N-acetylglucosaminyltransferase and high endothelial venule-restricted sulfotransferase collaboratively control lymphocyte homing. J Biol Chem 279:3058–3067 104. Broide DH, Miller M, Castaneda D, Nayar J, Cho JY, Roman M, Ellies LG, Sriramarao P (2002) Core 2 oligosaccharides mediate eosinophil and neutrophil peritoneal but not lung recruitment. Am J Physiol Lung Cell Mol Physiol 282:L259–L266 105. Smith MJ, Smith BR, Lawrence MB, Snapp KR (2004) Functional analysis of the combined role of the O-linked branching enzyme core 2 beta1-6-N-glucosaminyltransferase and dimerization of P-selectin glycoprotein ligand-1 in rolling on P-selectin. J Biol Chem 279:21984–21991 106. Li F, Wilkins PP, Crawley S, Weinstein J, Cummings RD, McEver RP (1996) Posttranslational modifications of recombinant P-selectin glycoprotein ligand-1 required for binding to P- and E-selectin. J Biol Chem 271:3255–3264 107. Kumar R, Camphausen RT, Sullivan FX, Cumming DA (1996) Core2 beta-1, 6-N-acetylglucosaminyl-transferase enzyme activity is critical for P-selectin glycoprotein ligand-1 binding to P-selectin. Blood 88:3872–3879 108. Kanda H, Tanaka T, Matsumoto M, Umemoto E, Ebisuno Y, Kinoshita M, Noda M, Kannagi R, Hirata T, Murai T, Fukuda M, Miyasaka M (2004) Endomucin, a sialomucin expressed in high endothelial venules, supports L-selectin-mediated rolling. Int Immunol 16:1265–1274 109. Prorok-Hamon M, Notel F, Mathieu S, Langlet C, Fukuda M, El-Battari A (2005) N-glycans of core2 beta(1, 6)-N-acetylglucosaminyltransferase-I (C2GnT-I) but not those of alpha(1, 3)-fucosyltransferase-VII (FucT-VII) are required for the synthesis of functional P-selectin glycoprotein ligand-1 (PSGL-1): effects on P-, L- and E-selectin binding. Biochem J 391:491–502 110. Kikuchi J, Ozaki H, Nonomura C, Shinohara H, Iguchi S, Nojiri H, Hamada H, Kiuchi A, Nakamura M (2005) Transfection of antisense core 2 beta1, 6-N-acetylglucosaminyltransferase-1 cDNA suppresses selectin ligand expression and tissue infiltration of B-cell precursor leukemia cells. Leukemia 19:1934–1940 111. Valenzuela HF, Pace KE, Cabrera PV, White R, Porvari K, Kaija H, Vihko P, Baum LG (2007) O-glycosylation regulates LNCaP prostate cancer cell susceptibility to apoptosis induced by galectin-1. Cancer Res 67:6155–6162 112. Nguyen JT, Evans DP, Galvan M, Pace KE, Leitenberg D, Bui TN, Baum LG (2001) CD45 modulates galectin-1-induced T cell death: regulation by expression of core 2 O-glycans. J Immunol 167:5697–5707
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113. Stone EL, Ismail MN, Lee SH, Luu Y, Ramirez K, Haslam SM, Ho SB, Dell A, Fukuda MF, Marth JD (2009) Glycosyltransferase function in core 2-type protein O gycosylation. Mol Cell Biol 29:3770–3782 114. Huang MC, Chen HY, Huang HC, Huang J, Liang JT, Shen TL, Lin NY, Ho CC, Cho IM, Hsu SM (2006) C2GnT-M is downregulated in colorectal cancer and its re-expression causes growth inhibition of colon cancer cells. Oncogene 25:3267–3276 115. Brockhausen I (2006) Mucin-type O-glycans in human colon and breast cancer: glycodynamics and functions. EMBO Rep 7:599–604 116. Ohyama C, Tsuboi S, Fukuda M (1999) Dual roles of sialyl Lewis x oligosaccharides in tumor metastasis and rejection by natural killer cells. EMBO J 18:1516–1525 117. Berg EL, Robinson MK, Mansson O, Butcher EC, Magnani JL (1991) A carbohydrate domain common to both sialyl Le(a) and sialyl Le(x) is recognized by the endothelial cell leukocyte adhesion molecule ELAM-1. J Biol Chem 266:14869–14872 118. Takada A, Ohmori K, Takahashi N, Tsuyuoka K, Yago A, Zenita K, Hasegawa A, Kannagi R (1991) Adhesion of human cancer cells to vascular endothelium mediated by a carbohydrate antigen, sialyl Lewis A. Biochem Biophys Res Commun 179:713–719 119. Kannagi R (1997) Carbohydrate-mediated cell adhesion involved in hematogenous metastasis of cancer. Glycoconj J 14:577–584 120. Hagisawa S, Ohyama C, Takahashi T, Endoh M, Moriya T, Nakayama J, Arai Y, Fukuda M (2005) Expression of core 2 beta1, 6-N-acetylglucosaminyltransferase facilitates prostate cancer progression. Glycobiology 15:1016–1024 121. Nakayama J, Shimizu F, Katsuyama T (2002) Glycosyltransferase genes as tumor marker. Rinsho Byori Suppl 123:142–148 122. Dalziel M, Whitehouse C, McFarlane I, Brockhausen I, Gschmeissner S, Schwientek T, Clausen H, Burchell JM, Taylor-Papadimitriou J (2001) The relative activities of the C2GnT1 and ST3Gal-I glycosyltransferases determine O-glycan structure and expression of a tumor-associated epitope on MUC1. J Biol Chem 276:11007–11015 123. Kim YJ, Borsig L, Varki NM, Varki A (1998) P-selectin deficiency attenuates tumor growth and metastasis. Proc Natl Acad Sci USA 95:9325–9330 124. Qian F, Hanahan D, Weissman IL (2001) L-selectin can facilitate metastasis to lymph nodes in a transgenic mouse model of carcinogenesis. Proc Natl Acad Sci USA 98:3976–3981 125. Cabrera PV, Amano M, Mitoma J, Chan J, Said J, Fukuda M, Baum LG (2006) Haploinsufficiency of C2GnT-I glycosyltransferase renders T lymphoma cells resistant to cell death. Blood 108:2399–2406 126. Xia B, Royall JA, Damera G, Sachdev GP, Cummings RD (2005) Altered O-glycosylation and sulfation of airway mucins associated with cystic fibrosis. Glycobiology 15:747–775 127. Iwai T, Kudo T, Kawamoto R, Kubota T, Togayachi A, Hiruma T, Okada T, Kawamoto T, Morozumi K, Narimatsu H (2005) Core 3 synthase is down-regulated in colon carcinoma and profoundly suppresses the metastatic potential of carcinoma cells. Proc Natl Acad Sci USA 102:4572–4577 128. Hounsell EF, Fukuda M, Powell ME, Feizi T, Hakomori S (1980) A new O-glycosidically linked tri-hexosamine core structure in sheep gastric mucin: a preliminary note. Biochem Biophys Res Commun 92:1143–1150 129. An G, Wei B, Xia B, McDaniel JM, Ju T, Cummings RD, Braun J, Xia L (2007) Increased susceptibility to colitis and colorectal tumors in mice lacking core 3-derived O-glycans. J Exp Med 204:1417–1429 130. McBride DS, Brockhausen I, Kan FW (2005) Detection of glycosyltransferases in the golden hamster (Mesocricetus auratus) oviduct and evidence for the regulation of O-glycan biosynthesis during the estrous cycle. Biochim Biophys Acta 1721:107–115 131. Beum PV, Basma H, Bastola DR, Cheng PW (2005) Mucin biosynthesis: upregulation of core 2 beta 1, 6N-acetylglucosaminyltransferase by retinoic acid and Th2 cytokines in a human airway epithelial cell line. Am J Physiol Lung Cell Mol Physiol 288:L116–L124 132. Beum PV, Bastola DR, Cheng PW (2003) Mucin biosynthesis: epidermal growth factor downregulates core 2 enzymes in a human airway adenocarcinoma cell line. Am J Respir Cell Mol Biol 29:48–56
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133. White SJ, Underhill GH, Kaplan MH, Kansas GS (2001) Cutting edge: differential requirements for Stat4 in expression of glycosyltransferases responsible for selectin ligand formation in Th1 cells. J Immunol 167:628–631 134. Ishibashi Y, Inouye Y, Okano T, Taniguchi A (2005) Regulation of sialyl-lewis x epitope expression by TNF-alpha and EGF in an airway carcinoma cell line. Glycoconj J 22:53–62 135. Guo P, Zhang Y, Shen ZH, Zhang XY, Chen HL (2004) Effect of N-acetylglucosaminyltransferase V on the expressions of other glycosyltransferases. FEBS Lett 562:93–98 136. Radhakrishnan P, Beum PV, Tan S, Cheng PW (2007) Butyrate induces sLex synthesis by stimulation of selective glycosyltransferase genes. Biochem Biophys Res Commun 359:457–462 137. Yanagihara K, Seki M, Cheng PW (2001) Lipopolysaccharide induces mucus cell metaplasia in mouse lung. Am J Respir Cell Mol Biol 24:66–73
Chapter 26
Studying Carbohydrate Self-Recognition in Marine Sponges Using Synthetic Aggregation Factor Epitopes Johannis P. Kamerling and Adriana Carvalho de Souza
Keywords Marine sponges • Microciona prolifera • Aggregation factors • Carbohydrate–carbohydrate interaction • Neoglycoconjugates • Gold glyconanoparticles • Surface plasmon resonance • Transmission electron microscopy • Atomic force microscopy Sponges (Porifera) are the simplest and earliest multicellular organisms. They do not produce complex patterned structures (i.e., organ systems). Epithelial cells (pinacocytes) line the outer surface and the internal system of openings, channels, and chambers, through which water is continuously pumped by flagellated cells called choanocytes. The generated water current supplies food particles and oxygen and removes metabolic waste products. The major part of the sponge biomass consists of a gelatinous extracellular matrix (ECM) containing a large number of highly motile cells. This part of the sponge body is called the mesohyl. The mesohyl also contains the skeletal elements of the sponge body: spicules (needle-like structures made of either silicon or calcium carbonate) and spongin (collagenous fibers). The complex ECM observed in sponges suggests that the system that mediates cell motility and adhesion in sponges is common to all multicellular animals, thus placing the developmental role of the ECM as an ancestral homology that unites all Metazoans [1]. Interactions between cells and extracellular molecules play a fundamental role in the development of “higher” animals. The same complex set of molecules found in vertebrate ECMs also appears to be involved in cell motility and development in sponges. As sponges are the simplest multicellular animals living today, they represent an ideal model system for studying the molecular mechanisms that guide cell recognition and adhesion in ancestral Metazoans. In 1907, Wilson pioneered the use of sponges as model animals for the study of cell adhesion. He described the existence of species-specific reaggregation of marine sponge cells that had been mechanically dissociated [2]. When sponges J.P. Kamerling (*) Department of Bio-Organic Chemistry, Bijvoet Center, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands e-mail:
[email protected] A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_26, © Springer Science+Business Media, LLC 2011
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were dissociated by squeezing through a fine-mesh cloth, single cells were produced that adhered to each other to form aggregates and eventually reorganized into perfect miniature sponges in a Ca2+-dependent process [3, 4]. When dissociated cells of different species with natural pigments of contrasting colors were mixed and allowed to aggregate, it was observed that cells from the same species would combine, but not cells from different species. These results led to the conclusion that specific surface antigens are essential for adhesion. In further studies of sponge cells dissociated in Ca2+-free seawater (referred to as “chemically dissociated cells”), extracellular particulates that were responsible for the species-specific cell aggregation (referred to as aggregation factors [AFs]) could be isolated [5–7]. These AFs were found to be a requirement for the aggregation of dissociated sponge cells. In summary, sponges are thought to have evolved from their unicellular ancestors about 1 billion years ago by developing cell recognition and adhesion mechanisms for discriminating against “non-self.”
26.1 Sponge AFs Porifera AFs have been identified in several marine sponge species [5, 6, 8–10]. All of them are proteoglycan-like macromolecular complexes with molecular masses in the order of several million daltons. They are composed mainly of protein and carbohydrate in variable but substantial amounts, with a high net negative charge [11, 12]. The cell aggregation activity of marine sponge AFs depends on a Ca2+ concentration of about 10 mM, similar to the concentration found in seawater. Other alkaline earth cations, Mg2+, Sr2+, and Ba2+, could not replace Ca2+ as an aggregation-mediating agent; however, two transition elements, Mn2+ and Cd2+, could partially replace Ca2+. Interestingly, lanthanides produced normal cell aggregation activity but only at 10- to 400-fold lower cation concentrations than Ca2+ [13]. Although AFs from freshwater sponges have been described and partially purified [14, 15], they are still poorly characterized. Freshwater sponge AFs are already active at Ca2+ concentrations of 1 mM [16], implying major differences when compared to their marine counterparts. To confirm the role of AFs in the first step of cell recognition and adhesion, the purified proteoglycan, called glyconectin [17] or spongican [18], of three different sponge species, Microciona prolifera, Halichondria panicea, and Cliona celata, attached to pink, yellow, and white latex-amidine beads, respectively, were used for aggregation studies [17]. After a short rotation of the three types of proteoglycan beads in seawater with 10 mM CaCl2, xenogeneic sorting and adhesion occurred. Replacement of Ca2+ with Mg2+ ions did not result in bead aggregation. In a control experiment, a mixture of yellow, pink, and white latex beads, all decorated with M. prolifera proteoglycan, yielded mixed orange-colored aggregates after a short rotation in the presence of 10 mM CaCl2. These experiments directly showed that
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species-specific proteoglycan-to-proteoglycan interactions are an example of a primordial self-recognition mechanism approaching the selectivity shown by the immunoglobulin superfamily in higher animals. The binding strength between two individual AFs in the presence of 10 mM CaCl2 was measured with atomic force microscopy (AFM) [19–21]. Under physiological conditions, the average adhesive forces of individual interaction sites in two AF molecules have been estimated to be 40 ± 15 pN, whereas the average forces measured between a single pair of molecules amounted to about 125 pN, with maxima up to 400 pN [19]. Initial characterization studies showed that the M. prolifera AFs (MAF) and the Geodia cydonium AFs had a sunburst shape in the electron microscope micrographs, with a central ring structure of about 130–200 nm in diameter and 16–25 radiating arms [9, 22]. AFs extracted from Halichondria bowerbanki, Terpios zeteki, Homalomena occulta, and H. panicea had similar size and form, but a linear backbone [22, 23]. The supramolecular structure of the circular proteoglycan MAF was elucidated by using immunochemical and electrophoretical procedures, combined with AFM imaging [18, 23–25]. MAF promotes adhesion of sponge cells through a two-step process involving (1) a Ca2+-dependent self-aggregation of the AF complexes and (2) a Ca2+-independent binding to cell surface receptors [26]. The main proteins of MAF, MAFp3 (ranging from 30 to 50 kDa) and MAFp4 (approximately 400 kDa), are extremely polymorphic carbohydrate-containing molecules [27]. Structural studies have shown that although MAFp3 and MAFp4 are not directly related to any other described proteoglycan component, the structure of MAF has characteristics that resemble the aggregates of cartilage proteoglycan [23]. However, unlike any proteoglycan described so far, the central backbone of MAF is circularized. The estimated total mass of MAF amounts to 2 × 104 kDa, with a carbohy drate content of 60–70% and a monosaccharide composition of glucuronic acid (GlcA):fucose (Fuc):mannose (Man):galactose (Gal):N-acetylglucosamine (GlcNAc) = 10:34:9:25:22 [28]. Twenty units from each of the two N-glycosylated proteins, MAFp3 and MAFp4, form the central ring and radiating arms of MAF, respectively (Fig. 26.1) [18, 23]. From the AFM images, it was estimated that each of the 20 MAFp4 arms is 140–180-nm long and has a beaded structure consisting of about 15 globules. A ~6-kDa N-glycan, g-6, involved in the binding of MAF to the cell receptor, is found exclusively at MAFp4. It was calculated that one MAF molecule has about 950 g-6 units [29], resulting in about 50 g-6 units in each of the MAFp4 arms. A ~200-kDa N-glycan, g-200, involved in the self-association of MAF [30], is found in the ring forming MAFp3. The same studies showed that one MAF has approximately 26 g-200 units. AFM imaging has shown that one or two short linear structures protrude from each of the 20 globular structures that form the ring of MAF. Whenever MAF rings were observed to form dimers or larger aggregates, they appear to be interconnected through these short chains, thus suggesting that they are g-200 glycans [23]. A simplified model of MAF-mediated sponge cell adhesion is depicted in Fig. 26.2. Ca2+-dependent self-interactions of the MAFp3bound g-200 glycan mediate self-binding of two AF complexes. MAFp3 and
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Fig. 26.1 AFM image of MAF showing the localization of MAFp3 in the ring (circumferences) and of MAFp4 in the arms (lines). From Fernàndez-Busquets and Burger [18], with permission
MAFp4 interact through a hyaluronan (HA)-like molecule [18]. The binding through the g-6 glycan to cell receptors probably involves other pericellular proteins [31–33]. For the g-200 self-interaction, a more physical model has also been proposed [21].
26.2 The N-Glycans of MAF The g-6 and g-200 N-glycans were isolated and purified after complete pronase digestion of MAF followed by polyacrylamide gel electrophoresis, gel filtration, and ion-exchange chromatography. Composition analysis of g-6 revealed the presence of GlcA, Fuc, Man, Gal, GlcNAc, sulfate, and asparagine (Asn) in a molar ratio of 7:3:2:5:14:2:1 [29]. Carbohydrate and amino acid analysis of the purified g-200 glycan showed the presence of GlcA, Fuc, Man, Gal, GlcNAc, and Asn in a molar ratio of 32:68:2:18:19:1 [30]. It should be noted that PNGase-F is able to release the g-6 N-glycans of MAF but not the g-200 N-glycans. In a more recent study [34], monosaccharide analysis of purified MAF (glyconectin) showed the presence of GlcA, Fuc, Man, Gal, GlcNAc, GalNAc, and Gal4,6Pyr in a molar ratio of 11:28:9:29:17:1:4, a carbohydrate content of 63%, and a sulfate content of 820 mol/mol. Another recent study focused on the g-200 glycan [35] revealed the occurrence of GlcA, Fuc, Man, Gal, GlcNAc, and GalNAc in a molar ratio of 9:29:9:32:18:3. In the context of revealing the molecular basis of MAF aggregation, two specific monoclonal antibodies raised against purified MAF, called Block 1 [28] and Block 2 [30], turned out to inhibit the Ca2+-dependent self-association process without
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Fig. 26.2 A proposed model of the MAF-mediated sponge cell adhesion. Only half of MAFp4 and of the g-200 glycans are represented. (a) Ca2+-dependent self-interactions of the MAFp3bound g-200 glycan; (b) MAFp3 and MAFp4 interact through an HA-like molecule; (c) Ca2+independent binding through the g-6 glycan to cell receptors. From Fernàndez-Busquets and Burger [18], with permission
interfering with the cell-binding activity. Binding studies of the antibodies to MAF demonstrated that the highly repetitive epitopes (1,100 antigenic sites for Block 1 Fab fragments [28] and 2,500 sites for Block 2 Fab fragments [30]) were located at the acidic g-200 glycan (Mr = 200 × 103 ± 40 × 103 kDa) [36]. However, in their monomeric form, g-200 glycans showed no measurable self-association or interaction with MAF in the presence of 10 mM CaCl2, supporting data that MAF–MAF association is based on multiple low-affinity carbohydrate–carbohydrate interactions; in fact, cross-linking with glutaraldehyde into polymers led to reconstitution of the Ca2+-dependent self-interaction. Also, glass aminopropyl beads coated with the g-200 glycans aggregated in the presence of 10 mM CaCl2 (but not 2 mM CaCl2), indicating again that the glycan, presented in a multivalent way, is capable
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a Na+ −OOC
b OO
O OH
O HO
OH
O HO
O
OH
O
OH
HO O
NHAc
OH HO Na+ −O3SO
O
OH OH
HO O
NHAc
Fig. 26.3 Chemical structure of the pyruvated trisaccharide (a) and the sulfated disaccharide (b), isolated from the MAF g-200 glycan
of mediating adhesion exclusively through homophilic carbohydrate–carbohydrate interactions [30, 36]. AFM studies with the g-200 glycan in the presence of 10 mM CaCl2 showed the binding forces for single glycans to be 310 ± 75 pN [36]. The epitopes, recognized by the monoclonal antibodies Block 1 and Block 2, were isolated after mild acid hydrolysis of the glycan fraction obtained via pronase digestion of MAF. Structural analysis of these epitopes, applying 1H nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry, showed two novel oligosaccharides, the pyruvated trisaccharide b-d-Galp4,6(R)Pyr-(1→4)-b-dGlcpNAc-(1→3)-l-Fucp [37] (recognized by Block 1) and the sulfated disaccharide b-d-GlcpNAc3S-(1→3)-l-Fucp [38] (recognized by Block 2) (Fig. 26.3). In the latter study, the structure of a third nonimmunoreactive oligosaccharide, a-d-Galp(1→2)-b-d-Galp3S-(1→4)-b-d-GlcpNAc-(1→3)-l-Fucp, was also determined [38]. In additional mass spectrometric studies of acid hydrolysates of purified MAF (glyconectin), besides GlcNAc3S-Fuc (see above), two other oligosaccharide fragments, Fuc-GlcNAcS-Fuc and GlcNAc-Fuc-GlcNAcS-Fuc, were identified, indicating that the sulfated disaccharide epitope may be part of a repetitive unit. Furthermore, besides Gal-GalS-GlcNAc-Fuc (see above), the novel fragments Gal-GalS-GlcNAc and GlcNAc-GalS-Fuc were established, whereas Gal4,6PyrGlcNAc-Fuc (see above) was found back in minor amounts [39]. In view of the observation of preferential cleavage of glycodomains rich in acid-labile linkages (note the absence of GlcA), it has been suggested that the polysaccharide moieties are probably constituted of external acid-labile domains and an acid-resistant backbone structure. Two-dimensional NMR studies on the native MAF g-200 glycan made the assignments of a-Fuc, a-Gal, b-Gal, and b-GlcNAc possible [34], demonstrating that on the polysaccharide level, Fuc occurs in the a-configuration.
26.3 Glycans of AFs of Other Species Besides the reports on the glycan moieties of MAF, attention was paid more recently to the mass spectrometric structural analysis of oligosaccharide fragments derived from the purified N-glycosylated AFs (glyconectins) of C. celata and H. panicea [34, 39].
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Monosaccharide analysis of the AF of C. celata revealed the presence of Ara: Fuc:Man:Gal:GalNAc:GlcNAc:GlcA in a molar ratio of 11:31:20:12:7:13:6, with a carbohydrate content of 36% and a sulfate content of 700 mol/mol. The AF contained one major 110-kDa acidic glycan (50%) together with a mixture of smaller glycans. Proposed structures of oligosaccharides obtained via partial hydrolysis comprise HexNAc-PentS-(dHex)0,1, HexNAc-dHexS-(dHex)0,1, HexNAc-PentSHexNAc-(dHex)0,1, HexNAc-dHexS-HexNAc-(dHex)0,1, and Hex-HexNAc-PentSHexNAc-dHex. Monosaccharide analysis of the AF of H. panicea revealed the presence of Fuc: Man:Gal:GalNAc:GlcNAc:Gal4,6Pyr:GlcA in a molar ratio of 7:16:43:1:14:16:3, with a carbohydrate content of 21% and a sulfate content of 620 mol/mol. The AF contained one major 180-kDa acidic glycan (60%) together with a heterogeneous population of acidic glycans with sizes 6 mg/mL by a vacuum centrifugal concentrator. After 1:1 dilution with buffer A (150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 20 mM Tris [pH 7.4]), each solution was incubated with ConA agarose (Sigma) overnight at 4°C with rotation. After incubation, ConA beads were washed twice with buffer A, and ConA-enriched plasma proteins were eluted by incubating the beads with 500 mM a-Me-d-Man in buffer A.
31.1.7 Tryptic In-Gel Digestions of 2-D PAGE-Resolved Proteins and MALDI–TOF Mass Spectrometry Protein spots were excised from the polyacrylamide gel and destained with 1% potassium ferricyanide and 1.6% sodium thiosulfate (Sigma). The proteins were then reduced with 25 mM NH4HCO3 containing 10 mM DTT (USB) at 56°C for 45 min and alkylated with 55 mM iodoacetamide (Promega) at room temperature in the dark for 30 min. The gel was washed once with 100 ml of 50% aceto nitrile/25 mM NH4HCO3 and dried using a SpeedVac concentrator. The dried gel pieces were swollen in 10 ml of 25 mM ammonium bicarbonate containing 0.1 mg of trypsin (Promega, Madison, WI, USA). The gel pieces were then crushed with a siliconized blue stick and incubated at 37°C for at least 16 h. The peptides were subsequently extracted with 20 ml of 50% acetonitrile/1% trifluoroacetic acid and stored at −20°C until needed for analysis. These proteins (typically one-twentieth) from nonseparated tryptic digests were cocrystallized in a matrix of a-cyano-4hydroxycinnamic acid on the MTP Anchorchip 600/384 TF (Bruker Daltonics) and analyzed using an Ultraflex (Bruker Daltonics, Bremen, Germany). The measured monoisotopic masses of peptides were analyzed using Internet search programs, such as Mascot, provided by EMBL (http://www.matrixscience.com), with an MSDB database. The search was performed using a mass uncertainty of ±50 ppm, 1 as the maximum number of missed tryptic cleavage, and a molecular weight range of ±10% from the Mr determined from 2-D electrophoresis (2-DE). The output consisted of a list of proteins ranked by statistical score.
31.2 Results 31.2.1 Histology of Allogeneic Liver Grafts During Rejected and Tolerant Periods Histological examination revealed the in situ immunological conditions following OLT. As shown in Fig. 31.1a, serious rejection was confirmed by dense cellular infiltration on day 14 and focal necrosis of hepatocytes scattered throughout the
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Fig. 31.1 Histology of allogeneic liver grafts: nonrejection in the combination DA into PVG. (a) At 2 weeks (POD 14) there is dense cell infiltration, mainly of the portal tract, and scattered focal necrosis of the liver cells with mononuclear cells throughout the sinusoids (see arrow); rejection grade 2. (b) By 9 weeks (POD 60), the liver is largely normal, apart from a few residual cells in the portal tracts and mild bile-duct proliferation; rejection grade 0
sinusoids. Alternatively, rejection was overcome and tolerance was induced at POD 60 of OLT (Fig. 31.1b). This was identified by regression to normal histological liver architecture without signs of inflammation.
31.2.2 Comparison of 2-DE Protein Profiles Between POD-14 and POD-60 Samples Based on the fact that serum from the OLT drug-free tolerant rat could avoid the immune attack, we hypothesized that some serum proteins may be released in cascades during the process of inducing tolerance. To evaluate this hypothesis, 2-DE was applied to separate serum proteins at different periods after OLT, with a mass range between 17 and 170 kDa and a pI range from 4 to 7. By using the ImageMaster 2-D Elite computerized program, a total of 114 common protein spots were counted for both POD-14 and POD-60 samples. Of these proteins, two spots (spots 1 and 2) that appeared in the POD-14 samples were markedly reduced or absent in the POD-60 samples. Conversely, one spot (spot 3) was found in more abundant quantity in POD 60 but markedly decreased or absent in the POD-14 serum samples. These three proteins were further identified by a peptide mass fingerprint (PMF) and are indicated with Arabic numerals on the silver stained gel in Fig. 31.2. Some differential protein spots were not identified by PMF because they gave poor spectra owing to their low abundance. The comprehensive results of spectrometric analyses were summarized in Table 31.1.
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Fig. 31.2 2-DE profiles of serum from OLT treatment model at POD 14 and POD 60. Serum proteins (100 mg) were focused on a 4–7 IPG and then migrated at a right angle in SDS–PAGE on a 10% polyacrylamide gel. The protein patterns were stained with silver staining. The identified proteins are annotated and indicated by number
31.2.3 Immunohistochemistry of Haptoglobin Expression in OLT Liver The proteomic results indicated that haptoglobin (Hp) was highly expressed in the rat serum during the tolerant period at POD 60, and various modifications of Hp were also found. To further demonstrate the significant difference in Hp expression and localization between the rejection and tolerant periods, immunohistochemistry with an Hp-specific antibody was performed. As shown in Fig. 31.3a, on day 2 following OLT, the expression of Hp is modest. After day 14 of OLT, the intensity of Hp staining increased significantly and was identified and scattered throughout the inside of the hepatocyte during rejection (Fig. 31.3b). This regulatory cycle recruited another high level of Hp on POD 40 (Fig. 31.3c), resulting in the upregulation of Hp on POD 60 with highly concentrated spots gathering in the hepatocyte Golgi organelle (Fig. 31.3d).
31.2.4 Detection of Glycosylation by Pro-Q Emerald 488 Glycosylation plays a prominent role in the biological functions of proteins in immune responses. As shown in Fig. 31.4, sera samples were separated by SDS– PAGE and stained directly with the Pro-Q Emerald 488 glycoprotein gel stain kit, which permits the detection of less than 5–18 ng of glycoprotein per band. Total protein profiles were subsequently detected using silver stain. The parallel images show several similarities and differences. While only 15 bands were detected on the Pro-Q Emerald 488 glycoprotein image, more than 20 bands were detected on the silver stained gel. Generally, more intense glycosylation of proteins was observed in tolerant samples than during a rejection period. Of note, a2-microglobulin,
T-Kininogen II (MAP1) P08932
T-Kininogen I a1-antitrypsin IgA
4
5 6 7
P01048 P17475 P01878
P06866
Haptoglobin
3
Q63041
a1-microglobulin
2
Table 31.1 List of identified protein spots Spot No Protein name Accession number 1 Albumin P02770
48.76/6.29 46.28/5.7 53.21/5.49
48.76/5.94
39.05/6.10
52.0/7.58
Mw/pI 70.67/6.09
123 (43%) 110 (49%) 85 (35%)
106 (50%)
167 (44%)
154 (50%)
Score (coverage) 168 (41%)
994/1535/1802 1153/1603 1389/2445
1010/1803/1816
1012/1373
870/1672
MS/MS 1479/1882
Inhibitor of serine proteases Ig alpha is the major immunoglobulin class in body secretions
Function Its main function is the regulation of the colloidal osmotic pressure of blood Is able to inhibit all four classes of proteinases by a unique “trapping” mechanism Haptoglobin combines with free plasma hemoglobin Kininogens are plasma glycoproteins in blood coagulation by helping to position optimally prekallikrein and factor XI next to factor XII
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Fig. 31.3 Localization of Hp in the tolerogenic rat OLT model. Hp was not detected at POD 2 (a) but appeared at POD 14 (b), POD 40 (c), and POD 60 (d). Hp was scattered throughout the inside of the hepatocyte during rejection on day 14 (b). In the tolerogenic liver, Hp appeared to be in highly concentrated spots and gathered in the hepatocyte Golgi organelle on hepatocyte (d)
albumin, and Hp were identified with significantly increased glycosylation at POD 60 compared with POD 14, suggesting that glycoproteins play a critical role in immune regulation.
31.2.5 Identification of ConA-Enriched Serum Proteome For whole serum proteome analysis, the dynamic ranges of proteins hamper in-depth analysis of protein profiles. Hence, ConA-enriched serum proteins were analyzed by 2-DE to distinguish differential glycoproteins and to allow for easier detection of proteins at lower concentrations. Reproducible gel patterns were observed between the replicates. More than 120 proteins were identified for both POD-14 and POD-60 samples (Fig. 31.5). We recovered several target proteins from 2-DE gels based on changes in spot intensity, and further identified the proteins by in-gel trypsin digestion and MALDI–TOF mass spectrometry (Fig. 31.6a). Five candidate spots generated from the 2-DE gels were also
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Fig. 31.4 Comparison of glycoprotein patterns detected by Pro-Q Emerald 488 methods in SDS–PAGE. S: silver stain
subsequently sequenced and identified as summarized in Table 31.1. Further characterization was performed by a postsource decay analysis of the mass peptide fragments (Fig. 31.6b).
31.3 Discussion By means of 2-DE analysis of serum samples of OLT animal models, the differential expression of proteins was evaluated, revealing 13 proteins with significant changes between rejection and tolerance periods after OLT and several proteins that might be associated with tolerance or rejection. Previous reports have proposed that carbohydrate-rich plasma proteins produced by the liver are located on the surface of cancer cells and allow them to escape the immunological attack of the host [17, 26]. These carbohydrate-rich plasma proteins are now identified as acute-phase reactants.
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Fig. 31.5 2-DE profiles of ConA-enriched serum l at POD 14 and POD 60. Serum proteins (100 mg) were focused on a 4–7 IPG and then migrated at a right angle in SDS–PAGE on a 10% polyacrylamide gel. The protein patterns were stained by silver staining. The identified proteins are annotated and indicated by number
During tolerance after OLT, Hp was highly expressed at POD 60, and Hp protein glycosylation – which may be involved in the mechanisms of immunomodulation – was also upregulated during the rejection period [27]. The major function of Hp is to bind to hemoglobin and has been found to be potentially suppressive on many cellmediated immune responses in patients with cancer. The regulation of Hp has also been reported to be essential in the sertoli cell of the testis for maintaining immune privilege. In our recent study, we showed the immunosuppressive effects of Hp on the allogenic DA splenocyte in vitro [17, 28]. In addition, our previous results demonstrated that T-cell apoptosis may contribute to the control of the immune response in the drug-free tolerant OLT model. CD8+ T cells may be susceptible to apoptosis by differential crosslinking of glycoproteins in the absence of interleukin-2 and T-cell receptor signaling and enable contraction upon attenuation of immune signaling [29, 30]. These evidences directed attention to Hp as an immunological suppressor in OLT. It has been shown previously that human a1-microglobulin protein carries several N-glycosidically linked oligosaccharides of the high-mannose type and inhibited the proliferative response of peripheral human blood leucocytes to the antigen [31]. This result is consistent with our findings that overexpression and glycosylation of a1-microglobulin might be associated with immunosuppression at POD 60 post-OLT.
31 Serum Glycoprotein Profiles in Liver Transplantation
595
a [Abs. Int. * 1000]
10
1010.577 316-323
923.467 208-214
∗
1816.847 44-58
∗
1803.716 215-229
9 8 1407.670 183-195
7 6 5
1295.692 172-182
4 3 2
2125.951 369-386 2109.947 369-386
1391.663 183-195
1130.555 66-75 1111.573 269-278
1653.777 98-113 1535.751 118-131
2438.230 279-299 2431.155 343-362 1944.959 43-58
1 1000
1 51 101 151 201 251 301 351 401
1500
MKLITILLLC GNQFLLYRVT EAATGECTTT DSSDLKPVLK VQTNCSKEDF PGDDLFSLLP DTVKKATSQV NANVYMRPWE LNSCEYKGRL
SRLLPSLAQE EGTKKDGAET LGKKENKFSV HAVEHFNNNT PFLREDCVPL KKCFGCPKNI VAGTKYVIEF NKVVPTVRCQ SKAGAGPAPD
2000 m/z
EGAQEMDCND LYSFKYQIKE ATQICNITPG KHTHLFALTE PYGDHGECRG PVDSPELKEA IARETNCSKQ ALDMMISRPP HQAEASTVTP
2500
ETVFQAVDTA GNCSVQSGLT KGPKKTEEDL VKSAHSQVVA HTYVDIHNTI LGHSIAQLNA TNTELTADCE GFSPFRLVQV
3000
LKKYNAELES WQDCDFKDAE CVGCFQPIPM GMNYKIIYSI AGFSQSCDLY QHNHLFYFKI TKHLGQSLNC QETKEGTTRL
Fig. 31.6 Identification of target proteins and tandem mass spectrometry (MS/MS) spectrum of T-kininogen II (MAP1). (a) MALDI–TOF spectrums were obtained by in-gel trypsin digestion of T-kininogen II (MAP1). (b) LIFT-TOF/TOF (MS/MS) spectra generated by an Ultraflex TOF/ TOF operated in LIFT mode. The parent ions m/z 1010.577 and 1816.847 were selected for further analysis by MS/MS. The amino acid sequences YVIEFIAR and YNAELESGNQFLLYR were both assigned to human T-kininogen II. A sequence was confirmed from the labeled b- and y-ions in the spectrum
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ConA-enriched serum profile was taken as a subserum proteome to be analyzed. In addition to the common and abundant proteins, several differential serum glycosylated proteins with a potential regulatory role in OLT were identified. T-kininogen I and T-kininogen II (MAP1) belong to the plasma kallikrein–kinin system and participate in the pathogenesis of inflammatory reactions involved in cellular injury, coagulation, fibrinolysis, kinin formation, complement activation, cytokine secretion, and allograft rejection [32, 33]. They were expressed in lower levels revealed by ordinary 2-DE analysis at POD 60 with respect to POD 14; however, these two proteins significantly increased under ConA enrichment at POD 60, indicating that glycosylation of T-kininogen I and T-kininogen II may be associated with spontaneous tolerance after OLT. In conclusion, this proteomics analysis has permitted a logical evaluation and the direct detection of differentially expressed protein patterns associated with OLT tolerance, including Hp, immunoglobulin, and several glycoproteins. By means of specific staining and enriched techniques, we confirmed that Hp, a1-microglobulin, and kininogen with glycosylated modifications are important molecules in regulating the immune response and tolerogenic liver transplantation without the need for immunosuppressive therapy. Our results offer the opportunity for making serum proteins as clinical biomarkers for liver transplantation. The link among glycosylated modifications, the tolerogenic model, and the pharmacological effect of these protein targets in vivo still requires further experimental exploration.
31.4 Summary Liver allograft transplantation is an effective treatment for patients with end-stage liver diseases, and much of the successful transplantation should be due to the inherently tolerogenic manner. Recent reports have indicated that glycoproteins play fundamental roles in complex immunological processes, such as the acquisition of self-tolerance, especially in the innate and adaptive immune response. Our efforts to establish the functional serum proteome have led to the identification and characterization of glycoproteins that might be responsible for spontaneous tolerance of OLT in rats. In this report, we have described the differential patterns of protein expression in serum samples extracted from transplanted animals on POD 14 and POD 60, with regard to rejection and tolerance, respectively. Various plasma proteins associated with immunosuppressive and immunoregulatory effects were identified as glycoproteins and are discussed as well. Soluble proteins from serum samples were analyzed by one-dimensional gel electrophoresis and staining of gels with dyes that bind to glycoproteins (Pro-Q Emerald). Glycoproteins were further confirmed by binding to an immobilized ConA column, and their identity was revealed by MALDI/TOF–TOF. These proteins, including T-kininogen I, T-kininogen II, and a1-antitrypsin, have biological functions associated with immune regulation.
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Taken together, the analyses of global glycoprotein expression profiles provide new insights into discovering potential protein targets involved in OLT tolerance and developing successful strategies for allograft survival in the future. Acknowledgments This work was supported by a grant from the National Science Council (NSC96-2320-B-182-023-MY2) and the Chang Gung Memorial Hospitals (CMRPD170191), Taiwan. We would like to thank colleagues at the Chang Gung University for their excellent skills in carrying out the MALDI–TOF.
References 1. Billingham RE, Brent L, Medawar PB (1954) Quantitative studies on tissue in transplant immunity. Proc R Soc Lond 143:58–80 2. Zimmermann FA, Davies HS, Knoll PP, Gokel JM, Schmidt T (1984) Orthotopic liver allografts in the rat. The influence of strain combination on the fate of the graft. Transplantation 37:406–410 3. Kamada N, Davies HS, Brons G (1981) Reversal of transplantation immunity by liver grafting. Nature 292:840–842 4. Kamada N, Sumimoto R, Baguerizo A, Yoshimatsu A, Teramoto K, Yamaguchi A (1988) Mechanisms of transplantation tolerance by liver grafting in rats: involvement of serum factors in clonal deletion. Immunology 64:315–317 5. Goto S, Lord R, Kobayashi E, Vari F, Edwards-Smith C, Kammada N (1996) Novel immunosuppressive proteins purified from the serum of liver retransplantation rats. Transplantation 61:1147–1151 6. Starzl TE, Demetris AJ, Murase N, Trucco M, Thomson AW, Rao AS (1996) The lost chord: microchimerism and allograft survival. Immunol Today 17:577 7. Bishop GA, McCaughan GW, Sun J, Aheil AG (1997) Microchimerism and transplant tolerance. Immunol Today 18:455–456 8. Riordan SM, Williams R (1999) Tolerance after liver transplantation: does it exist and can immuno-suppression be withdrawn? J Hepatol 31:1106–1119 9. Goddard S, Adams DH (2002) New approaches to immunosuppression in liver transplantation. J Gastroenterol Hepatol 17:116–126 10. Yan SF, Ramasamy R, Schmidt AM (2008) Mechanisms of disease: advanced glycation endproducts and their receptor in inflammation and diabetes complications. Nat Clin Pract Endocrinol Metab 4:285–293 11. Rinaldi L, Gallo P (2005) Immunological markers in multiple sclerosis: tackling the missing elements. Neurol Sci 26(Suppl 4):S215–S217 12. Li B, An HJ, Kirmiz C, Lebrilla CB, Lam KS, Miyamoto S (2008) Glycoproteomic analyses of ovarian cancer cell lines and sera from ovarian cancer patients show distinct glycosylation changes in individual proteins. J Proteome Res 7:3776–3788 13. Mehta A, Block TM (2008) Fucosylated glycoproteins as markers of liver disease. Dis Markers 25:259–265 14. Miller I, Haynes P, Eberini I, Gemeiner M, Aebersold R, Gianazza E (1999) Proteins of rat serum: III. Gender-related differences in protein concentration under baseline conditions and upon experimental inflammation as evaluated by two-dimensional electrophoresis. Electrophoresis 20:836–845 15. Saldova R, Wormald MR, Dwek RA, Rudd PM (2008) Glycosylation changes on serum glycoproteins in ovarian cancer may contribute to disease pathogenesis. Dis Markers 25:219–232
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16. Pussinen PJ, Paju S, Mäntylä P, Sorsa T (2007) Serum microbial- and host-derived markers of periodontal diseases: a review. Curr Med Chem 14:2402–2412 17. Pan TL, Wang PW, Huang CC, Goto S, Chen CL (2004) Expression, by functional proteomics, of spontaneous tolerance in rat orthotopic liver transplantation. Immunology 113:57–64 18. Bockhorn M, Fingas CD, Rauen U, Canbay A, Sotiropoulos GC, Frey U, Sheu SY, Wohlschläger J, Broelsch CE, Schlaak JF (2008) Erythropoietin treatment improves liver regeneration and survival in rat models of extended liver resection and living donor liver transplantation. Transplantation 86:1578–1585 19. Schlaf G, Altermann WW, Rothhoff A, Seliger B (1997) Soluble CD30 serum level – an adequate marker for allograft rejection of solid organs? Histol Histopathol 22:1269–1279 20. Wu J, Lenchik NJ, Pabst MJ, Solomon SS, Shull J, Gerling IC (2005) Functional characterization of two-dimensional gel-separated proteins using sequential staining. Electrophoresis 26:225–237 21. Hart C, Schulenberg B, Steinberg TH, Leung WY, Patton WF (2005) Detection of glycoproteins in polyacrylamide gels and on electroblots using Pro-Q Emerald 488 dye, a fluorescent periodate Schiff-base stain. Electrophoresis 24:588–598 22. Goto S, Kamada N, Delriviere L, Kobayashi E, Lord R, Ware F, Hara Y, Edwards-Smith C, Shimizu Y, Vari F (1995) Orthotopic liver retransplantation in rats. Microsurgery 16:167–170 23. Yang Z, Harris LE, Palmer-Toy DE, Hancock WS (2006) Multilectin affinity chromatography for characterization of multiple glycoprotein biomarker candidates in serum from breast cancer patients. Clin Chem 52:1897–1905 24. Wang L, Li F, Sun W, Wu S, Wang X, Zhang L, Zheng D, Wang J, Gao Y (2006) Concanavalin A-captured glycoproteins in healthy human urine. Mol Cell Proteomics 5:560–562 25. Yang Z, Hancock WS (2004) Approach to the comprehensive analysis of glycoproteins isolated from human serum using a multi-lectin affinity column. J Chromatogr A 1053:79–88 26. Sanlioglu AD, Dirice E, Elpek O, Korcum AF, Balci MK, Omer A, Griffith TS, Sanlioglu S (2008) High levels of endogenous tumor necrosis factor-related apoptosis-inducing ligand expression correlate with increased cell death in human pancreas. Pancreas 36:385–393 27. Huntoon KM, Wang Y, Eppolito CA, Barbour KW, Berger FG, Shrikant PA, Baumann H (2008) The acute phase protein haptoglobin regulates host immunity. J Leukoc Biol 84:170–181 28. Hsu LW, Goto S, Nakano T, Lai CY, Lin YC, Kao YH, Chen SH, Cheng YF, Jawan B, Chiu KW, Chen CL (2007) Immunosuppressive activity of serum taken from a liver transplant recipient after withdrawal of immunosuppressants. Transpl Immunol 17:137–146 29. Shah A, Lowenstein H, Chant A, Khan A (2006) CD52 ligation induces CD4 and CD8 down modulation in vivo and in vitro. Transpl Int 19:749–758 30. Nasr IW, Wang Y, Gao G, Deng S, Diggs L, Rothstein DM, Tellides G, Lakkis FG, Dai Z (2005) Testicular immune privilege promotes transplantation tolerance by altering the balance between memory and regulatory, T cells. J Immunol 174:6161–6168 31. Wester L, Fast J, Labuda T, Cedervall T, Wingårdh K, Olofsson T, Akerström B (2000) Carbohydrate groups of alpha1-microglobulin are important for secretion and tissue localization but not for immunological properties. Glycobiology 10:891–900 32. Zavasnik-Bergant T (2008) Cystatin protease inhibitors and immune functions. Front Biosci 13:4625–4637 33. Acuña-Castillo C, Leiva-Salcedo E, Gómez CR, Pérez V, Li M, Torres C, Walter R, Murasko DM, Sierra F (2006) T-kininogen: a biomarker of aging in Fisher 344 rats with possible implications for the immune response. J Gerontol A Biol Sci Med Sci 61(7):641–649
Part VII
Glycobiology of Cancer
Chapter 32
Hematogenous Metastasis: Roles of CD44v and Alternative Sialofucosylated Selectin Ligands Konstantinos Konstantopoulos and Susan N. Thomas
Keywords Metastasis • Colon carcinoma cells • Selectin • CD44 • Shear flow P-selectin, expressed on activated endothelial cells and platelets, is a transmembrane glycoprotein (GP) that mediates, among others, host cell–tumor cell adhesion relevant to the process of hematogenous metastasis. The most compelling evidence for a direct role of P-selectin in the metastatic process is the pronounced inhibition of metastasis in P-selectin-deficient mice compared to wild-type controls in a colon carcinoma cell model [1–3]. Along these lines, enzymatic removal of P-selectin ligands from the colon carcinoma cell surface results in a pronounced reduction of experimental metastasis [1]. Although molecules that bind P-selectin have previously been identified in tumor cell lines [4–6], their functional roles and biological significance have not been substantiated. As has been appropriately argued in the literature [7], distinctions must be made between molecules that can bind to P-selectin under static conditions in vitro, and functional ligands that do interact with P-selectin under fluid dynamic conditions in vivo. By identifying the functional P-selectin ligand(s) on colon carcinoma cells, using an integrated approach consisting of bioengineering tools and contemporary biochemistry and molecular biology techniques, we provide guidelines for engineering novel therapeutic agents that selectively block tumor cell ligand binding function and thus interfere with metastatic spread. Such a strategy may offer specific antimetastatic efficacy without impairing other important P-selectin-mediated physiological processes [8, 9]. Alternatively, these molecules could be utilized in a targeted drug-delivery approach, which would aim at selectively or preferentially eradicating colon carcinoma cells from the vasculature.
K. Konstantopoulos (*) Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, MD 21218, USA e-mail:
[email protected] A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_32, © Springer Science+Business Media, LLC 2011
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32.1 Cell Adhesion Molecules 32.1.1 Selectins: Structure, Tissue Distribution, Regulation, and Ligand-Binding Site Localization Selectins are transmembrane glycoproteins that mainly recognize specific glycoconjugates on apposing cell surfaces [7, 10, 11], thereby mediating cell–cell interactions that are important for the processes of inflammation, hemostasis/thrombosis, wound healing, and cancer metastasis. The selectins are a family of three structurally related cell adhesion molecules: P-, L-, and E-selectin. Each selectin has an amino terminal domain with a sequence similar to the calcium-dependent animal lectins (C-type lectins), followed by an epidermal growth factor (EGF)-like domain, variable numbers of short consensus repeat domains, a single-pass transmembrane domain, and a short cytoplasmic carboxyl terminal tail. While they share many common elements, the tissue distribution and regulation of the three selectins are quite different, possibly reflecting their crucial involvement in a number of pathophysiological processes. L-selectin is constitutively expressed on the surface of almost all types of leukocytes but gets rapidly shed upon cell activation with cytokines, chemokines, or formyl peptides. L-selectin binds to inducible ligands on endothelium at sites of inflammation [10], to constitutively expressed ligands on high endothelial venules (HEVs) of the peripheral lymph nodes [10], and to ligands on other leukocytes [12–15] and a variety of tumor cell lines [16–18]. E-selectin expression is induced on vascular endothelial cells, requires de novo messenger RNA (mRNA) and protein synthesis, and peaks 4–6 h after activation with an inflammatory stimuli, such as interleukin (IL)-1, tumor necrosis factor-alpha, or endotoxin in vitro. However, in vivo, E-selectin may be chronically expressed at sites of local inflammation, particularly in the blood vessels of skin during delayed hypersensitivity reactions [19]. P-selectin expression on both vascular endothelium and platelets is also inducible. However, P-selectin is stored preformed in the Weibel–Palade bodies of endothelial cells and alpha-granules of platelets, from which it can be rapidly mobilized (within minutes) to the plasma membrane upon activation. Additionally, P-selectin expression on vascular endothelium may also be regulated at the transcription level. Cytokines, such as IL-4 or oncostatin M, significantly upregulate P-selectin mRNA levels in cultured human endothelial cells in a delayed and sustained fashion [20]. Both E- and P-selectin bind to ligands on myeloid cells [10, 21], subsets of lymphocytes [10], and a variety of tumor cells [18, 22–25]. P-selectin may also interact with ligands on platelets [26], HEVs [27], and on activated endothelial cells [28]. Like all C-type lectins, the selectins bind to carbohydrate ligands in a calciumdependent manner. Detailed studies involving site-directed mutagenesis, domain swapping, and antibody inhibition have revealed that carbohydrate ligands bind to the lectin domain on a shallow region that overlaps a single calcium coordination site opposite, where the EGF domain is located [7, 10]. Several lines of evidence
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also suggest that the EGF domain and, to a lesser degree, the short consensus repeat domains contribute to ligand specificity [7, 10]. On the other hand, published findings illustrate that some anionic phospholipids can bind to L- and P-selectin (but not E-selectin) at a site distinct from the carbohydrate-binding site [7, 29].
32.1.2 P-Selectin Ligands The selectins share the ability to recognize the tetrasaccharide sialyl Lewis x (sLex; NeuAc a2,3 Gal b1,4 [Fuc a1,3] GlcNAc-R) and its isomer sialyl Lewis a (sLea) [7, 10]. sLex is a terminal component of glycans attached to glycoproteins and glycolipids on most circulating leukocytes and some endothelial cells. On the other hand, sLea, which has the attachment sites of the Gal and Fuc reversed (NeuAc a2,3 Gal b1,3 [Fuc a1,4] GlcNAc-R), is found on some tumor cells but not on normal leukocytes. The binding affinity of selectins for isolated monovalent sLex and sLea oligosaccharides is very low. Consequently, neither expression of the sLex nor the sLea groups per se correlates with the properties of endogenous selectin ligands on cellular targets. As pointed out by Varki [7], the challenge is to “… tell the difference between what can bind … in vitro, and what does bind under fluid dynamic conditions in vivo…” To this end, a functional selectin ligand should fulfill certain criteria; it should be expressed in the right place at the right time, removal or absence of the ligand should prevent interactions, and the ligand should bind with some selectivity and relatively high affinity. To keep this discussion focused, only putative ligands for P-selectin are reviewed here, although there are fascinating separate literatures on endothelial ligands for L-selectin and leukocyte ligands for E-selectin. Efforts to characterize and clone the expression of a P-selectin ligand have been successful [30, 31]. The P-selectin glycoprotein ligand-1, PSGL-1, is expressed on virtually all blood leukocytes, human hematopoietic progenitor cells, human promyelocytic leukemia (HL-60) cells [32], and, to a much lesser extent, on blood platelets [33]. PSGL-1 is a highly extended membrane glycoprotein that forms homodimers covalently linked by a disulfide bond between a single extracellular cysteine in each subunit. The extracellular domain of the mature protein (~60-nm long) has the hallmarks of a mucin since it is rich in serines, threonines, and prolines and includes 16 decameric repeats [32]. Detailed biochemical studies indicate that PSGL-1 has fucose- and sialic acid-containing polylactosamine side chains, most of which are presumably attached to serines and/or threonines of the decamer repeats and many of which terminate in sLex [32]. Consequently, PSGL-1 is susceptible to proteases, including P. hemolytica O-sialoglycoprotease, which only cleaves glycoproteins with heavily O-glycosylated sialylated domains. PSGL-1 also has two or three N-linked sugars that are not required for binding to P-selectin [32]. In addition to the presence of O-linked fucosylated and sialylated structures, high affinity binding of recombinant PSGL-1 to P-selectin requires three tyrosine sulfate residues located near the amino terminus of the mature glycoprotein [34].
604
K. Konstantopoulos and S.N. Thomas
Although PSGL-1 contains 80% NeuBu residues were effective inhibitors of 13D9 binding to the ManBu-treated tumor cells (Fig. 36.4). Conversely, only the homogeneous NeuAc-containing inhibitor was able to interfere with mAb 735 binding to the same treated tumor cells. This result indicated that 2.5
OD405
2.0 1.5
b NAcPSA-HSA NPrPSA-HSA NBuPSA-HSA
1.0 0.5 0.0 0.001
2.5 2.0
OD405
a
1.5
NAcPSA-HSA NPrPSA-HSA NBuPSA-HSA
1.0 0.5
0.01
0.1
mAb 735 (ug/ml)
1
0.0 0.001
0.01
0.1
mAb 13D9 (ug/ml)
1
Fig. 36.3 Binding characteristics of mAbs 735 and 13D9. The specificities of mAbs 735 (a) and 13D9 (b) were determined by indirect enzyme-linked immunosorbent assay against human serum albumin conjugates of PSA ( filled square), NPrPSA ( filled up triangle), and NBuPSA ( filled down triangle)
684
R.A. Pon et al.
native PSA and de novo NBuPSA exist as discrete polysaccharides on the surface of ManBu-treated cells and not as a single polysaccharide composed of varying levels of mixed NeuAc to NeuBu residues. An identical inhibition profile was obtained using mixed inhibitors composed of NeuPr:NeuAc residues on ManPr-treated tumor cells supporting this conclusion (Fig. 36.4). Although flow cytometric analysis indicated a relatively high surface density of de novo NBuPSA and residual PSA, the strong cytotoxicity responses of ManBu-treated tumor cells in the presence of either mAb 13D9 or 735 and a complement illustrated that the abundances of these two surface polysaccharides were sufficient to behave as functional determinants (Fig. 36.5). Although our studies indicated that an array of tumor cells were amenable to bioengineering with various N-acyl mannosamine sialic acid precursors, other groups [25, 26] reported that ManBu precursor treatment had the effect of abrogating PSA expression on human NT2-derived neurons. This observation has significant
70
% Inhibition
60
b mAb 735
50
% Inhibition
a
40 30 20 10 0 0 35 65 80 100
90 80 70 60 50 40 30 20 10 0
mAb 735 mAb 13D9
0 35 65 80 100
0
35
65
80
%NPr in NAcPSA %NBu in NAcPSA
%NPr in NAcPSA
RMA-s
NPrRMA-s
% Inhibition
c
90 80 70 60 50 40 30 20 10 0
100
mAb 735 mAb 13D9
0
35
65
80
100
%NBu in NAcPSA
NBuRMA-s Fig. 36.4 Surface epitope specificity on bioengineered RMA-s tumor cells. Mixed polysaccharide inhibitors of defined length and composition (NPr:NAcPSA; NBu:NAcPSA) were prepared [27] and used in competitive inhibition studies. RMA-s tumor cells were cultured for 3 days with media alone (a), 10 mM ManPr (b), or ManBu (c), stained with either mAbs 735 (a–c) or 13D9 (b, c) that were pretreated with 1 mg of the indicated mixed inhibitor, and evaluated by flow cytometry. The percent inhibition was calculated relative to the mean channel fluorescence values obtained from each respective mAb without polysaccharide inhibitors
36 Polysialic Acid Bioengineering of Cancer and Neuronal Cells
60 % Specific cytotoxicity
b
RMA-s cells- non-treated mAb (ug) 4 2 1 0.5 0.25 0.125 0.063 0.031 0.016 0.008 0.004
50 40 30 20 10 0
RMA-s cells- ManBu treated 60
% Specific cytotoxicity
a
685
50 40 30 20 10
mAb (ug) 4 2 1 0.5 0.25 0.125 0.063 0.031 0.016 0.008 0.004
0 mAb 735 mAb 13D9 mAb treatment
mAb 735 mAb 13D9 mAb treatment
Fig. 36.5 Complement-dependent cytotoxicity of native and ManBu precursor-treated RMA-s tumor cells with mAbs 735 and 13D9. RMA-s cells labeled for 1 h with Na2[51Cr]O4 were combined with graded amounts of mAb 735 or 13D9 in the presence of 5% baby rabbit complement for 4 h at 37°C. Released Cr-51 was measured by scintillation counting, and the percent specific cytotoxicity was calculated. Maximum release was determined in the presence of cetrimide and spontaneous release in the presence of medium alone and was consistently Et (ethyl) > Ph ~ p-Tol (p-toluenyl)” is suggested for the relative reactivity of different thio functional groups. The most commonly used promoters include methyl trifluoromethanesulfonate (MeOTf), dimethyl(methylthio) sulfonium trifluoromethanesulfonate (DMTST), methylsulfenyl trifluoromethanesulfonate, 1-benzenesulfinyl piperidine and trifluoromethanesulfonic anhydride, benzeneselenyl trifluoromethanesulfonate, iodonium di-sym-collidine perchlorate, and N-iodosuccinimide (NIS) with trifluoromethanesulfonic acid (TfOH) [56, 57]. Toward the preparation of the carcinoembryonic antigen, Verez-Bencomo has reported the synthesis of the SAa2,6Galb1,4GlcNHAc unit (Scheme 37.5) [71]. The lactosamine moiety (27) (81%) was generated via regioselective glycosylation of the 3,4-diol (26) with the galactose-derived imidate donor (25) in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) as a promoter. Acetylation of (27) yielded the ester (28) (quant.), which underwent sequential hydrazinolysis, acetylation, and hydrolysis to provide the desired disaccharide acceptor (29) (73% overall yield). Sialylation, activated by NIS and TfOH, was then carried out using the thioglycoside (30) as the donor to give the trisaccharide (31) (67%, a/b = 3/1). A similar preparative route has been described by Nifant’ev et al. (Scheme 37.6) [72]. The lactosamine part (34) was prepared in 37% yield by the CuBr2/AgOTf/
C.-C. Wang et al.
698 Ph Ph
O O AcO
OBn O
+
AcO O
O NPhth
CCl 3
O O
TMSOTf
O
HO HO
O
N3
81%
AcO
26
NH
OBn O
AcO
25
HO OH O AcO AcO
73%, in 3 steps
O
N3
27: R = H
Ac2O, pyridine
1. N 2H4 2. Ac2O, pyridine 3. 60% HOAc
O O NPhth
O RO
28: R = Ac (quant.)
OBn O AcO
O O NHAc
N3
O
29
OAc
AcO AcO
OAc MeO2C
O AcHN AcO AcO
+
SPh
29
NIS, TfOH 67%, α/β = 3/1
MeO 2C
O AcHN O AcO HO AcO
O
AcO
AcO
30
OBn O AcO
O O NHAc
O
N3
31
Scheme 37.5 The synthesis of the SAa2,6Galb1,4GlcNHAc unit
Ph
BzO
+
O
SEt
O
HO BnO
BzO
OBn
O
O BnO
AcO
O O(CH2)3NHCOCF3 NHAc 34 AcO
AcO
37%, in 2 steps
33
32 OH
90% TFA
O(CH2)3NHCOCF 3 NHAc
OAc
HO
CuBr2, AgOTf, n-Bu4NBr
OBn
O O
OAc MeO2C
O AcHN AcO AcO 30
SPh
+
34
NIS, TfOH CH3CN, 79%, α/β = 1.3/1
OAc MeO2C
O O AcHN AcOAcO HO BzO
OBn
O
O AcO BnO 35
Scheme 37.6 A similar preparative route described by Nifant’ev et al. [72]
O O(CH2)3NHCOCF3 NHAc
37 Synthesis of Hemagglutinin-Binding Trisaccharides
699
Bu4NBr-promoted assembly of the ethyl thiogalactoside donor (32) with the lactosamine acceptor (33), followed by the removal of the benzylidene acetal in 90% trifluoroacetic acid (TFA). Coupling of (34) with the thioglycoside (30) employing NIS and TfOH in acetonitrile gave the trisaccharide (35) in 79% yield (a/b = 1.3/1). 37.1.1.2 Path B Using a different approach, Kiso et al. [73] have reported the synthesis of the pentasaccharide SAa2,6Galb1,4GlcNHAcb1,3Galb1,4Glc. As summarized in Scheme 37.7, regioselective coupling of the S-methyl donor (36) and 2-(trimethylsilyl) ethyl 3-O-benzyl-b-D-galactopyranoside (37) at O6 using DMTST as the promoter led to the disaccharide (38) in 45% yield. In this strategy, the key step that sometimes causes drop in the overall yield is carried out at the early stage. Benzoylation of the 2,4-diol (38) yielded the ester (39) (61%), which was transformed into the AcO OAc
AcO
AcHN AcO
O
OH
HO
MeO2C SMe
+
O
SiMe3
OH
OAc
MeO2 C
O AcHN O AcO AcO RO
DMTST
O
BnO
OAc
45%
BnO
37
36
OAc
AcHN AcO AcO
Ac2O, BF3/OEt2 61%
AcO
MeO 2C O
MeSSiMe 3, BF3 /OEt2
O
BzO
39: R = Bz, 61%
OAc MeO 2C
OAc
BnO
OBz
41
+
HO AcO
O
O
NHAc
OBn O BnO
O
SMe
OBz
41
40 OBn BnO
SiMe 3
38: R =H
O AcHN O AcO AcO BzO
87%
O
BnO
O
OR BzCl, pyridine
AcO
O
OBn O
O BnO
O
OBn
DMTST SiMe 3
55%
42
AcO
OAc MeO 2C
O AcHN O AcO AcO BzO BnO
O
BzO
OBn BnO O AcO
O
O
NHAc
OBn O BnO
OBn O BnO
O OBn
O
SiMe 3
43
Scheme 37.7 Using a different approach, Kiso et al. [73] have reported the synthesis of the pentasaccharide SAa2,6Galb1,4GlcNHAcb,3Galb,4Glc
C.-C. Wang et al.
700
corresponding acetate (40) (62%) via treatment with Ac2O and BF3/OEt2. Anomeric replacement of (40) with MeSSiMe3 in the presence of BF3/OEt2 afforded the thioglycoside (41) (87%), which was coupled with the trisaccharide acceptor (42) using DMTST to give the desired pentasaccharide (43) in 55% yield. To further study the binding specificity of various HAs, Kiso et al. [74] have reported a total synthesis of the pentasaccharide probe Neu5Gca2,6 Galb1,4GlcNHAcb1,3Galb1,4Glc (Scheme 37.8). Similarly, the 2,6-glycosidic
OAc
AcO AcO O
MeO2C H O N SPh OAc OAc
OH
HO
NIS, TfOH
O
+ BnO
O
SiMe3
OH
44 OAc
AcO
AcO
MeO 2C
AcO O
H O O N OAc OAc RO BnO
TFA 97%
O
OAc MeO 2C H O N O OAc OAc BzO BnO
SiMe 3
O
OH
OBz
45 : R =H
47
46 : R = Bz, 91% OAc
AcO CCl3CN, DBU
AcO O
O OR
Bz2O, DMAP pyridine
67%
37
MeO2C H O O N OAc OAc BzO
AcO O
96%
BnO
O
BzO OC(NH)CCl
3
48 OBn BnO 48
+
HO BnO
O
OBn O
O
BnO
NHAc
OBn O BnO
TMSOTf
O
O
OBn
SiMe3
82%
49
AcO AcO O
OAc MeO 2C H O O N OAc OAc BzO BnO
O
BzO
OBn BnO O BnO
O
O
NHAc
OBn O BnO
OBn O BnO
O OBn
O
SiMe3
50
Scheme 37.8 To further study the binding specificity of various HAs, Kiso et al. [74] have reported a total synthesis of the pentasaccharide probe Neu5Gca2,6 Galb1,4GlcNHAcb1,3Galb1,4Glc
37 Synthesis of Hemagglutinin-Binding Trisaccharides
701
bond between Neu5Gc and Gal was built first. Regioselective glycosylation of the thiophenyl Neu5Gc donor (44) with the galactose-derived triol (37) in the presence of NIS and TfOH gave the disaccharide (45) (67%), which was benzoylated to furnish the fully protected disaccharide (46) (91%), having the C2 participating group to ensure the b-configuration in the succeeding glycosylation. Cleavage of the 2-(trimethylsilyl)ethyl group in (46) with TFA provided the alcohol (47) (97%), which was converted into the corresponding trichloroacetimidate (48) in 96% yield. The subsequent glycosylation reaction of the donor (48) with the trisaccharide acceptor (49) was carried out by using TMSOTf as activator, and the pentasaccharide (50) was obtained in 82% yield. A recent synthesis of the SAa,6Galb1,4GlcNHAcb1,3Galb1,4Glc pentasaccharide has been described by Seeberger and Hanashima (Scheme 37.9) [75]. OAc
AcO AcO
OAc
TrocHN AcO
OH
HO
MeO2C
OTDS
Ac2O, pyridine
TDS = thexyldimethylsilyl TCA = trichloroacetyl Aloc = allyloxycarbonyl
AcO
53: R = H 54: R = Ac, 71%, over 2 steps α/β = 6:1
OAc
AcO
MeO 2C
O TrocHN O AcO AcO AcO BzO
CF3C(NPh)Cl, Cs 2CO3
OAc MeO2C
O TrocHN O AcO AcO AcO
92%
O
BzO
OH
BzO
OBn BnO O O NHTCA
BzO
O
CF3
56
OBn O
NPh
O
BzO
55
HO 56 + LevO
OTDS
OBz
Troc = 2,2,2-trichloroethoxycarbonyl
80%
O
BzO
52
51
HF/pyridine
O
OAc RO
CH 3CN
OBz
OAc
O
TrocHN AcO
NIS, TfOH
O
SPh + BzO
O
MeO 2C
OBn O BzO
TMSOTf
O
O(CH 2)6NHAloc
OBz
90%
57
AcO
OAc MeO 2C
O TrocHN O AcO AcO AcO BzO
O
BzO
O LevO
OBn BnO O O NHTCA
OBn O BzO
OBn O BzO
O
O(CH2)6 NHAloc
OBz
58
Scheme 37.9 A recent synthesis of the SAa2,6Galb1,4GlcNHAcb1,3Galb1,4Glc pentasaccharide has been described by Seeberger and Hanashima [75]
C.-C. Wang et al.
702
The galactose-derived diol (52) was regioselectively sialylated with the N-Trocprotected thioglycoside (51) using the NIS/TfOH promoter system in acetonitrile to give the disaccharide (53) that, upon acetylation, provided the ester (54) in two steps in 71% overall yield (a/b = 6/1). Removal of the anomeric thexyldimethylsilyl (TDS) group led to the alcohol (55) (80% yield), which was transformed into the N-phenyl trifluoroacetimidate (56) in 92% yield. Coupling of compound (56) and the trisaccharide building block (57) catalyzed by TMSOTf afforded the pentasaccharide (58) in 90% yield. 37.1.1.3 One-Pot Glycosylation The first one-pot glycosylation using the sialyl donor as the starting sugar unit via chemical approach was reported by Takahashi et al. for synthesizing the branched 2,6-sialyl-T antigen [76, 77]. The first one-pot synthesis of the SAa2,6Galb1,4GlcNHAc trisaccharide unit was carried out by Hung et al. [78]. As illustrated in Scheme 37.10, the target molecule (1), presented as a linear trisaccharide, could be prepared via the assembly of building blocks (59, 60, and 61) in a one-pot manner. The challenge for this one-pot protocol was the selective activation of the leaving groups on the sialyl donor (59) and the thiogalactoside (60). Exploiting the difference in reactivity between S-benzoxazolyl (SBox) and S-toluenyl (STol), it was achieved by using silver trifluoromethanesulfonate (AgOTf) as the promoter in a mixed solvent, CH2Cl2/Et2O = 3/1, to drive the steric preference of the disaccharide intermediate from b- into the desired a-configuration (Scheme 37.11). After forming the SAa2,6Gal intermediate, the building block (61) was added into the same vessel without workup or purification, and the STol group of the disaccharide intermediate was sequentially activated by p-toluenesulfenyl triflate, generated in situ from p-toluenesulfenyl chloride and AgOTf, to trigger the coupling of the intermediate with the 4-alcohol (61). Owing to the neighboring group participation of the O2 benzoyl group in the galactose unit, the stereoselectivity of the second glycosylation is exclusively b, and the expected trisaccharide (65) was isolated in 63% yield (a/b = 2.7/1). The terminal acceptors (62, 63, and 64) were also employed in the one-pot strategy to afford the corresponding products (66) (a/b = 2.2/1), (67) (a/b = 2.1/1), and (68) (a/b = 2.2/1) in 61, 56, and 83% yields, respectively. HO
OH
AcO
HO2C
O AcHN O HO HO HO HO
O OH
AcHN AcO
OH O HO
OAc MeO 2C O AcO
O AcHN
SAα2,6Galβ1,4GlcNHAc
OMe
BnO
S O
N
OH O
+ BzO
OBz
OBz STol
+
O
HO BnO
N3 OMe
60 61 59
1
Scheme 37.10 The first one-pot synthesis of the SAa2,6Galb1,4GlcNHAc trisaccharide unit was carried out by Hung et al. [78]
37 Synthesis of Hemagglutinin-Binding Trisaccharides AcO
OAc
60 AgOTf
MeO 2C
AcHN AcO
O AcO
S
ROH, p -TolSOTf
703
O
MeO2C
AcHN AcO
CH2Cl2/Et2O, N (1/3) −40 oC
(α/β = 5/2)
OAc
AcO
O
OR
OBz 65-68 OBz O
HO BnO
N3 OMe
O BzO
65 63%, α/β = 2.7/1
BzO
OMe
O BnO
BzO OMe 66 61%, α/β = 2.2/1
BnO BnO
HO
H NHCbz OMe O
OMe
63
64
OBn
O N3 OMe
O
BnO BnO
62
OBz BnO
OH
OBn HO BnO
61
Product, R=
O
BzO
59
ROH =
O
BnO
AcO
O
BzO OMe 67 56%, α/β = 2.1/1
H NHCbz OMe O 68 83%, α/β = 2.2/1
Scheme 37.11 The challenge for this one-pot protocol was the selective activation of the leaving groups on the sialyl donor (59) and the thiogalactoside (60). Exploiting the difference in reactivity between SBox and STol, it was achieved by using AgOTf as the promoter in a mixed solvent, CH2Cl2/Et2O = 3/1, to drive the steric preference of the disaccharide intermediate from b- into the desired a-configuration
37.1.2 Chemical Synthesis of the SAa 2,3Gal b 1,4GlcNHAc Trisaccharide Unit The preparation of the SAa2,3Galb1,4GlcNHAc trisaccharide unit (69) via chemical approach can be classified into two pathways (Scheme 37.12). In pathway A, the target molecule (69) could be synthesized via coupling of the sialyl donor (2) and the lactosamine acceptor (70), bearing a free hydroxyl group at the C3¢ position of the galactose unit. The synthesis of (72) would be either from commercially available lactosamine or from the assembly of the galactose-derived donor (4) and the glucosamine-derived acceptor (5), followed by regioselective deprotection at C3¢. Via pathway B, the SAa2,3Gal donor (71) would be synthesized by coupling of the sialyl donor (2) and the galactose-derived acceptor (72), followed by subsequent anomeric transformation. The disaccharide donor (71) could be coupled with the glucosamine-derived acceptor (5). Again, despite the additional steps for changing the anomeric functionality, the stereoselective sialylation of the monosaccharide unit (72) at the early stage is believed to be easier than that of disaccharide (70) performed at the later synthetic steps.
C.-C. Wang et al.
704 OP6
P 4O
OP6
+
O
P3 O
LG
OP2
HO P3O
OP8 5
HO
OH
P HN P 7O P 4 O
A HO2C HO
O AcHN HO HO
O
OH O OH HO
O
P = Protecting group LG = Leaving group
LG
O
HO
P2 O
OP 6 O P3O
O
OR
P2HN
70
OR
AcHN
SAα2,3Galβ1,4GlcNHAc 69
+
2
OH
O
O
OP6
P4O
P1O2C
OR
5
4
P9 O
O P2HN
OP8
P9O
B
4 P 1O 2C P O
O P5HN P7O P4O
P9 O
OP8
2
LG
OP6
+
O OP
2
LG
+
HO P 3O
O
OR
P2HN 5
71
P4O
P1O2C
O P5HN P7O P4O
O
OP 6
HO
OP6 O OP 2
P1
72
Scheme 37.12 The preparation of the SAa2,3Galb1,4GlcNHAc trisaccharide unit (69) via chemical approach can be classified into two pathways
37.1.2.1 Path A The synthesis of the SAa2,3Galb1,4GlcNHAc trisaccharide unit using the sialyl dibenzyl phosphates was first introduced by Wong [79, 80] (Scheme 37.13). These highly reactive glycosyl donors can be activated by a catalytic amount of TMSOTf. Due to steric effect, the C3¢-OH is the most reactive among all of the secondary hydroxyl groups in the lactosamine derivative (74). Regioselective coupling of compound (73) with the tetraol (74) led to the desired trisaccharide (75) in good yield and stereoselectivity (78%, a/b = 6/1) [79]. In the synthesis carried out by Ogawa et al. (Scheme 37.14) [81], the lactosaminederived triol (79) was prepared from the galactose-derived thioglycoside (76) in two steps. Coupling (76) with the 4-alcohol (77) furnished the disaccharide (78) (82%), which underwent deacetylation to give the triol (79) in 74% yield. TMSOTf-catalyzed regioselective coupling of the diethyl b-sialyl phosphite (80) with (79) provided the trisaccharide (81), which was acetylated to give the corresponding ester (82) in two steps with 51% overall yield (a/b = 1.7/1). An alternative route is to use the sialyl thioglycoside (36) as the glycosyl donor. Coupling of (36) with (79) afforded the trisaccharide (81), which was subjected to acetylation to yield the product (82) (67%, a/b = 2.5/1).
37 Synthesis of Hemagglutinin-Binding Trisaccharides AcO
OAc OP(OBn) 2 O
AcHN AcO
CO 2Me
OTBDPS
HO
+
O
HO
HO
OAc
705
OTBDPS
TMSOTf
O
O HO
O NHAc
CH3CN, −42 oC, 78% (α/β = 6/1)
74
73
OAc
AcO
O
AcHN AcO
OTBDPS
MeO2C HO
O
O
O OH HO
OAc
OTBDPS O O NHAc
75
Scheme 37.13 The synthesis of the SAa2,3Galb1,4GlcNHAc trisaccharide unit using the sialyl dibenzyl phosphates was first introduced by Wong [79, 80]
AcO
OBn
OBn
O
O
AcO
SPh + HO BnO
AcO 76
OMP NDCPhth 77
DCPhth =
MP = Cl
82%
OBn
O
O OMP NDCPhth
O RO BnO 78: R = Ac 79: R = H
OMe
O AcO OP(OEt) 2 O
CO2Me
+ 79
TMSOTf CH3CN, −40 oC
OAc
CO2Me
OAc
MeO 2C RO
Ac2O, pyridine DMAP 51%, over 2 steps (α/β = 1.7/1)
OAc O
OAc
O AcHN AcO OAc
80
AcHN AcO
OBn
NaOMe, 74%
OAc
AcHN AcO
AcO
RO
O
Cl
AcO
RO NIS, TfOH
SMe
+ 79
NIS, TfOH CH3CN, −40 oC
81
Ac 2O, pyridine DMAP 67%, over 2 steps (α/β = 2.5/1)
O
OBn O
O RO BnO
OBn O
OMP NDCPhth
81: R = H
82: R = Ac
82
36
Scheme 37.14 The synthesis carried out by Ogawa et al. [81]
As discussed previously, the sialic acid-derived thioglycosides are widely used as donors in glycosylation reactions. Table 37.1 summarizes the synthesis of the trisaccharides via coupling of the donor (30) with a variety of acceptors (79, 83, 85, 87, 89, 91, and 93) employing NIS and TfOH as the promoters [81–88]. The trisaccharides (81, 84, 86, 88, 90, 92, and 94) were obtained in 65, 43, 61, 51, 45, 56, and 55%, respectively. A typical protection of the lactosamine acceptor, reported by Ogawa et al. [89], could be applied to prepare the trisaccharide unit (Scheme 37.15). The lactosamine derivative (95) was treated with 4-methoxyphenol and TfOH to afford compound
C.-C. Wang et al.
706 Table 37.1 Coupling of the thioglycoside (30) with various acceptors OAc
AcO
MeO2C
O AcHN AcO AcO
SPh
+ acceptor
AcO
NIS, TfOH
product
AcHN AcO
30
Entry Acceptor 1
Product
HO OBn O
HO
OBn O
O HO BnO
OMP NDCPhth
HO RO
OPiv O
OMe NPhth
O HO BnO
HO OClAc O RO O HO BnO
OPiv O
O HO HO
OBn NPhth
HO RO
OPiv O
O HO HO
HO
OBn O
OBn O
O BnO BnO
O(CH2)7CH3 NHAc
HO OBn O RO O BnO BnO
87 O O HO
O HO
OPiv O
O HO
OMe NPhth
O O RO
O
O HO HO
89
O HO
O HO
OTBDMS O O(CH2)7CH3 NHAc
OPiv O
OMe NPhth
43
-b
83
OPiv O
OBn NPhth
61
-b
84 85
OBn O
O(CH2)7CH3 NHAc
51
-b
86
OPiv O
OMe NHAc
45
-b
87
O O RO
O
O HO HO
92
OTBDMS O O(CH2)7CH3 NHAc
56
-b
88
55
-b
89
MP
MP O O HO
82
90
91
7
1.8/1
Ph
O O HO
65a
88
Ph
6
Ref.
Ph
Ph
5
α/β
86
85
4
OMP NDCPhth
*
OAc
84
HO OPiv O
HO
OBn O
O
83
3
O
81
HO OClAc O HO O HO BnO
HO
MeO2C
Yield (%)
OBn
79
2
OAc
R=
OBn O
O
O HO BnO
O(CH2)7CH3 NHAc
O O RO
O
O HO BnO
93
OBn O
O(CH2)7CH3 NHAc
94
a Determined after subsequent peracetylation. b Not reported.
(96) (91%), which was subsequently deacetylated by NaOMe to give the hexaol (97) in 84% yield. 3¢,4¢-Isopropylidenation of (97) with 2,2-dimethoxypropane in the presence of para-toluenesulfonic acid led to the tetraol (98) (83%), which was regioselectively protected with pivaloyl chloride at O6 and O6¢ to yield the diol (99) (83%). The isopropylidene group in (99) was removed by the treatment with 80% TFA to give the tetraol (100) in 78% yield. Sialylation of (100) with the S-methyl thioglycoside (36) using NIS/TfOH gave the trisaccharide (101), which was acetylated to provide the product (102) in two steps in 63% overall yield.
37 Synthesis of Hemagglutinin-Binding Trisaccharides AcO
OAc O
AcO
O AcO
AcO
O PhthN
RO
4-methoxyphenol, TfOH
OAc
NaOMe
OR
O
2,2-dimethoxypropane, PTSA
O
HO
+
100
97: R = H (84%)
O HO
OPiv
HO
OMP NPhth
O
HO
78%
OPiv O OMP NPhth
O HO
HO
100
99: R = Piv (83%)
OAc
AcO
CO2Me
96: R = Ac
80% TFA
O
OAc O
O OMP NPhth
98: R = H
PivCl, DMAP, pyridine
SMe
OR O RO
OR
O
83%
AcHN AcO
O RO
95
AcO
OR
RO
91%
OAc
707
NIS, TfOH
O AcHN AcO OAc
CH3 CN, −40 oC
OAc
O
O
RO
OPiv O OMP NPhth
O RO
101: R = H
Ac2O, pyridine DMAP 63%, over 2 steps
36
OPiv
MeO2C RO
102: R = Ac
Scheme 37.15 A typical protection of the lactosamine acceptor, reported by Ogawa et al. [89], could be applied to prepare the trisaccharide unit
AcO
OAc CO2R
AcHN AcO
O
SMe
OAc
36: R = Me
HO
+
HO
OBn
OBn
O
NIS, TfOH
O
O BnO BnO
CH3CN, −40 oC
O NPhth
104
103: R = CH2CH2CN
AcO
OAc
RO2 C HO
O AcHN AcO OAc
O
OBn O
O BnO BnO
OBn O
O NPhth
105: R = Me, 52% (α/β = 11:1) 106: R = CH2CH2CN, 54% (α/β = 19:1)
Scheme 37.16 Coupling of the lactosamine derivative (104) with compound (36) under typical NIS/TfOH conditions led to the trisaccharide (105) in 52% yield (a/b = 11/1)
To tackle the difficulties in controlling the stereochemistry of sialylation, the substituent effect of the C1 ester was examined [90]. The change of the C1 methyl into the cyanoethyl ester, which can be easily removed by the treatment with 1,8-diazabicyclo[5.4.0]undec-7-ene, has been reported by Ito and Ishiwata [91]. As illustrated in Scheme 37.16, coupling of the lactosamine derivative (104) with
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708
compound (36) under typical NIS/TfOH conditions led to the trisaccharide (105) in 52% yield (a/b = 11/1). When the C2-S-methyl,C1-cyanoethyl ester (103) was used, the a:b ratio was improved to 19:1. 37.1.2.2 Path B In pathway B, the sialic acid-derived donor and the galactose-derived acceptor are linked first. Boons et al. [92, 93] (Scheme 37.17) have applied the 2-S-methyl-5-N, N-diacetyl neuraminyl derivative (107) to couple with the 2,3,4-triol (108) and the 2,3-diol (109) to afford the disaccharides (110 and 111) in 72% and 81% yields, OAc
AcO
MeO2C
Ac2N AcO OBz
HO
SMe
Ph O O
107
O
HO
O OAc
O
OH
O
HO
SiMe3
108
NIS, TfOH
NIS, TfOH
CH3CN, −40 oC, 72%
CH3CN, −40 oC, 81%
O
SiMe3
OH 109 Ph
OAc
AcO
Ac2N AcO
O
AcO
OBz
MeO2C HO
O
O
O
OAc
O O
MeO2C
Ac2N AcO
SiMe3
OH
OAc O
AcO
OAc
Ac2N AcO
SiMe3
111 1. Ac2O, pyridine 2. Ac2O, BF3 /OEt2 3. TMSSMe, TMSOTf
88%, over 3 steps
MeO2C AcO O
O
OH
OAc
110 1. Ac2O, pyridine 2. Ac2O, BF3/OEt2 3. TMSSMe, TMSOTf
O
O
AcO
OBz
O
O
Ac2N AcO
SMe
OAc
OAc
OAc
85%, over 3 steps
MeO2C AcO O
OAc
O
SMe
OAc
OAc
112
O
113 OTr TrO BnO
MeOTf, 95%
O
MeOTf,
O
96%
PhthN 114
AcO
OAc
MeO2C AcO
O Ac2N AcO OAc
O
OBz O
O AcO BnO 115
AcO
OTr O PhthN
O
OAc
MeO2C AcO
O Ac2N AcO OAc
O
OAc O
O AcO BnO
OTr O
O
PhthN
116
Scheme 37.17 Boons et al. [92, 93] have applied the 2-S-methyl- 5-N,N-diacetyl neuraminyl derivative (107) to couple with the 2,3,4-triol (108) and the 2,3-diol (109) to afford the disaccharides (110) and (111) in 72 and 81% yields, respectively
37 Synthesis of Hemagglutinin-Binding Trisaccharides
709
respectively. Transformation of compounds (110 and 111) into the corresponding thioglycosides (112) (88%) and (113) (84%), individually, was carried out via acetylation (Ac2O, pyr.), acetolysis (Ac2O, BF3/OEt2), and anomeric substitution (TMSSMe, cat. TMSOTf). MeOTf-promoted coupling of the donors (112 and 113) with the pentenyl 4,6-di-O-tritylgalactopyranosyl acceptor (114) gave the desired trisaccharides (115 and 116) in high yields, respectively. The utilization of the S-methyl SAa2,3Gal building block (118) [94] to construct the SAa2,3Galb1,4GlcNHAc skeleton has been described by Kiso and Ishida (Scheme 37.18) [74, 95]. Coupling of the S-methyl sialyl donor (36) with the galactose-derived triol (108) in the presence of DMTST yielded the disaccharide (117) (43%) [96, 97], which underwent sequential benzoylation, acetolysis, and anomeric conversion to give the thioglycoside (118) (46%) in three steps. DMTST-activated assembly of the donor (118) with the alcohols (119 and 49) gave the desired trisaccharide (120) and the pentasaccharide (121) in 70 and 78% yields, respectively.
OAc
AcO
O
AcHN AcO
AcO
OBz
HO
MeO 2C
O
SMe + HO
DMTST
O
SiMe3
OH
OAc
43%
OAc
MeO 2C HO O
AcHN AcO
1. BzCl 2. Ac2O, BF3 /OEt2 3. TMSSMe, BF3 /OEt2
OAc
AcO
AcHN AcO
46%, over 3 steps
O
O
O
OH
OAc
108
36
OBz SiMe3
117
MeO2C BzO O
O
OBn O
HO PMBO
OBz O
C14H29
, DMTST
119
SMe
70%
OBz
OAc
C14 H29
O NHAc
118 OAc
AcO
AcHN AcO
MeO 2C BzO O
O
OBz O BzO
OAc
OBn
O PMBO
C14H29
O
O NHAc
C14 H29
120
OBn BnO 118
+
HO BnO
O
O NHAc
OBn O BnO
OBn DMTST
O
O BnO
O
OBn
SiMe3
78%
49
AcO
OAc
MeO2 C BzO
O AcHN OAc OAc
O
OBz O BzO
OBn BnO O BnO
O
O
NHAc
OBn O BnO
OBn O BnO
O OBn
O
SiMe 3
121
Scheme 37.18 The utilization of the S-methyl SAa2,3Gal building block (118) [94] to construct the SAa2,3Galb1,4GlcNHAc skeleton has been described by Kiso and Ishida [74, 95]
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710
Kiso and Ishida have also reported an alternative route using the glycosyl trichloroacetimidate for the coupling reaction (Scheme 37.19) [74]. The 2,3-diol (109) was glycosylated with the sialyl donor (44) under typical NIS and TfOH conditions to provide the disaccharide (122) (63%), which was subjected to hydrogenolysis to yield the triol (123) (74%). Perbenzoylation of compound (123) afforded the ester (124)
AcO AcO O
Ph
OAc
O O
MeO2C H O N OAc OAc
SPh +
NIS, TfOH O
HO
O
SiMe3
OH
44
CH3CN, −35 oC, 63%
109
Ph AcO AcO O
OAc
O O
MeO2C H O N OAc OAc
H 2, 10% Pd-C O
O
O
SiMe3
OH
AcOH, 74%
122
AcO AcO O
OAc
MeO2C RO H O N O OAc OAc
98%
OAc
AcO
O
TFA
O
SiMe3
OR
AcO
quant.
123: R =H
Bz2O, DMAP pyridine
CCl3CN, DBU
OR
OBz
MeO2C BzO H O O N OAc O OAc
O BzO
OH
125
124: R = Bz, 87%
AcO
OAc
MeO2C BzO OBz O H O N O BzO OC(NH)CCl3 O OAc OAc
AcO
126
126
+
HO BnO
OBn BnO O O NHAc
OBn O BnO
OBn O BnO
TMSOTf
O
O OBn
SiMe3
0 oC, 77%
49
AcO AcO O
OAc
MeO2C BzO OBz O H O O N O BnO OAc OAc BzO
OBn BnO O
O
NHAc
OBn O BnO
OBn O BnO
O
O
OBn
SiMe3
127
Scheme 37.19 Kiso and Ishida have also reported an alternative route using the glycosyl trichloroacetimidate for the coupling reaction [74]
37 Synthesis of Hemagglutinin-Binding Trisaccharides
711
(87%), which was treated with TFA to give the 1-alcohol (125) in quantitative yield. Transformation of (125) which was converted into corresponding imidate (126) was carried out in excellent yield (98%), and the subsequent coupling of (126) with the trisaccharide acceptor (49) in the presence of TMSOTf led to the pentasaccharide (127) in 77% yield. A recent report by Seeberger and Hanashima [75] describes a total synthesis of the SAa2,3Galb1,4GlcNHAcb1,3Galb1,3Glc pentasaccharide unit (Scheme 37.20). The galactose-derived 2,3-diol (129) was regioselectively coupled with the N-Trocprotected dibenzyl phosphite sialyl donor (128) at the O3 position in the presence of TMSOTf in CH3CH2CN to give the disaccharide (130) in 65% yield. After benzoylation at O2, the 4,6-benzylidene acetal of (131) was removed by the treatment with pyridinium p-toluenesulfonate, and the 4,6-diol (132) was peracetylated to yield the product (133). Cleavage of the anomeric TDS group in (133) led to the 1-alcohol (134) (93%), which was converted into the N-phenyl trifluoroacetimidate (135) in 85% yield. The TMSOTf-catalyzed coupling of the donor (135) with the alcohol (57) afforded the pentasaccharide (136) in 89% yield. Ph
OAc
AcO
OP(OBn)2
TrocHN AcO
O
CO2Me
Ph
O O
+
HO
OAc
AcO PPTS
OTDS
OH
128
AcO
TMSOTf
O
CH3CH2CN, −78 oC, 65%
MeO2C RO
TrocHN AcO
O
AcO
O
O
OTDS
HF/pyridine 93%
OBz
OAc 132: R = H
Ac2O, pyridine
O
130: R = H 131: R = Bz, 89%,
OAc
MeO2C AcO
TrocHN AcO
OTDS
OR
OAc O
O
O
BzO
OAc
CF3C(NPh)Cl, Cs2CO3
133: R = Ac, 92% in 2 steps
O
O
OAc
BzCl, pyridine
OR
O O
MeO2C
TrocHN AcO
129
OAc
OAc
OR
134: R = H 135: R = C(=NPh)CF3, 85%,
PPTS = pyridinium p-toluenesulfonate
OBn BnO 135
+
HO LevO
O
OBn O
O
NHTCA
OBn
BzO
TMSOTf
O
O BzO
O(CH2)6NHAloc
89%
OBz
57
AcO
OAc
TrocHN AcO
MeO2C AcO O OAc
O
OAc O BzO
OBn BnO O LevO
O
O
NHTCA
OBn O BzO
OBn O BzO
O
O(CH2)6NHAloc
OBz
136
Scheme 37.20 A recent report by Seeberger and Hanashima [75] describes a total synthesis of the SAa2,3Galb1,4GlcNHAcb1,3Galb1,3Glc pentasaccharide unit
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712
37.2 Enzymatic Synthesis Glycosyltransferases, employing a variety of sugar nucleotides as glycosyl donors, are a series of enzymes that catalyze different biosyntheses of oligosaccharides and glycoconjugates in mammals. The glycosylation reactions are highly regioselective and stereoselective, and tedious protection of the monosaccharide building blocks is not required. Owing to the increasing need of sugar materials for further studies, many glycosyltransferases have been applied to oligosaccharide synthesis [58, 98–101]. Paulson and Sabesan [102] have reported the enzymatic synthesis of the SAa2,6Galb1,4GlcNHAc and SAa2,3Galb1,4GlcNHAc trisaccharide units and their nuclear magnetic resonance spectral characterization (Scheme 37.21). Due to the high price of uridine diphosphate (UDP)-galactose (138), UDP-galactose 4¢-epimerase (UDPGE) was used to convert the inexpensive UDP-glucose (137) into UDP-galactose (138) in situ with an equilibrium of Gal/Glc = 1/3.5. The galactosyltransferase utilizes the so-formed (138) as the donor to couple with the N-acetyl glucosamine derivative (139) to give the lactosamine derivative (140). Glycosylation of compound (140) with cytidine monophosphate (CMP)-Neu5Ac catalyzed by the a2,6-sialyltransferase (EC 2.4.99.1) or a2,3-sialyltransferase (such as EC 2.4.99.5 and EC 2.4.99.6) furnished the trisaccharides (141) and (142), respectively. A one-pot, two-step enzymatic glycosylation to synthesize the
OH HO HO
OH
HO UDPGE
O
OH
O
+
HO HO OUDP
O
HO HO
OMe NHAc
HO OUDP
137
138
139
galactosyltransferase UDP UDPGE = UDP-galactose 4'-epimerase
HO
OH O
HO
OH
OH O
O HO
OMe
NHAc 140
α2,6-sialyltransferase, CMP-Neu5Ac HO
α2,3-sialyltransferase, CMP-Neu5Ac CMP
OH
CMP
HO 2C
O AcHN O HO HO HO HO
HO O
O OH HO
141
OH O
OMe NHAc
OH HO2C HO
O AcHN HO HO
OH O
O
OH
OH O HO
O
OMe
NHAc
142
Scheme 37.21 Paulson and Sabesan [102] have reported the enzymatic synthesis of the SAa2,6Galb1,4 GlcNHAc and SAa2,3Galb1,4GlcNHAc trisaccharide units and their nuclear magnetic resonance spectral characterization
37 Synthesis of Hemagglutinin-Binding Trisaccharides
713
SAa2,6Galb1,4GlcNHAc unit with different substituent groups, such as OH, N3 and short peptides, was carried out by the same research group [107]. The UDP and CMP, released from the sugar nucleotide donors and acting as inhibitors of sialyltransferases, were decomposed into uridine with two subsidiary equivalents of Pi and cytidine with one equivalent of Pi, respectively, by addition of calf intestinal alkaline phosphatase. This drives the reaction toward the formation of the product and improves the yield [103]. The enzymatic glycosylation was retarded by the fact that the required sugar nucleotides were not readily available. The enzymatic synthesis of lactosamine was no exception. Whitesides and Wong [104] provided a sophisticated system that regenerated UDP-galactose using the UDP released from UDP-galactose during the glycosylation reaction with N-acetylglucosamine (Scheme 37.22). Because both phosphoglucomutase and glucose 6-phosphate (145) are inexpensive and more stable than glucose 1-phosphate (146), compound (145) was used as the starting material to generate UDP-glucose (137) when it was treated with uridine 5¢-triphosphate OH O
HO HO
AcHN 143
OH
HO HO
OH O OH
OH O HO
O AcHN
OH
144
HO
galactotransferase
OH O
HO
UDP
HO OUDP
OPO3 H
138
CO2 pyruvate kinase
UDPGE
O
OH HO HO
O
CO2
UTP
HO OUDP 137
UDP-glucose pyrophosphorylase
OH 2Pi
PPase
PPi
HO HO
O
phosphoglucomutase
HO OPO H 3 146
OPO3H HO HO
O HO
OH
145
Scheme 37.22 Whitesides and Wong [104] provided a sophisticated system that regenerated UDP-galactose using the UDP released from UDP-galactose during the glycosylation reaction with N-acetylglucosamine
C.-C. Wang et al.
714
(UTP) and pyrophosphorylase. UDP-glucose (137) was then transformed into UDP-galactose (138) by UDPGE. After the galactosylation of N-acetylglucosamine (143) catalyzed by galactosyltransferase, pyruvate kinase/phosphoenolpyruvate converted the released UDP into UTP, which was then used to regenerate UDPgalactose (138) via the formation of UDP-glucose (137). N-acetyllactosamine (144) can be synthesized in gram scale using this methodology. The unavailability of CMP-sialic acid was also a bottleneck for the enzymatic sialylation. An efficient synthesis of CMP-sialic acid (Scheme 37.23) was also developed by Whitesides [105] and Wong [106–108]. The CMP released from the sialylation reaction was transformed into cytidine triphosphate (CTP) after a series of enzymatic reactions. CTP can react with Neu5Ac in the presence of CMPNeu5Ac synthetase to generate CMP-Neu5Ac in situ, making it available for sialylation with N-acetyllactosamine (144) by a2,6-sialyltransferase [106, 107] and a2,3-sialyltransferase [108] to afford the SAa2,6Galb1,4GlcNHAc trisaccharide (147) and the SAa2,3Galb1,4GlcNHAc trisaccharide (148), respectively. In this protocol, pure CMP-Neu5Ac need not be added in the reaction. Therefore, the synthesis and isolation of CMP-Neu5Ac were abridged [106–108]. A similar one-pot multienzyme system was also reported by Thiem [109, 110].
O
OPO3H
CO2
CO2 PK
OPO3H
CDP
Neu5Ac
PK O
CMP-Neu5Ac synthetase
NMK
PPi
ATP
CO2
HO
CTP
ADP
CO2
CMP
OH
HO OH
O
HO
O O OH HO AcHN 147
HO
OH HO2C HO
O AcHN HO HO
OH O OH
OH
or HO
2Pi
α2,6 or α2,3 sialyl-transferase
HO2C
O AcHN O HO HO HO
PPase
CMP-Neu5Ac
OH O HO
O AcHN
OH
144 NMK = nucleoside monophosphate kinase
OH O
O
OH 148
OH O HO
O AcHN
OH
Scheme 37.23 An efficient synthesis of CMP-sialic acid was also developed by Whitesides [105] and Wong [106–108]
37 Synthesis of Hemagglutinin-Binding Trisaccharides
715
The regeneration of CMP-Neu5Ac using N-acetylmannosamine as the starting material and the biosynthetic cycle of lactosamine via the regeneration of UDPgalactose were combined in a multienzyme system by Wong’s group [107]. Through this protocol, the trisaccharide (147) could be prepared in an efficient one-pot manner. The recombinant fusion protein that artificially links b-1,4-galactosyltransferase and UDP 4¢-galactose epimerase fusion protein (b4galT-galE fusion protein) was shown to be efficient for the enzymatic galactosylation. The incorporation of the b4galT-galE fusion protein and a2,6-sialyltransferase in a one-pot, two-step manner gave the SAa2,6 Galb1,4GlcNHAc trisaccharide unit in 85% overall yield [111]. The recombinant fusion protein that contains CMP-Neu5Ac synthetase and the a2,3-sialyltransferase from Neisseria meningitidis was also expressed in Escherichia coli, and various galactosyl acceptors could be sialylated in a large scale using the nucleotide cycle system [112]. The commonly used mammalian a2,6-sialyltransferase (EC 2.4.99.1) preferring Galb1,4GlcNHAc as the acceptor, with a much lower rate of incorporating Galb1,3GlcNHAc, Galb1,6GlcNHAc, and Galb1,4Glc (£1%), has been reported [113–116]. On the other hand, the mammalian a2,3-sialyltransferase (EC 2.4.99.5) recognizes both Galb1,4GlcNHAc and Galb1,3GlcNHAc as substrates and also accepts Galb1,4Glc, despite the lower affinity [113–116]. In spite of the specificity, the 2,6-sialyltransferases and 2,3-sialyltransferases accommodate the modification of the substituent groups linked to the disaccharide units. Although the a2,6-sialyltransferases do not accept the terminal Galb1,4GlcNHAc subunit of glycosphingolipid globosides [117], a series of Galb1,4GlcNHAc connecting various anomeric substituents, such as p-aminophenyl [118], p-nitrophenyl [119], 2-(trimethylsilyl)ethyl [120], propargyl [121], 2-hydroxy-5-nitrobenzyl [122, 123], 4-methylumbelliferyl fluorescent tag [124], polymer support [125–131], and gold nanoparticle [132], can be sialylated by the a2,6- and a2,3-sialyltransferases. Unlike the a2,6-sialyltransferase (EC 2.4.99.1) and a2,3-sialyltransferases (EC 2.4.99.5 and EC 2.4.99.6) from rat liver, the a2,6- or a2,3-sialyltransfera ses from bacteria have a broader specificity to acceptors and are able to catalyze sialylation of N-acetyllactosamine or modified N-acetyllactosamine. The a2, 6-sialyltransferase from Photobacterium damsela [133, 134] and the a2,3-sialyltransferases from N. meningitidis [112, 135, 136] and Neisseria gonorrhea [137] have been reported to have better acceptance of sialyl donors [138–140]. The a2,3-sialylation of N-acetyllactosamine with KDN, Neu5Gc, and 9-azido-9-deoxysialic acid catalyzed by N. meningitidis a2,3-sialyltransferase was studied [140]; it catalyzed the sialylation of N-acetyllactosamine using Neu5Gc as the donor, but not KDN and 9-azido-9-deoxysialic acid. In addition, the a2,3-sialyltransferase from N. gonorrhea does not use both CMP-KDN and CMP-Neu5Gc as the substrates [137]. Chen et al. [141, 142] (Scheme 37.24) have reported a one-pot, three-enzyme system using the recombinant P. damsela a-2,6-sialyltransferase (Pd2,6ST) [141] and a recombinant tPm0188Ph protein [142] from Pasteurella multocida as the a2,3-sialyltransferase that accommodates the modification of both sialyl donors and acceptors to synthesize natural and modified sialosides.
C.-C. Wang et al.
716 HO
NHAc O
HO HO
OH 149 O
O
CO2
CO2
Aldolase Neu5Ac CTP
NmCSS
Mg2+ PPi
CMP-Neu5Ac
Pd2,6ST
CMP
tPm0188Ph OH
HO
HO
O
HO
OH
OH
HO2C
O AcHN O HO HO HO HO
CMP
OH O HO
O OH
AcHN
144 HO O
O OH HO
147
OH HO2C HO
OH O AcHN
OH
AcHN HO HO
O
OH O
O
OH
OH O HO
O AcHN
OH
148
Scheme 37.24 Chen et al. [141, 142] have reported a one-pot, three-enzyme system using the recombinant P. damsela a-2,6-sialyltransferase (Pd2,6ST) [141] and a recombinant tPm0188Ph protein [142] from Pasteurella multocida as the a-2,3-sialyltransferase that accommodates the modification of both sialyl donors and acceptors to synthesize natural and modified sialosides
Its applications to synthesize the SAa2,6Galb1,4GlcNHAc trisaccharide (147) and the SAa2,3Galb1,4GlcNHAc trisaccharide (148) were also carried out (Scheme 37.24). Mannosamine (149) or C2-, C4-, C5-, and/or C6-modified mannose or mannosamine was used as the starting material and treated with pyruvate and aldolase to form Neu5Ac or C5-, C7-, C8-, and/or C9-modified sialic acid in situ. The CMP-sialic acid synthetase from N. meningitidis (NmCSS) has flexible substrate specificity and converts Neu5Ac or modified sialic acid into the corresponding CMP-sialic acid derivatives. Sialylation of CMP-sialic acid with N-acetyllactosamine (144) catalyzed by Pd2,6ST or tPm0188Ph protein was conducted in the same pot, and the SAa2,6Galb1,4GlcNHAc trisaccharide (147) and SAa2,3Galb1,4GlcNHAc trisaccharide (148) were obtained in good yields, respectively.
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37.3 Glycan Microarray Analysis of HA Specificity Glycan microarray technology, which allows surveillance of the couplings of hundreds of diverse glycan structures with proteins on a single chip, is an efficient and highthroughput tool for investigating the interactions of various glycan-binding proteins and their glycan ligands at a low cost [143–155]. The construction of glycan microarrays also enables scientists to decode the information contained in the binding of HA and its carbohydrate receptor via rapid screening of carbohydrate libraries with well-defined structures. Moreover, the binding specificity of new emerging viruses can be rapidly screened and monitored in a short time. A microarray study of a series of HAs was performed by Blixt et al. [156]. The microarray was prepared using robotic printing technology. The amine-functionalized glycans were covalently immobilized onto the commercially available amine-reactive N-hydroxysuccinimide-activated glass slides to form the arrays (Fig. 37.2) [149]. The tested HAs were expressed in the baculovirus system to acquire the functional trimers [29]. Because of the relatively weak binding affinity between HA and its receptors, a significant signal could only be observed when the recombinant HA was precomplexed with fluorescent-labeled mouse anti-His antibody and an extra fluorescent anti-mouse-IgG1 antibody (Fig. 37.3) [149]. The enhancement of the signal was believed to be caused by the strengthened multivalency effect after forming the complex and the two additional fluorescent sources. Several human and avian HA serotypes, including H1, H3, H5, and even mutated HA, have been analyzed using the glycan array technology to interpret their structural O Glycans
NH2
+
O N O
O N O
O O
O O
O N O
O O
O N O
O N O
O O
O O
N O
O O
NHS-activated glass surface
HN O
Glycans
HN O
Glycans
HN O
Glycans
HN O
Glycans
Glycans
Glycans HN
HN O
O
array surface
Fig. 37.2 Immobilization of amine-functionalized glycans on the N-hydroxysuccinimideactivated slide to form the sugar array
C.-C. Wang et al.
718 Fluorescent Tag
Mouse anti-His6 antibody
Fluorescent Tag Mouse anti-His6 antibody
Mouse anti-His6 antibody
His Tag
Recombinant Haemagglutinin with His Tag
HN O
Glycans
HN O
Glycans
HN O
Glycans
HN O
Glycans
HN O
Glycans
HN O
Glycans
HN O
Glycans
HN O
Glycans
HN O
Glycans
HN O
Glycans
Glycans
Glycans HN
HN O
O
array surface
Fig. 37.3 Because of the relatively weak binding affinity between HA and its receptors, a significant signal could only be observed when the recombinant HA was precomplexed with fluorescent-labeled mouse anti-His antibody and an extra fluorescent anti-mouse-IgG1 antibody [149]
and affinity relationships [41, 156]. After rapid screening with various glycans, it was revealed that the different HAs demonstrate distinct binding preferences, not only to the a2,3- or a2,6-linkages, but also to the modifications of the glycans, such as fucosylation, sulfations, and further sialylation. The human H1 and H3 and the avian H3 and H5 show preferences to 6-sulfated SAa2,6Galb1,4GlcNHAc and 6-sulfated SAa2,3Galb1,4GlcNHAc trisaccharide units, respectively, suggesting that these modifications are important in the interactions between viruses and hosts. In fact, the switch of a single amino acid between two strains of 1918 influenza viruses, A/South California/1/1918 (Asp225) and A/New York/1/1918, is enough to alter their binding preferences. The former exhibits a strict a2,6 preference; the latter, however, binds to both a2,6- and a2,3-linkages [156].
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37.4 Summary Significant advances in both chemical and enzymatic syntheses of the SAa2,6 Galb1,4GlcNHAc and SAa2,3Galb1,4GlcNHAc trisaccharide units have been reported. For the chemical approach, the glycosidic bonds between either sialic acid and galactose or galactose and glucosamine can be constructed first, and even the one-pot procedure has been achieved. Various sialyl donors, including chloride, thiophenyl, thiomethyl, SBox, 2-(dibenzyl)phosphite, and 2-(diethyl)phosphite, were used, and the sialylation showed good regioselectivity for the diol or triol present in the acceptor. However, the major difficulty still lies in the stereoselectivity of the sialylation, which reduces the efficiency and the overall yields. For the enzymatic preparation, the use of a2,6- and a2,3-sialyltransferases is appealing since lactosamine is their natural substrate. The in situ regeneration of CMPNeu5Ac and the one-pot, three-enzyme system are attractive owing to their high yield and efficiency. A combination of synthesis and microarray technology has allowed scientists to get a deeper insight into the structure and affinity relationships of the binding between the HAs of a virus and the glycans of its host. The change of specificity can be rapidly monitored so that uncovering the pathway by which viruses cross the species barrier becomes increasingly possible.
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Chapter 38
Fabrication and Applications of Glyconanomaterials Po-Chiao Lin, Avijit Kumar Adak, and Chun-Cheng Lin
Keywords Nanoparticle • Multivalent interaction • Protein detection • Site specific Nanomaterials have unique optical, electronic, or magnetic properties, thus explaining their potential applications in complex biosystems when coupled with biomolecules, such as DNA, peptides, proteins, or carbohydrates. With a large surface-tovolume ratio and homogeneity in aqueous solutions, various biomolecule-conjugated nanomaterials are exploited for elucidating biological interactions. During the past decade, biomolecule-conjugated nanoparticles (NPs) have been prepared and used in diagnostics [1], creative therapeutics [2], biomolecular interactions [3], and in vivo cell imaging [4, 5]. For example, Mirkin et al. developed an ultrasensitive bio-barcode detection method based on oligonucleotide-conjugated gold NP (AuNP) for biomarkers in small amounts in complex biofluids [6]. Weissleder et al. fabricated antibody-conjugated iron oxide NPs and used them to enhance T2 signals in magnetic resonance imaging [7]. Since they have unique magnetic properties, diverse functionalized magnetic nanoparticles (MNPs) have been designed and prepared to purify target proteins from crude cell lysate by simple magnetic separation. Recently, the authors combined antibody-conjugated MNP with matrixassisted laser desorption/ionization–time of flight (MALDI–TOF) mass spectrometry (MS) as a rapid and cost-effective detection method for diagnosing disease markers in human sera [8–11]. Biomolecule-modified quantum dots (QDs) have also been demonstrated as having promising applications in in vivo imaging, including cell trafficking and targeting. Besides metallic NPs, carbon nanotubes (CNTs) have also been demonstrated to be powerful carriers and to be useful in biological systems because of their high surface utilization efficiency and good size uniformity. C.-C. Lin (*) Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan and Chemical Biology and Molecular Biophysics, Taiwan, International Graduate Program, Academia Sinica, Taipei, Taiwan e-mail:
[email protected] A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_38, © Springer Science+Business Media, LLC 2011
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Although many biomolecules, such as oligonucleotides, peptides, and proteins, have been assembled on NPs and used in various biological systems, carbohydratemodified NP was not extensively studied before 2001. Glycans comprise covalently bonded sugars (oligosaccharides or polysaccharides) in either free form or complexed with proteins or lipids (also named as glycoconjugates). Most glycans are present as membrane-bound glycoconjugates or as secreted molecules, which are regarded as integral to the host extracellular matrix. These glycans in specific positions regulate cell adhesion, motility, and intracellular signaling events [12]. Tumors aberrantly express various glycans that are believed to regulate various aspects of tumor progression, including proliferation, invasion, angiogenesis, and metastasis. Glycoproteins and glycosphingolipids can potentially activate growth factor receptors and tyrosine kinase and also function as coreceptors for soluble tumor growth factors, which may result in the proliferation of tumor cells. Additionally, some specific glycosyltransferases expressed by tumor cells induce the formation of tumor-related glycans, which facilitate invasion. Some glycans such as gangliosides, one class of glycosphingolipids, can be shed into the bloodstream and detected. Therefore, they can serve as markers of disease in screening cancers as well as in tracking responses to therapy. Although carbohydrates are important in the diagnoses of many diseases, only a few glycan-based targeting strategies [12] have been tested in clinical trials. The authors believe that the diverse glyco-NPs formed by combining carbohydrates with nanocarriers are a powerful platform for studying the complex carbohydrate-involving interactions. Penadés et al. first reported the use of glyco-NPs to study multivalent interaction [13]. Based on a transmission electron microscopy (TEM) image, in the presence of Ca2+, Lex assembled AuNPs (Lex-AuNPs) to form aggregates by carbohydrate– carbohydrate interaction. These results demonstrated that Lex-AuNP successfully mimicked the glycosphingolipid cluster at the cell surface. This initial work suggested that carbohydrate-modified NPs can be designed as a practical platform to imitate the cell surface receptor interaction. Since the size of the NPs can be controlled, they could be prepared in the sizes suitable for studying interactions with cells, bacteria, and viruses. Therefore, the complex cell surface interactions could be reduced and studied specifically under simple conditions. Advances in nanotechnology have led to the reporting of many biomolecule-conjugated NPs [14]. This article focuses on applications for carbohydrate-functionalized nanomaterials, such as gold, semiconductors (QDs), and MNPs, as well as CNTs.
38.1 Fabrication and Application of Glyco-AuNPs AuNPs are the most used metal NPs and have many interesting properties, including diverse molecule assembly and extraordinary size-related electronic/optical properties (quantum size effect). Over the past decade, AuNPs have been used in chemical and biological systems [6]. Many methods for producing colloid AuNPs, based on the method of Brust et al. [15], have been employed to prepare various
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AuNPs for various research purposes. The general method is to use a reducing agent (such as NaBH4) to reduce gold salt (AuCl4−) to metallic Au (AuNP) in the presence of surfactant or thiolated compounds. The newly formed AuNP surface is protected by the surfactant and can be further exchanged with thiolated compounds to yield the desired ligands. Since both Au and S are soft, the strong Au–S bond protects and stabilizes the Au core. The carbohydrate-immobilized AuNP (glycoAuNP) was the first reported example of a carbohydrate-assembled metallic NP for use in carbohydrate-related studies.
38.1.1 Importance of Glyco-AuNP Recent studies have demonstrated that the specific interactions between carbohydrate/carbohydrate and carbohydrate/lectin (carbohydrate-binding protein) govern cell–cell interactions. The strength of this interaction depends on the multivalent effect, a “cluster glycoside effect,” which involves multiple carbohydrates of a particular type and orientation. The high surface-to-volume ratio of the metal NPs causes the carbohydrate-assembled NPs to increase the carbohydrate density on the particle surface and may mimic the microdomain of the glycoside cluster and induce a multivalent effect. Since glyco-AuNP may mimic the carbohydrate antigen on a cell surface [16], many efforts have been made to prepare complex glycan AuNPs, and their functions have been studied extensively under realistic conditions. Svarovsky et al. synthesized Thomsen–Friedenreich disaccharides, human tumor-associated carbohydrate antigens that are present in approximately 90% of carcinomas [17] and are rarely expressed in normal tissues, and assembled them on the AuNP surface [18]. Patz et al. synthesized Ley-functionalized AuNPs [19]. A sulfated disaccharide (GlcpNAc3S(b1-3)Fucp) and its derivatives have been synthesized and conjugated on AuNP to study the recognition and adhesion of marine sponge cells [20]. Penadés et al. incorporated carbohydrate antigens (sialyl Tn and Lewisy), helper T-cell peptides, and glucose on the AuNP with various combinations of ratio, as presented in Fig. 38.1, to exploit the multifunctional AuNP as a potential cancer vaccine [21]. Varying the ratios of carbohydrate ligands during preparation causes the modulated carbohydrate ligand density on AuNP to exhibit effective properties against colon, liver, prostate, and ovarian carcinomas. The carbohydrate-functionalized AuNPs can be characterized by TEM, scanning electronic microscopy (SEM), Fourier transform infrared spectroscopy, and nuclear magnetic resonance (NMR) spectrometry. The strength of carbohydrate–carbohydrate/carbohydrate–protein interaction depends strongly on the presence of multiple carbohydrates of the correct type and orientation [22]. Therefore, a high affinity is attributable to the increase in carbohydrate density on the interaction microdomain. However, only a few works have addressed the “multivalent effect” of glyco-NPs. Spain et al. adopted the reversible addition-fragmentation chain transfer polymerization, an effective and convenient
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Fig. 38.1 Multifunctional glyco-NP preparation (from [21], by permission)
synthesis, to prepare glycopolymers using 2-(b-D-galactosyloxy)ethyl methacrylate monomer [23], as presented in Fig. 38.2. Optical microscopy was employed to observe the multivalent interaction between peanut agglutinin (PNA)-coated agarose beads and galactosyl polymer AuNPs
38.1.2 Glyco-AuNP–Carbohydrate Interactions Besides protein–protein and protein–carbohydrate interactions, considerable evidence supports the claim that cells use glycosphingolipid microdomains for intercellular adhesion and recognition. Both homophilic (same carbohydrates) and heterotopic (different carbohydrates) interactions are important to many cell behaviors, such as
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Fig. 38.2 Preparation of glycopolymer-stabilized gold nanoparticles (from [23], by permission). Spain et al. adopted the reversible addition-fragmentation chain transfer polymerization, an effective and convenient synthesis, to prepare glycopolymers using 2-(b-d-galactosyloxy)ethyl methacrylate monomer [23]
melanoma cell metastasis and the binding of sperm to egg membranes [24]. Numerous carbohydrate–carbohydrate interactions depend on divalent metal cations and involve very low affinity between two carbohydrate monomers. However, the multivalence of carbohydrates can overcome this low-affinity interaction. Atomic force microscopy (AFM) has been used to measure the adhesive force in the carbohydrate–carbohydrate interaction between a two-dimensional (2-D) gold surface and an AFM tip [25, 26]. The AFM tip and self-assembled monolayer gold chip were modified using thiol-derived oligosaccharides. The attractive force of carbohydrate–carbohydrate produced a stepwise profile because of the successive binding of molecules along the force–distance curve. In 2001, de la Fuente et al. synthesized glyco-AuNPs and used them to investigate the carbohydrate– carbohydrate interaction to elucidate the difference between the interaction of a 2-D gold monolayer and the globular 3-D NPs [16]. Since the glyco-AuNPs are globular and have a high surface-to-volume ratio, they can be regarded as having been the practical basis of the discovery of the cell–cell adhesion process. In the cited studies, two carbohydrates, lactose- and Lex-AuNPs, were prepared to investigate the specific Ca2+-mediated carbohydrate–carbohydrate interaction. In a TEM image, the clear aggregation of Lex-AuNPs in the presence of Ca2+ cations (10 mM CaCl2) indicated the unique Lex–Lex interaction. After ethylenediaminetetraacetic acid (EDTA, a Ca2+ chelator) was added, the aggregated Lex-AuNPs were redispersed in aqueous solution (Fig. 38.3). In contrast, the lactose-AuNPs remained well dispersed in 10 mM CaCl2, suggesting an absence of carbohydrate–carbohydrate interaction. Isothermal titration calorimetry was performed to provide thermodynamic evidence of Ca2+-mediated self-aggregation of glyco-AuNPs [27]. The heat released by glycol AuNP aggregation in the presence of 10 mM solutions of various cations (Ca2+, Mg2+, or Na2+) was measured. The enthalpy of Lex-AuNP aggregation for 10 mM CaCl2 was −160 ± 30 kcal/ml, and values that were many times lower
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Fig. 38.3 TEMs and core size-distribution histograms: (a) Lex-AuNP in water; (b) lactose-AuNP in water; (c) Lex-AuNP (0.1 mg/mL) in 10 mM CaCl2 solution; (d) Lex-AuNP (0.9 mg/mL) in 10 mM CaCl2 solution; (e, f) mean (c, d) added with EDTA; (g) lactose-AuNP (0.1 mg/mL) in 10 mM CaCl2 solution; (h) lactose-AuNP (0.9 mg/mL) in 10 mM CaCl2 solution (from [16], by permission)
(−30 ± 20 and −50 ± 30 kcal/ml) were obtained in a magnesium chloride and sodium chloride solution. De Souza et al. prepared a series of molecular aggregation factorrelated, sulfated disaccharide-conjugated AuNPs to mimic cell aggregation in
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O O
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Fig. 38.4 Illustration of the AuNP core and the linker with fluorescent probe (from [29], by permission)
nature and elucidate glycan-induced aggregation on a cell surface [28]. Rojas et al. synthesized fluorescein isothiocyanate- and lactose (or Lex)-hybridized AuNPs (Fig. 38.4) to generate a bifunctional AuNP. These bifunctional glyco-AuNPs exhibited similar aggregation phenomena in the presence of Ca2+. The presence of fluorescein on the AuNP made the aggregation easy to observe [2]. AuNP aggregation can also be detected by measuring the shift of the AuNP absorption peak. The ultraviolet (UV)-visible absorption wavelength shifted from that of monodispersed AuNP at 520 nm to that of aggregated AuNP at 650 nm [30]. Unlike Panedés, Reynolds et al. used the absorption shift to demonstrate that a linker between carbohydrate ligand and NP surface modulated a specific Ca2+-mediated lactose–lactose interaction. The short linker (single ethylene glycol) was more sensitive to the Ca2+ concentration than the long linker (triple ethylene glycol). Surface plasma resonance (SPR) is a powerful tool for low-affinity interaction analysis to examine the kinetics of a carbohydrate–carbohydrate interaction. In early works, the interaction of carbohydrate-assembled gold sensor chips with glycoconjugates was studied by SPR [31, 32]. Using SPR analysis and glyco-AuNPs,
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Fig. 38.5 Schematic representation of our binding event strategy with a typical sensorgram. Gray circles mean AuNPs, black circles mean Ca2+ (from [33], by permission)
Hernaiz et al. proposed an NP-based SPR to analyze the kinetic homophilic interactions of Lex and lactose, as shown in Fig. 38.5 [33]. The results revealed that LexAuNP exhibited much greater affinity (Kd 5.4 × 10−7 M) than that of monovalent Lex (Kd 5.7 × 10−3 M), suggesting the superiority of AuNP as a carrier of carbohydrate ligands. This work also demonstrated that glyco-AuNP can mimic the microdomain in which the multivalent effect is evident in a carbohydrate–carbohydrate interaction.
38.1.3 Glyco-AuNP–Protein Interactions Cell surface glycan and membrane protein interactions govern many important biological processes, including cell adhesion, recognition, and infection [12]. Therefore, complex glycans are promising targets in developing new pharmaceutical agents. Overcoming fundamental challenges to clarify the structure–activity relationship of a glycan is key to the discovery of many drugs. However, the affinity between a monomer carbohydrate ligand and its receptor is, in general, low (Kd at mM range). Nature overcomes the associated weak interaction by using a multivalent interaction that increases the affinity, resulting in high specificity. To study the multivalent interaction, many schemes, such as SPR [34] and microarray-based chemistry [22], have been developed as reliable methods for detecting carbohydrate–protein interaction. As stated in earlier sections, glyco-AuNPs exhibit a strong multivalent effect that increases carbohydrate binding affinity. Therefore, glyco-AuNPs should also interact strongly with target lectins. The specific interaction between Concanavalin A (ConA) and a-d-mannose/a-d-glucose is a well-defined and reliable model system for studying carbohydrate–protein multivalent interactions. In this context,
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Fig. 38.6 (a) Schematic illustration of the interactions of carbohydrate and AuNP, and Concanavalin A (ConA) on the biosensor chip used in competition assays. (b) Inhibition of 0.5 mM ConA binding to the chip by mannose-AuNP. A set of inhibition curves for 0, 0.175, 0.5, and 1 mM mannose-AuNP (top to bottom) (from [35], by permission)
diverse glyco-AuNPs with various types of carbohydrates, linker lengths, and particle sizes exhibited various binding affinities toward ConA in SPR studies, as shown in Fig. 38.6 [35]. Guo et al. developed a method that involved assembling N-acetylglucosamine glycolipid AuNPs on a sensor chip to recognize ConA by measuring the redshift in the UV-visible spectrum, resulting in an ultrasensitive assay for carbohydrate–lectin interaction [36]. The fabricated AuNP chip was very sensitive to ConA, and the minimum detection concentration of ConA was 0.1 nM. A competitive colorimetric assay was presented to evaluate the carbohydrate–protein interaction by titrating the mannose–AuNP/ConA complex with other lectins [37]. Besides ConA–mannose/ glucose interaction, Recinus communis agglutinin (RCA120), a bivalent lectin, is also well known to be a specific lectin for lactose/galactose recognition. Kataoka et al. synthesized stable polyethylene glycol (PEG)-AuNPs by the in situ aqueous reduction of HAuCl4 in the presence of a-acetal-w-mercapto-PEG followed by immobilization of lactose. The lactose-PEG-AuNP facilitated RCA120 lectin aggregation, and the aggregate was dissociated by adding galactose. Therefore, the amount of dissociated lectin was quantified by measuring the changes in absorption intensity and was observed easily by the naked eye (red → purple → red), as shown in Fig. 38.7 [38]. Additionally, the optimal lactose density on AuNPs for aggregation assay should be higher than 20% to ensure detectable particle aggregation [39]. Gervay-Hague et al. focused on the study of the human immunodeficiency virus (HIV) entry recognition mechanism between the carbohydrate and its particular host cell receptors to understand the fundamental and molecular adhesion processes that are involved in the viral recognition of host cells [40]. The interaction between the viral surface gp120 and the T-cell surface CD4 domain protein is known to initiate the HIV infection process. Recent studies [41] have found that gp120 can also interact with galactose-containing ceramides, indicating that the initial-stage adhesion of HIV infection may involve a carbohydrate–carbohydrate or protein interaction. Various galactose/glucose-conjugated AuNPs have been synthesized, and their binding affinity with gp120 was examined by biotin neutravidin adhesion assay [42].
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Fig. 38.7 Schematic representation of the reversible aggregation-dispersion behavior of lactosePEGylated AuNPs by sequential addition of RCA120 lectin and galactose (from [38], by permission)
The galactose/glucose-AuNPs had a much higher affinity (>300 times) than that of the corresponding disulfide dimers. The enhanced binding affinity of glyco-AuNPs may be associated with the basic changes in the recognition of divalent to polyvalent interactions [43]. Given their evident superiority in multivalent binding affinity enhancement, glyco-AuNPs actually constitute an excellent platform for carbohydrate–protein interaction.
38.1.4 Other Interesting Applications of Glyco-AuNPs To extend the use of glyco-AuNPs to the cell level, we were the first to address specific recognition of target bacterium with glyco-AuNPs (Fig. 38.8) [44]. The glyco-AuNPs were tested to determine their capacity to bind mannose-specific adhesin FimH of type-1 pili in Escherichia coli. Two E. coli strands, ORN 178 (expressing FimH on pili) and ORN 208 (mutant without FimH gene), were used to verify the specific binding of mannose-AuNP to FimH. The TEM image revealed that the mannose-AuNP exhibited satisfactory specificity toward the target ORN 178 strain, indicating that the biomolecule-conjugated NPs provide a relatively easy and direct method for visualizing the target receptors on the cell surface under an electron microscope. Excellent specificity and sensitivity enabled further design of the galactose and trisaccharide Pk antigen (Gala-1-4Galb1-4Gal)-conjugated
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Fig. 38.8 TEM images of sectioned areas of (a) pili of E. coli ORN 178 strain bound with mannose-AuNPs and (b) E. coli ORN 208 strain deficient of the fimH gene without mannoseAuNP binding (from [44], by permission)
AuNPs as a Pseudomonas aeruginosa-I lectin probe for specific protein purification [45]. Following mass analysis, the target protein was directly identified by MALDI-TOF MS. The captured protein was digested by protease and then analyzed by MALDI MS/MS (Fig. 38.9) to elucidate the carbohydrate–protein binding interaction. This method established the feasibility of carbohydrate-functionalized glyco-AuNPs for the simultaneous enrichment and isolation of target proteins from the mixture at the femtomole level, and subsequent protein identification and mapping of the bindingepitope-containing peptides with minimum sample handling. Additionally, AuNP can be directly analyzed by MS without the need to elute the captured protein because of the electrical conductivity of metal NPs. Therefore, the tedious desalting process before mass analysis can be simplified by washing out the salt using water. Glyco-AuNPs also have promising clinical applications as potential therapeutics for suppressing cancer metastasis, as demonstrated by Rojo et al. and displayed in Fig. 38.10 [46]. In metastasis, tumor cells detach from the primary tumor and travel through the lymphoid or blood vessels to their target at specific location. One of the critical steps in metastasis is the adhesion of the tumor cells to the vascular endothelium. Thus, the abnormal glycosylation [12] of tumor cell surface glycoproteins and glycolipids is considered to be a significant factor in the development of new therapies. To study the cell surface glycan-induced tumor cell adhesion and metastasis, the great “multivalent effect” presented by glyco-AuNPs established an effective method for elucidating the low-affinity carbohydrate interaction. Many studies have demonstrated that cell aggregation is related to the expression of gangliosides, GM3 (NeuNAc2a3Galb4GlcbCer) and lactosylceramide (Galb4GlcbCer), on the cell surface. Therefore, lactose- and maltose-conjugated AuNPs were prepared, and their capacity to inhibit the metastasis and progression of murine melanoma cell line (B16) in the lung was examined. Lactose-conjugated
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Fig. 38.9 The analytic scheme of the nanoprobe-based affinity mass spectrometry (NBAMS) technique for the specific capture of target proteins and the rapid mapping of binding-epitopecontaining peptides (from [45], by permission)
5 min, 37°C Glyconanoparticles
B16F10 Cells
1000 rpm, RT
Glyconanoparticlestreated B16F10
Control B16F10
TC 1 week
3 weeks
G (gluco-GNP) 3L (lacto-GNP) 9L (lacto-GNP)
Cell Viability
Lung tumoral foci score/ Anatomopathologic studies
Fig. 38.10 Schematic representation of the experimental design for evaluating the antimetastatic potential of lacto-glyco-NPs (from [46], by permission)
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glycosyltransferase gene transfected E.coli
a
b [3 + Na]+ 1058
homogenize
[4 + Na]+ 1221
S
S
Au
i)
glycosyl acceptor on gold nanoparticle
ii)
S
S
Au
1000
1200 1100 m/z
1300
crude cell extract
Fig. 38.11 Direct monitoring of bacterial b-1,4-GalT activity in a cell-free extract with glycoAuNP residues as the acceptor substrates. (3) means the GlcNAc residue on AuNPs, and (4) means the Gal-b-1,4-GlcNAc on AuNPs (from [48], by permission)
AuNPs inhibit tumor metastasis in mouse lung, demonstrating the potential use of glyco-AuNPs in anticancer therapy. As the effective multivalent ligand carrier, AuNP can be regarded as an ideal solid support for the synthesis of carbohydrate. Nishimura et al. described two methods for elongating carbohydrates on AuNP by either chemical or enzymatic reactions. In chemical synthesis, glucose-conjugated AuNP was used as the starting material in extending the sugar chain by complex chemical manipulations. After a series of protection–deprotection and glycosylation steps, the lactose-AuNP was synthesized, and the carbohydrate product on the NP surface was directly identified by MALDI-TOF MS without any pretreatment [47]. The oligosaccharide on the NP surface was also constructed by performing enzymatic reactions. In enzymatic synthesis, sugars were added to N-acetyl glucosamine-conjugated AuNP (GlcNAcAuNP), using b-1,4-galactosyltransferase and a-1,3-fucosyltransferase to yield Lex-AuNP. The GlcNAc-AuNP serves as a probe for detecting the activity of glycosyltransferase in a cell-free extraction. Coincubation of the extracted cell lysate and GlcNAc-AuNP in the presence of b-1,4-galactosyltransferase formed disaccharide (Gal-b-1,4-GlcNAc) on AuNP. The product was identified by its mass, as shown in Fig. 38.11 [48].
38.2 Fabrication and Applications of Glyco-QDs QDs are semiconductor nanocrystals with unique optical properties, such as narrow size-dependent emission, high luminescence, and low photobleaching. These significant features of QDs make them important biolabeling candidates. However, their poor
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solubility in water limits their use in bioimaging. Hence, surface modification of QDs to improve their solubility in water is important for practical bioapplications of QD. The QD surface was modified by a coating with polymer [49], micellar emulsion [50], or conjugation with thiol-containing hydrophilic derivatives [51]. Although many significant biomolecules, such as peptides, proteins, and DNA, have been assembled on QDs, the carbohydrate-assembled QDs (glyco-QDs) have seldom been investigated. This section discusses the fabrication of glyco-QDs and their bioapplications.
38.2.1 Fabrication of Glyco-QDs The preparation of QDs, such as CdS, CdTe, and CdSe/ZnS core-shell QDs, has been thoroughly described in the literature [52–54]. Most QDs are prepared using trioctylphosphine oxide (TOPO) as the surfactant and are insoluble in water. Watersoluble and functionalized QDs are formed by exchanging the surface TOPO (or similar organic ligands) with target molecules by the methods described earlier [55]. Chen et al. described the first development of glyco-QDs that are suited to biomolecule conjugation while maintaining high emission efficiency [56]. The layer-by-layer glyco-QD modification was achieved by the electrostatic interaction of positively charged polylysine cross-mediated with negatively charged QD surface and the subsequent coupling with carboxymethyl dextran (CM-dextran), as shown in Fig. 38.12. The thiol-containing carbohydrate can also be regarded as the surface surfactant in the control and stabilization of the size of the QDs. De la Fuente et al. expanded their glyco-AuNP synthetic method to the preparation of glyco-QDs. The disulfide-linked Lex antigen and maltose were reduced in the presence
Fig. 38.12 Structures of negatively charged mercaptosuccinic acid-modified CdSe-ZnS QDs, positively charged polylysine, and negatively charged CM dextran (from [56], by permission)
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of Cd(NO3)2 and Na2S, respectively. This method directly forms glyco-QD. Like glyco-AuNPs, glyco-QDs can be characterized by NMR, UV-vis spectrometry, and TEM imaging [57].
38.2.2 Applications of Glyco-QDs Exploiting the unique optical properties of QDs and their resistance to photobleaching, Niikura et al. synthesized various neoglycolipid-coated CdTe QDs using the surface-exchange method and investigated their behaviors in digitonin-treated HeLa cells with or without adenosine triphosphate (ATP) [58]. The use of QDs as a probe had at least two advantages: first, the nanoscale of QDs makes them an effective platform for revealing how multivalent carbohydrates mimic natural proteins. Second, since the size of QDs is similar to that of conventional proteins, the behavior of glyco-QDs is similar to that of glycoproteins. Niikura et al. used a confocal-laser scanning microscope to demonstrate that N-acetylglucosamine (GlcNAc)-QDs were specifically accumulated in the endoplasmic reticulum only in the presence of ATP. The b-GlcNAc is also important in cellular recognition during fertilization [59, 60]. Accordingly, the GlcNAc unit (thought to be an essential component of the egg surface [zona pellucida] ZP3 membrane protein) has been helping it to bind with the complementary sugar-binding enzyme on the surface of sperm. Robinson et al. developed the GlcNAc-encapsulated CdSe/ZnS core-shell QD to examine the specific carbohydrate–sperm interaction [61]. As shown in Fig. 38.13, GlcNAc-QDs exhibit excellent specificity and efficiency under a confocal microscope. Osaki et al. prepared sugar-conjugated QDs of three sizes, 5, 15, and 50 nm, as tracing markers to monitor the uptake of QDs by cells and thus
Fig. 38.13 Confocal microscope imaging for the staining of sperm with glyco-QDs. (a) selective QDGLN labeling on the heads of sea urchin sperm (scale bar = 20 mm), (b) closeup of QDGLNlabeled sea urchin sperm, and (c) closeup of QDMAN-labeled mouse sperm (from [61], by permission)
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elucidate the effect of the size of QDs in cell endocytosis [62]. The uptake of cells by QDs depended strongly on the size of the NPs (50 » 15 » 5), and only slight aggregation of the glyco-QDs was observed within the cell. Like the unique fluorescent property of QD, the small size and high surfacearea-to-volume ratio of QDs have attracted the attention of scientists in many fields. Dai et al. designed an NP-based biosensor for detecting carbohydrate by measuring the interaction of glyco-QD with a lectin-coated Au chip [63]. The target lectin was covalently conjugated on an Au surface by forming an amide bond and was identified using the probe glyco-QD. In this work, the electrochemical readout was defined as the detection platform because of its benefits of miniaturization and low cost. Strong binding with the specific GalNAc-QD significantly affects the electrochemical readout of a lectin-Au surface, and the detection limit of the target PNA was found to be 0.1 mM. Babu et al. used lactose-, melibiose-, and maltotrioseconjugated QDs to evaluate their detection specificity and efficiency with ConA [64]. Based on the scattering of light at 600 nm by the lectin-induced QD aggregation, in the study of the interaction between maltotriose and ConA, the detection limit was 100 nM of lectin.
38.3 Carbohydrate-Assembled Glyco-MNPs and Their Applications Magnetic materials exhibit magnetic resonance effects and undergo easy and rapid magnetic separation. Recently, various MNPs have been designed as powerful carriers for use in diverse studies [65], such as protein purification [66], pathogen detection [67], and antigen diagnosis [8]. However, the carbohydrate-conjugated magnetic materials have been less commonly examined and have been used in few practical applications. Sun et al. coated the target carbohydrate on a magnetic bead (MB, 1 mm) and used it as a probe to study the carbohydrate–lectin interaction [68]. The biotinylated glycopolymers were anchored on the MB–streptavidin surface via the noncovalent biotin–streptavidin interaction and used to recognize the complementary lectin. The interaction of b-d-galactose with RCA120 revealed that the glyco-MB is a probe that could be directly analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and MALDI-TOF analysis. De la Fuente et al. prepared gold–iron oxide glyco-NPs (glyco-AuFeNPs) to develop nanoscale magnetic materials. The newly formed NPs were magnetic, and their surfaces were easily modified by the formation of an Au–S bond [69]. The glyco-AuFeNP was synthesized by reducing FeCl3 and HAuCl4 with NaBH4 in the presence of the disulfide glycoconjugate in water. The physical and chemical features of glyco-AuFeNP were characterized by 1H NMR spectrometry, TEM, AFM, and superconducting quantum interference device magnetometry. Lactose and maltose disaccharides with various linkers were immobilized on AuFeNP. The shape, magnetic behavior, and SPR of the resulting AuFeNPs had very different properties from those of glyco-AuNPs.
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To exploit the metallic NP as a biocompatible targeting agent, the NP must not be allowed to agglomerate in aqueous solution but can be taken up intracellularly. Therefore, surface modification of NP is key to enhancing its permeability through the cell membrane. Horák et al. developed new iron oxide MNPs for stem cell labeling [70]. Initially, they used dextran-coated monocrystalline MNPs to penetrate the cell membrane, but the uptake efficiency obtained by endocytosing the cells was very low. The MNPs were coated with D-mannose, which interacts with the target receptor on the cell surface (mannose-MNP) and was used to improve their cellular uptake. Two mannose-MNPs were fabricated by an in situ coating procedure to yield 2 nm of MNP and by the postsynthesis coating procedure to yield 6 nm of MNP. Mannose-MNPs markedly outperformed dextran-coated MNPs (Endorem) and unmodified MNPs in bone marrow stromal cell labeling, as shown in Fig. 38.14. A promising probe for labeling living cells, mannose-MNP also could be a potential diagnostic and therapeutic agent because of its high relaxivity, which is responsible for the contrast in MR images, and easy internalization by cells.
Fig. 38.14 Microscope observation of bone marrow stromal cells (a) labeled with Endorem (control experiment), (b) primary uncoated 6.05-nm iron oxide NPs, (c) undiluted (0.2 mg/mL) and (d) diluted (0.02 mg/mL) d-mannose-modified 1.6-nm iron oxide NPs (from [70], by permission)
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38.4 Fabrication and Applications of Glyco-CNTs 38.4.1 Introduction and Scope of CNTs Since their discovery, CNTs have shown promise in many applications. CNTs have a wide range of properties, such as a high surface-area-to-volume ratio, a high mechanical strength with ultralight weight, favorable electronic properties, and excellent chemical and thermal stability, making them well suited to diverse applications in a wide range of fields. The lengths of the CNTs are on the nanometers-to-micrometer scale, with diameters from 0.4 to 2.0 nm for single-walled (SW) and 2 to 100 nm for multiple-walled CNTs. CNTs are characterized by hexagonally arranged sp2-hybridized carbon atoms that have been “rolled up” into a cylinder. The range of applications of CNTs is currently expanding rapidly, as they are used not only as electronic, but also as chemical or biological materials. A special issue [71] of Accounts of Chemical Research and many excellent review articles and monographs have provided a comprehensive overview of the field [72–78]. This section reviews the surface modification of CNTs with carbohydrates and their applications in various fields. The notoriously poor solubility of CNTs in aqueous media has greatly limited the use of CNTs in bioapplications. The highly hydrophobic nature of CNTs enables them to disperse only slightly in aqueous solution under ultrasonication and causes them to reprecipitate as soon as this process is interrupted. Therefore, many methods of chemical modification have been developed to produce soluble CNTs.
38.4.2 Surface Modification of CNTs Many methods for modifying the surfaces of CNTs have been developed, including the covalent and noncovalent functionalization of sidewalls, and endohedral functionalization [71–78]. In noncovalent functionalization, supramolecular adducts on the surface are easily formed with surfactants or polymers, maintaining the structure and original properties of the CNTs. All of the currently known techniques for preparing CNTs yield only 70% CNT by weight, with other contaminants of primarily amorphous carbon and catalytic particles. In the purification of raw CNTs by oxidative methods, the ends of the CNTs are oxidized, resulting in the formation of small holes and the incorporation of oxygenated functional groups, mainly carboxyl and hydroxyl groups, on the surface [79]. Thus, the CNTs can be modified at these defect sites by coupling with amines, as shown in Fig. 38.15 [80].
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COOH R-NH2 COOH
CONHR
Activating agent e.g. diimide
SOCl2
R-NH2 COCl COCl
Fig. 38.15 Functionalization of CNTs through the defect sites of the graphitic surface
38.5 Fabrication of Glyco-CNTs Cyclodextrins (CDs) are macrocyclic oligosaccharides that are known to selectively form inclusion complexes with a wide range of guest molecules. Dodziuk et al. [81] exploited this feature of CDs to prepare an aqueous, soluble, SW CNT (h-CD-SWCNT) by forming a pseudorotaxane-type complex with h-CD. Resasco and Pompeo were the first to describe the grafting of glucosamine SW-CNT by the amidation of carboxy groups of CNT with d-glucosamine (Fig. 38.16) [83]. The water solubility of the resulting glyco-SW-CNT was increased from 0.1 to 0.4 mg/mL. This method was adopted to synthesize b-galactose–modified SW-CNT (Gal-SW-CNT) [82]. Gal-SW-CNT formed a turbid aqueous dispersion by self-aggregation. However, its solubility in water was dramatically increased by adding b-galactose–specific PNA. Surface galactoses of CNT were wrapped by PNA lectins, forming a supramolecular network structure and increasing the solubility in water. Stoddart et al. employed amylose, a linear component of starch, to produce supramolecular helical complexes on the SW-CNT in aqueous solution (Fig. 38.17) [84]. Iodine facilitated the formation of CNT-inserted helical amylose (amyloseSW-CNT) as revealed by the fact that the soluble CNT was generated using starchiodine complex aqueous solution but not starch solution. A solution of amylose-iodine complex allows SW-CNTs to displace iodine inside the preorganized amylose helix by a “pea-shooting” mechanism upon sonication, as shown in Fig. 38.17c. Amylopectin, a highly branched component of starch, does not dissolve SW-CNTs in water because it has a high molecular weight and a dendrimer-like shape, which interrupt the formation of iodine complex in aqueous solution. Kim et al. investigated the behavior of water-soluble amylose-SW-CNT [85] and found that the diameter of amylose-SW-CNT was ~30 nm and formed aggregated bundles. Although functionalization of CNTs with carbohydrate polymer is a very popular method of generating water-soluble CNT, no direct evidence of the formation of a truly helical structure from SW-CNTs and water-soluble polymers is available.
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HOCO HOCO
OH O
HO HO
HOCO
OH O
+ HO
HOCO
EDC, HOBt, Et3N
O
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HO HO
NH 2
water
HO
1
HO
O
OH
OH O
O
OH OH O
HNCO HOCO HNCO HOCO
O
OH
HOCO HNCO
Fig. 38.16 Preparation of b-Gal-SW-CNT (from [82], by permission)
a c Blue Amylose lodine Complex 2 HO 1
3
OH
4 o
o 5 o
6 OH
Sonication with SWNTs
n
Grey Amylose SWNTs Complex
b OH O O HO
OH O
OH O HO
OH O HO
OH O OH n
amylose Fig. 38.17 Schematic representation of (a) helix adopted by amylose, (b) repeating unit of amylose structure, and (c) Stoddart’s “pea-shooting” mechanism, where CNTs displace iodine from a preorganized amylose helix (from [84], by permission)
In 2004, Shinkai et al. demonstrated that CNT induced the formation of a “periodical” helical structure of natural polysaccharides, such as schizophyllum (SPG) and curdlan (Fig. 38.18) [86]. SPG and curdlan are similarly composed of highly branched glucose repeating units, but curdlan lacks the side chain of glucose. They both form a triple-stranded helix (t-SPG) in water and random coiled single-strands (s-SPG) in dimethyl sulfoxide. The helical nature of the main chain b-1,3-glucan of s-SPG
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Fig. 38.18 (a) Polysaccharides used in the helix formation, (b) a representative model of the s-SPG triple helix, and (c) and (d) magnified AFM images of SW-CNTs/curdlun and SW-CNTs/ SPG composite, respectively (from [86], by permission)
and curdlan clearly reveals the helical structures of polysaccharide-SW-CNTs. The helical twining of water-soluble polymers on CNT was confirmed by AFM, as shown in Fig. 38.18c.
38.5.1 CNT as Multivalent Carbohydrate Ligand Carrier Until recently, little attention had been paid to the integration of CNTs as synthetic multivalent carbohydrate ligands, but CNTs have been extensively investigated as potential biosensors and biomolecule transporters over the past decades [87, 88]. Clearly, various carbohydrate-modified CNTs can participate in important interactions between carbohydrates and their receptors. Shinkai et al. were the first to synthesize the lactoside-conjugated SPG-SW-CNT and used it as a multivalent carbohydrate ligand to interact with RCA120, as presented in Fig. 38.19 [89]. Bertozzi et al. were the first to demonstrate the molecular recognition process of mucin-like CNT with a target receptor [90]. The a-GalNAc glycolipid that contained a C18 lipid chain at the reducing end was self-assembled on the SW-CNT surface (forming GalNAc-SW-CNT) by hydrophobic interaction, as displayed in Fig. 38.20. GalNAc-SW-CNT is water soluble and can be directly characterized by AFM, SEM, and TEM. The multivalent interaction of this mucin-mimic CNT with the target receptor Helix pomatia agglutinin, a hexavalent a-GalNAc residue– specific lectin, was evaluated under a fluorescence microscope. The unmodified SW-CNT and GalNAc-SW-CNT were coincubated with Chinese hamster ovary (CHO) cells to elucidate the biocompatibility and cytotoxicity of SW-CNTs [91]. Observations of the cell number and viability of cultured cells revealed that GalNAc-SW-CNT was nontoxic, while unmodified CNT inhibited cell growth (Fig. 38.21). Sun et al. [92] functionalized SW-CNTs by amide bond formation of the surface carboxyl group of CNT with 2¢-aminoethyl-b-dgalactopyranoside to yield a unique scaffold with multivalent carbohydrate ligands. This galactose-conjugated SW-CNT served as a polyvalent ligand that interacted with a target receptor (d-galactose binding protein) on pathogenic E. coli, and the resulting complex was visualized by SEM analysis, as shown in Fig. 38.22.
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a
HO OH O
HO HO
HO HO HO
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OH O
O OHO O OH
O
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HO OH OH O O OH
OH O
O HO HO
HN
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H N
OH O
O
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OH O
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c
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OH O
O
OH
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546.67 nm
b
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0 nm 0 nm
546.67 nm
1093.33 nm
Fig. 38.19 Structures of (a) SPG-Lac, (b) SPG-Lac-wrapped SW-CNTs for studying interactions with lectin, and (c) interactions of SPG-Lac/CNT composites with the lectin RCA120 by AFM (from [89], by permission)
The same group also studied the aggregation associated with the multivalent interaction between Bacillus anthracis spores and derivatized mannose- or galactosecoated SW-CNTs. Sun et al. [93] nicely demonstrated that monosaccharide-coated SW-CNTs exhibited specific binding activities to B. anthracis. In the presence of a divalent cation (such as Ca2+), B. anthracis aggregated with mannose-coated SW-CNTs immediately. The aggregates were visible by the naked eye, as shown in Fig. 38.23 (right). The addition of EDTA to the aggregation solution caused the redispersion of both spores and CNTs. These results indicated that the CNT represents a unique multivalent display of monosaccharide ligands. Shinohara et al. recently developed CNT-based water-soluble photoluminescent multivalent carbohydrate glycoconjugate polymer-coated SW-CNTs (Fig. 38.24) [94]. The photoluminescence of the a-glucose-conjugated SW-CNT was measured as a contour plot in D2O solution under well-defined conditions.
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α-GaINAc
C18 lipid
HO HO
OH O AcHN
H3C-(CH2)17
CN
H N O
C18-α-MM
HO HO
OH O
AcHN
O N
O N
N N AcHN O AcHN O HO HO O O HO OH HO OH
b
Fig. 38.20 (a) Structural features of synthetic mucin mimic (C18-a-MM); O-linked a-GalNAc residues are attached to the Ser/Thr residues of the polypeptide containing the C18 lipid tails. (b) A model representation of the self-assembly of C18-terminated a-GalNAc–conjugated mucin mimic on the CNT surface (C18-a-MM-CNT) (from [90], by permission)
Fig. 38.21 (a) Binding of C18-a-MM coated to CHO cells; (b) effect of glycopolymer-coated and unmodified CNTs on the growth of CHO cells (from [91], by permission)
+ E.coli
Gal-swnt
=
E.coli
OH OH O
HO
OH
O O
NH
= D-galactose-binding protein
Fig. 38.22 Sun’s galactose-bound CNTs as multivalent carbohydrate ligand carriers (left) and SEM image of their multivalent interactions with E. coli (from [92], by permission)
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Fig. 38.23 Mannose- and galactose-coated SW-CNTs (left) and optical micrographs (right) showing their unique binding interactions with B. anthracis spores to form aggregates (from [93], by permission)
Fig. 38.24 Schematic presentation of the glycoconjugate polymer-coated SW-CNTs (from [94], by permission)
38.6 Summary This article has presented simple and effective methods for preparing glyconanomaterials as multivalent ligands. The carbohydrate-conjugated metal NPs, including AuNPs and MNPs, exhibited high affinity and specificity in carbohydrate– carbohydrate and protein interactions, and were useful in many applicat ions, including binding-epitope mapping, cell recognition, and enzyme activity analysis, among others. In in vivo imaging, glyco-QDs perform excellently in cell labeling and tracing. Additionally, many examples of the formation and application of glyco-SW-CNTs were presented. Despite their importance in various fields of research, the cytotoxicity and solubility of nanomaterials remain major concerns regarding their use as tools for biological applications. Further development of synthetic strategies, such as carbohydrate-conjugated nanomaterials with greater water solubility and lower cell toxicity, may yield candidate materials for biomedical applications. Acknowledgments The authors would like to thank the National Science Council of the Republic of China, Taiwan, for financially supporting this research under contract.
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Chapter 39
Glycan Arrays to Decipher the Specificity of Plant Lectins Els J.M. Van Damme, David F. Smith, Richard Cummings, and Willy J. Peumans
Keywords Carbohydrate-binding activity • Glycan array • N-glycan • Specificity In recent years, evidence has been accumulating that protein–carbohydrate interactions play an important role in host–pathogen interaction(s), development, cell–cell communication, and cell signaling. To study the protein–carbohydrate recognition phenomena that take place within or at the surface of a cell, it is requisite to have the appropriate tools for dissecting this type of interaction. During the past decade, microarray technology has successfully been introduced into the field of glycobiology. These carbohydrate or glycan microarrays allow the rapid and comprehensive screening of carbohydrate-binding proteins for interaction with a large set of carbohydrate structures and characterization of their carbohydrate-binding properties.
39.1 Glycan Arrays Since the discovery that plant lectins bind glycoconjugates, methods to monitor protein–glycan interactions have been in development [13, 23, 38]. Classical methods were based on observing glycoprotein or polysaccharide precipitation or, more commonly, agglutination of erythrocytes or other cell types. The carbohydrate specificities of lectins were explored by indirect methods, including exoglycosidase digestion of intact cells to determine what terminal monosaccharides were involved in binding and inhibition of precipitation or cellular agglutination by hapten sugars or glycoconjugates. These methods were often effective, and much of our current knowledge of the carbohydrate specificity of plant lectins is based on hapten inhibition studies. Inhibition studies, however, require that each hapten be E.J.M. Van Damme (*) Laboratory of Biochemistry and Glycobiology, Department of Molecular Biotechnology, Ghent University, Coupure Links 653, 9000 Gent, Belgium e-mail:
[email protected] A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_39, © Springer Science+Business Media, LLC 2011
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prepared at a known concentration and assayed in separate experiments, which is a labor-intensive activity that requires relatively large amounts of lectin and hapten. As a general approach, interrogating a solid phase with lectins or glycan-binding proteins (GBPs) is more efficient and amenable to a high-throughput approach. An early example of such an approach was an enzyme-linked immunosorbent assay (ELISA)-based test to detect antibodies against bacterial polysaccharides that had been adsorbed to plastic [8]. Another important solid-phase assay for GBPs was the glycolipid overlay method; in this approach, glycolipids, separated on a thin layer chromatogram, were probed or interrogated with 125I-labeled toxins, anticarbohydrate antibodies, and lectins [17, 18, 27]. This innovation permitted the analysis of multiple glycolipid targets from natural sources with extremely low concentrations of protein. A modification of this overlay method was to prepare neoglycolipids (NGLs) by reducing glycans using reductive amination with dipalmitoylphosphatidyl ethanolamine [29]. The resulting NGLs could then be used in a variety of formats, including separation by thin-layer chromatography, incorporation into liposomes, or adsorption on microtiter plates, where they were available for probing with GBPs [11]. NGLs prepared from defined glycans are used in glycan microarrays, where they are noncovalently bound after printing on nitrocellulose-derivatized glass slides [12, 16]. A number of glycan array formats were subsequently developed [12, 16]. In 2001, the Consortium for Functional Glycomics (CFG) was funded by the National Institutes of Health/National Institute of General Medical Sciences to establish a publicly available array. The first publicly available resource was an ELISA-type format, which initially contained several dozen synthetic, biotinylated glycans. It was later expanded to well over 100 glycans. The biotinylated glycans were robotically added in replicates of four to commercial streptavidin-coated 96-well microtiter plates and interrogated by adding GBPs at known concentrations to the individual wells. GBPs bound to glycans in the wells were detected by fluorescence measurements of either directly or indirectly labeled GBP, using a microplate fluorimeter. The ELISA glycan array was used in studies on the glycan specificities of many plant and animal GBPs as project requests carried out in collaboration with the Protein–Carbohydrate Interaction Core (Core H) of the CFG [1, 6, 9, 10, 14, 15, 19, 22, 26, 30, 37]. The results of many of these analyses can be found among the public data available through the CFG Web site (http://www.functionalglycomics.org). More recently, the CFG developed a covalent glycan microarray by directly coupling glycans with an aliphatic amine spacer to commercial glass slides containing N-hydroxysuccinimide (NHS) coupling chemistry on a polyethylene glycol surface. In addition to using NHS chemistry, which is highly reactive toward primary aliphatic amines, this microarray could be prepared by conventional contact printing approaches that are used for DNA and protein microarrays [5]. Robotic pin printers generate six replicates at 0.6 nL of 10- and 100-mM stock glycan solutions as 100–150-mm spots on the surface of NHS-derivatized slides to form microarrays. After the capture reaction occurs, the printed slides are blocked with ethanolamine to eliminate any residual reactive groups. The printed slides are washed with appropriate buffer components, rinsed with water, dried, and stored desiccated at room temperature until used [5, 28, 39].
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To prepare GBPs for analysis on the printed glycan microarray, the proteins can be directly labeled by any appropriate procedure, including commercially available kits. For analysis on the array, GBPs are prepared in isotonic-binding buffer containing divalent cations that may be required for binding: 1–3% bovine serum albumin (BSA) or other blocking protein and 0.05% Tween 20 to prevent nonspecific binding. After the dried slides are rehydrated in buffer, the GBP is added at 1–200 mg/mL. The concentration used depends upon the affinity of the GBP for the corresponding coupling glycan(s). The total volume of analysis on the slide surface is 50–70 mL and is carried out under a cover slip. Alternatively, the array may be contained on a slide within a barrier drawn with a hydrophobic pen; in that case, approximately 1 mL of GBP solution may be added to the total surface of the slide. GBPs that are directly labeled with a fluorescent tag can be analyzed after a one-step process of binding. This is followed by gently immersing the slide into a series of buffer solutions containing no BSA, followed by washing with buffer lacking detergent, and finally washing with distilled water. The slide is then dried to remove excess water by spinning in a slide centrifuge or using a gentle stream of nitrogen. Unlabeled GBPs bound to the array may be detected using any indirect immunochemical fluorometric technique. To quantify the relative binding of GBP to glycans, the slides are processed in a ProScanArray fluorescence scanner (PerkinElmer). The fluorescence of four replicates (following the elimination of the highest and lowest values of each glycan) represented on the array are reported as the average of relative fluorescence units (RFU). Analysis of the fluorescence image is carried out using IMAGENE image analysis software (BioDiscovery, El Segundo, CA) [5], and data are presented in tables using Microsoft Excel. These tables list the glycan structures on the microarray, their corresponding average RFU values of binding by the GBP, the standard error of the mean, and the coefficient of variation reported in percent. The results are presented in a histogram. The current glycan array (v4.2) available from the CGF comprises 511 glycan targets. These are considered representative of many nonreducing termini of Nand O-glycans in glycoproteins and glycolipids. A complete list of the glycans in all versions of the glycan microarray of the CFG is available on the Web site at the following address: http://www.functionalglycomics.org/static/consortium/ resources/resourcecoreh8.shtml. Like the ELISA glycan array, the printed glycan microarray provided by the CFG is publicly available upon request. Cores D and H of the CFG have analyzed hundreds of different samples, including GBPs, viruses, bacteria, and sera for investigators.
39.2 Use of Glycan Arrays to Study Carbohydrate-Binding Properties of Plant Lectins The group of plant lectins comprises “all plant proteins possessing at least one noncatalytic domain, which binds reversibly to a specific mono- or oligosaccharide.” Structural and molecular studies have confirmed the occurrence of different types of
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plant lectins with distinct molecular structures and carbohydrate-binding properties [33, 34, 36]. In the past, several classification systems have been proposed to subdivide the seemingly very heterogeneous group of plant lectins into the so-called natural groups, e.g., based on sugar specificity. However, it turned out that there is no direct link between the molecular properties of a lectin and its carbohydratebinding specificity. Even very closely related plant lectins can exhibit different carbohydrate-binding properties. For example, within the legume family, the carbohydrate-binding activity of the different lectins covers a very broad range, from specificity toward mannose/glucose, fucose, GlcNAc, Gal/GalNAc, and sialic acid to very complex specificity. Hitherto, twelve different sugar-binding domains/motifs have been identified with certainty in plants. For some lectin families, structure–function relationships – in terms of specific recognition of monosaccharides – are reasonably well understood. However, though many plant lectins bind with a fairly high affinity to specific monosaccharide(s) and by virtue of this activity are widely used as versatile tools in biological and biomedical research, the biological relevance of the recognition and binding of a particular monosaccharide is probably very low because simple sugars are not considered natural receptors for any plant lectin. Moreover, specificity studies clearly demonstrated that all plant lectins exhibit a much higher affinity for oligosaccharides/complex glycans than for any monosaccharide. Structural analyses not only confirmed that the monosaccharide-binding site of plant lectins usually accommodates a single well-defined sugar unit of N- or O-linked glycans but also indicated that other amino acid residues located in the vicinity of the primary site participate in the binding of other sugar units so that a more extended carbohydrate-binding site is created. Over the years, several methods have been established to study the carbohydratebinding properties of plant lectins and their interactions with different glycan structures. Over the past few years, it has become possible to study the specificity/ interaction of plant lectins with both natural and synthetically derived glycan structures in array formats [5, 20]. Glycan array analyses of a representative set of plant lectins confirmed the preferential binding of most lectins to oligosaccharides and glycans rather than to monosaccharides. In addition, the glycan array analyses allowed refining the specificity of many plant lectins and, what is more, provided evidence that the “canonical” specificity of some plant lectins no longer holds true. Furthermore, the introduction of glycan arrays greatly facilitated the dissection of the specificity of those plant lectins that possess multiple carbohydrate-binding sites interacting with different carbohydrate structures.
39.2.1 Lectin Specificity Refined and Redefined Lectins from a variety of plant species have been reported to possess activity against human immunodeficiency virus (HIV) [2–4]. Especially those carbohydrate-binding proteins that specifically recognize mannose (Man) and N-acetylglucosamine
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(GlcNAc) exhibit remarkable anti-HIV activity in cell culture. These lectins qualify as potential anti-HIV microbicide drugs because they not only inhibit infection of cells by cell-free virus (in some cases in the lower nano- or even subnanomolar range), but they can also efficiently prevent virus transmission from virus-infected cells to uninfected T lymphocytes. It was shown that the most likely mechanism of antiviral action is the interruption of virus entry (i.e., fusion) into its target cell. Lectins presumably act by directly binding to the glycans that are abundantly present on the HIV-1 gp120 envelope [4]. Detailed analysis of the Galanthus nivalis (snowdrop) agglutinin (GNA) and Urtica dioica (stinging nettle) agglutinin (UDA), two lectins with potent anti-HIV activity, on the glycan array revealed that both lectins strongly interact with N-glycans. GNA was first reported in 1987 as a lectin with a unique specificity for mannose [31]. More detailed studies of the carbohydrate-binding properties of this lectin were performed using quantitative precipitation, hapten inhibition, and affinity chromatography of glycan structures on immobilized lectin columns. Shibuya et al. [25] reported that GNA precipitated highly branched yeast mannans but did not react with most glucans. In addition, hapten inhibition experiments showed that d-mannose is an inhibitor of GNA–mannan interaction, but neither N-acetyl-dmannosamine nor d-glucose is an inhibitor. Hapten inhibition with various sugars showed that GNA requires the equatorial hydroxyl groups at the C-3 and C-4 positions and an axial group at the C-2 position of the d-pyranose ring. Furthermore, the lectin requires a nonreducing terminal d-mannose residue for the interaction of oligosaccharides, and oligosaccharides with terminal Man(a-1-3)Man units showed the highest inhibitory potency among the manno-oligosaccharides tested. Glycopeptides that carry Man(a1-3)Man units were retarded on the immobilized GNA column, whereas those lacking this unit or with hybrid-type glycan chains were not retarded on the column [25]. These results are in very good agreement with the recent results obtained when analyzing the interaction of GNA with the glycan array (Fig. 39.1). GNA shows strong interaction with Man9, Man8, Man7, Man6, and Man5. The lectin also interacts with Man3-chitobiose but shows low reactivity toward chitotetraose and chitotriose, indicating that the lectin specifically recognizes the mannose part of the glycan. The reactivity of the snowdrop lectin toward complex N-glycans is also very low. In 1984, Peumans et al. [21] reported an unusual lectin from stinging nettle rhizomes. The interaction of the stinging nettle lectin (UDA) with carbohydrates was studied in detail using quantitative precipitation assays and hapten inhibition. The carbohydrate-binding site of UDA was shown to be complementary to an N,N¢,N″-triacetylchitotriose unit. It was proposed that the carbohydrate-binding site consists of three subsites, each of which has a slightly different binding specificity. Furthermore, equilibrium dialysis and ultraviolet difference spectroscopy revealed that UDA has two carbohydrate-binding sites per monomer [24]. Analysis of the carbohydrate-binding properties of UDA on the glycan array revealed that UDA interacts strongly with (GlcNAc)3-oligomer as well as with high mannose N-glycans, e.g., Man9, Man8, Man6, and Man5 (Fig. 39.1). These results suggest that UDA not only interacts with GlcNAc-oligomers, but also recognizes high-mannose
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RFU
GNA
Glycan No.
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Glycan No. Fig. 39.1 Interaction of AlexaFluor 488-labeled GNA and UDA on the printed array, version 1
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N-glycans. These results indicate that UDA is not interacting with the terminal sugar residues of the N-glycan but rather recognizes the GlcNAc residues of the N-glycan. The results of the glycan array analysis with GNA and UDA explain why both UDA and GNA show potent anti-HIV activity. It can be envisaged that both lectins can interact with the same N-glycans present on gp120. Whereas GNA specifically recognizes the outer branches of the glycan, UDA recognizes the GlcNAc residues at the base of the N-glycan.
39.2.2 Lectins Possessing Different Carbohydrate-Binding Sites with Distinct Specificity Carbohydrate-binding proteins comprising one or two domains equivalent to GNA form one of the major plant lectin families [33, 34]. A reinvestigation of the occurrence and taxonomic distribution of proteins built up of protomers consisting of two tandem-arrayed domains equivalent to the GNA revealed that these are widespread among monocotyledonous plants. Phylogenetic analysis of the available sequences indicated that these proteins do not represent a monophylogenetic group but most probably result from multiple independent domain-duplication/in-tandem-insertion events. To corroborate the relationship between interdomain sequence divergence and the widening of specificity range, a detailed comparative analysis was made of the sequences and specificity of a set of two-domain GNA-related lectins. Glycan microarray analyses combined with frontal affinity chromatography and surface plasmon resonance measurements demonstrated that the two-domain GNA-related lectins acquired a marked diversity in carbohydrate-binding specificity that strikingly contrasts the canonical exclusive specificity toward mannose of their singledomain counterparts [35]. Moreover, it appears that, in contrast to GNA, most two-domain GNA-related lectins interact with both high mannose and complex N-glycans. This dual specificity relies on the simultaneous presence of at least two different independently acting binding sites. The combined phylogenetic, specificity, and structural data strongly suggest that plants used domain duplication followed by divergent evolution as a mechanism for generating multispecific lectins from a single mannose-binding domain.
39.2.3 Some Lectin-Related Proteins Presumably Devoid of Sugar-Binding Activity Specifically Interact on Glycan Array In 1997, we described the isolation and molecular cloning of a novel type-2 ribosomeinactivating protein (RIP) from elderberry (Sambucus nigra) bark that was apparently devoid of agglutinating activity [32]. Attempts to bind this protein on immobilized sugars (e.g., galactose, N-acetylgalactosamine, and N-acetylneuraminic
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acid) or glycoproteins (e.g., fetuin, asialofetuin, mucin, asialomucin, ovomucoid, and thyroglobulin) that were successfully used for the isolation of other type-2 RIPs failed. Based on these findings, it was concluded that the elderberry type-2 RIP was devoid of sugar-binding activity, and accordingly it was named “Sambucus nigra lectin-related protein” (SNLRP). Molecular modeling and docking studies (using the B-chain of ricin as a model) confirmed that, due to the replacement of some critical amino acids, none of the potential binding sites of SNLRP can accommodate either Gal or GalNAc. Accordingly, SNLRP was considered an example of a natural carbohydrate-binding-deficient type-2 RIP. However, a reinvestigation of SNLRP on the glycan array revealed a strong interaction with GlcNAc-oligomers (pentamer, hexamer, trimer) as well as with many glycan structures containing GlcNAc residues substituted with Gal residues or sialic acid.
39.3 Conclusions A major advantage of the glycan microarray is the availability of many glycans that can be interrogated in a single analysis using very little GBP and very little immobilized glycan. The slide format and the speed of analysis make this a relatively high-throughput technique. Glycan microarrays printed on microscope slides have considerable advantage over the earlier microplate-based arrays. Although the printing and analysis of the array on a microscope slide require specialized equipment, the final printed array requires only 50–70 mL of GBP solution for each analysis, which permits the study of recombinant proteins that can only be expressed in small quantities. The glycan libraries can be maintained in small volumes, and only very small quantities of glycan derivatives are required for printing each slide. Thus, a glycan library can be maintained and expanded as glycans become available. Clearly, the ideal glycan array would be one that represented all known glycan structures. Because of the complexity and large numbers of naturally occurring glycans, current synthetic methods are not able to support such a comprehensive library of glycans. The glycan array supported by the CFG attempts to represent many of the major nonreducing-terminal glycan determinants on animal cell glycolipids and glycoproteins, with an emphasis on human and mouse glycans. Recent approaches to derivatization of naturally occurring free glycans, or glycans released from glycoconjugates, have been developed to support expansion of the glycan microarrays. These new developments include the coupling of free glycans to 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine to form neoglycolipids that can be printed on nitrocellulose [12] and derivatization of free glycans with a bifunctional spacer that contains a methyl-N,O-hydroxylamine for coupling to the reducing sugars and a primary amine group suitable for coupling to NHS-activated surfaces [7]. The recent development of fluorescent bifunctional diamino spacers, such as 2,6 diaminopyridine [28, 39], is particularly attractive for developing microarrays of naturally occurring glycans because they provide a sensitive tag to
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monitor and quantify nanomolar amounts of derivatized free glycans during their purification and structural definition, and permit the glycans to be printed as microarrays. The expansion of the glycans available on microarrays are important in the identification of glycan structure, and the construction of natural glycan libraries from specific glycoconjugates, cells, and tissues provides useful tools for the discovery and specificity analysis of novel GBPs. Acknowledgments The financial support of the Research Council of Ghent University and the Fund for Scientific Research-Flanders (FWO grants G.0201.04 and G.0022.08) is gratefully acknowledged. This work was supported in part by the Consortium for Functional Glycomics under National Institutes of Health/National Institute of General Medical Sciences Grant GM62116.
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Chapter 40
Targeting C-Type Lectin for the Treatment of Flavivirus Infections Szu-Ting Chen, Yi-Ling Lin, Ming-Ting Huang, Ming-Fang Wu, and Shie-Liang Hsieh
Keywords Dengue virus • Fingerprinting • Innate immunity receptor • Polysaccharide
40.1 Flavivirus-Mediated Human Diseases The genus Flavivirus contains approximately 70 viruses, and the major flaviviruses that cause human diseases are yellow fever virus (YFV), dengue virus (DV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus [1]. The flaviviral particles contain single-stranded, positive-sensed RNA genome packaged within an icosahedral capsid formed by the capsid protein. The genome-containing capsid is surrounded by a host-derived lipid bilayer bearing dimers of the viral envelope protein and the membrane protein. The sizes of flavivirus virions are approximately 37–50 nm. Thus, the antigenic, genetic, and three-dimensional structures of all the flaviviruses are similar to each other. Most flavivirus infections are relatively benign, but serious aseptic meningitis and encephalitic or hemorrhagic disease can occur. The encephalitis viruses include St. Louis encephalitis virus, WNV, JEV, Murray Valley encephalitis virus, and Russian spring-summer encephalitis virus. Approximately 20% of individuals infected with WNV develop West Nile fever, and 1% may develop encephalitis, meningitis, or meningoencephalitis [2]. The hemorrhagic flaviviruses are DV and YFV. DV is a major worldwide problem, with up to 100 million cases of dengue fever (DF) and 300,000 cases of dengue hemorrhagic fever (DHF) occurring per year. The incidence of the more serious DHF has quadrupled since 1985. DF is also known as breakbone fever; the symptoms and signs consist of high fever, headache, rash, and back and bone pain that last 6–7
S.-L. Hsieh (*) Department and Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_40, © Springer Science+Business Media, LLC 2011
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days. Secondary infection with another serotype of the four related strains of DV might cause DHF and dengue shock syndrome (DSS). Nonneutralizing crossreactive antibodies resulting from previous DV infection promote uptake of the virus into macrophages, a phenomenon known as antibody-dependent enhancement, which is one of the major factors leading to vasculature rupture, internal bleeding, and loss of plasma [2]. YFV infections are characterized by severe systemic disease, with degeneration of the liver, kidney, and heart, as well as hemorrhaging. Liver involvement causes the jaundice from which the disease gets its name. The mortality rate associated with yellow fever during an epidemic is as high as 50%. No treatment exists for flavivirus infection other than supportive care. A live vaccine against YFV and killed vaccines against JEV and Russian spring-summer encephalitis virus are available. A tetravalent vaccine against DV is being developed to prevent the risk of immune enhancement of the disease on subsequent challenge. However, whether it can stimulate equal responses to all four serotypes in people who have been exposed to one DV serotype is unknown, and its long-term and short-term efficacy need to be further confirmed.
40.2 Glycosylation and Flavivirus Virulence Numerous evidences have shown that the glycosylation status of envelope (E) protein is involved in the pathogenesis of flavivirus infections. The DV E protein contains two N-linked glycosylation sites, at Asn-67 and Asn-153. The glycosylation site at position 153 is conserved in most flaviviruses while the site at position 67 is thought to be unique for DVs [3]. However, this is in conflict with previous observations that the DV serotype-2 (DV2) E protein is only glycosylated at Asn-67 while the DV1 E protein is glycosylated at both Asn-67 and Asn-153 [4]. Whether both Asn-67 and Asn-153 are glycosylated in all the DV serotypes needs to be further confirmed. It has been shown that Asn-67 of the DV E protein is essential during viral propagation. DV2 lacking Asn-67 was able to infect mammalian cells and translate and replicate the viral genome, but the production of new infectious particles from mammalian BHK cells was dramatically impaired, while DV secretion from the mosquito cell lines C6/36 was not affected. Furthermore, DVs lacking the Asn-67 showed reduced infection of immature dendritic cells, suggesting an interaction between this glycan and the dendritic cell-specific intercellular adhesion molecule3-grabbing nonintegrin (DC-SIGN) or C-type lectin domain family 4, member L (CLEC4L) [5]. This is in accord with the finding that CLEC4L/DC-SIGN, which is abundant in immature dendritic cells, binds to DV2 (strain PR159-S1) as determined by cryo-electron microscopy [6]. A previous study indicated that the carbohydrate moiety at Asn-153 extends across the dimer interface covering the fusion peptide [7]. An N153Q DV2 mutant, which abolishes N-glycosylation at Asn-153 on the E protein, has a deleterious
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effect on viral infectivity to mammalian BHK cells (but not to mosquito C6/36 cells). This suggests that the glycan at Asn-153 has a differential modulatory effect on infectivity in mammals and mosquitoes [5]. In contrast, glycosylation at Asn154 of WNV decreased WNV infectivity in BHK and C6/36 cells (corresponding to Asn-153 in DV) [8]. Together, the results indicate that the glycans at positions 153 and 154 play different roles in DV and WNV, respectively. For DV4, the mouse-adapted strain H241 is highly neurovirulent for mice, whereas its non-mouse-adapted parent is rarely neurovirulent. The single substitution of Ile for Thr-155 or Ala-144, which ablated the glycosylation of Asn-153 in the E protein of the parent strain, yielded a virus that was almost as neurovirulent as the mouse-adapted mutant [9]. This indicates that Asn-153 is also involved in viral virulence in mice. For WNV, the E protein glycosylation status of the New York strain is an important determinant of virus neuroinvasiveness. To elucidate the determinant of the difference between E protein-glycosylated and nonglycosylated WNV infections, the cytokine expression of murine peritoneal macrophages infected with each virus was examined. Tumor necrosis factor (TNF)-a and interleukin (IL)-1b were upregulated with replication of the E protein-glycosylated virus, suggesting that TNF-a and IL-1b expression are related to the virulence of E protein-glycosylated WNV [10]. It is interesting to note that the virions and subviral particles are bound to CLEC4L/DC-SIGN and CLEC4M/DC-SIGN receptor (DC-SIGNR) [11]. Unlike the two glycosylation sites found in the DV E protein, the WNV E protein only contains Asn-153, indicating that the interaction of WNV with CLEC4L and CLEC4M might be via Asn-153. Thus, DV and WNV seem to use distinct glycans to interact with DC-SIGN and DC-SIGNR.
40.3 C-Type Lectins and Flavivirus Infection Even though in vitro studies demonstrate that the target cells of flaviviruses include monocytes, macrophages, dendritic cells, endothelial cells, and others, the information regarding in vivo replication of flaviviruses in host cells is very limited. Recently, several studies demonstrated the importance of myeloid cells in the pathogenesis of dengue infection. DV was shown to replicate in human dermal dendritic cells, the Langerhans cells [12], and monocytes/macrophages [13] isolated from human samples during acute infectious status. Replication of the flavivirus produces a double-stranded RNA replicative intermediate, which then stimulates dendritic cells and macrophages to secret both inflammatory cytokines and interferons (IFN). However, the details about how DV enters dendritic cells and monocytes/macrophages, and the machinery involved in DV recognition, were unknown until very recently. Several C-type lectin receptors, such as CLEC4L/DC-SIGN/CD209, CLEC4M/ DC-SIGNR/CD299 [14, 15], and CLEC13D/mannose receptor/CD206 [16], have been shown to interact with DV directly and play important roles in DV’s entry into
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dendritic cells and macrophages. To further investigate the role of the innate immunity receptors involved in DV-mediated disease, 22 fusion proteins comprising the Fc portion of human immunoglobulin G1 and the extracellular domain of innate immunity receptors – including Toll-like receptors (TLRs), C-type lectins, triggering receptor expressed on myeloid cells (TREMs), and TREM-like transcripts (TLTs) – were tested for their binding specificity to DV (Fig. 40.1). In addition to CLEC4L and CLEC4M, CLEC5A was found to bind to DV specifically in a Ca++independent manner [17]. CD14+ monocytes
Purity > 98%
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Fig. 40.1 Flow chart for the production of innate receptor Fc fusion proteins
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CLEC5A is a type II transmembrane protein belonging to the C-type lectin superfamily and contains a charged residue in the transmembrane region that enables it to pair with a 12-kDa DNAX-activating protein (DAP12). DAP12 contains an immunoreceptor tyrosine-based activation motif-bearing signaling molecule that is noncovalently associated with activating isoforms of major histocompatibility complex class I receptors on natural killer cells [18]. Unlike the classic C-type lectin domain, CLEC5A contains a “natural killer T-cell C-type lectin domain” that binds sugar independently of Ca++ [17]. It is interesting to note that CLEC7A/Dectin-1, a C-type lectin receptor for fungal cell wall b-glucan that plays an essential role in immune responses against fungi [19], also contains a natural killer T-cell C-type lectin domain and binds to glycans independently of Ca++ and Mg++ [20]. In contrast to CLEC4L and CLEC4M, the CLEC5A–DV interaction does not result in viral entry. In contrast, DV induces DAP12 phosphorylation and stimulates the release of proinflammatory cytokines via CLEC5A. Blockade of the CLEC5A–DV interaction suppresses the secretion of proinflammatory cytokines without affecting the release of IFN-a, supporting the notion that CLEC5A acts as a signaling receptor for proinflammatory cytokine release. To study the role of CLEC5A in the pathogenesis of DHF/DSS, DV2 (either New Guinea C-N or PL046 strain) was inoculated into signal transducer and activator of transcription-1 (STAT1)-deficient mice to induce a severe inflammatory reaction and a massive cytokine release (also called a cytokine storm), which is believed to be one of the major contributory factors for the pathogenesis of DHF/DSS [21]. Severe subcutaneous hemorrhage, neuronal damage, and shock were observed. The severe symptoms observed in STAT1-deficient mice can be attributed to at least three aspects: (1) STAT1 is essential for the activation of IFN signaling [22]; (2) the negative feedback of the TAM receptor tyrosine kinase is STAT1 dependent [23]; and (3) the binding of the WNV E protein to DC-SIGN-downregulated TLR3 expression and reduced cytokine secretion via a STAT1-mediated pathway; thus, the deficiency of STAT1 enhances the TLR3-mediated signaling cascade to augment cytokine release [24]. Under such severe inflammatory reactions, injection of anti-CLEC5A monoclonal antibodies (mAbs) still inhibits DV-induced plasma leakage, as well as subcutaneous and vital-organ hemorrhaging, and reduces the mortality of DV infection by about 50% in STAT1-deficient mice. This observation indicates that blockade of CLEC5A-mediated signaling attenuates the production of proinflammatory cytokines by macrophages infected with DV (either alone or complexed with an enhancing antibody), without damping the secretion of IFN-a, which is essential for antiviral immunity [17].
40.4 C-Type Lectin and the Severity of DV Infections After incubation of the DV2 PL046 strain with CD14+-derived macrophages randomly obtained from the blood bank, variations in the infection rate and cytokine release were observed. Among the 200 samples tested, the infection rates can be
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classified into three categories: (1) 80–50% (approximately 50 out of 200 cases); (2) 20–40% (approximately 100 out of 200 cases); and (3) less than 10% (approximately 50 out of 200 cases). This suggests that certain unidentified host factors might determine the different DV infection rates in human macrophages. Given that the glycosylation status of DV influences virus replication and propagation; that members of C-type lectins (CLEC4L [14], CLEC4M [15], and CLEC13D [16]) are involved in DV entry; and that CLEC5A is responsible for DV-induced inflammatory reactions [17], it is reasonable to speculate that the polymorphism of C-type lectins might play an important role in DV infections. It has been demonstrated that variation of the CLEC4L promoter (G allele at 336) correlates with dominant protection against DF but not DHF. The expected decrease in expression of CLEC4L carrying the G allele at CLEC4L-336 may result in a lower susceptibility of dendritic cells to DV in the early stages of infection [25]. However, the correlation between dengue infections and CLEC4M, CLEC13D, and CLEC5A has not been investigated yet.
40.5 Targeting CLEC5A for the Treatment of DV Infections The dilemma to control severe inflammatory reactions is that suppression of inflammatory cytokine release also attenuates antiviral immunity. Steroid treatment is ineffective at controlling viral spread and decreasing mortality in victims of severe acute respiratory syndrome. TLRs are involved in the production of both proinflammatory (such as TNF-a) and antiviral (such as IFN) cytokines, thus apparently they are not ideal candidates for controlling virus-induced inflammatory reactions. In contrast, members of C-type lectins are not involved in IFN production. Therefore, blockade of the virus–lectin interaction offers a promising strategy for alleviating tissue damage and increasing the survival of patients suffering from DHF and DSS, and possibly even other virus-induced inflammatory diseases (Fig. 40.2).
40.6 Summary It becomes clear that multiple receptors and intracellular sensors are involved in host–pathogen interactions. In addition to TLRs, the non-TLRs – including C-type lectins, TREMs, and TLTs – are promising candidates for recognizing pathogenassociated molecular patterns. Considering that glycosylation is one of the most common and complex posttranslational modifications and that pathogens are covered by glycans, it is obvious that the first contact between innate immunity receptors and pathogens depends on a protein–carbohydrate interaction. Among the acute viral infections, the pathogenesis of viral hemorrhagic fever is still unclear, but a cytokine storm is believed to play a critical role in such a highly lethal disease. Our recent experiments demonstrate that the dengue virion acts as a ligand to bind CLEC5A (also know as myeloid DAP-12-associating lectin), and possibly other
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Fig. 40.2 Inhibition of proinflammatory cytokine secretion by antagonistic anti-CLEC5A mAb without attenuating IFN-a secretion
non-TLRs, directly. DV induces massive cytokine secretion via CLEC5A, and blockade of CLEC5A attenuates inflammatory reactions without damping antiviral immunity. Moreover, anti-CLEC5A antagonistic mAbs protect mice from DV-induced hemorrhaging and shock syndrome. The segregation of proinflammatory reaction from antiviral immune response provides a great chance to design a better strategy for treating viral infections in the future. Acknowledgments This work is supported mainly by the National Research Program for Genomic Medicine, National Science Council, Taiwan (NSC-95-3112-B-010-0171 & NSC 96-3112-B-010-2), and in part by the National Yang-Ming University, Taiwan (96A-D-D132 from MOE), the Taipei Veterans General Hospital (V97S5-001), and Academia Sinica. We are grateful to the resources and collaborative efforts provided by the RNAi Consortium, Academia Sinica, Taiwan, and the Consortium for Functional Glycomics, funded by NIGMS-GM62116 USA.
References 1. Barrett A, Weaver S (2002) Arboviruses: alphaviruses flaviviruses and bunyaviruses. Med Microbiol 482–494 2. Murray P, Rosenthal K, Pfaller M (2005) Togaviruses and flaviviruses. Med Microbiol 619–628 3. Heinz FX, Allison SL (2003) Flavivirus structure and membrane fusion. Adv Virus Res 59:63–97
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4. Johnson AJ, Guirakhoo F, Roehrig JT (1994) The envelope glycoproteins of dengue 1 and dengue 2 viruses grown in mosquito cells differ in their utilization of potential glycosylation sites. Virology 203:241–249 5. Mondotte JA, Lozach PY, Amara A, Gamarnik AV (2007) Essential role of dengue virus envelope protein N glycosylation at asparagine-67 during viral propagation. J Virol 81:7136–7148 6. Pokidysheva E, Zhang Y, Battisti AJ et al (2006) Cryo-EM reconstruction of dengue virus in complex with the carbohydrate recognition domain of DC-SIGN. Cell 124:485–493 7. Modis Y, Ogata S, Clements D, Harrison SC (2003) A ligand-binding pocket in the dengue virus envelope glycoprotein. Proc Natl Acad Sci USA 100:6986–6991 8. Hanna SL, Pierson TC, Sanchez MD, Ahmed AA, Murtadha MM, Doms RW (2005) N-linked glycosylation of west nile virus envelope proteins influences particle assembly and infectivity. J Virol 79:13262–13274 9. Kawano H, Rostapshov V, Rosen L, Lai CJ (1993) Genetic determinants of dengue type 4 virus neurovirulence for mice. J Virol 67:6567–6575 10. Shirato K, Miyoshi H, Kariwa H, Takashima I (2006) The kinetics of proinflammatory cytokines in murine peritoneal macrophages infected with envelope protein-glycosylated or nonglycosylated West Nile virus. Virus Res 121:11–16 11. Davis CW, Nguyen HY, Hanna SL, Sanchez MD, Doms RW, Pierson TC (2006) West Nile virus discriminates between DC-SIGN and DC-SIGNR for cellular attachment and infection. J Virol 80:1290–1301 12. Wu SJ, Grouard-Vogel G, Sun W et al (2000) Human skin Langerhans cells are targets of dengue virus infection. Nat Med 6:816–820 13. Chen YC, Wang SY (2002) Activation of terminally differentiated human monocytes/macrophages by dengue virus: productive infection, hierarchical production of innate cytokines and chemokines, and the synergistic effect of lipopolysaccharide. J Virol 76:9877–9887 14. Navarro-Sanchez E, Altmeyer R, Amara A et al (2003) Dendritic-cell-specific ICAM3grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue viruses. EMBO Rep 4:723–728 15. Tassaneetrithep B, Burgess TH, Granelli-Piperno A et al (2003) DC-SIGN (CD209) mediates dengue virus infection of human dendritic cells. J Exp Med 197:823–829 16. Miller JL, deWet BJ, Martinez-Pomares L et al (2008) The mannose receptor mediates dengue virus infection of macrophages. PLoS Pathog 4:e17 17. Chen ST, Lin YL, Huang MT et al (2008) CLEC5A is critical for dengue-virus-induced lethal disease. Nature 453:672–676 18. Bakker AB, Baker E, Sutherland GR, Phillips JH, Lanier LL (1999) Myeloid DAP12associating lectin (MDL)-1 is a cell surface receptor involved in the activation of myeloid cells. Proc Natl Acad Sci USA 96:9792–9796 19. Brown GD, Taylor PR, Reid DM et al (2002) Dectin-1 is a major beta-glucan receptor on macrophages. J Exp Med 196:407–412 20. Brown GD, Gordon S (2001) Immune recognition. A new receptor for beta-glucans. Nature 413:36–37 21. Pang T, Cardosa MJ, Guzman MG (2007) Of cascades and perfect storms: the immunopathogenesis of dengue haemorrhagic fever-dengue shock syndrome (DHF/DSS). Immunol Cell Biol 85:43–45 22. Theofilopoulos AN, Baccala R, Beutler B, Kono DH (2005) Type I interferons (alpha/beta) in immunity and autoimmunity. Annu Rev Immunol 23:307–336 23. Rothlin CV, Ghosh S, Zuniga EI, Oldstone MB, Lemke G (2007) TAM receptors are pleiotropic inhibitors of the innate immune response. Cell 131:1124–1136 24. Kong KF, Delroux K, Wang X et al (2008) Dysregulation of TLR3 impairs the innate immune response to West Nile virus in the elderly. J Virol 82:7613–7623 25. Sakuntabhai A, Turbpaiboon C, Casademont I et al (2005) A variant in the CD209 promoter is associated with severity of dengue disease. Nat Genet 37:507–513
Appendix
MICC-1 Contents I. ANTIBODY SPECIFICITY, EPITOPE AND LECTINOLOGY I-1
Antibody Combining Sites: How Much of the Antibody Repertoire are We Seeing? How does it Influence Our Understanding of the Structural and Genetic Basis of Antibody Complementarity? Elvin A. Kabat
1
I-2
Sialic Acids as Antigenic Determinants of Complex Carbohydrates Roland Schauer
47
I-3
Blood Group Active Haptens in Urine and Faeces Arne Lundblad and M. Alan Chester
73
I-4
The Separation of Immunocyte Subpopulations by Use of Various Lectins Toshiaki Osawa
83
I-5
Mannose-binding Proteins of Animal Origin Y.C. Lee
105
I-6
Sialic Acid and N-Acetylgalactosamine Specific Bacterial Lectins of Enterotoxigenic Escherichia coli (ETEC) Mats Lindahl, R. Brossmer, and T. Wadström
123
I-7
Interaction of Viruses, Bacteria and Bacterial Toxins with Host Cell Surface Glycolipids. Aspects on Receptor Identification and Dissection of Binding Epitopes Klaus Bock, K.-A. Karlsson, N. Strömberg, and S. Teneberg
153
I-8
Membrane Glycoproteins and Plant and Animal Proteins with Lectin or Lectin-Like Properties Z.W. Shen
187
A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6, © Springer Science+Business Media, LLC 2011
777
778 I-9
Appendix
Differential Binding Properties of GalNAc and/or Gal Specific Lectins Albert M. Wu and Shunji Sugii
205
II. COMPLEX CARBOHYDRATES AS ANTIGENS
II-1
Antigenic Properties of Human Erythrocyte Glycophorins Elwira Lisowska
265
II-2
Carbohydrate Structures as ONCO-Developmental Antigens and Components of Receptor Systems Ten Feizi
317
II-3
Chemistry of Human Erythrocyte Polylactosamine Glycopeptides (Erythroglycans) as Related to ABH Blood Group Anitgenic Determinants Roger A. Laine and Jeffrey S. Rush
331
II-4
Blood Group Antigens and the Enzymes Involved in their Synthesis: Past and Present (Abstract) Winifred M. Watkins
349
II-5
Structural Concepts of the Human Blood Group A, Lea, Leb, I, and i Active Glycoproteins Purified from Human Ovarian Cyst Fluid Albert M. Wu
351
II-6
Biochemistry and Lectin Binding Properties of Mammalian Salivary Mucous Glycoproteins Anthony Herp, Carol Borelli, and Albert M. Wu
395
II-7
New Trends in Ganglioside Chemistry Sandro Sonnino, R. Ghidoni, G. Gazzotti, D. Acquotti, and G. Tettamanti
437
II-8
Structural Aspects of Blood Group Glycosphingolipids in the Gastrointestinal Tract Gunnar C. Hansson
465
II-9
Chemically Modified Capsular Polysaccharides as Vaccines H.J. Jennings
495
II-10
Structural and Immunochemical Aspects of Brucella Abortus Endotoxins Albert M. Wu, Neil E. Mackenzie, L. Garry Adams, and Roberta Pugh
551
II-11
Chemical and Immunochemical Studies on Lipopoly-saccharides of Coxiella Burnetii Phase I and Phase II Hubert Mayer, J. Radziejewska-Lebrecht, and S. Schramek
577
Appendix
779
II-12
Structural Comparisons of Streptococcus pneumonaiae Specific Polysaccharides of Group 9 (9N, 9V, 9L, 9A) Related to the Choice of Vaccine Components (Abstract) James C. Richards and M.B. Perry
593
II-13
Elucidation and Comparison of the Chemical Structures of the Specific Capsular Polysaccharides of Streptococcus pneumoniae Groups 11 (11F, 11B, 11C, 11A) (Abstract) James C. Richards, M.B. Perry, and M. Moreau
595
II-14
Application of Two Dimensional NMR Methods to the Structural Elucidation of Complex Polysaccharide Antigens. The Structure of the Capsular Polysaccharide of Streptococcus pneumoniae Type 22F (Abstract) James C. Richards and M.B. Perry
597
II-15
Structural and Immunochemical Investigations on Snail Galactans (Abstract) Hägen Bretting, G. Jacobs, U. Knels, J. Thiem, and W. Konig
599
III. GLYCOCONJUGATES AS CANCER/OR TUMOR ANTIGENS III-1
Tumor-Associated Blood Group Antigen Expressions and Immunoglobulins Associated with Tumors Byron Anderson, L.E. Davis, and M. Venegas
601
III-2
The Chemical Basis for Expression of the Sialyl-Lea Antigen David Zopf and Gunnar C. Hansson
657
III-3
Glycoconjugates and Tumor Metastasis T. Irimura, M. Nakajima, T. Yamori, D.M. Ota, K.F. Cleary, and G.L. Nicolson
677
III-4
Glycolipids and Glycoproteins in Plasma Membrane of Hepatocellular Cancer Tian-Jue Gu
705
III-5
Cancer-Associated Carbohydrate Antigens in Mucins (Abstract) John L. Magnani
723
III-6
N-Glycolylneuraminic Acid-Containing Gangliosides as a Tumor Associated Antigen in Human: Expression of HunganutziuDeicher Antigen Active Gangliosides on Human Colon Carcinoma and Melanoma Tissues (Abstract) Yoshio Hirabayashi, M. Matsumoto, Hideyoshi Higashi, and Shiro Kato
725
780
Appendix
IV. STRUCTURAL ANALYSIS OF HAPTEN MOIETY OF COMPLEX CARBOHYDRATE ANTIGENS IV-1
Structural Elucidation of Complex Carbohydrates Vernon N. Reinhold and S. Santikarn
727
IV-2
Analysis of Complex Carbohydrate Primary and Secondary Structure Via Two-Dimensional Proton Nuclear Magnetic Resonance Spectroscopy Theodore A.W. Koerner, Robert K. Yu, J. Neel Scarsdale, Peter C. Demou, and James H. Prestegard
759
IV-3
Structural Studies on the Carbohydrate Chains of Glycoproteins (Abstract) J.F.G. Vliegenthart
785
IV-4
The Use of Enzymes for Structural Determination of Complex Carbohydrates Y.-T. Li and S-C. Li
787
IV-5
Mass Spectrometry of Hexosamine Containing Oligosaccharides as Permethylated N-Trifluoroacetyl Derivatives Bo Nilsson
803
IV-6
Immunochemical Analysis of Lipopolysaccharides with 2-D Gel Electrophoresis and Monoclonal Antibodies (Abstract) Blair A. Sowa, R.P. Crawford, F.C. Heck, J.D. Williams, A.M. Wu, K.A. Kelly, and L.G. Adams
815
APPENDIX
A-I
A Guide for Carbohydrate Specificities of Lectins Albert M. Wu, Shunji Sugii, and Anthony Herp
819
CONTRIBUTORS
849
INDEX
855
Appendix
781
MICC-2 Contents I. ANTIBODY SPECIFICITY, EPITOPE AND LECTINOLOGY I-1
Antibody Combining Sites: How Much of the Antibody Repertoire are We Seeing? How does it Influence Our Understanding of the Structural and Genetic Basis of Antibody Complementarity? Elvin A. Kabat
1
I-2
Sialic Acids as Antigenic Determinants of Complex Carbohydrates Roland Schauer
47
I-3
Blood Group Active Haptens in Urine and Faeces Arne Lundblad and M. Alan Chester
73
I-4
The Separation of Immunocyte Subpopulations by Use of Various Lectins Toshiaki Osawa
83
I-5
Mannose-binding Proteins of Animal Origin Y.C. Lee
105
I-6
Sialic Acid and N-Acetylgalactosamine Specific Bacterial Lectins of Enterotoxigenic Escherichia coli (ETEC) Mats Lindahl, R. Brossmer, and T. Wadström
123
I-7
Interaction of Viruses, Bacteria and Bacterial Toxins with Host Cell Surface Glycolipids. Aspects on Receptor Identification and Dissection of Binding Epitopes Klaus Bock, K.-A. Karlsson, N. Strömberg, and S. Teneberg
153
I-8
Membrane Glycoproteins and Plant and Animal Proteins with Lectin or Lectin-like Properties Z.W. Shen
187
I-9
Differential Binding Properties of GalNAc and/or Gal Specific Lectins Albert M. Wu and Shunji Sugii
205
II. COMPLEX CARBOHYDRATES AS ANTIGENS
II-1
Antigenic Properties of Human Erythrocyte Glycophorins Elwira Lisowska
265
II-2
Carbohydrate Structures as ONCO-Developmental Antigens and Components of Receptor Systems Ten Feizi
317
782
Appendix
II-3
Chemistry of Human Erythrocyte Polylactosamine Glycopeptides (Erythroglycans) as Related to ABH Blood Group Anitgenic Determinants Roger A. Laine and Jeffrey S. Rush
331
II-4
Blood Group Antigens and the Enzymes Involved in their Synthesis: Past and Present (Abstract) Winifred M. Watkins
349
II-5
Structural Concepts of the Human Blood Group A, Lea, Leb, I, and i Active Glycoproteins Purified from Human Ovarian Cyst Fluid Albert M. Wu
351
II-6
Biochemistry and Lectin Binding Properties of Mammalian Salivary Mucous Glycoproteins Anthony Herp, Carol Borelli, and Albert M. Wu
395
II-7
New Trends in Ganglioside Chemistry Sandro Sonnino, R. Ghidoni, G. Gazzotti, D. Acquotti, and G. Tettamanti
437
II-8
Structural Aspects of Blood Group Glycosphingolipids in the Gastrointestinal Tract Gunnar C. Hansson
465
II-9
Chemically Modified Capsular Polysaccharides as Vaccines H.J. Jennings
495
II-10
Structural and Immunochemical Aspects of Brucella Abortus Endotoxins Albert M. Wu, Neil E. Mackenzie, L. Garry Adams, and Roberta Pugh
551
II-11
Chemical and Immunochemical Studies on Lipopoly-saccharides of Coxiella Burnetii Phase I and Phase II Hubert Mayer, J. Radziejewska-Lebrecht, and S. Schramek
577
II-12
Structural Comparisions of Streptococcus pneumonaiae Specific Polysaccharides of Group 9 (9N, 9V, 9L, 9A) Related to the Choice of Vaccine Components (Abstract) James C. Richards and M.B. Perry
593
II-13
Elucidation and Comparison of the Chemical Structures of the Specific Capsular Polysaccharides of Streptococcus pneumoniae Groups 11 (11F, 11B, 11C, 11A) (Abstract) James C. Richards, M.B. Perry, and M. Moreau
595
Appendix
783
II-14
Application of Two Dimensional NMR Methods to the Structural Elucidation of Complex Polysaccharide Antigens. The Structure of the Capsular Polysaccharide of Streptococcus pneumoniae Type 22F (Abstract) James C. Richards and M.B. Perry
597
II-15
Structural and Immunochemical Investigations on Snail Galactans (Abstract) Hägen Bretting, G. Jacobs, U. Knels, J. Thiem, and W. Konig
599
III. GLYCOCONJUGATES AS CANCER/OR TUMOR ANTIGENS III-1
Tumor-Associated Blood Group Antigen Expressions and Immunoglobulins Associated with Tumors Byron Anderson, L.E. Davis, and M. Venegas
601
III-2
The Chemical Basis for Expression of the Sialyl-Lea Antigen David Zopf and Gunnar C. Hansson
657
III-3
Glycoconjugates and Tumor Metastasis T. Irimura, M. Nakajima, T. Yamori, D.M. Ota, K.F. Cleary, and G.L. Nicolson
677
III-4
Glycolipids and Glycoproteins in Plasma Membrane of Hepatocellular Cancer Tian-Jue Gu
705
III-5
Cancer-Associated Carbohydrate Antigens in Mucins (Abstract) John L. Magnani
723
III-6
N-Glycolylneuraminic Acid-Containing Gangliosides as a Tumor Associated Antigen in Human: Expression of Hunganutziu-Deicher Antigen Active Gangliosides on Human Colon Carcinoma and Melanoma Tissues (Abstract) Yoshio Hirabayashi, M. Matsumoto, Hideyoshi Higashi, and Shiro Kato
725
IV. STRUCTURAL ANALYSIS OF HAPTEN MOIETY OF COMPLEX CARBOHYDRATE ANTIGENS IV-1
Structural Elucidation of Complex Carbohydrates Vernon N. Reinhold and S. Santikarn
727
784
Appendix
IV-2
Analysis of Complex Carbohydrate Primary and Secondary Structure Via Two-Dimensional Proton Nuclear Magnetic Resonance Spectroscopy Theodore A.W. Koerner, Robert K. Yu, J. Neel Scarsdale, Peter C. Demou, and James H. Prestegard
759
IV-3
Structural Studies on the Carbohydrate Chains of Glycoproteins (Abstract) J.F.G. Vliegenthart
785
IV-4
The Use of Enzymes for Structural Determination of Complex Carbohydrates Y.-T. Li and S-C. Li
787
IV-5
Mass Spectrometry of Hexosamine Containing Oligosaccharides as Permethylated N-Trifluoroacetyl Derivatives Bo Nilsson
803
IV-6
Immunochemical Analysis of Lipopolysaccharides with 2-D Gel Electrophoresis and Monoclonal Antibodies (Abstract) Blair A. Sowa, R.P. Crawford, F.C. Heck, J.D. Williams, A.M. Wu, K.A. Kelly, and L.G. Adams
815
APPENDIX A-I
A Guide for Carbohydrate Specificities of Lectins
819
Albert M. Wu, Shunji Sugii, and Anthony Herp CONTRIBUTORS
849
INDEX
855
Appendix
Adv Exp Med Biol (1988); 228 (MICC-1)
785
786
Adv Exp Med Biol (2001); 491 (MICC-2)
Appendix
Index
A Aberrant glycosylation caveolar and noncaveolar domains, cell surface caveolin–1, 666 ceramide, 668 characterization, 664 description, 663 domains, 666 GN11 and GT1, 665 growth factor receptor signaling and, 665–666 integrin signaling, 667 lipid raft/lipid-rich membrane domain, 667 “lipid rafts”, 664 low-density membrane fraction, 665 Neu3, 667–668 palmitoylation, 666–667 plasma membrane structures, 663–664 proteins and growth factor receptors, 663 sphingolipid-enriched membrane domains, 664–665 Src-family kinases, 667 UDP-Gal-4-epimerase-deficient ldlD14 cell line, 668 caveolin–1, tumor progression (see Caveolin–1) description, 653 glycosphingolipid (GSL) expression, 654–655 metabolic mechanisms, 653–654 tumor cell phenotype caveolar membrane fraction, 661 CD9 and tetraspanins, 658 glycosynapse, 659 GM2 and GM3, 658 integrin signaling, 660
microdomains, 660–661 noninvasive cell line (KK47) and YTS1, 659–660 tumor-host interactions, GSLs extracellular matrix, 655 GD3, GD2 and GM3 gangliosides, 656–657 glycosynapse concept, 658 GM3 ganglioside, 657 GSL-GSL interaction, 657 motility and metastatic potential, 657–658 sphingolipids, 655–656 b-Actin, 428–429 Adhesion/growth-regulatory galectins CG–1A vs. CG–1B binding properties, 136 CRDs, 137 immunohistochemical profile, 134 vs. natural glycoproteins reactivity, 135 reactivity, 135 relative potency, oligosaccharides, 135–136 sequences indication, 134 transcription factors, 129–133 natural glycoproteins (gps) chicken galectins, 125, 128 codes and structural units, 123 b-galactoside-binding, 123–124 inhibition assays, 124 lectin binding, 122 rat galectin–5 to glycotopes, 124 prototype CGs galectin–glycan binding assays, 128 gene expression, 128 HADDOCK, 128
787
788 Adhesion/growth-regulatory galectins (cont.) rGal–4N vs. rGal–5 binding properties, 127 plant lectin (ricin) reactivity, 125 relative potency, 126 sugar code potent translators, 118–122 principles, 117–118 AFM. See Atomic force microscopy AFs. See Aggregation factors Aggregation factors (AFs) MAFs (see Microciona prolifera) sponge, 504–506 Allograft tolerance, 595, 606 Alzheimer’s disease (AD) presenilin 1and presenilin 2 gene, 430–431 P-tau and PHF-tau, 432 b-and g-secretases, 431 tau kinase, 431 b-Amyloid peptide amino-acid sequence, 430 APP, 431 secretion, 431 b-site and g-site, 430–431 Angiogenesis capillary endothelial cell line, 462 factor VIIIc expression, 469 microenvironment and extracellular signaling, 463 Anti-a-galactosyl antibodies, human serum anti-Gala1–3Gal galactose residues, 583–584 homologous genomic sequence, 582 immunoglobulin G (IgG) binding, 582 immunotherapy, cancer, 584 specificity, 583 xenotransplantation, 583 anti-NOR antibodies (see Anti-NOR antibodies) Gala1–3Gal–Sepharose, 589 immunoglobulins binding, 588 oligosaccharides, 588, 589 Anti-Gala1–3Gal antibodies cryptic glycotopes, 584 galactose residues, 583–584 homologous genomic sequence, 582 immunotherapy, cancer, 584 specificity, 583 xenotransplantation, 583 Antiganglioside antibodies C. jejuni, 360–361 cross-reactive, production, 364 GM1 and GM3, 365 NMJ, 362, 368
Index production, 365 voltage-gated Na channel, NSC–34 cells, 366 Anti-NOR antibodies blood group A and B antigens, 586 erythrocytes, 585 Gala1–4Gal reactivity, 586 glycolipids, 585 immunoglobulins binding, 587 polyagglutination, 584–585 reactivity, 588 specificity, 586 Apoptosis ceramide-induced, 633 deregulation, 633 galectins (see Galectins) GD3 (see GD3) inducers, 633 inhibition, cancer cells, 634 inhibitor of apoptosis (IAP), 634 signaling pathways, anticancer agents, 632 Apoptosis-inducing factor (AIF), 323 Atomic force microscopy (AFM), 517 Avian egg whites, bacterial lectins and ConA interactions binding, 193–194 decreasing orders, chicken, 193 immature progeny, 192 interactions, comparison, 194 RSL inhibition, 193 Avian flu, 703 Avian influenza virus host range, 451–452 intermediary hosts, 455 receptor sialo-sugar chains, humans fatality rate, 456 mutations, 457 Neu5Aca2–3Gal, 456–457 B Bacterial lectins adhesins, 156 animal cells, interaction human blood platelets, 174–175 human peripheral leukocyte, 175 human spermatozoa, 174 in vitro and in vivo tissues, 178–180 malignant tumor cells, 175–178 RBCs, 173–174 avian egg white glycans, Con A binding, 193–194 decreasing orders, chicken, 193 immature progeny, 192
Index interactions, comparison, 194 RSL inhibition, 193 beehive honey and RJ bovine and human milk, 198 comparative Western blotting, 196 ConA inhibition, 197 floral nectars, 198 hemagglutinating activities inhibition, 195, 196 parental brood care, 194 production, honey, 195–196 RSL and RS-IIL, 197 therapeutic purposes, 196 UEA-I, 197 binding-site amino acids, 160 biochemistry laboratory, Bar-Ilan University, 156, 157 blocking, human body fluid SRMDGs fucophilic and mannophilic bacterial lectins, 186–187 galactophilic interactions, 185–186 hemagglutination inhibition, 185 immune cells and components, 184 probes, 185 secretors’ and nonsecretors’ body fluids, differentiation, 188 blocking, mammalian milk SRMDGs inhibition comparison, 188–189 interactions, ConA, 189–192 crystallographic X-ray studies, 159 fucose/mannose-binding, 249 gene data, 239 gene sequence, 158 inhibition, 240 lectinology, 249 monosaccharide specificities, 159 PA-IL and PA-IIL, 158 PA interaction Archaea, 171–172 autologous bacterial cells, 170 heterologous bacteria, 171 unicellular organisms, 172 PA lectin-inhibiting glycans, anti-adhesion treatment coupling, hydrophobic component, 169 flow cytometry, 170 in vitro models, 169–170 pathogenic interactions, diverse glycoconjugates anti-adhesion strategy, 160 CV-IIL, 168–169 PA-IIL, 163–165 PA-IL, 160–162
789 plant and microbial polysaccharides, 162–163 RS-IIL, 168 RSL, 166–168 purified, probes, 239 soil bacteria, 156 specificity, human RBC antigens blood group typing, 180 fucose-and mannose-binding, 184 low-temperature-favored PA-IL interaction, 183 PA-IL interactions, 181–183 system antigens, blood group, 181 synthetic glycodendrimers, 240 virulence factors (VIFs), 158 Beehive products ConA inhibition, 198 SRMDGs, 201 therapeutic purposes, 196 Bioengineering, PSA. See Polysialic acid bioengineering Blood group A and B antigenicity, human RBC, 91–92 endo-b-galactosyl linkage, 89 glycotopes, 89 Blood groups ABH antigens and bacterial lectins, 184 antigenic activity, 192, 194 epitopes, gps and polysaccharides, 162 H blood group epitopes, 188 PA-IIL interaction, 165 PA-IL interactions, 161 system antigens and detection, 181 typing, 180 Body fluids PA-IL interactions, 185 plant lectin inhibition, 186 secretors’ and nonsecretors’, differentiation, 188 SRMDGs and bacterial lectin blocking glycans, 185 human milk carbohydrates, 184–185 infection-blocking service, newborns, 185 probes, 184 Bone marrow (BM), 326, 327 Branching enzymes, mucin O-glycan. See Mucin O-glycan Breast carcinomas BCRP localization, 633 cells, culturing and synchronization, 639, 640 GLT assays, 645 incubation, 638 morphological cell changes, cisplatin, 639
790 C Campylobacter jejuni AMAN patients, 357 ganglioside-mimicking, 364 and GBS development, 364 LOSs, 360, 361, 366 molecular mimicry, 362 poultry, 365 serotypes, LOSs, 361 Cancer KDN biological significance, human, 684–685 biosynthesis, mammalian cells, 681–682 expression, human tissues, 682 free, abnormal conditions, 683 glycan-bound and free, 684 serum mannose, mammalian organs, 683–684 PSA bioengineering (see Polysialic acid bioengineering) Cancer cells agglutination and adsorption tests, 201 human ovarian IGROV–1, 178 PA lectins, 176 Cancer metastasis acidic antigens, 67 disialyl function, 68 E-/P-and L-selectins, 67–68 H7721 cells, 66 homing receptor, 68 malignant cells, 68 malignant transformation, 67 sialyl appearance, 67 SLex, 66 specimens, 65 surface expression, 65–66 variety, 65 Capillary endothelial cell angiogenesis in vitro, 462 8Br-cAMP effect, 469 factor VIIIc-like protein expression immunoprecipitation, 464 perinuclear localization, 463–464 GRP–78 expression, 463 insulin, 466 invasion, factor VIIIc role antifactor, monoclonal antibody, 470 MMPs, 469–470 N-glycosylation, 467 Carbohydrate binding amaranthin and surface analysis, 151 Gleheda, 148 lectin–carbohydrate interactions, 258, 267 Morniga G, 149 VVLB4, 146, 147
Index Carbohydrate-carbohydrate interactions AFM, 517 epitope and variants, 510–512 molecular level, 509–510 SPR spectroscopy, 513–514 TEM, 514–517 UV spectroscopy, 512–513 Carbohydrate recognition domains (CRDs) CG–1A (C–16) and CG–1B (C–14), 137 galectins, 439, 440 Cathepsin D, aging concanavalin A (ConA) staining, 429 1D-PAGE, 429 rat brain analysis, 429–430 Caveolin–1 caveolae and, 665–666 GN11 and GT1 cells, 665 human melanoma cells, overexpression, 668 MDCK, 666 oligomeric, membrane domain characterization, 664 tumor progression description, 661 human breast and colon carcinoma, 662 mammary carcinoma, 662 ovarian carcinoma, 661–662 phosphorylation, Tyr14, 663 sphingolipid-enriched domain, 663 Src kinases, cell-cell adhesion, 662–663 at Tyr14, 667 CD44 cancer metastasis biological processes, 616 molecular action types, 615–616 SDS-PAGE, 615 upregulation, 616–617 selectin-mediated adhesion isoforms, 623–624 sialylated glycoproteins, 621–622 Cell motility B11 cells, 346 GM3 ST3GAL5 gene, 345, 346 transfectants, 346 Transwell experiments, 345, 346 GM6001, inhibition, 352 MMP–9, B16 cell motility, 351 C2GnT estrous cycle, 490 expression regulation C2GnT-L and C2GnT-M, 490–491 conditions, 491 IL–12, 491 upregulation and downregulation, 491 genomic organization
Index bovine structure, 483 C2GnT-T gene characterization, 484 chromosomal localization, human b6GlcNAc, 483 human IGnT, 484 open reading frame (ORF), 481, 483 six exons, mouse and human, 483 structures and human expression, 482 transcripts, 483–484 immune function activity and polylactosamine, 485–486 CD43, CD44 and CD45, 485 cell function, 484–485 mucin core–2 structure, isozymes, 486–487 PSGL–1 dimerization, 486 T-cell maturation, 486 mucin glycan branch structure, human cancer colorectal, 490 core 3, 489–490 function, 489 Childhood acute lymphoblastic leukemia (ALL) malignant transformation, 333 sialic acids 9-O-acetylation role, 325 Chitin oligomers chitin-binding receptors, LYM molecules, 527–528 docking, LysM-RLKs chitin tetrasaccharide, 525, 527 Medicago truncatula, 526 O6-linked sulfate group, 527 Chlamydia trachomatis, 22–24 Choline (Ch), 241–242 Chromobacterium violaceum (CV) CV-IIL agglutination, 173 Fuc affinity, 159–160 inhibition, human lactoferrin, 189 interactions, diverse glycans, 168–169 sensitivity, saliva, 186 discovery, 159 Cisplatin apoptosis, 647 caspase–3 activation, 644 MDA–468 cells, 644 morphological changes, apoptotic breast cancer cells, 639 Classification, lectins, 279 Clostridium perfringens preparation commercial sialidase, 82 genomic DNA, 86–87 purification, nEndo-ABase, 86 CMP. See Cytidine monophosphate
791 Colon carcinoma cells CD44v, selectin-mediated adhesion isoforms, 623–624 sialofucosylated O-linked carbohydrate structures, 621–622 LS174T binding adhesion model, 619 immobilized platelets, 618 protease sensitivity and O-glycandependent binding activity, 620 sialylated molecule CD24, 619–620 GPIb/IX complex, 619 PSGL–1, 619 CRDs. See Carbohydrate recognition domains Cutaneous lymphocyte-associated antigen (CLA), 561 Cytidine monophosphate (CMP) Neu5Ac regeneration, 725 sialic acid synthesis, 724 synthetase, 726 sugar nucleotide donors, 723 D DEAE. See Diethylaminoethyl Deaminated neuraminic acid Neu5Gc, 679–681, 683, 685 nonulosonate skeletal structures, 679–680 Dengue virus (DV) Asn–153, 780 CLEC5A–DV interaction, 783 dendritic cells, 782 DHF and dengue shock syndrome (DSS), 780, 784 E protein, 780, 781 infections, 783–784 recognition, 781 replication, human, 781 serotype–2 (DV2), 780 treatment, 784 Diethylaminoethyl (DEAE), 18, 84 Dimethyl (methylthio) sulfonium trifluoromethanesulfonate (DMTST) promoter, 709–710 S-methyl sialyl donor coupling, 719 Disaccharide Galb1–2Gal, 120–121 DMTST. See Dimethyl (methylthio) sulfonium trifluoromethanesulfonate DNA microarray agents, 638 GLTs RNA levels, 645 transcriptional regulation, 649
792 E ELIA. See Enzyme-linked lectin assay ELLSA. See Enzyme-linked lectin sorbent assay Encephalitis virus, 779, 780 Endo-ABase gene application agglutination modification, human RBCs, 90 antigen expression, 91–92 immunohistochemical analysis, 92 indirect immunofluorescence method, 90–91 BLASTN, FASTA and BLAST searches, 92 cloning Clostridium perfringens genomic DNA, 86 genomic library construction, 87 purification, nEndo-ABase, 86, 88 rEndo-ABase purification, 87–88 SDS-PAGE, nEndo-ABase and rEndo-ABase, 85, 88 XL1-Blue MRF and SOLR cells, 87 properties, 88 purification centrifugations, 83 ConA sepharose chromatography, 84 crude enzyme preparation, 84 fractogel DEAE and SP chromatography, 84 mono Q chromatography, 84–86 sephacryl S–200 gel filtration, 84 substrate specificity A-Penta vs. B-Penta, hydrolysis rate, 89, 90 Galili pentasaccharide, 89–90 TLC analysis, 88–89 Endo-b-galactosidase classes, 82 cyanogen bromide, 86 Endo-ABase gene application, 90–92 cloning, 86–88 vs. other proteins, 92 properties, 88 purification, 83–86 substrate specificity, 88–90 enzyme assay, 83 glycoside hydrolase family, 93 observation, 82–83 Enzyme-linked lectin assay (ELIA) biotinylated lectins, binding, 259 jacalin binding, 260
Index microtiter plate, 259 monosaccharides, 261 VVA, 260, 261 Enzyme-linked lectin sorbent assay (ELLSA), 107, 108 Erythrocytes, 9-O-AcGD3 caspase–3 activation antiapoptotic role and tumor progression, 333 apoptosis, 332 membrane alterations and PCD, anti–9-O-AcGD3 mAb, 332 hydrophobicity, 328–330 membrane osmotic fragility, 327–328 morphological changes, 330 PS externalization annexin V, 331 anti–9-O-AcGD3 mAb death induction, 331 senescence process, 330–331 Erythropoiesis glycophorin A, 326 mean fluorescence intensity (MFI), 327, 328 9-O-Acetyl GD3 status, 326, 327 F Fabrication and application glyco-AuNPs analytic scheme, NBAMS, 748 bacterial b–1,4-GalT activity, 749 carbohydrate interactions, 740–744 cell aggregation, 747 colloid AuNPs, 738–739 E. coli, 746 importance, 739–740 metal NPs, 738 protein interactions, 744–746 pseudomonas aeruginosa-I lectin probe, 746–747 glyco-CNTs amylose and behaviour, 755–756 amylose schematic representation, 756 b-Gal-SW-CNT preparation, 756 glucosamine SW-CNT, 755 helix formation, polysaccharides, 756 macrocyclic oligosaccharides, 755 multivalent carbohydrate ligands, 757–760 polysaccharides use, helix formation, 757 scope, 754 surface modification, 754–755 glyco-MNPs, 752–753 glyco-QDs, 749–752
Index Factor VIIIc-like glycoprotein, angiogenesis capillary endothelial cell line expression, 463–464 in vitro, 462 capillary invasion MMPs, 469–470 monoclonal antibody effect, 470 endothelial, asparagine-linked, 464–465 expression, 469, 470 microenvironment and extracellular signaling, 463 N-glycosylation regulation, transmembrane signaling, 466–468 physiological function deficiency, 462 description, 461 sequential domains, 461–462 Fertilization cumulus cells, 406 sperm binding, 409 Filament lectin filamin, 218 fractions, 216, 218 purification scheme, 217 separation, 214 Flavivirus characterization, 780 glycosylation, 780–781 infections, 779 particles, 779 treatment, 780 Fluorescein isothiocyanate-labeled Phaseolus vulgaris leucoagglutinin (FITC-L-PHA), 404–405 Fucosyltransferases (Fuc-T) VII gene, 560–561 G a-Galactosyl glycotopes cancer immunotherapy, 584 immunogenicity, 582 Galactosyltransferase (GalT) GLT-GalTs, posttranslational activities GalT–4 and GalT–5, 645–646 GalT5 inhibition, 646 Lewis glyco-epitope synthesis, 59–60 GALAXY database, 26, 27 Galectin–1 in vitro studies cells apoptosis, 442 neurons degeneration, 443 T cells, 442, 443
793 in vivo studies apoptosis-inducing activity, 443 Th1 immune response, 443 Galectins apoptosis regulators, 447 calcium influx and apoptosis induction, 446 cell growth/apoptosis, 445–446 surface receptors, 447 cellular homeostasis, 446 characteristics extracellular functions, 440–441 intracellular functions, 441–442 intracellular proteins, 440 oligovalency and lattice, 440 CRDs, 439 description, 439 galectin–1 (see Galectin–1) galectin–2, 445 galectin–7, 445 galectin–8, 445–446 galectin–9, 445 galectin–3, studies cells, transferred DNA, 444 galectin-3-deficient mice, 444 soluble recombinant, 444 GalNAc residue. See N-Acetylgalactosamine Ganglioside GM1 galectin’s binding site, 118 pentasaccharide bioactive conformers, 120 positioning, contact site, 119 Ganglioside O-acetylated GD3 CD95-and ceramide-mediated apoptosis, 550–551 description, 546 melanoma cells, immune, 547 7-O-acetyl GD3 carriers, 549 structure, 542 9-O-acetyl GD3 carriers, 549 structure, 542 Gangliosides biosynthetic pathway, mono-and disialosyl, 645 cell-surface composition, 307 COS–7 cells, 311 exogenous administration, 302–303 ganglioside–protein complex, cytosol, 301 GM3, 304 in situ sialylation, 308–309 lactonization, 309–310 SAT–4 and SAT–4’, 647
794 Gangliosides and plasma membrane-associated sialidase biological functions, glycosphingolipids cellular/animal models, 301 epidermal growth factor receptor (EGFr), 303–304 fumonisin B1, 305 “lipid rafts”, 302 neuroblastoma cell lines, 303 rat cerebellar neurons, 305 sphingolipid-and cholesterol-enriched membrane domains, 304 synthetic inhibitors, 303 modulation, glycosphingolipid composition ceramide, hydrophobic character, 307 cultured hippocampal neurons, 308 dexamethasone treatment, 309 flip-flop process, 307 glycosylation and deglycosylation pathways, 309–310 b-hexosaminidase A, 308 lactonization/delactonization, 310 metabolic remodeling, 310 molecular heterogeneity, 305 Neu3 activity, 308 Neu3 expression, 307–308 sphingomyelin–ceramide interconversion, 306 sphingomyelin hydrolysis, 306 regulation, glycosphingolipid composition biosynthesis and degradation, 299 degradation, lysosomes, 301 de novo biosynthetic pathway, 300 glucosylceramide, 300 metabolic pathways, 300 GD3 AIF, 323 enforced expression, 323 ganglioside biosynthesis, 322, 323 human tumor lymphoid and myeloid cell lines, 323 9-O-acetylation, 324 9-O-AcGD3, 324 tumor-associated ganglioside, 322 Glycan arrays advantage, 774 carbohydrate-binding activity devoid sugar-binding activity, 773–774 distinct specificity, 773 glycan structures, 770 lectin refined and redefined, 770–773 plant lectins types, 770 sugar-binding domains/motifs, 770 CFG, 768
Index current targets, 769 ELISA-type format, 768 GBPs, 768–769 ideal glycan, 774–775 microarrays, 774 solid-phase assay, 768 Glycans avian egg-white, 192–194 CV-IIL interactions, 168–169 EGF-R, 71 fucosylated, 70 intracellular binding, galectins, 441, 447 PA-IL interactions, 160–162 RS-IIL interactions, 168 RSL interactions, 166–168 sequence diversity, 117 Glycoamidase A, 15, 16 Glyco-CNTs, fabrication amylose and behaviour, 755–756 amylose schematic representation, 756 b-Gal-SW-CNT preparation, 756 glucosamine SW-CNT, 755 helix formation, polysaccharides, 756 macrocyclic oligosaccharides, 755 multivalent carbohydrate ligands C18-a-MM coated to CHO cells, 759 EDTA addition, 758 glycoconjugate polymer-coated SW-CNTs, 760 mannose-and galactose-coated SW-CNTs, 760 molecular recognition process, 757 SW-CNT and GalNAc-SW-CNT, 757–758 synthetic mucin mimic structure, 759 polysaccharides use, helix formation, 757 scope, 754 Glycogerontology Alzheimer’s disease, 419–420 b-amyloid peptide (Ab), 430–431 biosynthesis, sugar chains, 420 cytosolic cathepsin D, aging 1D-PAGE and 2D-PAGE, 429 rat brain, histochemical analysis, 429–430 dementia, 430 glycoconjugates altered sialylation, brain hippocampal synaptosome proteins, 428–429 MAL and SSA, 428 glycoproteins, 420 IgG, sugar chain structures changes bio-gel P–4 column elution profiles, 422 C1q binding, 422 Fc fragment and Fcg receptors, 425
Index galactose residues, 421–422 H and L chains, 420–421 human serum, N-linked sugar chain, 421 hydrazinolysis, 421 N-glycosylation, 420 oligosaccharide, 421 RCA-I and PVL column, 424 rheumatoid factor (RF), 422, 424 sera IgGs, PVL binding, 424–425 U937, 424 N-linked sugar chains alteration, glycoproteins age-related alterations, 426 gp30 structure, 426–427 Po, 427–428 tissues homogenization, 425–426 paired helical filaments (PHFs), 431 b-and g-secretases, 431 ST6Gal1 overexpression, 431 sugar chains, 420 tau, 431–432 Glycolipids (GLs), thermophilic bacteria biological membrane structure, 373 biosynthesis, 649 CpxA-CpxR, 374 GLT assays, 645 HSR bacteria, 373–374 “homeoviscous adaptation”, 374 yeast cells, 373 roles, biological activities, and perspectives a-configuration, 379 fatty acid chain length, 379 fatty acids, 377 glycoglycerolipids, 378, 379 innate and adaptive immune systems, 378 lipid A, 379–380 LPSs, 377–378 membranes, 379 PGL1/PGL2 mixture, 378–379 sturcture, thermodynamics and biosynthesis Acholeplasma laidlawii, 376 carbohydrate part, 377 glucosyltransferases characterization, 376–377 Hex hexose, HexNH hexosamine, R alkyl chain, 375 membrane lipid, 377 microorganisms, pH value, 374 phosphoglycolipids PGL1 and PGL2, R alkyl chain, 376 phospholipids, 377 polar lipids, 375 Thermus and Meiothermus species, 375
795 Glycomic mapping asialo HOC chains, 40 fluid glycoprotein interactions, 41–42 HOC 350, 43 Glyconanomaterials assemble biomolecules, 738 bio-barcode detection, 737 biomolecule-modified quantum dots (QDs), 737 carbon nanotubes (CNTs), 737 glyco-AuNPs carbohydrate interactions, 740–744 colloid AuNPs, 738–739 importance, 739–740 metal NPs, 738 other applications, 744–746 protein interactions, 744–746 glyco-CNTs amylose and behaviour, 755–756 amylose schematic representation, 756 b-Gal-SW-CNT preparation, 756 glucosamine SW-CNT, 755 helix formation, polysaccharides, 756 macrocyclic oligosaccharides, 755 multivalent carbohydrate ligands, 757–760 scope, 754 surface modifications, 754–755 glyco-MNPs and applications, 752–753 glyco-QDs applications, 751–752 fabrication, 749–751 multivalent interaction, 738 particles, 737 transmission electron microscopy (TEM), 738 tumors, 738 Glycoproteins investigation, A. thaliana binding, specificity, 258 delipidated cells, 259 ELIA (see Enzyme-linked lectin assay) lectin affinity chromatography, 262 lectin blotting, 261–262 monosaccharide composition analysis, 262–263 lectin–plant, specificity, 258 and lectins, tobacco seeds and seedlings blotting, 264 sialidase treatment, MAA and SNA-I, 265 VVA–biotin pretreatments, 265–266 serum profiles, tolerance (see Serum glycoprotein profiles, liver allograft transplantation)
796 Glyco-QDs applications, 751–752 fabrication, 749–751 Glycosidase classification system, 93 occurrence, 82 Glycosylation description, 257 glycomics, 258 O-glycosylation, 143 proteins, plants, 258 Glycosyltransferase (GlcT) gene regulation, apoptotic breast cancer cells caspase–3 activation dose-dependent, 644 MDA–468 cells, 643–644 caspase categories, 634 cell cultures morphological change, cisplatin, 639 phosphate-buffered saline (PBS), 638 ceramide-induced cytochrome c release, 633 “death domain”, 632 “extrinsic pathway”, 631 fluorescence staining and examination apoptotic cells, 639–640 image capture and processing, 640 organelle membrane changes, 640–643 glycobiology microarray, L-PPMP, 647–648 glycosphingolipid GLT assays, 644 posttranslational activities, 645–646 inhibitor of apoptosis (IAP), 634 multidrug-resistant (MDR) proteins, 632–633 reagents antibodies, 635 apoptotic agents, 635 cell culture supplies, 635–636 DNA microarray, 638 GDP-[3H]-Fucose, 637 Western blot supplies, 636 signaling pathways, anticancer agents, 632 Glycotopes blood group A and B, 88–89 disaccharide, 82 trisaccharide, 92 Glycotope structures and intramolecular affinity factors Forssman antigen, 143–144 O-glycosylation, 143 submolecular requirements, Tn and T antigen amaranthin, 150 disaccharide, 149 docking, 147, 148, 151
Index GalNAc residue, 148 Gal residue, 149–150 H-bonding, 146, 149 peanut and homodimeric lectin, 150 SER residue, 148–149 Tyr127 structural analyses, 146 VVLB4, 146 Tn and T plant lectins description, 144 family and species, 145 glycotopes, 145 isolation and purification, 144, 146 Tn antigen, conversion, 144 GM3 A431 and MDCK cells, 666 bladder cancer, 657 caveolar membrane fraction, 661 CD9/a3-integrin association, 660 GM3-Gg3 interaction, 657–658 mouse melanoma, 656 GM6001 cell motility inhibition, 352 gelatin zymography, 350 TNF-a, 351 GM3 upregulation cell lines and culture, 343 cell motility B4GALT6 sense cDNA transfectant, 345 transfectants, 346 Transwell experiment, 345, 346 chemicals and antibodies, 343 endogenous, MMP–9 synthesis gelatin zymography, 347–348 RT-PCR, 347 exogenous addition/depletion, 348 ganglioside extraction and HPTLC, 344 gangliosides, 342 gelatin zymography, 344–345 MMP–9, 341 MMP–9 and B16 cell motility, 351 PI3K/AKT pathway B16 cells, 350 MMP–9 expression, 349 PI3K cell lines, 341 RNA extraction and RT-PCR, 343–344 siRNA, 344 TNF-a expression, 342 transwell experiments, 345 Gold glyconanoparticles, 510, 511 Guillain–Barré syndrome (GBS) anti-GSL antibodies antibody titers, 357
Index gangliosides, 357–358 markers, 359–360 putative infectious pathogens and symptoms, 358–359 sensory signs, 360 TLC, 357 TLC immunostaining, 359 ganglioside mimicry and neuromuscular junction antiganglioside antibody, voltage-gated Na channel, 366 anti-lipid antibody, 365 autoimmune attack, 361 autopsy, 363–364 campylobacteriosis, 365 C. jejuni infection, 364 disialogangliosides, 363 functional inhibition, sodium channels, 366 ligand-binding studies, 363 monoclonal IgM antibody, 362 TLC immuno-overlay, LOS, 364 voltage-gated sodium channels (VGSCs), 363 ganglioside molecular mimicry and Campylobacter jejuni LOSs, 362 C. jejuni, 360 hypothesis, 360 muscle weaknesses, 365, 368 serotypes, 361 neuropathies AIDP and CIDP, 356 AMAN and AMSAN, 356–357 subtypes, 356 symptoms, 356 Gynaecin carbohydrate specificity, 221 and filamin, 225, 226 inhibition, 223 interaction fetuin, 224 gynaecin-fetuin, 221, 222 isolation, chromatography, 226 Neu5Ac methyl ester, 222 relative binding affinity, 223 specificity, sialic acid linkage, 224 tentative model, carbohydrate-binding site, 225 Gynoecium gynoecium lectin (GL) isolation, 218 purification, 218 separation, S. indica, 214
797 H HADDOCK. See High ambiguity-driven docking Heat-shock response (HSR), 373 HeLa cells, 24 Hemagglutinin amino acids, 455 function, host cell membranes, 454 H3 hemagglutinin, 454 H5N1 virus, 457 host receptor recognition specificity, 453 Hemagglutinin (HA)-binding trisaccharides antiviral drugs, 702 “bird flu”, 701–702 chemical synthesis SAa2,3Gal b1,4GlcNHAc, 713–721 SAa2,6Gal b1,4GlcNHAc, 703–713 enzymatic synthesis CMP and UDP role, 723 CMP-sialic acid, 724 glycosyltransferases, 722 NmCSS, 726 NMR spectral characterization, 722–723 one-pot, three-enzyme system, 725–726 recombinant fusion protein, 725 UDP-galactose regeneration system, 723–724 glycan microarray analysis amine-functionalized glycans immobilization, 727 human and avian serotypes, 727–728 and receptor binding affinity, 727, 728 technology and construction, 727 H1N1 and H2N2 virus, 703 influenza/flu disease, 701 influenza virus, human and poultry, 702–703 M2 blockers and NAIs, 703 replication cycle, viral RNA, 702 High ambiguity-driven docking (HADDOCK), 128 High-mannose oligosaccharides (M7), 24–26 High-performance liquid chromatography (HPLC), 17 H5N1 infection, human intestinal tract, 457 mutation, 455 Neu5Aca23Gal, 457 tigers, 452 HOC 350 gylomic mapping, 43 internal structure, 48 native and asialo, 47 short type chain, 48
798 Homodimeric human galectin–1, 119 Human blood group ABH/Lewis antigens ABH-active glycoproteins, 35 A and B group, 33 12 blood group identification, 35 definition, 33 Le and Se gene, 36 structure, carbohydrate chains, 34 type 1, 2 and 3 disaccharides, 33 biosynthesis, 35 expression, 35–36 structure, HOC glycoproteins carbohydrate chains, 38–39 cyst core, 39 interactions, 39–40 Human IgG, 21–22 Human ovarian cyst (HOC) glycoproteins A/A-Leb/y and B/B-Leb/y blood group identification HOC 89 and Cyst 19 binding profiles, 46 interaction profile, 44 O-glycan structures, 43, 45 structure carbohydrate chains, 38–39 cyst core, 39 interactions, 39–40 ABH, Sialyl and Lewis role, 38 biosynthesis, 35 core structure, 48 expression, 35–36 glycomic mapping asialo HOC chains, 40 fluid glycoprotein interactions, 41–42 HOC 350 glycomic mapping, 43 internal structure, 48 native and asialo, 47 and short type chain, 48 pseudomucinous-type, 46 I IgG C1q binding, 422, 423 description, 420–421 ELISA, 424 galactose residues, human, 421–422 oligosaccharide, 421 properties, 425 PVL binding, 425 rabbit, Fc portion, 424 rheumatoid factor (RF), 422, 424 U937, human Fc receptors, 424
Index Immunoregulation sialic acid O-acetylation carbohydrate-lectin interactions, 546 carriers, 549 CD60b and-c, 549–550 chicken erythrocytes, 548 function, intestine, 547 gangliosides, 550 GD3, 546–547, 550–551 glycobiology, 546 glycoproteins, 547 human milk, 548 mucin, beneficial effects, 548 native CD34+ hematopoietic precursor cells, CD60 epitopes, 551 natural antibodies, 549 tumor cells, 548–549 Inflammatory cytokines and interferons (IFN), 781 Influenza virus binding trisaccharide, 702–703 “bird flu”, 701–702 H1N1 and H2N2, 703 host range sialo-sugar chains, 451 A viruses, 452 molecular basis, receptor binding specificity selection hemagglutinin glycoprotein, 454 influenza A virus, 454 mutation, pathogenic avian H5N1 virus, 455, 456 receptor binding specificity animal species, 452 molecular basis, selection, 454–456 sialyl lactosamine chains, 453 subtype H9N2, 453–454 receptor sialo-sugar chains amino acids, 457 fatality rate, 456 Neu5Aca2–3Gal, 456–457 replication cycle, 702 types, 701 Innate immunity receptor C-type lectins, 782 protein–carbohydrate interaction, 784 toll-like receptors (TLRs), 782 TREM-like transcripts (TLTs), 782 triggering receptor expressed on myeloid cells (TREMs), 782 J Japanese encephalitis virus (JEV), 779
Index K KDN biological significance, humans free KDN, 684–685 glycan formation, 685 Neu5Ac, 685 biosynthesis mechanism, mammalian cells CMP-KDN, 681 enzyme catalyzing reaction, 681 KDN/free vs. membrane-bound Neu5Ac, 682 Man–6-P, 682 rainbow trout testis reaction, 681 description, 679 expression, cancer cells, 683 glycan-bound and free, 684 human tumor tissues, 682 mammals, 680 properties, Neu5Ac, 679–680 serum mannose, mammalian organs description, 683 oral ingestion, 683–684 L Lectin–glycan interactions characterization factors, 112–113 criteria, biomedical applications, 99–100 functional roles, 99 grouping, monosaccharides and oligosaccharides applied lectins, 100 binding properties, Gal/GalNAc, 105–106 degree of specificity, 100–101 GalNAc-specific lectins, 102 Gal-specific lectins, 104–105 GlcNAc, 105 Glc-specific lectins, 105 LFuc and sialic acid specific lectins, 105 mammalian glycoconjucates structural units, 101, 103–104 oligosaccharide structures, 101–102 polyvalency effect, glycotopes biochemical recognition processes, 107 carbohydrate–LFuc specific lectins, 110, 112 ELLSA method, 107, 108 oligo-antennary N-glycans, 107, 109 recognition factors, Gal/GalNAcspecific lectins, 110, 111 Lectin production regulation, PA and CV beneficial contribution, 230 infection blocking, anti-PA-lectin antibodies
799 lungs, 236 PLP application, 236 natural protection, binding bacterial blocking, 241 bacterial lectin inhibition, 240 glycans, 239 human milk glycans, 240 PA-IL and PA-IIL genes discovery and 3-D crystal structures, 237–239 interactions, diverse cells, 234–235 linkage, VIF production, 232–234 specificity and function, 231–232 QS (see Quorum sensing) stimulation, osmoprotectants, 244 VIFs, 230 wilting symptoms, 231 Lectins. See also Plant lectins A. dichotoma, 220 carbohydrate-binding specificity, 225 elderberry bark, 214 ELIA, 259–261 isolation, S. indica filament, 216–218 gynoecium, 218 seed integument, 215–216 linkage specificity, 220 microorganisms, 225 mushroom, 214 noncarbohydrate-mediated interaction (see Noncarbohydrate-mediated binding) purification, gynoecium, 219 purified, homogeneity, 215 saracin detection, 214 Lens culinaris agglutinin (LCA) glycopeptide fraction, 427 sugar chains, rat P0, 427 Lewis glyco-epitopes (Le) antigens and cell apoptosis, 73–74 antigens detection, 57 biosynthesis epitopes, 56 fucosyltransferase, 60–61 galactosyltransferase, 59–60 monoclonal antibodies use, 57 N-acetylglucosaminyltransferase, 57–59 neutral and sialylated antigens, 62–64 requirement, 56–57 sialyltransferase, 61–62 sulfotransferase role, 64–65 and cancer metastasis, 65–68 carbohydrate antigens, 76 cell adhension, migration and expression, SLex, 74–75
800 Lewis glyco-epitopes (Le) (cont.) embryo implantation and Ley, 69–70 glycobiology research, 75 identification, 53 Lewis B and H. pylori infection, 68–69 localization and antibodies detection, 56 nucleotide sequence, 75 proteomics and glycomics research, 75 SLex cell signaling and growth CDK2 activation, 72–73 EGF-R, 71 expression on cell surface, 70–71 FucT-VII cDNA, 71–72 retinoblastoma protein (Rb), 72 structures ABH(O) blood group, 56 long-chain antigens, 55 sugar chains, 54 sulfated antigens, 55 type 1 a/b series and type 2 x/y series, 53–54 uncommon Lewis antigens, 55 sugar determinant, 53 Lipooligosaccharides (LOSs), Neisseria species analysis (SDS-PAGE) N. meningitidis and N. gonorrhoeae, 388 patterns, 388, 389 profiles, N. canis and N. mucosa, 389, 390 glycosyltransferase gene studies distribution, 392, 393 genetic characteristics, N. polysaccharea strains, 392, 394 lgtB and lgtE, 391, 392 N. meningitidis and lgtH mutant characterization, 394, 395 oligosaccharide biosynthesis, 391–392 PCR method, 392 human, 387 meningococcal disease, 387–388 polysaccharea, 388 profiles, polysaccharea strains heterogeneity and molecular size, 390–391 SDS-PAGE analysis, 389–390 serological cross-reactivities antimeningococcal antisera, 395–396 4.1-kDa and 4.0-kDa species, 396 N. meningitidis vs. N. polysaccharea, 397 N. polysacchareae and rabbit antiserum, 396
Index Lipopolysaccharides (LPSs) C. jejuni, 362 molecular-mimicry hypothesis, 360 LOS glycosyltransferase (lgt) genes described, 391–392 distribution, Neisseria strains, 392, 393 L-threo–1-phenyl–2-palmitoylamino–3morpholino–1-propanol (l-PPMP) apoptotic activity, 647 GlcT inhibition, 648 GSL biosynthesis, 633 MDA cells caspase–3 activation, 644 cisplatintreatment, MDA–468 cells, 644 GalT–5 activity, 646 PSS–380 and AKS–0 staining, 641 SKBR–3 cells, PSS–380 and AKS–0 staining, 641 Lymphoblasts ALL, 325, 327 Neu5,9Ac2-GPs functional implication, 333 Lymphocyte homing central helper memory T cells molecules masking and unmasking, 563–565 selectin-mediated, human, 563 selectin ligand expression Fuc-T VII, 566 GATA–3, 566–567 GlcNAc6ST–1 and HEC-GlcNAc6ST, 567–568 skin, T cells expressing modified sialic acid peripheral blood, 566 sialyl 6-sulfo Lewis x, 565–566 Th2 cells, 568 T lymphocytes, sulfated ligands, 561–562 Lymphocytes human tonsillar B, 551 T and B, CD60b and-c, 549 LysM-receptor kinases, LysM-RLK LysM domains, 524–525 M. truncatula, 523 NFP vs. LYK3, 523–524 M Maackia amurensis lectin (MAL), 428 Marine sponges. See Microciona prolifera Matrix metalloproteinase–9 (MMP9) B11 cells, 347 exogenous GM3 addition, 348 expression, regulation, 348 gangliosides, 342, 343 stimulation, 341 synthesis, endogenous GM3, 347–348
Index Membrane fluidity cells, 374 STRE, 373 Membrane hydrophobicity, 328–330 Membrane osmotic fragility cell death, 328 mature erythrocytes sensitization, 9-O-AcGD3 mAb, 329 pathological conditions, 327 Memory T lymphocytes Fuc-T VII gene transcription, 560–561 homing, selectin sulfated ligands CLA, 561–562 sialyl 6-sulfo Lewis x, 561 masking and unmasking, 563–565 selectin-mediated homing, 563 skin-homing modified sialic acid, 565–566 selectin ligand expression, 566–568 Metastasis biochemical characterization, P-selectin LS174T binding, 618–619 protease sensitivity and O-glycandependent binding activity, 620 sialylated molecule, 619–620 CD44v, selectin-mediated adhesion isoforms, 623–624 sialofucosylated O-linked carbohydrate structures, 621–622 cell adhesion molecules CD44, 615–617 P-selectin ligands, 613–615 selectins, 612–613 P-selectin blood-borne, 617 platelet-tumor cell adhesive interactions, 618 sialylated fucosylated glycans, 617 Microbial typing agglutination and adsorption tests, 201 archaea, 172 human blood group, 180 PA lectins, 171 Microciona prolifera aggregation factors (MAFs) AFM imaging, 505–506 carbohydrate-carbohydrate interactions (see Carbohydrate-carbohydrate interactions) cell recognition and adhesion, 504–505 characterization, 505 description, 504 glycans, 508–509 N-glycans, 506–508
801 supramolecular structure, 505 total mass, 505 cell adhesion, 503–504 description, 503 ECM, 503 Milk saccharides, 164 Molecular docking, LysM-RLKs chitin tetrasaccharide, 525, 527 Medicago truncatula, 526 O6-linked sulfate group, 527 Molecular mimicry ganglioside antiganglioside complex-positive (+) groups, 361 and Campylobacter jejuni LOSs, 362 C. jejuni, 360 hypothesis, 360 muscle weaknesses, 365, 368 serotypes, 361 pathogenesis, 367 Mucin O-glycan biosynthesis, b6GlcNAc transferases categories, acceptor, 475 core 4, 475 core 1-and core 2, 476 structures, 474–475 carbohydrate, 473 C2GnT gene, genomic organization bovine structure, 483 C2GnT-T gene characterization, 484 chromosomal localization, human b6GlcNAc, 483 human IGnT, 484 open reading frame (ORF), 481, 483 six exons, mouse and human, 483 structures and human expression, 482 transcripts, 483–484 description, 473 enzyme activity, core structure, 474 functions, 473 human C2GnTs role cancer, 487–490 estrous cycle, 490 expression regulation, 490–491 immune function, 484–487 loss, muc2-gene-knockout mice, 473–474 membrane-tethered mucins, 474 protein structures, b6GlcNAc transferases amino acid sequences, 476–479 binding, amino acids, 481 cDNAs cloning, 476 C–6 hydroxyl group activation, 481 GT-A-and GT-B-fold, 480–481 mC2GnT-L and bC2GnT-M, 479–480 N-glycosylation site, 479
802 Multiple-oocyte follicles (MOFs), 408–409 Multivalent interaction glyco-AuNP, 739, 744 glyco-NPs, 738 multivalent carbohydrate ligand, 757–759 N N-Acetylgalactosamine (GalNAc) docking, 148 H-bonds, 151 N-acetyl group, 146 pyranose ring, interaction, 146, 148 b6N-acetylglucosa minyltransferase mucin O-glycan biosynthesis (see Mucin O-glycan) protein structures, 476–481 NAIs. See Neuraminidase inhibitors Naïve B lymphocytes CD22/siglec–2, 564–565 extravascular tissue infiltration, 574–576 siglecs, sialylated and sulfated ligands CD22/siglec–2, 570–571 ITIM domains, 571, 573 a2→6 sialylated 6-sulfo-LacNAc glycans, 572–574 Nanoparticles (NPs) gold, 738 magnetic, 737 Neisseria polysaccharea children, 388 vs. N. meningitidis, 388 serological cross-reactivities, 395–397 strains genetic characteristics, 392, 394 LOS profiles, 389–391 PCR method, 392 Neoglycoconjugates, 170 Neuraminidase inhibitors (NAIs), 702, 703 N-glycans high-mannose, 771–773 and O-glycans oogenesis, fertilization and embryonic development, 413 sperm binding, 410 sperm receptors, 409–410 SSEA, 411–412 oogenesis female fertility, 406–407 germ cell, 406 Mgat1-null eggs, 407 zona glycoproteins, 406 reactivity, 771 residues, 773
Index targeting complex and hybrid Cre-loxP recombination technology, 405–406 folliculogenesis and ovulation, 405 GlcNAcT-I, 403–404 Mgat1-null embryos, 404–405 N-iodosuccinimide (NIS) sialylation, 707 and TfOH promoter system, 712, 715 thioglycoside coupling, 709 N-linked oligosaccharide comprehensive analysis, 15 development methods glycoamidase A, 16 glyco tree diagram, 20 sulfooligosaccharides, 21 three-dimensional mapping, 18 trisialyl and triantennary N-glycans, 20–21 two-dimensional mapping, 17 unit contribution concept, 19 2-D mapping technique, 16 structures a2,3 and a2,6 sialic acids, 25–27 chlamydia trachomatis, 22–24 M7, 25 N-glycans from human IgG, 21–22 squid rhodopsin, 25 N-linked sugar chain glycoproteins, aging brain gp30 structure, 426–427 P0, 427–428 spinal cord, 426 tissues homogenization, 425–426 IgG, 420–421 structures and molar ratio, 432 NMR spectroscopy. See Nuclear magnetic resonance spectroscopy Nodulation (Nod) factors chitin-binding receptors, LYM molecules, 527–528 docking, LysM-RLKs chitin tetrasaccharide, 525, 527 Medicago truncatula, 526 O6-linked sulfate group, 527 electrostatic potentials, 529 fatty acid chains recognition, 528 homology modeling, 528–529 LYK3 and NFP, 521–522 LysM-RLK receptors LysM domains, 524–525 M. truncatula, 523 NFP vs. LYK3, 523–524 membrane-associated proteins, 522
Index as signal molecules NodSm-IV, 522 S. meliloti mutants, 522–523 silico docking, 528 structure, 522 Noncarbohydrate-mediated binding glycoproteins (see Glycoproteins) glycosylation, 257 lectin–carbohydrate interactions, 258 lectins and glycoproteins, tobacco seeds and seedlings blotting, 264 sialidase treatment, 265 VVA–biotin, pretreatments, 265–266 rice prolamin, jacalin endo-a-N-acetylgalactosaminidase, 267 SDS-PAGE analyses, 266 Nonmammalian glycoconjugates, 258 NOR glycosphingolipids, 585 Nuclear magnetic resonance (NMR) spectroscopy, 118–120 Nucleocytoplasmic protein animal cells, 286 CBP70, 286–287 comitin, 287 families, 274 O 9-O-acetylated sialoglycoproteins ALL, peripheral blood mononuclear cells, 325 lymphocytes and erythrocytes, 333 9-O-Acetyl GD3, lymphoid and erythroid cells anti–9-O-AcGD3, erythrocyte membrane hydrophobicity (see Membrane hydrophobicity) morphological changes, 330 osmotic fragility (see Membrane osmotic fragility) childhood ALL, 333 death signals, 334 erythrocytes (see Erythrocytes, 9-O-AcGD3) erythroid cells, 325 erythroid progenitor populations identification MNC, 325 status, erythroid maturation, 325, 326 erythropoiesis (see Erythropoiesis) ganglioside GD3 structure, 322 GD3 and 9-O-Acetyl GD3, apoptosis (see GD3) O-acetylation, 321
803 PCD caspase–3 activation, 332–333 PS externalization, 331 sialic acids, 321, 333 O-glycans. See also N-glycans core 1-derived mucin target E12 and E14, 408 GalNAc, 407 maternal and zygotic C1galt-/-embryos, 408 MOFs, 408–409 fucose targeting notch pathway genes, 412–413 pofut1-/-embryos, 412 receptor, notch, 412 O-glycosylation, 143 OLT. See Orthotopic liver transplantation Orthotopic liver transplantation (OLT) 2-DE analysis, animal models, 603 ether anesthesia, 596 Hp localization, tolerogenic rat model, 602 liver, haptoglobin expression, 601 serum, 599 tolerance, 595–596, 606 P P0
APP sugar chains, 431 description, 427 homophilic adhesion, cell line, 427–428 PA. See Pseudomonas aeruginosa Pandemic influenza, 456, 703 Penicillin dissolve bacteria, 6 heparin structure, 7 notatum, 6 penicillin and lysozyme actions, 7 Physiological role carbohydrate-binding activity, 273 inducible plant lectins carbohydrate-binding protein, 283 nucleocytoplasmic transport, 284 plant defense, 284–285 receptor glycans, 283–284 Plant lecting specificity, 767–775 Plant lectins biomedical applications carbohydrate-binding properties, 286 Morniga M, 285–286 Nictaba, 285 proteins, purification, 286
804 Plant lectins (cont.) classical and inducible amaranthin-like sequences, 282–283 cytoplasmic, EUL domain, 281–282 cytoplasmic GNA, 280–281 jacalin-related, 274–276 Nictaba domain, 276–280 definition, 271 physiological role, inducible carbohydrate-binding protein, 283 nucleocytoplasmic transport, 284 plant defense, 284–285 receptor glycans, 283–284 T antigen structural requirements amaranthin, benzyl-T antigen, 150 disaccharide, 149 Gal residue, 149–150 H-bonds, 151 peanut and homodimeric lectin, 150 Tyr127 structural analyses, 146 Tn and T specific affinity, glycotopes, 145 description, 144 family and species, 145 isolation and purification, 144, 146 Tn antigen binding structural requirements docking, 147, 148 GalNAc residue, 148 H-bonding, 146, 149 Tyr127 analyses, 146 VVLB4, 146 Plant polysaccharides branched galactans, 162 galactomannans, 162–163 guar and locust gum, 163 Plant protein. See Noncarbohydrate-mediated binding Plasma membrane modulation, glycosphingolipid composition, 305–310 regulation biosynthesis and degradation, 299 degradation, lysosomes, 301 de novo biosynthetic pathway, 300 glucosylceramide, 300 metabolic pathways, 300 trans and cis functional interactions, 302 Polar lipids, 374, 375 Polyagglutination Cad, 581 NOR, 585 Polymerase chain reaction (PCR) method, 392
Index Polysialic acid (PSA) bioengineering, cancer and neuronal cells description, 689–690 expression, RMA-s, 690 GD3 structure, 691 mAb 735, 695 ManBu precursor treatment, 694–695 ManPr, 692 N-acyl mannosamine precursors, 691 N-butanoyl mannosamine (ManBu), 692 N-butanoyl polysialic acid (NBuPSA), 693 neuAc-containing inhibitor, 693–694 NPrPSA, 693 RMA-s, ManBu precursor treatment, 693–694 Pregnant mare’s serum gonadotrophin (PMSG), 406–407 Programmed cell death (PCD), 332 Pro-Q Emerald 488 glycoprotein gel stain, 596 glycosylation detection, Pro-Q Emerald, 488 glycoproteins, 602 immune responses, 601 Proteomics functional, 595–596 haptoglobin (Hp), 601 Psathyrella velutina lectin (PVL), 214, 424–425 P-selectin. See also Metastasis biochemical characterization, colon carcinoma cells LS174T binding, 618–619 protease sensitivity and O-glycandependent binding activity, 620 sialylated molecule, 619–620 ligands CD24, 614 heparan sulfate glycosaminoglycans, 615 platelet glycoprotein Ib/IX/V complex, 614 PSGL–1, 613–614 Pseudomonas aeruginosa (PA). See also Bacterial lectins; Lectin production regulation, PA and CV infection blocking, anti-PA-lectin antibodies lungs, 236 PLP application, 236 lectin discovery beneficial contribution, 230 C. violaceum infections, 231 PA-IIL discovery, 158 encoding gene, 158
Index PA-IL and PA-IIL interactions, diverse cells, 234–235 linkage, VIF production, 232–234 specificity and function, 231–232 Q Quorum sensing (QS) and choline-dependent regulation, PA lectin production factors, PA-IL discovery, 241 LuxI box-like 20-bp elements, 242 RpoS consensus sequences, 242 transcriptional regulating signal circuits, 241 VIFs, 242 molecular information, 234 R Ralstonia solanacearum lectin discovery beneficial contribution, 230 virulence factors (VIFs), 230 wilting symptoms, 231 levels, 249–250 LuxI box-like 20-bp elements, 242 PA-IIL, 249 RSL, RS-IIL revelation alanine–alanine–asparagine triad, 239 amino acid homology, 237 3-D structure, 238 sugar specificities, 238 stimulation, RSL, RS-IIL lectin production, 243 trehalose-induced, 245 Ralstonia solanacearum (RS) CV genomic ORFs, 168 human RBC antigens, 184 RS-IIL, 168 RSL discovery, 159 interaction, diverse glycans, 166–168 low activity, cow cells, 174 Se and sese milk, 188 Receptor binding specificity, influenza viruses host cell membrane receptors, 453 influenza A viruses, 452 molecular basis, selection, 454–456 subtype H9N2, 453–454 sialo-sugar chains, pathogenic avian influenza viruses amino acids, 457
805 fatality rate, 456 Neu5Aca2–3Gal, 456–457 Red blood cells (RBCs), 33 Rheumatoid arthritis (RA) IgG samples, 424 sera IgGs, PVL binding, 425 S SAa2,3Gal b1,4GlcNHAc trisaccharide synthesis path A lactosamine derivative coupling, 717–718 lactosamine derived triol preparation, 714, 715 protection, lactosamine acceptor, 715–716, 717 sialylation, 717–718 sialyldibenzyl phosphates, 714, 715 thioglycoside coupling, 715, 716 path B DMTST, 719 glycosyl trichloroacetimidate use, 720–721 N-diacetyl neuraminyl coupling, 718–719 pentasaccharide unit, 721 S-methyl SAa,3Gal building block, 719 pathways, 713–714 SAa2,6Gal b1,4GlcNHAc trisaccharide synthesis anomeric isomers, 704 coupling reaction, 703–704 one-pot glycosylation sialyl donor and thiogalactoside, 712, 713 terminal acceptors, 712 path A Ag2CO3 promoter, 707 anomeric thio groups, 707 carcinoembryonic antigen preparation, 707, 708 isopropylidenation, 706 lactosamine preparation, 707–709 Paulsen and Tiez work, 705 path B binding specificity, HAs, 710–711 NIS/TfOH promoter system, 712 pentasaccharide unit, 709, 711 retrosynthesis, pathways, 704–705 Saccharin production, 5 toluene–2-sulfonamide oxidation, 4 Sambucus sieboldiana agglutinin (SSA), 428
806 Saraca indica detection, lectin saracin, 214 lectin isolation filament, 216–218 gynoecium, 218 seed integument, 215–216 seed integuments purification, 216 scraping, 214 SDS-PAGE. See Sodium dodecyl sulfate polyacrylamide gel electrophoresis Seed integument, lectin hemagglutination activity, 215 purification, 215–216 saracin, 226 Selectin binding activity, sialyl 6-sulfo Lewis x, 565 description, 612 homing, human central helper memory T cells, 563 L-selectin and E-selectin, 612 P-selectin (see P-selectin) and siglecs, biological significance, 568–570 skin-homing T cells, 566–568 sulfated ligands sialylated and B lymphocytes, 570–574 T lymphocyte homing, 561–562 Serendipity cluster effect, 11 common sugars, 11 description, 3 d-sugars, 13 GlcNAc, 8–9 heparin structure, 5–6 hepatic carbohydrate receptor, 7 ManNAc and GlcNAc, 10 Morgan–Elson reaction, 10 neoglycoproteins preparation, 10–11 NeuAc oxidation, 8 penicillin, 6–7 saccharin, 4 sialic acid and NANA, 8 sialic acid configuration, 9 Simhaladvipa, 3 Splenda, 5 sucralose structure, 5 sugar series configuration, 12 toluene–2-sulfonamide oxidation, 5 Serological cross-reactivities, 395–397 Serum glycoprotein profiles, liver allograft transplantation allogeneic liver grafts histology 2-DE profiles, OLT treatment model, 599 rejection, 598, 599
Index animals, 596 carbohydrate-rich plasma proteins, liver, 603 ConA-enriched serum proteins, 602–603, 605 2-DE protein profiles, POD–14 and POD–60 peptide mass fingerprint (PMF), 601 protein spots, 600 serum proteins, 599 gel staining and protein detection, 597 glycosylation detection, Pro-Q Emerald 488 glycoproteins, 602 immune responses, 601 haptoglobin (Hp) expression, immunohistochemistry, 601 regulation, 604 a1-microglobulin protein, 604 orthotopic liver transplantation, 596 plasma protein sample preparation, ConA-enriched, 597–598 POD 60, 606 sample preparations, 596–597 tryptic in-gel digestions, 598 two-dimensional electrophoresis, 597 Shear flow endothelial cells, 614 free-flowing colon carcinoma cells, 618 P-selectin, 620 Sialate-O-acetylesterases (SOATs) bovine submandibular gland, 538 eukaryotic, 540 O-acetyl migration, 541 9-O-acetyltransferase activity, 538–539 sialate–4-O-acetyltransferase, 539–540 Sialic acid chemical degradation, 9 cleavage, 8 configuration, 9 description, 689 enzyme permissiveness, 690 exocyclic chain, 7 NPrPSA control tumor migration, antibodies, 691 precursor therapy, 690–691 PSA bioengineering (see Polysialic acid bioengineering) pyruvic acid and N-acetyl-glucosamine, 8 specificity, lectins carbohydrates, 218–225 elderberry bark lectins, 214 immobilized form, 213 isolation, S. indica, 215–218 materials and methods, 214–215 WGA, 213–214 splitting, 8
Index Sialic acid O-acetylation analysis and occurrence histochemistry, 537 microorganisms and deuterostome lineage, 537–538 bacteria, 545 biological roles, mammalian cells antioxidative effect, 545 ligand function, 544 biosynthesis, 535–536 definition, 535 degradation catabolism, enzymes, 541 mammalian esterases, 541–542 Sia-degrading esterases, 541, 542 sialidases, 543–544 immunological implications carbohydrate-lectin interactions, 546 carriers, 549 CD60b and-c, 549–550 chicken erythrocytes, 548 function, intestine, 547 gangliosides, 550 GD3, 546–547, 550–551 glycobiology, 546 glycoproteins, 547 human milk, 548 mucin, beneficial effects, 548 native CD34+ hematopoietic precursor cells, CD60 epitopes, 551 natural antibodies, 549 tumor cells, 548–549 metabolism, SOATs eukaryotic, 540 O-acetyl migration, 541 9-O-acetyltransferase activity, 538–539 sialate–4-O-acetyltransferase, 539–540 Sialidase Clostridium perfringens, 303 colon and renal cancer, 310 plasma membrane-associated, 307, 308 Sialylation glycoconjugates, aging brain 2D-PAGE, 428–429 MAL and SSA, 428 a2,6-sialylation, APP, 431 Sialyl-fucosyl-neolactotetraosyl ceramide (SA-LeX), 637 Sialyl Lewis X (SLex) cell adhension, migration and expression, 74–75 cell signaling and growth CDK2 activation, 72–73 EGF-R, 71
807 expression on cell surface, 70–71 FucT-VII cDNA, 71–72 retinoblastoma protein (Rb), 72 Sialyltransferase (SAT) posttranslational activities, apoptotic cells, 645–646 SAT–4’ inhibition, 646 Siglec and selectins, biological significance, 568–570 sialylated and sulfated ligands B lymphocytes, 570–574 malignant B cells, 574–576 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) homogeneity, purified lectins, 215 lectin blotting, 261–262 LOS/LPS analysis lgtH mutant size, 394, 395 Neisseria polysaccharea strains, 389–391 Neisseria species, 388–389 nEndo-ABase and rEndo-ABase, 85, 87, 88 peptide separation, 86 SP. See Sulfopropyl Specificity amaranthins, 282 carbohydrate-binding, 281 glycan arrays, 279 ligand, 280 mannose, 274, 280 Orysata, 283 physiological role, plant lectins, 273 Splenda, 5 Squid rhodopsin, 25 Stage-specific embryonic antigens (SSEA) Lewis X and Y antigen, 411 LeY, 411–412 Stress-response promoter element (STRE), 373 Sucralose. See Splenda Sugar code potent translators disaccharide Galb1–2Gal, 121 galectins, 121–122 ganglioside GM1 bioactive conformers, 120, 121 pentasaccharide chain, 119 prototypes sequences, 122 ribbon structure, 118–119 therapeutic agents, 121 principles glycans coding capacity, 117 glycosidic linkages, 118 histo-blood group epitope, 118 sequence diversity, 117–118
808 Sulfated disaccharide probe, MAF AFM, 517 epitope and variants BSA conjugate, 511 gold glyconanoparticles (GNPs), 511–512 sodium salt, 510 molecular level, 509–510 SPR spectroscopy polyvalent multilayer formation, 1a-BSA, 514 protein-protein and protein-carbohydrate interaction, 513–514 TEM Au–2 and Au–3, 517 GNPs, 514–515, 517 UV spectroscopy 1a-BSA, Glc6S-BSA and BSA, 513 BSA, 511, 512 Sulfooligosaccharides, 21 Sulfopropyl (SP), 84 Surface plasmon resonance polyvalent multilayer formation, 1a-BSA, 514 protein-protein and protein-carbohydrate interaction, 513–514 T Targeting C-type lectin CLEC5A, DV infections treatment, 784 cytokine secretion, 785 flavivirus, 779–780 and flavivirus infection CLEC5A role, 783 inflammatory reactions, 783 innate receptor production, 782 myeloid cells, 781 receptors, 781–782 replication, 781 type II transmembrane, 783 glycosylation and flavivirus virulence, 780–781 host–pathogen interactions, 784 severity of DV infections, 783–784 Tau cathepsin D, 430 N-linked sugar chains, 432 PHF-tau, 431–432 T cells galectin–1, 442 galectin–2, 445 galectin–3, 444 TfOH. See Trifluoromethanesulfonic acid
Index T glycotope, 145 Therapeutic saccharides antibodies, 168 beehive products, 196 Thin-layer chromatography (TLC) analysis, A+-PGM and B+-HOCG hydrolysis, 88–89 silica gel-coated plate, 83 Tick-borne encephalitis virus Murray Valley, 779 Russian spring-summer, 779 St. Louis, 779 TLC. See Thin-layer chromatography Tn glycotope plant lectins affinity, 145 recognition, 151 Traffic and homing, T and B lymphocytes biological significance, selectins and siglecs nonsulfated sialyl Lewis x, 568–570 protective effect, skin homing., 571 sialyl 6-sulfo Lewis x glycan, 570 B lymphocytes, sialylated and sulfated ligands a2→6-sialylated 6-sulfo-LacNAc expression, 572–574 CD22/siglec–2, 570–571 ITIM domains, 571–573 masking and unmasking CD22/siglec–2, 564 dual homing processes, 565 glycan-binding activity, 563 two-way traffic, 564–565 selectin-mediated, 563, 564 sialylated and sulfated ligands, malignant B cells anti-CD22 antibodies, 576 a2→6-sialylated 6-sulfo-LacNAc glycan, 574, 575 CD22/siglec–2, 574, 576 sialyl Lewis x, T-helper–1 cell, 560–561 skin, sialic acid moiety modification, 565–566 peripheral blood, 566 sialyl 6-sulfo Lewis x, 566 sulfated ligands CLA, 561 CLA-positive lymphocytes, 562 sialyl 6-sulfo Lewis x, 561, 562 Th2 cells, bronchial asthma and atopic dermatitis, 568 Transmission electron microscopy (TEM), sulfated disaccharide probe Au–2 and Au–3, 517 GNPs, 514–515, 517
Index Trehalase activity, PA, 244 trehalose hydrolysis, 245 Trehalose decomposition, 244 hydrolysis, trehalase, 245 Trifluoromethanesulfonic acid (TfOH) promoter system, 712 sialylation, 707 Trisialyl and triantennary N-glycans, 20–21 Tumor cell motility B16 melanoma cells, 657–658 breast, c-Src and FAK, 661 CD9/a3-integrin, 660 GSLs, 657 laminin–5-dependent, ldlD14 cells, 659 YTS1 bladder, 661 Tumor necrosis factor-alpha (TNF a) B16 cells incubation, 350 GM3, 342 MMP–9 synthesis, 342 mRNA values, 344 rapamycin, 342
809 U UDP. See Uridine diphosphate Uridine diphosphate (UDP) galactose 4’-epimerase (UDPGE), 722 epimerase fusion protein, 725 regeneration system, 723–724 sugar nucleotide donors, 723 V Vicia villosa B4(VVLB4), Tn antigen carbohydrate binding site, 147 Tn-binding, 146 WBAI, 146 Virulence factors (VIFs), 158, 230, 545 W West Nile virus (WNV), 779 Wheat germ agglutinin (WGA), 213–214 Y Yellow fever virus (YFV), 779
