Peptidases and Neuropeptide Processing (Methods in Neurosciences)

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Methods in Neurosciences Volume 23

Peptidases and Neuropeptide Processing

Methods in Neurosciences Editor-in-Chief

P. Michael Conn

Methods in Neurosciences Volume 23

Peptidases and Neuropeptide Processing

Edited by A. Ian Smith Peptide Biology Laboratory Baker Medical Research Institute Prahran, Victoria Australia

ACADEMIC PRESS San Diego New York

Boston

London

Sydney Tokyo Toronto

This book is printed on acid-free paper.

Copyright 9 1995 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Academic Press, Inc. A Division of Harcourt Brace & Company 525 B Street, Suite 1900, San Diego, California 92101-4495

United Kingdom Edition published by Academic Press Limited 24-28 Oval Road, London NW1 7DX

International Standard Serial Number: 1043-9471 International Standard Book Number: 0-12-185293-8

PRINTED IN THE UNrIED STATES OF AMERICA 95 96 97 98 99 00 EB 9 8 7 6

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4

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Table of Contents

Contributors to Volume 23 Preface Volumes in Series

ix ~ 1 7 6 1 7 6

Xlll XV

Section I Molecular Approaches for the Study of Intracellular Processing Enzymes 1. Molecular Strategies for Identifying Processing Enzymes Nabil G. Seidah 2. In Situ Hybridization Techniques to Map Processing

Enzymes 16

Martin K.-H. Schiifer and Robert Day

3. Analysis of Ontogeny of Processing Enzyme Gene Expression and Regulation 45

Min Zheng and John E. Pintar

4. Use of Vaccinia Virus Vectors to Study Neuropeptide Processing 65

Judy K. VanSlyke, Laurel Thomas, and Gary Thomas

5. Overexpression of Neuropeptide Precursors and Processing Enzymes 94

Iris Lindberg and Yi Zhou

6. Use of Antisense RNA to Block Peptide-Processing Enzyme Expression Richard E. Mains

109

Section II Immunological and Biochemical Approaches to the Study of Peptide-Processing Pathways 7. Combination of High-Performance Liquid Chromatography and Radioimmunoassay for Characterization of Peptide-Processing Pathways A. Ian Smith and Rebecca A. Lew

125

8. Development and Use of Two-Site Immunometric Assays for Examining Peptide-Processing Pathways Steven R. Crosby

140

vi

TABLE OF CONTENTS 9. Methods for Identification of Neuropeptide-Processing Pathways Paul Cohen, Mohamed Rholam, and Hamadi Boussetta

155

10. Immunological and Related Techniques for Studying Neurohypophyseal Peptide-Processing Pathways Harold Gainer, Mark O. Lively, and Mariana Morris

195

11. Approaches to Assessing Ontogeny of Processing Enzymes Richard G. Allen and Julianne Stack

208

12. Measurement, Distribution, and Subcellular Localization of Peptide-Amidating Activity Rebecca A. Lew and A. Ian Smith

219

13. Methods for Studying Carboxypeptidase E Lloyd D. Fricker

237

14. Characterization of Endothelin-Converting Enzymes Terry J. Opgenorth, Sadao Kimura, and Jinshyun R. Wu-Wong

251

15. In Vivo Approaches for Studying Peptide Processing Arthur Shulkes

266

Section III Identification and Characterization of Extracellular Processing Enzymes in the Central Nervous System 16. Identification and Characterization of Central Nervous System Peptidase Activities John R. McDermont and Alison M. Gibson

281

17. Strategies for Characterizing, Cloning, and Expressing Soluble Endopeptidases Marc J. Glucksman and James L. Roberts

296

18. Proteolytic Processing and Amyloid Protein Precursor of Alzheimer's Disease D. H. Small, G. Reed, S. J. Fuller, A. Weidemann, K. Beyreuther, and C. L. Masters

317

19. Strategies for Measurement of Angiotensin and Bradykinin Peptides and Their Metabolites in Central Nervous System and Other Tissues Duncan J. Campbell, Anne C. Lawrence, Athena Kladis, and Ann-Maree Duncan

328

20. Distribution and Roles of Endopeptidase 24.11 Anthony J. Turner and Kay Barnes

344

TABLE OF CONTENTS

vii

21. Identification and Distribution of Endopeptidase 24.16 in the Central Nervous System F. Checler, P. Dauch, H. Barelli, V. Dive, Y. Masuo, B. Vincent, and J. P. Vincent

363

22. Autoradiographic Techniques to Map Angiotensin-Converting Enzyme in Brain and Other Tissues Siew Yeen Chai and Frederick A. O. Mendelsohn

Index

383 399

This Page Intentionally Left Blank

Contributors to Volume 23

Article numbers are in parentheses following the names of contributors. Affiliations listed are current.

RICHARD G. ALLEN (11), Center of Research on Occupational and Environmental Toxicology, Oregon Health Sciences University, Portland, Oregon 97201 H. BARELLI (21), Institut de Pharmacologie Mol6culaire et Cellulaire, Centre National de la Recherche Scientifique, 06560 Valbonne, France KaY BARNES (20), Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom K. BEYREtJTHEI~ (18), Center for Molecular Biology, University of Heidelberg, D-6900 Heidelberg, Germany HaMaoi BOUSSETTA (9), Biochimie des Signaux R6gulateurs Cellulaires et Mol6culaires, Universit6 Pierre et Marie Curie, F-75006 Paris, France DUNCAN J. CAMVBEU~ (19), St. Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia SIEW YEEN CI-IAI (22), Department of Medicine, University of Melbourne, Austin Hospital, Melbourne, Victoria 3048, Australia F. CI-IECLErt(21), Institut de Pharmacologie Mol6culaire et Cellulaire, Centre National de la Recherche Scientifique, 06560 Valbonne, France PAUL COHEN (9), Biochimie des Signaux R6gulateurs Cellulaires et Mol6culaires, Universit6 Pierre et Marie Curie, F-75006 Paris, France Sa'wVEr~ R. CROSBY (8), School of Biomolecular Sciences, Liverpool John Moores University, Liverpool L3 3AF, United Kingdom P. Dauci-i (21), Institut de Pharmacologie Mol6culaire et Cellulaire, Centre National de la Recherche Scientifique, 06560 Valbonne, France ROBERT DAY (2), Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, Quebec, Canada H2W 1R7 V. D I w (21), CEN de Saclay, 91191 Gif s/s Yvette, France ANN-MArtEE DUNCAr~ (19), St. Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia

ix

CONTRIBUTORS TO VOLUME 23

LLOYD D. FRICKER (13), Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461 S. J. FULLER (18), Department of Pathology, University of Melbourne, Parkville, Victoria 3052, Australia HAROLD GAINER (10), Laboratory of Neurochemistry, National Institute of Neurological Disorders and Strokes, National Institutes of Health, Bethesda, Maryland 20892 ALISON M. GIBSON (16), Medical Research Council, Neurochemical Pathology Unit, Newcastle General Hospital, Newcastle Upon Tyne NE4 6BE, United Kingdom MARC J. GLUCKSMAN (17), Fishberg Research Center in Neurobiology, Mount Sinai School of Medicine, New York, New York 10029 SADAO KIMURA (14), Center for Biomedical Science, School of Medicine, Chiba University, Chiba 260, Japan ATHENA KLADIS (19), St. Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia ANNE C. LAWRENCE (19), Department of Biology, Medawar Building, University College London, London WC1E 6BT, United Kingdom REBECCA A. LEW (7, 12), Peptide Biology Laboratory, Baker Medical Research Institute, Prahran, Victoria 3181, Australia IRIS LINDBERG (5), Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans, Louisiana 70112 MARK O. LIVELY (10), Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27157 RICHARD E. MAINS (6), Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 C. L. MASTERS (18), Department of Pathology, University of Melbourne, Parkville, Victoria 3052, Australia Y. MASUO (21), Takeda Chemical Industries, Ltd., Pharmaceutical Group, Tsukuda 300-42, Japan JOHN R. MCDERMONT (16), Medical Research Council, Neurochemical Pathology Unit, Newcastle General Hospital, Newcastle Upon Tyne NE4 6BE, United Kingdom


xi

FREDERICK A. O. MENDELSOHN (22), Department of Medicine, University of Melbourne, Austin Hospital, Melbourne, Victoria 3048, Australia MARIANA MORRIS (10), Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27157 TERRY J. OPGENORTH (14), Aging and Degenerate Disease Research, Abbott

Laboratories, Abbott Park, Illinois 60064 JOHN E. PINTAR (3), Department of Neuroscience and Cell Biology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854 G. REED (18), Department of Pathology, University of Melbourne, Parkville, Victoria 3052, Australia MOHAMED RHOLAM (9), Biochimie des Signaux R6gulateurs Cellulaires et Mol6culaires, Universit6 Pierre et Marie Curie, F-75006 Paris, France JAMES L. ROBERTS (17), Fishberg Research Center in Neurobiology, Mount Sinai School of Medicine, New York, New York 10029 MARTIN K.-H. SCH,g,FER (2), Department of Anatomy and Cell Biology, Phillips University of Marburg, D-35037 Marburg, Germany NABIL G. SEIDAH (1), Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, Quebec, Canada H2W 1R7 ARTHUR SHULKES (15), Department of Surgery, University of Melbourne, Melbourne, Victoria 3084, Australia D. H. SMALL (18), Department of Pathology, University of Melbourne, Parkville, Victoria 3052, Australia A. IAN SMITH (7, 12), Peptide Biology Laboratory, Baker Medical Research Institute, Prahran, Victoria 318 l, Australia JULIANNE STACK (11), The Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201 GARY THOMAS (4), Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201 LAUREL THOMAS (4), Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201

~176

Xll


ANTHONY J. TURNER (20), Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom JUDY K. VANSLYKE (4), Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201 B. VINCENT (21), Institut de Pharmacologie Mol6culaire et Cellulaire, Centre National de la Recherche Scientifique, 06560 Valbonne, France J. P. VINCENT (21), Institut de Pharmacologie Mol6culaire et Cellulaire, Centre National de la Recherche Scientifique, 06560 Valbonne, France A. WEIDEMANN (18), Center for Molecular Biology, University of Heidelberg, D-6900 Heidelberg, Germany JINSHYUN R. Wu-WON6 (14), Aging and Degenerative Diseases Research, Abbott Laboratories, Abbott Park, Illinois 60064 MIN ZHENG (3), Department of Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York, New York 10032 YI ZHOU (5), Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans, Louisiana 70112

Preface

The generation of bioactive peptides from inactive precursor molecules involves a series of highly ordered, enzyme-mediated processing events. The posttranslational modifications can occur within the cell at the point of secretion or postsecretion. The precise pattern of processing for any given precursor also can vary, depending on the site of expression and/or stage of development, reflecting the differential expression of processing enzymes. The last decade or so has seen the characterization of both peptide products and the majority of the processing enzymes involved in their production, thus facilitating the development of various biochemical, immunological, and molecular probes necessary to characterize these complex pathways in greater detail. The aim of this volume in the Methods in Neurosciences series is to describe in a very practical way the "state-of-the-art" technology being developed and applied in the field of peptidases and neuropeptide processing. It is divided into three sections. The first, "Molecular Approaches for the Study of Intracellular Processing Enzymes," covers strategies for the molecular characterization of processing enzymes, including cloning, expression, localization by in situ hybridization, and the use of antisense mRNA to block enzyme expression. The second, "Immunological and Biochemical Approaches to the Study of Peptide-Processing Pathways," describes the combination of more classical approaches such as immunoassays, HPLC, and the use of specifically modified substrates to characterize both the precise pattern of peptide products in a given tissue and the regulation and distribution of the enzymes involved in their generation. Finally, the last section, "Identification and Characterization of Extracellular Processing Enzymes in the Central Nervous System," is designed to provide an insight into, as well as strategies for, the investigation of this exciting and developing area in which extracellular enzymes can generate, modulate, or terminate peptide signals in the central nervous system. In this book, like others in the series, the authors have been encouraged to provide chapters that reflect the latest techniques being developed in their laboratories, with their own specific scientific interests providing the practical application. Each chapter provides sufficient detail to allow the experimental procedures to be easily duplicated, although, for practical reasons, lengthy operating procedures for common laboratory equipment have been omitted. Absolute conditions for any given experiment are inevitably determined empirically; however, it is hoped that this volume will provide both

xiii

xiv

PREFACE

the student and experienced researcher a valuable starting point in developing strategies for the study of peptidases and neuropeptide processing. I would like to express my appreciation to the Baker Medical Research Institute for supporting the production of this work. Appreciation is also expressed to my fellow authors for the high standard of their contributions and for meeting their deadlines. A. IAN SMITH

Methods in Neurosciences

Volume 1 Gene Probes Edited by P. Michael Conn Volume 2 Cell Culture Edited by P. Michael Conn Volume 3 Quantitative and Qualitative Microscopy Edited by P. Michael Conn Volume 4 Electrophysiology and Microinjection Edited by P. Michael Conn Volume 5 Neuropeptide Technology: Gene Expression and Neuropeptide Receptors Edited by P. Michael Conn Volume 6 Neuropeptide Technology: Synthesis, Assay, Purification, and Processing Edited by P. Michael Conn Volume 7 Lesions and Transplantation Edited by P. Michael Conn Volume 8 Neurotoxins Edited by P. Michael Conn Volume 9 Gene Expression in Neural Tissues Edited by P. Michael Conn Volume 10 Computers and Computations in the Neurosciences Edited by P. Michael Conn Volume 11 Receptors: Model Systems and Specific Receptors Edited by P. Michael Conn Volume 12 Receptors: Molecular Biology, Receptor Subclasses, Localization, and Ligand Design Edited by P. Michael Conn Volume 13 Neuropeptide Analogs, Conjugates, and Fragments Edited by P. Michael Conn Volume 14 Paradigms for the Study of Behavior Edited by P. Michael Conn Volume 15 Photoreceptor Cells Edited by Paul A. Hargrave Volume 16 Neurobiology of Cytokines (Part A) Edited by Errol B. De Souza Volume 17 Neurobiology of Cytokines (Part B) Edited by Errol B. De Souza Volume 18 Lipid Metabolism in Signaling Systems Edited by John N. Fain Volume 19 Ion Channels of Excitable Membranes Edited by Toshio Narahashi

XV

xvi

VOLUMES IN SERIES

Volume 20 Pulsatility in Neuroendocrine Systems Edited by Jon E. Levine Volume 21 Providing Pharmacological Access to the Brain: Alternate Approaches Edited by Thomas R. Flanagan, Dwaine F. Emerich, and Shelley R. Winn Volume 22 Neurobiology of Steroids Edited by E. Ronald deKloet and Win Sutanto Volume 23 Peptidases and Neuropeptide Processing Edited by A. Ian Smith Volume 24 Neuroimmunology (in preparation) Edited by M. Ian Phillips and Dwight E. Evans Volume 25 Receptor Molecular Biology (in preparation) Edited by Stuart C. Sealfon Volume 26 PCR in Neuroscience (in preparation) Edited by Gobinda Sarkar

Section I

Molecular Approaches for the Study of Intracellular Processing Enzymes


[1]

Molecular Strategies for Identifying Processing Enzymes Nabil G. Seidah

Introduction In the early 1960s it was proposed that polypeptide hormones are first synthesized as inactive precursors that require specific cleavage after pairs of basic residues (such as LysArg-, ArgArg-, LysLys-, and ArgLys-) in order to release the active hormone. Since then this model has been extended to other precursors, as it is also applicable to progrowth factors, proneurotrophic factors, hormonal receptors, adhesion molecules, retroviral surface glycoproteins, proenzymes, and even certain protoxins. The elaboration of the structures of many precursors as well as their biosynthetic products also revealed that processing C terminal to single basic residues such as Arg(and less frequently Lys-) as well as after multiple basic residues (three or more) occurs in about 20% of the processed sites utilized in vivo. Therefore, it was of great interest to identify the proteinase(s) responsible for such proprotein processing and to define whether cleavage after monobasic residues and C terminal to pairs of basic residues was performed by the same enzyme(s). The search for the physiologically important processing enzymes, termed "proprotein convertases" or "PCs," was laborious and a number of laboratories, including our own, participated actively in this hunt (1). The major breakthrough came in 1984, with the molecular identification of the convertase responsible for the activation of the yeast a-mating factor and killer toxin. The proteinase identified by genetic complementation of a K E X 2 mutant strain was found to be a subtilisin-like serine proteinase (2, 3) and is now called "kexin." The search for the mammalian counterpart of kexin took about 5 years, before it was realized by computer database searches for sequence identity to kexin that a partial human genomic sequence encoding a protein called furin had already been reported by Roebroek et al. in 1986 (4). In the reported DNA sequence only the active site serine and the catalytically important asparagine residue found in all subtilisin-like proteases were identified. The complete sequence of the 5' end of the gene was completed in 1989 and it comprised the other two active site residues, aspartate and histidine (5).

Discovery of PC1 and PC2 Alignment of the amino acid sequences of furin and kexin within their catalytic domains revealed a number of segments exhibiting a high degree of Methods in Neurosciences, Volume 23

Copyright 9 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

4

I

MOLECULAR APPROACHES hFurin

SGVTQRDLNVKAAWAQGYTGHGIVVSILDDGIEKNHPDLAGNYDPGASFD

174

yKexin

PSFPGSDINVLDLWYNNITGAGVV~~D_CzLDYENEDLKDNFCAEGSWD

196 224

9Z. . . .

J:ll"

I "-'Jl

I:l''J'llJ::

~:'11

:J s : : : l Z l

hFurin

VNDQDPDPEPRYTQMNDNRHGTRCAGEVAAVANNGVCGVGVAYNARIGGV

yKexin

F N D N T N L P K P R . . .L S D D Y H G T R C A G E I A A K K G N N F C G V G V G Y N A K I SG I 243

hFurin

R M L D G E V T D A V D A R S L G L N P N H I H I Y S A S W G P D D D G K T V H G P A R L A E E A F 274

yKexin

RI L S G D I T T E D E A A S L I Y G L D V N D IYS C S W G P A D D G R H L Q G P S D L V K K A L

hFurin

II o~ F R G V S Q G R G G L G S I F V W A S G N G G R E H D S C N C D G Y T N S IYTLS I S S A T Q F G

yKexin

V K G V T E G R D S KGAI Y V F A S G N G G T R G D N C N Y D G Y T N S I YS I T I GAI D H K D

9II..

I.II

I'I.I-'I..

:.I:

@

IIIIIIII:II

:I II ..

9:II.,II'-I.I'I'IIIIII

AS

@

.I..IIIII:III:I:I:

9 .III.IIII.III-"II.

I...I:

I.II-IIIIIIII.:.I'..:

(.-m I

(..

293

9

324 343

hFurin

NVPWYSEACSSTLATTYSSGNQNEKQ IVTTDLRQKCTESHTGTSASAPLA

374

yKexin

LHPPYSEGCSAVMAVTYSSG.. SGEYIHSSDINGRCSNSHGGTSAAAPLA

391

I III'II..'I.IIIII

.'. I ..I.'..'I.-II.IIII.IIII

FIG. 1 Alignment of the amino acid sequences of human furin and yeast kexin within the catalytic domain. The active site residues Asp", His", and Sera are emphasized, as well as the catalytically important Asne. The sense (S) and antisense (AS) oligonucleotides used to identify human PC2 are shown, as well as the primers (I and II) that were first used to identify mouse PC1 and PC2.1, Identical sequence; :, highly similar in sequence.

sequence identity (Fig. 1). In 1989, the partial sequence of furin (from the catalytically important Asn* up to the C terminus) (4) and the full sequence ofkexin (3) were known. Accordingly, on the basis of the concept of sequence conservation around the active sites of serine proteinases, polymerase chain reaction (PCR) amplification of mRNA (reverse transcriptase-PCR or RTPCR) allowed two laboratories simultaneously to isolate for the first time other mammalian homologs of kexin, known as PC1 (6, 7) and PC2 (6, 8), representing the first endocrine and neuroendocrine processing enzymes molecularly characterized in mammalian tissues. Polymerase chain reaction amplification of a cDNA synthesized from human insulinoma total RNA, using degenerate oligonucleotides encoding the consensus sequence surrounding the active site residues Asp" (oligo S; Fig. 1) and His" (oligo AS; Fig. 1) in kexin and related subtilisins, gave a 150-bp probe. The latter was used to screen a human insulinoma library and to isolate a full-length cDNA encoding a novel convertase called PC2 (8). Independently, PCR applied to cDNA obtained from mouse pituitary total RNA using oligonucleotides encoding the sequence around the catalytically important Asn* and the active site Ser u of human furin (oligos I and II, Fig. 1), allowed the isolation of a 260-bp probe (6, 7). Screening mouse pituitary and mouse insulinoma libraries

[1] PROPROTEIN CONVERTASES

5

with this probe led to the isolation of full-length cDNA clones encoding mouse PC2 (6) and also another convertase that was named PC1 [(6, 7); also called PC3 in Smeekens et al. (9)].

Polymerase Chain Reaction Procedure The PCR methodology used (5, 6) consisted first of reverse transcribing about 1-5/zg of total RNA obtained from tissues of interest (e.g., pituitary or cell lines) and then performing 30 cycles of PCR amplification using 100 pmol of each primer (e.g., oligos I and II; Fig. 1), 2.5 units of Taq DNA polymerase in 10 mM Tris (pH 8.3), 50 mM KCI, 1.5 mM MgC12, and 200 ~M dNTPs. The original cycling PCR program used consisted of successive 1-min incubations at temperatures of 94, 53, and 72~ in a Perkin-Elmer (Norwalk, CT) model 480 cycler. The amplified products were digested with restriction enzymes, for which sites were already encoded at the 5' ends of the chosen oligonucleotides. The digested products were then purified on a preparative 2% (w/v) agarose gel, size selected, and then subcloned in a vector of choice. This cumbersome cloning procedure has now been replaced by a simpler version, whereby the amplified products are directly ligated in the PCRII vector (Stratagene, La Jolla, CA) without restriction enzyme digestion. This is possible because the Taq polymerase always adds an extra A nucleotide at the 5' ends of the amplified cDNA and, hence, the use of a vector with T overhangs permits a rapid subcloning procedure. We recommend this protocol because it saves time and also circumvents the problem of having to add, at the 5' ends of the primers used in the PCR reaction, a restriction site that may also be present within the amplified segment.

D i s c o v e r y of P C 4 a n d PC5 Analysis of the deduced sequence homology between mammalian convertases PC1, PC2, and furin revealed that other segments are also conserved. In an effort to isolate other convertases, we developed a procedure that allowed us to identify three more members of this subtilisin/kexin-like family called PC4 (10) and PC5 (11) as well as rodent homologs of human PACE4 (12). As shown from the homology of the sequences of the six known convertases (Fig. 2), highly conserved segments are also found in regions other than those encompassing the active site Ser" and the catalytically important Asn ~ We have chosen a set of two degenerate oligonucleotides, one preceding the catalytically important asparagine (sense oligo IV) and the other following the active site serine (antisense oligo Ill) (Figs. 2 and 3). We found

6

I

M O L E C U L A R APPROACHES mPCI

Consensus

KeRsKRsVqk fdRkKRgyRd KRRtKRdVyq rRRvKRslv, KRRvKRqVR. KkRtKRdydl KRR-KR-VR-

dsalDL.FND ineiDinmND dPt ...... D vPt ...... D sdpQaLYFND sraQstYFND -P-QDLYFND

PmWnqQWYLq dTrmtaalpk PIFtkQWYLf nTgqadgtpg PkFpqQWYL ..... sgvtqr PwFskQWYM ..... nkeieq PiWsnmWYLH CgDknsrcrS PkWpsmWYMH CsDnthpcqS P .... QWYLH CTD ...... S

LDLhVipvWe LDLNVaeAWe .DLNVkAAWa .DLNIlkvWn .EMNVqAAWk .DMNIegAWk LDLNV-AAW-

mPCl mPC2 hfurin rPC4-A hPACE4 rPC5 Consensus

kGiTGKGVVI IGYTGKGVtI qGYTGhGIVV qGITGrGVVV rGYTGKnVVV rGYTGKnIVV -GYTGKGVVV

TVLDDGIEWN gIMDDGIDYI sILDDGIEkN sILDDGIEkd TILDDGIERN TILDDGIERt TILDDGIERN

HtDiyANYDP HPDLAyNYna HPDLAgNYDP HPDLwANYDP HPDLApNYDs HPDLmqNYDa HPDLAANYDP

eASYDfNDND dASYDfssND gASFDvNDqD IASYDfNDyD yASYDvNgND IASCDvNgND -ASYD-NDND

hDPFPRYdlt PyPYPRYtdd PDPePRYtqm PDPqPRYtpn yDPsPRYdAS IDPmPRYdAS PDP-PRY-AS

mPCl mPC2

rPC4-A hPACE4 rPC5 Consensus

NENKHGTRCA wfNsHGTRCA NDNrHGTRCA dENrHGTRCA NENKHGTRCA NENKHGTRCA NENKHGTRCA

GEIAmqANNh GEVsAaAsNn GEVAAvANNg GEVsATANNg GEVAAsANNs GEVAATANNs GEVAATANN-

kCGVGVAYNs iCGVGVAYNs vCGVGVAYNA fCGaGVAFNA yCiVGIAYNA hCtVGIAFNA -CGVGVAYNA

KVGGIRMLDG KVaGIRMLDq rIGGVRMLDG rIGGVRMLDG KIGGIRMLDG KIGGVRMLDG KIGG-RMLDG

i.VTDaIEAs pfmTDIIEAs E.VTDaVDAr a. ITDIVEAq D.VTDVVEAk D.VTDmVEAk D-VTDIVEA-

mPCI mPC2 hfurin rPC4-A hPACE4 rPC5 Consensus

SigFNPgHVd SishmPQIId SIgLNPnHIh SIsLqPQHIh SlgirPnyId SvsYNPQHVh S--LNPQHI-

IYSASWGPnD IYSASWGPtD IYSASWGPDD IYSASWGPED IYSASWGPDD IYSASWGPDD ~ D D

DGKTVEGPGR nGKTVDGPre DGKTVhGPaR DGrTVDGPGI DGKTVDGPGR DGKTVDGPap DGKTVDGPGR

LaQkAFEyGV LtlQAmadGV LaeeAFfrGV LtQeAFrrGV LakQAFEyGI LtrQAFEnGV L-QQAFE-GV

KqGRQGkGSI nKGRgGkGSI sqGRgGLGSI tKGRQGLGtl KKGRQGLGSI rmGRrGLGSV KKGRQGLGSI

mPCI mPC2

FVWASGNGGR YVWASGdGG. FVWASGNGGR FIWASGNGGI FVWASGNGGR FVWASGNGGR ~ R ~II~

qgDNCdCDGY syDdCNCDGY ehDsCNCDGY hyDNCNCDGY egDyCsCDGY skDhCsCDGY --DNCNCDGY

TdSIYTISIS asSmWTISIn TNSIYTISIS TNSIhTISVg TNSIYTISVS TNSIYTISIS TNSIYTISIS

SAsqQGIsPW SAindGRtal SATqfGnvPW StTrQGRvPW SATenGykPW StaesGkkPW SAT-QGR-PW

YaEkCSSTLA YdEsCSSTLA YsEaCSSTLA YsEaCaSTFt YIEeCaSTLA YIEeCSSTLA Y-E-CSSTLA

TsYSSGDYtD sTFSnGrkrn TTYSSGnqnE TTFSSGvvtD TTYSSGaFyE TTYSSGEsyD TTYSSG---D

qr..ItsaDL peagVaTTDL kq..IVTTDL pq..IVTTDL rk..IVTTDL kk..IITTDL .... IVTTDL

hndCTEtHTG TSASAPLAAG ygnCTlrHsG TSAaAPeAAG RQkCTEsHTG TSASAPLAAG hhqCTDkHTG TSASAPLAAG RQRCTDgHTG TSvSAPMvAG RQRCTDnHTG TSASAPMAAG RQRCTD-HT~t__~G (- I (-

IfALALEANP VfALALEANI IIALtLEANk mIALALEANP IIALALEANs IIALALEANP IIALALEANP

nLTWRDMQHL dLTWRDMQHL nLTWRDMQHL ILTWRDLQHL qLTWRDvQHL fLTWRDvQHv - L ~ (-III(-

VVWTSeydpL tViTSkrnqL VVqTSkPAHL VVRaSRPAqL IVkTSRPAHL IVRTSRagHL VVRTSRPAHL

asN.pgWKkN hdevhqWrrN NAN..DWatN qAe..DWriN kAs..DWKvN NAN..DWKtN NAN--DWK-N

GaGLmVnsrF GvGLefnHLF GvGrKVSHsW GvGrqVSHhY GaGhKVSHFY aaGFKVSHLY G-GLKVSHLY

GFGLLnAkAL GYGvLDAGAM GYGLLDAGAM GYGLLDAGIL GFGLvDAeAL GFGLMDAeAM G-GLLDAGA-

mPCl mPC2 hfurin rPC4-A hPACE4 rPC5 Consensus

VDLAdpRTwr VkMAkdW..k VaLAqnWT.. VDLArvWl.. VveAkKWT.. VmeAeKWT.. VDLA-KWT--

nVPekkeCVV TVPerfhCVg TVapQrKCII ptkpQkKCtI aVPsQhmCVa TVPqQhvCVe TVP-Q-KCVI

kdnnfEPral gsvq.nPekI dilt.EPkdI rvvh.tPtpI asdk.rPrsI stdr.qiktI ..... EP--I

kangEVivei PPtgklvlTl gkrlEVRKT, iPrmlVpKn, PlvqvlRtTa rPnsaVRsiy PP--EVRKT-

pTrACEgqEN kTnACEgkEN vTaclgepnh vTvcCDgsrr iTSACaehsd kaSgCsdnpN -TSACE--EN

mPC1 mPC2 hfurin rPC4-A hPACE4 rPC5 Consensus

a. IksLEHVQ .FVRYLEHVQ ..ItrLEHaQ rLIRsLEHVQ qrVvYLEHVv hhVnYLEHVv ---RYLEHVQ

feaTIeYsRR GDLhVtLTSa aviTVnatRR GDLnInMTSP aRITISYnRR GDLAIhLvSP VqlslSYsRR GDLeIFLTSP VRtsIShpRR GDLqIYLvSP VRiTIthpRR GDLAIYLTSP VR-TISY-~YLTSP (-V~

vGTstvLLAe MGTkSiLLsr MGTRStLLAa MGTRStLvAi sGTkSqLLAk sGTRSqLLAn MGTRS-LLA-

Rer.DtSpnG RPrdDdSkvG RPh.DySaDG RPL.DiSgqG RIL.DlSnEG RIF.DhSmEG RPL-D-S-EG

mPC3

hfurin rPC4-A hPACE4 rPC5

hfurln

hfurin

rPC4-A hPACE4 rPC5

Consensus mPCI mPC2

hfurin

rPC4-A

hPACE4

rPC5 Consensus mPCl mPC2 hfurin rPC4-A hPACE4

rPC5 Consensus

VI

e

IV~

[1]

PROPROTEIN Ile

mPCI mPC2 hPC2 hFur mFur rFur rPC5 mPC5 rPC4 mPC4 hPACE4

Tyr Ser Ala

ATT

TAC

- -C

.....

--C

--C --C - -C

--C

AGT

GCA

C

- -C

Consensus

ATC

Ser

AGC

Trp G l y P r o

TGG

C . . . . . C --C --T G-.....

C

GGC

--C --C

T

TAC

AG-

C

SAnme

C C

C

-C-

C - -T

A

C

C

C

G AG-

T

TGG

Oliaonucleotide

GGC

CC

IV

Thr Arg

Cys A l a

G l y Glu

ACA

GGA

GAA

--C

--C

C --T

TGT

hPace4

--C --C ...... G

--T --C

--G --G

----G

Consensus

T C TAG C A - G G - A C - -GC A ACA

rPC5

RGD

S*nse

--G C-G C-C C-T C-C

..... C --C --T ..... G ..... C

TGT

A A GC- GGG C

Oliaonucleotide

VI

-T

C-G

- -A

Met

ATG

C-G C-G C-G

ACC

TGG

AA - G- G A C C G

Gln

CAA

.....

G G

Leu CTG

His

CAT C

G G G .... G G .... G C .... G C-A --G G-C --G

--C --C ---

A -T G

T CA- CTG

G

G CAA

----G-T G-T -------

--C --C --C

C

300,000, Sigma, St. Louis, MO) for 10 min, followed by additional overnight drying at 50~ A simplified slide subbing procedure has been used, in which both treatments with gelatin and poly-IAysine are replaced by a 30-sec dip in a siliconizing silane solution [2% (v/v) 7-aminopropyltriethoxysilane prepared in acetone; Pierce, Rockford, IL). This is followed by two additional rinses in acetone and one rinse in diethylpyrocarbonate-treated H20 (DEPC-H20). Thus, the whole process of treating a batch of slides can be completed within 1 hr, with the slides subsequently allowed to dry at room temperature overnight. In our experience, slides coated with silane offer equivalent efficiency of tissue adhesion as gelatin and poly-IAysine subbing. Coated slides can be stored for several months in a dust-free environment. Both sagittal and transverse sections (8-12 ~m thick) of the embryonic tissues are prepared in a cryostat (Hacker Instruments, Inc., Fairfield, NJ) for in situ hybridization experiments. Tissue sections are thaw mounted on slides and stored at -80~ until use.

Preparation o f Probes Both sense and antisense [35S]UTP-labeled cRNAs are prepared using an in vitro transcription system (Promega, Madison, WI). The transcription mixture in a total volume of 10/xl is assembled at room temperature, containing the transcription buffer [40 mM Tris (pH 7.5), 6 mM MgC12,2 mM spermidine, and 10 mM NaC1], 10 mM dithiothreitol (DTT), 40 units of ribonuclease

48

I

MOLECULAR APPROACHES

inhibitor (RNasin), 160/zCi of speed-vacuum dried [35S]UTP (specific activity, 1000-1500 Ci/mmol; DuPont-New England Nuclear, Boston, MA), 0.5 mM each of unlabeled nucleotide triphosphates (ATP, GTP, and CTP), 1/zg of linearized DNA template, and 20 units of bacteriophage RNA polymerase. The transcription reaction is allowed to proceed at 37~ for 60 min. The DNA template is removed by digestion with 1 unit of RNase-free DNase at 37~ for 20 min. Size-exclusion chromatography is used to remove unincorporated nucleotides, by passing probes through a Sephadex G-50 RNase-free spin column (Boehringer-Mannheim, Indianapolis, IN) after phenol-chloroform extraction. The specific activity of recovered cRNA is measured by scintillation counting. Riboprobes are aliquoted and stored at -80~ The sense probes are used as a negative control.

In Situ Hybridization Protocol In situ hybridization experiments are performed following protocols previously described (6), with certain modifications. The following is a summary of the procedure currently in use. Tissue slides are retrieved from the -80~ freezer and fixed in 4% (w/v) paraformaldehyde at room temperature for 10 min. The slides are rinsed in DEPC-H20 for 5 min, dehydrated in ethanol, and air dried for 10 min. Slides are subsequently rehydrated and treated with acetic anhydride to suppress nonspecific electrostatic binding of probes to the tissue and slide coating (7). In this procedure, after a brief pretreatment of slides with 0.1 M triethanolamine-0.05 M acetic acid, slides are rinsed vigorously in freshly prepared 0.2% (v/v) acetic anhydride diluted with triethanolamine. After a further 10-min incubation, slides are rinsed in 0.2x SSC (1.0• SSC is 0.15 M NaC1 plus 0.015 M sodium citrate), dehydrated, and air dried. To be able to apply different probes to multiple tissue sections on a slide and help to retain the applied solution via capillary tension, a rubber cement ring is carefully applied, circling each section. The prehybridization is performed at room temperature for 1-3 hr in a buffer containing 50% (v/v) deionized formamide (Boehringer-Mannheim), 10 mM Tris (pH 7.5), 1 mM ethylenediaminetetraacetic acid (EDTA), 600 mM NaC1, Denhardt's solution [0.02% (w/v) Ficoll, 0.02% (w/v) polyvinylpyrrolidone, and 0.02% (w/v) bovine serum albumin], and heat-denatured heterologous nucleic acids [0.05% (w/v) yeast total RNA, 0.005% (w/v) yeast tRNA, and 0.05% (w/v) salmon sperm DNA]. The riboprobes are heated at 85~ for 5 min and then chilled on ice (to disrupt the possible formation of RNA secondary structure). These are diluted to an activity of---4 x 10 7 dpm/ml (confirmed by scintillation counting) in the hybridization buffer. The hybridization buffer is similar to the buffer used for prehybridization, except that 10% (w/v) dextran sulfate

[3]

ONTOGENY OF PROCESSING ENZYME EXPRESSION

49

(Mr 8000; Sigma), 10 mM DTT, and 0.1% (w/v) sodium dodecyl sulfate (SDS) are added. Prehybridization solution is replaced by 25-60/~1 of the hybridization buffer, depending on the size of the tissue section. The slides are placed in a moist chamber and hybridization is carried at 50~ for 12 hr (overnight). Slides are then washed at 50~ for 30 min in a solution containing 50% (v/v) deionized formamide, 1 x SSC, and 10 mM DTT. This is followed by a rinse in 0.5 x SSC and treatment at room temperature for 30 min with RNase A [0.1 mg/ml, diluted in 10 mM Tris (pH 8), 1 mM EDTA, 500 mM NaC1] to degrade nonhybridizing single-stranded probes (8). The final wash is performed with 0.2x SSC at 50-60~ for 2 hr with gentle stirring. Slides are dehydrated in an ethanol series and subjected to X-ray autoradiography. This is followed by nuclear emulsion autoradiography for higher resolution of signal detection. Slides are coated with a 1:1 (v/v) dilution of Kodak NTB2 emulsion (Eastman Kodak, Rochester, NY) and exposed at 4~ in the dark. Usually at least a threefold longer time of exposure for emulsioncoated slides is needed compared to a sufficient X-ray film exposure. Finally, dipped slides are developed with Kodak D19 developer and stained with hematoxylin-eosin (HE). The slides are examined in Wild and Leitz microscopes (Leica, Heerbrugg, Switzerland) under bright-field, dark-field, and epipolarized illumination.

Discussion o f Methodology Rat embryonic development proceeds relatively rapidly. Both tissue morphology and the functional status of specific cells change significantly during organogenesis. The precise determination of developmental stage of embryo, therefore, is of paramount importance in the study of expression of genes involved in developmental processes. Although the experimental animals used to obtain embryos are "timed-pregnant" and gestational stages inferred from observed copulation plug date, sometimes embryos are obtained that are different from their expected age because plugs were missed. Furthermore, because of differences in fertilization time of individual oocytes, individual embryos within the same litter are at different developmental stages and indeed can differ in developmental age by as much as 24 hr. In establishing a more precise staging system, we have adopted a procedure of Wanek et al. (5), which is based on evaluating the gross external morphology of the mouse limb at different developmental stages. Corresponding ages for rat embryos of equivalent age are deduced from the listed mouse stages by adding 1-1 89days. The rat embryo is rapidly isolated from the uterus and the morphological characteristics of both forelimb and hindlimb are evaluated under a dissecting microscope (with the hindlimb usually 1 day behind the forelimb in development). If necessary, embryonic limbs are dissected and

50

I


placed in a petri dish containing PBS solution for better viewing. Gross morphology of the limbs is compared to a chart detailing the morphological characteristics of the limb at various developmental stages and the appropriate gestational stage is assigned to the embryo. Sophisticated stereotypic development of the limb ensures that this staging system can effectively encompass a large developmental window (e 10-e ! 8). The application of limb bud staging system ensures a consistency in determining rapidly the precise age of each embryo. In general, e12-e18 embryos are embedded in OCT compound (directly fresh frozen). We have found that sections derived from fresh-frozen embryos consistently give a higher signal-to-noise ratio than those from fixed embryos. This is particularly advantageous in the detection of low-abundance transcripts such as processing enzyme mRNAs and has therefore been used in most experiments reported here. In contrast, prefixed tissues compromise the signal labeling intensity for better morphology. Fixation of the embryo helps to identify regions and cell types of gene expression, such as those of early postimplantation staged embryos, with greater precision and often in conjunction with immunocytochemistry. We have, therefore, used paraformaldehyde prefixation only for embryos younger than el2, although freshfrozen sections have also been prepared. Owing to the small size of the uterus in these stages and the accessibility of its intrauterine cavity when decidua are isolated individually, intracardial perfusion of adult rat is not necessary. Although cDNA and oligonucleotide probes have been used, the most common probes for in situ hybridization remain radiolabeled cRNAs (riboprobes), which can readily be labeled to high specific activities. To ensure success and consistency during in situ hybridization experiments, the quality of newly synthesized probes should always be evaluated via gel electrophoresis prior to use. Probes of high quality usually result after an efficient transcription reaction (over 108 dpm/labeling reaction, using 1 /zg of plasmid template) and appear as a major band of appropriate size when electrophoresed on a denaturing polyacrylamide gel. Probes of relatively short length (riboprobes varying in length from 320 to 372 bp are used in the present study), can be used directly for hybridization experiments. Substantially longer probes should be shortened after synthesis by alkaline hydrolysis to allow their efficient penetration into the tissue sections (2). The extent of hydrolysis should be carefully controlled and the average length of probe monitored by electrophoresis. Alternatively, sections can be pretreated with proteinase to facilitate probe penetration. Although the possibility of RNase contamination may exist, inefficient transcription is usually due to the precipitation of DNA template by the spermidine present in the stock concentration of transcription buffer (9). To avoid this problem, all the components of the

[3] ONTOGENY OF PROCESSING ENZYME EXPRESSION

51

reaction may be added separately to the side wall of an Eppendorf tube and mixed by a brief spin on a microcentrifuge. Additional pipetting of the mixture is recommended to ensure thorough dispersion of all the components, especially the bacteriophage polymerase. To compare informatively the relative abundance of different processing enzyme gene mRNAs in development, probes of similar length and specific activity should be used. Colocalization of a processing enzyme with a proprotein is indicative of a possible enzyme-substrate relationship, whereas the coexistence of distinctive types of processing enzymes suggests their possible collaboration in the completion of posttranslational modification processes. Although such information can sometimes be deduced by comparing the expression of different genes on serial sections, unequivocal demonstration of coexpression of two genes requires their covisualization on a single tissues section. One way to achieve this goal is to apply two different cRNA probes to the same section, which are subsequently distinguished by virtue of the distinct nature of probe labeling. Besides the commonly used [35S]UTP-labeled probes, we have also used digoxigenin-UTP to synthesize cRNA probes, using commercially available reagents (Genius RNA labeling kit; Boehringer Mannheim). Digoxigenin-UTP-labeled probes are subsequently detected on the tissue sections by an enzyme immunoassay. Owing to the similar nature of nucleotide hybridization with both kinds of cRNA probes, we have been able to apply both probes simultaneously, with minimum change in the basic in situ hybridization protocol. In our experience, the hybrids formed by the [35S]UTP-labeled probes are stable during the subsequent color visualization of digoxigenin-UTP-labeled probes. Prehybridization is performed in a buffer containing 50% (v/v) deionized formamide, 5 • SSC, Denhardt's solution, 0.025% (w/v) yeast tRNA, 10% (w/v) dextran sulfate, 0.01% (w/v) Nlauroylsarcosine, and 0.02% (w/v) SDS. The hybridization buffer is similar to the prehybridization buffer, except for the addition of 10 mM DTT. Digoxigenin-UTP-labeled probe is empirically diluted to the final concentration of 1-100 pg/ml in the hybridization buffer, depending on the abundance of the transcripts to be detected; higher transcript concentrations, although required for the detection of low-abundance mRNA, can also result in higher nonspecific background. After hybridization, slides are washed under identical stringency conditions and digested with RNase A as described above. Slides are then treated with nonfat dry milk (1 mg/ml), which is dissolved in a buffer containing 100 mM Tris (pH 7.5) and 150 mM NaCI (buffer I). This is followed by incubation with a 1 : 1000 (v/v) dilution in buffer I of alkaline phosphatase-conjugated anti-digoxigenin antibody (Fab fragment; Boehringer Mannheim) at room temperature for 1-3 hr. If the signals are low, lengthening the incubation time usually enhances the antibody binding and should be performed at 4~ Slides are then washed in the buffer I for

52

I

M O L E C U L A R APPROACHES

10 min twice and equilibrated with buffer II [100 mM Tris (pH 9.5), 100 mM NaC1, and 50 mM MgCI2]. Digoxigenin-UTP-labeled probe is visualized by a 2-hr to overnight incubation of slides in chromogen solution in the dark at room temperature. The chromogen solution is freshly prepared in buffer II and consists of 150 ~g of nitroblue tetrazolium [NBT, stock solution 75 mg/ ml, prepared in 70% (v/v) dimethylformamide] per milliliter, 100/~g of 5bromo-4-chloro-3-indolylphosphate (X-phosphate; stock solution 50 mg/ml, prepared in straight dimethylformamide) per milliliter, and 25 ~g of L[- ]-2,3,5,6-tetrahydro-6-phenylimidazo[2,1-b]thiazole (levamisole; Sigma) per milliliter. The color reaction is terminated by incubating slides in a solution consisting of 10 mM Tris (pH 8), 1 mM EDTA, and 100 mM NaC1. Slides are then dehydrated in an ethanol series. After exposure to X-ray film, slides are emulsion dipped in Ilford L4 emulsion (Polysciences, Inc., Warrington, PA), developed, and mounted in Permount without counterstain. Digoxigenin-UTP labeling usually appears as bluish deposits, which form high contrast to the silver grains derived from [35S]UTP-labeled probes on the same section (see Fig. 2c and d). Besides its greater speed compared to using conventional radiolabeled probes (lengthy emulsion dipping is not needed), digoxigenin-UTP labeling in situ hybridization offers satisfactory sensitivity in mapping gene expression in development. Using the same preparation of digoxigenin-UTP-labeled proopiomelanocortin (POMC) probe as well as [35S]UTP-labeled POMC probe, for instance, we have obtained comparable results in detecting prenatal POMC gene expression just following the closure of Rathke's pouch. In addition, there is an increasing number of reports that have used "whole-mount in situ hybridization" to detect specific mRNAs in vertebrate embryos at early postimplantation ages (10).

Examples Expression of Furin and Carboxypeptidase E in Early Rat Embryogenesis Several mammalian endoproteases have been successfully identified, including furin, PC1, PC2, PC4, and PACE4 (11, 12). Among these, furin has been shown to cleave preferentially those proproteins maturing from the constitutive secretory pathway. The proteolytic cleavage of proproteins by furin exposes the dibasic residues at the C termini, which are usually absent from the mature protein and thus are removed by subsequent exoproteolytic cleavage. The only known enzyme capable of performing such a trimming reaction is carboxypeptidase E [CPE (13)]. It is thus possible that furin and CPE may functionally collaborate to complete the proteolytic processing of common proproteins in embryogenesis and, if so, would be expected to be

[3]

O N T O G E N Y OF PROCESSING ENZYME EXPRESSION

53

expressed in both a spatially and temporally correlated manner. To compare the expression domain of these two genes in development, we have performed in situ hybridization experiments on serial transverse sections of el2 rat embryo, with furin and CPE probes applied on adjacent sections. The results are shown in Fig. 1 (14). Positive labeling appears as a bright region owing to the reflection of autoradiographic grains under dark-field illumination. At this stage, in agreement with its general involvement of constitutive proprotein processing, furin mRNA is expressed at low but detectable levels in nearly all tissues and structures throughout the embryo. Severalfold higherthan-basal levels of expression, however, are observed in heart and liver primordium, as well as in the body wall surrounding the umbilical vein (Fig. l c, f, and i). In contrast, the developing nervous system noticeably lacks detectable levels of furin gene expression. In comparison, CPE mRNA is prominently expressed throughout the mantle layer of the neural tube, as well as in cranial and peripheral ganglia. In addition, a significant level of CPE expression is also observed in the embryonic heart, in the epithelium of the mandibular component of first bronchial arch, and the mesentery surrounding the gut (Fig. l d, g, and j). Although the expression domains of both genes overlap, with the embryonic heart being a clear example, several regions where furin is expressed at high levels lack CPE expression, such as the body wall surrounding the umbilical vein (compared Fig. li and j) and the liver primordium (data not shown). Applying both furin and CPE probes in parallel on adjacent tissue sections has thus enabled direct comparison of their spatial expression at this stage. Careful comparison of their expression on serial transverse sections shows that the expressions of furin and CPE only partially overlap. This indicates that if furin is functional at this stage, additional types of carboxypeptidase(s) may be recruited for exoproteolytic processing in such regions as liver primordium. The molecular identity of such a processing enzyme remains to be elucidated.

PC2 Mediation of Developmentally Regulated Proopiomelanocortin Proteolytic Processing in Anterior Rat Pituitary In the rat pituitary, the polyprotein precursor proopiomelanocortin (POMC) is synthesized by all parenchymal cells in the intermediate lobe (IL) and by 5-10% of cells in the anterior lobe (AL) [(15), see also Fig. 2a]. Distinctive posttranslational modifications, however, occur in these two cell populations. Proopiomelanocortin undergoes limited proteolytic cleavages in the AL, generating mainly adrenocorticotropic hormone (ACTH) and/3-1ipotropin (/3-LPH). In contrast, further cleavages inside both ACTH and/3-LPH occur in the IL, thus generating smaller peptide products such as c~-melanocytestimulating hormone (c~-MSH), y-LPH, and/3-endorphin (/3-EP) (15). PC1 and PC2 have been shown to be expressed distinctly in pituitary: specifically,

Hb

b, c, d

a

b

d

C

e

. w

Uv

h

.j

[3] ONTOGENY OF PROCESSING ENZYME EXPRESSION

55

PC1 is expressed in both the AL and IL, whereas PC2 is mainly expressed in the IL [(16), see also Fig. 2b]. These patterns, when correlated with results of gene transfer experiments (17, 18), indicate that the distinct expression of PC1 and PC2 is responsible for the observed lobe-specific proteolytic processing of POMC. Therefore, POMC is processed to a limited extent by PC 1 in the AL, whereas collaboration of both PC 1 and PC2 in the IL results in more extensive POMC processing. Interestingly, fetal and early postnatal AL have been shown to process POMC more extensively, generating a peptide profile similar to that of the IL (19, 20). This developmental plasticity could be mediated by the presence of additional proteinaceous modulators, alterations of the processing microenvironment, or regulation of the expression of distinct processing enzymes. To address this question, we examined the expression of PC1 and PC2 on serial ages of postnatal pituitary. In contrast to the findings in adult, the AL contains significant levels of PC2 mRNA during these stages, reaching a peak at postnatal day 15 (p15) (Fig. 2d). In situ hybridization was performed with digoxigenin-UTP-labeled POMC probe and [35S]UTP-labeled PC2 probe on the same pituitary section, using protocols described above. This allows covisualization of both probes. The POMC transcripts are revealed as areas of blue deposits. The cytoplasm of POMC-expressing cells in both the IL and AL are clearly delineated, whereas the nuclear regions are largely devoid of labeling (Fig: 2c). This indicates that the majority of the POMC transcripts are processed to their mature form and exported out of the nuclei. The extremely fine deposit of nonradioactive labeling products allows the resolution of this method to rival that of immunocytochemistry. [35]UTP-labeled PC2 hybrids appear as autoradiographic grains. Aggregates of the grains are scattered in both the AL and IL, indicating that PC2 is expressed in both lobes at this stage (Fig. 2d). The majority of the POMC-expressing cells in the IL and some of the cells in the AL

FIG. 1 Comparison of furin and CPE gene expression in the 12-day rat embryo. (a) Line drawing of an e 12 rat embryo, indicating the approximate planes of section. Bright-field (b, e, and h) and dark-field (c, d, f, g, i, and j) micrographs of corresponding adjacent sections are shown. Adjacent sections are hybridized with furin cRNA (c, f, and i) and CPE cRNA (d, g, and j), thus allowing the comparison of their spatial patterns of expression. Furin is expressed at a low level in most tissues, but is undetectable in the neural tube. In contrast, CPE is expressed in the newly differentiated neurons in the diencephalon (Di), hindbrain (Hb), trigeminal ganglia (Tg), and spinal cord (Sc). Both furin and CPE are expressed in embryonic heart (H). Note that furin is expressed at a significant level in the body wall surrounding the umbilical vein (Uv), whereas CPE is expressed at the adjacent mesentery (Ms) surrounding the midgut. Exposure time for autoradiography is 6 weeks. Magnification: x8.5.

i~~,~ ~I ', ~i........... ~,

b

Ir

~

jr

~ii I'illI i~

.,~lt

a~

to

[3] ONTOGENYOF PROCESSING ENZYME EXPRESSION

57

possess both types of labeling products, indicating that POMC and PC2 transcripts can coexist. This is in contrast to the lack of apparent PC2 expression in the AL in adult pituitary. This suggests that enhanced expression of PC2 in AL POMC-expressing cells may be responsible for the developmental switch in the POMC proteolytic processing pattern.

RNase Protection Assay The RNase protection assay (solution hybridization, RNase mapping) detects the presence and abundance of mRNA transcripts by virtue of hybridization with the complementary ribonucleotide probes in an aqueous phase. The probes annealing to the homologous sequence on the transcripts are protected from subsequent ribonuclease digestion and are detected by gel electrophoresis. This technique was pioneered in the early 1980s by Zinn et al. (21) and has been refined for studying the neuroendocrine systems (22). Over the years the RNase protection assay has been perfected to be one of the most sensitive methods in evaluating levels of gene expression. In the following sections we describe the methods utilizing the RNase protection assay in combination with a short-term tissue culture system, which can be used to study the regulation of processing enzyme gene expression in rat pituitary development.

Fit:;. 2 Comparison of POMC and PC2 expression in adult pituitary (a and b) and postnatal day 15 (p15) pituitary (c and d). Adjacent frontal sections of adult pituitary are hybridized with POMC (a) and PC2 (b), respectively, using [35S]UTP-labeled riboprobes. Micrographs are taken under dark-field illumination. Note that the anterior lobe (AL) expresses only a low level of PC2 compared to the intermediate lobe (IL), whereas the posterior lobe (PL) does not express PC2. Exposure times are 4 days (a) and 6 weeks (b). Magnification: x8.4. (c and d) Colocalization ofdigoxigenin-UTPlabeled POMC transcripts with [35S]UTP-labeled PC2 transcripts in the p l5 rat pituitary. POMC-expressing cells are outlined by areas of chromagen deposits in the cytoplasm [(c), bright-field illumination; section without counterstain]. Nearly all cells in the IL and scattered cells in the AL express POMC. In (d), the same section has been photographed under dark-field illumination and PC2-expressing cells are identified by aggregates of fine autoradiographic grains. Note that a considerably higher level of PC2 is expressed in the AL at this stage compared to that in adult [compare (b) and (d)]. Some aggregates of autoradiographic grains in both the IL and AL overlap with the chromagen deposits (arrows), indicating coexpression of PC2 with POMC. Note that there is some chromagen interference with optimal photography of autoradiograph grains. The slide was incubated for 3 hr in the chromogen reaction, followed by 6 weeks of autoradiographic exposure. Magnification: x 35.

58

I MOLECULAR APPROACHES

Primary Tissue Culture Rat pituitaries of various postnatal ages are rapidly isolated, and the anterior lobe (AL) and neurointermediate lobe (NIL, intermediate lobe and posterior lobe) of each are separated under a dissecting microscope. Six ALs or NILs are pooled per sample and collected in 1 ml of ice-cold medium containing Dulbecco's modified Eagles' medium (D-MEM; GIBCO-BRL, Gaithersburg, MD), 1% (v/v) heat-inactivated fetal bovine serum, and kanamycin (50/zg/ ml). Each sample is washed in 1 ml of this medium for 5 min and transferred to 24-well plates (16-mm well diameter) containing 0.5 ml of medium with or without a desired regulatory factor. Samples are incubated at 37~ for 1-24 hr in a chamber supplied with a 95% air/5% CO2 mixture.

RNA Isolation Pituitary samples either freshly isolated or harvested from the primary culture are collected and homogenized in 200/zl of ice-cold buffer containing 10 mM Tris (pH 8), 3 mM CaC12, 2 mM MgC12, 0.15% (v/v) Triton X-100, and 0.3 M sucrose. Homogenization is carried out in a Dounce (Wheaton, Millville, NJ) all-glass homogenizer. Typically 25 strokes are sufficient to disrupt the cell membrane integrity. Homogenates are then layered onto a 300-/zl cushion of the same buffer, except that 0.3 M sucrose is replaced by 0.4 M sucrose. Intact nuclei are separated from the cytoplasmic fraction by centrifugation at 1000 g for 10 min at 4~ Cytoplasmic RNA is purified from the supernatant by digestion with proteinase K (100/zg/ml) at 42~ for 1 hr in the presence of 0.1 vol of digestion buffer, which contains 10 mM Tris (pH 8), 5 mM EDTA, and 1% (w/v) SDS. This is followed by phenol-chloroform extraction and 2-propanol precipitation. Pelleted cell nuclei are resuspended in 60/xl of buffer containing 50 mM Tris (pH 8), 0.1 mM EDTA, 5 mM MgC12, and 40% (v/v) glycerol. Nuclear samples are then treated with 30 units of RNasefree DNase, followed by digestion with proteinase K (100/xg/ml) at 42~ for 30 min in the presence of digestion buffer. Nuclear RNA is purified by phenol-chloroform extraction and ethanol precipitation. Both nuclear and cytoplasmic RNA are resuspended in 5-10/xl of TE [10 mM Tris (pH 7.6), 1 mM EDTA] and the combined total RNA is measured by ultraviolet (UV) absorbance at 260 nm.

Preparation of Probes [a-32p]UTP-labeled runoff transcripts are synthesized from cDNA templates, using an in vitro transcription system (Promega). The plasmid clone is linear-

[3]


59

ized inside the cDNA template at a desired site downstream of the bacteriophage promoter. Transcription is initiated by adding the appropriate bacteriophage RNA polymerase and terminated by the removal of DNA templates by adding 1 unit of RNase-free DNase. Twenty micrograms of carrier tRNA is added and probes are extracted with phenol-chloroform. To remove incomplete runoff transcripts that may contribute to smearing background in the hybridization, riboprobes are electrophoresed on a 5% (w/v) denaturing polyacrylamide gel. Gel strips corresponding to the full-length probes are excised and eluted in a buffer containing 10 mM Tris (pH 7.5), 1 mM EDTA, 0.1% (w/v) SDS, and 500 mM ammonium acetate. The labeled RNA is isolated by precipitation with 2-propanol in the presence of 2.5 M ammonium acetate and resuspended at saturating levels in the hybridization buffer (see below) to a final concentration of 2 x 10 7 dpm/ml (for detecting abundant transcripts, such as POMC) or 5 x 10 6 dpm/ml (for detecting processing enzymes).

RNase Protection Assay Protocol The RNase protection assay protocol is based on those previously reported (22-25). The hybridization mixture in the volume of 30/zl is freshly prepared, which contains 80% deionized formamide, 40 mM piperazine-N,N'-bis(2ethanesulfonic acid) (PIPES; Sigma), 0.4 M NaC1, and 1 mM EDTA. Five microliters of each RNA sample (1.5-4/zg, diluted in TE) and diluted probes (5-20 x 106 dpm/ml) are added. The hybridization mixture is heat denatured at 85~ for 5 min and hybridization is allowed to proceed at 45~ for 12 hr (overnight). To remove single-stranded probes, samples are digested with RNase A (40/zg/ml) and RNase T1 (2 /xg/ml) at 30~ for 1 hr in a buffer containing 100 mM Tris (pH 8), 5 mM EDTA, and 300 mM NaC1. The digestion is terminated by adding 20/xl of 10% (w/v) SDS, 20/xg of proteinase K, and incubating at 37~ for 20 min. Samples are phenol-chloroform extracted and ethanol precipitated, with 5 /xg of carrier tRNA added. The pellet is resuspended in 10/xl of gel loading buffer [TE containing 1% (w/v) bromphenol blue and 0.1% (w/v) xylene cyanol], heated at 70~ for 5 min, and electrophoresed on a 5% (w/v) nondenaturing polyacrylamide gel. The gel is heat dried under vacuum and exposed to Kodak X-Omat film (Eastman Kodak) with a calcium tungstate intensifying screen at -80~ for 2 hr to 3 days, depending on the abundance of the transcripts to be detected and level of background. The gel strips corresponding to protected bands by different probes are excised and radioactivity measured by liquid scintillation counting.

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Data Analysis A cRNA probe for the ubiquitously expressed gene encoding cyclophilin has been included in all experiments performed. Cyclophilin gene expression has been shown to be inert to a variety of exogenous treatments (26). Since small variations of total tissue RNA applied in each reaction usually occur, the level of cyclophilin RNA is assayed simultaneously to serve as an internal standard, reflecting the actual amount of total RNA in each sample. The level of t h e expression of gene of interest can then be normalized to the amount of cyclophilin RNA. Results obtained from a treated group are then converted to the percentage of those of a control group. These are subject to one-way analysis of variance, followed by a Student's t test to evaluate the difference in processing enzyme RNA level between treated and control untreated groups.

Discussion o f Methodology Preparation of probes for RNase protection assay is essentially the same as for in situ hybridization, except that [a-32p]UTP is used. A specific activity of over 1 x 10 9 dpm//~g of RNA probes, representing incorporation of over 60%, is typically achieved in a single reaction. In linearizing the DNA template for synthesizing the runoff transcripts, restriction enzymes generating a 5' overhang or blunt end should be used. This can avoid possible synthesis of sense strand sequences, which can initiate at a 3' overhang sequence (27). In our procedure, however, the choice of restriction enzyme for DNA template linearization is not necessarily restricted, because only full-length transcripts initiated from the promoter are isolated from the polyacrylamide gel. Although in this case the transcription efficiency may be compromised by ectopic initiation of transcription, the yield of riboprobe is still sufficient for multiple rounds of RNase protection assays. For RNA purification, we have used a protocol [modified from Autelitano et al. (23-25)] in which nuclear and cytoplasmic RNAs are isolated separately. Separation of these two species of RNA, in conjunction with the use of intron-exon splice junction probes, allows the effect of exogenous factors on nuclear and cytoplasmic RNA levels be evaluated separately [(23, 25), also see Fig. 3]. If sufficient RNA species are detected in a subsequent RNase protection assay, changes in heteronuclear RNA (hnRNA) level are usually easier to observe than those of mRNA after short-term treatment, as the more abundant mRNA requires a longer time of treatment to exhibit significant change. If nuclear and cytoplasmic RNAs are to be isolated separately, care must be taken to avoid excessive tissue homogenization that

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would disrupt nuclear integrity, and to avoid transferring any of the nuclear pellet into cytoplasmic fraction. In the absence of DNase digestion, a low level of contamination of DNA in the cytoplasmic fraction may significantly inflate the overall 0D260reading of the cytoplasmic RNA sample. A common problem encountered with the RNase protection assay is the appearance of multiple shorter band species, protected by the probe, that can accompany the "appropriate-sized" band. This is most often a concern when a lengthy X-ray exposure time is required such as when detecting lowabundance transcripts, such as an endopeptidase. This problem is primarily due to the hybridization and protection from RNase digestion of shortened radiolabeled cRNA or partially degraded RNA transcripts. Care, therefore, must be exercised to avoid ribonuclease contamination prior to hybridization. In addition, incomplete runoff transcripts in the probe labeling reaction will contribute significantly to the hybridization background but can be readily removed by isolating only the full-length transcripts from a denaturing polyacrylamide gel. Even purified full-length probes, however, will eventually break down to smaller species owing to autoradiolysis. Therefore, the best hybridization results are obtained when probes are used on the day they are prepared or within 2-3 days when stored at -80~ Probes stored for longer than 1 week should not be used. If the high background problem persists, other steps may be modified empirically to optimize the procedure for a particular probe. These include (a) determining the minimum but sufficient amount of probe to be used, (b) varying the hybridization temperature, (c) varying the amount of ribonucleases used, digestion temperature, or digestion time, and finally, (d) redesigning a shorter length version of the probe.

Example: Hormonal Regulation of Processing Enzyme Gene Expression in Developing Rat Pituitary Proopiomelanocortin synthesized in pituitary is under complex hormonal regulation, most of which is exerted at least in some part at the transcriptional level (23). Corticotropin-releasing factor (CRF), for instance, elicits an increase in both biosynthesis and release of POMC-derived peptides, with an accompanying change in POMC mRNA levels (28). Activation of the pituitary-adrenal axis, mediated principally by ACTH, results in elevated levels of circulating glucocorticoids, which in turn inhibits the biosynthesis and release of POMC peptides in the AL, but not the NIL. Previous work in the laboratory has shown that the transcriptional responsiveness of POMC to CRF is established by as early as el5 (25). Interestingly, in the NIL a transient inhibitory effect of glucocorticoids on POMC transcription exists at late prenatal and early postnatal ages, which is lost by pl0. As a first step

62

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AL

NIL

1

2

3

4

PC1 (200 bp) ~

PC2 (136 bp)

/'~'~

POMC mRNA(97 bp)

6

7

8

,~

POMC hnRNA(160 bp)

Cyclophilin (111 bp)

5

f/

',,

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,,,,~,~~ -~~2e

~

:)

~

~

-"

FIG. 3 Distribution of POMC and PC transcripts in different adult rat pituitary lobes as determined by RNase protection assay. Autoradiogram of RNA samples from neurointermediate lobe (NIL) (2 /zg; lanes 1-4) and AL (3 /zg; lanes 5-8) after hybridization with POMC exon 1/intron A splice junction probe (lanes 1 and 5), PC 1 probe (lanes 2, 4, 6, and 8), PC2 probe (lanes 3, 4, 7, and 8), and RNase A/T1 digestion. Rat cyclophilin cRNA is also included in each lane as a standard to monitor the amount of total RNA applied. The position and length of each protected transcript are shown (arrows indicate the positions of protected PC2 transcripts). Use of POMC splice junction probe allows separate detection of hnRNA and mRNA, by virtue of the different-sized transcripts protected [see Scott and Pintar (25) for details]. Note that multiple probes protecting different lengths of specific mRNA can be incubated with a single RNA sample and subsequently separated by gel electrophoresis (compare lanes 2, 3, 6, and 7 with lanes 4 and 8). The autoradiographic film was exposed for 2 days at -80~ with an intensifying screen.

to investigate the possible mechanisms of how POMC-expressing cells in pituitary cope with altered demand of POMC processing under hormonal regulation, we have established the RNase protection assay to quantify the expression of processing enzymes PC1 and PC2 in the pituitary. Figure 3 shows that in adult pituitary, PC1 probe protects the corresponding transcripts in the appropriate 200-bp band, which are detected in RNA samples derived from both the NIL and AL. In contrast, the protected PC2 transcripts (136 bp) mainly appear in the NIL. This is in agreement with the abovedescribed results on lobe-specific expression of PC1 and PC2 RNA by in situ hybridization experiments. Although both PC1 and PC2 transcripts are apparently expressed in less abundance than POMC, the sensitivity of the assay allows a reliable quantitation of their expression level. This makes it possible to apply this method in conjunction with the short-term tissue culture

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procedure to determine whether the level of expression of PC1 and PC2 mRNA changes in concert with POMC in response to hormonal influence, and whether these responses are established in development.

Conclusions As a sensitive and reliable tool, in situ hybridization allows the visualization of the spatial pattern of gene expression. This technique can be readily applied to the study of the temporal pattern of processing enzyme gene expression in embryogenesis. This is aided by devising a reliable developmental staging system, allowing the determination of the precise age of each embryo under study. To assess the possible contribution of processing enzymes to the maturation process of an individual proprotein, both can be covisualized at the cellular level by virtue of dual labeling of probes for the processing enzyme and the potential substrate. On the other hand, to study the regulation of processing enzyme gene expression, isolation of the tissue of interest away from the in vivo context is usually advantageous. Various regulatory factors can be applied and their effects assayed by a quantitative measure, such as an RNase protection assay. Three particular examples are given to illustrate how an appropriate application of these techniques can advance our understanding of molecular mechanisms of proprotein processing in development.

Acknowledgments The authors are pleased to thank Dr. Nabil Seidah for providing the PC1, PC2, and furin probes, and Dr. Lloyd Fricker for providing the CPE probe used in this work. We also wish to thank Drs. James Roberts, Roderick Scott, Randal Streck, Joseph Cerro, and Teresa Wood for technical advice and assistance during these studies. This work was supported by Research Grants HD-18592 and DA-08622 from the National Institutes of Health to J.E.P.

References 1. R. E. Mains, E. I. Cullen, V. May, and B. A. Eipper, Ann. N. Y. Acad. Sci. 493, 278 (1987). 2. K. H. Cox, D. V. DeLeon, L. M. Angerer, and R. C. Angerer, Dev. Biol. 101, 485 (1984). 3. J. G. Gall and M. Pardue, Proc. Natl. Acad. Sci. U.S.A. 63, 378 (1969).

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I MOLECULARAPPROACHES H. A. John, M. L. Birnstiel, and K. W. Jones, Nature (London) 223, 582 (1969). 5. N. Wanek, K. Muneoka, D. G. Holler-Dinsmore, R. Burton, and S. V. Bryant, J. Exp. Zool. 249, 41 (1989). D. I. Lugo, J. L. Roberts, and J. E. Pintar, Mol. Endocrinol. 3, 1313 (1989). 7. S. Hayashi, I. C. Gillam, A. D. Delaney, and G. M. Tener, J. Histochem. Cytochem. 26, 677 (1978). L. H. Tecott, J. H. Eberwine, J. D. Barchas, and K. L. Valentino, in "In Situ Hybridization: Applications to Neurobiology" (K. L. Valentino, J. H. Eberwine, and J. D. Barchas, eds.), p. 3. Oxford Univ. Press, New York, 1987. P. A. Krieg and D. A. Melton, in "Methods in Enzymology" (R. Wu, ed.), Vol. 155, p. 397. Academic Press, Orlando, FL, 1987. 10. D. G. Wilkinson, in "In Situ Hybridization: A Practical Approach" (D. G. Wilkinson ed.). IRL Press, Oxford, 1992, p. 75. 11. P. J. Barr, Cell (Cambridge, Mass.) 66, 1 (1991). 12. W. J. M. Van de Ven, A. J. M. Roebroek, and H. L. P. Van Duijnhoven, CRC Crit. Rev. Oncogen. 4, 115 (1993). 13. L. D. Fricker, Annu. Rev. Physiol. 50, 309 (1988). 14. M. Zheng, R. D. Streck, R. E. M. Scott, N. G. Seidah, and J. E. Pintar, J. Neurosci., 14, 4656 (1994). 15. B. A. Eipper and R. E. Mains, Endocrin. Rev. 1, 1 (1980). 16. R. Day, M. K.-H. Schafer, S. J. Watson, M. Chr6tien, and N. G. Seidah, Mol. Endocrinol. 6, 485 (1992). 17. S. Benjannet, N. Rondeau, R. Day, M. Chretien, and N. G. Seidah, Proc. Natl. Acad. Sci. U.S.A. 88, 3564 (1991). 18. L. Thomas, R. Leduc, B. A. Thorne, S. P. Smeekens, D. F. Steiner, and G. Thomas, Proc. Natl. Acad. Sci. U.S.A. 88, 5297 (1991). 19. S. M. Sato and R. E. Mains, Endocrinology 117, 773 (1985). 20. J. E. Pintar and D. I. Lugo, Annu. N. Y. Acad. Sci. 512, 318 (1987). 21. K. Zinn, D. DiMaio, and T. Maniatis, Cell (Cambridge, Mass.) 34, 865 (1983). 22. M. Blum, in "Methods in Enzymology" (P. Conn, ed), Vol. 168, p. 618. Academic Press, San Diego, 1989. 23. D. J. Autelitano, M. Blum, M. Lopingco, R. G. Allen, and J. L. Roberts, Neuroendocrinology 51, 123 (1990). 24. R. E. M. Scott, D. J. Autelitano, D. I. Lugo, M. Blum, J. L. Roberts, and J. E. Pintar, Mol. Endocrinol. 4, 812 (1990). 25. R. E. M. Scott and J. E. Pintar, Mol. Endocrinol. 7, 585 (1993). 26. P. E. Danielson, S. Forss-Petter, M. A. Brow, L. Calavetta, J. Douglass, R. J. Milner, and J. G. Sutcliffe, DNA 7, 261 (1988). 27. E. T. Schenborn and R. C. Mierendorf, Jr., Nucleic Acids Res. 13, 6223 (1985). 28. J. L. Roberts, N. Levin, D. Lorang, J. R. Lundblad, S. Dermer, and M. Blum, Handb. Exp. Pharmacol. 104, 347 (1992). ,

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Use of Vaccinia Virus Vectors to Study Neuropeptide Processing Judy K. VanSlyke, Laurel Thomas, and Gary Thomas

Introduction Advances in cell biology have been greatly assisted by the application of vaccinia virus (VV) as an eukaryotic expression vector. Recombinant VV have been used successfully to express foreign proteins from viral, bacterial, protozoan, fungal, and metazoan sources (1, 2). Polypeptides of cellular origin include voltage-gated ion channels, G proteins, cell surface receptors, growth factors, neuropeptides, and proteinases from the regulated and constitutive secretory pathways of eukaryotic cells (3-11). Especially in the study of prohormone processing, VV recombinants have been useful in answering complex biological questions. Recombinants have been used to study the fates of prohormones and prohormone convertases in heterologous cell types (12). Large quantities of the prohormone and proprotein convertase are often produced and this feature has been exploited for purification of these molecules for in vitro analysis (13-16). Recombinant VV have been used in concert to reconstitute proprotein processing pathways in suitable mammalian cells in order to mimic the processes that occur in both neuroendocrine, endocrine, and nonendocrine cells (9-11, 17-20). These vectors have also been used in the study of targeting and localization of proproteins and proteinases in cellular compartments (8, 21-23). A gene transfer method to facilitate the study of the cell biology and enzymology of neuropeptide processing must meet several criteria. First, variability inherent to the expression system should be minimal. Second, reasonably high levels of expression are required to facilitate biochemical characterization of processed peptides (preferably >0.1 pmol/106 cells). Third, a study of tissue-specific processing requires the ability to express the prohormone in a variety of distinct cell types. Methods for gene transfer into mammalian cells can be divided into two general categories: those that result in stable integration of the foreign gene into the genome of heterologous cells, and those that produce transient expression. Each method has unique advantages and disadvantages (12). Expression systems based on random integration of foreign DNA into the host genome are generally subject to clonal variations. These may result from differences in expression of foreign protein as well as phenotypic drift Methods in Neurosciences, Volume 23


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frequently associated with transformed cell lines. Isolation of stable transformants is both a labor-intensive and time-consuming process. Conversely, transient transfection systems do not suffer from clonal variation and results can be obtained expediently. Plasmid transfection efficiencies, however, vary with cell type and frequently low levels of expressed protein are obtained in these types of preparations. Typically, transfection approaches are limited to only a select cell type. By contrast, vaccinia virus does satisfy each of the above criteria. The construction of a vaccinia recombinant is rapid and not difficult. For example, purified recombinant virus can be prepared in less than 2 weeks. The vaccinia genome can accommodate large and/or multiple DNA inserts (24). Foreign genes are inserted by homologous recombination, precluding clonal variations. High levels of foreign protein can be obtained routinely by cloning the cDNAs along with vaccinia promoters (on the order of picomoles per 10 6 cells, using the VV 7.5K promoter) (25, 26). Infection with vaccinia is efficient. Between 80 and 100% of the cells in a population can be made to express the foreign protein. The broad host range of vaccinia allows information to be shuttled readily and rapidly between a variety of mammalian cell types and species. Importantly, experiments can be performed with primary cultures as well as established cell lines. Because cells can be infected simultaneously with multiple vaccinia recombinants, interactions among a group of foreign proteins can be studied in a defined cell system. Finally, vaccinia expression experiments can be performed within 24 hr. The development of gene transfer strategies has greatly enhanced the elucidation of the function of many molecules in higher eukaryotic cells. The goal of this chapter is to describe one gene transfer approach, recombinant vaccinia virus, in understanding the cell biology and enzymology of neuropeptide processing.

Vaccinia Virus Life Cycle The reasons why VV is a favorable expression vector become apparent when one considers the unique features surrounding the viral life cycle and genetic makeup. Vaccinia virus, a member of the orthopoxvirus family, is a doublestranded DNA (dsDNA) virus that replicates solely within the cytoplasm of an infected host cell (27). The 192-kbp genome encodes approximately 198 polypeptides that are expressed in a regulated fashion throughout the virus life cycle (28). After the virus enters the cell, partial uncoating of the virion takes place and early gene expression begins. The enzymes necessary for transcription (29) are packaged inside the virus particle and carried into the host cell during infection. Early gene expression produces the enzymes

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necessary for DNA replication and intermediate gene expression. As the life cycle proceeds, a second and complete uncoating event takes place, releasing the genomic material. As DNA synthesis begins, early gene expression ceases and intermediate genes are transcribed and translated. The polypeptides synthesized from these open reading frames include the transcription factors necessary for late gene expression. Subsequently, late genes are transcribed and translated, producing the constituents necessary for the virus particle formation.The infectious life cycle of vaccinia virus results in the formation of mature virions within the cytoplasm, referred to as intracellular enveloped virus (IEV), and extracellular enveloped forms (EEV) outside the host cell. Both forms of the virus are infectious, but EEV is thought to be the chief cause of cell-to-cell spread. The IEV form, however, is the one usually manipulated for expression vector work and the strains used in most laboratories produce large amounts of this virus (27).

Strategies for Making Vaccinia Virus Expression Vectors Recombinant DNA technology can be applied so that recombinant VV vectors can be tailored specifically for the desired application. For instance, VV recombinants can be constructed that will produce large amounts of a desired protein during viral infection (30, 31) or express a foreign gene only when an appropriate inducer is present (32, 33). Generally, a strategy for making a VV expression vector begins with selection of a recombinant plasmid that contains an appropriate viral promoter and genomic sequences that will allow homologous recombination to take place with the viral genome. [An example of such a plasmid, pZVneo (34), is represented in the top part of Fig. 1.] Generally, the promoter from the 7.5K gene of VV has been used in most expression vectors because it directs the efficient production of the gene of interest throughout the viral life cycle (35). Another important aspect to consider before making a recombinant VV is the method(s) for selecting the new virus away from the parental wild type. Insertional inactivation of a viral gene results in a loss of function that in some cases can be used as a means for selecting a recombinant. For instance, inactivation of the thymidine kinase (TK) gene can be selected for by growing virus in the presence of bromodeoxyuridine (BUdR), a lethal metabolite, in the presence of an active TK enzyme (36). Similarly, insertion into the hemagglutinin (HA) gene can be screened for by using an agglutination assay of erythrocytes (37). Most commonly the positive recombinant is selected out of a background of wild-type VV by screening thousands of plaques with a radioactively labeled DNA probe. With the development of liposomes for delivering plas-

68

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mid DNA into an infected host cell, the initial step in producing a recombinant virus (marker transfer) is easier and the transfer of DNA is more efficient (38). More powerful methods for selecting recombinants have been developed (2, 35). Genes that confer resistance to antibiotics or other drugs or have a reporter function have been used to select recombinant VV. To use this mechanism of selection to make a recombinant virus that expresses a foreign gene, dual cassette plasmids have been constructed and used (Fig. 1). Plasmids that contain two open reading frames, one being the gene for expression behind the VV 7.5K promoter and the other being the resistance or reporter gene under the direction of another vaccinia promoter, are flanked by portions of a VV gene. The promoters are abutted so that transcription is driven in opposing directions. These plasmids have been used successfully in constructing recombinant VV; selectable markers such as the neomycin transferase (39), guanine phosphoribosyltransferase (40), and fl-galactosidase genes (41) have improved the process of isolating the recombinants from parental virus. Described in detail below is a method for making a VV expression vector using the neomycin resistance gene in the recombinant plasmid pZVneo and selection with the antibiotic G418 sulfate (Geneticin; GIBCO-Bethesda Research Laboratories, Gaithersburg, MD). A schematic diagram of this procedure is represented in Fig. 1; however, procedures for growing, isolating, and manipulating vaccinia virus will be described initially to familiarize the reader with basic techniques that will be used throughout the process of isolating a recombinant virus.

Vaccinia Virus Methodology

General Vaccinia Virus Maintenance It is important to remember that vaccinia virus, although relatively harmless, is still a human pathogen. However, with a few safety precautions, potential hazards can be easily avoided. Virus can be killed with bleach, surfaces that have been near virus (such as counter tops and pipetting instruments) should be disinfected with Lysol, and disposable items should be autoclave sterilized. When working with virus, the researcher should always wear gloves, a laboratory coat, and protective eyewear. Most research institutions have guidelines for working with an infectious agent. The National Institutes of Health (NIH, Bethesda, MD) recommends routine vaccinations for laboratory personnel and safety level 2 procedures must be followed (42). With these guidelines in mind, the following protocols can be employed to maintain and utilize vaccinia virus in the laboratory.

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Infection of Cells with Vaccinia Virus Although stock preparations of vaccinia virus may vary, the basic protocol for infecting cells is the same. The general procedure is outlined below, but the variations will be described when appropriate. 1. Count the cells on a replicate plate: The cells are trypsinized from the plate, diluted with medium, and counted under the microscope with a hemocytometer. 2. Calculate the amount of virus to use to give a desired multiplicity of infection (MOI), which is recorded in plaque-forming units (PFU) per cell: With the known cell count, the titer of the virus (described below), the amount of virus to use can be calculated as follows: (No. of cells/plate)(PFU/cell)(103/A/ml) = virus (/A)/plate Viral titer (PFU/ml) 3. Prepare viral inoculum: A virus stock is thawed and sonicated (use a bath sonicator) for two 8- to 10-second intervals to disrupt aggregates (be careful not to overheat; place on ice intermittently). The appropriate amount of virus is diluted in PBS + MB [phosphate-buffered saline plus 1 mM MgC12 plus 0.1% (w/v) bovine serum albumin]. The thawed virus stock is stored at 4~ for 4-8 weeks and should be sonicated briefly before each use. 4. Infect cells with diluted virus: The cells are washed with PBS+M (phosphate-buffered saline plus 1 mM MgCI2), warmed to 37~ and overlaid with viral inoculum (just enough to cover the plate, i.e., 0.5 ml for 35- or 60-mm plates, 1.5 ml for 100-mm plates, and 5 ml for 150-mm plates). The cells are kept at room temperature for 30 min (or in some cases the cells are placed at 31~ for 2 hr); rocking at least once during the incubation is advisable to ensure that the cells remain covered with liquid. 5. Replace the medium: The viral inoculum is removed and the cells are washed once or twice with PBS+M and overlaid with culture medium, prewarmed to 37~

Large-Scale Preparation of Partially Purified Vaccinia Virus Most manipulations of vaccinia virus are executed from a central stock of partially purified virus. This form of the virus can be diluted easily and prepared for inoculating cells. To make a sizable quantity of virus, four confluent 150-mm plates of BSC-40 cells are routinely used for growth of the virus. 1. Infect plates at an MOI of 0.01 PFU/cell: Viral inoculum is prepared


71

in 5 ml of PBS +MB per plate. The cells are watched between day 2 and 3 postinfection to ensure that they are harvested when approximately all of the cells have rounded up, but are still attached to the plate. 2. Harvest the infected cells: The cells should detach easily when gently triturated with the surrounding medium, using a serological pipette, but if they are still attached to the plate they can be scraped with a sterile rubber policeman. This procedure should be done gently to avoid cell rupture. 3. Pellet and wash the infected cells: The cells suspended in medium are pelleted in 50-ml conical tubes at 200 g for 5 min. The medium is removed and discarded, the cells are resuspended in 10 ml (per pellet) of PBS+M, collected in one tube, and centrifuged again. All of the following procedures are performed at 0-4~ 4. Hypotonically swell and disrupt the cells: After the wash solution is removed and discarded, the cells are resuspended in 10 ml of cold 10 mM Tris-HC1, pH 9, placed on ice for 10 to 30 min, and resuspended occasionally. This step causes the cells to swell and makes them more susceptible to mechanical rupture. The suspension is placed in a sterile Dounce (Wheaton, MiUville, NJ) homogenizer, with a tight pestle, and the cells are disrupted with 25 strokes, keeping the apparatus on ice the entire time. 5. Pellet the nuclei: After the cells are ruptured, the suspension is placed in a 15-ml conical tube and centrifuged at 800 g for 5 min at 4~ to pellet the nuclei. The resulting supernatant should be milky in appearance with a soft, white pellet at the bottom. The supernatant is transferred to a new tube, without disturbing the pellet. The pellet is then resuspended in 2 ml of 10 mM Tris-HC1, pH 9, and subjected to 25 additional pestle strokes. The mixture is recentrifuged and the supernatant removed and added to the portion collected from the first spin. 6. Pellet VV through a sucrose cushion: Two ultracentrifuge tubes (14 x 89 mm), each containing 6 ml of 36% (w/v) sucrose (in 10 mM TrisHC1, pH 9) and chilled to 4~ are carefully overlaid with the collected supernatant, placing 6 ml on each sucrose shelf. The tubes are balanced and placed in an SW41 rotor (prechilled) and centrifuged at 18,000 rpm for 80 min at 4~ 7. Harvest the virus pellet: The interface between sample and sucrose, which contains cell debris, is removed and discarded first so that it does not mix the pellet. The rest of the supernatant is discarded and the pellet that contains virus is resuspended in 800 ~1 of 10 mM Tris-HC1, pH 9. 8. Homogenize the virus suspension: The viscous suspension is placed in a sterile Duall homogenizer (Kontes Glass, Vineland, NJ) and the centrifuge tube is then washed with 100/~1 of 10 mM Tris, pH 9, which is added to the homogenizer. The virus mixture is homogenized with eight strokes of the pestle (the viscosity of the solution makes this difficult) to disrupt aggregates

72


and returned to the ultracentrifuge tube. The homogenizer is then washed with 100/zl of 10 mM Tris, pH 9, and this is added to the partially purified virus stock. 9. Aliquot the partially purified virus preparation: The virus is dispensed into 25- to 40-/xl aliquots and stored at -70~

Titering Virus Stock To determine the titer of a particular vaccinia virus stock, confluent cell monolayers are needed for infection with various dilutions of the virus. Sixtymillimeter dishes are sufficient for determining viral titer. 1. Make a series of 10-fold dilutions of the viral stock: Ten microliters of a partially purified virus stock is placed into 990/zl of PBS + MB, sonicated, vortexed, and labeled as a 10-2-fold dilution. From this solution, virus is diluted sequentially in 10-fold increments 8 more times, vortexing between each dilution. 2. Infect confluent monolayers with the diluted virus solutions: Confluent monolayers are covered with 0.5 ml of three sequentially diluted virus solutions (e.g., 10 -7, 10 -8, and 10 -9 from a partially purified virus stock; 10 -6, 10 -7, and 10-8 from a crude virus stock). For more accurate determinations, the infections should be done in duplicate. Thirty minutes after inoculation, medium is replaced and the cells are incubated as previously described. 3. Approximately 36 hr postinfection, stain the plaques formed on the monolayers: The medium from each well or plate is removed and the cells are covered with 1 ml of a 0.5% (w/v) methylene blue solution in 50% (v/v) methanol. After about 5 min, the stain is removed and cells are gently rinsed two or more times with a 10% (v/v) solution of methanol until plaques are easily visible. 4. Count plaques and calculate the viral titer: A plate that has at least 30 plaques on it (but not more than 100) is selected for counting. After the number is determined, the titer is calculated as follows: (No. of plaques/0.5 ml)(2)(1/10 -n) = No. of plaque-forming units/ml where n is the dilution factor of the viral inoculum used on the monolayer of cells counted (step 2 above) and the number 2 corrects the value to a 1-ml volume.

Isolation of Vaccinia DNA Often it is necessary to obtain a large preparation of vaccinia virus genomic DNA. The following procedure describes isolation of VV DNA from one 100-mm plate of virally infected cells, but it can be scaled up for bigger preparations.

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VACCINIA VIRUS VECTORS

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1. Infect a 100-mm plate of confluent BSC-40 cells with VV at an MOI of 0.01. Virus is diluted into a 1.5-ml inoculum volume for overlaying the cells. 2. Remove the inoculum and replace with medium warmed to 37~ 3. Two days after infection, harvest the infected cells: Remove cells as described for making a large virus preparation, depending on whether they are floating or still attached to the plate. 4. Pellet the infected cells and resuspend them in 600/zl of PBS+M in a microfuge tube, using the same procedure as previously described. 5. Swell the cells by adding the following reagents to the cell suspension and place on ice for 10 min: 30/zl of 10% (v/v) Triton X-100, 1.5 ~1 of 2-mercaptoethanol (2-ME), 48/xl of 250 mM ethylenediaminetetraacetic acid (EDTA), pH 8. (Mix the reagents together first, then add to cells.) Vortex occasionally. 6. Pellet the nuclei in a microcentrifuge for 2 min at 3000 rpm. 7. Pellet the virus: The supernatant is removed (avoiding the pellet) to a new microfuge tube and centrifuged at 15,000 rpm for 10 min. 8. Gently resuspend the virus pellet in 100/zl of TE buffer, pH 8 (10 mM Tris-HCl, 1 mM EDTA, pH 8). Do not vortex the sample from this step forward. 9. Incubate the resuspended virus pellet with proteinase K. The following reagents are added to the virus suspension and gently mixed by flicking or rocking the tube: 1.5 tzl of a 10-mg/ml stock of proteinase K, 6.7/zl of 3 M NaC1, 0.3/xl of 2-mercaptoethanol, 10/xl of 10% (w/v) SDS. The solution is incubated at 50~ for 30 min and mixed occasionally. 10. Extract the mixture twice with phenol-chloroform (CHC13)-isoamyl alcohol (IAA) (made at a ratio of 25:24:1, v/v). a. An equal volume of phenol-CHC13-IAA is added to the mixture. b. The tube is shaken vigorously by hand 50 times. c. The suspension is centrifuged at 15,000 rpm for 2 min. d. The top layer is removed (slowly and avoiding the interphase) and placed in a new microfuge tube. Steps a through d are repeated with this solution. 11. Precipitate the DNA with ethanol and NaC1 twice. a. Four/zl of 3 M NaC1 and 250/zl of cold 100% ethanol are added to the solution (top layer from the last phenol extraction) and the mixture is mixed and placed at -70~ for 30 min. b. The precipitated DNA is centrifuged at 15,000 rpm for 15 min and the supernatant is removed and discarded (avoiding the pellet). c. The DNA is resuspended in 100/xl of distilled water and precipitated again with 5 ~1 of NaC1 and 250/xl of ethanol. Steps a and b are repeated.

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12. Rinse the pellet with cold 80% ethanol. a. Five hundred microliters of 80% ethanol is added to the tube with the pellet. The tube is centrifuged at 15,000 rpm for 30 sec and the supernatant is removed. b. The tube is dried completely under vacuum. c. The DNA is allowed to resuspend in 20/~1 of distilled water for at least several hours and is stored at 4~ The preparation is more viscous when there is more DNA present. 13. Determine the DNA concentration by optical density.

Making Recombinant Vaccinia Virus DNA Preparation The recombinant plasmid pZVneo contains a multiple cloning site featuring unique restriction enzyme recognition sites for insertion of the gene that is desired to be expressed. The foreign insert needs to contain a functional start and stop codon and should be oriented so that the 5' end of the open reading frame lies closer to the 7.5K promoter. Selection of recombinant plasmids can utilize the ampicillin resistance gene supplied by the Bluescript (Strategene, La Jolla, CA) backbone or the neomycin resistance gene abutted to the VV 11K promoter. Apparently the VV promoter is recognized by the Escherichia coli RNA polymerase and confers resistance to kanamycin to the bacterium housing the plasmid. This feature is used to select pZVneo away from any other contaminating plasmids (e.g., an uncut version of the plasmid utilized to isolate the insert DNA). After the insert is cloned into the plasmid and the DNA sequence checked for spontaneous mutations, a large-scale preparation of plasmid DNA is made using either CsCI gradients or polyethylene glycol (PEG) precipitation.

Marker Transfer Protocol HeLa or BSC-40 cells are prepared in advance so that they are 50 to 80% confluent in a 35-mm plate. HeLa cells are cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) fetal calf serum (FCS) at 37~ and 5% CO2 and BSC-40 cells are grown under the same conditions except that MEM is used. 1. Determine the number of cells on a replicate plate. 2. Infect cells with wild-type virus (MOI = 0.5 or 1.0): Virus is diluted in 0.5 ml of PBS+MB and sonicated to disrupt aggregates. 3. Prepare the DNA-lipid mixture: Fifteen minutes before the end of


75

inoculation time period (30 min), the lipofectin-DNA mixture is prepared by bringing 5 /~g of plasmid DNA and 1 /~g of wild-type VV DNA (both resuspended in sterile distilled H 2 0 ) up to a total volume of 50/A in water. (VV DNA is included for optimal recombination efficiency, but is not absolutely required.) In a polystyrene tube, 20 ~1 of lipofectin (Cat. No. 8292A; GIBCOBRL) is mixed with 30/A of sterile water. The DNA is then gently added to the lipofectin mix and the tube is gently flicked to mix. This mixture is left at room temperature for 15 min while the liposome-DNA complexes form. 4. Overlay infected cells with the transfection medium: The inoculum is removed and the cells are washed three times with PBS + M warmed to 37~ Nine hundred microliters of the appropriate medium (depending on the cell type) minus serum is added to the polystyrene tube, mixed gently, and overlaid on the infected cells. 5. Allow the transfection to proceed for 3 to 4 hr and then add medium supplemented with serum and G418 sulfate: The cells are placed in the incubator for 3 to 4 hr, at which time 1 ml of the appropriate medium plus 10% (v/v) fetal calf serum plus G418 sulfate (4 mg/ml) is added to the plate (without removing anything first) and mixed gently. The plate is returned to the incubator. The concentration of G418 sulfate refers to total weight. Although the active weight (recorded on the label) varies from bottle to bottle, it usually comprises ->50% of the total weight. The antibiotic is added to medium, allowed time to dissolve, and the medium is sterilized by passing through a syringe filter (0.22-/~m pore size) before applying to infected cells. 6. Harvest the cells from the marker transfer step: After 24 hr, if the cells are still adherent to the bottom of the plate, they are scraped up with a sterile rubber policeman. Cells are collected by centrifugation as previously described, the medium is removed, and the cells are washed with 2 ml of PBS+ M before centrifuging again. All procedures are performed at 4~ 7. Prepare a crude stock: Cells are resuspended in 0.5 ml of PBS+M and a crude stock is prepared by putting the suspension through three freeze/ thaw cycles with liquid N2 and a 37~ water bath. The crude virus stock should be stored at -70~ until use. At this point the crude stock will have an approximate titer of 1 • 105 PFU/ml and recombinant viruses should constitute approximately 5% of the population. A marker transfer reaction without pZVneo present can be included and carried along through the process of selecting a recombinant virus to ensure that the drug selection is working.

Drug Selection The crude viral stock is grown in BSC-40 cells in the presence of the G418 sulfate (2 mg/ml) to select for viruses that contain the neomycin resistance gene.

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1. Infect cells at an MOI of 0.01 from the crude stock made from the marker transfer reaction: A 35-mm dish with a confluent monolayer (cell count known) is infected in the same way as described before for the marker transfer procedure. 2. Overlay infected cells with medium containing G418 sulfate: MEM plus 10% (v/v) FCS plus G418 sulfate (2 mg/ml) is prepared as previously described. After the viral inoculum has been on the cells for 30 min, it is removed and the cells are washed with PBS+M warmed to 37~ which is replaced with medium containing antibiotic. 3. Harvest the cells and make a crude stock: After 2 or 3 days, if cytopathic effects of the viral infection are clearly visible (cells are rounded up), cells can be harvested in the same way as described for the marker transfer protocol. At this point the titer of the virus stock will be around 5 x 10 6 PFU/ml and between 30 and 50% of the population will be bona fide recombinants. Titering the crude stocks is recommended to ensure that the drug selection is working and to determine titers for calculating the amount to use in generating a specific multiplicity of infection, but estimated titers can be used so that the selection process is not delayed. Another round of growth in the presence of the antibiotic will increase the titer and the proportion of recombinants in the total virus population. However, if time is of the essence, this is not absolutely necessary.

Methods for Plaque Purification of Recombinant Virus An isolated plaque should represent a clonal population of virus originating from a single infectious virion. With this in mind, the next step in purification involves generation of well-isolated plaques and isolation of the recombinant virus. If G418 sulfate selection is used, plaques are isolated through agarose overlays containing antibiotic. If drug selection cannot be used, isolated plaques are screened and recovered from filter lifts prior to a final purification from agarose overlays. Both methods are described here. Isolation from under Agarose Overlays At this point, the crude stocks that have been passaged through cells in the presence of G418 sulfate can be plated on cell monolayers for plaque purification. Confluent monolayers of BSC-40 cells in 100-mm dishes are prepared. 1. Calculate the amount of viral inoculum to use: Keeping in mind both the titer of virus in the last crude stock and the percentage of the population

[4]

2.

3.

4.

5.


77

that is actually recombinants, dilutions are made so that about 50 recombinant viruses will infect 1 plate. (It is best to try plating out several different dilutions to ensure that there will be enough plaques.) Infect confluent monolayers with virus from the crude stock produced at the last drug selection step: The infection protocol is performed as already described. Prepare agarose medium overlay containing G418 sulfate. a. Agarose mixture: SeaPlaque agarose (FMC, Philadelphia, PA) is mixed with distilled water (0.12 g/5 ml) and sterilized by autoclaving for 15 min only. [A larger sterile preparation of 2.4% (w/v) agarose can be made ahead of time and stored until needed. Once autoclaved, agarose can be melted in a microwave.] b. Medium mixture [20% (v/v) FCS, G418 sulfate (4 mg/ml)]: Prepare 4 ml of 2x strength sterile MEM plus 1 ml of fetal calf serum plus 20 mg of G418 sulfate (dissolved completely). Filter sterilize and place in a 37~ water bath. c. Mix agarose and medium in a 1" 1 ratio" Once the agarose solution is cooled to about 45~ it is mixed with the above medium. After removing the viral inoculum and washing the cell monolayer, 8 ml of medium plus agarose is carefully layered over the cells. The agarose is allowed to solidify at room temperature (without rocking the plate) and placed in the 37~ incubator. After 48 hr, stain the plaques grown under agarose: Prepare sterile molten 1% (w/v) SeaPlaque agarose in PBS. After the solution is cooled to 45~ a sterile stock solution of Neutral Red is added (final concentration is 0.02%, v/v). The agarose is carefully overlaid on the preexisting agarose, beginning in the center of the plate. Again the plate is allowed to sit at room temperature for 5 min before transferring to a 30~ incubator. The plaques are usually visible within 2 hr, but selection can be done as late as the next day. They will appear small (if compared to virus grown under agarose without drug selection) but only the recombinant virus should grow in the presence of the drug. Pick plaques from under the agarose overlay: Tubes containing 0.25 ml of PBS§ are prepared for as many plaques as will be selected. A pipettor (20-200 /~1, with the dial set at 20 /~1) is used and the tip is exchanged between each selection. Virus is isolated by punching the pipette tip through the agarose directly above a well-isolated plaque and suctioning up and down gently a few times, trying to capture a little of the cell layer at the edge of plaque. The pipette tip is then placed in a tube of PBS+MB and the solution is used to rinse the tip by pipetting action. Clearly established plaques should be preferentially selected. Isolated plaques should be frozen and thawed three

78

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times and stored at -70~ immediately.

if not taken through an amplification step

Isolation from Filter Lifts Confluent monolayers of BSC-40 cells in 100-mm dishes are required for this procedure. 1. Infect monolayers with virus from the crude stocks from the marker transfer step or the drug selection step: Infect cells as previously described with a dilution that will generate around 100 plaques/plate. 2. Replace viral inoculum with medium: Remove viral inoculum, wash cells with PBS+M warmed to 37~ and overlay with MEM plus 10% (v/v) FCS. 3. After 48 hr, stain the cells with Neutral Red: Prepare a room temperature solution of PBS plus 0.01% (v/v) neutral red and add 5 ml directly to the medium on plates of infected cells. Transfer the plates to a 30~ incubator. 4. Transfer plaques to a filter. a. Prepare 100-mm dishes for storing filter lifts: Invert the dishes and place a Whatman (Clifton, NJ) 3MM filter circle in the lid. Add 1 ml of PBS + M to each filter. b. Remove medium from the culture plate. c. Carefully place a nylon filter on the monolayer of cells. Do not let it slip after contact with the plate surface. Press a Kimwipe dampened with PBS+M on any area of the filter that is dry and not absorbed to the cell monolayer. d. Lift the filter off the plate and place it plaque side up on the moistened Whatman circle. e. Immediately make a replicate filter lift: Place a replicate nitrocellulose filter on the plaque side of the nylon filter and press them together. (Again, do not move after contact has occurred.) With a paper punch, make holes in the paired filters for orientation later. With forceps, separate the two filters and place the nitrocellulose filter on the Whatman circle in the dish and place the nylon filter on paper toweling to dry. Seal the dish containing the filter with Parafilm and store at -70~ 5. Prepare nylon filter lifts for hybridization. a. Float the filters on denaturing solution for 10 min. Denaturing solution: 1.5 M NaC1, 0.5 M NaOH

[4]


79

b. Float the filters on neutralizing solution for 2 min. Repeat. Neutralizing solution: 3.0 M NaCI, 0.5 M Tris-HC1, pH 7.5 c. Wash the filters (submerge) briefly in SSC. SSC: 0.3 M NaC1, 30 mM sodium citrate d. Let the filters air dry, then bake them for 20 min at 80~ e. Incubate the filters in proteinase K buffer plus proteinase K (50 ~g/ ml) enough to cover the filters, at 50-55~ for 30 min. Proteinase K buffer: 100 mM Tris-HC1, pH 8, 150 mM NaC1, l0 mM EDTA, 0.2% (w/v) SDS. Proteinase K is added just before incubation period 6. Prehybridize the filters for 2 to 4 hr at 37~ Prehybridization solution: 50% (v/v) deionized formamide, 1 M NaC1, 10% (w/v) dextran sulfate, 1% (w/v) SDS, 25 /~g of sheared salmon sperm DNA (heated to 100~ for 10 min immediately before adding to the prehybridization solution) 7. Hybridize overnight at 37~ Add denatured radioactive probe (randomprimed DNA labeling kit; Boehringer Mannheim, Indianapolis, IN) directly to prehybridization solution. 8. Wash the filters in the following manner: a. Submerge in SSC at room temperature for 10 min. Repeat. b. Incubate in SSC plus 1% (w/v) SDS at 65~ for 30 min. 9. Dry the filters. Mark orientation marks with luminescent dye. Place under X-ray film. 10. Isolate "positive" plaques from the nitrocellulose filter. a. Line up a positive signal on the film with a plaque on the nitrocellulose filter. b. With a sterile paper punch or razor blade, excise the area containing the plaque and put it in a tube containing 200 ~1 of PBS+MB. c. Freeze/thaw the preparation three times and store at -70~ if not proceeding through the amplification step immediately.

Amplification of Plaque-Purified Vaccinia Virus Isolate When the plaques are ready for selection, a 24-well dish or 100-mm plates should be ready with confluent monolayers of BSC-40 cells. 1. Infect the 24-well plate with isolated plaques.

80


a. From agarose overlay: After all the desired plaques are selected, the medium on the 24-well plate is aspirated. A 1-ml pipettor is used to homogenize the agarose plug in PBS + MB, by passing it up and down through the pipette tip multiple times, and deliver the solution to an individual well. The tip is exchanged after each inoculation and each well is inoculated with a different viral isolate. b. From filter lift: Infect 1 well of a 24-well plate with 100/xl of the frozen and thawed viral inoculum. If a larger crude stock is desired, dilute 5 to 20/xl of the viral inoculum up to 1.5 ml with PBS+MB and infect a 100-mm dish of cells. Replace with regular medium. 2. Replace viral inoculum with medium containing antibiotic. a. From a 24-well plate: Prepare MEM plus 10% (v/v) FCS plus G418 sulfate (2 mg/ml). After 30 min, the viral inoculum is removed from each well, using a Pasteur pipette with a 20- to 200-/zl pipette tip on the end. The tip is replaced after each aspiration. The cells are then overlaid with medium containing G418 sulfate, avoiding any crosscontamination between the wells. The infections are allowed to proceed for 2 days before cell harvest. b. From a 100-mm plate: Remove the viral inoculum and replace with regular medium warmed to 37~ 3. After 2 days, harvest the cells and make crude stocks: Prepare crude stocks of the virally infected cells in the 24-well plate. Aspirate the medium (using a new pipette tip each time), overlay the cells with 0.3 ml ofPBS+ M per well, and perform three freeze/thaw cycles by placing the plate alternately at -70 and 37~ Screen for desired recombination events and purity of the virus stock by methods described below.

Screening for Recombinants and Purity Multiple crude stocks are screened for the presence of recombinant VV and the purity of the stock. Four methods of doing this are discussed below. Slot (Dot)-Blot Screening A portion of the crude stocks (100/xl) is transferred to a nylon filter through a slot-blot manifold and standard Southern analysis (43) procedures are followed to screen for the presence of the insert DNA. This screening method will identify crude stocks containing recombinant virus and the intensity of the radioactive signal will indicate the extract of enrichment. It will not show if the recombinant virus stock is still contaminated with wild-type VV. DNA Isolation and Southern Analysis Purity of the crude stock can be determined by analyzing the genomic VV DNA. A large preparation of VV DNA is isolated from an infected 100-mm plate, cleaved with the appropriate restriction enzyme (usually HindlII), and


81

subjected to electrophoresis in a 0.7% (w/v) agarose gel (43). Wild-type DNA is included for comparison so that the change in band patterns can be examined. The DNA is transferred to a nylon filter and subsequently probed with appropriate radiolabeled nucleic acids to determine the presence of foreign DNA and absence of wild-type viral DNA.

Immunoblot Analysis Another method of screening for the desired recombinants requires immunological reagents that recognize the foreign protein expressed from the recombinant virus in an immunoblot analysis. If this method is used, a replicate infected 24-well plate is preferable so that the cells can be harvested directly in a buffer compatible with SDS-polyacrylamide gel electrophoresis (43). This method may also indicate which stocks are at higher titers than others. However, virus purity is not discernible with this approach.

Polymerase Chain Reaction Analysis The last method outlined for screening multiple virus stocks utilizes the sensitivity and speed of the polymerase chain reaction (PCR) (43, 44). DNA is isolated from crude stocks by the method described for the VV DNA isolation procedure, beginning with the proteinase K treatment. Three primers, one complementary to the 5' end of the TK gene, one complementary to the 3' end of TK, and one complementary to the foreign DNA of interest, are used to prime the PCR reaction (Fig. 2A). The primers are oriented such that if the TK gene has not been inactivated, a small fragment will be generated. If foreign DNA had been inserted then the primer corresponding to the 3' end of the TK gene and the insert primer will amplify a somewhat larger PCR product. The product that would be made from the two TK primers across the insert is much too large to occur efficiently. Therefore, a putative recombinant viral stock could potentially generate a small product, a larger product, or both, as seen in the samples screened and analyzed by gel electrophoresis (Fig.2B). The presence of both a smaller and a larger PCR product indicates either that the crude stock still has wild-type VV or that a single-site cross-over event has occurred during homologous recombination (e.g., Fig. 2B, lane 2). However, a sample that generates only a larger PCR product represents a purified recombinant virus stock (e.g., Fig.2 B, lanes 3 and 4). The differential size is important so that the results are easily visualized on an agarose gel. The PCR products are all kept around or under 1 kb to maintain efficiency, with the band that denotes the presence of wildtype VV DNA being the smallest. This ensures the sensitivity for the presence of contaminating wild-type virus.

82

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FIG. 2 Polymerase chain analysis ofVV DNA from putative recombinant virus crude stocks. (A) Potential VV DNA templates present in a crude stock of recombinant virus are shown with the three primers (discussed in text) aligned next to their complementary regions within the DNA. The arrows indicate the direction in which DNA synthesis will occur. Filled bars below the template and primers represent the potential products generated by PCR when either VV DNA is present (**, recombinant VV DNA specific product; *, wild-type VV DNA-specific product). (B) The dsDNA products generated by PCR reactions containing plasmid DNA (pZVNEO construct) used to make the recombinant virus (lane 1), VV DNA from three different crude stocks of recombinant virus (lanes 2-4), or wt VV DNA (lane 5) are separated on a TBE-agarose gel. The positions of nucleic acid markers are indicated by size (in bp) on the left-hand side of the photograph and the two potential PCR products are marked as noted in (A) on the right.


83

Experimental Strategies to Investigate Neuropeptide Processing Using Vaccinia Recombinants To illustrate the utility and efficacy of vaccinia technology in addressing the cell biology and enzymology of neuropeptide processing, a summary of the work that utilized this gene transfer vector to elucidate many of the molecular mechanisms that govern the tissue specificity of proopiomelanocortin (POMC) processing is presented. The studies are divided into three sections, each exploiting features unique to the vaccinia expression system. First, we show the use of vaccinia technology to assess the ability of heterologous cell types to mimic the tissue-specific processing of POMC. Second, we show how vaccinia can be used to express multiple foreign genes simultaneously to permit study of their interactions. Specifically, we show the reconstitution of POMC processing by the PC2 and PC3 (also called PC1) proprotein convertases. Third, we show how the vaccinia vector can be used to express significant levels of bona fide prohormone to use as a substrate for prohormone processing in vitro.

Expression of Proopiomelanocortin in Heterologous Cells Using a Vaccinia Recombinant To identify heterologous cell types that mimic the tissue specificity of POMC processing as it occurs in the anterior (AL) and neurointermediate (IL) lobes of the pituitary (Fig. 3), a VV recombinant that directs the synthesis of mouse POMC (VV : mPOMC) was constructed and used to express the prohormone in a variety of endocrine and nonendocrine cell types (18). The cell lines studied include several nonendocrine [e.g., BSC-40 (African Green monkey kidney epithelium) and Ltk- (mouse fibroblast)] and several endocrine and neuroendocrine [e.g., G H 4 C 1 (rat somatomammotroph), NG108 (mouse neuroblastoma x rat glioma), Rin m5F (rat insulinoma)] cell lines. In addition, the expression and processing of POMC was studied in primary cultures of bovine adrenomedullary (BAM) cells. For most cell lines, infections were performed as described in General Vaccinia Virus Maintenance, above (MOI = 5). However, for some cell types (e.g., Rin m5F and BAM), the inoculum was applied to the cells for 2 hr at 31~ and then the infected cultures were refed with medium containing 2% (v/v) serum. The efficiency of infection in all cases is high; between 80 and 100% of the cells in a culture will express the foreign protein. After 16 hr the media were removed and frozen and the rinsed cells were extracted with 1 M acetic acid (pH 1.9). The level of POMC expression as measured by an adrenocorticotropin (ACTH)

84

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FIG. 3 Cell type-specific processing of mouse preproopiomelanocortin (mPOMC). Shown are the primary POMC peptides produced in pituitary anterior lobe corticotrophs and neurointermediate lobe melanotrophs. Also depicted are the primary POMC processing products produced in heterologous cells expressing mPOMC with a vaccinia vector. Cell types that express only the prohormone ("maturation deficient") include BSC-40, Ltk-, HeLa, PC12, GH4C1, and P388D1 (an interleukin 1-secreting monocyte line). Primary cultures of bovine adrenomedullary chromaffin cells (BAM) process mPOMC just as do anterior lobe corticotrophs, whereas the rat insulinoma


85

radioimmunoassay (RIA) ranged between 0.7 and 3.7 pmol of ACTH per 106 cells in cell extracts and between 5 and 22 pmol of ACTH per 106 cells in the medium samples, depending on the cell type examined. Peptide analysis showed that vaccinia-mediated expression of POMC in a number of cell lines including BSC-40, NG 108, and GH4C 1 resulted in the efficient production of bona fide POMC (see, e.g., Fig. 4A) (8, 45). Studies of stimulated secretion showed the prohormone was packaged into the regulated pathway in GH4C 1 cells (8). None of these cell types was capable of processing the prohormone; hence they were termed "maturation deficient" (for neuroendocrine peptide precursors). In contrast, expression of POMC in the rat insulinoma (Rin m5F) resulted in the efficient processing of the ACTH and/3-1ipotropin (/3-LPH) domains in the regulated pathway to sets of peptides reminiscent of neurointermediate lobe melanotrophs. Both the ACTH and/3-LPH domains were efficiently processed to corticotropin-like intermediate lobe peptide (CLIP), 7-LPH, and/3-endorphin (1-31) (8, 45). Expression of POMC in primary cultures of BAM cells resulted in the targeting of the prohormone to the regulated pathway (23). Stimulation of secretion with 3 mM BaCI 2 elicited an 11.5-fold increase in the secretion of POMC immunoreactivity (IR) in 30 min, with greater than 50% of the intracellular contents being released. Peptide analysis showed that the BAM cell endoproteases processed POMC to generate peptides present in anterior lobe corticotrophs; ACTH and/3-LPH were the primary peptide products with limited cleavage of/3-LPH to y-LPH and/3-endorphin (1-31). These studies exemplify several prominent features of the vaccinia system. First, a wide variety of cell types, both established cell lines and primary cultures, can be screened rapidly to assess their processing phenotypes. Second, sufficiently high levels of foreign protein are expressed that a complete characterization of product peptides can be performed from as little as two 150-mm plates of cells (---3 • 107 cells). Third, vaccinia does not affect the ability of the prohormone to be targeted to the regulated pathway, where it is correctly and efficiently processed.

Rin m5F processes mPOMC in a manner similar to the neurointermediate lobe melanotrophs (no detectable processing at Lys232Lys233).( | ) cleavage site; P, phosphorylation; (73), O-linked carbohydrate; ( 9 N-linked carbohydrate. Note that open symbols represent partial glycosylation whereas closed symbols denote complete glycosylation. The amino acid numbering of the prohormone begins with the initiator methionine. Data describing these gene transfer studies have been previously reported (8, 23, 45).

86


retention time (min)

1 0 2 0 3 0 4 0 5 0 6 0 7 0

retention time (mln)


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Reconstitution of Prohormone-Processing Pathways Unique features of vaccinia biology afford a powerful gene transfer approach to assess the ability of several candidate processing enzymes to cleave precursor proteins in the secretory pathway. This approach exploits the maturationdeficient cell lines described above as well as the ability to use VV recombinants to express multiple foreign genes simultaneously in a cell population. Vaccinia offers two strategies for coexpression of multiple foreign genes in heterologous cells. These are either coinfection with multiple vaccinia

FIG. 4 Processing of mPOMC by Kex2, PC3, and PC2 in vivo and in vitro. (A) Replicate 150-mm plates of BSC-40 cells (---2 x 107 cells) were coinfected with VV recombinants expressing VV : mPOMC (2 PFU/cell) and VV : WT, VV : KEX2, VV : mPC3, and VV :hPC2 (each at 5 PFU/cell, 7 PFU/cell total), or VV :mPC3 and VV :hPC2 together (each at 3 PFU/ceI1, 8 PFU/cell total). After 16 hr, the medium was removed and replaced with serum-free minimal essential medium-0.07% (w/v) bovine serum albumin. After an additional 2 hr, this medium was recovered, adjusted to 0.2% (v/v) trifluoroacetic acid, and applied to a C4 reversed-phase column. Retained material was eluted with a gradient of acetonitrile and 1-min fractions were collected and assayed for ACTH (+)-,/3-endorphin (O)-, or y-LPH (O)-immunoreactive material. Elution positions of mPOMC-derived peptides from the mouse corticotroph cell line AtT-20 are indicated. [From Thomas et al. (9); a description of the methods used to characterize each POMC peptide fully is presented in references (8, 9, 16, 18, 19, 23). (B) Conversion of fl-LPH to y-LPH and fl-endorphinl_31 in the regulated pathway. At 36 hr after plating, replicate plates of chromaffin cells were coinfected with VV : mPOMC (2 PFU/cell) and either VV :WT, VV : KEX2, or VV : hPC2, each at 5 PFU/cell (7 PFU/cell total). Sixteen hours after infection, cells were washed and incubated for 2 hr in medium containing cycloheximide at 50/zg/ml. The cells were again washed and incubated 45 min in Ca2+-free balanced salt solution containing cycloheximide with or without 3 mM BaC12. This medium was collected, adjusted to 0.1% (v/v) trifluoroacetic acid, and resolved on the C4 column, as described in (A). Aliquots from 1-min fractions were assayed for fl-endorphin- and y-LPHimmunoreactive material. No corresponding y-LPH IrM peptides were detected in control samples (no Ba2+; top panel). (C) Radioimmunoassay analysis of mPOMC processing products generated by insulin secretory granule endopeptidase activity. mPOMC processing by Triton X-114 aqueous phase-extracted material from insulin secretory granule lysate over 3.5 hr at 30~ was assessed as described (16). The mPOMC products were resolved by Vydac reversed-phase C4 HPLC, 5-/zl aliquots of the 1-ml fractions were assayed by specific RIA to regions of mPOMC, and the total amount of product per fraction was calculated. The elution positions of various POMC processing product standards are indicated. The RIA analysis was for flendorphin (0), y-LPH (O), and ACTH (+).

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recombinants or construction of vaccinia recombinants containing two (dual recombinant) or three (triple recombinant) foreign genes as vectors. Marker transfer plasmids that direct recombination into either the VV HindIII J (e.g., pZVneo), C, or A regions have been reported (35) and each has been used successfully to construct dual or triple recombinants (B. A. Thorne, L. Thomas, and G. Thomas, unpublished results, 1994). In most experiments, however, the use of multiple single recombinants is satisfactory. To begin identification of the mammalian processing endoproteases, we developed a strategy based on knowledge gained from studies in Saccharomyces cerevisiae that provided the first unequivocal identification of the gene encoding a eukaryotic processing endoprotease, KEX2 (46). In our initial studies we showed that coexpression of POMC and Kex2p in any of the maturation-deficient cell lines shown in Fig. 3 resulted in the efficient processing of the prohormone on the C-terminal side of-LysArg- doublets to yield fl-endorphin (1-31) and y-LPH (18) (see, e.g., Fig. 4A, POMC/WT vs POMC/KEX2). The studies on KEX2 showed that this endoprotease is functionally and, thus, structurally similar to the mammalian processing endoprotease(s). Indeed, a number of DNA sequences encoding a family of higher eukaryotic KEX2-1ike endoproteases has been reported (47, 48). The possibility that two of these DNAs, PC2 and PC3, are prohormone convertases became readily apparent because they (a) are KEX2-1ike, (b) are neuroendocrine specific, and (c) their relative RNA distribution in different tissues matched what our gene transfer studies predicted-both AL and BAM preferentially express PC3 RNA compared to PC2 RNA whereas both IL and Rin cells preferentially express PC2 RNA compared to PC3 RNA. Vaccinia recombinants expressing mouse PC3 and human PC2 were constructed as described earlier and were used in expression studies to examine their ability to cleave mPOMC (9). Cells were then coinfected with two or three vaccinia recombinants, using the standard inoculation protocol. The multiplicity of infection was adjusted such that any cell expressing the substrate will also express the enzyme (see caption to Fig. 4A). Depending on the cell type and endoprotease used, processed peptides are isolated from either cell extracts or from the culture medium. For example, with KEX2 sufficient quantities of processed peptide accumulate within cellular compartments to allow peptide identification directly from cell extracts. Similarly, with PC2 expressed in BAM cells, sufficient quantities of processed peptide accumulate in secretory granules to allow peptide identification from cell extracts. By contrast, with PC2 and PC3 expressed in BSC-40 cells, insufficient amounts of cellular peptides accumulate to perform biochemical characterization. Therefore peptides are isolated from the culture medium. For these experiments, secreted peptides are collected either for 16-18 hr in a


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serum- and Phenol Red-free defined medium, MCDB 202, or for 2 hr (beginning 16 hr postinfection) in MEM containing 0.07% (w/v) bovine serum albumin. When secreted material is analyzed, it is critical to perform a mixing experiment as a control to ensure that the processed peptides were generated in the cell by the candidate endoprotease rather than artifactually by extraneous proteases in the medium. Coexpression of POMC and PC3 in BSC-40 cells results in the production of peptides similar to those of anterior lobe corticotrophs; ACTH and/3LPH were the prominent processing products (Fig. 4A, POMC/PC3). Coexpression of POMC and PC2 resulted in the efficient excision of/3-endorphin (1-31) and a processing intermediate containing the ACTH and ),-LPH sequences (ACTH/3,-LPH, open arrowhead) (Fig. 4A, POMC/PC2). However, coexpression of PC3 and PC2 together with POMC in BSC-40 cells resulted in ACTH and an insulinoma-like pattern of cleavages in the/3-LPH domain, with efficient processing to ~/-LPH and /3-endorphin (1-31) (Fig. 4A, POMC/PC3+PC2). The efficient processing of/3-LPH to ~/-LPH and/3-endorphin (1-31) by PC2 in BSC-40 cells suggested that this enzyme is capable of catalyzing one insulinoma (IL) cleavage but the conversion of ACTH to a-MSH was not detected. Therefore we chose to determine whether coexpression of PC2 and POMC in cells containing a regulated pathway (BAM cells) would enable PC2-directed processing of ACTH. In addition, these studies would allow us to determine whether PC2 could cleave POMC in the regulated pathway~a requirement for a prohormone-processing endoprotease. As a negative control, POMC was coexpressed with Kex2p, which also efficiently cleaved/3LPH in BSC-40 cells but, being a yeast enzyme, should not be capable of regulated pathway processing. Finally, these experiments allowed us to determine whether the tissue specificity of prohormone processing can be explained simply by a differential expression of PC3 and PC2. If so, then overexpression of PC2 in BAM cells should result in an insulinoma-processing phenotype. Briefly, replicate plates of BAM cells were coinfected with VV : mPOMC and either VV" WT, VV" KEX2, or VV" hPC2. Sixteen hours postinfection, cells were washed and incubated for 2 hr in medium containing cycloheximide (50/xg/ml). This allowed any processed peptides present in the constitutive pathway to be secreted while those present in the regulated pathway are retained. The cells were again washed and incubated for 45 min in Ca2+-free balanced salt solution containing cycloheximide with or without 3 mM BaC12 (Fig. 4B). This medium was collected and analyzed for POMC peptides by high-performance liquid chromatography (HPLC) (reversed phase and cation exchange) coupled with domain-specific radioimmunoassay. The results show that coexpression of POMC with PC2, but not Kex2p, resulted in

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processing of/3-LPH to y-LPH and /3-endorphin (1-31) in the regulated pathway, demonstrating that PC2 has specialized properties that enable it to be routed to, and function in, this compartment (compare Fig. 4B, bottom, with second and third panels). In addition, PC2 was able to cleave ACTH to a-MSH correctly. The vaccinia reconstitution system has also been used to show that PC3 and PC2 together, and not separately, are capable of cleaving proinsulin correctly when coexpressed in maturation-deficient cells (20). Similarly, PC2 cleaves pro-LHRH to luteinizing hormone-releasing hormone (LHRH) and GAP peptides (49).

Processing o f Vaccinia-Derived Proopiomelanocortin Insulinoma Secretory Granule Type H Activity in Vitro The efficient expression of bona fide prohormone in VV : mPOMC-infected BSC-40 cells (22 pmol of secreted ACTH IR/10 6 cells) affords the ability to prepare significant quantities of POMC to be used as a substrate for processing reactions in vitro. Depending on the requirements, the prohormone can be prepared as a nonradiolabeled [approximately 40/zg of mPOMC can be prepared from 1 x 108 cells (5 • 150-mm plates)] or a radiolabeled [3 x 10 6 cpm are incorporated into mPOMC (4000 cpm/ng)] substrate. Methods describing the production of each have been described (16). To compare cleavage site specificity of the insulin secretory granule type II endopeptidase in vitro with that of the endogenous endoproteases in insulinoma (8) (Fig. 3) and bovine adrenal medullary (BAM) cells (23) (Fig. 3), as well as transfected PC2 and PC3 (9), 10/~g of nonradiolabeled mPOMC was incubated (30~ for 3.5 hr) with an aqueous-phase Triton X-114 extract of insulin secretory granules prepared from an insulinoma propagated in NEDH (New England Deaconess Hospital strain) rats (16). This preparation is highly enriched for the type II (PC2) activity, with lesser amounts of the type I (PC3) activity (---10-30%). The digested sample was resolved by reversed-phase HPLC and the resultant fractions assayed with domain-specific RIAs as previously described (8, 9, 23, 45) [see, e.g., Fig. 4C; Note, compare with the profile of intact POMC shown in Fig. 4A (POMC/WT)]. Analysis showed that the processing in vitro of POMC by the insulinoma PC2 preparation resulted in the excision of a set of peptides highly similar to that produced by Rin m5F cells expressing mPOMC and BSC-40 cells expressing POMC, PC2, and PC3 [compare with Fig. 3 and 4A (POMC/PC3 + PC2)]. Briefly, the major fl-LPH domain peptides identified were 7-LPH and a 7-LPH cleavage product (cleavage at the Leu~~ ~ bond in 7-LPH) as well as native and oxidized fl-endorphin

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(1-31). Thus, like the endogenous Rin m5F cell endoproteases (8) and transfected PC2 (11, 24), the type II endopeptidase efficiently cleaves the Lysl63-Arg 164site at the ACTH//3-LPH and LysZ~ TM y-LPH//3-endorphin junctions but not the LysZ32-Lys233 at the C terminus of/3-endorphin. Prominent ACTH domain peptides included c~-MSH, CLIP, ACTH, and the ACTH/y-LPH processing intermediate. Thus, like PC2 and PC3 expressed in transfected BSC-40 cells (9), the type II activity correctly cleaves at the LyslZZ-Arg 123 site between J peptide/ACTH junction (Fig. 1). Furthermore, like PC2 expressed in transfected cells (9), the type II endopeptidase excised the ACTH/y-LPH processing intermediate from mPOMC.

Acknowledgments The authors thank Dr. Steve Arch for critical reading of the manuscript. This work was supported by NIH Grants DK-44629 and DK-37274. J.V. is supported by NIH Neuroendocrinology Training Grant DK-07680.

References 1. C. Flexner and B. Moss, Annu. Rev. Immunol. 5, 305 (1987). 2. D. E. Hruby, Clin. Microbiol. Rev. 3, 153 (1990). 3 R. J. Leonard, A. Karschin, S. Jayashree-Aiyar, N. Davidson, M. A. Tanouye, L. Thomas, G. Thomas, and H. A. Lester, Proc. Natl. Acad. Sci. U.S.A. 86, 7629 (1989). 4. F. Quan, L. Thomas, and M. A. Forte, Proc. Natl. Acad. Sci. U.S.A. 88, 1898 (1991). 5. A. Karschin, B. Y. Ho, C. Labarca, O. Elroy-Stein, B. Moss, N. Davison, and H. A. Lester, Proc. Natl. Acad. Sci. U.S.A. 88, 5694 (1991). 6. M. Chinkers and E. M. Wilson, J. Biol. Chem. 267, 18589 (1992). 7. R. H. Edwards, M. J. Selby, W. C. Mobley, S. L. Weinrich, D. E. Hruby, and W. J. Rutter, Mol. Cell. Biol. 8, 2456 (1988). 8. B. A. Thorne, L. W. Caton, and G. Thomas, J. Biol. Chem. 264, 3545 (1989). 9. L. Thomas, R. Leduc, B. A. Thorne, S. P. Smeekens, D. F. Steiner, and G. Thomas, Proc. Natl. Acad. Sci. U.S.A. 88, 5297 (1991). 10. P. A. Bresnahan, R. Leduc, L. Thomas, J. Thorner, H. L. Gibson, A. J. Brake, P. J. Barr, and G. Thomas, J. Cell Biol. 111, 2851 (1990). 11. S. Benjannet, N. Rondeau, R. Day, M. Chr6tien, and N. G. Seidah, Proc. Natl. Acad. Sci. U.S.A. 88, 3564 (1991). 12. G. Thomas, B. A. Thorne, and D. E. Hruby, Annu. Rev. Physiol. 50, 323 (1988). 13. K. I. Andreasson, W. W. Tam, T. O. Feurst, B. Moss, and Y. P. Loh, FEBS Lett. 248, 43 (1989). 14. I. Lindberg and G. Thomas, Endocrinology (Baltimore) 126, 480 (1990).

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I MOLECULAR APPROACHES 15. S. S. Molloy, P. A. Bresnahan, S. H. Leppla, K. R. Klimpel, and G. Thomas, J. Biol. Chem. 267, 16396 (1992). 16. C. J. Rhodes, B. A. Thorne, B. Lincoln, E. Nielsen, J. C. Hutton, and G. Thomas, J. Biol. Chem. 268, 4267 (1993). 17. S. Hallenberger, V. Bosch, H. Angliker, E. Shaw, H.-D. Klenk, and W. Garten, Nature (London) 360, 358 (1992). 18. G. Thomas, B. A. Thorne, L. Thomas, R. G. Allen, D. E. Hruby, R. Fuller, and J. Thorner, Science 241, 226 (1988). 19. L. Thomas, A. Cooper, H. Bussey, and G. Thomas, J. Biol. Chem. 265, 10821 (1990). 20. S. P. Smeekens, A. G. Montag, G. Thomas, C. Albiges-Rizo, R. Carroll, M. Benig, L. A. Phillips, S. Martin, S. Ohagi, P. Gardner, H. H. Swift, and D. F. Steiner, Proc. Natl. Acad. Sci. U.S.A. 89, 8822 (1992). 21. E. B. Stephens, R. W. Compans, P. Earl, and B. Moss, EMBO J. 5, 237 (1986). 22. G. Seethaler, M. Chaminade, R. Vlasak, M. Ericsson, G. Griffiths, O. Toffoletto, J. Rossier, H. G. Stunnenberg, and G. Kreil, J. Cell Biol. 114, 1125 (1991). 23. B. A. Thorne, O. H. Viveros, and G. Thomas, J. Biol. Chem. 266, 13607 (1991). 24. G. L. Smith and B. Moss, Gene 25, 21 (1983). 25. G. Thomas, L. Thomas, B. A. Thorne, and E. Herbert, Biochimie 70, 89 (1988). 26. G. Thomas, E. Herbert, and D. E. Hruby, Science 232, 1641 (1986). 27. B. Moss, in "Virology" (B. N. Fields and D. M. Knipe, eds.), Vol. 2, p. 2079. Raven Press, New York, 1990. 28. S. J. Geobel, G. P. Johnson, M. E. Perkus, S. W. Davis, J. P. Winslow, and E. Paoletti, Virology 179, 247 (1990). 29. B. Moss, Annu. Rev. Biochem. 59, 661 (1990). 30. T. Fuerst R. E. G. Niles, F. W. Studier, and B. Moss, Proc. Natl. Acad. Sci. U.S.A. 83, 8122 (1986). 31. O. Elroy-Stein, T. R. Fuerst, and B. Moss, Proc. Natl. Acad. Sci. U.S.A. 86, 6126 (1989). 32. T. R. Fuerst, M. P. Fernandez, and B. Moss, Proc. Natl. Acad. Sci. U.S.A. 86, 2549 (1989). 33. W. A. Alexander, B. Moss, and T. R. Fuerst, J. Virol. 66, 2934 (1992). 34. J. S. Hayflick, W. J. Wolfgang, M. A. Forte, and G. Thomas, J. Neurosci. 12, 705 (1992). 35. P. L. Earl, N. Cooper, and B. Moss, Curr. Prot. Mol. Biol. Supplement 15, p. 16.15.1 (1991). 36. M. Mackett, G. L. Smith, and B. Moss, J. Virol. 49, 857 (1984). 37. H. Shida, T. Tochikura, T. Sato, T. Konno, K. Hirayoshi, M. Seki, Y. Ito, M. Hatanaka, Y. Hinuma, M. Sugimotor, F. Takahashi-Nishimaki, T. Maruyama, K. Miki, K. Suzuki, M. Morita, H. Sashiyama, and M. Hayami, EMBO J. 6, 3379 (1987). 38. P. L. Feigner, T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J. P. Northrop, G. M. Ringold, and M. Danielsen, Proc. Natl. Acad. Sci. U.S.A. 84, 7413 (1987). 39. C. A. Franke, C. M. Rice, J. H. Strauss, and D. E. Hruby, Mol. Cell. Biol. 5, 1918 (1985). 40. F. G. Falkner and B. Moss, J. Virol. 62, 1849 (1988).


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41. S. Chakrabarti, K. Brechling, and B. Moss, Mol. Cell. Biol. 5, 3403 (1985). 42. J. H. Richardson and W. E. Barkley, "Biosafety in Microbiological and Biomedical Laboratories." Health and Human Services Publication (NIH) 88-8395 (1988). 43. J. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual," Vols. 1-3. "Cold Spring Harbor Lab., Painview, NY, 1989. 44. D. R. O'Reilly, L. K. Miller, and V. A. Luckow, "Baculovirus Expression Vectors: A Laboratory Manual," Vol. 1, p. 161. Freeman, New York, 1992. 45. B. A. Thorne and G. Thomas, J. Biol. Chem. 265, 8436 (1990). 46. D. Julius, A. Brake, L. Blair, R. Kunisawa, and J. Thorner, Cell (Cambridge, Mass.) 1075 (1984). 47. D. F. Steiner, S. P. Smeekens, S. Ohagi, and S. J. Chan, J. Biol. Chem. 267, 23435 (1992). 48. P. J. Barr, Cell (Cambridge, Mass.) 66, 1 (1991). 49. W. C. Wetsel, L. Thomas, J. S. Hayflick, H. R. Rivera, N. Lautermilch, and G. Thomas, Endocr. Soc. Abstr. 74, 453 (1992).

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Overexpression of Neuropeptide Precursors and Processing Enzymes Iris Lindberg and Yi Zhou Although the existence of neuropeptide precursors has been recognized for over 10 years, few neuropeptide precursors have as yet been overexpressed. The overexpression of both neuropeptide precursors and their processing enzymes represent important endeavors for several reasons. These studies will allow the production of enough material for protein crystallization. Obtaining sufficient quantities of different neuropeptide precursors and the common processing enzymes will also permit specificity studies using a variety of precursors. Both of these efforts should ultimately provide the information needed to address the general principles underlying differential processing of neuropeptide precursors by a restricted number of processing enzymes. Overexpression of recombinant proteins in a stable mammalian system has many advantages over other methods of overexpression. Mammalian cells contain enzymes capable of performing appropriate posttranslational modifications such as glycosylation and phosphorylation; disulfide bond formation is also correct. These properties mean that the expressed protein is likely to possess biological activity. We have chosen the Chinese hamster ovary/dihydrofolate reductase (CHO/DHFR) system (1) in order to supply milligram quantities of recombinant secreted proteins in a soluble form. One of the advantages of this system is the ability of these eukaryotic cells to make appropriate posttranslational modifications. The final cell line produced represents a stable system, ensuring that multiple harvests can be obtained from the same roller bottles (which provides cost effectiveness). In addition, supertransfection using other selectable markers (such as G418) is feasible using this method; it is thus possible to add other proteins to the cell line already overexpressing one protein. Another advantage of the CHO/DHFR system is that the final result is a series of cell lines each expressing higher levels of the desired protein; the effect of overexpression dosage can thus be explored. A disadvantage of the DHFR method is the length of time required to obtain the overexpressing cell lines, usually no less than 6 months. The principle of the method revolves around the requirement of all cells for the enzyme dihydrofolate reductase for nucleoside synthesis; when this enzyme is lacking, cells must be growth in medium containing nucleosides. Transfection of a CHO cell line lacking the endogenous DHFR gene with a mixture of cDNAs encoding DHFR and the desired protein results in cointegration in the genome of these two cDNAs. The addition of the drug methotrexate (MTX), a tightly binding inhibitor of DHFR, depletes the cell

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Methods in Neurosciences, Volume 23 Copyright 9 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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of active enzyme. Amplification of the transfected DHFR, together with the cotransfected cDNA, then ensues in a subpopulation of cells, resulting in the generation of clones expressing higher levels of DHFR and of the desired cDNA. Progressive introduction of these new cell lines to higher levels of MTX forces overexpression to its maximum, reached when the transport of MTX across the cell membrane is limiting. The method described below is adapted from procedures described in Ausubel et al. (2); further information can also be found in Kaufman (3). Overexpression and characterization of recombinant proenkephalin and the prohormone convertase PC1 have been published (4, 5).

T r a n s f e c t i o n of Cells Before beginning the procedure, nucleoside-free fetal bovine serum must be prepared. This serum is obtained by thorough dialysis, which is extremely important for the success of the procedure (L. Chasin, personal communication, 1994). The 500-ml bottle of serum [ICN (Costa Mesa, CA) or other source] is first treated at 60~ for 20 min. We then dialyze it against three changes of 10 liters each of Dulbecco's phosphate-buffered saline [made from GIBCO (Grand Island, NY) stock powder; no calcium added] at 4~ Buffer changes are made at 8- to 16-hr intervals as convenient, and penicillin-streptomycin is included at standard concentrations in the last dialysis buffer change in order to guard against microbial growth. The serum is sterilized by filtration and stored frozen at -20~ in 45-ml aliquots. All glassware is washed by rinsing; no detergents are used. Chinese hamster ovary cells that have the endogenous gene for DHFR deleted (DG44) (6) are obtained from L. Chasin (Department of Biological Sciences, Columbia University, New York, NY). The DHFR vector can be obtained from the Genetics Institute (Cambridge, MA). Both of these sources retain rights to the expressed materials. DG44 cells are grown in Ham's F12 medium with 10% (v/v) fetal calf serum at 37~ in an atmosphere of 5% CO2. Split 2-5 x 10 6 cells into 10-cm dishes the day before transfection; cells should be about 40% confluent with plenty of space between them. For maximum amplification leverage, the ratio of the plasmid bearing the cDNA to be overexpressed to the DHFR-containing plasmid should be high. We find that ratios much greater than 100:1 result in few clones and ratios less than 50"1 result in hundreds of clones. It should be borne in mind, however, that having few clones is not necessarily bad. With the proenkephalin overexpression, two parallel transfections using different amounts of the DHFR vector yielded 1 clone in one transfection and about 100 in the other. After screening by radioimmunoassay, it was found that the single clone had

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a higher initial expression than any one clone obtained from the other transfection. Transfections should be carried out using the calcium phosphate technique, following established methods (3, 4), but using 5-20/zg/10-cm plate of the plasmid bearing the cDNA of interest (which must represent a eukaryotic expression plasmid, purified using a Qiagen kit (Chatsworth, CA), cotransfected with 0.1-0.5/zg of DHFR-coding plasmid. We obtained PC1 and PC2 expression plasmids from N. Seidah (IRCM, Montreal, Canada) (7), and constructed proenkephalin-containing expression vectors from plasmids and inserts provided by others (4). Two days after transfection, cells are selected for the expression of DHFR by splitting into six to ten 10-cm dishes containing Alpha minus minimal essential medium, which lacks nucleosides (GIBCO), containing 10% (v/v) well-dialyzed fetal calf serum (FCS). Two to four T-150 flasks with peelaway tops (Costar, Cambridge, MA) can also be used. DG44 cells should also be split into this medium in order to provide a control for cellular selection; no DG44 cells should survive transfer to nucleoside-free medium. If the cDNA vector encoding the desired protein also contains the gene for G418 resistance, cells can first be selected in G418 (600/zg/ml; 50% active; GIBCO) and selected clones or pools of clones subsequently placed into Alpha minus medium. While this procedure was followed for the PC 1 amplification with good results, it was not particularly successful for our first PC2 expression attempt. The plates are fed twice weekly with flesh medium until the appearance of clones 10 days to 2 weeks later. Between 15 to 30 large, isolated clones are randomly picked using either glass cloning rings (Bellco, Vineland, NJ) or the agarose-overlay method. The latter procedure involves overlaying the cells with 3-5 ml (for a 10-cm plate; 25 ml for a 150-cm flask) of a fleshly prepared 1:1 mixture of 2% (w/v) agarose (autoclaved and maintained at 50~ and 2x Dulbecco's modified Eagle's medium (DMEM) containing 2 • trypsin-EDTA at room temperature; circled clones are picked through the gel layer with a pipettor and sterile 200-/zl truncated pipette tips. Clones are placed into either 48- or 24-well plates (depending on the number of clones selected) containing 1 or 2 ml of medium per well, respectively. When these wells are about 70% confluent, each well is expanded to a 35mm well (in 6-well plates) or to a T-25. When these are again near confluence, a portion is frozen down [in Alpha minus medium with 10% (v/v) serum and 10% (v/v) dimethyl sulfoxide (DMSO)], a portion is maintained in singlicate, and a 10-cm dish or 35-mm well is set up for screening. Almost any plate arrangement should work as long as precautions are taken to guard against loss of clones by contamination, by maintaining duplicates and/or by immediate freezing. The maintenance of clones prior to obtaining screening results can be extremely time consuming; it is sometimes preferable to freeze the

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clones until all of the screening data are in, and then bring up the selected clone.

Screening If antisera are available that are known to react with the protein, screening of secretion medium by Western blotting is rapid and convenient. The wells/ plates are washed with phosphate-buffered saline (PBS) to remove serum proteins and then placed into an appropriate volume (1 ml/well of a 6-well plate; 6 ml for near-confluent 10-cm dish) of Opti-MEM (GIBCO) containing sterile aprotinin (100/~g/ml; Sigma, St. Louis, MO) and allowed to secrete at 37~ overnight. At this stage allowances can be made for clonal growth variations by scoring cells (heavy, medium, light) and adjusting the volume of Opti-MEM appropriately; minimum volumes that allow covering the bottom of the well must be used. The following morning, the conditioned medium is removed, centrifuged to remove any floating cells, and a 1/10 volume of 10 x Laemmli sample buffer is added. The samples are boiled and subjected to electrophoresis on an appropriate percentage sodium dodecyl sulfate (SDS)-polyacrylamide gel [15% (w/v) was used for proenkephalin, 8.8% for PC1] and Western blotting for analysis of expression of an appropriately sized protein. Another screening method involves radioimmunoassay of conditioned medium; for overexpression of proenkephalin, clones were screened using radioimmunoassay of the overnight-conditioned Opti-MEM for Metenkephalin-Arg-Phe immunoreactivity (this peptide represents the carboxylterminal heptapeptide of proenkephalin). For overexpression of prodynorphin, we used a Leu-enkephalin radioimmunoassay (following digestion of the conditioned medium with trypsin and carboxypeptidase B to release this cryptic peptide). We also used a mixture of antisera directed against the amino and carboxyl termini of this protein in Western blots. If no antisera of the desired protein are available, clones can also be screened by preparing RNA and performing Northern blots. Owing to the length of time required for growth of all of the clones, preparation of RNA, and Northern blotting, the clones are stored frozen until all of the data are obtained. We prepare a 10-cm dish of cells from each clone; when 70-80% confluence is reached, the cells are scraped into a small volume of PBS, pelleted, and RNA is prepared from the pellet using the detergent lysis method (2). The pellet can also be stored at -70~ until RNA preparation is convenient. Twenty micrograms of RNA from each clone is subjected to Northern blotting on formaldehyde gels, using standard procedures (2); the blot is photographed under ultraviolet (UV) light in order to provide a record of the relative amounts of rRNA per lane. Following prehybridization at

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42~ in formamide-containing buffer (2), 1-2 • 10 6 cpm/ml of random-primed labeled cDNA probe is included in the 42~ hybridization, and the blot is then washed and exposed to film. With the PC enzymes, for which no antisera were available until well into the amplification, considerable clonal variation in the amount of RNA expression was observed between clones or cell pools derived from early, but generally not late, amplifications. The highest expressing clone is brought up from the freezer into a T-25 flask. When this flask reaches near confluence, it is trypsinized and split 1:6 into two 10-cm plates containing methotrexate at the next highest concentration (see below); the remainder of the cells is frozen again at this time.

Amplification During amplification, the drug methotrexate (MTX) [Sigma, known as (+)amethopterin], which represents a tightly binding inhibitor of DHFR, is applied to the cells in gradually increasing doses. Cellular survival requires the presence of active DHFR and thus amplification of the DHFR gene and coamplification of the gene of interest occur simultaneously following exposure to MTX. For each amplification series, MTX should be purchased in several 100-mg bottles of the same specific activity. This compound is kept frozen in sterile 5-ml aliquots as a stock solution in Alpha minus medium lacking serum (the solution should be warmed and well mixed prior to sterilization by filtration to ensure that all material is soluble). Because MTX is considered hazardous, we do not weigh out this material but instead dissolve the entire contents of the bottle in 42 ml of medium (5 mM stock). The amplification procedure cannot be performed quickly because cells must be slowly adapted to increasing concentrations of MTX. We split cells 1 96 or 1 98 into a four- to fivefold higher concentration of MTX. The addition of increased MTX generally slows cell growth such that the cells do not overgrow the dishes; however, occasionally the cells express sufficient DHFR such that they grow more rapidly and need to be split again within a few days. Under the proper conditions for DHFR starvation, the cells take on a flat, stretched look and begin dying. The plates are fed twice a week with fresh medium containing MTX at the appropriate concentration. Within 10 days to 2 weeks, clones will begin to form, and 12 of these clones are then grown up and again screened for production of the protein of interest. Often, however, clonal growth does not occur; instead, the cells grow slowly over the entire dish. In this case, the cells are expanded and screened as a pool. Simultaneous amplification of six such pools has been recommended by others (3). Provided that expression of the desired protein has been verified at a previous step, this method usually works well at later steps and saves

[5]


99

considerable hands-on time in screening clones. However, we always compare conditioned medium obtained from clones at various levels of amplification in order to ensure that increasing expression is being obtained over several concentrations of methotrexate. While occasionally no increase is observed from one step to another, over several stages of amplification increasing expression should be observed; otherwise the final cell pool may exhibit low expression (this was the case for PC2). In this case, it may be possible to perform clonal selection at the stage where increasing expression is lost, with the idea of potentially locating a higher expressing subclone among the cells at a particular amplification level. The following amplification steps (concentrations of methotrexate) are employed; 5 nM, 20 nM, 100 nM, 500 nM, 2.5 tzM, 10 tzM, and 50 tzM. Cells must be growing well before the next level is attempted (i.e., attain near confluence from a 1:8 split in 3-4 days) and this usually takes 3-4 weeks. The entire amplification procedure takes about 6 months. It is extremely important to freeze cells at each level of amplification in order to guard against the loss of time by accidental contamination of the current line. For the proenkephalin overexpression, expression of immunoreactive Met-enkephalin-Arg-Phe increased steadily from about 1 pmol/106 cells at 5 nM MTX to 380 pmol/10 6 cells at 50 tzM MTX. However, we have observed a great variability in the expression of various proteins; PC2 was expressed at about 0.5 ~g/ml, PC1 at 2/~g/ml, and proenkephalin at 30 izg/ml. The reason for this variability is unknown but is potentially related to integration-dependent expression as well as possible toxicity of the expressed protein.

Collection of Conditioned Medium The collection schedule of conditioned medium is important. First, the optimal length of incubation for each harvest needs to be determined. Generally, the longer the cells are incubated, the more protein will be secreted into the medium; however, this does not necessarily result in a higher level of the intact protein or greater amounts of enzymatic activity in the medium. Protein degradation is more apparent during longer incubations, especially for smaller peptide precursors; thus the presence of certain proteinase inhibitors or serum proteins in the medium is helpful. For proenkephalin, we found 2% serum to exhibit a protective effect with regard to degradation (4); however, as described below, we now employ Opti-MEM containing aprotinin because it is easier to purify proenkephalin from this solution. Because medium conditions do not resemble intracellular conditions, not only stability but also enzymatic activity may decrease during prolonged incubations. We find that PC1 activity does not always increase in parallel with increasing

100

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FIG. 1 Incubation time course: enzymatic activity of PC1 does not always correlate with incubation time. CHO/PC1 cells were cultured in regular medium containing serum in roller bottles, until 80% confluence was reached; bottles were then washed and incubated in serum-free Opti-MEM (120 ml/roller bottle) for 24 hr. One milliliter of medium was removed from each roller bottle at each time point, centrifuged, and the supernatant kept at 4~ until the last time point was collected. PC1 enzymatic activity was then assayed in all aliquots using the RSKR-AMC fluorogenic substrate in the presence of an enzyme inhibitor cocktail to block nonspecific proteinases. incubation time, suggesting that PC1 is inactivated during prolonged incubation (Fig. 1). Western blotting and radioimmunoassay data indicate, however, that immunoreactive PC 1 continuously increases over the same time period. This apparent inactivation of PC 1 is in large measure due to protein aggregation (Y. Zhou, unpublished results, 1994). Second, one needs to determine how many harvests can be carried out using the same flask or roller bottles. Normally, CHO cells can be continuously cultured in serum-free medium from several days to several weeks, depending on the particular cell line and the culture conditions. However, longer culturing in serum-free medium, as compared to regular medium, can sometimes cause cells to detach, possibly due to the rolling movement of the roller bottle. In addition, CHO cells continue to divide slowly in serumfree medium; thus overcrowding may also contribute to cell detachment. Probably because of this cellular detachment and damage, nonspecific proteinase activities (distinguished from PC1 by differential sensitivity to proteinase inhibitors and neutral pH optimum) are increased in PC 1-expressing CHO cell-conditioned medium at later harvests. In contrast, proenkephalinconditioned medium has been successfully harvested from the same roller bottle up to 13 times. Therefore, to achieve the highest yields of the recombinant protein (or activity), a time course experiment such as the one shown in Fig. 1 should be performed to determine the optimal harvest pattern.

[5]


101

Collection of conditioned medium is performed from roller bottles containing cells at about 80% confluence. CHO cells producing the highest yield of either PC 1 or proenkephalin are cultured in roller bottles with MEM Alpha medium and 10% FBS (MTX is not necessary). To minimize contamination by serum proteins, serum-free medium, Opti-MEM (GIBCO), is used during the collection phase. After two successive washes with 40 ml each of warmed PBS, 100-120 ml of Opti-MEM containing aprotinin (100 txg/ml; Miles Labs, Kanakee, IL) is added. Aprotinin is used to prevent degradation of secreted proenkephalin and prodynorphin; however, this proteinase inhibitor does not appear to be necessary for protection of PC1. To collect PC1, roller bottles are incubated for 12 hr at 37~ to collect proenkephalin, bottles can be incubated up to 24 hr. Increasing degradation of proenkephalin in this medium is seen at 48 hr. These degradation products are extremely difficult to purify away, therefore it is worth preventing their appearance. The freshly collected conditioned medium is centrifuged at 1000 rpm for 15 min to eliminate remaining cells and stored frozen. For proenkephalin, we remove a 1-ml aliquot of each harvest prior to freezing for quality control of harvests by Western blotting; PC1 and nonspecific proteinase activity in each harvest are monitored by performing an enzyme assay in the presence and absence of an inhibitor cocktail (see below). We generally store proenkephalincontaining medium frozen prior to purification. PC 1-containing conditioned medium is also stored frozen; after one freeze-thaw cycle, PC1 activity is decreased 20-40%.

Purification of Secreted Recombinant Protein To obtain secreted recombinant protein from large quantities of conditioned medium, purification should be initiated with a step that affords concomitant concentration. Generally, precipitation methods, ultrafiltration, or certain chromatographical methods are used for this purpose. However, when conditioned medium is concentrated more than 10-fold, protein degradation is dramatically increased, even when procedures are performed at 4~ therefore, adding proteinase inhibitors (such as 1 mM phenylmethylsulfonyl fluoride) is necessary during and after direct concentration of conditioned medium. We have attempted to avoid ultrafiltration because we have observed major degradation of proenkephalin during this procedure. Although they are more time consuming, chromatographic methods appear to be preferable to concentrate both proenkephalin and PC1. The major contaminants in the conditioned medium are residual serum proteins and inactive or degradation fragments of the recombinant proteins. Even when cells are well washed with PBS, trace quantities of serum proteins,

102

I MOLECULARAPPROACHES predominantly albumin and immunoglobulin, still remain bound to the cells. Opti-MEM contains about 50/zg of protein per milliliter (mainly transferrin and insulin), while the concentration of recombinant protein will range between 1 and 30/zg/ml. On the basis of the extent of overexpression achieved, the biochemical properties, and the biological activity of the particular protein overexpressed, specific purification strategies must be devised for each recombinant protein.

Purification of Proenkephalin The highest resolution technique for the purification of recombinant proenkephalin is reversed-phase chromatography. If no serum has been used in the collection of medium, the chromatofocusing step previously employed (4) can be omitted. Using a preparative C4 column (Vydac, 2.2 • 25 cm; Separations Group, Hesperia, CA) it is possible to purify proenkephalin to homogeneity from conditioned medium in one step; substantial, although not complete, separation of glycosylated from unglycosylated proenkephalin is also achieved in this step. This gradient is based on the method of Thomas et al. (8). One hundred and fifty to 200 ml of thawed medium is acidified by the addition of 0.1% (v/v) trifluoroacetic acid and centrifuged for 20 min at 10,000 g. The supernatant is filtered by hand through a 45-/zm Rainin (Ridgefield, NJ) Nytran filter into a 60 ml syringe attached to pump A, and pumped directly onto the HPLC column [equilibrated with buffer A, 0.1% (v/v) trifluoroacetic acid (TFA)] at 6 ml/min. The column is washed with 16% (v/v) buffer B [80% (v/v) acetonitrile in 0.1% (v/v) TFA] until the UV absorbance at 280 nm returns to 0, and a gradient consisting of the following steps is then applied at 4 ml/min: to 35% B in 5 min, to 51% B in 75 min, and to 90% B in 10 min. Fractions of 4 ml are collected and 50-/zl aliquots of each fraction are dried into small Eppendorf tubes, using a Speed-Vac (Savant, Hicksville, NY) and reconstituted in Laemmli sample buffer. Electrophoresis on a 15% polyacrylamide gel followed by Coomassie staining is used to verify the position of proenkephalin (56 min), which depending on the degree of expression may represent the major secreted protein. Phenol red elutes at about 20 min; bovine serum albumin (BSA) elutes at about 69 min; immunoglobulin G (IgG) elutes at 98 min; proenkephalin fragments elute just prior to proenkephalin. The fractions containing proenkephalin are pooled and lyophilized (although variable loss occurs during drying); alternatively, they can be dialyzed against an appropriate buffer. Proenkephalin is very soluble (at least 10 mg/ml in water) but sticky and it is advisable to keep stock solutions at a high protein concentration (> 1 mg/ml). If it is desired to more completely separate glycosylated proenkephalin from

[5]


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unglycosylated proenkephalin, further purification of fractions containing unglycosylated proenkephalin can be performed on a semipreparative C4 column (Vydac, 1 x 25 cm). Pooled fractions are diluted at least threefold with buffer A, 0.2% heptafluoroacetic acid (HFBA; Pierce, Rockford, IL), and applied to the column by repetitive injection through a 5-ml loop. The column is washed with 16% buffer B (100% acetonitrile containing 0.2% HFBA) until the absorbance returns to 0 and is then eluted with a linear gradient to 51% B in 50 min, to 62% in 25 min, 70% in 15 min, and 100% in 10 min. Unglycosylated proenkephalin elutes around 86 min. In both HPLC systems, glycosylated proenkephalin elutes earlier than unglycosylated proenkephalin. Using only the first column, we obtain between 4 and 5 mg of recombinant proenkephalin per preparation. Quality control of the recombinant protein should be performed by amino-terminal sequencing.

Purification of PC1 PC 1, an 87-kDa glycoprotein, has been implicated in the proteolytic maturation of proopiomelanocortin and thus represents a likely candidate for a neuropeptide precursor convertases (9-11). Owing to its size and its enzymatic lability, it is impossible to purify PC1 by reversed-phase HPLC and maintain its biological activity; therefore, FPLC (fast protein liquid chromatography) methods are used. Like HPLC, FPLC provides high resolution, rapid performance, and reproducible conditions; however, because organic solvents are not employed, protein conformation is not altered and enzymatic activity is better maintained during purification. During purification, by optimal arrangement of purification procedures, concentration and desalting of the protein can be accomplished during each chromatographic step, which will minimize time requirements. The entire purification takes less than 12 hr, and enzymatically active PC1 is purified about 19-fold from the conditioned medium to homogeneity (Table I). To prevent degradation and stabilize the protein, all of the procedures are performed at 4~

Enzyme Assay PC 1 activity is monitored by the cleavage of a custom-synthesized fluorogenic substrate; Cbz-Arg-Ser-Lys-Arg-aminomethylcoumarin (RSKR-AMC; Enzyme Systems Products, Dublin, CA). Alternatively, Pyr-Arg-Ser-Lys-ArgAMC (Peptides International, Lexington, KY) can also be used. Similar fluorogenic compounds may also represent appropriate substrates for PC2 (12) and furin (13, 14) respectively. Assays are conveniently carried out in Parafilm-wrapped polypropylene microtiter plates (Costar), but can be

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TABLE I Purification of Recombinant PC1 Protein from Conditioned Medium of CHO/PC1 Cells

Step

Enzymatic activity (units) ~

Protein amount (/xg)

Yield (%)

Specific activity (units//zg)

Purification (-fold)

1050 937 743

5180 4860 444

100 89 70

0.19 0.20 1.65

1 1 8.7

56 330

40 90

1.40 3.67

7.4 19

Conditioned medium DEAE-Blue cartridge Phenyl-Superose (I) Mono Q Peak (I) Peak (II) a

5.3 31

One unit is 1 nmol of AMC/hr.

performed in Eppendorf tubes at pH 5.5 with 10 mM calcium chloride and 0.2 mM substrate (final concentrations). Ten-microliter aliquots of each fraction from the purification are included and the final volume is brought up to 50 /zl with distilled water. Conditioned medium samples and harvest-quality control samples are assayed at the same concentrations of substrate and calcium, but are assayed both in the presence and absence of a proteinase inhibitor cocktail of 2.5 I~M trans-epoxysuccinic acid (E-64), 1/xM pepstatin, tosyl lysyl chloromethyl ketone (TLCK) (50/xg/ml), and tosyl phenyl chloromethyl ketone (TPCK) (100 /~g/ml). Activity measured in the absence of the inhibitor cocktail indicates the presence of nonspecific proteinases, and the harvest is not used if high levels (10-20% of total) of such nonspecific proteinases are detected. This inhibitor cocktail is not required in assays following the hydrophobic interaction purification step. The pH of column fractions is adjusted by adding a 1/10 vol of 1 M Bis-Tris buffer, pH 5.5. After 10 to 16 hr of incubation at 37~ cleavage of the peptide bond is detected by measuring the concentration of free AMC, which is highly fluorescent, with a standard spectrofluorometer or a microtiter plate fluorometer (Cambridge Biotechnology, Cambridge, MA) at 380-nm excitation, 460-nm emission. The latter instrument has the advantage that time course experiments can be performed using the same plate incubated for varying lengths of time. Fluorescence is compared to a standard curve of 1-100/zM AMC (Peninsula, Belmont, CA). PC1 immunoreactivity in aliquots of column fractions is analyzed by SDS-PAGE on 8.8% polyacrylamide gels and Western blotting (Mini-PROTEAN system; Bio-Rad, Richmond, CA) using polyclonal PC1 antiserum directed against residues 84-100 (representing the presumed amino terminus of the mature protein) (15).

[5] OVEREXPRESSION OF PROTEIN PRECURSORS

105

Concentration by DEAE-Blue Chromatography Three hundred and fifty milliliters of conditioned medium, representing several successive harvests, is recentrifuged at 10,000 rpm for 20 min. The supernatant is then diluted with an equal volume of 20 mM Bis-Tris buffer (pH 6.5) and is loaded onto a 5-ml DEAE-Blue Econo-cartridge column (BioRad) at a flow rate of 2 ml/min. The column is washed with at least 20 ml of the same buffer and is then isocratically eluted with 30 ml of 1 M sodium acetate in 20 mM Bis-Tris, pH 6.5. The effluent is monitored at 280 nm by UV absorbance and a 20-ml peak of UV-absorbing material is collected. During column loading, most of the phenol red and minor contaminant proteins are removed because they do not bind to the column. This relatively inexpensive column concentrates the medium about 18-fold in 7 hr, and prevents the more expensive FPLC columns from becoming contaminated with phenol red. It can be reused several times before discarding (due to flow-through of enzyme).

Purification by Hydrophobic Interaction Chromatography The peak of UV absorbance at 280 nm, which is obtained in 1 M sodium acetate, 20 mM Bis-Tris buffer (pH 6.5), is applied directly to a phenylSuperose column (HR5/5; Pharmacia, Piscataway, NJ) without further manipulation. Elution of proteins is performed using an 18-ml linear gradient from 1 M sodium acetate, 20 mM Bis-Tris, pH 6.5 to 20 mM Bis-Tris, pH 6.5, followed by an 18-min isocratic elution with 20 mM Bis-Tris buffer, pH 6.5. One-milliliter fractions are collected at a flow rate of 0.5 ml/min. A single broad peak of proteinase activity against the fluorogenic substrate is detected, and this peak should overlap with PC1 immunoreactivity. During this chromatographic step, albumin and most serum proteins do not bind to the column under the loading conditions, while inactive PC1, mostly represented by aggregated forms, elutes later than the peak of enzymatically active PC1. Thus by using this hydrophobic interaction column, active PC 1, which consists predominantly of the 87-kDa form, is purified from the major contaminants and from inactive forms (Fig. 2).

Purification by Anion-Exchange Chromatography Enzymatically active fractions obtained from the phenyl-Superose column are pooled, diluted with 2 vol of 20 mM Bis-Tris, pH 6.5 buffer, and applied to a Mono Q column (HR5/5; Pharmacia). Proteins are eluted with a linear gradient from 20 mM Bis-Tris, 0.1% Brij 35, pH 6.5 to 1 M sodium acetate, 20 mM Bis-Tris, 0.1% Brij 35, pH 6.5. The flow rate is 0.5 ml/min and 1-ml fractions are collected. All of the fractions are screened by enzyme assay and Western blotting. On the anion-exchange column, the single active peak

106

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MOLECULAR APPROACHES 0.5 E c

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FIG. 2 Purificationprofile using hydrophobic interaction chromatography. The curve represents UV absorbance and the bars depict enzymatic activity against the fluorogenic substrate.

from the phenyl-Superose column is separated into two peaks (Fig. 3A), both of which overlap with the peak of immunoreactive 87-kDa PC 1. Because the protein in the first active peak is not homogenous (as shown by SDSPAGE analysis and Coomassie staining; Fig. 3B), further analysis and characterization are performed using the second peak of active PC1 (5). We have further purified the second peak by gel-filtration chromatography (Superdex G-200; Pharmacia) and verified that a single UV absorbance peak was found that overlapped with both enzymatic activity and 87-kDa PC1 immunoreactivity (5). Owing to losses of activity, this gel-filtration step is now not routinely employed in the purification. The final enzymatically active fractions from the anion-exchange column are pooled, brought to 10% glycerol, and aliquoted. The aliquots can be stored frozen at -20 or -70~ Spontaneous protein aggregation was observed to occur in unfrozen samples kept at initial protein concentrations, resulting in a diminution of activity.

Conclusions We have found the DHFR-coupled amplification method a reliable and relatively straightforward way to overexpress a variety of different protein precursors. To date, we have used the method to overexpress proenkephalin, prodynorphin, a mutated proenkephalin, PC1, and PC2. Expression levels

[5]

A


107

300

0.i0

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250 o~" m

0 O0 Cq

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peak II

u 0.05 t-

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20

97 kDa--

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30

FIG. 3 Purification profile using anion-exchange chromatography and SDS-PAGE analysis. (A) The curve represents UV absorbance and the bars depict enzymatic activity against the fluorogenic substrate. (B) SDS-PAGE analysis and Coomassie staining of the applied sample and of peaks I and II.

for these proteins have ranged from 0.5 to 30/xg/ml. Interesting differences have emerged from these various amplifications; for example, we have consistently found prodynorphin more difficult to recover intact than proenkephalin; and PC2, but not PC 1, is secreted as a zymogen rather than as the active enzyme (17). Using the DHFR-coupled amplification method, neuropeptide precursors can often be obtained in yields appropriate for crystallization efforts, while processing enzymes that are constitutively secreted (i.e., do not possess a membrane-spanning domain) can be enzymatically characterized. Further refinements of this overexpression method should lead to better methods of maintaining enzyme activity, because at present the amount of enzymatically active PC1 recovered and the specific activity of PC 1 are low compared to the amount of enzyme protein secreted. With the overexpression of several different neuropeptide precursors, interesting comparisons of processing specificity of the new neuroendocrine convertases will become possible.

Acknowledgments The work reported herein was supported by NIH Grant DA 05084. I. Lindberg was supported by an RCDA from the NIDDK.

108


References 1. R. J. Kaufman and P. A. Sharp, J. Mol. Biol. 159, 601 (1982). 2. F.M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. A. Sith, J. G. Seidman, and K. Struhl, eds., "Current Protocols in Molecular Biology." Wiley, New York, 1987. 3. R. J. Kaufman, in "Methods in Enzymology" (D. Goeddel et al., eds.), Vol. 185, p. 537. Academic Press, San Diego, 1990. 4. I. Lindberg, E. Shaw, J. Finley, D. Leone, and P. Deininger, Endocrinology (Baltimore) 128, 1849 (1991). 5. Y. Zhou and I. Lindberg, J. Biol. Chem. 268, 5615 (1993). 6. G. Urlaub, P. J. Mitchell, E. Kas, and L. A. Chasin, Somatic Cell Mol. Genet. 12, 555 (1986). 7. S. Benjannet, T. Reudelhuber, C. Mercure, N. Rondeau, M. Chr6tien, and N. G. Seidah, J. Biol. Chem. 267, 11417 (1992). 8. G. Thomas, E. Herbert, and D. E. Hruby, Science 232, 1641 (1986). 9. S. Benjannet, N. Rondeau, R. Day, M. Chr6tien, and N. G. Seidah, Proc. Natl. Acad. Sci. U.S.A. 88, 3564 (1991). 10. L. Thomas, R. Leduc, B. A. Thorne, S. P. Smeekens, D. F. Steiner, and G. Thomas, Proc. Natl. Acad. Sci. U.S.A. 88, 5297 (1991). 11. B. T. Bloomquist, B. A. Eipper, and R. E. Mains, Mol. Endocrinol. 5, 2014 (1991). 12. I. Lindberg, B. Lincoln, and C. J. Rhodes, Biochem. Biophys. Res. Commun. 183, 1 (1992). 13. S. M. Molloy, P. A. Bresnahan, S. H. Leppla, K. R. Klimpel, and G. Thomas, J. Biol. Chem. 267, 16396 (1992). 14. K. Hatsuzawa, M. Nagahama, S. Takahashi, K. Takada, K. Murakami, and K. Nakayama, J. Biol. Chem. 267, 16094 (1992). 15. O. Vindrola and I. Lindberg, Mol. Endocrinol. 6, 1088 (1992). 16. F. S. Shen, N. G. Seidah, and I. Lindberg, J. Biol. Chem. 268, 24910-24915 (1993).

[6]

Use of Antisense RNA to Block PeptideProcessing Enzyme Expression Richard E. Mains

Introduction Antisense RNA and DNA have become useful tools with which to investigate the function of specific enzymes in peptide processing. Figure 1 depicts a secretory granule and a lysosome in a neuroendocrine cell; even in a cell synthesizing a great deal of a given peptide, such as a pancreatic/3 cell making insulin or an intermediate pituitary cell making melanotropin, most of the protease activity in a crude cell extract is not found in secretory granules. Thus, the goal in antisense technology is to determine which of the endogenous enzymes are actually involved in peptide processing and how those enzymes work. Overexpression of an exogenous candiate endoprotease may result in the appearance or enhancement of a specific cleavage; this, however, would not prove that the candidate processing enzyme normally carries out that cleavage step. In its simplest form, the basic idea of the antisense method is to lower the rate of synthesis of one specific target protein and then to investigate the consequences for peptide processing in a neuroendocrine cell that is synthesizing biologically interesting peptides. The method may involve the introduction into cells of RNA complementary to the normal coding or sense strand of RNA; the RNA can be introduced into the cell directly or made by the cell that is expressing an appropriate DNA vector, and the antisense RNA (which we will call AS-RNA for convenience) will then pair with only the targeted endogenous mRNA (as for PC1 in Fig. 2). Alternatively, a synthetic oligonucleotide (DNA) is introduced into the cell and pairs with the endogenous sense RNA (as for PC2 in Fig. 2). The hope in either approach is that the antisense RNA or DNA will alter expression of only the targeted protein. In the case of peptidylglycine a-amidating monooxygenase (PAM; EC 1.14.17.3), AS-RNA indeed lowered the level of PAM protein in a stable cell line (1). As a comparison, PAM activity was also blocked in cells by a drug treatment that chelated copper ions essential for PAM activity (2) but such a drug treatment could alter other cellular functions as well. Antisense treatment also avoids the problem of inaccessibility of the peptide-processing enzyme; guanidinoethylmercaptosuccinic acid (GEMSA) is a potent and specific inhibitor of carboxypeptidase H (EC 3.4.17.10), but it is ineffective Methods in Neurosciences, Volume 23


109

110

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secr.____etory granule/trans-Golgi lysosomal proteases

novel

I| C~

CPH

endoproteases N U

C

f-"~PC1 C

N ~J [ ) /

N

l

t

cyt~

FIG. 1 Use of antisense technology to identify processing enzymes. Various cell organelles contain peptide-processing enzymes and other proteases. Prohormone convertases PC1 and PC2 are shown, along with an unidentified convertase called PCX; the Golgi endoprotease furin, which is closely related to PC1 and PC2, is shown; enzymes acting later in the biosynthetic pathway, such as CPH and PAM, are also shown (1, 3, 6-8, 16, 51, 52). from outside the cell (3). Another advantage of the antisense approach is that it can be adapted to block expression of one member of a closely related family of proteins when known drugs cannot distinguish the family members adequately (4, 5). The antisense method provides an answer to the question of what consequences a selective drop in the expression of a particular enzyme has on peptide biosynthesis. Frequently, selective overexpression of the same enzyme can provide additional important data on changes in peptide processing. It is important to consider that other members of a related family of proteins may also be affected by an antisense treatment. For example, because PC1 and PC2 have some stretches of nearly identical nucleotide sequences (6-8), a poorly chosen antisense oligonucleotide might block expression of both PC1 and PC2. Any one chosen treatment might inadvertently block more than one targeted protein, for example, blocking expression of an additional putative prohormone convertase ("PCX" in Figs. 1 and 2) by an antisense method supposedly targeting PC1 or PC2.

[6]

A N T I S E N S E RNA BLOCKING ENZYME EXPRESSION

111

PC1 ~ nuclease Ii11/1111111111111111111111111111 PC1 -AS-RNA

PCX

0 0 ~0 0 ks-~176 ribosome

FIG. 2 Antisense techniques reduces expression of a single mRNA: endogenous mRNAs (arrows indicate 5'-to-3' direction) in the cytosol of a neuroendocrine cell, with the PC1 mRNA paired with antisense RNA (AS-RNA) about to be destroyed by a nuclease, and the PC2 mRNA paired with an antisense oligodeoxynucleotide blocking the ability of ribosomes to translate the PC2 mRNA. The other cellular mRNAs are unaffected by the antisense treatments.

Antisense Method" Mode of Action There are several distinct ways that AS-oligonucleotides and AS-RNA have been shown to lower the level of synthesis of the targeted protein. One mode of action involves blocking synthesis of the targeted protein by binding tightly to the region of the initiation codon and blocking the initiation of protein synthesis (PC2 in Fig. 2). Another mode of action involves the destruction of the endogenous mRNA and a net lowering of the level of the targeted mRNA (PC1 in Fig. 2) (5, 9-13). In theory, antisense methods might also block transcription or interfere with correct splicing of RNA, but the evidence for such actions is still rather limited (5, 9, 10, 12, 14). In the case of ASRNA and simple DNA AS-oligonucleotides, the destruction of the endogenous mRNA is produced by endogenous nucleases that destroy doublestranded RNA and R N A - D N A hybrids (5, 9-11). Even a single cleavage in the mRNA for a targeted protein could be adequate to precipitate the destruction of much of the endogenous mRNA, because the nicked mRNA would

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have lost its poly(A) tail and would presumably become unstable (13). Certain derivatized oligonucleotides, such as methylphosphonate oligonucleotides, block translation when binding to the initiation codon but do not initiate destruction of the targeted mRNA (10, 12, 15).

Antisense Method: Requirements An antisense approach is more likely to be effective, first, if the targeted protein is rate limiting (5, 9, 10). For targeted enzymes present in excess, dramatic reductions in expression are required before functional consequences can be observed. In the case of peptide-processing enzymes, the prohormone convertases and PAM are clearly rate limiting, because there can be significant amounts of the substrates for these enzymes (high molecular weight precursors; glycine-extended peptides) remaining in tissue extracts (3, 7, 8, 16, 17). By comparison, carboxypeptidase H is probably not rate limiting, because there are usually negligible levels of peptides with COOHterminal basic resides in tissue extracts. Second, a convenient cell or animal system is needed. The system can be primary tissue cultures, cell lines, transgenic animals, or embryos that can be manipulated. In each case, the neuroendocrine target cells must synthesize peptides efficiently enough and in large enough quantity so that rigorous examination of changes in peptide processing is possible. Along with the neuroendocrine tissue, there must be a reliable method of delivering the ASRNA or oligonucleotide to the inside of the cells. These methods usually involve uptake of synthetic oligonucleotides or AS-RNA with a transient disruption of normal peptide biosynthetic processing, or else transient or stable expression of a DNA vector that encodes the AS-RNA. Third, the nucleotide sequence of the targeted protein needs to be known. For the synthetic oligonucleotide approach, a published sequence is all that is needed to choose a short sequence to use as the target. For the AS-RNA method, a cloned fragment of DNA is required, although the cloned fragment need not be the full coding region of the protein, and fragments derived from polymerase chain reaction (PCR) (even containing a few errors) are perfectly suitable. For either approach, the potential for confusion from related members of a family of proteins must be considered carefully (4, 6, 8). Finally, assays are needed to ascertain the success of the antisense treatment, preferably several independent assay methods. Northern analyses of the endogenous mRNA are essential, as is some sort of assay for the protein target. The assays for lowered protein levels can be enzyme assays and Western blot analyses, as in the case of PAM (1). Immunostaining could

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also work, but detecting a loss in immunostaining signal for a rate-limiting protein requires high-quality antisera. Clearly, rigorous assays of the changes in peptide processing are needed, with microsequencing and peptide mapping often being crucial to allow full interpretation of the results.

Advantages and Disadvantages of Antisense Oligonucleotide Method The most immediate advantage of this method is that one can test a hypothesis a few days after a nucleotide sequence is established, if the neuroendocrine target tissue and the assay methods are ready. The method can work effectively using primary cultures (5, 18-21), which means that the limited number of stable, differentiated neuroendocrine cell lines is not an impediment. The controls for the method are simple and direct, because the sense oligonucleotide should not show the inhibition seen with the appropriate antisense oligonucleotide; in fact, a single base mismatch in an antisense 15-mer is usually enough to eliminate the effect seen with the correct antisense oligonucleotide (20-22). The fact that a single base mismatch can make an antisense oligonucleotide ineffective can be a serious limitation, however, because it means that one cannot be certain that an antisense oligonucleotide designed against the nucleotide sequence for a peptide-processing enzyme in one species should work in a second species. The most commonly used oligonucleotides are 15-mers, because they are long enough to have the requisite specificity and still enter cells well;larger oligonucleotides have also been used successfully (5, 9, 10, 23). A major problem with the oligonucleotide approach is that even a trial experiment costs hundreds of dollars, because the oligonucleotides must be used at concentrations in the 10-100/~M range (9, 12, 15, 21, 24). Another problem is that the synthetic oligonucleotides are often unstable in culture medium unless serum is excluded or vigorously heat inactivated, which may introduce additional confusing factors (9, 10, 25). The breakdown products from oligonucleotides used at such high levels can also accumulate inside cells and be toxic (12, 26). Various chemically modified oligonucleotides are more stable in the medium and gain entry into cells more easily, but on a molar basis are usually less effective at suppressing synthesis of the targeted protein (12, 15). To add to the potential confusion, there are several examples in which the level of the targeted mRNA increased rather than decreased after treatment with antisense oligonucleotides (27). Owing to limitations in space, this chapter focuses on the vector methods, and readers are referred to excellent reviews on the antisense oligonucleotide methods (5, 12, 15, 21, 28).

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Advantages and Disadvantages of Antisense RNA Method Stable cell lines expressing AS-RNA to PAM and PC1 have been used to examine the role of those enzymes in peptide biosynthesis (1, 6). For other targeted proteins, it is clear that AS-RNA stably expressed from the appropriate vector can effectively lower the level of a protein when the ASoligonucleotide method did not work in the same system (5, 9, 10, 29). One clear advantage of creating stable cell lines or transgenic animals expressing a given AS-RNA is that a wide range of experiments can then be performed on the same cells, allowing much more complete testing of potential artifacts than is possible with transient experiments. Another advantage is that the method tolerates a mismatch of up to 15-20% of the nucleotide sequence (10, 11), which means that using the established nucleotide sequence of a protein in one species to inhibit expression in a related species has a good chance of success (as for PAM and PC1, where the rat clone was used to block expression of the mouse enzymes) (1, 6). The property of tolerating mismatches makes the choice of the nucleotide region to target much more crucial in the case of proteins that are members of a family, such as the PCs. One obvious disadvantage of this method is the need to clone a region of the cDNA for the targeted protein, but given the advent of the polymerase chain reaction, any published nucleotide sequence can be used to create a cDNA fragment that may work. While transient expression of vectors encoding a new protein can work using primary cultures (30, 31), there are not yet any reports of the transient method working to disrupt peptide biosynthesis using AS-RNA. Another problem with the AS-RNA approach is that there may be one or more open reading frames in the AS-RNA and thus the AS-RNA might encode a protein that could have its own side effects (9, 10, 32). In addition, the presence of double-stranded RNA in a cell can set off a number of self-destructive reactions, such as the phosphorylation of factors crucial to protein synthesis, which have nothing to do with the desired ablation of the one targeted protein (5, 9, 10). There are also a number of reports of apparently stable cell lines that have lost the ability to express AS-RNA (5).

Choice of Strategy For antisense oligonucleotides, it is clear that targeting the initiation codon is the best initial guess for what region of the nucleotide sequence to block, because binding an oligonucleotide to the initiation codon can block translation and also precipitate destruction of the mRNA (PC2 in Fig. 2) (9, 10, 23). For AS-RNA, the 5' end of the targeted mRNA is usually unsurpassed

[6]

ANTISENSE RNA BLOCKING ENZYME EXPRESSION Met 1 i 3'-"

s,ro

mRNA for enzyme, propeptide

I antisense transcript

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5' Northern analyses with RNA Probes

Expression Vector for Antisense RNA

wt

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AS

wt

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FIG. 3 A vector encoding an antisense RNA and a drug resistance gene is diagrammed, along with possible results of Northern analyses with sense and antisense RNA probes. in effectiveness by vectors aimed at other regions of the mRNA (PC1 in Fig. 2) (9, 10, 23, 33). Using a fragment that is shorter than the full-sized endogenous mRNA creates the opportunity that the endogenous mRNA and the added AS-RNA might be distinguishable on the same Northern analysis probed with a cDNA probe (which would detect both the endogenous sense mRNA and the added AS-RNA; the sum of the patterns for sense and antisense RNA probes in Fig. 3) (1, 34-37). Another crucial choice is whether to employ an inducible or a constitutive promoter for expressing the ASRNA. Inducible promoters, such as metallothionein or mouse mammary tumor virus, provide the advantage that the antisense approach can then be used for targeted proteins whose expression is essential for cell viability, but the induction itself will necessitate additional control experiments (1, 6, 20, 38, 39). Constitutive promoters have a corresponding set of advantages and problems (5).

Determining Whether Antisense Method Is Working Clearly, the most direct measures of whether the antisense method is working involve direct measurements of the targeted protein. These can involve Western analyses and enzyme assays and immunostaining. The peptides

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made in the neuroendocrine tissue must also be analyzed rigorously, to determine precisely which step(s) in peptide processing have been disrupted, if any. Northern analyses are essential to be certain that the decrease in the targeted protein is not merely the result of a clonal variant, but rather was indeed caused by expression of the AS-RNA. This is not a trivial worry; before we succeeded in establishing AtT-20 lines stably expressing PAM AS-RNA, we encountered several AtT-20 cell lines with low PAM activity that seemed to be clonal variants (1). Even when the AS-RNA is designed to be different in size from the endogenous mRNA, detecting the AS-RNA in a Northern analysis is frequently difficult. The AS-RNA to PAM was designed to be much smaller than the endogenous PAM mRNA, and the AS-RNA was detected with a cDNA probe in samples from stably transfected AtT-20 cells (1). However, in AtT20 cells expressing AS-RNA to PC1, the PC1 AS-RNA was designed to be the same size as the endogenous PC1 mRNA, and the AS-RNA was not distinguishable using a cDNA probe (6). The literature has several examples of AS-RNAs that were readily detected using cDNA probes (9, 36, 37) and of AS-RNAs that were undetectable using cDNA probes (10, 33, 35, 40). A more sensitive approach is to use a sense strand RNA probe, which will detect only the AS-RNA (Fig. 3), but even that approach does not always visualize the AS-RNA because of its extremely rapid turnover (5, 35). An additional complexity is added by the existence in cells of endogenous "unwindase" activity, which is now recognized to have both unwinding and modifying activity (5, 11). Unwindase activity unwinds double-stranded RNA, which at first would seem to prevent the successful action of ASRNA to bring about the destruction of the endogenous mRNA. It is now clear, however, that the unwindase also modifies the paired RNAs by changing up to half of the A residues into I residues, which are then read as G residues during protein synthesis (5, 11). This means that apparently intact RNA, as judged by Northern analysis, would encode a thoroughly altered protein, because many codons would no longer result in the insertion of the normal amino acid in the protein during synthesis. This is probably an explanation for the finding that the targeted protein level can sometimes be depressed far more than Northern analyses would predict (5, 11). This also suggests that the use of solution hybridization-RNase protection assays instead of Northern analyses would be a more quantitative and reliable means of determining how much unaltered endogenous mRNA remains after treatment with AS-RNA. The unwinding and modifying activity is thought to be important in the block of fibroblast growth factor expression in developing Xenopus oocytes, because the oocytes naturally express an antisense RNA to the fibroblast growth factor RNA at a set time during development

(5, lO).

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The use of antisense RNA to alter targeted protein expression can occasionally be confused by the fact that some tissues express natural RNAs that are made from the strand opposite the one normally expected. Examples include the Xenopus oocyte as cited above, gonadotropin hormone-releasing hormone and certain transcriptional factors (10), and insulin-like growth factor II (41). In addition, the problem mentioned above of open reading frames in the AS-RNA may be important if the protein encoded can interact with the native targeted protein. This concept is still controversial, with data supporting the notion of antisense peptides and proteins interacting with natural proteins (42-44) and data refuting any important interactions (32, 45, 46).

Detailed Methodology for Antisense RNA Blocking Enzyme Expression On the basis of the literature cited above and our experience with PAM and PC1 AS-RNA, the following protocol is recommended as the most likely to yield positive results in initial experiments. First, roughly the 5' half of the mRNA should be used to construct the antisense vector (Fig. 3), with careful selection to avoid long open reading frames. This region can be cut from the full cDNA with the appropriate restriction enzymes or created using published sequences and reverse transcription-polymerase chain reaction, starting with RNA from the tissue richest in the targeted protein. The cDNA is then inserted into the expression vector in the antisense direction. We have had success with both inducible promoters (1, 6) and constitutive promoters (A. Zhou and R. E. Mains, unpublished observations, 1994). For the overexpression of sense RNAs, encoding a functional protein, we have had success with drug selection built into the vector and on a separate plasmid (47-50); for antisense work, the best results have come using vectors that have drug resistance closely linked to the antisense sequence of interest (Fig. 3). The plasmid needs to be purified by banding on a CsC1 gradient before use; other methods of plasmid preparation, such as polyethylene glycol precipitation, have yielded significantly fewer stable transformants. A 60-mm dish containing a monolayer of cells (about 1 mg of cell protein or 3 x 106 cells) is rinsed in protein-free medium for 30-60 min, to remove as much protein as practical, because protein inhibits the transfection process. During this rinse period, 30 txg of the plasmid is added to 350 txl of 4 M ammonium acetate, pH 5, and then precipitated using 1 ml of ethanol. The precipitate is collected using a refrigerated microcentrifuge, and washed with 70% ethanol. The 70% ethanol is removed completely in a tissue culture hood, and the precipitated DNA is allowed to dry for 20-30 min. The DNA

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is then dissolved in 30/zl of protein-free culture medium. We have had good success with the calcium phosphate precipitation method (47), but more recently the lipofection technique with cationized lipids has given better results (31, 48-50). The dissolved DNA is mixed with 30/zl of lipofectin reagent (Bethesda Research Laboratories, Gaithersburg, MD). The proteinfree rinse medium on the cells is discarded and replaced with 3 ml of fresh protein-free medium. The DNA-lipid mixture is then transferred into the dish of cells and the cells allowed to take up the DNA at 37~ for 4-5 hr (longer times can be toxic to the cells). The cells are then removed from the dish with trypsin and plated into several 96-well plates, using 100/xl of regular growth medium per well. The manufacturer has changed the recommended protocol for this procedure several times; however, all the variations seem to work well. After 24 hr, the wells are overlaid with an additional 100/zl of medium containing the selection drug (e.g., G418 at 0.5 mg/ml or hygromycin at 200 U/ml, depending on the vector used). Cultures are then fed daily until nontransfected cells begin to die off, after which the feeding schedule is reduced to twice or even once a week. The amount of medium to remove and replace depends on the rate at which the cells are metabolizing; the goal is to prevent the wells from ever becoming acidic, yet to keep some of the conditioned medium in each well to help the establishment of resistant clones. After 3-4 weeks, colonies become apparent to the naked eye when the plates are viewed from below; the colonies metabolize rapidly enough to change the color of fresh medium after an overnight incubation. At this point, the cells are trypsinized from the original plate and equal aliquots are plated into duplicate 24-well plates; one 24-well plate will be used for the initial biochemical screening (Northern analyses, Western analyses, or enzyme assays) and the other plate serves as the stock of cells for further expansion. Typically three-quarters of the clones survive this initial expansion step. After this initial selection, we tend to keep all lines that grow vigorously and show any promising results at all in the biochemical analyses, because subsequent analyses of well-established lines sometimes differ on which lines are best. We also freeze aliquots of promising clones before going further in the analyses. It takes 1-2 months from the time of transfection to have established several stable cell lines expressing the desired plasmid. About half the cell lines showing good growth in the selection medium can be expected to express the transfected DNA at clearly detectable levels; we have found that the best results invariably come from the first wave of cell lines that show good growth in the drug selection medium. For several cell types (AtT-20, hEK-293, and GH 3) we have found that treating the 96-well and 24-well plates with the lid removed for 30 min and the ultraviolet light in the tissue culture hood on, greatly improves initial


119

cell attachment. We have also found that the use of high serum levels in the initial plating (up to 30% serum, heat inactivated at 56~ for 30 min) greatly increases the number of stable cell lines established. The number of 96-well plates to use must be determined empirically for each cell line; the basic goal is to have enough plates so that the probability of a drug-resistant well being clonal is high (perhaps a maximum of 10 drug-resistant wells per 96well plate), yet to have the cell density high enough in each well so that there is an adequate feeder layer for the few individual cells that stably incorporate DNA and eventually grow into stable lines. Another variable to determine for each line is the feeding schedule and the amount of medium to replace at each feeding, which must be adjusted for each transfection. Cross-feeding with conditioned medium from wild-type AtT-20 or GH3 cells can also be helpful. For AtT-20 cells, the six or eight 96-well plates from one transfection are usually fed with a nearly complete change of medium (remove and replace 150-175/zl of the 200/xl of medium) each day for 7-10 days, after which the feeding schedule drops to twice-a-week removal and replacement of 100/zl of medium. After the establishment of stable cell lines, we have often found that subcloning is essential to ensure that a clone is being studied (49), especially for the slowly growing antisense lines. A confluent 60-mm dish of the desired line is trypsinized and suspended in 10 ml of growth medium. Then 50 Izl of the cells is combined with 12.5 ml of growth medium containing the selection drug and one 96-well plate is seeded with 100/zl of the diluted cell jypension per well. The remainder of the diluted suspension is mixed with an additional 10 ml of medium and a second plate is seeded, continuing to eight plates with serially diluted cells. A dense 60-mm dish of wild-type GH 3 or hEK293 cells is then trypsinized and the cells suspended in 80 ml of selection medium; a 100-tzl aliquot of the wild-type cells is added to the serially diluted clone of interest. The wild-type cells provide a feeder layer of morphologically distinguishable cells that die at a rate that usually matches the rate of expansion of the stably transfected clones. Clones of stably transfected cells should be visually identified and marked after 10 days to 2 weeks, and are expanded when dense enough. Sometimes a feeder layer of nontransfected cells has also proved useful for the initial stage of transfection.

Acknowledgments This work was supported by Public Health Service Grants DK-32948, DA-00266, and DA-00097. I thank Drs. Betty Eipper, Brian Bloomquist, Sharon Milgram, and An Zhou for critical reading of the manuscript.

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References

10. ll. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

R. E. Mains, B. T. Bloomquist, and B. A. Eipper, Mol. Endocrinol. 5, 187 (1991). R. E. Mains, L. P. Park, and B. A. Eipper, J. Biol. Chem. 261, 11938 (1986). L. D. Fricker, B. Das, R. S. Klein, D. Greene, and Y. K. Jung, NIDA Res. Monogr. 111, 171 (1991). A. J. Baertschi, Y. Audigier, P. M. Lledo, J. M. Israel, J. Bockaert, et al., Mol. Endocrinol. 6, 2257 (1992). R. Baserga and D. T. Denhardt, eds., Ann. N. Y. Acad. Sci. 660, 1 (1992). B. T. Bloomquist, B. A. Eipper, and R. E. Mains, Mol. Endocrinol. 5, 2014 (1991). N. G. Seidah and M. Chr6tien, Trends Endocrinol. Metab. 3, 133 (1992). S. P. Smeekens, Biotechnology 11, 182 (1993). D. A. Melton, ed., "Antisense RNA and DNA." Cold Spring Harbor Lab., Cold Spring Harbor, NY, 1988. J. A. H. Murray and N. Crockett, in "Antisense RNA and DNA" (J. A. H. Murray, ed.), p. 1. Wiley, New York, 1992. B. L. Bass, in "Antisense RNA and DNA" (J. A. H. Murray, ed.), p. 159. Wiley, New York, 1992. J. J. Toulme, in "Antisense RNA and DNA" (J. A. H. Murray, ed.), p. 175. Wiley, New York, 1992. M. Y. Chiang, H. Chan, M. A. Zounes, S. M. Freier, W. F. Lima, et al., J. Biol. Chem. 266, 18162 (1991). C. Robinson-Benion, N. Kamata, andJ. T. Holt, Antisense Res. Dev. 1, 21 (1991). D. M. Tidd, in "Antisense RNA and DNA" (J. A. H. Murray, ed.), p. 227. Wiley, New York, 1992. D. F. Steiner, S. P. Smeekens, S. Ohagi, and S. J. Chan, J. Biol. Chem. 267, 23435 (1992). R. E. Mains, I. M. Dickerson, V. May, D. A. Stoffers, S. N. Perkins, et al., Front. Neuroendocrinol. 11, 52 (1990). W. Gerdes, W. Brysch, K. H. Schlingensiepen, and W. Seifert, NeuroReport 3, 43 (1992). A. Ferreira, J. Niclas, R. D. Vale, G. Banker, and K. S. Kosik, J. Cell Biol. 117, 595 (1992). R. H. Selinfreund, S. W. Barger, M. J. Welsh, and L. J. Van Eldik, J. Cell Biol. 111, 2021 (1990). F.J. Mangiacapra, S. L. Roof, D. Z. Ewton, and J. R. Florini, Mol. Endocrinol. 6, 2038 (1992). L. Neyses, J. Nouskas, and H. Vetter, Biochem. Biophys. Res. Commun. 181, 22 (1991). S. A. Liebhaber, F. Cash, and S. S. Eshleman, J. Mol. Biol. 226, 609 (1992). C. Boiziau and J. J. Toulme, Biochimie 73, 1403 (1991). J. C. Larcher, M. Basseville, J. L. Vayssiere, L. Cordeau-Lossouarn, B. Croizat, et al., Biochem. Biophys. Res. Commun. 185, 915 (1992). A. C. Yu, Y. L. Lee, and L. F. Eng, J. Neurosci. Res. 30, 72 (1991). L. C. Yeoman, Y. J. Danels, and M. J. Lynch, Antisense Res. Dev. 2, 51 (1992).


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28. L. Neckers, L. Whitesell, A. Rosolen, and D. A. Geselowitz, CRC Crit. Rev. Oncogen. 3, 175 (1992). 29. A. Ao, R. P. Erickson, A. Bevilacqua, and J. Karolyi, Antisense Res. Dev. 1, 1 (1991). 30. J. M. Burrin and J. L. Jameson, Mol. Endocrinol. 3, 1643 (1989). 31. R. A. Maurer, BRL Focus 11, 25 (1989). 32. A. N. Eberle, R. Drozdz, J. B. Baumann, and J. Girard, Pept. Res. 2, 213 (1989). 33. M. C. Moroni, M. C. Willingham, and L. Beguinot, J. Biol. Chem. 267, 2714 (1992). 34. M. I. Munir, B. J. F. Rossiter, and C. T. Caskey, in "Antisense RNA and DNA" (J. A. H. Murray, ed.), p. 97. Wiley, New York, 1992. 35. S. R. Rodermel and L. Bogorad, in "Antisense RNA and DNA" (J. A. H. Murray, ed.), p. 121. Wiley, New York, 1992. 36. S. M. Kaiser, P. Laneuville, S. M. Bernier, J. S. Rhim, Kremer, R., et al., J. Biol. Chem. 267, 13623 (1992). 37. M. Kimura, M. Sato, and M. Katsuki, in "Antisense RNA and DNA" (J. A. H. Murray, ed.), p. 109. Wiley, New York, 1992. 38. M. J. Smith and E. V. Prochownik, Blood 79, 2107 (1992). 39. T. Liu, J. G. Williams, and M. Clarke, Mol. Biol. Cell 3, 1403 (1993). 40. H. Yamada, S. Koizumi, M. Kimura, and N. Shimizu, Exp. Cell Res. 184, 90 (1989). 41. E. R. Taylor, E. A. Seleiro, and P. M. Brickell, J. Mol. Endocrinol. 7, 145 (1991). 42. D. W. Pascual and K. L. Bost, Immunol. Invest. 19, 421 (1990). 43. G. Fassina, M. Zamaia, M. Brigham-Burke, and I. M. Chaiken, Biochemistry 28, 8811 (1989). 44. T. S. Elton, L. D. Dion, K. L. Bost, S. Oparil, and J. E. Blalock, Proc. Natl. Acad. Sci. U. S. A. 85, 2518 (1988). 45. A. N. Eberle and M. Huber, J. Recept. Res. 11, 13 (1991). 46. U. B. Rasmussen and R. D. Hesch, Biochem. Biophys. Res. Commun. 149, 930 (1987). 47. I. M. Dickerson, J. E. Dixon, and R. E. Mains, J. Biol. Chem. 262, 13646 (1987). 48. A. Zhou, B. T. Bloomquist, and R. E. Mains, J. Biol. Chem. 268, 1763 (1993). 49. S. L. Milgram, R. C. Johnson, and R. E. Mains, J. Cell Biol. 117, 717 (1992). 50. F. A. Tausk, S. L. Milgram, R. E. Mains, and B. A. Eipper, Mol. Endocrinol. 6, 2185 (1992). 51. M. C. Kiefer, J. E. Tucker, R. Joh, K. E. Landsberg, D. Saltman, et al., D N A Cell Biol. 10, 757 (1991). 52. O. Vindrola and I. Lindberg, Mol. Endocrinol. 6, 1088 (1992).


Section II

Immunological and Biochemical Approaches to the Study of Peptide-Processing Pathways


[7]

Combination of High-Performance Liquid Chromatography and Radioimmunoassay for Characterization of Peptide-Processing Pathways A. Ian Smith and Rebecca A. Lew

Introduction The accurate quantitation of often very low levels of biologically important peptides in tissue or body fluid extracts is generally possible only by using immunologically based assay systems. Given that active peptides are generated from higher molecular weight precursors (which can be differentially processed in different tissues) and that peptides with varying biological functions can contain common amino acid sequences, these assays are often complicated by the lack of uniquely specific antisera. Strategies such as twosite immunoassays ([8] in this volume) can often overcome some of these problems; however, these assays are restricted only to well-characterized peptides, take time to develop, and allow the measurement of only one peptide per assay. An alternative approach is to use antisera with broad specificity to assay the fractions generated following the chromatographic separation of cross-reacting components. The separation of polypeptides has traditionally proved difficult as they are complex molecules that vary in size, charge, solubility, and solution conformation. Over the last decade or so the development and application of reversed-phase high-performance liquid chromatography (RP-HPLC) has to a large extent overcome these difficulties. This chapter focuses on the development of strategies that combine RPHPLC and radioimmunoassay (RIA) to identify and characterize peptides in tissue extracts. In addition, examples are given of how these techniques can be applied to characterize peptide-processing pathways and to show that in addition to expression and secretion, the precise pattern of peptide processing can be influenced by physiological regulators.

Reversed-Phase High-Performance Liquid Chromatography In this chapter particular emphasis is placed on the development and application of HPLC strategies. The generation of antibodies and their use in radioimmunoassay is well described in a previous volume in this series (Methods in Neuroscience, Vol. 13, Chapters 20-22) and in this volume ([8 and 19]). Methods in Neurosciences, Volume 23 Copyright 9 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Reversed-phase HPLC has become the method of choice for separating biologically active peptides. The separations are achieved following the partitioning of the peptide solute with the hydrophobic stationary phase in an aqueous buffer (solvent A). The peptides are then sequentially eluted, their retention dependent on the overall hydrophobicity of the peptide, by decreasing the aqueous component while increasing the organic (hydrophobic) component (solvent B) of the mobile phase. The widespread use of RP-HPLC for the separation of biologically active peptides reflects not only the stability and, importantly, the predictable retention characteristics of the stationary phases, but also their ability to handle a wide spectrum of polar, nonpolar, large, and small peptide solutes. In addition, the versatility of RP-HPLC arising from its ability to modulate solute retention via selective ion suppression, and via solvophobic and ion pair alterations, allows the separation of even very closely related peptides.

Sample Preparation The preparation of samples prior to injection represents the crucial first step in any HPLC analysis. The goal of the chromatogapher is to achieve high recovery of the solute in an injectable volume, free of organic solvent and particulate material. In the case of biological extracts, samples must also be free from protease activity, lipid, high molecular weight protein, or any other substance(s) that may bind irreversibly to the stationary phase.

Tissue Extraction The efficient extraction of peptides or peptide fragments from flesh or frozen tissues requires the removal of protein and lipid, and the inactivation of proteases. Extractants generally fall into one of two categories: (a) aqueous acid and (b) organic solvent. HC1 at 0.1 M has proved a highly effective extractant in terms of extraction efficiency (typically >90%); however, one disadvantage is that labile modifications can be cleaved during extraction, for example, prolonged exposure can deamidate glutamine and asparagine residues. Acetic acid at 2 M, perhaps not so efficient, is, however, a more gentle extractant but should be used only in conjunction with protease inhibitors. One effective extractant developed by Bennett (1) consists of 5% (v/v) formic acid, 1% (v/v) trifluoroacetic acid (TFA), 1% (w/v) NaC1 in 1 M hydrochloric acid. This cocktail, although harsh, has the advantage of precipitating most cellular proteins and would potently inhibit most proteases. Acidified organic solvent, for example, 80% methanol containing

[7] COMBINATION OF HPLC AND RIA

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0.1 M HC1, gives a clean protease-free supernatant; however, for larger peptides (>2500 Da) recoveries are low (30%) C18 columns, such as the Ultracarb C~8 (Phenomenex, Torrance, CA). Third, the separation of very hydrophobic peptides can be achieved by substituting CN (~Bondapak CN; Waters Assoc., Milford, MA) for C~8; this lowers the overall hydrophobicity of the stationary phase, thus allowing the separation of small hydrophobic molecules. One criterion of peptide purity is its elution as a single Gaussian peak in two distinct HPLC systems. Changing solvents and ion-pair agents, as discussed below, is useful, as is changing the stationary phase. However,

[7]

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6

AO[

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.J 5

lb TIME

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FIG. 1 Separation of standard synthetic peptides. Solvent A was 0.08% TFA, solvent B was 0.08% TFA in 70% ACN, and a 20-min linear gradient from 5 to 70% B was used. Peak 1, His-Pro-diketopiperazine; peak 2, TRH; peak 3, TRH-OH; peak 4, GnRH; peak 5, substance P; peak 6, somatostatin; peak 7, CCK-8 (all 2.5/zg, except peaks 1 and 3, which are 10 ~g); 0.2 AUFS at 206 nm.

changing both column type and mobile phase is optimal in confirming the purity of an isolated or synthetic peptide. Finally, the selection of column size is of course largely dependent on the particular application, although in some cases choice may be limited by the output of the high-pressure pumping system. Preparative HPLC columns (500 • 25 mm), used to purify up to 10 g of peptide per injection, normally run at flow rates in excess of 10 ml/min, which is beyond the capacity of most analytical HPLC systems. The modern analytical HPLC system is designed to generate flows of between 100/zl and 10 ml/min, which is sufficient to drive semipreparative columns (25 x 1 cm) in which sample loads may reach 100 mg/injection. The development of microbore column technology has allowed the peptide chemist to analyze very low levels (_ Lys-Lys Arg-Lys). In contrast, a number of features suggested that the structural basis for the recognition of processing sites by the relevant proteolytic enzyme machinery was more complex. Indeed, the following observations were made (2, 3). Methods in Neurosciences, Volume 23


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1. All potential cleavage sites are not processed in vivo. Less than 60% of the dibasic moieties present in precursors are indeed cleaved. Unprocessed basic residues are recovered within the bioactive (or connecting) fragments resulting from proteolytic processing. 2. Examination of the amino acid sequences situated around the dibasic cleavage sites did not reveal the existence of a unique consensus sequence. Only a few preferences for some residues in given positions appeared and might play a role in enzyme/subsite recognition. This indicated that the conservation of basic residues as signals for endoprotease recognition was not correlated with the existence of a single consensus primary sequence. 3. Although both monobasic and dibasic sites are encountered in proforms there appears to be a hierarchy within the amino acid residues that are cleaved. Arginine is more frequently used as a processing site than lysine, and the Lys-Arg doublet represents about 68% of the moieties that are recognized as processing sites in vivo, versus the Arg-Arg (18%), Arg-Lys (5%), and Lys-Lys (9%) arrangements. 4. Predictive methods to analyze the possible secondary structure around the dibasic cleavage sites in a database of prohormones and proproteins (70 precursor sequences) indicated that these processing loci are preferentially situated in, or in the immediate vicinity of, privileged structures constituted by 13 turns (2) or alternatively larger loops (4). Several questions then arose on the mechanisms underlying these important biological events. These include the following: (a) What are the enzymes involved in these processes? (b) What are the enzyme mechanisms that govern recognition of the substrates? and (c) What is the chronology and topology of these processing reactions? Answering these questions implies the identification of processing intermediates and of the cellular and the subcellular localization of processing events. Therefore, on the basis of these considerations, two main experimental strategies were adopted by workers in the field. One classic strategy, mainly based on biochemical grounds, was to detect, purify, isolate, and then characterize endoproteases exhibiting this type of selectivity toward basic residues included in peptide, or protein, substrates. The next step was to attempt the obtention of partial structural information to achieve cDNA cloning and complete determination of enzyme primary structures. This approach has turned out to be tedious and difficult for a number of reasons: (a) because of the inherent properties of these proteases and/or peptidases, which are poorly represented in the producing tissues, and (b) because proper substrates could not be defined unequivocally in all cases. The other approach was based on the knowledge of a processing endoprotease, the product of the S. cerevisiae KEX2 gene, which is involved in the

[9] IDENTIFYING NEUROPEPTIDE-PROCESSING PATHWAYS

157

maturation of pro-a mating factor and of the prokiller toxin (5). This subtilisinlike endoprotease, whose sequence has been determined (6), was used as a probe to clone by homology-related cDNAs present in endocrine and exocrine tissues of higher organisms. The deduced sequences of the endoproteases related to the "furin" gene, were classified under the generic term of PCEs (prohormone-converting enzymes) or "kexins." In some cases the corresponding gene expression products, that is, the active enzymes, were obtained by using adequate systems (baculovirus or/ and Cos cell expression). Unfortunately, at the present time, these compounds exhibit little enzyme activity in vitro, a major drawback in obtaining quantitative data on their kinetic properties toward well-defined substrates. In any case, the design and use of suitable substrates to monitor the enzymes and to analyze their mode of action has proved useful. Various substrates have been made use of, including fluorogenic derivatives of amino acids or of small peptides; synthetic peptides of various lengths reproducing, or mimicking, precursor sequences around the cleavage sites; and "fulllength" precursors obtained by hemisynthesis or by use of recombinant DNA techniques. To answer the questions relative to the chronology and topology of biosynthetic events the most successful methods were based on the following strategies" (a) identification of biosynthetic intermediates by the combined use of high-performance liquid chromatography (HPLC) and of selective antibodies against well-defined domains of the precursor; and (b) identification of processing events in situ by use of antibodies directed against precursor epitopes that were unmasked after the proper proteolytic reaction had occurred. In the present chapter, we discuss essentially those methods that have proved useful and efficient (a) in the detection and identification of processing intermediates of the precursor biosynthetic pathway; (b) in the detection and characterization of processing enzymes; and (c) in the identification and localization of posttranslational events occurring in the secretory machinery of producing cells.

Identification of P r e c u r s o r - P r o c e s s i n g Intermediates To establish the biosynthetic pathway of a given peptide it is necessary to identify with precision the precursor as well as the fragments that are generated after the action of one, or many, processing enzyme(s). The task can be considerably facilitated by the knowledge of the complete amino acid sequence of the precursor obtained from cDNA cloning. This allows the

158

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design of adequate immunochemical tools to monitor the resulting fragments after the careful separation by chromatographic and/or electrophoretic methods. In some cases, partial or extensive amino acid sequencing by conventional or micromethods may be needed. This can be illustrated in the case of the biosynthetic pathways of prooxytocin-neurophysin in the corpus luteum and in the hypothalamoneurohypophyseal tract.

Prooxytocin-Neurophysin Maturation The brain nonapeptide oxytocin (OT) and its associated neurophysin (Np) are present in the ovarian corpus luteum of the cow as well as in the sheep and human. They both derive from a common precursor in which the Nterminal hormone sequence is separated from the C-terminal neurophysin domain by a "processing sequence" Glyl~ 12, which is indeed eliminated on complete processing of the precursor (Fig. 1). To detect and identify the possible processing intermediates and putative corresponding processing enzymes, a strategy was developed on the basis of the following principles (7, 8): (a) a highly enriched preparation of granules was made from bovine corpora lutea and used as a source of enzymes; and (b) the fresh tissues were used as a separate source of peptides that were identified by radioimmunoassay (RIA) following HPLC separation with refer-

[ OT L-Gly-Lys-Arg~

Np

Endoprotease

! OT l_GlyTLys.~Arg + [ , : I

I

I I

I

.P',

I

I Carboxypeptidase B-like I OT LGly + Lys + Arg

I Amidating enzyme i OT I.NH 2 FIG. 1 A schematic representation of prooxytocin-neurophysin processing in the corpus luteum. [From Clamagirand (46).]

[9] IDENTIFYINGNEUROPEPTIDE-PROCESSINGPATHWAYS

159

ence to synthetic standards. The following peptides were produced by solidphase synthesis: pro-OT/Np(1-20) (peptide I) and the corresponding N- and C-terminal fragments pro-OT/Np(1-12)(OT-Gly-Lys-Arg ~2)(peptide II), proOT/Np(1-11)(OT-GlyLys 1~) (peptide V), pro-OT/Np(1-10)(OT-Gly) (peptide IV), and pro-OT/Np(13-20)(Ala ~3~ Arg 2~ (peptide III).

Experimental Procedure Granule Preparation Cows, superovulated during the luteal phase by follicle-stimulating hormone (FSH) and prostaglandin F2~ (PGF2~) injections, and bred by two successive artificial inseminations at 12 and 24 hr after estrus, (INRA, Jouy en Josas, France), are used as a source of corpus luteum. Animals are sacrificed 7-8 days after the heat period and ovaries are immediately collected and rapidly transported on ice to the laboratory. Fifteen corpora lutea are dissected and the 32 g of fresh tissues is homogenized in 20 mM Tris-HC1 (pH 7.0), 250 mM sucrose buffer (1 g of tissue per 10 ml of solution). The homogenate is then subjected to differential centrifugation to yield a granule pellet. Secretory granules are then purified by a gradient centrifugation run in 33% (w/v) Percoll-250 mM sucrose at 64,000 g for 10 min. Each 1-ml fraction of the gradient is measured for its refractive index, oxytocin immunoreactivity, and acid phosphatase activity (9). The oxytocin-containing fractions with a refractive index ranging from 1.3506 to 1.3464 (fractions 6 through 16) are pooled and used subsequently.

Enzyme Fractionation A lysate of purified granules (a total of 13.7 mg of proteins) is obtained by osmotic shock followed by three successive freeze/thaw cycles, then submitted to molecular sieve filtration on Sephadex G-150 (65 x 1 cm) in 50 mM ammonium acetate, pH 7.0, 4~ Fractions of 1 ml are analyzed for enzyme activity (see below). Isoelectric focusing is conducted (on a total of 0.487 mg of proteins) according to the classic method of an LKB (Bromma, Sweden) apparatus (110 ml) using a pH 3.5 through 10 gradient (10, 11).

Peptide Synthesis All peptides used in this work are synthesized by the solid-phase method. Purification and physicochemical analysis are as in Nicolas et al. (12). The following compounds are used in this work, either as substrates or as standards.

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II


Peptide I: pro-OT/Np(1-20), i.e., CysITyrlleGlnAsnCysProLeuGlyGlyl~

Peptide Peptide Peptide Peptide

2~

II: pro-OT/Np(1-12), i.e., C y s l ~ Arg 12 (OT-GlyLysArg) III: pro-OT/Np(13-20), i.e., Ala 13--~ Arg 2~ IV: pro-OT/Np(1-10), i.e., C y s I ~ Gly 1~(OT-Gly) V: pro-OT/Np(1-11), i.e., C y s 1 --~ Lys l~ (OT-GlyLys)

Peptide Isolation and Identification Bovine corpora lutea are first homogenized (0~ then extracted overnight (4~ in 0.1 N HC1 (10 ml/g fresh tissue). After centrifugation (30 min, 12,000 g, 4~ the supernatant is filtered through a Millipore (Bedford, MA) filter (0.45-/~m pore size), then applied to a Sep-Pak C18 cartridge (Waters, Milford, MA) previously washed with methanol, water, then 0.1% trifluoroacetic acid (TFA) successively. After peptide and protein adsorption (extracted from 3 g of fresh tissues) the cartridge is washed with 32 ml of 0.1% (v/v) TFA and peptides are eluted with 4 ml of 50% (v/v) acetonitrile in 0.1% (v/v) TFA. After evaporation, peptides are further analyzed by HPLC, thin-layer chromatography (TLC), and radioimmunoassay. Thin-layer chromatography of the peptides recovered from HPLC is performed on HP-KF silica gel plates (Whatman, Clifton, NJ), using as eluent the upper phase of the mixture [butanol-pyridine-H20-0.1% AcOH, 50:30:110, v/v).

Enzyme Assay Endoprotease and carboxypeptidase B-like activities are monitored by using peptide I substrate and measuring the production of both peptides II and III (endoprotease) and of both peptides IV and V (carboxypeptidase B-like). Routinely, 20/zg of either substrate is incubated with an aliquot of the fraction to be tested (containing, on average, 6/zg of protein) in ammonium acetate (50 mM, pH 7.0) for 24 hr at 37~ After acidification the remaining substrate and the products of reaction are analyzed by HPLC [~Bondapak C18 column eluted isocratically with 20% (v/v) acetonitrile in H20, TFA 0.1%, (v/v), then by a 20-40% (v/v) acetonitrile gradient in the same aqueous TFA solution]. Production of peptides II and III from peptide I is quantified by ultraviolet (UV) absorbance using a D-2000 (Merck, Rahway, NJ) integrator coupled to the HPLC. Endoprotease activity was expressed as quantities of either peptide II or III produced (in micrograms) (Fig. 2).

Neurophysin Isolation and Characterization Cows, superovulated and bred as previously described (7), are used as a source of corpus luteum. Animals are sacrificed at different stages after estrus and ovaries are immediately collected and transported on ice to the

[9]

161

I D E N T I F Y I N G NEUROPEPTIDE-PROCESSING PATHWAYS

Q.

O o O

L._

I

Z~ o.,

IIIL

...:]~o-'

~_

o~

o.1

|n

l

T.,

0

.

.

.

.

.

.

1,,1

"

~'

Jl~ .

9

~g

II •

I 0

T.

~ ~g

~ .

.

.

.

.

30 Retention

..

,.'111

Ill

/

60

0

time, min

FIG. 2 Identification ofprooxytocin-neurophysin(1-20) (peptide I) and its fragments by HPLC: separation and detection at 220 nm: Peptide II, Cys 1 --* ArgO2; peptide III, Ala ]3 ~ Arg2~ peptide V, Cys 1~ Lys 11. Upper trace: Elution positions of the peptides used as references (peptides II, V, III, and I). L o w e r trace: Elution of the fragments generated after exposure of peptide I to pro-OT/Np convertase, the putative processing endoprotease isolated from bovine corpus luteum and neurohypophysis. [From Clamagirand et al. (10).] Copyright 1987 American Chemical Society.

laboratory. Neurophysins, small proteins of Mr -" 10,000, are extracted from corpora lutea as usual (11), in the presence of protease inhibitors [aprotinin (4/zg/ml), pepstatin (1 /zg/ml), and 5 mM phenylmethylsulfonyl fluoride], and obtained hormone free with others proteins of similar molecular weight by two successive molecular sieve filtrations. Proteins (Mr "~10,000) are analyzed by isoelectric focusing (IEF) using the Phast System apparatus (Pharmacia, Uppsala, Sweden) with Phast Gel IEF 4-6.5, as recommended by the manufacturer. The proteins are transferred by diffusion to nitrocellulose membrane (Hybond C; Amersham, UK) at 4~ for 90 min in the presence of 25 mM Tris, 150 mM glycine, pH 8.3. After transfer the membrane is treated with 3% (w/v) bovine globulin-flee albumin (Sigma, St. Louis, MO)

162

II


in 10 mM phosphate buffer, pH 7.5, containing 130 mM NaC1. It is then incubated with rabbit anti-bovine neurophysin I serum (618-11) prepared in the laboratory. Goat anti-rabbit IgG-alkaline phosphatase conjugate and color development reagents 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt and nitroblue tetrazolium chloride are used to visualize neurophysin-anti-neurophysin complexes (Bio-Rad, Richmond, CA) To characterize prooxytocin-neurophysin, proteins of Mr "10,000 prepared from corpora lutea 2 or 3 days after estrus are subjected to enzyme cleavage by trypsin and by prooxytocin convertase. One microgram of Ntosyl-L-phenylalanine chloromethyl ketone (TPCK)-treated trypsin (Sigma) is incubated with proteins, free of protease inhibitors by previous filtration on Sephadex G-25 and containing 2/xg of immunoreactive neurophysin species, in 100 mM ammonium bicarbonate, pH 8, at 37~ for 60 min. The reaction is stopped by addition of 0.1 N HC1. Immunoreactive oxytocin is measured by RIA and the released peptides are identified by HPLC. Pr0oxytocin convertase is purified by isoelectric focusing as previously described (10). Proteins of Mr "~10,000 containing 9/zg of immunoreactive neurophysin are incubated with the convertase in 100 mM ammonium acetate, pH 7, at 37~ for 16 hr. After acidification, the reaction mixture is analyzed by HPLC. Granulosa Cell Culture Cows, superovulated and bred as previously described (7), are used as a source of granulosa cells. Animals are sacrificed 40 hr after the beginning of estrus. Ovaries are immediately collected, washed with sterile 0.9% (w/v) NaC1, and immersed in cold culture medium before transportation to the laboratory. Nonovulated follicles are used. At first, follicular fluid is removed by aspiration with a 27.5-gauge needle and 5-ml syringe and replaced by culture medium. The granulosa cells are harvested through a slit by gentle scraping of the inner wall, using a microinoculation loop. Medium and cells are recovered with a Pasteur pipette and centrifuged (150 g, 5 min, 4~ The cell pellet is washed with culture medium, and the cells are resuspended in culture medium, counted, and distributed into Nunclon Delta 24-well plates (Nunc, Copenhagen, Denmark): 0.3 to 1 • 10 6 cells in 1 ml of culture medium per well. The culture medium is Dulbecco's modified Eagle's medium and Ham's mixture F12 (DMEM/F12) containing penicillin (100 U/ml), streptomycin (0.1 mg/ml), (Eurobio, Paris, France), insulin (2/zg/ml), and without fetal calf serum. The cells are incubated at 37~ in a 5% CO2 humidified atmosphere (Sanyo incubator, Tokyo, Japan). Two to 4 hr after incubation, attached cells are washed with culture medium to eliminate dead cells and erythrocytes. Cultures are carried out in the presence or absence of the following hormones"


163

FSH (NIH-FSH-S 1), 1.5 mU per milliliter of culture medium, 0.5 IxM testosterone, or 0.5 txM dehydrotestosterone. Culture media are changed daily. Cell number is deduced from DNA concentration. DMEM/F12 and hormones are supplied by Sigma.

Neurophysin and Oxytocin Isolation from Culture Media Before RIA, immunoreactive neurophysin and oxytocin secreted into the culture media are isolated using Sep-Pak C~8 cartridges (Waters Assoc.). One milliliter of culture medium is filtered through the cartridge previously washed with methanol, water, and 0.1% (v/v) trifluoroacetic acid, and elution is obtained with 3 ml of 40% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid. Eluate is evaporated in a Speed-Vac concentrator (Savant Instrument Co., Hicksville, NY), then dissolved into RIA buffer. HPLC identification of oxytocin peptides is also performed on culture media purified using Sep-Pak C18 cartridges. The procedure is the same, but elution is obtained with 20% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid. The recovery yield of oxytocin-Gly-Lys-Arg, oxytocin-Gly-Lys, oxytocin-Gly, and oxytocin-NH2 is determined as 83, 77, 67, and 69%, respectively.

Neurophysin, Oxytocin, and Progesterone Radioimmunoassay Neurophysin RIA is carried out with purified bovine neurophysin I (11) as standard and rabbit anti-neurophysin I serum (618-12) prepared in the laboratory and used at a final dilution of 1 : 75,000. Separation of free from bound neurophysin is obtained by zirconyl phosphate. Sensitivity is 40 pg and 50% displacement of the 125I-labeled neurophysin I is obtained by either 0.6 ng of neurophysin I or 200 ng of neurophysin II. Oxytocin RIA is carried out as previously described (7). Oxytocin and Cterminally extended oxytocin peptides used as reference in RIA or HPLC are synthesized (see above). A minimum of 2 pg of oxytosin is detected and 50% displacement of the tracer (~25I-labeled oxytocin) is obtained with 75 pg of oxytocin, 300 pg of oxytocin-Gly-Lys-Arg, 250 pg of oxytocin-Gly-Lys, and 210 pg of oxytocin-Gly, thus allowing a sensitive detection of the mature oxytocin and the corresponding extended forms. Concentrations of progesterone in culture media are directly determined using an RIA kit (PROGCT) (CIS; Bio Industries, Paris, France). Sensitivity is 15 pg.

Results This system is particularly interesting because in this organ the appearance of a peak of pro-OT/Np mRNA at days 1-4 after the heat period is followed by the delayed appearance of free and active oxytocin at days 8 to 13 of the

164

II


cow estrous cycle. The measurements made indicated clearly the presence of a set of C-terminally extended forms of OT, that is, OT-Gly-Lys-Arg, OTGly-Lys, and OT-Gly, the substrate for the c~-amidating enzyme producing OT(NH2) (Fig. 2). A similar pattern was obtained in human granulosa cells in culture (Fig. 2). interestingly, the neurophysin-like material produced in corpus luteum 3-4 days after heat contained a higher molecular weight species with a pHi of 4.7. This material was indistinguishable from hemisynthetic pro-OT/Np (13) and was converted by a putative pro-OT/Np convertase from either neurohypophysis or corpus luteum into stoichiometric amounts of mature neurophysin and OT-Gly-Lys-Arg, which was identified by HPLC (as described above). Analysis of the corpora lutea 4 to 7 days after estrus revealed the presence of "Np-like" material, including the pHi 4.3 "OT-associated" Np, indicating that precursor processing had taken place. These data (8) unequivocally demonstrated that pro-OT/Np mRNA, which appears as early as day 1 after estrus, is translated into the precursor but that the latter is not processed further before day 4 after estrus. Moreover, the production of C-terminally extended forms of OT may indicate that processing is incomplete because mature OT(NH 2) appeared later. This might suggest a physiological role for these intermediary processing forms that remains, as yet, unidentified in ovary.

Detection and Characterization of Processing Enzyme Activities The major difficulty in detecting proteolytic enzymes in tissue, or cell, extracts resides in the adequate definition of a specific substrate. Homogenization of biological materials results in removal of cellular barriers, which add to the complexity of the resulting proteolytic activities in crude extracts. Therefore, the definition of a specific activity at the early stages of purification of a given, specific, proteolytic activity remains awkward. In attempting to quantitate the presence of a processing activity in biological materials the researcher should in general perform a first fractionation step before meaningful numbers may be obtained (14). Because cleavage of propeptides generally occurs at basic residues, the various substrates used by several workers are either basic amino acids or peptides bearing in their sequence the monobasic or dibasic moieties.

Use of Fluorogenic Substrates Fluorogenic substrates are the most convenient tools for the detection of proteolytic activities and for the rapid determination of kinetic constants. Either derivatives of arginine and lysine or of dibasic moieties Lys-Arg, Arg-

[9] IDENTIFYINGNEUROPEPTIDE-PROCESSING PATHWAYS

165

Arg, Arg-Lys, and Lys-Lys can be used. Because of the lack of solution structure of these substrates, they may be recognized by several proteolytic activities with a "basic residue" selectivity. Therefore they are not recommended for use at the early stages of protease purification, but can be efficient tools to determine enzyme parameters on purified preparations of processing endoproteases (or exoproteases). This can be illustrated in the cases of enzymes that cleave on the carboxyl side of paired basic residues (15, 16). In the first case the substrate used is Boc-Gln-Arg-Arg-MCA, a tert-butyloxycarbonylmethylcoumarin derivative of Gln-Arg-Arg. In a typical assay the synthetic substrate (obtained by solidphase synthesis) is incubated (20 nmol) in the presence of the S. cereoisiae enzyme in a final volume of 250/zl of solution containing 0.2 M Tris-HC1 buffer (pH 7.0) and 0.1% (v/v) Lubrol and 1 mM CaC12 (because the enzyme is a membrane-bound species, activated by Ca 2§ The reaction is kept for 1-24 hr at 37~ and at the end of the incubation 3 ml of H20 is added and the amounts of 7-amino-6 methylcoumarin (AMC) released from the substrate are measured with a fluorometer (excitation at 380 nm and emission at 460 nm). An arbitrary unit system can be defined as the enzyme quantity that can release, in 1 hr of incubation under the standard conditions, a certain quantity (in nanomoles) of AMC. To attempt the definition of an enzyme specificity, various derivatives are designed (see Table I) and percent activity is measured. It would be preferable in either case to measure the Km and Vmax from Line-weaver-Burk or Hofstee-Eadie plots. A comparable approach was used by Brenner and Fuller (16) to analyze the behavior of the Ca 2+-dependent, subtilisin-like endoprotease product of the KEX2 gene of S. cereoisiae. Boc or Ac derivatives of tetra- or pentapeptides were designed in which the C terminus is derivatized by a methylcoumarinamide group (see Table II). This study analyzed the influence of the doublet on Kex2 specificity. Lys-Arg, Arg-Arg, and Lys-Lys derivatives were compared, but not the Arg-Lys arrangement (Table II). Because the P'I and other P' positions are not filled by amino acid residues, only the P1, P2, P3, P4, and, in a few cases, P5 positions were analyzed with respect to the kinetics of Arg-MCA (or Lys-MCA) bond hydrolysis. A typical standard assay is as follows: 100/xM Boc-peptide-AMC in 50 /xl of 200 mM Bis-Tris-HC1 (pH 7.0), 1 mM CaC12, and 0.5% (v/v) dimethyl sulfoxide (DMSO) in 0.01% (v/v) Triton X-100. Initiation of the reaction is achieved by adding ---75 units of the enzyme and incubation is performed for 4 min at 37~ then stopped by placing tubes in ice water and adding 950/~1 of 0.125 M Z n S O 4. The AMC released from digested substrates is evaluated by fluorometry [excitation (hex) 385 nm; emission (hem) 465 nm]; 1 unit of Kex2 is defined as the release of 1 pmol of AMC/min.

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II

I M M U N O L O G I C A L AND B I O C H E M I C A L APPROACHES TABLE I

E n d o p r o t e a s e Specificity t o w a r d Various Boc Peptidyl-MCA or Aminoacyl-MCA a

Substrate b

Activity (%)

Boc-Gln-Arg-Arg-MCA Boc-Leu-Arg-Arg-MCA Boc-Gly-Arg-Arg-MCA Boc-Leu-Lys-Arg-MCA Boc-Gly-Lys-Arg-MCA Boc-Val-Pro-Arg-MCA Boc-Ala-Pro-Arg-MCA Boc-Glu-Lys-Lys-MCA Pro-Phe-Arg-MCA Z-Phe-Arg-MCA Bz-Arg-MCA Arg-MCA Leu-MCA

100.0 97.8 14.3 118.1 74.9 51.4 25.3 0.8 0.5 0.3 1.1 1.1 0.5

a Using a Ca: §-dependent membrane-bound enzyme from yeast (15). Protease activity for Boc-Gln-Arg-Arg-MCA was taken as 100%. Each substrate (20 nmol) was incubated for 10 hr with the enzyme at 37~ (as described in Use of Fluorogenic Substrates). MCA released was measured by fluorometry. b Boc, tert-butyl oxycarbonyl; Z, benzyloxycarbonyl; Bz, benzyl.

TABLE II

S t e a d y S t a t e K i n e t i c P a r a m e t e r s for C l e a v a g e o f Boc-peptidyl-MCA Substrates a Substrate

P5

P4

Ac-Pro- MetBocBocBocB o c - Arg Boc BocBocBocBoc a

P3 Tyr LeuLeu Gin Val Val GlnLeuGin Glu -

P2

P1

P'I

LysLysArgArgArgPro Ala ThrGly Lys -

Arg-MCA Arg-MCA Arg-MCA Arg-MCA Arg-MCA Arg - M C A Arg-MCA Arg-MCA Arg-MCA Lys - M C A

By Kex 2 endoprotease from S. cerevisiae (16).

kcat

gm

kcat/grn

(sec_l)

(/zM)

(sec -1 M -1)

25 23 45 21 21 31 26 18 6.1 0.19

2.2 3.9 17 13 19 150 210 800 320 55

11,000,000 5,900,000 2,600,000 1,600,000

1,100,000 210,000 120,000 22,000 19,000 3,500


167

Results This technique allowed convenient, rapid determination of steady state kinetics, optimal pH, and of kcat, gm, and k c a t / g m ratios (Table II). However, with respect to analyzing the influence of substrate structure on these parameters, one major drawback arose from the following features: (a) the relatively short length of the substrates and the absence of ordered peptide structure in solution, and (b) the absence of amino acid sequence or the C terminus of the dibasic and principally the lack of P'I residue, which turned out to be critical in processing at the dibasic-Xaa (P' 1) site (3).

Use of Synthetic Peptides Some laboratories have used a different approach, taking into account the basic considerations about the expected substrate requirements of the endoproteolytic processing enzymes (General Considerations, above). Therefore, synthesized peptides included in their sequence the mono- or dibasic moieties, which constitute a part of the putative signals for enzyme recognition. This strategy can be illustrated in the case of prooxytocin-neurophysin (proOT/Np) (7) and the prosomatostatin family (17). In a typical experiment, the substrate reproducing or mimicking the precursor sequence around the monoor dibasic cleavage site is exposed to the putative processing endoproteolytic activity. At the end of the incubation period, the remaining substrate and the generated fragments are separated by HPLC and identified by either (or a combination) of the following methods: (a) by reference to synthetic peptide standards, (b) by RIA with the appropriate antibodies, and (c) by amino acid composition and/or sequencing. Because the unequivocal identification of fragments is critical, some variance in the experimental procedure was designed; that is, either the substrate or the generated fragments are derivatized using an amino-terminal reagent absorbing in the visible region (e.g., DABITC) (18) (see Fig. 3).

Unlabeled Peptides A typical illustration of this technique is provided by pro-OT/Np(1-20), the N-terminal domain of the common precursor to oxytocin and neurophysin. To detect a putative processing endoprotease capable of cleaving at the Lys~-Arg 12 pair, a 20-amino acid residue synthetic peptide reproducing the N-terminal sequence of pro-OT/Np was synthesized by the Merrifield technique (Fig. 4). It was used to monitor the purification and characterization of a metalloendoprotease detected in bovine neurohypophysis and in the corpus luteum (14).

168

II IMMUNOLOGICAL AND BIOCHEMICAL APPROACHES

A LysArgI

I

Endoprotease

LysArg +

I

LysArg

I I

DABITC

LysArg

Endoprotease

LysArg +

C

I

LysArg

I I

Endoprotease

.LysArg +

DABITC

LysArg +

FIG. 3 Schematic representation of the various methods used for analysis of a peptide substrate and its fragments generated after endoproteolytic cleavage at the Lys-Arg $ Xaa dibasic site. (A) The peptide fragments were analyzed after HPLC by UV (Azz0nm). (B) The peptide substrate was prederivatized with DABITC and both the intact precursor and its N-terminal fragment were identified by HPLC and detection at 436 nm. (C) Both the N- and C-terminal peptide fragments were derivatized after endoproteolytic cleavage by DABITC and detected, after HPLC separation, by their A436n m values.

[9]

IDENTIFYING NEUROPEPTIDE-PROCESSING PATHWAYS

Cs~Tyr-I l e -lev-Glanl-Asn-Ct - L eSus-Pr~ -Asp-Leu-Asp-val-ArgS 1

10

11

12

10

11

12

I I

I I

13

169 20

Endopeptidase I

13

20

Cys-Tyr-lle-Gln-Asn-Cys-Pro-Leu-Gly-Gly~Lys~Arg + Ala-Val-keu-Asp-keu-Asp-Val-Arg

l--s

s

I

Carboxypeptidase B-like I

1o

Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-Gly IS s~l . I

I Amidating enzyme 1

9

Cys-Tyr-lle-Gln-Asn-Cys-Pro-Leu-Gly-NH 2 Oxy tocin

FIG. 4 A schematic view of prooxytocin-neurophysin(1-20) processing by enzyme activities from secretory granules lysates. [From Clamagirand (7).]

In a standard assay, 20 txg of the reference substrate is incubated with an aliquot of enzyme preparation (representing 1 txg of protein) in 100 mM ammonium acetate buffer, pH 7.0, in a final volume of 200 txl for 24 hr at 37~ The resulting fragments are analyzed by HPLC after acidification with 15 lxl of 1 N HC1, using a Cl8 column (Whatman), and eluted by a gradient of 0.05% (v/v) TFA in acetonitrile on an LKB apparatus. Peptide elution is monitored at 220 nm. Both the (1-12) and (13-20) fragments are identified by reference to synthetic standards and by amino acid composition (Fig. 4). The identification of both N- and C-terminal peptides resulting from the ArglZ-Ala ~3 cleavage is essential because contaminating exoproteases such as amino- and/or carboxypeptidase(s) can further degrade the generated products. Quantitation of the enzyme reaction can be achieved by several methods, including (a) evaluation of the disappearance of substrate, and (b) measurement of the generated N- and/or C-terminal fragments.

170

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[9]


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A comparison of the numbers obtained via the two techniques may be useful to reveal contaminating activities that may affect the stability of either, or both, generated products during the incubation conditions. This technique may also be applied to a number of derivatives of the reference sequence to study the role of the dibasic moiety, of peptide length, secondary structure, and of given amino acid residues in the kinetics of peptide cleavage and in substrate recognition (13, 19). However, careful interpretation of the kinetic data suggests that a certain number of conditions be respected. These include the following: (a) only peptides of similar length should be compared, and (b) structural information on the solution conformation of the peptide substrates should be obtained by circular dichro~sm, Fourier transform-infrared, and possibly one- and two-dimensional highresolution ~H nuclear magnetic resonance (NMR) spectroscopies (19, 20). Prederivatization of Peptide Substrate In this case the N terminus of the synthetic substrate is first reacted with a chromophore reagent [such as dimethylaminoazobenzene isothiocyanate (DABITC)] to generate the dimethylaminoazobenzene thiocarbamoyl peptide (DABTC-peptide). This is well illustrated in the case of the detection and purification of the "RXVRG" endoprotease from skin exudate of Xenopus laevis (Fig. 5). There the substrate was obtained by reaction of DABITC (21) with a tetradecapeptide (peptide I) mimicking the conserved region of X. laevis skin hormonal precursors, that is,

DABTC-peptide I = DABTC-AspValAspGluArgAspValArgGlyPheAlaSerPheLeu-NH2 Both amino- and C-terminal blockage prevent the substrate from amino- and carboxypeptidase(s) attack. The preparation of substrate and steps of the enzyme assay are as follows. 1. The substrate (peptide I) and the fragments generated by its cleavage at the R-G (i.e., a = AspValAspGluArgAspValArg) or R-D (i.e., b = Asp-

FIG. 5 An enzyme test to detect "RXVRG" endoprotease from X. laevis skin secretions. The peptide used as substrate, called "Kermit," mimicked the consensus sequence of hormone precursors around a processing locus (R $ G)-DABTC-Kermit = DABTC-D1VDERSDVR8GFASFL-NH2). Detection of the remaining substrate as well as of its generated fragments by cleavage at R8 (a) and R5 (b) was made after HPLC separation by A436n m measurements. (c) Elution position of the unmodified DABTC-Kermit. [From Kuks et al. (21).]

172

II


ValAspGluArg) or S-F (i.e., c = AspValAspGluArgAspValArgGlyPheAlaSer) bonds are reacted with DAB ITC. 2. Fifty micrograms of peptide is derivatized with dimethylaminoazobenzene isothiocyanate as described (18) and purified by HPLC (Milton Roy) on a Nucleosil 5-tzm C~8 column (146 x 4.6 mm) eluted with 35% (v/v) acetonitrile in aqueous 1% (v/v) N-methylmorpholine acetate buffer at pH 5.3 (flow rate, 1 ml/mn). DABTC-peptides (retention time, 25-40 min) are collected, dried, and then redissolved in acetonitrile-water (2:1, v/v) and stored at -20~ 3. Fifty picomoles of DABTC-peptide I is incubated with 12.5 Izl of enzyme preparation for 2 hr. The reaction is stopped by the addition of 12.5 Izl of pyridine and the mixture is analyzed by HPLC [30% (v/v) acetonitrile; flow rate, 1.5 ml/min]. All cleavage fragments elute separately within 5 min. 4. The fractions that generate the fragment DABTC-peptide I-(1-8) with little or no DABTC-peptide I-(5) (Fig. 5) are defined as active. 5. Effects of amino acid substitution on the endoprotease action are monitored by a competition test with the reference standard (21).

Postderivatization of Generated Fragments This technique can be illustrated in the case of the detection and characterization of an arginine-specific monobasic endoprotease from rat intestinal mucosa (22, 23). In this case a synthetic peptide (LeuGlnArgSerAlaAsnSerNH2) is synthesized corresponding to the prosomatostatin(62-68) region as a carboxamidated form: that is, pro-S(62-68)-NH2 is used as substrate and exposed to the enzyme. Then the generated fragments and the recovered intact substrate are subjected to N-terminal derivatization with DAB ITC, and the derivatized fragments are analyzed by HPLC separation (Fig. 6). The standard assay is as follows. Aliquots of enzyme are incubated with 2 nmol of pro-S(62-68)-NH2 in 10 mM sodium phosphate, pH 7.4, for 1 hr at 37~ in a final volume of 15 tzl. Controls establish that the enzyme activity is perfectly stable for at least 5 hr at 37~ The reaction is stopped by addition of dimethylaminoazobenzene isothiocyanate in pyridine (2 mg/ml) and incubated at 70~ for 50 min. The excess reagents are extracted with heptane-ethyl acetate (2 : 1). The aqueous phase is dried and redissolved in 50 ~1 of pyridine-H20 (1 : 1), and one-tenth is analyzed by HPLC using a $5ODS2 column (4.6 x 250 mm) (Prolabo, Paris, France) eluted with triethylamine acetate [1% (v/v) buffer], pH 5.3, containing 36% (v/v) acetonitrile, at a flow rate of 1 ml/mn. The dimethylaminoazobenzene thiocarbamoyl peptides (DABTC-peptides) are monitored at 436 nm. Under these conditions, DABTC-pro-S(62-64), DABTC-proS(65-68)-NH2, and the DABTC-substrate are visualized on a single run in 12 min (see Fig. 6).

[9]


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FIG. 4 Brain (left) and neural lobe extracts (right) (postnatal days 7-21) analyzed by HVE for fully and partially processed forms of OT. Brain and pituitary supernatants were applied on an HVE plate, after a Sep-Pak step, and electrophoresed as described in the caption to Fig. 2, except that in this experiment a 100-/xl aliquot (brain) and a 2-/xl aliquot (neural lobe) of the 500-/xl cellulose-HCl extract were analyzed for OT and OT-X immunoreactivity. Oxytocin immunoreactivity (dashed line) was determined using OT-MM antiserum (1 : 100,000), and OT-X immunoreactivity (solid line) was calculated from the difference between the immunoreactivities of OT-VA17 (1 : 60,000) and OT-MM. Electrophoretic positions of standards (arrows) were visualized by fluorescamine and measured by means of RIA as described in text. The value of OT-GK was corrected for the cross-reactivity of anti-OT-VA17 with OT-GK. Abbreviations: OT-G, OT-glycine; OT-GK, OT-glycine-lysine; OT-GKR, OT-glycine-lysine-arginine. [From Altstein and Gainer (7).]

ml of cold 4% acetic acid, followed by 5 ml of distilled water. The peptides are eluted with 4 ml of 75% (v/v) acetonitrile-25% (v/v) of 4% acetic acid. The eluant is lyophilized and stored at -70~ The plasma is applied directly to the column while the tissue samples are sonicated in cold 0.1 N HC1, with a ratio of 10" 1 solvent to tissue. The extract is centrifuged at 3500 rpm for

[10] NEUROHYPOPHYSEAL PEPTIDE PROCESSING

203

20 min at 4~ with the supernatant further purified by the ion-exchange method described above.

High-Performance Liquid Chromatography Peptide separations are performed using an automated gradient chromatography system from Waters Chromatography (Division of Millipore, Milford, MA). This system consists of a model 845 chromatography workstation with a VaxStation 4000 computer for control and data collection. The chromatograph is equipped with a WISP model 712 automated sample injector, two model 510 pumps, a model 484 variable-wavelength detector, and a temperature control oven for the columns. Peptides and tissue extracts are separated using reversed-phase chromatography with a trifluoroacetic acid (TFA)-CH3CN system on a Spheri-5 RP-18 column (4.6 • 230 mm, 5-mm C18 silica particles; Applied Biosystems, Inc., Foster City, CA). Standard samples of authentic OT-amide (Bachen California, Torrance, CA), OT-GlyLys (H. Gainer, NIH, Bethesda, MD), and OT-Gly-Lys-Arg (synthesized in our laboratory) are eluted using the following gradient. The flow rate is constant at 1 ml/min with ultraviolet (UV) detection at 215 nm. The column is initially equilibrated in 100% solvent A (0.1% aqueous TFA). Following injection, the mobile phase is maintained at 100% solvent A for 5 min. From 5 to 12 min, the concentration of solvent B (70% CH3CN in 0.1% TFA) is increased linearly to 7%, then to 35% over the following 40 min. Fifty-two minutes after injection, the concentration of solvent B in the mobile phase is increased to 100% over the next 3 min. Prior to injection, lyophilized extracts of tissue or plasma prepared using the Sep-Pak method are dissolved in 800 ml of 0.1% TFA, filtered through a Nylon 66 membrane (0.22-mm pore size; CentriFree, Millipore, Baltimore, MA). Fractions (1 ml) are collected and reduced to dryness by vacuum centrifugation. The fractions are redissolved in RIA buffer containing BSA (1 mg/ml), then analyzed by RIA. Table II shows the separation of peptide standards as monitored by UV detection (215 nm) and RIA of the HPLC fractions. Using a gradient HPLC system, there was a clear separation of the peptides; OT eluted first, followed by OT-GK and OT-GKR. OT-G was shown to migrate closely to OT in previous work. A similar HPLC and RIA combination can be used to separate the AVP family of amidated and C-terminally extended peptides. These methods have been used for the study of the oxytocin peptide forms present in fetal sheep plasma and hypothalamus (Figs. 5 and 6). In the late gestation fetal sheep (134 days, term of a approximately 142 days) there is evidence for the presence of three forms of oxytocin in the circulation. A comparison of the results with the two OT assays [OT-MM, which is specific

204

II IMMUNOLOGICAL AND BIOCHEMICAL APPROACHES TABLE II

High-Performance Liquid Chromatography Separation of Oxytocin Peptide Standards a Peptide peak HPLC fraction number Peptide

OD

RIA

OT-NH2 OT-GK OT-GKR

49 53 56

50 54 58

Peptide standards were separated by a gradient HPLC method and evaluated by UV absorbance (215 nm) and RIA of the HPLC fractions. The HPLC fraction number for the peptide peak is provided. The separation system is the same as that described in text, using a C~8 silica column and a TFA-CH3CN gradient.

200 '

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HPLC FRACTION

FIG. 5 Measurement of OT forms in fetal sheep plasma. Plasma (5 ml) from a 134day sheep fetus was purified using a Sep-Pak C18 cartridge with acetonitrile-acetic acid elution. The lyophilized extract was resuspended in 0.1% TFA and separated by HPLC on a C18 column with a TFA-CH3CN gradient (described in text). The fractions were lyophilized, resuspended in RIA buffer, and measured by two RIAs with different specificities. OT-MM (11) is specific for OT, whereas OT-X ([2) recognizes OT and the C-terminal extended forms. [125I]OT was used as the tracer with the antisera used at final dilutions of 280K for OT-MM and 48K for VA-17 (OT-X). The arrows indicate the elution pattern of the peptide standards OT, OT-NH2, OT-GK (OT-glycine-lysine), and OT-GKR (OT-glycine-lysine-arginine).

[10]

NEUROHYPOPHYSEAL

PEPTIDE

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205

PROCESSING

OT-GKR

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8 16 24 32 4{) 45 47 49 51 53 55 57 59 61 63 65 HPLC FRACTION

FIG. 6 Measurement of OT forms in fetal sheep hypothalamus and posterior pituitary. The medial basal hypothalamus (0.6 g) and the posterior pituitary from a 134day-old sheep fetus were sonicated in 0.1 NHC1 and purified by C18Sep-Pak extraction and HPLC separation as described in the caption to Fig. 5. The HPLC fractions were lyophilized and measured by RIA using OT-X, which cross-reacts with OT and the C terminal-extended OT peptides. (n) Hypothalamus; ([]) posterior pituitary. The arrows indicate the elution positions of the peptide standards OT-NH2 and OT-GKR.

for amidated OT, and VA-17, which cross-reacts with the amidated and the C-terminal extended forms (OT-X)] shows that there was a single peak with OT-MM and three peaks with OT-X. The peak eluting at fraction 48 is amidated OT, with similar amounts measured with the two assays. The two later peaks are thought to be OT-GK and OT-GKR on the basis of the chromatographic migration of peptide standards. These HPLC-RIA data confirm and extend our previous results that demonstrated by assay subtraction methods that fetal sheep plasma contained high levels of the extended OT peptides (9, 12). The existence of these alternative OT peptides was first suggested by the work of Amico and colleagues (13-15). Using different OT antisera, they found evidence for the secretion of C-terminal extended OT during pregnancy and after estrogen stimulation. They reported that the primary plasma form in humans and primates was OT-G (15). However, the identity of the circulating OT form(s) may be questioned because the antisera used in this study could not detect OT-GK or OT-GKR. HPLC separation of fetal sheep hypothalamus and posterior pituitary revealed different patterns of peptide expression (Fig. 6). There were two peaks in the hypothalamic extract, comigrating with OT and OT-GKR. The levels of the amidated and extended peptide were essentially equal. The

206

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I M M U N O L O G I C A L AND BIOCHEMICAL APPROACHES

posterior pituitary showed one major peak that was coincident with OT. These results are consistent with studies in the rat (Fig. 4), which showed that the alternative OT forms, primarily OT-GKR, were present in the hypothalamus, but not the posterior pituitary. Although Amico did not detect the extended OT forms in a variety of primate tissues, this was likely the result of the use of an antiserum that was specific for OT-G (15). Indeed, there is strong evidence for the presence of the C-terminal extended OT peptides in peripheral tissues, including the ovary (16), corpus luteum (17), and thymus (18). We have also used HPLC separation and RIA quantitation to study the OT forms in peripheral tissues from fetal and maternal sheep. The results indicate that the adrenal, thymus, chorion, and amnion all contain the alternative OT forms, OT-GKR and OT-GK (unpublished data).

Conclusions In this chapter we have illustrated uses of antibodies that were specific for the neurohypophyseal peptides (OT or AVP) but did not distinguish between the intermediate and amidated forms of the peptides. Combined with an appropriate separation technique and RIA procedures, these antibodies could be used to distinguish between the intermediate forms. We have also found these antibodies useful for immunoprecipitation and immunocytochemical procedures (not illustrated). Another alternative, not described here, is to make peptide antibodies that are specific for each intermediate form of the peptide. For the OT and AVP peptides this should be relatively easy by linkage of the amino termini of these peptides to carriers (by glutaraldehyde, etc.) before immunization. Given such antibodies, assays of the intermediate forms could be performed in a single step, that is, by RIA or immunoprecipitation. The biological significance of the presence of stable "intermediate" forms of OT but not AVP peptides in three species (rodents, sheep, and primates) remains unclear at present. Is this due to the intrinsic nature (structure) of the OT-prohormone (versus the VP prohormone), to different convertases in the cells containing these peptides, or to different microconditions in subcellular organelles where the processing occurs? Future experiments using these and other techniques will be necessary to answer these questions.

Acknowledgments We would like to acknowledge the assistance of Drs. James Rose and K. Tsai. This work was supported by Grants HL43178 (M.M.) and HDll210 (J.R.).

[10] NEUROHYPOPHYSEAL PEPTIDE PROCESSING

207

References

.

,

8.

9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

H. Gainer, Prog. Brain Res. 60, 205 (1983). R. Ivell, H. Schmale, and D. Richter, Neuroendocrinology 37, 235 (1983). E. Breslow and S. Burman, Adv. Enzymol. 63, 1 (1990). H. Gainer, J. T. Russell, and Y. P. Loh, Neuroendocrinology 40, 171 (1985). M. K. H. Sch~ifer, R. Day, W. E. Cullinan, M. Chr6tien, N. G. Seidah, and S. J. Watson, J. Neurosci. 13, 1258 (1993). M. Altstein, M. H. Whitnall, S. House, S. Key, and H. Gainer, Peptides (N. u 9, 87 (1988). M. Altstein and H. Gainer, J. Neurosci. 8, 3967 (1988). W. G. North, E. F. O'Conner, and C. B. Gonz~dez, Peptides (N. Y. ) 13, 395 (1992). M. Morris, S. W. Stevens, and M. R. Adams, Biol. Reprod. 23, 782 (1980). G. Moore, A. Lutterodt, G. Burford, and K. Lederis, Endocrinology (Baltimore) 101, 1421 (1977). P. R. P. Salacinski, C. McLean, J. E. Sykes, V. V. Clement-Jones, and P. J. Lawrey, Anal. Biochem. 117, 136 (1981). M. Morris, M. Castro, and J. C. Rose, Am. J. Physiol. (Regulatory Integrative Comp. Physiol.) 32, R738 (1992). J. A. Amico, M. G. Ervin, F. M. Finn, R. D. Leake, D. A. Fisher, and A. G. Robinson, Metab. Clin. Exp. 35, 596 (1986). J. A. Amico, M. G. Ervin, R. D. Leake, D. A. Fisher, F. M. Finn, and A. G. Robinson, J. Clin. Endocrinol. Metab. 60, 5 (1985). J. A. Amico, in "Recent Progress in Posterior Pituitary Hormones" (S. Yoshida and L. Share, eds.), p. 207. Elsevier, New York, 1988. M. D. Guillou, N. Barre, I. Bussenot, I. Plevrakis, and C. Clamagirand, Mol. Cell. Endocrinol. 83, 233 (1992). C. Clamagirand, M. Camier, C. Fahy, C. Clavreul, C. Creminon, and P. Cohen, Biochem. Biophys. Res. Commun. 143, 789 (1987). V. Greenen, F. Robert, H. Martens, A. Benhida, G. De Giovanni, M. P. Defresne, J. Boniver, J. J. Legros, J. Martial, and P. Franchimont, Mol. Cell. Endocrinol. 76, C27 (1991).

[11]

Approaches to Assessing Ontogeny of Processing Enzymes Richard G. Allen and Julianne Stack

Introduction

Posttranslational Processing of Preproopiomelanocortin Preproopiomelanocortin (POMC) is one of the most thoroughly characterized prohormones (12, 25, 30). The posttranslational processing of POMC is complex and varies in different cell types (13, 18). Each cryptic peptide encoded in POMC is flanked by two or more basic amino acid residues, for instance, -Lys-Arg-(KR), -RR-, -RK-,-KK-, a motif found in essentially all prohormones (21). Liberation of the bioactive peptides is a two-step process (29): the precursor is cleaved at the carboxyl side of the basic residues by a prohormone convertase (PC) (11, 17) and the remainder of basic residues exposed on the carboxyl-terminal end is removed by an enzyme with carboxypeptidase B activity (15). The biochemical basis for the tissue specificity of the proteolytic processing reactions is not completely understood. Several factors may be involved, including selective expression of the distinct PCs, differential compartmentation of either one or more proteases or the precursor, and modulation of cleavage-site accessibility by differential modification of the precursor (27).

Preproopiomelanocortin Processing: Cell and Tissue Specific Cell populations residing in the anterior and intermediate lobes of the pituitary gland process the common precursor prohormone POMC to different peptide end products (13, 19). In the rodent and monkey (1, 4, 16), anterior lobe corticotropes process POMC to predominantly/3-1ipotropin (/3-LPH), /3-endorphin(1-31), and adrenocorticotropin [ACTH(1-39)]; thus posttranslational processing stops at a certain proteolytic cleavage in the anterior lobe and does not proceed to the additional cleavages and biochemical modifications that define POMC-derived peptides in the melanotrope. Simply stated, the intermediate lobe (IL) POMC end products [a-melanocyte-stimulating hormone (a-MSH), N-acetylated and carboxy-shortened /3-endorphins, and ACTH(18-39) (CLIP)] are smaller (and further biochemi208


[11] ONTOGENY OF PROCESSING ENZYMES

209

cally modified by a-N-acetylation, a-N,O-diacetylation, carboxy-terminal shortening, c~-amidation, and phosphorylation) when compared to the major POMC-derived end products ACTH(1-39) and/3-LPH produced in the anterior lobe (AL) corticotrope (13, 19). Thus,/3-endorphin(1-31) and ACTH(1-39) serve as biosynthetic intermediates requiring further endoproteolytic cleavages by PCs to reach their final forms. Many pulse-labeling schemes have been used to define the order of POMC-processing steps in the corticotrope and melanotrope (2, 14, 31). A strict order of cell-specific cleavages of precursor and intermediates has been assigned (31).

Preproopiomelanocortin Processing during Development Because the cells destined to secrete/3-endorphins, ACTH, and a-MSHs emanate from a common embryonic structure (Rathke's pouch), the POMC system continues to be an interesting model of cell differentiation and development that can be studied at the molecular level (1, 4, 16, 26). Over the last several years this laboratory has been studying POMC processing during fetal pituitary development, in both the monkey and rat (1, 4, 16). Here, we would like to present new data using reversed-phase high-performance liquid chromatography (RP-HPLC) fractionation methods combined with immunoassay of specific POMC-derived peptides, addressing the ontogeny of POMC processing, and discuss these findings in light of what is now known about the prohormone convertases and their endoproteolytic specificities. Methods

Tissue Procurement and Preparation Pituitary tissues obtained at different stages of prenatal [embryonic day 15 (el5)-birth] and postnatal (P1, P2 etc.) development are dissected with the aid of a dissecting microscope and homogenized in ice-cold 30% (v/v) acetic acid containing bovine serum albumin (BSA; 0.5 mg/ml) and phenylmethylsulfonyl fluoride (PMSF; 0.3 mg/ml). After freeze/thawing three times, the insoluble material is removed by centrifugation, an aliquot is taken for total POMC peptide immunoactivity, and the supernatants are diluted, lyophilized, and frozen at -80~ until fractionation by RP-HPLC.

Fractionation by RP-HPLC After lyophilization, samples are redissolved in 0.2-0.5 ml of buffer A and injected onto a Vydac RP-HPLC column (C4,300-/~ pore size; the Separa-

210

II

I M M U N O L O G I C A L AND B I O C H E M I C A L APPROACHES

m

~

~

~

~

~

~

m

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n

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10

~

20

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30

40

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50

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60

70

80

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FIG. 1 Fractionation of peptides by RP-HPLC. Various combinations of 1-2 p,g of each peptide were dissolved in HPLC-grade H20 containing 0.1% TFA and injected onto a Vydac (Hesperia, CA) RP-HPLC column (C4, 300-,~ pore size). A Waters HPLC system with a fixed-wavelength UV detector (214 nm) was used to determine the elution positions of POMC-derived peptides. The heavy solid line shows the linear gradient of 0.1% TFA in HPLC-grade H20, and 80% CH3CN containing 0.1% TFA, that was used to elute peptides. The initial loading conditions were 17% solvent B with a brief "step up" to 23% solvent B. The flow rate was 1 ml/min. The marker peptides were as follows: (1) deacetyl-a-MSH, ACTH(1-13)NH2; (2) monoacetyl-a-MSH, a-N-acetyl-ACTH(1-13)NH2; (3) diacetyl-ct-MSH, a-N,O-diacetylACTH(1-13)NH2; (4) CLIP, human ACTH(18-39); (5) human ACTH(1-39); (6) fl-endorphin(1-31); (7) fl-endorphin(1-27); (8) a-N-acetyl-fl-endorphin(1-31); (9) fl-endorphin(1-26); (10) a-N-acetyl-fl-endorphin(1-27); (11) a-N-acetyl-fl-endorphin(1-26). All fl-endorphin peptides were the camel amino acid sequences, which correspond to rat fl-endorphins. tions Group, Hesperia, CA) and a Waters (Milford, NJ) HPLC system with a fixed-wavelength UV detector (214 nm) is used to fractionate the peptides. A linear gradient (shown in Fig. 1) of acetonitrile (CH3CN) in 0.1% trifluoroacetic acid (TFA) is used to elute peptides. The flow rate is 1 ml/min and 1-min fractions are collected. Buffer A is 0.1% TFA in HPLC-grade H20 (Baker, Phillipsburg, NJ); buffer B contains 80% CH3CN and 0.1% TFA. Synthetic peptides (1-2 g) are obtained from both Peninsula Laboratories (Belmont, CA) and Bachem (Torrance, CA) and used to determine the elution times of the POMC peptides shown in Fig. 1.

Radioimmunoassay of Preproopiomelanocortin Peptides All basic assay procedures have been described (1-3, 16). The 125I-labeled peptides used in the immunoassays are generated by either the hypochlorite

211

[11] ONTOGENY OF PROCESSING ENZYMES 1.2 A,

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2% will be apparent as a new band, and by Western immunoblotting (if a specific antibody is available). Functionality can be evaluated if one has an assay for enzymatic activity to test a panel of samples. Common features are shared among expression vectors (see Fig. 1). 1. Selectable phenotype: This is a genetic utility for the large-scale screening of putative positive clones. Often a marker such as drug immunity is employed. The most commonly used is ampicillin resistance (AmpR). 2. Promoter: This element consists of an RNA polymerase-binding site for tight regulation and is one of the most important determinants of efficient transcription~the frequency of RNA polymerase initiation. These promoters are usually induced by the addition of IPTG (isopropyl-/3-D-thiogalactopyranoside) to the media; IPTG stimulates transcription of the fused lac operon. 3. Ribosome-binding site: The ribosome-binding site is involved in the initiation of mRNA translation in Escherichia coli by the Shine-Dalgarno sequence (complementary to a sequence within the 16S rRNA), bringing the ribosome in close proximity to the initiator codon and an appropriate AUrich translational spacer of four to nine nucleotides. The site is designed for optimum recognition and binding. 4. Purification aids (polylinker, fused affinity tag, protease site): The polylinker introduces restriction endonuclease sites for convenient directional

309

[17] CLONING AND EXPRESSION OF ENDOPEPTIDASES

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I

~/cleavage purification PURIFIED ENDOPEPTIDASE PROTEIN

FIG. 1 Anatomy of an expression system. Top: Elements involved in a plasmid construct designed to produce high amounts of a specific protein. RBS, Ribosomal binding site; AUG and UGA, translational start and stop codons, respectively. The other elements are described in text. All sequences are written 5' to 3' and correspond to the coding strand. Initiation of transcription and translation are controlled by the promoter and the ribosomal binding site, respectively. Middle: Elements comprising the mRNA. Bottom: Fused protein. Although this depiction is for a prokaryotic system there are analogous sequences in the eukaryotic vector, and the same principles apply. subcloning of enzyme DNA for expression in-frame. Depending on the system employed (described below), a fused affinity tag aids in the purification. To liberate a fusion sequence from the enzyme, a sequence encoding a cleavage site for a protease such as thrombin, factor X, or enterokinase is incorporated. 5. Coding sequence: This portion of the construct encodes, in-frame, the endopeptidase of interest from the first amino acid through the stop codon. 6. Transcription terminator" A transcription terminator is included in the vector to prevent unneeded read-through transcription. This element usually consists of a putative stem-loop structure in the transcript, which aborts transcription. 7. (HIS), tag: Six to eight histidines, synthesized using the two codons, can be placed on the amino or carboxy terminus of the protein of interest,

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Protein Expression Systems

System [prokaryotic (P)/ eukaryotic (E)]

trpE (P) GST/MalE (P) pET (P) Baculovirus (E) Pichia pastoris (E) CHO/COS cells (E)

Fused/unfused

Glycosylation

Fused Fused/unfused Fused/unfused Fused/unfused Fused/unfused Fused

No No No Yes a Yes a Yes

Glycosylation of the core sugars is performed, but is not the same as in mammalian systems. Subcellular targeting must be confirmed.

a

regardless of the vector utilized for expression. Once the protein is expressed, selective purification is achieved through a remarkable affinity of the tag to a resin containing nickel nitrilotriacetic acid (Ni-NTA). Many laboratories are equipped to perform molecular biology and rudimentary protein purification, which is adequate for a bacterial expression system. Lower eukaryotes such as Pichia will perform posttranslational modifications and are amenable to manipulations in a laboratory setting without the high startup costs required for tissue culture facilities. Many of these protein expression systems are available in the form of commercial kits. Representative systems that are most prevalent are summarized in Table III and described below.

trpE Fusion Vectors The prokaryotic trpE fusion vector system was one of the first used on a large scale. It is relatively easy to insert the gene into the polylinker region of the vector. The gene expressing the protein of interest is placed under the strict transcriptional and translational control mechanisms recognized by the bacterial host in order to synthesize fused proteins efficiently. There is often basal expression of the cloned gene owing to the enormous strength of the promoter. Induction can occur by a temperature jump (to 42~ or chemically, by tryptophan starvation or addition of fl-indole acetate. The product can be detected by enzyme assay or antibody to the inserted gene (if available), or by commercially available antibody directed toward the fused portion of the gene, acting as an antigenic marker (trpE) to monitor purification and conjugation. Often the prodigious amounts of protein pro-


311

duced partition into inclusion bodies and are thus contained in the insoluble fraction of the cell when lysed. p E T Vectors

pET vectors (Novagen, Madison, WI) utilize the bacteriophage T7 RNA polymerase and the simpler promoter sequences have several advantages compared to E. coli. The T7 system can synthesize longer transcripts, and is more efficient in initiating transcription and translation. It can carry out this synthesis at a fourfold higher rate. Some of the plasmid constructs are inducible by IPTG. A yield of greater than 10% of the cloned gene translated into cellular protein is common, after a few hours of induction. Another feature of using this promoter is that the genes under control ofT7 polymerase are relatively transcriptionally silent, so that there is little protein production in the absence of induction. This is useful for potentially toxic genes, although soluble endopeptidases are not usually included in this class. The original EP 24.15 cloning and expression (3) were performed in a derivative of this system, the Bluescript vector (Stratagene). Sequences are available for cleavage by proteases to yield unfused product. The presence of a filamentous bacteriophage origin allows single-stranded plasmid DNA to be produced for DNA sequencing and site-directed mutagenesis. Glutathione S-Transferase or MalE

Glutathione S-transferase (GST) (Pharmacia-LKB, Piscataway, NJ) or Maltose binding protein (MalE) (New England BioLabs) systems are based on the same principle: fusion to a portion of a protein that binds tightly to a chemical moiety in an affinity resin to allow single-step purification. The pGEX system utilizes a chromatography matrix composed of glutathione coupled to Sepharose. The glutathione has a high affinity for the glutathione S-transferase/enzyme fusion protein. Thrombin is added and incubated in the column to yield free protein, which is then eluted. The MalE system fuses the protein of interest to maltose-binding protein, and signals cytoplasmic expression. The resultant malE-protein fusion binds with high afinity to amylose-conjugated resin. The plasmid utilizes a strong, inducible tac promoter (fused to the lacZ gene and thus inducible with IPTG) and malE initiation translation sequences to yield high amounts of expression. The vector also contains a sequence encoding specific cleavage by protease factor Xa, allowing for nonfused gene product. The vectors also include the filamentous bacteriophage DNA origin of replication, allowing production of single-stranded DNA for both sequencing and site-directed mutagenesis. Elution is conducted with free maltose. In both cases efforts must be made to avoid contamination with the protease utilized for the cleavage of the

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EXTRACELLULAR PROCESSING ENZYMES IN THE CNS

fused protein to the enzyme of interest. This is the current expression system employed for use with EP 24.15 (29). Baculovirus The baculovirus system (Invitrogen) has been reviewed comprehensively (30). Neutral endopeptidase (enkephalinase, EP 24.11), an endopeptidase closely related to EP 24.15 but found as an integral membrane protein, was expressed in a functionally soluble form utilizing an insect cell line infected with baculovirus (31). This eukaryotic expression system, in contrast with the bacterial systems, increases the likelihood of obtaining large quantities of target proteins requiring extensive post- and cotranslational modifications in a biologically active form. A "Baculo-GEX" system has been produced that combines this eukaryotic insect cell line (ligated to the polyhedrin promoter) with the advantages of the GEX system described above for a one-step purification coupled with specific proteolytic cleavage resulting in unfused protein (32). Pichia pastoris Pichia pastoris (Invitrogen) (33) is a eukaryotic expression system, inroduced in the last year and closely related to S. cerevisiae; it is reported to express up to 10 g/liter of a recombinant protein introduced via plasmid into a transformed spheroblast of this methylotropic (fermentation induction by methanol for high-volume, foreign protein productivity), yeastlike organism. An extremely efficient promoter (alcohol oxidase, AOX1) is utilized, which normally allows the organism to process methanol as the sole carbon source. Additionally, the vector contains a coupled HIS4 gene, allowing for screening of recombinants that will grow by plating on histidine-deficient medium. Expression is allowed in either the secreted or intracellular form. Because the medium is virtually protein free, and there are signal sequences in the vector for secretion, purification of the protein is simplified. Expression in yeast requires no additional equipment or media other than that present in common microbiological facilities. CHO or COS Cells In the study of endopeptidases, Chinese hamster ovary (CHO) or African green monkey kidney (COS-CV1) cells [American Type Culture Collection (ATCC), Bethesda, MD] have been used to express a soluble and fully active form of rabbit neutral endopeptidase, which was secreted utilizing transfection of a recombinant expression vector fusing the ectodomain of neutral endopeptidase (NEP) to a cleavable signal peptide (34).


313

Hisn Protein Purification Many of the vectors described above include (or the user can add) the oligonucleotide encoding six to eight histidine residues (using a mixture of the two codons, CAC/CAT). The poly(His) can be inserted in-frame at the amino or carboxy terminus (to assure fully terminated proteins), and does not usually interfere with the activity of the cloned, expressed gene, because there is no net charge difference at physiological pH. This enables a rapid purification (--~95% purity achieved in one step) of the protein of interest by selective chelation on a nickel nitrilotriacetate resin by conventional chromatography under native or denaturing conditions. The protein is then eluted from this column under gentle conditions of imidazole as a competitor, or by reducing the pH to about 5.5. Column binding is unaffected by small concentrations of ionic/non-ionic detergents, reducing agents, or strong denaturants. Maximizing heterologous expression levels of recombinant proteins (proteins not native to the particular host organism) in prokaryotes or eukaryotes may present a problem with their solubility. Because high amounts of expression lead to cells that produce several percent of their total protein as foreign biomolecules, these foreign proteins are partitioned, at high concentration, into inclusion bodies (dense aggregates of insoluble, misfolded protein). Often, changing the host strain is all that is needed to change expression and increase partitioning into a soluble fraction. In certain cases this is desirable, because this may simplify obtaining a homogeneous product. The first step of purification involves washing away cytoplasmic proteins and the major contaminants that are misfolded, proteolyzed, or oligomeric forms of the protein of interest. One must be aware that it is possible to produce cytotoxic material deleterious to the viability of the organism. All of these points notwithstanding, the problems with the inclusion body approach deal with issues of purpose and purity. If the purpose is to study a native, folded, intact molecule by biophysical approaches such as NMR or X-ray crystallography, or by an elicited activity in an assay system, then inclusion bodies pose serious problems. With purity as a criterion, the nature of the contaminants as often poorly soluble, hydrophobic membrane components must be considered; variants of the expressed protein are difficult to purify. Refolding proteins quantitatively on a large scale proves a vexing problem. One simple way to avoid aggregates of a soluble recombinant protein in an intracellular system such as E. coli is to lower the temperature (35) to 28-30~ at which the majority of EP 24.15 was found in the soluble form (10). Other solutions involve the choice of expression vector coupled to a secretion system. The addition of protease inhibitors, or detergents, or highdensity growth (as well as optimizing the temperature), can improve secreted yields.

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It is important that after cloning and expressing an enzyme a comparison be made, utilizing enzymological criteria such as catalytic properties toward substrate and inhibitors, with the purified wild-type enzyme derived from a tissue source.

Endopeptidase 24.15 as Example A plasmid vector obtained via library screening and without previous modification for expression is usually not suitable if an unfused protein product is required. The insert containing the soluble endopeptidase must thus be subcloned into an alternative vector. The original recombinant EP 24.15 clone (3) lacked the coding potential for the first two amino acids and was modified by site-directed mutagenesis to yield the complete unfused EP 24.15 with a BamHI site. A 39-nucleotide oligomer was synthesized containing 14 nucleotides identical to the vector, 6 nucleotides identical to the BamHI site, and 19 nucleotides identical to the amino terminus of the protein. The construct was transformed and bacterial cultures containing either plasmid with or without cloned insert, was grown in 100 ml of medium with antibiotic selection and diluted into 1000 ml to an absorbance reading of 0.6 at 600 nm. Expression of the fusion gene was induced with 0.4 mM IPTG, and the growth of properly folded fusion gene product proceeded at 30~ for 3 hr. The bacterial suspension was centrifuged for 5 min at 3000 g and the pellet was resuspended in 20 ml of 50 mM Tris-HCl, pH 7.0. Bacteria were then lysed by two cycles of freezing and thawing followed by sonication. Bacterial debris was removed by centrifugation (10,000 g for 10 min). The bacterial supernatant was then incubated with glutathione-Sepharose beads, and incubated with thrombin to cleave at the junction of the two genes. Thrombin, which contaminates the column eluate by 0.02% (w/w), can be easily removed by exhaustive filtration with a Centricon 50 (Amicon, Beverly, MA) which quantitatively removes thrombin to the filtrate. This yielded pure protein (2.5 mg/liter culture) as assayed by native and SDS-polyacrylamide gel electrophoresis.

Conclusion The cloning of the cDNA encoding an endopeptidase becomes a crucial step in explicating the role that the peptidase plays in nervous system function. Ultimately, elucidating the function and structure of one such protease can aid in understanding the regulation of neuropeptide function by these enzymes as a class. The peptidases can be targeted for pharmacological inter-


315

vention through the use of specifically designed modulatory ligands, either agonistic or antagonistic. Examples using this rationale involve the development of inhibitors of the human immunodeficiency virus (HIV) aspartic protease as a treatment for human immunodeficiency virus (36), inhibitors of angiotensin-converting enzyme, such as captopril, to treat hypertension (37), and inhibitors of enkephalinase as a treatment for congestive heart failure and as a nonaddictive analgesic (38).

Acknowledgments M.J.G. thanks the Revson Foundation for Biomedical Research and a grant from the SEED program of Mount Sinai Medical Center for support of this work.

References M. Orlowski, C. Michaud, and T. G. Chu, Eur. J. Biochem. 135, 81 (1983). 2. T. G. Chu and M. Orlowski, Biochemistry 23, 3598 (1984). 3. A. Pierotti, K. W. Dong, M. J. Glucksman, M. Orlowski, and J. L. Roberts, Biochemistry 23, 10323 (1990). G. R. Acker, C. Molineaux, and M. Orlowski, J. Neurochem. 48, 284 (1987). 5. M. J. Glucksman, N. X. Barrezueta, A. Pierotti, N. S. Bengani, S. Greene, and J. L. Roberts, Endocrinology (1994) (in press). A. Devault, C. Lazure, C. Nault, H. Le Moual, N. G. Seidah, M. Chr6tien, P. Kahn, J. Powell, J. Mallet, A. Beaumont, B. P. Roques, P. Crine, and G. Boileau, EMBO J. 6, 1317 (1987). K. M. Carvalho and A. C. M. Camargo, Biochemistry 20, 7082 (1982). 8. U. Tisljar and A. J. Barrett, Arch. Biochem. Biophys. 274, 138 (1989). 9. J. R. McDermott, J. A. Biggins, and A. M. Gibson, Biochem. Biophys. Res. Commun. 185, 746 (1992). 10. Z. Zeng and M. J. Glucksman, in preparation (1994). 11. B. L. Valee and D. S. Auld, Proc. Natl. Acad. Sci. U.S.A. 87, 220 (1990). 12. M. J. Glucksman, M. Cascio, M. Orlowski, and J. L. Roberts, in press (1994). 13. N. Sugira, H. Hagiwara, and S. Hirose, J. Biol. Chem. 267, 18067 (1992). 14. S. Kawabata, K. Nakagawa, S. Iwanaga, and E. W. Davie, J. Biol. Chem. 268, 12498 (1993). 15. S. G. Oliver, Q. J. M. van der Aart, M. L. Agostini-Carbone, M. Aigle, L. Alberghina, D. Alexandraki, G. Antoine, R. Anwar, J. P. Ballesta, P. Benit, et al., Nature (London) 357, 38 (1992). 16. C. A. Hrycyna and S. Clarke, Biochemistry 32, 11293 (1993). 17. G. Isaya, F. Kalousek, and L. E. Rosenberg, Proc. Natl. Acad. Sci. U.S.A. 89, 8317 (1992). 18. F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. ~

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19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38.

Smith, and K. Struhl, eds., "Current Protocols in Molecular Biology," Vol. 2. Wiley, New York, 1992. M. J. Glucksman, M. Orlowski, and J. L. Roberts, Biophys. J. 62, 119 (1992). J. Devereux, P. Haeberli, and O. Smithies, Nucleic Acids Res. 12, 387 (1984). C. Gaboriaud, V. Bissery, T. Benchetrit, and J. P. Mornon, FEBS Lett. 224, 149 (1987). T. Benchetrit, V. Bissery, J. P. Mornon, A. Devault, P. Crine, and B. P. Roques, Biochemistry 27, 592 (1988). S. M. Muskal and S. H. Kim, J. Mol. Biol. 225, 713 (1992). S. R. Holbrook, I. Dubchak, and S. H. Kim, BioTechniques 14, 984 (1993). J. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Lab., Cold Spring Harbor, NY, 1989. T. Maruyama, T. Gojobori, S. Aota, and T. Ikemura, Nucleic Acids Res. 14, r151 (1986). D. H. Mack and J. J. Sinisky, Proc. Natl. Acad. Sci. U.S.A. 85, 6077 (1988). E. Ohtsuka, S. Matsuki, M. Ikchara, Y. Takahashi, and D. J. Matsubasu, J. Biol. Chem. 260, 2605 (1985). R. A. Lew, T. Tetaz, M. J. Glucksman, J. L. Roberts, and A. I. Smith, J. Biol. Chem. 269 (1994) (in press). M. Cascio, in "Methods in Neurosciences, Volume 25: Receptor Molecular Biology" (Stuart C. Sealfon, ed.) (in press). F. Fossiez, G. Lemay, N. Labont6, F. Parmentier-Lesage, G. Boileau, and P. Crine, Biochem. J. 284, 53 (1992). A. H. Davies, J. B. M. Jovett, and I. M. Jones, Bio/Technology 11, 933 (1993). J. M. Cregg, T. S. Vedvick, and W. C. Raschke, Bio/Technology 11, 905 (1993). G. Lemay, G. Waksman, B. P. Roques, P. Crine, and G. Boileau, J. Biol. Chem. 264, 15620 (1989). C. H. Schein and M. H. M. Noteborn, Bio/Technology 6, 291 (1988). T. L. Blundell, R. Lapatto, A. F. Wilderspin, A. M. Hemmings, P. M. Hobart, D. E. Danley, and P. J. Whittle, Trends Biochem. Sci. 15, 425 (1990). M. J. Antonaccio and D. W. Cushman, Fed. Proc., Fed. Am. Soc. Exp. Biol. 40, 2275 (1981). E. G. Erdos and R. A. Skidgel, FASEB J. 3, 145 (1989).

[18]

Proteolytic Processing of Amyloid Protein Precursor of Alzheimer's Disease D. H. Small, G. Reed, S. J. Fuller, A. Weidemann, K. Beyreuther, and C. L. Masters

Introduction The two major pathological features in the brain of patients with Alzheimer's disease are neurofibrillary tangles and amyloid plaques. The production of the amyloid plaques is thought to be directly related to the underlying pathogenic mechanism. One line of evidence for this assumption is that the frequency of amyloid plaques correlates approximately with the extent of cognitive impairment (1, 2). The major protein component of the amyloid plaques is a polypeptide known as the amyloid or/3A4 protein (3, 4). Amino acid sequencing of this polypeptide led to the cloning of its precursor, a much larger protein known as the Alzheimer's disease amyloid protein precursor or APP (5). Although the production of amyloid plaques is associated with Alzheimer's disease, APP is a constituent of many normal cell types (6). The identification of rare familial diseases involving point mutations in the APP gene has established the importance of APP in the pathogenesis of Alzheimer's disease (7-9). Although the production of the amyloid protein from APP is linked to the pathogenesis of Alzheimer's disease, the precise relationship between APP and the disease process is unclear. One hypothesis to explain this relationship is that the/3A4 amyloid protein is neurotoxic (10). However, other hypotheses also need to be considered. For example, the disruption of the normal function of APP, caused by inappropriate proteolytic processing, could also contribute to neurodegeneration.

Structure and Function of Amyloid Protein Precursor Multiple molecular weight forms of APP result from alternative mRNA splicing of the APP gene product (11). The major APP mRNA expressed in the brain encodes a protein containing 695 amino acids (5). Two other major transcripts encoding forms with 751 and 770 amino acid residues have been identified (12-14). APP TM is identical to APP 695, except for an extra 56-residue domain homologous to members of the Kunitz family of protease inhibitors. Methods in Neurosciences, Volume 23 Copyright 9 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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The presence of this Kunitz protease inhibitor (KPI) domain confers the ability to inhibit a few serine proteases (15, 16). APP 77~contains an additional 19-residue domain homologous to the OX-2 antigen (17). The amyloid sequence itself is contained in a stretch of 43 amino acid residues that comprises a small portion of the ectodomain of APP and extends into the transmembrane domain (Fig. 1). Minor forms of APP involving other spliced products have also been identified. A P P 714 contains the OX-2, but lacks the KPI domain (18). A P P 365 and A P P 563 do not contain the transmembrane domain and are therefore thought to be secreted forms (19, 20). Finally, many cells express large amounts of a transcript lacking exon 15 ( A P P 733 o r L-APP) (21). The amyloid protein precursor is posttranslationally modified by N- and O-linked glycosylation, sulfation, and phosphorylation. There are two potential N-linked glycosylation sites close to the transmembrane domain (Fig. 1). Although the sites of O-linked glycosylation have not been identified, a cluster of threonine residues (found next to the acidic region) is a consensus sequence for O-glycosylation (22). The amyloid protein precursor can also be phosphorylated by one or more serine kinases (23); however, the function of this phosphorylation is unknown. As APP is phosphorylated in the ectodomain, this suggests that the phosphorylation of APP could regulate its interaction with extracellular molecules, such as the extracellular matrix (24). The function of APP is still unknown. In the central and peripheral nervous system, APP may be involved in the regulation of neurite outgrowth (24-28). In the embryonic chick brain, APP expression increases during the major phase of neurite outgrowth (24). Other studies (25-28) suggest that APP can directly stimulate neurite outgrowth from neurons or neuronal cell lines in culture. The involvement of APP in the development of the nervous system is also suggested by genetic studies. Drosophila lacking a gene homologous to the human APP gene possess a behavioral deficit that can be corrected with the human APP gene (29).

Proteolytic Processing of the Amyloid Protein Precursor Some of the proteases reported to cleave APP are shown in Table I (30-39). The identification of specific APP-processing enzymes is not an easy task as many different proteases may have the required specificities for cleavage. It seems likely that in vivo, specificity is defined not only by the amino acid sequence around the cleavage site, but also by cellular and subcellular compartmentation of the enzymes with APP. Several criteria should be fulfilled for the unequivocal identification of an APP-processing enzyme. These criteria are as follows.

[18]

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P R O T E O L Y T I C PROCESSING OF APP

~ch

s fate']

hepaHn-binding site

~-- Zn binding site ACIDIC THR-rich

KPI ~me~ 9

domain/ i

T~~

LLLLLIJ,,L

FIG. 1 Diagrammatic representation of the structure of APP 695, including the portion of the amyloid protein sequence containing the cleavage site for the APP secretase (Lys-16 in the amyloid sequence). Full-length APP possesses a large ectodomain containing the N terminus, a single transmembrane domain of 24 amino acid residues (residues 625-648 in the APP 695 sequence), and a short cytoplasmic tail of 24 amino acid residues containing the C terminus. Amyloid protein precursor contains two potential N-linked glycosylation sites and a number of domains, including cysteinerich, threonine-rich, and acidic (aspartate and glutamate-rich) domains. Heparinbinding and zinc-binding domains have been identified, along with a growth-promoting domain. APP TM and APP 77~contain an extra sequence with homology to the Kunitz family of proteinase inhibitors (KPI domain) inserted close to the threonine-rich region. APP 77~has an additional domain with homology to the OX-2 antigen. Failure to cleave APP at Lys-16 of the amyloid sequence by the APP secretase results in the preservation of the amyloid sequence. Amyloidogenic forms of APP may subsequently be degraded by amyloid-generating enzymes (AGEs), which cleave adjacent to the methionine residue at position - 1.

1. The protease should show the expected cleavage specificity. For example, the protease should be able to cleave synthetic peptides with sequences homologous to known cleavage sites in APP.

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TABLE I Putative Amyloid Protein Precursor Processing Enzymes Protease

Proposed cleavage site

Ref.

1. Calcium-activated serine protease 2. Multicatalytic protease (calcium regulated)

N-terminus of flA4 Glnl5-Lys 16of/3A4 N terminus of flA4 N terminus of flA4 C terminus of/3A4 Lys16-Leu17of flA4 Lysl6-Leu 17 of flA4 Lys16-Leu17 of flA4 Glu3-Phe4 of flA4 Multiple sites in APP ArgSl~ TM of APP

30 31 31 32 33 34 35 36 37 38 39

3. 4. 5. 6. 7. 8. 9. 10.

Chymotrypsin-like protease (clipsin) Prolyl endopeptidase Acetylcholinesterase-associated protease Cathepsin B Gelatinase A Multicatalytic protease (ingesin) Calpain I Thrombin

2. The spatiotemporal expression of the enzyme should match that of APP or its cleavage products. For example, increased expression of the protease should be associated with increased cleavage of APP, and the tissue distribution of the protease should to some extent reflect the distribution of its substrate. 3. Inhibitors of the protease should inhibit the processing of APP in situ. This would include the use of antisense oligonucleotide methodology. 4. The most stringent criterion for the identification of a processing enzyme is to show that in organisms (or cells in culture) engineered for a deletion in the processing enzyme, there is a reduction or failure to cleave APP. A major route of APP processing involves an enzyme called the "APP secretase," which cleaves APP between Lys-16 and Leu-17 in the flA4 amyloid sequence (40). The resulting C-terminally truncated APP possesses a molecular mass that is approximately 10 kDa lower than the transmembrane protein and is subsequently secreted from the cell (41). Since the secretase cleavage site was first identified, other potential secretase cleavage sites have been found (42, 43). Studies by Sisodia (44) using site-directed mutagenesis have shown that the APP secretase is probably not highly specific in the type of peptide bond it is able to cleave. Instead, the distance of the peptide bond from the plasma membrane is the most critical factor that defines the secretase specificity. Cole et al. (45) provided evidence for the processing of APP through the lysosomal system. Studies by Golde and co-workers (43) suggest that the processing of APP through the endosomal-lysosomal system could result in the production of a complex series of C-terminal derivatives. Some of these

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derivatives retain the amyloid sequence intact and may therefore be amyloidogenic; many cells may secrete these amyloidogenic products. In microglia and astrocytes, amyloidogenic cleavage may be a major pathway of processing (46). The presence of a consensus sequence (NPXY) for coated pit-mediated internalization (47) supports the proposition that APP may undergo endocytosis. The APP secretase is important for understanding the pathogenesis of Alzheimer's disease. Cleavage at Lys-16 destroys the amyloid sequence and thus prevents flA4 amyloid formation. In many cell types, the APP secretase may represent the major processing route. Furthermore, there is some evidence to suggest that a failure to cleave APP at the secretase step could result in amyloidogenic processing. Although candidate enzymes have been proposed (34, 35), the APP secretase has not yet been identified. As with the neuropeptide-processing endopeptidases, a molecular genetic approach may be the only way to clearly identify the secretase. Nonetheless, cell lines that are transfected with APP cDNA constructs are useful for studying the processing of APP through amyloidogenic and nonamyloidogenic pathways. Some general methods for studying the processing of APP are described in the following sections. These methods can be usefully adapted to studying a range of different aspects of APP processing and secretion. The monoclonal antibody (clone 22C11, which can be obtained from Boehringer Mannheim, Indianapolis, IN) used in these procedures recognizes a domain close to the N terminus (residues 66-81) (48). With the identification of other members of a now-expanding APP gene family (49, 50), it is possible that the 22C11 antibody may recognize other APP-like proteins. Thus, the specificity of the antibody must be demonstrated in each tissue under examination. Studying the processing of APP in cells transfected with an APP cDNA expression plasmid eliminates this problem.

T r a n s f e c t i o n of H e L a Cells with an A m y l o i d P r o t e i n P r e c u r s o r Expression Plasmid The method for transfecting HeLa cells with an expression plasmid employs the standard procedure of calcium phosphate coprecipitation. We have used an expression vector (pAPP-695) derived from pUC in which the A P P 695 sequence is inserted at the B a m H I site. The procedure is essentially as described by Weidemann et al. (41). 1. Approximately 24 hr prior to transfection, HeLa cells are split into 75flasks at a density of 1.5 x 10 6 cells/flask. Cells are cultured in 10 ml/

cm 2

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flask of Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) fetal calf serum. At least 3 hr before transfection, the growth medium is replaced with fresh medium. 2. Plasmid DNA (24/zg) is precipitated with ethanol and air dried, and then dissolved in 438/zl of 10 mM Tris-HC1 buffer (pH 7.6). Then 62/zl of 2 M CaC12 is added under sterile conditions and the solution allowed to incubate for 10 min at room temperature. 3. During the incubation period, 500/xl of fresh 2 • HeBS (280 mM NaC1, 1.5 M Na2HPO4,50 mM HEPES, titrated to pH 7.13 with NaOH) is aliquoted into 5- or 10-ml round-bottom tubes. The DNA solution is added dropwise to the 2 • HeBS solution with stirring over a period of about 15 sec. The solution is allowed to incubate under sterile conditions at room temperature for 10-30 min, and then the DNA-CaC12 solution is added gently and evenly over the cells and the flask gently agitated. 4. The cells are incubated at 37~ in an atmosphere of 5% CO2 overnight (16-20 hr); the precipitated DNA is then removed, and the cells washed twice with Ca 2§ Mg2+-free phosphate-buffered saline (PBS). Finally the cells are incubated in 10 ml of DMEM containing 10% (v/v) fetal calf serum for 24-48 hr. During this period, the cells should express A P P 695 maximally. Approximately 5-10% of the cells should be transfected using this protocol. The expression and secretion of A P P 695 from transfected HeLa cells can be monitored both by immunoprecipitation and by Western blotting, using a commercially available monoclonal antibody (clone 22C11).

I m m u n o p r e c i p i t a t i o n of the A m y l o i d P r o t e i n P r e c u r s o r after Pulse-Chase Labeling The procedure for immunoprecipitation has been described previously (51). 1. Cells are labeled with [35S]methionine in methionine-free medium in the absence of serum for the required length of time (e.g., 20 min to 1 hr), using any one of several standard protocols. At the end of the pulse, the cells are washed once with methionine-containing medium, chased in methioninecontaining medium for 15 min to 2 hr, and then resuspended in 180 ~1 of Ca 2+ , Mg2+-free PBS containing 2 mM phenylmethylsulfonyl fluoride (PMSF). The cells are disrupted by adding 20/~1 of 10% (w/v) sodium dodecyl sulfate (SDS) and the proteins denatured in a boiling water bath for 5 min. At the end of this period, 200/zl of neutralization buffer [6% v/v) Nonidet P-40

[18] PROTEOLYTIC PROCESSING OF APP

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(NP-40), 200 mM Tris-HC1 buffer (pH 7.4), 300 mM NaC1, 10 mM EDTA, 4 mM NAN3] is added. The solution is sonicated (Branson sonifier on setting 1, four short bursts at 50% intermittency) and then centrifuged in a microfuge at 12,000 rpm for 10 min at 4~ and the pellet discarded. 2. The monoclonal antibody (22C 11 from Boehringer Mannheim) is added (10/zl of a 60-/zg/ml solution) and the tubes are incubated for 1 hr at room temperature with gentle shaking. 3. Protein A-Sepharose (PAS; Pharmacia-LKB, Piscataway, NJ) (7 mg/ incubation tube) is hydrated in STEN buffer [50 mM Tris-HC1 buffer (pH 7.5), 150 mM NaC1, 2 mM EDTA, 0.2% (v/v) Nonidet P-40] for 1 hr and then washed six times with 1 ml of STEN buffer. The PAS is then resuspended in 1 ml of STEN and 1.0 /zl of rabbit anti-mouse immunoglobulin G (IgG) (DAKO Corp., Carpinteria, CA) is added for every milligram of PAS and the mixture incubated for 60 min at room temperature with gentle shaking. The PAS is washed a further three times with STEN, followed by two washes in high-salt STEN (STEN buffer with 500 mM NaC1 instead of 150 mM NaC1) and one wash with STEN. 4. For each incubation tube, 7 mg of anti-mouse IgG-coupled PAS is resuspended in 50/zl of STEN and added to the samples, which are then incubated for 1 hr at room temperature with gentle shaking. After incubation, the gel is washed three times with STEN, twice with high-salt STEN, and once with 10 mM Tris-HCl buffer, pH 7.5. The immunoprecipitated APP can then be analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) followed either by fluorography or analysis with a PhosphorImager (Fig. 2).

Western Blotting of Amyloid Protein Precursor If a nonquantitative assessment of APP levels in a tissue sample is required, Western blotting is an appropriate procedure. Western blotting provides information not only on the level of APP immunoreactivity, but also on the various molecular weight forms of APP in a tissue. The procedure is similar to the procedure of Weidemann et al. (41). 1. Fractions are normally analyzed on 10% (w/v) polyacrylamide gels in the presence of 0.1% (w/v) SDS. Electrophoresis is normally performed with 0.5-mm-thick minigels, using the Bio-Rad (Richmond, CA) Mini-PROTEAN system. After electrophoresis, the proteins are electrophoretically blotted onto polyvinylidene difluoride (PVDF) or nitrocellulose membrane. The PVDF membranes are presoaked in 100% methanol. We transfer at 300 mA for 16 hr, with cooling in a Bio-Rad Trans-Blot cell with plate electrodes.

324

III


B

A

Mr

Mr 2007-

69-

46-

200-

r ~'

110 kDa

110 kDa

97 - ~ - 69--

~100

kDa

9

6-

FIG. 2 Immunoprecipitation of [35S]methionine-labeled APP from PC12 cells (A) and Western blot analysis of a cell homogenate and conditioned medium from HeLa cells (B). (A) PC12 cells were labeled with [35S]methionine (70/zCi/ml) for 1 hr and then the APP immunoprecipitated from a detergent extract of the cells as described in text. The immunoprecipitated [35S]APP was analyzed by SDS-PAGE on a 8.5% (w/v) polyacrylamide gel. (B) HeLa cells were transfected with pAPP-695, using the calcium phosphate coprecipitation method. Proteins were separated by SDS-PAGE, transferred onto nitrocellulose, and stained with a monoclonal antibody (clone 22C 11). Most of the cellular APP is in full-length form and migrates with an apparent molecular mass of l l0 kDa. The secreted form of APP695 in the conditioned medium is C-terminally truncated by the APP secretase and possesses an apparent molecular mass of 100 kDa.

The transfer buffer contains 25 mM Tris base, 190 mM glycine, and 20% (v/v) methanol. 2. After electroblotting, nonspecific binding sites are blocked by incubating for 2 hr at room temperature with a buffer containing 0.1 M Tris-HC1 buffer (pH 7.4), 150 mM NaCI, 0.25% (w/v) bovine serum albumin (BSA), 0.05% (v/v) Tween 20, 2 mM MgC12. The PVDF membrane is incubated with 22C 11 antibody (2/zg/ml) in blocking buffer for 2 hr at room temperature. The membrane is then washed three times with TBST [10 mM Tris-HC1 (pH 8.0), 150 mM NaC1, 0.05% (v/v) Tween 20] and incubated for 2 hr at room temperature with an alkaline phosphatase-conjugated anti-mouse IgG (normally 1" 10,000 dilution) in TBST. 3. After washing three times with TBST, the immunoreactive bands are visualized by staining with a chromogenic buffer for alkaline phosphatase, such as naphthol AS-MX/Fast Red [1 part naphthol AS-MX phosphate (0.4 mg/ml) in water mixed with 1 part Fast Red TR (6 mg/ml) in 0.2 M TrisHC1 (pH 8.0), containing 2 mM MgC12]. Full-length forms of APP are seen

[18] PROTEOLYTICPROCESSING OF APP

325

as multiple broad bands with apparent molecular weights of 100,000 to 130,000 (Fig. 2).

References

10. 11. 12. 13.

14.

15. 16. 17. 18.

M. Roth, B. E. Tomlinson, and G. Blessed, Nature (London) 209, 109 (1966). E. K. Perry, B. E. Tomlinson, G. Blessed, K. Bergmann, P. H. Gibson, and R. H. Perry, Br. Med. J. 2, 1457 (1978). G. G. Glenner and C. W. Wong, Biochem. Biophys. Res. Commun. 120, 885 (1984). C. L. Masters, G. Simms, N. A. Weinman, G. Multhaup, B. L. McDonald, and K. Beyreuther, Proc. Natl. Acad. Sci. U.S.A. 82, 4245 (1985). J. Kang, H. G. Lemaire, A. Unterbeck, J. M. Salbaum, C. L. Masters, K. H. Grzeschik, G. Multhaup, K. Beyreuther, and B. M011er-Hill, Nature (London) 325, 733 (1987). S. Sinha and I. Lieberburg, Neurodegeneration 1, 169 (1992). A. Goate, M. C. Chartier-Harlin, M. Mullan, J. Brown, F. Crawford, L. Fidani, L. Giuffra, A. Haynes, N. Irving, L. James, R. Mant, P. Newton, K. Rooke, P. Roques, C. Talbot, M. Pericak-Vance, A. Roses, R. Williamson, M. Rossor, M. Owen, and J. Hardy, Nature (London) 349, 704 (1991). M. C. Chartier-Harlin, F. Crawford, H. Houlden, A. Warren, D. Hughes, L. Fidani, A. Goate, M. Rosor, P. Roques, J. Hardy, and M. Mullan, Nature (London) 353, 844 (1991). M. Mullan, F. Crawford, K. Axelman, H. Houlden, L. Lilius, B. Winblad, and L. Lannfelt, Nat. Genet. 1, 345 (1992). B. A. Yankner, L. K. Duffy, and D. A. Kirschner, Science 250, 279 (1990). R. E. Tanzi, A. I. McClatchey, E. D. Lamperti, L. Villa-Komaroff, J. F. Gusella, and R. L. Neve, Nature (London) 331, 528 (1988). N. Kitaguchi, Y. Takahashi, Y. Tokushima, S. Shiojiri, and H. Ito, Nature (London) 331, 530 (1988). P. Ponte, P. Gonzalez-DeWhitt, J. Schilling, J. Miller, D. Hsu, B. Greenberg, K. Davis, W. Wallace, I. Lieberburg, F. Fuller, and B. Cordell, Nature (London) 331, 525 (1988). W. E. Van Nostrand, S. L. Wagner, M. Suzuki, B. H. Choi, J. S. Farrow, J. W. Geddes, C. W. Cotman, and D. D. Cunningham, Nature (London) 341, 546 (1989). R. P. Smith, D. A. Higuchi, and G. J. Broze, Jr., Science 248, 1126 (1990). H. Kido, A. Fukutomi, J. Schilling, Y. Wang, B. Cordell, and N. Katunama, Biochem. Biophys. Res. Commun. 167, 716 (1990). M. J. Clarke, J. Gagnon, A. F. Williams, and A. N. Barclay, EMBO J. 4, 113 (1985). T. E. Golde, S. Estus, M. Usiak, L. H. Younkin, and S. G. Younkin, Neuron 4, 253 (1990).

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III EXTRACELLULAR PROCESSING ENZYMES IN THE CNS 19. J. S. Jacobsen, H. A. Muenkel, A. J. Blume, and M. P. Vitek, Neurobiol. Aging 12, 575 (1991). 20. F. De Sauvage and J. N. Octave, Science 245, 651 (1989). 21. G. K6nig, U. M6nning, C. Czech, R. Prior, R. Banati, U. Schreiter-Gasser, J. Bauer, C. L. Masters, and K. Beyreuther, J. Biol. Chem. 267, 10804 (1991). 22. I. B. H. Wilson, Y. Gavel, and G. von Heijne, Biochem. J. 275, 529 (1991). 23. C. Haass, A. Y. Hung, M. G. Schlossmacher, T. Oltersdorf, D. B. Teplow, and D. J. Selkoe, Ann. N.Y. Acad. Sci. 695, 109 (1993). 24. D. H. Small, V. Nurcombe, R. Moir, S. Michaelson, D. Monard, K. Beyreuther, and C. L. Masters, J. Neuroscience 12, 4143 (1992). 25. E. M. Milward, R. Papadopoulos, S. J. Fuller, R. D. Moir, D. Small, K. Beyreuther, and C. L. Masters, Neuron 9, 129 (1992). 26. A. C. LeBlanc, D. M. Kovacs, H. Y. Chen, F. Villar6, M. Tykocinski, L. AutilioGambetti, and P. Gambetti, J. Neurosci. Res. 31, 635 (1992). 27. J. S. Whitson, Neurosci. Lett. 110, 319 (1990). 28. D. H. Small, V. Nurcombe, G. Reed, H. Clarris, R. Moir, K. Beyreuther, and C. L. Masters, J. Neurosci. 14, 2117 (1994). 29. L. Luo, T. Tully, and K. White, Neuron 9, 595 (1992). 30. C. R. Abraham, J. Driscoll, H. Potter, W. E. Van Nostrand, and P. Tempst, Biochem. Biophys. Res. Commun. 174, 790 (1991). 31. S. Kojima and M. Omori, FEBS Lett. 304, 57 (1992). 32. R. B. Nelson and R. Siman, J. Biol. Chem. 265, 3836 (1990). 33. S. Ishiura, T. Tsukahara, T. Tabira, T. Shimizu, K. Arahatz, and H. Sugita, FEBS Lett. 260, 131 (1990). 34. D. H. Small, R. D. Moir, S. J. Fuller, S. Michaelson, A. I. Bush, Q. X. Li, E. Milward, C. Hilbich, A. Weidemann, K. Beyreuther, and C. L. Masters, Biochemistry 30, 10795 (1991). 35. K. Tagawa, T. Kunishita, K. Maruyama, K. Yoshikawa, E. Kominami, T. Tsuchiya, K. Suzuki, T. Tabira, H. Sugita, and S. Ishiura, Biochem. Biophys. Res. Commun. 177, 377 (1991). 36. K. Miyazaki, M. Hasegawa, K. Funahashi, and M. Umeda, Nature (London) 362, 839 (1993). 37. S. Ishiura, T. Tsukahara, T. Tabira, and H. Sugita, FEBS Lett. 257, 388 (1989). 38. R. Siman and G. Christoph, Biochem. Biophys. Res. Commun. 165, 1299 (1989). 39. K. Igarishi, H. Murai, and J. Asaka, Biochem. Biophys. Res. Commun. 185, 1000 (1992). 40. F. S. Esch, P. S. Keim, E. C. Beattie, R. W. Blacher, A. R. Culwell, T. Oltersdorf, D. McClure, and P. J. Ward, Science 248, 1122 (1990). 41. A. Weidemann, G. K6nig, D. Bunke, P. Fischer, J. M. Salbaum, C. L. Masters, and K. Beyreuther, Cell (Cambridge, Mass.) 57, 115 (1989). 42. C. Haass, M. G. Schlossmacher, A. Y. Hung, C. Vigo-Pelfry, A. Mellon, B. L. Ostaszewski, I. Lieberburg, E. H. Koo, D. Schenk, D. B. Teplow, and D. J. Selkoe, Nature (London) 359, 322 (1992). 43. T. E. Golde, S. Estus, L. H. Younkin, D. J. Selkow, and S. G. Younkin, Science 255, 728 (1992). 44. S. S. Sisodia, Proc. Natl. Acad. Sci. U.S.A. 89, 6075 (1992).

[18] PROTEOLYTIC PROCESSING OF APP 45. 46. 47. 48.

327

G. M. Cole, T. V. Huynh, and T. Saitoh, Neurochem. Res. 14, 933 (1989). C. Haass, A. Y. Hung, and D. J. Selkoe, J. Neurosci. 11, 3783 (1991). W. J. Chen, J. L. Goldstein, and M. S. Brown, J. Biol. Chem. 265, 3116 (1990). C. Hilbich, U. M6nning, C. Grund, C. L. Masters, and K. Beyreuther, J. Biol. Chem. 35, 26571 (1993). 49. W. Wasco, K. Bupp, M. Magendantz, J. F. Gusella, R. E. Tanzi, and F. Solomon, Proc. Natl. Acad. Sci. U.S.A. 89, 10758 (1992). 50. C. A. Sprecher, F. J. Grant, G. Grimm, P. J. O'Hara, F. Norris, K. Norris, and D. C. Foster, Biochemistry 32, 4481 (1993). 51. G. L. Caporaso, S. E. Gandy, J. D. Buxbaum, and P. Greengard, Proc. Natl. Acad. Sci. U.S.A. 89, 2252 (1992).

[19]

Strategies for Measurement of Angiotensin and Bradykinin Peptides and Their Metabolites in Central Nervous System and Other Tissues Duncan J. Campbell, Anne C. Lawrence, Athena Kladis, and Ann-Maree Duncan

Introduction Whether angiotensin and bradykinin are neuropeptides is a subject of continuing debate. The strength of the evidence for or against such a proposition is dependent on the methodological basis for such evidence. Rather than address this issue directly, in this chapter we describe some of the methodologies we have developed for the measurement of angiotensin and bradykinin peptides and their metabolites in the central nervous system (CNS) and other tissues. In the past, radioimmunoassay (RIA) of angiotensin and bradykinin peptides was based on the use of carboxy (C) terminal-directed antisera. This was due in large part to the ease with which a peptide may be coupled via its amino (N) terminus to carrier proteins for the purpose of immunization. However, for both angiotensin and bradykinin peptides important processing events take place toward the C terminus of the molecule (1-3)- (Figs. 1 and 2). For example, the decapeptide angiotensin I (Ang I) is cleaved between residues 8 and 9 by angiotensin-converting enzyme (ACE, kininase II, EC 3.4.15.1, peptidyl-dipeptidase A) to release angiotensin II (Ang II), and both Ang I and Ang II are cleaved between residues 7 and 8 by a number of endopeptidases to release angiotensin(1-7) [Ang(1-7)]. Both Ang II and Ang(1-7) are bioactive. An alternative pathway of conversion of Ang I to Ang II may involve the sequential cleavage of the two C-terminal residues of Ang I by carboxypeptidase activity (1). Moreover, the nonapeptide bradykinin(1-9) [BK(1-9)] is cleaved between residues 8 and 9 by carboxypeptidases N (kininase I) and M to release bradykinin(1-8) [BK(1-8)], and between residues 7 and 8 by ACE and other endopeptidases to release bradykinin(1-7) [BK(1-7)]. Both BK(1-9) and BK(1-8) are bioactive. When these differentially processed peptides are separated by high-performance liquid chromatography (HPLC), it is of assistance if the peptides of interest 328


[19] MEASUREMENTOF ANGIOTENSIN AND BRADYKININ

Mast cell chymase Neutrophil cathepsin G

Aminopeptidase A 1

2

Chymotrypsin

3

4

A s p-A~/~-V ~ a, ,-Ty ~ T rypsi n

6

329

AC E Humanheartchymase

Tonin

7

10

,e-H i s - ~ - P he-~~Le u

~Endopeptidase , %

24.15 ~

" E n d o p e p t i d a s e 24.11

Carboxypeptidases Prolylendopeptidase

FIG. 1 Diagrammatic representation of cleavage sites of angiotensin I by different enzymes. Both endopeptidases 24.11 and 24.15 cleave angiotensin I between residues 4 and 5, and between residues 7 and 8; in addition, endopeptidase 24.11 cleaves angiotensin I between residues 2 and 3. After removal of the amino-terminal aspartic acid by aminopeptidase A, the Arg2 residue can be cleaved by aminopeptidase N. For angiotensin II, the sites of cleavage by endopeptidases are the same as those shown for angiotensin I, except that endopeptidases 24.11 and 24.15 do not cleave between residues 7 and 8 of angiotensin II. ACE, Angiotensin-converting enzyme.

can be measured with the same RIA. To this end, we established N terminaldirected RIA for the measurement of angiotensin and bradykinin peptides and their C-terminal truncated metabolites. In previous attempts to raise N terminal-directed antisera to angiotensin peptides, although the peptides were coupled to the carrier protein via the C terminus, the antisera raised were predominantly directed to the C terminus (4, 5). However, Nussberger et al. (4) found that when Asn ~, VaP-Ang II was acetylated at the N terminus and coupled via the C terminus for immunization, they readily achieved N terminal-directed antisera. This result suggests that acetylation of the N terminus of a peptide renders the N terminus more immunogenic. We used this approach to raise N terminal-directed antisera against N-acetylated angiotensin and bradykinin peptide analogs

Endopeptidase24.15

KininaseII (ACE)

Arg(?-Pro-G ly~ he-Ser-Pro-P he-Arg AminopeptidaseP

~Endopeptidase 2 4 . 1 1 ~ ~ P ~ ~

\

l KininaseI (CarboxypeptidaseN) Prolylendopeptidase Carboxypeptidase M

FIG. 2 Diagrammatic representation of cleavage sites of bradykinin by different enzymes. ACE, Angiotensin-converting enzyme.

330

III


(3, 6), with the intention of acetylating peptides extracted from biological samples before RIA with these antisera.

Preparation of Antisera For the preparation of antisera directed against the N terminus of Ang II, angiotensin III (Ang III), and B K(1-9), the following peptides are synthesized: N-acetyl-AspArgValTyrIleHisProPheLys (N-Ac-Lysg-Ang II), N-acetyl-ArgValTyrIleHisProPheLys (N-Ac-Lys8-Ang III), and N-acetyl-ArgProProGlyPheSerProPheLys [N-Ac-Lys9-BK(1-9)]. Peptides are synthesized from tert-butoxycarbonyl-protected amino acids, using an Applied Biosystems (Foster City, CA) 430A automated peptide synthesizer. Acetylation of a-amino groups of Lysg-Ang II, Lys8-Ang III, and Lys9-BK(1-9) is performed on the protected resin before hydrogen fluoride treatment (6). Peptides are coupled to bovine thyroglobulin via the C-terminal lysine residue with glutaraldehyde (7), and antisera are raised in rabbits (8). Six rabbits are immunized with each peptide and the best antiserum against each peptide is subsequently used to establish an RIA.

Description of Radioimmunoassays All peptide concentrations are determined by amino acid analysis, using stocks of approximately 1 mg/ml in 20% (v/v) acetic acid in water, and stored at -30~ Working solutions [1 /~M in lysozyme (1 mg/ml), 10 mM acetic acid] are stored at -30~ and discarded after thawing once. All RIA components are diluted with casein phosphate buffer [casein (1 g/liter), 100 mM sodium phosphate, 10 mM disodium ethylenediaminetetraacetic acid (EDTA), sodium azide (1 g/liter), 154 mM sodium chloride, pH 7.0]. A pH optimum of 7.0 has been shown for each of the three assays described. Initially, the total RIA assay volume was 500 /A (6), but this has since been reduced to 250/~1 to increase sensitivity. Although initially prepared on ice, assays are now prepared at room temperature. Each assay tube contains 50/A of diluted antibody, 50/~1 of tracer (---2500 cpm), 50/~1 of standard or unknown peptide solution, and 100/~1 of buffer. Usually the assays are incubated at 4~ for 48 hr before separation of free from bound radioactivity. For the antibody A41 assay, addition of tracer is delayed t~or 48 hr, and the assay is incubated at 4~ for a further 24 hr before separation of free from bound radioactivity. Separation of free from bound radioactivity with albumin/dextran-coated charcoal is performed using a modification (9) of the method described by

[19] MEASUREMENT OF ANGIOTENSIN AND BRADYKININ

331

Herbert et al. (10). Stock dextran-coated charcoal [Norit A charcoal (25 g/liter), dextran T10 (2.5 g/liter), 7.1 mM sodium barbitone, 7.1 mM sodium acetate, adjusted to pH ---7.4 with hydrochloric acid] is stirred with bovine serum albumin (BSA, 10 mg/ml) for 1-24 hr at 4~ and then diluted with 4 vol of 150 mM sodium chloride immediately before use. One milliliter of albumin/dextran-coated charcoal is added to each tube at 4~ and, after standing at 4~ for 10 min, the assay tubes are centrifuged at 5000 g for 10 min at 4~ the supernatants rapidly aspirated, and the charcoal pellets counted. Tracer peptides are iodinated with 125I using chloramine-T (11), and the monoiodinated peptides are purified by HPLC on a C18 column, using a gradient of acetonitrile in 0.1% (v/v) trifluoroacetic acid (TFA), and stored in aliquots at -30~ Tracer peptides can be stored for up to 2 months without deterioration in assay performance. Antibody A41 was raised against N-Ac-Lys9-Ang II. The antibody A41 assay uses N, O-diacetyl-Ang II (Ac-Ang II, acetylated as described below) as standard peptide and mono[~ZSI]iodo-Ac-Ang II as tracer. At a dilution of 1 : 270,000, binding of tracer is approximately 50%, and 50% displacement is obtained with ---8 fmol of Ac-Ang II/tube, with a detection limit of ---0.25 fmol/tube. The within-assay coefficient of variation is 6% and the betweenassay coefficient of variation is 19%. Antibody A41 was initially studied using N-Ac-Lys9-Ang II as standard and 125I-labeled N-Ac-Lys9-Ang II as tracer; however, displacement of 125I-labeled N-Ac-Lys9-Ang II by N-AcLys9-Ang II and Ac-Ang II was not superimposable, with incomplete displacement by Ac-Ang II, indicating that a proportion of the antibody population of A41 was specific for N-Ac-Lys9-Ang II. Consequently, N-Ac-Lys 9Ang II cannot be used as standard for the measurement of Ang II levels in biological samples; instead, Ac-Ang II must be used as standard. The use of 125I-labeled Ac-Ang II as tracer has the advantage that Ac-Ang II produces complete displacement of tracer. Antibody A52 was raised against N-Ac-Lysg-Ang III. The antibody A52 assay uses N-Ac-LysS-Ang III as standard peptide and 125I-labeled N-AcLysS-Ang III as tracer. At a dilution of 1 : 48,500, binding of tracer is approximately 50%; assays with antibody A52 have been performed using a total assay volume of only 500 ~1, and 50% displacement is obtained with --~16 fmol of N-Ac-LysS-Ang III/tube, with a detection limit of ---1.0 fmol/tube. The between-assay coefficient of variation is 12%. In contrast to the antibody A41 assay, displacement of 125I-labeled N-Ac-Lys8-Ang III by N-Ac-Lys 9Ang III is identical to that produced by N,O-diacetyl-Ang III (Ac-Ang III), and this assay can be used to measure Ang III in biological samples. Antibody B24 was raised against N-Ac-Lysg-BK(1-9). The antibody B24 assay uses N-Ac-Lys9-BK(1-9) as standard peptide. 125I-Labeled Tyr 8BK(1-9) is acetylated as described below before purification by HPLC, and

332

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E X T R A C E L L U L A R PROCESSING ENZYMES IN THE CNS

mono[125I]iodo-Ac-Tyr8-BK(1-9) is used as tracer. At a dilution of 1 : 267,000, binding of tracer is approximately 50%; and 50% displacement is obtained with ---2 fmol of N-Ac-Lys9-BK(1-9)/tube, with a detection limit of---0.05 fmol/tube. The within-assay coefficient of variation is 14.5%. Displacement of nSI-labeled Ac-Tyr8-BK(1-9) by N-Ac-Lys9-BK(1-9) is identical to that produced by N,O-diacetyl-BK(1-9) [Ac-BK(1-9)], and this assay can be used to measure B K(1-9) in biological samples.

Characterization of Antisera A complete description of the specificities of the antisera is given elsewhere (3, 6). For all antisera, cross-reactivity studies revealed an absolute requirement for acetylation of the N terminus. For antisera A41 and B24 crossreactivities were 100% for peptides of eight or more residues, 75-80% for peptides of seven residues, and correspondingly less for shorter peptides. For antibody A52, cross-reactivity of Ac-Ang(2-7) was 87.5% of that for AcAng III, with a correspondingly lower cross-reactivity for shorter peptides. In practice, antibody A41 can be used for the measurement of Ac-Ang I, AcAng(1-9), Ac-Ang II, and Ac-Ang(1-7); antibody A52 can be used for the measurement of Ac-Ang(2-10), Ac-Ang(2-9), Ac-Ang III, and Ac-Ang(2-7); antibody B24 can be used for the measurement of Ac-B K(1-9), Ac-B K(1-8), and Ac-BK(1-7).

Acetylation of Peptides The method of acetylation is based on the procedure described by Dobson and Strange (12). Peptides or peptide extracts are taken to dryness in siliconized 13 x 100 mm borosilicate glass tubes, using a vacuum centrifuge (Savant Instruments, Hicksville, NY), then acetylated by sequential addition of 100 /A of water, 10 ~1 of triethylamine, and 5/A of acetic anhydride, with mixing by vortex after each addition. After centrifugation to remove particulate material, the sample is injected directly onto the chromatograph. Alternatively, the acetylated samples may be taken to dryness under vacuum and then dissolved in 120/A of 20% (v/v) acetic acid before centrifugation and injection onto the chromatograph (6). As described below, the acetylation procedure results in the acetylation of residues in addition to the a-amino group of each peptide. We have not identified these other acetylated residues, but they probably include Oacetylation of Try 4 of angiotensin and Ser 6 of bradykinin. In contrast to the N-acetyl group, these O-acetyl groups are labile and can be hydrolyzed by

[19] MEASUREMENT OF ANGIOTENSIN AND BRADYKININ

333

treatment with 10% (v/v) piperidine (3). Samples to be treated with piperidine are taken to dryness following acetylation, then dissolved in 100/xl of 10% piperidine in water and allowed to stand at room temperature for 60 min before evaporation to dryness again, dissolution in 120/zl of 20% acetic acid in water, centrifugation, and injection onto the chromatograph. S e p a r a t i o n of A c e t y l a t e d P e p t i d e s by H i g h - P e r f o r m a n c e Liquid Chromatography All samples are transferred to siliconized microfuge tubes and centrifuged in a microfuge at top speed (15,850 g) for 5 min at room temperature to remove particulate material before the supernatant is injected onto the chromatograph. All separations are performed on a 100 x 4.6 mm Brownlee RP-18 Spheri5 column preceded by a 15 x 3.2 mm RP-18 guard column (Applied Biosystems). The HPLC system consists of two pumps (model 6000A; MilliporeWaters, Milford, MA), an automated gradient controller (model 680; Millipore-Waters), and an injector (Rheodyne, Inc., Cotati, CA) with a 200-/zl sample loop. Solvent A is 0.1% TFA and 0.15 M NaC1 in water; solvent B is 0.1% TFA and 90% acetonitrile in water. Peptides are currently eluted by a linearly increasing gradient of 21-41% solvent B over 30 min, and this may need to be adjusted when the column is changed. The flow rate is 1 ml/min and 0.5-min fractions are collected into 12 x 75 mm borosilicate glass tubes containing 50/zl of protease-free bovine serum albumin (5 mg/ml; (Miles Diagnostics, Kankakee, IL) in water. The solvent blank prepared for assay tubes of the RIA standard curves is 0.5 ml of 31% solvent B in solvent A, added to 50 tzl of bovine serum albumin (5 mg/ml). Fractions and solvent blank tubes are evaporated to dryness under vacuum, and then dissolved in water immediately before RIA. When assayed with one RIA, fractions are dissolved in 120/zl of water and two 50-/zl aliquots taken for RIA of each fraction. When fractions are assayed with more than one RIA, the fractions are dissolved in a correspondingly greater volume of water before RIA. The elution positions of standard angiotensin peptides that were acetylated as described above are shown in Fig. 3A; those that were acetylated and then piperidine treated before HPLC are shown in Fig. 3B. An excellent separation of the different angiotensin peptides is obtained, with N-acetylated peptides (piperidine treated) eluting earlier than N,O-diacetylated peptides. A similar result was obtained for bradykinin peptides (Fig. 4A and B). L a b i l i t y of A c e t y l a t e d P e p t i d e s The first N terminal-directed RIAs we developed were for angiotensin peptides. During the development of these assays we did not suspect that acetyla-

334

III EXTRACELLULAR PROCESSING ENZYMES IN THE CNS 400

Ac Ang (1 7)

A

300

Ac-Ang II

200

Ac-Ang-(1-9)

~

.

100 0

5

400

10

15

20

25

30

35

40

45

50

55

50

55

B N AcAng (1 7 )

300

~

N AcAng II

N Ac Ang I .(1 9)

200

N AcAng I

100

5

10

15

20

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FIG. 3 Elution positions on HPLC of standard angiotensin peptides that were either N,O-diacetylated as described in text (A), or N-acetylated (piperidine-treated) (B). HPLC fractions were assayed by RIA with antibody A41, and data have not been corrected for recoveries or cross-reactivity with the antibody. N,O-Diacetylated peptides are designated Ac-; N-acetylated peptides are designated N-Ac-. AcAng(1-7), N,O-diacetylangiotensin(1-7); Ac-Ang(1-9), N,O-diacetylangiotensin(l-9); Ac-Ang II, N, O-diacetylangiotensin II; Ac-Ang I, N,O-diacetylangiotensin I; N-Ac-Ang(1-7), N-acetylangiotensin(1-7); N-Ac-Ang(1-9), N-acetylangiotensin(l-9); N-Ac-Ang II, N-acetylangiotensin II; N-Ac-Ang I, N-acetylangiotensin I.

tion of residues other than the N terminus was occurring, in that the acetylated products appeared to be completely homogeneous, with an efficiency of acetylation of--~100% (6). However, during subsequent development of the N terminal-directed RIA for bradykinin peptides, it was apparent that the acetylated product was not homogeneous. In Fig. 4A it can be seen that small peaks of immunoreactivity elute in the position ofN-acetylated peptides

[19] MEASUREMENTOF ANGIOTENSIN AND BRADYKININ 35o-

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[22] AUTORADIOGRAPHY OF ACE

391

125I-labeled 351A binding to human substantia nigra and Fig. 1B shows the adjacent section, which had been stained with thionin. As can be seen, nSI-labeled 351A binding is confined to the reticular part of the substantia nigra (15). In some instances, the adjacent sections were stained for another enzyme, for example, acetylcholinesterase. Its distribution in the brain is well mapped and extensively studied. Figure 2A shows an autoradiographic image of ~25Ilabeled 351A binding to human caudate nucleus and Fig. 2B shows the adjacent section, which had been stained for acetylcholinesterase. The patches of higher ~25I-labeled 351A binding corresponded to the regions of lower acetylcholinesterase activity, confirming that ACE is more concentrated in the acetylcholinesterase-poor striosomes (15).

Localization of Angiotensin-Converting Enzyme in Brain The distribution of ACE in the rat brain, as detected by in vitro autoradiography using nSI-labeled 351A, could be broadly divided into five categories (14). First, it was found on the endothelial surface of moderate-sized cerebral vessels, as in the vasculature of all organs. Second, a high density of ACE was detected in the choroid plexus (Fig. 3), where it was shown by immunohistochemical studies to occur on the brush border of the epithelial cells. Third, high concentrations of ACE were detected in all forebrain circumventricular organs, where the enzyme could convert circulating angiotensin I to angiotensin II to act on the local high densities of angiotensin II receptors present in these structures. Fourth, ACE was found in sites that correspond to the distribution of angiotensin II immunoreactivity and angiotensin II receptors, such as the hypothalamic neurosecretory nuclei and the dorsal vagal complex. At these sites, ACE may participate in the local formation of angiotensin II. Fifth, ACE was also detected in brain sites that were not thought to be rich in angiotensin II or its receptors, for example, the basal ganglia, hippocampal formation, cerebellar cortex (Fig. 3), and inferior olivary nucleus. Angiotensin-converting enzyme at these sites could be involved in processing neuropeptides other than angiotensin. Although we were unable to demonstrate angiotensin II receptors in the basal ganglia of the rat, in the

FIG. 2 Autoradiographic image of ACE distribution in the human caudate nucleus (A) and the adjacent section, which has been stained for acetylcholinesterase (B). The arrows indicate the striosomal patches, which contain high concentrations of ACE and low levels of acetylcholinesterase. CN, Caudate nucleus; ic, internal capsule.

392

III

E X T R A C E L L U L A R PROCESSING E N Z Y M E S IN THE CNS

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human brain these structures contained moderate to high densities of the receptors (22). A similar overall pattern of distribution of ACE was observed in other mammalian species (Fig. 4). The distribution of ACE in the monkey Macaca fascicularis (23) and human striatum (15) was heterogeneous. Angiotensinconverting enzyme was enriched in the striosomes, which are defined by patches of low acetylcholinesterase activity (Fig. 2). In contrast to the rat, in rabbit, monkey (23), and human brains (15) a moderate to high density of ACE was found throughout the cerebral cortex (Fig. 4). Mapping of brain ACE with [3H]captopril (16) gave a pattern that was broadly consistent with results obtained with 125I-labeled 351A. High concentrations of [3H]captopril binding were found in the choroid plexus, basal ganglia, and hypothalamic neurosecretory nuclei. However, 125I-labeled 351A detected the presence of ACE in many other sites in the amygdaloid complex, hippocampus, thalamus, cerebellum, and brainstem (14). This is because the


393

iodinated radioligand provided higher resolution autoradiographs that enabled more detailed anatomical localization of brain ACE. Moreover, it required shorter exposure times for its autoradiographs because of its higher specific activity. Applications

Accurate Quantitation o f Small Brain Nuclei The in vitro autoradiographic mapping of ACE in rat (12-14) and human (15, 24) brains revealed high concentrations of the enyzme in basal ganglia structures, including the caudate, putamen, internal and external globus pallidus, entopeduncular nucleus, and substantia nigra pars reticulata. These structures appear to be connected by a continuous pathway. Indeed, in the human basal ganglia, ACE is present in fibers in the internal capsule and cerebral peduncles (Fig. 5, color plate). Selective excitotoxin lesion of the rat striatum or 6-hydroxydopamine lesion of the substantia nigra pars compacta was carried out to investigate if ACE was associated with descending striatonigral or ascending nigrostriatal projections. In rats that had N-methyl-o-aspartic acid injected into the right striatum, ACE was decreased in the caudate putamen, globus pallidus, entopeduncular nucleus, and substantia nigra pars reticulata (Fig. 6, color plate). By contrast, 6-hydroxydopamine lesion of the substantia nigra, to lesion the ascending nigrostriatal dopaminergic system selectively, did not affect ACE levels in these structures (25). The high levels of ACE in other nuclei not associated with the basal ganglia were also not affected by either of the neurotoxins. These lesion studies confirmed that ACE is associated with neurons within the striatopallidal, striatonigral, and pallidonigral systems. Similarly, ACE was decreased in these basal ganglia structures in Huntington's disease but not in Parkinson's disease, confirming this assignment in the human brain (26). In vitro autoradiography enabled the detection of ACE in very small brain nuclei and even within neuronal fibers. Moreover, accurate quantitation of enzyme levels after chemical or other physical intervention can be carried out within a small brain nucleus because the autoradiograph can be overlaid on top of a stained section to determine the boundary of the nuclei.

Drug Penetration Studies Angiotensin-converting enzyme inhibitors have been successfully used in the treatment of hypertension and heart failure. However, the sites of action of these drugs are not clearly understood. Although ACE inhibitors

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were initially thought to mediate their effect via blockade of formation of circulating angiotensin II, their actions are more extensive than previously suspected. The hypotensive effect of these drugs outlasts inhibition of plasma ACE and they are effective in non-renin-dependent hypertension. Many investigators now believe that ACE inhibitors mediate their longterm hypotensive effects via inhibition of tissue ACE. The technique of in vitro autoradiography has also been used successfully to assess the sites and degree of inhibition of ACE inhibitors administered in vivo. This is possible because many of these drugs exhibit high affinity and tight binding to the active site of ACE, with relatively slow dissociation rates (27-29). The time course of tissue ACE inhibition was assessed by gavage-feeding rats with a particular dose of the drug followed by tissue collection at various time intervals (1-48 hr). The tissues were then sectioned and subjected to in vitro autoradiography as described above, except that the preincubation step was omitted. Trunk blood was collected for measurement of plasma ACE and drug levels. Similarly, dose-response studies were also carried out in which rats were administered different doses of a drug and tissue ACE inhibition measured by in vitro autoradiography. Acute administration of a single dose of an ACE inhibitor (lisinopril, perindopril, or benazepril) all produced varying degrees of tissue ACE inhibition (27-30). The difference in tissue ACE inhibition is probably due to tissue bioavailability and lipophilicity of the drug. Angiotensinconverting enzyme, which is located in brain structures other than the circumventricular organs, was not inhibited by an acute oral dose of most ACE inhibitors studied (Fig. 7, color plate) and the testicular enzyme was not blocked by any of the inhibitors administered. Angiotensinconverting enzyme at these sites is protected by the blood-testis or blood-brain barrier because it has been shown in membrane-binding studies that the enzyme in both these tissues is equally susceptible in vitro to all ACE inhibitors tested. The duration of tissue ACE inhibition was more prolonged than the suppression of plasma ACE and appeared to correlate more closely with inhibition of the pressor effect to exogenous angiotensin I (27). This

FIG. 4 Autoradiographic image of ACE distribution in a coronal section through the macaque monkey (A) and human (B) diencephalon, showing dense levels of ACE in the caudate nucleus (CN), putamen (Pu), nucleus acumbens (Acb), and external globus pallidus (GPe). Moderate densities of ACE are also present throughout the cerebral cortices, cc, corpus callosum; MS, medial septum; DB, diagonal band of Broca; ic, internal capsule; CI, claustrum.

396

III EXTRACELLULARPROCESSING ENZYMES IN THE CNS observation supports the hypothesis that the more long-term antihypertensive effect of an ACE inhibitor is due to tissue ACE inhibition and not just to the suppression of circulating angiotensin II levels.

Chronic Angiotensin-Converting Enzyme Inhibition Studies The chronic administration of an ACE inhibitor has been shown to cause a marked increase in plasma ACE level. The technique of in vitro autoradiography has been adapted to investigate this phenomenon. Angiotensin-converting enzyme belongs to a group of zinc metallopeptidases that are dependent on Zn 2§ for their catalytic activity. This property of ACE has enabled the design of experiments to evaluate tissue ACE inhibition and induction simultaneously. Tissue ACE inhibition was assessed as described in the previous section. In addition, tissue ACE induction was measured as follows" the enzyme was reversibly inactivated with ethylenediaminetetraacetic acid (EDTA), which chelated the Zn 2§ from the active site, resulting in dissociation of the bound inhibitor from the enzyme. Angiotensin-converting enzyme was then reactivated by the removal of EDTA and the active site replenished by the addition of Zn 2§ in the incubation together with the radioligand to measure total ACE in the tissue (31). The chronic administration of the ACE inhibitor lisinopril significantly increased ACE levels in plasma and lung but not in other tissues studied. This finding agreed with an earlier study that demonstrated increased ACE activity in the lung after chronic captopril treatment but not in the testis, kidney, or small intestine (32). The precise mechanism of plasma and tissue ACE induction, and its differential effect in the various tissues, are not known. FIG. 5 Computer-generated pseudocolor image of ACE distribution in a coronal section through the human diencephalon, showing high concentrations of ACE in the caudate nucleus, putamen, and globus pallidus, and in fibers projecting to the substantia nigra (arrows). Moderate densities of ACE are also present in the hippocampus (Hi) and insular cortex (ICx). The color scale is as follows: red represents high densities of ACE, yellow and green moderate, and blue low to undetectable levels of ACE. CN, Caudate nucleus; Pu, putamen; GPe, external globus pallidus; GPi, internal globus pallidus. FIG. 7 Computer-generated pseudocolor images of ACE distribution in coronal sections through the diencephalon of a control rat (A) and a rat 4 hr after an oral dose of lisinopril (10 mg/kg), showing blockade of ACE in the organum vasculosum of the lamina terminalis (OVLT) but not in the caudate putamen (CPu) or the choroid plexus (ClaP); ac, anterior commisure.

[22] AUTORADIOGRAPHYOF ACE

397

Conclusion The in vitro autoradiographic localization of ACE in the rat brain revealed the presence of the enzyme in many previously unreported sites and extended the mapping in sites where the enzyme was known to occur. Moreover, this technique provided the first detail mapping of ACE in the human brain. The extension of this technique to assess in vitro the degree of tissue ACE inhibition after ex vivo administration of ACE inhibitors provided more accurate and new information on the differential effect of these drugs in the various tissues. In the brain, the degree of blockade of ACE in structures within the blood-brain barrier appears to depend on the lipophilicity of the drugs. In addition, using in vitro autoradiography, we were able to assess accurately the degree of tissue ACE induction.

Acknowledgment These studies were supported by grants from the National Health and Medical Research Council and the National Heart Foundation of Australia and the Austin Hospital Medical Research Foundation. Siew Yeen Chai is supported by a National Health and Medical Research Council Australian Postdoctoral Fellowship. We gratefully acknowledge the generosity of Dr. C. Sweet of the Merck Institute for Therapeutic Research for the gift of 351A.

References 1. R. A. Skidgel, R. Defendini, and E. G. Erdos, in "Neuropeptides and Their Peptidases" (A. J. Turner, ed.), p. 165. Ellis Horwood, Chichester, 1987. 2. F. Soubrier, F. Alhenc-Gelas, C. Hubert, J. Allegrini, M. John, G. Tregear, and P. Corvol, Proc. Natl. Acad. Sci. U.S.A. 85, 9386 (1988). 3. L. Wei, F. Soubrier, P. Corvol, and E. Clauser, J. Biol. Chem. 266, 9002 (1991). 4. R. B. Perich, B. Jackson, F. Rogerson, and F. A. O. Mendelsohn, Mol. Pharmacol. 4211, 280 (1992). 5. R. B. Perich, B. Jackson, M. R. Attwood, K. Prior, and C. I. Johnston, Pharm. Pharmacol. Lett. 1, 41 (1991). 6. R. S. Kumar, J. Kusari, S. N. Roy, R. L. Softer, and G. C. Sen, J. Biol. Chem. 264, 16754 (1989). 7. H. Y. T. Yang and N. H. Neff, J. Neurochem. 19, 2443 (1972). 8. H. J. Wigger and S. A. Stalcup, Lab. Invest. 38(5), 581 (1978). 9. E. Rix, D. Ganten, G. Stock, and R. Taugner, Exp. Brain Res. S4, 126 (1982). 10. M. S. Brownfield, I. A. Reid, D. Ganten, and W. F. Ganong, Neuroscience 7(7), 1759 (1982).

398

III EXTRACELLULAR PROCESSING ENZYMES IN THE CNS 11. R. Defendini, E. A. Zimmerman, J. A. Weare, F. Alhenc-Gelas, and E. G. Erdos, Neuroendocrinology 37, 32 (1983). 12. F. A. O. Mendelsohn, Clin. Exp. Pharmacol. Physiol. 11, 431 (1984). 13. F. A. O. Mendelsohn, S. Y. Chai, and M. Dunbar, J. Hypertens. 2(s3), 21 (1984). 14. S. Y. Chai, F. A. O. Mendelsohn, and G. Paxinos, Neuroscience 20, 615 (1987). 15. S. Y. Chai, J. S. McKenzie, M. J. McKinley, and F. A. O. Mendelsohn, J. Comp. Neurol. 291, 179 (1990). 16. S. M. Strittmatter, M. M. S. Lo, J. A. Javitch, and S. H. Snyder, Proc. Natl. Acad. Sci. U.S.A. 81, 1599 (1984). 17. W. M. Hunter and F. C. Greenwood, Nature (London) 194, 495 (1962). 18. P. J. Munson and D. Rodbard, Anal. Biochem. 107, 220 (1980). 19. G. Waksman, E. Hamel, M. C. Fournie-Zalusky, and B. P. Roques, Proc. Natl. Acad. Sci. U.S.A. 83, 1523 (1986). 20. J. Friedland and E. Silverstein, Am. J. Clin. Pathol. 66, 416 (1976). 21. E. F. Hartree, Anal. Biochem. 48, 422 (1972). 22. A. M. Allen, G. Paxinos, M. J. McKinley, S. Y. Chai, and F. A. O. Mendelsohn, J. Comp. Neurol. 312, 291 (1991). 23. S.Y. Chai, M. J. McKinley, G. Paxinos, and F. A. O. Mendelsohn, Neuroscience 42, 483 (1991). 24. A. M. Allen, S. Y. Chai, J. Clevers, M. J. McKinley, G. Paxinos, and F. A. O. Mendelsohn, J. Comp. Neurol. 269, 249 (1988). 25. S. Y. Chai, M. J. Christie, P. M. Beart, and F. A. O. Mendelsohn, Neurochem. Int. 10, 101 (1987). 26. A. M. Allen, D. P. MacGregor, S. Y. Chai, G. A. Donnan, S. Kaczmarczyk, K. Richardson, R. Kalnins, J. Ireton, and F. A. O. Mendelsohn, Ann. Neurol. 32, 339 (1992). 27. K. Sakaguchi, S. Y. Chai, B. Jackson, C. I. Johnson, and F. A. O. Mendelsohn, Neuroendocrinology 48, 223 (1988). 28. K. Sakaguchi, S. Y. Chai, B. Jackson, C. I. Johnston, and F. A. O. Mendelsohn, Hypertension (Dallas) 11, 230 (1988). 29. K. Sakaguchi, B. Jackson, S. Y. Chai, F. A. O. Mendelsohn, and C. I. Johnston, J. Cardiovasc. Pharmacol. 12, 710 (1988). 30. S. Y. Chai, R. S. Perich, B. Jackson, F. A. O. Mendelsohn, and C. I. Johnston, Clin. Exp. Pharmacol. Physiol. 19(s19), 7 (1992). 31. M. Kohzuki, C. I. Johnston, S. Y. Chai, B. Jackson, R. Perich, D. Paxton, and F. A. O. Mendelsohn, J. Hyperten. 9, 579 (1991). 32. F. Fyhrquist, T. Forslund, I. Tikkanen, and C. Gronhagen-Riska, Eur. J. Pharmacol. 67, 473 (1980).

Index

ACE, s e e Angiotensin-converting enzyme a-N-Acetylendorphin HPLC, 131-132 processing, 131-133 ACTH, s e e Adrenocorticotropin Adrenocorticotropin, s e e a l s o Proopiomelanocortin HPLC, 134-135,212-213 processing, 83, 85, 89-91 radioimmunoassay, 135, 212-213 two-site immunometric assay, 150, 152-153 Affinity chromatography carboxypeptidase E, 247-248 proprotein convertase 1, 105 Alzheimer's disease /3-amyloid protein role, 281, 317 endopeptidase 24.15 role, 290 secretase role, 320-321 Amidation, s e e Peptidyl-glycine a-amidating monooxygenase Amyloid precursor protein biological function, 318 glycosylation, 318 homology with protease inhibitors, 317-318 immunoprecipitation, 322-323 phosphorylation, 318 point mutations, 317 processing site, 320-321 proteolytic processing, 281,290-291,294, 317318, 320 secretase, 320-321 size, 317 structure, 317-319 transfection of HeLa cells, 321-322 Western blotting, 323-325 fl-Amyloid protein neurotoxicity, 317 role in Alzheimer's disease, 281

Angiotensin antisera characterization, 332 preparation, 330 cleavage sites, 328-329 epitopes, 329-330 peptides acetylation, 332-333,338 extraction from biological samples, 336-340 lability, 333-336, 338-339 separation by HPLC, 333,338, 340 radioimmunoassay, 328-332, 338, 342 Angiotensin-converting enzyme autoradiography anatomical localization of enzyme, 389, 391 chronic inhibition studies, 396 drug penetration assessment, 393,395-397 film processing, 386-387 nonspecific binding, 387 quantitation of enzyme activity, 387, 389 quantitation of small brain nuclei, 393 radioligand sensitivity, 389 tissue preparation, 386 biological function, 383 inhibitors availabilitry, 273,384 characterization, 384-385 radiolabeling, 384 localization in brain, 391-393,397 membrane association, 344, 383 neurotensin processing, 273 size, 383 tissue distribution, 383-384 Antibody, s e e a l s o Two-site immunometric assay characterization by radioimmunoassay, 197-198, 210-211 generation, 196-197

399

400

INDEX

Antibody ( c o n ' t ) peptide haptens, 196-197 proprotein processing assays, 176-177 proinsulin, 177-179 proopiomelanocortin, 185, 187, 190-192 prosomatostatin, 179-185 protein requirements for production, 308 purification, 197 Antisense RNA assay systems, 112-113, 115-116 blocking of protein expression, 109-110 cell transfection, 117-119 cost of methods, 113 mechanism of action, 111-112 mismatch toleration, 113-114 Northern analysis, 115-116 plasmid preparation, 117 probe selection concentration, 113 sequence, 110, 112-115 size, 113, 117 promoter selection, 115 requirements for protein targeting, 112-113 selection of protein targets, 112 subcloning of stable cell lines, 119 APP, s e e Amyloid precursor protein Autoradiography, s e e Angiotensin-converting enzyme; Liquid emulsion autoradiography Baculovirus, protein expression system, 312 Bradykinin antisera characterization, 332 preparation, 330 cleavage sites, 328-329 peptides acetylation, 332-333,338 extraction from biological samples, 336-340 lability, 333-336, 338-339 separation by HPLC, 333,338, 340 radioimmunoassay, 328-332, 338, 342 Captopril, angiotensin-converting enzyme inhibition, 273,392 Carboxypeptidase E affinity chromatography, 247-248 assays fluorescence, 237-239, 241-244 Northern blot, 237-238,248 radioactive, 237, 244-246

sensitivity, 245 Western blot, 237-238, 248 expression in embryogenesis, 52-53 immunoprecipitation, 247 inhibitors, 238,244, 246 pH optimum, 238-239 precursor, 248 species distribution, 237 substrate specificity, 238-239, 245 Carboxypeptidase H, s e e Carboxypeptidase E Cathepsin D, processing of endothelin, 254 Chinese hamster ovary cell, protein overexpression system, 94-95,312 amplification, 98-99 collection of conditioned medium, 99-101 screening, 97-98 transfection, 95-96 CLIP, HPLC, 134-135 Cloning, s e e Endopeptidase 24.15, cloning Corticotropin-releasing factor, regulation of proopiomelanocortin, 61 COS cell, protein expression system, 312 DABTC, s e e Dimethylaminoazobenzine thiocarbamate Dansyl-Phe-Ala-Arg carboxypeptidase substrate, 238-239 protease assay, 241-244 synthesis, 239-241 Digoxigenin immunological detection, 31, 51-52 probe labeling, 20-21, 51 Dihydrofolate reductase methotrexate binding, 94-95 overexpression system, 94-95 Dimethylaminoazobenzine thiocarbamate, peptide derivatization, 171-172, 176 DNA, s e e Oligonucleotides ECE, s e e Endothelin-converting enzyme Embryo in s i t u hybridization, 46-47, 49-51 staging in rat, 49-50 Endopeptidase, insulin secretory granule type II, s e e Insulin secretory granule type II endopeptidase Endopeptidase 24.11 immunostaining antibody specificity, 350, 352

INDEX dual localization of antigens, 359-360 labeling immunogold, 356-359 peroxidase, 354-356 tissue preparation cryostat sections, 353 fixatives, 352-353 reagent penetration, 354 vibratome sections, 353-354 inhibitors, 386 mechanism, 344 membrane association, 344-346, 360 reconstitution, 346-347 subcellular fractionation, 347-348, 360 substrates, 345, 363 tissue distribution, 344-345 Endopeptidase 24.15 cloning amino acid sequence from pure enzyme, 301 antibody screening, 301-302 by homologous nucleic acid sequences, 302303 library preparation, 300-301 library screening oligonucleotide screening, 304-305 polymerase chain reaction, 305-307 sequence identification, 300-304 strategies, 299-300, 314 tissue selection, 300 related peptidases, 297-299 role in Alzheimer's disease, 290 secondary structural motifs, 303-304 solubility, 297 species distribution, 298 substrate specificity, 297, 363,372-375 tissue distribution, 297 Endopeptidase 24.16 antibody purification, 378 assays fluorimetric, 367 HPLC, 368-369 cleavage site specificity, 363-364 cloning, 381 distribution in central nervous system, 375378 immunoprecipitation, 379 inhibitors dipeptides, 370 phosphodiepryl 03,370, 372 immunoglobin G, 378-379

401 neurotensin processing, 370, 372-374 polyacrylamide gel electrophoresis, 379-380 purification, 378 substrate specificity, 363,365, 367, 372-375 Western blotting, 379-380 /3-Endorphin, s e e a l s o Proopiomelanocortin HPLC, 137-138, 212 processing, 85, 89-91 radioimmunoassay, 212 Endothelin biological function, 251 cloning, 252 homology with sarafotoxins, 252, 258 immunoassay, 259-260 inhibition of release, 256 processing, 252, 256-257 sequence, 257 Endothelin-converting enzyme aspartyl protease inhibition, 253 pH optimum, 253-254 size, 254 assay bioassay, 261-262 fluorescence energy transfer, 260 HPLC, 258-259 immunoassay, 259-260 scintillation proximity assay, 260-261 sensitivity, 260 cleavage site specificity, 253,256-257 expression in transfected cells, 262 intracellular localization, 252 kinetic parameters, 258 metalloprotease cloning, 256 glycosylation, 255 inhibitors, 255-256 pH optimum, 254-255 purification, 255-256 size, 255 pH dependence, 257 substrate recognition, 257-258 Enkephalin convertase, s e e Carboxypeptidase E ET, s e e Endothelin Expression vector affinity tag, 308-309 phenotype selection, 308 promoter, 308 ribosome-binding site, 308 transcription terminator, 309

402

INDEX

Fluorescence energy transfer, endothelin-converting enzyme assay, 260 Fluorogenic substrates, s e e a l s o Dansyl-Phe-AlaArg amino acid composition, 164-165 peptidase assay, 165-167, 238-239, 284-285, 287-288 solubility, 239 synthesis, 239-241 Formaldehyde, preparation of fixing solution, 25 Furin cleavage site specificity, 155-156 discovery, 3 expression in embryogenesis, 52-53 riboprobe synthesis, 22 sequence, 4 in situ hybridization, 37, 41-42 substrate specificity, 13-14, 16, 52 Gastrin, processing effects of hypersecretion, 273-274 parasecretory, 272 GEMSA, s e e Guanidinioethylmercaptosuccinic acid Glutathione S-transferase proprotein convertase fusion protein, 11-12 protein expression system, 311 Gonadotropin-releasing hormone, HPLC, 131-132 Granulosa, cell culture, 162-163 Guanidinioethylmercaptosuccinic acid, carboxypeptidase inhibition, 238, 244, 246, 289 High-performance liquid chromatography ion-exchange chromatography separation of peptidases, 291-292 reversed-phase chromatography analytical column types, 128-129 column size selection, 129-130 criteria of peptide purity, 128 flow rates, 129-130 ion pair strategies, 133-135 sample preparation prechromatography cleanup, 127-128 tissue extraction, 126-127, 201-203 separation of peptides, 125-126, 203-206, 209210, 287-288 solvent systems, 130-131,203,287 size-exclusion chromatography calibration curve, 137

separation of peptidases, 292-293 solvent systems, 135, 137 Histidine tag expression vectors, 309-310 protein purification, 313 HIV, s e e Human immunodeficiency virus HPLC, s e e High-performance liquid chromatography Human immunodeficiency virus, protease inhibitor development, 315 Hybridization, in s itu , s e e I n s itu hybridization Immunogold, labeling of ultrathin frozen sections, 191 Immunometric assay, two-site, s e e Two-site immunometric assay Immunostaining antibody specificity, 350, 352 dual localization of antigens, 359-360 labeling immunogold, 356-359 peroxidase, 354-356 proinsulin, 177-179 proopiomelanocortin, 185, 187, 190-192 prosomatostatin, 179-185 tissue preparation cryostat sections, 353 fixatives, 352-353 reagent penetration, 354 vibratome sections, 353-354 Inosine, nucleotide base pairing, 307 Ion-exchange chromatography, separation of peptidases, 291-292 I n situ hybridization, s e e a l s o Oligonucleotides; Riboprobes detection of hybridization signal liquid emulsion autoradiography, 29-31, 49 nonradioactive detection, 31, 51-52 X-ray film, 29 double-labeling techniques, 33, 51-52 embryo tissue, 46-47, 49-51 hybridization conditions, 27-28 mixing solution preparation, 27, 48-49 prehybridization treatment, 48 acetylation, 26 dehydration, 26 delipidation, 26 denaturation, 26 proteinase K treatment, 26

INDEX hybridization (con't) probe selection oligonucleotides, 18-19, 28 riboprobes, 17-18, 28, 50 processing enzyme localization, 35, 37, 41-42 quantitative densitometry computer analysis, 34 sampling, 35 standard curve construction, 34-35 sensitivity, 45 specificity controls negative, 32-33 positive, 32 subbing of slides gelatin coating, 24, 47 lysine coating, 24, 47 tissue fixation, 25 preparation, 24, 47 storage, 25 Insulin secretory granule type II endopeptidase, cleavage specificity, 90 In situ

Kexin cleavage site specificity, 155-156 discovery, 3 expression in recombinant vaccinia virus, 88 fluorescence assay, 165-166 kinetic parameters, 166-167 prohormone processing, 88 sequence, 4 substrate specificity, 165-166 fl-Lipotropin, s e e a l s o Proopiomelanocortin HPLC, 137-138 processing, 85, 89-91, 137 two-site immunometric assay, 150 Liquid emulsion autoradiography counterstaining, 30 developing, 30 dipping of slides, 30 emulsion preparation, 29-30 mounting, 30 photography, 30-31 fl-LPH, s e e fl-Lipotropin MalE system, protein expression system, 311-12 a-Melanocyte-stimulating hormone, s e e a l s o Proopiomelanocortin HPLC, 213-215 processing, 90 radioimmunoassay, 213-215

403 fl-Melanocyte-stimulating hormone, s e e a l s o Proopiomelanocortin HPLC, 212 radioimmunoassay, 212 Methotrexate application in overexpression systems, 94-95, 98 inhibition of dihydrofolate reductase, 94-95 Microdialysis, in v i v o flow rates, 275 osmotic pressure, 276 principles, 275 probes, 275-276 sampling at peptide secretion site, 274-275 MSH, s e e Melanocyte-stimulating hormone Multicatalytic endopeptidase, substrate specificity, 291 NacEP, s e e a-N-Acetylendorphin Neprilysin, s e e Endopeptidase 24.11 Neurolysin, s e e Endopeptidase 24.16 Neurophysin cleavage site, 158 fractionation, 159 isolation, 160-163 processing, 158-159, 163-164 radioimmunoassay, 163 synthetic peptide synthesis, 159-160 Neurotensin HPLC, 364, 367 parasecretory processing, 272-273 proteolytic processing, 363,381 Oligonucleotides labeling, 23-24 screening of cDNA libraries, 304-305 in situ hybridization, 28 synthesis, 18-19 Oxytocin, s e e a l s o Prooxytocin-neurophysin antibodies, 195 isolation, 163 peptides extraction from tissue, 198-199, 201-203 HPLC, 203,205-206 high-voltage electrophoresis, 199-200 radioimmunoassay, 203,206 processing, 158-159, 163-164 radioimmunoassay, 163 PACE4 reverse transcriptase-polymerase chain reaction, 8-9

404

INDEX

PACE4 ( c o n ' t ) sequence, 6-7 in s i t u hybridization, 37, 41-42 PAM, s e e Peptidyl-glycine a-amidating monooxygenase PC1, s e e Proprotein convertase 1 PC2, s e e Proprotein convertase 2 PC3, s e e Proprotein convertase 1 PC4, s e e Proprotein convertase 4 PC5, s e e Proprotein convertase 5 PCR, s e e Polymerase chain reaction Peptidases, central nervous system activators, 289 affinity chromatography, 283 biological function, 281,296 cloning, s e e Endopeptidase 24.15 fluorimetric assay, 284-285, 287-288 HPLC analysis of peptide degradation, 287-288 identification from cDNA libraries, 283 inhibitors, 289 mechanistic classes, 288-289, 296 pH optimum, 288-289, 292-293 polyacrylamide gel electrophoresis, 282-283 purification, 293-294 role in disease, 281 solubility, 296-297 substrate specificity, 281-282 synthetic peptide substrates size, 284 synthesis, 284-285 terminal blocking, 284 tissue collection brain fractionation, 286 peptidase stability, 285-286 preparation of synaptosomes, 286-287 Peptide processing, in v i v o animal models, 266-267 animal preparation, 267, 270 calculations half life, 269-270 metabolic clearance rate, 269 organ extraction, 270-271 production rate, 269 volume of distribution, 270 experimental design, 268-269 parasecretory processing, 272-273 pathways, 266 peptide infusion, 268 plasma stability, 271-272 sampling at secretion site, 274-276

Peptidyl-glycine a-amidating monooxygenase antisense RNA methods, 114, 116 assay applications, 230, 235 incubation time, 222 optimization, 225-227 principles, 219-223 product separation, 222-223 substrates, 220, 222 tissue preparation, 221-222 brain enzyme assay, 231 blood collection, 231 distribution of activity, 231-232, 235 hypothalamic enzyme kinetic parameters, 232233 plasma levels, 233 tissue homogenization, 230-231 cofactors, 222, 226-227 heart enzyme assay, 224-225 effect of chronic corticosteroid activity, 228230 subcellular distribution, 227-228 tissue collection, 223-224 tissue homogenization, 224 peptide substrates, 219 Phosphodiepryl 03, peptidase inhibition, 370, 372374 P i c h i a p a s t o r i s , protein expression system, 312 Polymerase chain reaction codon degeneracy in primer design, 305-306 primer selection, 9 size, 306 product purification, 306-307 proprotein convertases, 4-5, 8-9 screening cDNA libraries, 305-307 vaccinia virus recombinants, 81-82 POMC, s e e Proopiomelanocortin Preproopiomelanocortin, s e e Proopiomelanocortin Prodynorphin, overexpression system, 107 Proenkephalin glycosylation, 102-103 purification of overexpressed protein collection from medium, 100-101 reversed-phase chromatography, 102-103 solubility, 102 Progesterone, radioimmunoassay, 163

INDEX Proinsulin cleavage sites, 177-178 immunostain processing assay, 177-179 structure, 177-178 Proopiomelanocortin coexpression with processing enzymes, 88-89 epitopes, 152 expression in embryogenesis, 53, 55, 57 processing, 16, 83-86, 133, 152, 208-209 AtT-20 cells, 190-192 cleavage sites, 187, 190, 208 developmental changes, 209, 212-217 dopamine role in processing, 133 immunostain assay, 185, 187, 190-192 reconstitution of pathways, 87-90 subcellular localization, 191-192 tissue specificity, 83-86, 208 vaccinia virus recombinant protein, 83-86, 9091 regulation by corticotropin-releasing factor, 61 two-site immunometric assay, 150, 152-153 Prooxytocin-neurophysin cleavage sites, 158, 167-169, 196 fractionation, 159 processing assay, 169, 171-176 cleavage, 158-159, 163-164 purification, 174 radioimmunoassay, 162 synthetic peptide chromophore derivative, 171-172 synthesis, 159-160, 167-168 Proprotein convertase 1 activity assay, 103-104 aggregation in overexpression systems, 99-100 antisense RNA methods, 114 catalytic residues, 5, 7-8 coexpression with substrates, 12-14, 89 discovery, 4-5 expression in embryogenesis, 53, 55, 57, 216 expression in recombinant vaccinia virus, 88 fusion protein generation, l 1-12 purification of overexpressed protein affinity chromatography, 105 anion-exchange chromatography, 105-106 collection from medium, 100- l01 fast protein liquid chromatography, 103 hydrophobic interaction chromatography, 105 yield, 107

405 reverse transcriptase-polymerase chain reaction, 4 ribonuclease protection assay, 62-64 riboprobe synthesis, 22 sequence, 6-7 in situ hybridization, 37, 41-42 substrate specificity, 13-14, 16 Xenopus laeois gene cloning, 10 sequence, 11 Proprotein convertase 2 catalytic residues, 5, 7-8 cloning, Aplysia californica gene, 10 coexpression with substrates, 12-14, 89 discovery, 4-5 expression in embryogenesis, 216 expression in recombinant vaccinia virus, 88 fusion protein generation, 1l - 12 overexpression system, 107 reverse transcriptase-polymerase chain reaction, 4 ribonuclease protection assay, 62-64 riboprobe synthesis, 22 sequence, 6-7 in situ hybridization, 37, 41-42 substrate specificity, 13-14, 16 Proprotein convertase 4 catalytic residues, 5, 7-8 gene splicing, 14 reverse transcriptase-polymerase chain reaction, 8 sequence, 6-7 Proprotein convertase 5 catalytic residues, 5, 7-8 gene splicing, 14 reverse transcriptase-polymerase chain reaction, 8 riboprobe synthesis, 23 sequence, 6-7 in situ hybridization, 37, 41-42 Prosomatostatin cleavage sites, 179-180 immunostain processing assay, 179-185 processing L. piscatorius pancreatic islets, 18 l, 184-185 rat brain cortical cells, 179-180, 184 purification, 173 rat brain protein blockage of intracellular transport, 184 peptide identification, 183

406

INDEX

Prosomatostatin ( c o n "t) subcellular distribution, 182-183 subcellular fractionation, 182 Radioimmunoassay adrenocorticotropin, 135,212-213 angiotensin, 328-332, 338, 342 application with HPLC, 135, 138-139, 203-206, 212-217 bradykinin, 328-332, 338, 342 characterization of antibodies, 197-198, 210-211 fl-endorphin, 212 melanocyte-stimulating hormone, 212-215 progesterone, 163 proopiomelanocortin, 210-211 prooxytocin-neurophysin, 162-163,203,206 vasopressin, 203,206 RIA, s e e Radioimmunoassay Ribonuclease protection assay data analysis, 60 evaluation of gene expression, 57 gel electrophoresis, 59 hybridization mixture, 59 probe preparation, 58-60 RNA isolation, 58 tissue culture, 58 Riboprobes, s e e a l s o Antisense RNA hydrolysis, 21-22, 50 labeling nonradioactive, 20-21 radioactive, 19-20, 60 purification, 21, 50, 60-61 in s i t u hybridization, 28, 50 synthesis, 17-20, 22-23, 47-48, 60 RNA, antisense, s e e Antisense RNA Scintillation proximity assay, endothelin-converting enzyme assay, 260-261 Size-exclusion chromatography, s e e High-performance liquid chromatography, size-exclusion chromatography Somatostatin, HPLC, 131-132 Substance P, HPLC, 131-132 Synaptosome membrane isolation, 287 preparation from human brain, 286-287,348-350 Thyrotropin-releasing hormone, HPLC, 1311 3 2 t r p E fusion vector, protein expression system, 310-311

Two-site immunometric assay antibody characterization, 143-145 concentration, 148 generation, 143 radiolabeling, 145 selection, 140-142, 147 solid phase coupling, 145-146 antigen selection, 142, 152 assay format, 142-143 calibration, 151 incubation time, 149 optimization, 147-149 peptide interference, 150-151 principles, 140-142 sensitivity, 140 specificity, 149-150 stability of peptides, 151 standards, 146

Unwindase biological functions, 116 effect on antisense RNA experiments, 116

Vaccinia virus DNA isolation, 72, 74 expression vector amplification, 79 construction, 67-69 DNA preparation, 74 drug selection, 75-76 marker transfer protocol, 74-75 plaque purification agarose overlays, 76-78 filter lifts, 78-79 screening slot blot, 80 Southern analysis, 80-81 immunoblot, 81 polymerase chain reaction, 81-82 genome, 66-67 infection cell culture, 70 efficiency, 66, 83 large-scale preparation, 70-72 life cycle, 66-67 recombinant protein expression, 65-66, 83, 85, 87-91

INDEX reconstitution of prohormone processing pathways, 87-90 safety in handling, 69 titering of stock solutions, 72 Vasopressin antibodies, 195 peptides extraction from tissue, 198-199, 201-203 HPLC, 203,205-206 high-voltage electrophoresis, 199-200

407 radioimmunoassay, 203,206 processing, 195-196 Western blot amyloid precursor protein, 323-325 carboxypeptidase E, 237-238 endopeptidase 24.16, 379-380 X-ray crystallography, protein requirements, 308


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FIG.6 Computer-generated pseudocolor images of ACE distribution in coronal sections through a normal rat basal ganglia (A-D) and through a rat basal ganglia 2 weeks after N-methyl-D-aspartic acid lesion of the right caudate nucleus (E-H). In the lesioned brain, the right caudate putamen (CPu), globus pallidus (GP), entopeduncular nucleus (EP), and substantia nigra pars recticulata (SNR) contain appreciably less ACE than the structures on the left side or in the control brain.

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