Table Of ContentBRAK
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 Recently, there have been two other submissions to
is ubiquitously expressed in all normal tissue. the databasethat are identical to the original BRAK.
Although present in selected tumor tissue, it is Neither of these has been published. The first sub-
expressed at a significantly lower level. The structure mission describes BRAK isolation from a squamous
of BRAK is considerably divergent from all other cell carcinoma of the head and neck (accession
chemokines, and its activity is not yet well defined. numberAF144103).Thissubmissiontermstheencoded
protein NJAC. The second submission describes
BRAKasatranscriptthatisupregulatedinthekidney
BACKGROUND of adenine phosphoribosyltransferase-deficient mice
(accession number AF192557). These mice develop
Discovery kidneyfailuresecondarytoanoverproductionof2,8-
dihydroxyadenine stones (Stockelman et al., 1998).
This submission terms the encoded protein KEC, for
BRAK was first cloned from breast tissue using
kidney-encoded chemokine.
PCR (Hromas et al., 1999). Initially, two human
expressed sequenced tags (ESTs) were identified that
had distant homology to the CXC chemokine family Structure
(GenBank accession numbers: Breast AA514740,
kidney AA865643). Sequencing these cDNAs, it was
BRAK has two transcripts by northern analysis,
found that they did not contain the entire coding
one 2.5kb and another 0.6kb. The complete cDNA
sequence. They terminated prior to the N-terminus
of the shorter BRAK transcript is 420 nucleotides
of the protein-coding sequence. A 50 RACE PCR
in length. The longer transcript may represent
reaction was used to clone the 50 end of the cDNA.
alternative splicing in the untranslated portion of
The completed cDNA was assembled using PCR and
the cDNA. The short 50 untranslated region of 34
sequenced to assure accuracy. This novel CXC
nucleotides does not contain an inframe translational
chemokine was called BRAK for its initial identifica-
stopcodon.Thesequence50 oftheATGthatinitiates
tion in breast and kidney tissue.
translation fitsthe Kozakconsensusat 9of14amino
Using the human sequence as a template, two
acids.
murine EST sequences were identified (AA048803,
AA017998) that were also lacking the N-terminus
of the coding sequence of the signal secretion Main activities and
peptide. Assembling murine BRAK using PCR
pathophysiological roles
revealed that it differed from human BRAK in only
two amino acids. These changes were conservative in
nature. The main function of BRAK is not known.
CytokineReference Copyright#2001AcademicPress
2 Robert Hromas
GENE AND GENE REGULATION The four cysteines that participate in the disulfide
bonds that define this family are also conserved in
Accession numbers BRAK as are several other highly conserved residues
(Figure 1). Murine BRAK is most closely related to
MIP2(cid:11) and (cid:12), with 30% identity of amino acids and
Human: AF073957
55% similarity when conservative changes are taken
Mouse: AF152377
into account.
BRAK has many of the conserved amino acid fea-
Chromosome location turesoftheotherhumanCXCchemokines(Figure1),
thereareseveralunusualcharacteristicsofBRAKthat
areworthnoting.IthasashortN-terminus(Ser–Lys)
BRAK is located in its entirety on LBNL BAC
prior to the invariant CXC sequence compared with
genomic clone 7g12 (D5S471–D5S393 (129.6–140.8
other CXC chemokines. BRAK has a VSRYR insert
cM) accession number AC005738), which is from
starting at position 63 that is not seen in any other
human chromosome 5q31. The genomic structure of
CXC chemokines. Like other chemokines BRAK is
BRAK reveals that it is spread over 6.6kb in four
highly basic, for potential immobilization on nega-
exons (Hromas et al., 1999). There are no canonical
CCAAT or TATA boxes upstream of the 50 end of tivelychargedendothelialcellsurfacepolysaccharides.
Mature human BRAK differs from mature murine
the BRAK cDNA. There do appear to be sites that
BRAKatonlytwoaminoacids.ThereisanIletoVal
could bindSP-1. Given that the expression of BRAK
changefromhumantomouseatposition58,andaVal
is so widespread, it is possible that the BRAK
toMetatposition63.Theseareconservativechanges.
promoter is constitutively active.
While BRAK shares many of the conserved
sequence features of this family, it has some notable
exceptions.BRAKhasafiveaminoacidinsertstarting
PROTEIN
at position 63 not seen in any other members of this
family. This region is between the third and fourth
Sequence
cysteines in a predicted triple-stranded (cid:12) sheet. It
would be predicted to affect dimerization or receptor
See Figure 1. interactionbasedonthecrystallographicstructuresof
IL-8(Baldwinetal.,1990).Thesestructuresmayalso
be important in the overall function of this family,
Description of protein
perhapsbymaintainingappropriatetertiarystructure,
or mediating dimerization.
BRAK protein has 97 amino acids. This is consistent
with other members of the CXC chemokine family
Important homologies
(Figure 1). The first 22 amino acids of BRAK are
mainly hydrophobic, and presumably make up the
signal peptide. Using pSORT or SignalP software, BRAKhassimilaritytotheother(cid:12) chemokinefamily
these amino acids have a high probability that they membersinthepositioningoftheconservedcysteines
are the signal peptide, and the best cleavable site is that participate in the disulfide bonds that define this
between amino acids 22 and 23. This is the only family. However, its predicted N-terminus is shorter
statistically acceptable cleavable signal peptide than that of other CXC chemokines. The protein
sequence. BRAK does not contain an ELR motif at structure has not been solved, although it is likely to
its N-terminal like the angiogenic CXC chemokines. 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 system and a synthetic peptide with appropriate
hydrolyzed disulfide bonds (Hromas et al., 1999).
Using transwell chemotactic assays and flow cyto-
There is no evidence for any posttranslational
metry we did not find any chemotactic activity for
modifications.
BRAK in human or murine T cells, B cells,
monocytes, NK cells, or granulocytes. Neither of
thetwoBRAKproteinpreparationsinhibitedhuman
CELLULAR SOURCES AND
or murine CFU-GM, BFU-E, or CFU-GEMM in
TISSUE EXPRESSION
erythropoeitin/IL-3/GM-CSF/SCF-stimulated col-
onyformationassays.Neitherpreparationstimulated
Cellular sources that produce CFU-GM hematopoietic colony formation when
stimulated with M-CSF or GM-CSF alone.
BRAK is highly expressed in all normal tissue tested Although the synthesized BRAK had the correct
by northern analysis (Hromas et al., 1999). These sequence and was of the appropriate size, it is pos-
include heart, brain, placenta, lung, liver, skeletal sible that both of these proteins lacked a critical
muscle, kidney, and pancreas. BRAK is expressed at structural determinate, such as the proper amino
a somewhat lower level in normal lung than in other acids at the N-terminus of the protein. It is also
tissues. It is present as two transcripts, one approxi- possible that BRAK’s activities are quite different
mately 0.5kb and another approximately 2.5kb. The from known chemokine activities, and remain to
largertranscriptisexpressedatahigherlevelthanthe be defined. We also tested whether BRAK could
smaller transcript. inhibit the activity of SDF-1 chemotaxis on B cells
BRAK is expressed in only two of 18 cancer and T cells. We found that BRAK had no inhibitory
cell lines analyzed by northern blot (Hromas et al., activity on SDF-1. BRAK was also tested for any
1999). Of eight cancer cell lines BRAK is only ability to inhibit the angiogenic signal of VEGF in
expressed in colon adenocarcinoma cells (SW 485). It an in vitro endothelial cell tubule formation
was not expressed in HL60 promyelocytic leukemia, assay. No inhibition of angiogenesis could be
HeLa cervical carcinoma, K562 chronic myelo- demonstrated.
genous leukemia, Molt-4 T cell leukemia, Raji B cell However, we recently found that both BRAK
lymphoma,A549lungcarcinoma,orG361melanoma protein preparations were able to chemoattract
cells. Of 10 breast cancer cell lines, BRAK is only small but reproducible percentages (5–10%) of both
expressed in one, the breast cancer cell line MDA resting and activated normal human NK cells.
MB 435. Human NK cells activated by incubation with IL-2
Recently, a report described BRAK as being over- had increased chemotaxis as compared with
expressed in the normal tissue surrounding a tumor resting NK cells. Since this activity was blocked
(Frederick et al., 2000). Using in situ cRNA hybridi- by antisera to the BRAK protein, it is likely that
zation,BRAKwaspoorlyexpressedinvarioushuman there is a subset of NK cells that respond to
malignant tissues, but expression appeared to be BRAK.
induced in the normal tissue surrounding the tumor. Recently, another group reported that recombi-
This raises the question whether BRAK may play a nantmurineBRAKwasabletostimulatechemotaxis
role in the inflammatory response of normal tissue to of the murine B cell lines CESS and A20, and the
local tumor invasion. human monocyte cell line THP1 (Sleeman et al.,
2000).
RECEPTOR UTILIZATION
The receptor BRAK uses is not known currently.
IN VIVO BIOLOGICAL
ACTIVITIES OF LIGANDS IN
IN VITRO ACTIVITIES ANIMAL MODELS
In vitro findings Normal physiological roles
Mature BRAK protein was synthesized using two There are no known in vivo biological activities of
different methods, the pQE2 bacterial expression BRAK.
4 Robert Hromas
PATHOPHYSIOLOGICAL ROLES Crystallization of human interleukin-8. A protein chemotactic
for neutrophils and T-lymphocytes. J. Biol. Chem. 265, 6851–
IN NORMAL HUMANS AND
6853.
DISEASE STATES AND Frederick, M. J., Henderson, Y., Xu, X., Deavers, M. T.,
Sahin, A. A., Wu, H., Lewis, D. E., El-Naggar, A. K., and
DIAGNOSTIC UTILITY 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.
Normal levels and effects
Hromas, R., Broxmeyer, H., Kim, C., Nakshatri, H.,
Christopherson, K., Azam, M., and Hou, Y.-H. (1999).
There are no known roles for BRAK in human Cloning of BRAK, a novel divergent CXC chemokine prefer-
entially expressed in normal versus malignant cells. Biochem.
pathology.
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
IN THERAPY Kumble,K.D.(2000).Bcell-andmonocyte-activatingchemo-
kine (BMAC), a novel non-ELR alpha-chemokine. Int.
Immunol.12,677–689.
There is no published evidence that BRAK has a Stockelman, M. G., Lorenz, J. N., Smith, F. N., Boivin, G. P.,
therapeutic role in human disease. Sahota, A., Tischfield, J. A., and Stambrook, P. J. (1998).
Chronicrenalfailureinamousemodelofhumanadeninephos-
phoribosyltransferasedeficiency.Am.J.Physiol.275,154–163.
References
Baldwin, E. T., Franklin, K. A., Appella, E., Yamada, M.,
Matsushima, K., and Gronenborn, A. M. (1990).
