BRAK

BRAK Robert Hromas* Indiana University Medical Center and the Walther Oncology Center, The Indiana Cancer Research Institute, 1044 W. Walnut Street, Indianapolis, IN 46202, USA * corresponding author tel: 317-274-3589, fax: 317-274-0396, e-mail: [email protected]. DOI: 10.1006/rwcy.2001.10013.

SUMMARY

Alternative names

BRAK is a recently described CXC chemokine that is ubiquitously expressed in all normal tissue. Although present in selected tumor tissue, it is expressed at a significantly lower level. The structure of BRAK is considerably divergent from all other chemokines, and its activity is not yet well defined.

Recently, there have been two other submissions to the database that are identical to the original BRAK. Neither of these has been published. The first submission describes BRAK isolation from a squamous cell carcinoma of the head and neck (accession number AF144103). This submission terms the encoded protein NJAC. The second submission describes BRAK as a transcript that is upregulated in the kidney of adenine phosphoribosyltransferase-deficient mice (accession number AF192557). These mice develop kidney failure secondary to an overproduction of 2,8dihydroxyadenine stones (Stockelman et al., 1998). This submission terms the encoded protein KEC, for kidney-encoded chemokine.

BACKGROUND

Discovery BRAK was first cloned from breast tissue using PCR (Hromas et al., 1999). Initially, two human expressed sequenced tags (ESTs) were identified that had distant homology to the CXC chemokine family (GenBank accession numbers: Breast AA514740, kidney AA865643). Sequencing these cDNAs, it was found that they did not contain the entire coding sequence. They terminated prior to the N-terminus of the protein-coding sequence. A 50 RACE PCR reaction was used to clone the 50 end of the cDNA. The completed cDNA was assembled using PCR and sequenced to assure accuracy. This novel CXC chemokine was called BRAK for its initial identification in breast and kidney tissue. Using the human sequence as a template, two murine EST sequences were identified (AA048803, AA017998) that were also lacking the N-terminus of the coding sequence of the signal secretion peptide. Assembling murine BRAK using PCR revealed that it differed from human BRAK in only two amino acids. These changes were conservative in nature.

Cytokine Reference

Structure BRAK has two transcripts by northern analysis, one 2.5 kb and another 0.6 kb. The complete cDNA of the shorter BRAK transcript is 420 nucleotides in length. The longer transcript may represent alternative splicing in the untranslated portion of the cDNA. The short 50 untranslated region of 34 nucleotides does not contain an inframe translational stop codon. The sequence 50 of the ATG that initiates translation fits the Kozak consensus at 9 of 14 amino acids.

Main activities and pathophysiological roles The main function of BRAK is not known.

Copyright # 2001 Academic Press

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Robert Hromas

GENE AND GENE REGULATION

Accession numbers Human: AF073957 Mouse: AF152377

Chromosome location BRAK is located in its entirety on LBNL BAC genomic clone 7g12 (D5S471±D5S393 (129.6±140.8 cM) accession number AC005738), which is from human chromosome 5q31. The genomic structure of BRAK reveals that it is spread over 6.6 kb in four exons (Hromas et al., 1999). There are no canonical CCAAT or TATA boxes upstream of the 50 end of the BRAK cDNA. There do appear to be sites that could bind SP-1. Given that the expression of BRAK is so widespread, it is possible that the BRAK promoter is constitutively active.

PROTEIN

Sequence See Figure 1.

Description of protein BRAK protein has 97 amino acids. This is consistent with other members of the CXC chemokine family (Figure 1). The first 22 amino acids of BRAK are mainly hydrophobic, and presumably make up the signal peptide. Using pSORT or SignalP software, these amino acids have a high probability that they are the signal peptide, and the best cleavable site is between amino acids 22 and 23. This is the only statistically acceptable cleavable signal peptide sequence. BRAK does not contain an ELR motif at its N-terminal like the angiogenic CXC chemokines.

The four cysteines that participate in the disulfide bonds that define this family are also conserved in BRAK as are several other highly conserved residues (Figure 1). Murine BRAK is most closely related to MIP2 and , with 30% identity of amino acids and 55% similarity when conservative changes are taken into account. BRAK has many of the conserved amino acid features of the other human CXC chemokines (Figure 1), there are several unusual characteristics of BRAK that are worth noting. It has a short N-terminus (Ser±Lys) prior to the invariant CXC sequence compared with other CXC chemokines. BRAK has a VSRYR insert starting at position 63 that is not seen in any other CXC chemokines. Like other chemokines BRAK is highly basic, for potential immobilization on negatively charged endothelial cell surface polysaccharides. Mature human BRAK differs from mature murine BRAK at only two amino acids. There is an Ile to Val change from human to mouse at position 58, and a Val to Met at position 63. These are conservative changes. While BRAK shares many of the conserved sequence features of this family, it has some notable exceptions. BRAK has a five amino acid insert starting at position 63 not seen in any other members of this family. This region is between the third and fourth cysteines in a predicted triple-stranded sheet. It would be predicted to affect dimerization or receptor interaction based on the crystallographic structures of IL-8 (Baldwin et al., 1990). These structures may also be important in the overall function of this family, perhaps by maintaining appropriate tertiary structure, or mediating dimerization.

Important homologies BRAK has similarity to the other chemokine family members in the positioning of the conserved cysteines that participate in the disulfide bonds that define this family. However, its predicted N-terminus is shorter than that of other CXC chemokines. The protein structure has not been solved, although it is likely to be similar to that of other CXC chemokines.

Figure 1 Amino acid sequences for human and mouse BRAK compared with those of nine other CXC chemokines.

BRAK 3

Posttranslational modifications There is no evidence for any posttranslational modifications.

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce BRAK is highly expressed in all normal tissue tested by northern analysis (Hromas et al., 1999). These include heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. BRAK is expressed at a somewhat lower level in normal lung than in other tissues. It is present as two transcripts, one approximately 0.5 kb and another approximately 2.5 kb. The larger transcript is expressed at a higher level than the smaller transcript. BRAK is expressed in only two of 18 cancer cell lines analyzed by northern blot (Hromas et al., 1999). Of eight cancer cell lines BRAK is only expressed in colon adenocarcinoma cells (SW 485). It was not expressed in HL60 promyelocytic leukemia, HeLa cervical carcinoma, K562 chronic myelogenous leukemia, Molt-4 T cell leukemia, Raji B cell lymphoma, A549 lung carcinoma, or G361 melanoma cells. Of 10 breast cancer cell lines, BRAK is only expressed in one, the breast cancer cell line MDA MB 435. Recently, a report described BRAK as being overexpressed in the normal tissue surrounding a tumor (Frederick et al., 2000). Using in situ cRNA hybridization, BRAK was poorly expressed in various human malignant tissues, but expression appeared to be induced in the normal tissue surrounding the tumor. This raises the question whether BRAK may play a role in the inflammatory response of normal tissue to local tumor invasion.

system and a synthetic peptide with appropriate hydrolyzed disulfide bonds (Hromas et al., 1999). Using transwell chemotactic assays and flow cytometry we did not find any chemotactic activity for BRAK in human or murine T cells, B cells, monocytes, NK cells, or granulocytes. Neither of the two BRAK protein preparations inhibited human or murine CFU-GM, BFU-E, or CFU-GEMM in erythropoeitin/IL-3/GM-CSF/SCF-stimulated colony formation assays. Neither preparation stimulated CFU-GM hematopoietic colony formation when stimulated with M-CSF or GM-CSF alone. Although the synthesized BRAK had the correct sequence and was of the appropriate size, it is possible that both of these proteins lacked a critical structural determinate, such as the proper amino acids at the N-terminus of the protein. It is also possible that BRAK's activities are quite different from known chemokine activities, and remain to be defined. We also tested whether BRAK could inhibit the activity of SDF-1 chemotaxis on B cells and T cells. We found that BRAK had no inhibitory activity on SDF-1. BRAK was also tested for any ability to inhibit the angiogenic signal of VEGF in an in vitro endothelial cell tubule formation assay. No inhibition of angiogenesis could be demonstrated. However, we recently found that both BRAK protein preparations were able to chemoattract small but reproducible percentages (5±10%) of both resting and activated normal human NK cells. Human NK cells activated by incubation with IL-2 had increased chemotaxis as compared with resting NK cells. Since this activity was blocked by antisera to the BRAK protein, it is likely that there is a subset of NK cells that respond to BRAK. Recently, another group reported that recombinant murine BRAK was able to stimulate chemotaxis of the murine B cell lines CESS and A20, and the human monocyte cell line THP1 (Sleeman et al., 2000).

RECEPTOR UTILIZATION The receptor BRAK uses is not known currently.

IN VITRO ACTIVITIES

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

In vitro findings

Normal physiological roles

Mature BRAK protein was synthesized using two different methods, the pQE2 bacterial expression

There are no known in vivo biological activities of BRAK.

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PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Normal levels and effects There are no known roles for BRAK in human pathology.

IN THERAPY There is no published evidence that BRAK has a therapeutic role in human disease.

References Baldwin, E. T., Franklin, K. A., Appella, E., Yamada, M., Matsushima, K., and Gronenborn, A. M. (1990).

Crystallization of human interleukin-8. A protein chemotactic for neutrophils and T-lymphocytes. J. Biol. Chem. 265, 6851± 6853. Frederick, M. J., Henderson, Y., Xu, X., Deavers, M. T., Sahin, A. A., Wu, H., Lewis, D. E., El-Naggar, A. K., and Clayman, G. L. (2000). In vivo expression of the novel CXC chemokine BRAK in normal and cancerous human tissue. Am. J. Pathol. 156, 1937±1950. Hromas, R., Broxmeyer, H., Kim, C., Nakshatri, H., Christopherson, K., Azam, M., and Hou, Y.-H. (1999). Cloning of BRAK, a novel divergent CXC chemokine preferentially expressed in normal versus malignant cells. Biochem. Biophys. Res. Commun. 255, 703±706. Sleeman, M. A., Fraser, J. K., Murison, J. G., Kelly, S. L., Prestidge, R. L., Palmer, D. J., Watson, J. D., and Kumble, K. D. (2000). B cell- and monocyte-activating chemokine (BMAC), a novel non-ELR alpha-chemokine. Int. Immunol. 12, 677±689. Stockelman, M. G., Lorenz, J. N., Smith, F. N., Boivin, G. P., Sahota, A., Tischfield, J. A., and Stambrook, P. J. (1998). Chronic renal failure in a mouse model of human adenine phosphoribosyltransferase deficiency. Am. J. Physiol. 275, 154±163.