DNA encoding BEHAB, a brain hyaluronan-binding protein, and recombinant expression systems for production of BEHAB polypeptides

A gene encoding mammalian brain enriched hyaluronan binding (BEHAB) protein is isolated and characterized from brain tissue and found to have a high degree of sequence homology to members of the proteoglycan tandem repeat family of hyaluronan binding proteins. Unlike other members of the family, however, the expression of the gene is restricted to the central nervous system. BEHAB is expressed in markedly increased levels in human glioma tissue, so that the polypeptide can be used as a marker for diagnostic purposes.

DESCRIPTION 
1. Technical Field of the Invention 
This invention relates to a gene encoding a hyaluronan-binding protein that 
is restricted to the central nervous system, the polypeptide encoded by 
the gene, and methods for using the polypeptide. 
2. Background of the Invention 
The central nervous system extracellular matrix consists of a heterogenous 
mixture of glycoconjugates, many of which are proteoglycans (Jaworski, D. 
M., et al., J. Cell Biol. 125:495-509 (1994), the full text of which is 
hereby incorporated herein in its entirety by reference). Proteoglycans 
are complex macromolecules that consist of a core protein modified with 
one or more types of glycosaminoglycan chains. 
Many functional properties of proteoglycans have been ascribed to 
glycosaminoglycans (ibid.). Glycosaminoglycans have been reported to 
exhibit both adhesive and repulsive properties and, as such, have been 
suggested to mediate neuronal migration and axon guidance. 
Glycosaminoglycans are believed to regulate the local cellular environment 
primarily by serving as selective filters, facilitating permeability and 
retention of low molecular weight solutes, including growth factors, while 
excluding other macromolecules. 
Hyaluronan (also called hyaluronic acid or hyaluronate, and herein 
abbreviated HA) is particularly suited to this function because of its 
charge density and hydroscopic nature. HA is a negatively charged 
high-molecular-weight linear polysaccharide built from repeating 
disaccharide units (Laurent, T. C., and Fraser, J. R. E., FASEB (Fed. Am. 
Soc. Expo Biol.) 6:2397-2404 (1992)). Hyaluronan is ubiquitously 
distributed in the extracellular matrices of all tissues, including brain, 
and is believed to have several functions, including the organization of 
water and extracellular proteins (ibid.). During development, HA plays a 
role in the regulation of morphogenesis and differentiation of neural 
tissues. 
Because HA is ubiquitously present in extracellular space, cell type 
specific functions attributed to HA may be mediated through its 
interaction with HA-binding proteins, which not only bind HA but can also 
contain potential binding sites for other molecules. Several HA-binding 
proteins in the brain have been reported, a subset of which have a high 
degree of sequence similarity to one another, including versican 
(Zimmermann, D. R., and Ruoslahti, E., EMBO (Eur. Mol. Biol. Organ.) J. 8: 
2975-2981 (1989)), link protein (Doege, K., et al., Proc. Natl. Acad. Sci. 
USA 83:3761-3765 (1986)), neurocan (Rauch, U., et al., J. Biol. Chem. 267: 
19536-19547 (1992)), glial hyaluronate binding protein (GHAP, Perides, G., 
et al., J. Biol. Chem. 264:5981-5987 (1989)), and CD44 (Culty, M., et al., 
J. Cell Biol. 111: 2765-2774 (1990)). These have been called the 
proteoglycan tandem repeat (PTR) family of HA-binding protein. 
The spatial distribution and temporal expression of neural extracellular 
matrix proteoglycans and HA-binding proteins indicate that they may be 
involved in many events in the development and function of the mammalian 
central nervous system (Jaworski, et al., cited above) and in the 
modulation of cell-cell and cell-matrix interactions. While some 
HA-binding proteins represent general components of the extracellular 
matrix, others have a restricted pattern of expression on subsets of 
neurons. In addition, while some extracellular matrix molecules are 
transiently expressed during embryogenesis, others are first expressed 
late in the postnatal period, coincident with the decline in developmental 
synaptic plasticity. 
It would be desirable to isolate an HA-binding protein specific to a 
particular tissue or organ, especially where expression of the protein 
varied with pathological states so that it could be used as a marker for 
diagnostic purposes. 
SUMMARY OF THE INVENTION 
It is an object of the invention to provide a gene encoding a mammalian 
hyaluronan-binding protein and to elucidate the relationship of the 
structure of the protein encoded by the gene to other polypeptides, 
especially other hyaluronan-binding proteins. 
It is another and more specific object of the invention to provide a gene 
encoding a mammalian hyaluronan-binding protein that is restricted to 
central nervous system tissue and the polypeptide encoded by the gene. 
These and other objects are accomplished by the present invention which 
provides purified and isolated DNA fragments comprising DNA sequences 
encoding mammalian brain enriched hyaluronan binding protein (herein 
denoted BEHAB), the polypeptide structures they encode, and the 
relationship of the structures to other polypeptides. Also provided are 
RNA sequences corresponding to the DNA sequences of the genes, 
biologically functional plasmids or vectors comprising the DNA or RNA 
sequences, and procaryotic or eucaryotic host cells transformed or 
transfected with the plasmids or vectors in a manner allowing the host 
cell to express the polypeptides. 
DNA sequences encoding rat and cat BEHAB are cloned, characterized, and 
sequenced, and the putative amino acid sequences of the polypeptides 
encoded by the open reading frame are determined (SEQ ID NOs 1 and 2) and 
human BEHAB partially sequenced (SEQ ID NO 7). The sequence exhibits long 
stretches of identity between species, suggesting that the encoded protein 
is functionally important. Unlike other hyaluronan-binding proteins, the 
expression of BEHAB DNA is restricted to the central nervous system, and 
markedly increases in glioma. Thus, the protein can be employed as a 
diagnostic marker for the detection of brain tumors and other 
neuropathological states, and the invention encompasses methods of 
detection of BEHAB in biological samples.

DETAILED DESCRIPTION OF THE INVENTION 
This invention is based upon the identification of a new hyaluronan-binding 
protein, denoted BEHAB for Brain Enriched Hyaluronan Binding protein, that 
is restricted to the brain. 
By "hyaluronan-binding" protein is meant a protein that binds hyaluronan, a 
viscous mucopolysaccharide having the structure [D-glucuronic acid 
(1-.beta.-3)N-acetyl-D-glucosamine(1-.beta.-4)].sub.n (Laurent and Fraser, 
cited above). As described in the Examples that follow, the 
hyaluronan-binding proteins of this invention are restricted to central 
nervous system tissues, found in both white and gray matter, and are not 
detected in liver, kidney, spleen, lung, muscle or other tissues. 
Expression is elevated in human brain glioma, but is not detected in 
non-brain tumors, including breast, lung, and colon. The BEHAB gene 
encodes a neural specific protein that binds hyaluronan but lacks a 
transmembrane domain. 
The expression of BEHAB mRNA is developmentally regulated; expression is 
first detected in the late embryonic period and peaks during the first two 
postnatal weeks. In the embryo, BEHAB is expressed at highest levels in 
mitotically active cells. The size and sequence of BEHAB are consistent 
with the possibility that it could serve a function like link protein, 
stabilizing interactions between hyaluronan and brain proteoglycans. 
Sequence analyses of rat and cat BEHAB (SEQ ID NOs 8 and 9 and FIG. 1) show 
a substantial degree of amino acid identity to other members of the PTR 
family, which includes rat aggrecan, SEQ ID NO 3 (48%); rat neurocan, SEQ 
ID NO 4 (48%); human versican, SEQ ID NO 5 (46%); and rat link protein, 
SEQ ID NO 6 (42%). The NH.sub.2 -terminal domain of this family is defined 
by two structural motifs, (a) an immunoglobulin (Ig) fold (denoted FIG. 
1A) and (b) two PTR folds (PTR1 and PTR2, FIGS. 1B and 1C, respectively). 
The PTR folds have been suggested to mediate binding to HA. The Ig domain 
contains two clusters of conserved amino acids around the cysteine 
residues which generate the disulfide bond of the loop. The consensus 
sequence YxCxVxH in the COOH-terminal cluster is present in all 
immunoglobulin and major histocompatability complex proteins, and is also 
present in BEHAB (FIG. 1). The most conserved region of the PTR family's 
HA-binding protein domain is the sequence CDAGWL(A/S)D(Q/G)(T/S)VRYPI (SEQ 
ID NO 11, but using single letter nomenclature defined above) found in 
PTR1 and PTR2. Two copies of this sequence are also found in BEHAB. The 
degree of identity of BEHAB between rat and cat is high (84% overall), 
with the greatest conservation in PTR1. The identity in PTR1 is 95% over 
the entire domain and 100% over 44 amino acids of the domain. PTR2 shows 
the next highest homology (86%), followed by the Ig domain (84%). The 
relative degree of homology between the PTR1, PTR2, and Ig domains 
observed in rat and cat is also observed between BEHAB and other members 
of the PTR family. Human human BEHAB is also highly conserved in the PTR1 
domain. 
This invention provides purified and isolated DNA fragments comprising DNA 
sequences encoding mammalian brain enriched hyaluronan binding protein, 
and purified and isolated DNA fragments comprising DNA sequences which 
hybridize under stringent conditions with sequences encoding the protein. 
Also provided are RNA sequences corresponding to the DNA sequences. 
In one embodiment, the invention provides a purified and isolated DNA 
fragment derived from rat brain tissue comprising the nucleotides numbered 
251 to 1363 of SEQ ID NO 1, and DNA sequences that hybridize under 
stringent conditions with the sequence. In another embodiment, the 
invention provides the purified and isolated DNA fragment derived from cat 
brain tissue comprising the nucleotides numbered 270 to 1403 of SEQ ID NO 
2, and DNA sequences that hybridize under stringent conditions with the 
sequence. In a third embodiment, the invention provides a purified and 
isolated DNA fragment derived from human brain tissue comprising 
nucleotides of SEQ ID NO 7, and DNA sequences that hybridize under 
stringent conditions with the sequence. 
Encompassed by this invention are cloned sequences defining BEHAB of this 
invention, which can then be used to transform or transfect a host cell 
for protein expression using standard means. Also encompassed by this 
invention are DNA sequences homologous or closely related to complementary 
DNA described herein, namely DNA sequences which hybridize to BEHAB cDNA, 
particularly under stringent conditions that result in pairing only 
between nucleic acid fragments that have a high frequency of complementary 
base sequences, and RNA corresponding thereto. In addition to the 
BEHAB-encoding sequences, DNA encompassed by this invention may contain 
additional sequences, depending upon vector construction sequences, that 
facilitate expression of the gene. Also encompassed are sequences encoding 
synthetic BEHAB proteins exhibiting activity and structure similar to 
isolated or cloned BEHAB. These are referred to herein as "biological 
equivalents". 
Because of the degeneracy of the genetic code, a variety of codon change 
combinations can be selected to form DNA that encodes hyaluronan-binding 
protein of this invention, so that any nucleotide deletion(s), 
addition(s), or point mutation(s) that result in a DNA encoding the 
protein are encompassed by this invention. Since certain codons are more 
efficient for polypeptide expression in certain types of organisms, the 
selection of gene alterations to yield DNA material that codes for the 
protein of this invention are preferably those that yield the most 
efficient expression in the type of organism which is to serve as the host 
of the recombinant vector. Altered codon selection may also depend upon 
vector construction considerations. 
DNA starting material which is employed to form DNA coding for BEHAB 
proteins of this invention may be natural, recombinant or synthetic. Thus, 
DNA starting material isolated from tissue or tissue culture, constructed 
from oligonucleotides using conventional methods, obtained commercially, 
or prepared by isolating RNA coding for BEHAB, and using this RNA to 
synthesize single-stranded cDNA which is used as a template to synthesize 
the corresponding double stranded DNA, can be employed to prepare DNA of 
this invention. 
DNA encoding the proteins of this invention, or RNA corresponding thereto, 
are then inserted into a vector, e.g., but not limited to, a p series 
plasmid such as pBR, pUC, pUB or pET, and the recombinant vector used to 
transform a microbial host organism. Example host organisms useful in the 
invention include, but are not limited to, bacterial (e.g., E. coli or B. 
subtilis), yeast (e.g., S. cerevisiae) or mammalian (e.g., mouse 
fibroblast or other tumor cell line). This invention thus also provides 
novel, biologically functional viral and circular plasmid RNA and DNA 
vectors incorporating RNA and DNA sequences describing BEHAB generated by 
standard means. Culture of host organisms stably transformed or 
transfected with such vectors under conditions facilitative of large scale 
expression of the exogenous, vector-borne DNA or RNA sequences and 
isolation of the desired polypeptides from the growth medium, cellular 
lysates, or cellular membrane fractions yields the desired products. 
The present invention thus provides for the total and/or partial 
manufacture of DNA sequences coding for BEHAB, and including such 
advantageous characteristics as incorporation of codons preferred for 
expression by selected non-mammalian hosts, provision of sites of cleavage 
by restriction endonuclease enzymes, and provision of additional initial, 
terminal or intermediate DNA sequences which facilitate construction of 
readily expressed vectors. Correspondingly, the present invention provides 
for manufacture (and development by site specific mutagenesis of cDNA and 
genomic DNA) of DNA sequences coding for microbial expression of BEHAB 
analogues which differ from the forms specifically described herein in 
terms of identity or location of one or more amino acid residues (i.e., 
deletion analogues containing less than all of the residues specified for 
the protein, and/or substitution analogues wherein one or more residues 
are added to a terminal or a medial portion of the polypeptide), and which 
share the biological properties of BEHAB described herein. 
DNA (and RNA) sequences of this invention code for all sequences useful in 
securing expression in procaryotic or eucaryotic host cells of polypeptide 
products having at least a part of the primary structural conformation, 
and one or more of the biological properties of BEHAB which are 
comprehended by: (a) the DNA sequences encoding BEHAB as described herein, 
or complementary strands; (b) DNA sequences which hybridize (under 
hybridization conditions) to DNA sequences defined in (a) or fragments 
thereof; and (c) DNA sequences which, but for the degeneracy of the 
genetic code, would hybridize to the DNA sequences defined in (a) and (b) 
above. Specifically comprehended are genomic DNA sequences encoding 
allelic variant forms of BEHABs included therein, and sequences encoding 
RNA, fragments thereof, and analogues wherein RNA or DNA sequences may 
incorporate codons facilitating transcription or RNA replication of 
messenger RNA in non-vertebrate hosts. 
The invention also provides the BEHAB proteins encoded by the above 
described DNA and/or RNA, obtained by isolation or recombinant means. In 
one embodiment, for example, the invention provides a polypeptide having 
an amino acid sequence depicted in residues numbered 1 to 371 of SEQ ID NO 
8 or a biological equivalent thereof. In another embodiment, the invention 
provides a polypeptide having the amino acid sequence depicted in residues 
numbered 1 to 378 of SEQ ID NO 9 or a biological equivalent thereof. In a 
third embodiment, the invention provides a polypeptide set out in SEQ ID 
NO 7 or a biological equivalent thereof. 
Isolation and purification of proteins provided by the invention are by 
conventional means including, for example, preparative chromatographic 
separations such as affinity, ion-exchange, exclusion, partition, liquid 
and/or gas-liquid chromatography; zone, paper, thin layer, cellulose 
acetate membrane, agar gel, starch gel, and/or acrylamide gel 
electrophoresis; immunological separations, including those using 
monoclonal and/or polyclonal antibody preparations; and combinations of 
these with each other and with other separation techniques such as 
centrifugation and dialysis, and the like. 
It is an advantage of the invention that the isolation and purification of 
BEHAB provides a polypeptide marker for diagnostic purposes. Since BEHAB 
is neural-specific, it can be used as a diagnostic agent for brain or 
other central nervous system tumors or other neuropathological states. 
Expression of BEHAB is markedly increased in human brain glioma. Thus, 
this invention provides novel diagnostic methods employing biochemical 
markers for BEHAB, such as specific and sensitive immunoassays for the 
detection of BEHAB and patterns of its distribution in samples, to provide 
not only an indication of ongoing pathological processes in central 
nervous system tissue, but also differential diagnoses of pathological 
processes involving specific areas of the central nervous system. 
In the practice of the invention, the presence or absence of BEHAB, and/or 
relative concentrations of BEHAB, are assayed in biological samples 
obtained from animals or human beings. Typical samples include, but are 
not limited to, cerebrospinal fluid, serum, urine or tissue homogenates 
such as those obtained from biopsies. Serum and cerebrospinal fluid are 
particularly preferred. 
For diagnostic purposes, any method may be employed to assay for BEHAB 
protein. Assay methods include, but are not limited to, Western blots, 
Northern blots, Northern dot blots, enzyme-linked immunosorbent assays, 
radioimmunoassays, or mixtures of these. 
For example, one embodiment employs an enzyme-linked immunosorbent assay 
(ELISA). ELISAs typically utilize an enzyme such as horseradish 
peroxidase, urease, or alkaline phosphatase conjugated to an antibody or 
conjugated with a tag that interacts with a correspondingly tagged 
antibody. Example tags, where employed, are avidin and biotin. Test sample 
is incubated in the wells of microtiter plates with conjugated antibody. 
If the serum contains BEHAB antigen, the conjugated antibodies adhere to 
it. Subsequent measurement of enzyme activity estimates how much tagged 
antibody is present and bound to BEHAB. From that, amounts of BEHAB in the 
original test sample are calculated. Preferred ELISAs employ substrates 
known to those skilled in the art to be easily measurable, for example, by 
viewing color development in comparison with standards or by employing a 
spectrophotometer. These and other variations on ELISA protocols known by 
those skilled in the art are encompassed by the invention. 
Most preferred substrates are chromophoric or yield chromophoric products, 
so that enzyme activity can be readily measured by the appearance or 
disappearance of color. Examples of enzyme substrates include 
p-nitrophenyl phosphate for alkaline phosphatase, bromocresol purple and 
urea for urease, p-nitrophenyl-.beta.-galactopyranoside for 
.beta.-galaactosidase, and the like. Horseradish peroxidase requires 
hydrogen peroxide in addition to another substrate that serves as a 
hydrogen donor including, for example, 
2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid), 5-aminosalicylic 
acid, o-diaminobenzidine, 3,3'-dimethoxybenzidine, o-phenylenediamine 
(free base or dihydrochloride), 3,3',5,5'-tetramethylbenzidine (base or 
dihydrochloride), and the like chromogens. 
An alternate embodiment employs a radioimmunoassay (RIA). Typical RIAs 
employ antigens radiolabelled with .sup.125 I, .sup.3 H or other isotope 
that can be easily detected. For example, .sup.125 I-labelled BEHAB can be 
employed. Antibody is titrated with labelled antigen, and the activity and 
sensitivity of the antiserum is determined. A dilution series of samples 
to which known amounts of antigen have been added are distributed in wells 
of microtiter plates. Antibody is added, the well material and/or the 
supernatants analyzed for radioactivity after incubation, and compared to 
a standard curve prepared using pure antigen. Amounts of unlabelled 
antigen bound are calculated by difference. These and other variations on 
RIA protocols known by those skilled in the art are encompassed by this 
invention. 
The following examples are presented to further illustrate and explain the 
present invention and should not be taken as limiting in any regard. 
EXAMPLES 
Example 1 
Rat and cat cDNA clones encoding BEHAB from the two species are prepared in 
this example. 
To isolate rat cDNA clones encoding HA-binding proteins involved in neural 
development, an unamplified postnatal day 12 rat brain .lambda.gt10 cDNA 
library is screened with rat aggrecan clone pRCP 4 encoding the HA-binding 
region (described by Doege, K., et al., J. Biol. Chem. 262:17757-17767 
(1987)). A total of 3.2.times.10.sup.5 recombinants are screened resulting 
in two positives. The library is rescreened with one of these clones, 
resulting in 15 additional clones. 4.times.10.sup.4 phage (per 150 mm 
plate) are plated with E. coli C600 bacteria, immobilized onto 
nitrocellulose filters, and prepared for hybridization using standard 
techniques. Filters are prewashed for 1 hour in 1M NaCl, 0.1% sodium 
dodecyl sulfate (SDS), 20 mM Tris-HCl (pH 8.0) and 1 mMEDTA at 65.degree. 
C. Filters are then prehybridized for an additional 4 to 6 hours in 50% 
formamide, 5.times.SCC (1.times.SCC=0.15M sodium chloride, 0.015M sodium 
citrate), 1% SDS, 1.times.Denhardt's (0.02% Ficoll, 0.02% bovine serum 
albumin (BSA, Fraction V), 0.02% polyvinylpyrrolidone), 50 mM sodium 
phosphate (pH 6.7), and 100 .mu.g/ml salmon sperm DNA at 37.degree. C. 
Hybridization is carried out in the identical solution with the inclusion 
of 10.sup.6 cpm pRCP 4 probe/ml for 24 hours at 37.degree. C. For all 
experiments, radiolabelled probes (.sup.32 P-dCTP, Amersham) are prepared 
by random priming (Boehringer Mannheim Corp., Indianapolis Ind.) gel 
purified cDNA inserts, followed by the removal of unincorporated 
radionucleotides (NICK column, Pharmacia). One post hybridization wash is 
in 2.times.SSC, 0.1% SDS and one in 0.2.times.SSC for 1 hour each are 
performed at room temperature. Phage DNA is isolated using DE52 (Whatman) 
and the cDNA insert excised by EcoRI digestion. The insert size of the 
clones are determined and partial restriction maps are prepared to 
eliminate redundant clones. The cDNA is gel purified (Gene-Clean.RTM., Bio 
101), eight clones subcloned into pBluescript.RTM. KS+ (Stratagene, 
LaJolla, Calif.) and transformed into DH5.alpha. (GIBCO BRL, Gaithersburg, 
Md.). 
To isolate cat cDNA clones, random nonamers (1.4 mg) are used to synthesize 
first cDNA from 5 .mu.g poly A.sup.+ RNA isolated from P39 cat cortex, 
cDNA synthesis is performed according to manufacturer's instructions for 
the production of nondirectional libraries (Stratagene) and 
size-fractionated by column chromatography (GIBCO BRL). 50 ng of cDNA is 
ligated to 1 .mu.g EcoRI cut, phosphatized Lambda Zap.RTM. II vector and 
packaged into phage (Gigapack II Gold.RTM., Stratagene). This yields 
0.5.times.10.sup.6 recombinants when transfected into XL1-Blue.RTM. 
(Stratagene). The unamplified library is screened with rat clone H1. 
Hybridization is performed in 6.times.SSC, 0.1% SDS, 1.times.Denhardt's 
and 100 .mu.g/ml salmon sperm DNA at 65.degree. C. Filters are washed 
twice in 2.times.SSC, 0.1% SDS and twice in 0.2.times.SSC at 65.degree. C 
for 20 minutes. A total of 3.2.times.10.sup.5 recombinants are screened, 
resulting in 5 positives. cDNA inserts of plaque-purified positive clones 
are isolated in pBluescript.RTM. SK.sup.- by in vivo excision. 
Example 2 
DNA clones prepared in Example 1 are sequenced and compared with previously 
reported sequences in this Example. 
DNA sequencing is performed by the dideoxy chain termination method using 
Sequenase.RTM. (U.S. Biochemical, Cleveland, Ohio). Bluescript SK/KS 
primers or cDNA specific 20-mers are used. Sequence is verified from 
overlapping clones or by sequencing both strands of DNA. Sequence 
compressions are resolved using dITP nucleotides. After labelling, the 
reactions are incubated at 37.degree. C. for 30 minutes in the presence of 
1.times. reaction buffer, 1 mM dNTPs (pH 7.0) and 0.5 U terminal 
deoxynucleotidyl transferase to prevent premature termination caused by 
the use of dITP. Sequence analyses are performed using the University of 
Wisconsin Genetics Computer Group programs. 
For the rat BEHAB sequence, the composite sequence obtained from the 
overlapping clones identified after subcloning into pBluescript.RTM. KS+ 
as described in the previous Example is used (SEQ ID NO 1; sequence data 
are recorded in EMBL/GenBank/DDBJ under accession number Z28366). The 
complete BEHAB coding sequence is 1,113 base pairs. The nucleotide 
sequence preceding the first AUG contains a consensus sequence for 
translation initiation. In the 3' untranslated region, only that sequence 
verified from three clones is presented. The deduced amino acid 
composition of the BEHAB protein is comprised of 371 amino acids and 
includes a putative signal peptide cleavage site at Ala-22. The resulting 
mature protein has a predicted molecular mass of 38,447 kD. Analysis of 
the deduced amino acid sequence indicates the presence of two NX(S/T) 
consensus sequences for potential N-glycolsation. 
Similarly, the composite cat BEHAB sequence is obtained from the 
overlapping clones obtained in the pBluescript.RTM. SK.sup.- excision as 
described in the above Example. The results are set out in SEQ ID NO 2 
(sequence data are recorded in EMBL/GenBank/DDBJ under accession number 
Z28367). The complete coding sequence for cat BEHAB is 1,134 base pairs. 
The first AUG is preceded by both an in-frame termination codon and the 
translation initiation consensus sequence. The cat BEHAB sequence encodes 
378 amino acids which, like the rat, contains a 22 residue signal peptide. 
However, cat BEHAB contains 6 additional amino acids at the carboxy 
terminus, resulting in a predicted molecular mass of 38,955 kD. In the 
cat, Trp-373 is encoded by TGG, while the corresponding rat sequence of 
TAG results in the termination. This termination sequence is verified in 
three rat clones and by sequencing both strands of a cat clone. Cat BEHAB 
also contains one additional site for potential N-glycosylation not 
present in the rat. 
Database analyses at both the nucleic acid and amino acid levels indicate 
that BEHAB is a previously unreported member of the PTR family of 
HA-binding proteins. BEHAB has a substantial degree of amino acid identity 
to the other members of the PTR family, which includes rat aggregan, SEQ 
ID NO 3 (48%); rat neurocan, SEQ ID NO 4 (48%); human versican, SEQ ID NO 
5 (46%); and rat link protein, SEQ ID NO 6 (42%). See FIG. 1. The NH.sub.2 
-terminal domain of this family is defined by two structural motifs, (a) 
an immunoglobulin (Ig) fold and (b) two PTR folds (PTR1 and PTR2). The PTR 
folds have been suggested to mediate binding to HA. The Ig domain contains 
two clusters of conserved amino acids around the cysteine residues which 
generate the disulfide bond of the loop. The consensus sequence YxCxVxH in 
the COOH-terminal cluster is present in all immunoglobulin and major 
histocompatability complex proteins, and is also present in BEHAB (FIG. 
1). The most conserved region of the PTR family's HA-binding protein 
domain is the sequence CDAGWL(A/S)D(Q/G)(T/S)VRYPI found in PTR1 and PTR2. 
Two copies of this sequence are also found in BEHAB. The degree of 
identity of BEHAB between rat and cat is high (84% overall), with the 
greatest conservation in PTR1. The identity in PTR1 is 95% over the entire 
domain and 100% over 44 amino acids of the domain. PTR2 shows the next 
highest homology (86%), followed by the Ig domain (84%). The relative 
degree of homology between the PTR1, PTR2, and Ig domains observed in rat 
and cat is also observed between BEHAB and other members of the PTR family 
(Table I and FIG. 1). 
TABLE I 
______________________________________ 
Percent Identity of rat BEHAB to Other Members 
of the PTR Family of HA-Binding Proteins 
Protein Ig PTR1 PTR2 
______________________________________ 
Cat BEHAB 84% 95% 86% 
Aggrecan 40% 60% 51% 
Neurocan 37% 56% 57% 
Versican 36% 59% 48% 
Rat Link 34% 48% 53% 
CD44 22% 
______________________________________ 
Sequence homology is similarly observed for human BEHAB (SEQ ID NO 7). To 
determine the human BEHAB sequence, total RNA is extracted from a sample 
of human brain and reverse transcriptase polymerase chain reactions (PCR) 
performed using degenerate oligonucleotide primers corresponding to the 
ends of the PTR1 domain in rat and cat. PCR products are subcloned into 
the TA vector and sequenced by the dideoxy chain termination method 
described above. 
Example 3 
In this Example, tissue distribution of BEHAB mRNA is determined by 
Northern blot analysis and the spatial distribution, by in situ 
hybridization on central nervous system tissue sections. 
For Northern analysis, 25 .mu.g total RNA is denatured in 2.2M 
formaldehyde, 50% formamide, 1.times.MOPS (3-(N-morpholino)propanesulfonic 
acid) buffer at 65.degree. C. for 15 minutes. The RNA is electrophoresed 
on a 1.0% agarose-formaldehyde gel with 1.times.MOPS buffer at 50 V with 
buffer recirculation. The gel is briefly neutralized in transfer buffer 
(20.times.SSC) and RNA blotted to Zetaprobe.RTM. (BioRad Labs., Hercules 
Calif.) by capillary transfer. Filters are rinsed briefly in 2.times.SSC, 
and RNA is immobilized both by UV cross-linking and baking in vacuuo 
(80.degree. C. for 1 hour). Hybridization in 7% SDS, 1% BSA, 0.5M 
phosphate buffer (PB, pH 6.8), 1 mM EDTA and 0.5-2.5.times.10.sup.6 cpm 
rat H1 probe/ml are carried out for at least 8 hours at 65.degree. C. 
Filters are washed twice in 5% SDS, 0.5% BSA, 40 mM PB, 1 mM EDTA and 
twice in 1% SDS, 40 mM PB, 1 mM EDTA at 65.degree. C, and exposed to film 
(Hyperfilm, Amersham) at -70.degree. C. Molecular sizes are determined 
relative to RNA molecular weight standards (GIBCO BRL) and 28S and 18S 
ribosomal RNA observed during UV illumination. The ubiquitously expressed, 
non-developmentally regulated gene cyclophilin is used to determine equal 
loading of lanes. Densitometry is performed using the NIH Image program. 
The two clones recognize the same size mRNA transcript. 
Tissue distribution of rat BEHAB mRNA using this procedure shows a single 
3.9-kb mRNA transcript detected in adult rat cortex, spinal cord and 
cerebellum. This transcript is not detected in liver, kidney, spleen, lung 
or muscle, even with long film exposures. Observed amounts of human BEHAB 
mRNA is markedly (i.e., at least about four-fold) higher in brain glioma 
tissue in comparison to what is seen in normal brain tissue using the 
procedure. Moreover, BEHAB is not detected in non-brain tumor tissues, 
including breast, lung, or colon tumors. 
These observations are confirmed by in situ hybridization to whole embryos, 
which show that BEHAB expression is restricted to the central nervous 
system. In situ hybridization is performed on 12 to 14 micron thick frozen 
sections thaw-mounted onto gelatin-coated slides and postfixed in 0.1M 
sodium phosphate buffered 4% paraformaldehyde (pH 7.4). Sections are 
rinsed in 1.times.PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na.sub.2 PO.sub.4, 
1.8 mM KH.sub.2 PO.sub.4) 2.times.SSC and acetylated with 0.5% acetic 
anhydride in 0.1M triethanolamine (pH 8.0). Sections are then rinsed in 
2.times.SSC, 1.times.PBS, dehydrated in ethanol and delipidated in 
chloroform. Sections are prehybridized in 2.times.SSC, 50% formamide at 
50.degree. C. for 1 hour, and then hybridized in 0.75 M NaCl, 50% 
formamide, 1.times.Denhardt's, 10% dextran sulfate, 30 mM DTT, 10 mM 
Tris-HCl (pH 7.5), 1 mMEDTA, 100 .mu.g/ml salmon sperm DNA, 0.5 mg/ml 
yeast tRNA and 10.sup.6 cpm probe per slide at 50.degree. C. for 12 to 15 
hours. (.sup.35 S)-CTP (New England Nuclear, Boston Mass.) labelled cRNA 
probes are synthesized using T3 (GIBCO BRL), SP6, and T7 RNA polymerases 
(New England Biolabs inc., Beverly, Mass.). After hybridization, sections 
are washed in 2.times.SSC, 50% formamide, 0.1% BME 
(.beta.-mercaptoethanol) at 50.degree. C. for 1 hour and treated with 20 
.mu.g/ml RNase A in 0.5M NaCl, 10 mM Tris-HCl (pH 8.0) at 37.degree. C. 
for 30 minutes. Sections are then washed in 2.times.SSC, 50% formamide, 
0.1% BME at 58.degree. C. for 30 minutes and 0.1.times.SSC, 0.1% BME at 
63.degree. C. for 30 minutes and dehydrated. For initial localization of 
probe, the slides are exposed to film (Hyperfilm, Amersham) for 4 days. 
Autoradiograms are used as negatives for prints. For higher resolution, 
the slides are dipped in NTB-2 emulsion (Kodak), developed after 5 days 
and counterstained with cresyl violet. Neurofilament-middle (NF) antisense 
and rat clone sense probes are used as positive and negative controls, 
respectively. 
The spatial distribution of BEHAB mRNA within the nervous system is 
determined at higher resolution by in situ hybridization on tissue 
sections from P21 rat forebrain, brainstem, spinal cord, and cerebellum. 
Near adjacent sections are probed with an antisense cRNA probe of a rat 
clone and positive and negative controls. Using these procedures, BEHAB 
expression is found to be widely distributed in the brain, in both gray 
and white matter. The cortex exhibits diffuse hybridization with no 
laminar specification. Hybridization is detected in white matter tracts, 
including the corpus callosum, the fimbria of the hippocampus, and the 
anterior commissure. In the hippocampus, the most intense hybridization is 
present over neurons; it is highest in the CA1 subfield. The pattern of NF 
hybridization in the hippocampus is essentially reciprocal to that of 
BEHAB; the NF probe hybridizes most intensely in subfields CA2, CA3, and 
in the dentate gyrus. BEHAB hybridization is also seen throughout the 
inferior colliculus and less intensely in the superior colliculus. In 
addition to the hippocampus, BEHAB hybridization in gray matter is most 
intense in the substantia nigra. The rat sense probe generates almost no 
signal in most of the brain, but a low level of hybridization is seen in 
the hippocampus and dentate gyrus. 
In the brainstem, BEHAB is expressed throughout the reticular formation. 
Several brainstem nuclei also express BEHAB, including the superior 
olivary nucleus, the vestibular nuclei, the abducens nucleus and the 
dorsal column nuclei. A similar hybridization pattern is observed with NF, 
while no hybridization signal is detected with the sense probe. 
BEHAB expression in the spinal cord is greater in the gray matter than in 
white matter. In the gray matter, BEHAB expression is slightly greater in 
the ventral than in the dorsal horn. BEHAB hybridization is lacking in the 
substantia gelatinosa. In the ventral horn, hybridization is seen over 
motor neurons. In the spinal cord white matter, the size of labelled cells 
and their distribution indicates that BEHAB is expressed by glial cells. 
Like BEHAB, NF expression is greater in the ventral horn than in the 
dorsal horn; however, unlike BEHAB, NF is not detected in the spinal white 
matter. As observed in the brainstem, no hybridization signal is detected 
in the spinal cord with the sense probe. 
In the cerebellum, BEHAB expression is greatest in the deep cerebellar 
nuclei. In the cerebellar cortex, labeling is detected in all three 
cortical layers. In the molecular layer, the distribution of silver grains 
parallels the distribution of basket and stellate cells. In the Purkinje 
cell layer, labeling is clustered over Purkinje cells and, in the granule 
cell layer, it is clustered over Golgi II cells. The white matter of the 
cerebellar cortex also shows hybridization signal. NF is primarily 
expressed by Purkinje cells and by cells of the deep cerebellar nuclei. 
The sense probe generates a low level of diffuse hybridization signal 
throughout the granule cell layer. 
To determine the temporal regulation of BEHAB mRNA expression, Northern 
blot analysis is performed using total RNA from embryonic and postnatal 
rat cortex and spinal cord. The non-developmentally regulated gene 
cyclophilin is used as a control probe to verify equal loading. Unlike 
actin and tubulin, which exhibit variation of abundance with development, 
cyclophilin maintains a constant relative abundance throughout the central 
nervous system with development. The Northern blots are analyzed by 
densitometry, and band intensity of BEHAB is standardized by calculating a 
ratio of the abundance of BEHAB to cyclophilin at each developmental age. 
In the cortex, BEHAB recognizes a single 3.9-kb mRNA transcript. BEHAB 
expression is detected at embryonic day 17 and gradually increases to 
attain adult levels by postnatal day 21. In the spinal cord, BEHAB also 
recognizes a 3.9-kb mRNA transcript. At all ages except the adult, BEHAB 
expression is greater in the spinal cord than in the cortex. Like the 
cortex, BEHAB is present in the spinal cord at embryonic day 17 and 
gradually increases with age until reaching a maximal level at postnatal 
day 14. Unlike the cortex, BEHAB expression in the spinal cord then 
declines slightly. 
The expression of BEHAB in the embryo, like in the postnatal animal, is 
restricted to the central nervous system. BEHAB expression is absent in 
dorsal root ganglia, a peripheral nervous system structure. Tissues in the 
embryo that express high levels of closely related genes such as cartilage 
(which expresses aggrecan) also show no hybridization signal for BEHAB. 
The distribution of BEHAB expression in the embryonic central nervous 
system differs slightly from the postnatal brain. The highest levels of 
BEHAB expression are found in regions that contain mitotically active 
cells, such as the ventricular zone of the medulla, midbrain, and spinal 
cord. Expression of BEHAB is heterogenous in the developing brain. 
The above description is for the purpose of teaching the person of ordinary 
skill in the art how to practice the present invention, and it is not 
intended to detail all those obvious modifications and variations of it 
which will become apparent to the skilled worker upon reading the 
description. It is intended, however, that all such obvious modifications 
and variations be included within the scope of the present invention as 
defined in the appended claims. The claims are meant to cover the claimed 
components and steps in any sequence which is effective to meet the 
objectives there intended, unless the context specifically indicates the 
contrary. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 11 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1520 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: 
(A) DESCRIPTION: DNA encoding a protein 
(v) FRAGMENT TYPE: entire sequence 
(vi) IMMEDIATE SOURCE: rat brain 
(ix) FEATURE: 
(A) NAME/KEY: rat BEHAB 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
CGAGACCCGCGCAGAGAAGGGAGCGGGTCCCGTGACCGCGCA42 
GAGCCCCCCACGCGGCCAAAGGCCGGGGACGCGGGGAAGGCGGGGCGCGT92 
GGGAAGAAACCCCCTTTTGTGCGGCTCCCGGCGAGCTGGCGCCCCCGTCT142 
GCGTCCCGCGCGCCCGGCCCTGCTCGCGCCCGCGCATTGCCGCAGTCTCG192 
GCTGCGTGCGGGACGCGGTGTGTGGAGGGGACCTCACAAGTTCTTCCAAG242 
TTTGCAGCATGATCCCATTGCTTCTGTCCCTGCTGGCAGCTCTG286 
MetIleProLeuLeuLeuSerLeuLeuAlaAlaLeu 
510 
GTCCTGACCCAAGCCCCTGCAGCCCTCGCTGATGACCTGAAA328 
ValLeuThrGlnAlaProAlaAlaLeuAlaAspAspLeuLys 
152025 
GAAGACAGCTCAGAGGATCGAGCCTTTCGGGTGCGCATCGGT370 
GluAspSerSerGluAspArgAlaPheArgValArgIleGly 
303540 
GCCGCGCAGCTGCGGGGTGTGCTGGGCGGTTGGGTGGCCATC412 
AlaAlaGlnLeuArgGlyValLeuGlyGlyTrpValAlaIle 
4550 
CCATGCCACGTCCACCACCTGAGGCCGCCGCCCAGCCGCCGG454 
ProCysHisValHisHisLeuArgProProProSerArgArg 
556065 
GCCGCGCCGGGCTTTCCCCGAGTCAAATGGACCTTCCTGTCC496 
AlaAlaProGlyPheProArgValLysTrpThrPheLeuSer 
707580 
GGGGACCGGGAGGTGGAGGTGCTGGTGGCGCGCGGGCTGCGC538 
GlyAspArgGluValGluValLeuValAlaArgGlyLeuArg 
859095 
GTCAAGGTAAACGAAGCCTATCGGTTCCGCGTGGCGCTGCCT580 
ValLysValAsnGluAlaTyrArgPheArgValAlaLeuPro 
100105110 
GCCTACCCCGCATCGCTCACAGATGTGTCTTTAGTATTGAGC622 
AlaTyrProAlaSerLeuThrAspValSerLeuValLeuSer 
115120 
GAACTGCGGCCCAATGATTCCGGGGTCTATCGCTGCGAGGTC664 
GluLeuArgProAsnAspSerGlyValTyrArgCysGluVal 
125130135 
CAGCACGGTATCGACGACAGCAGTGATGCTGTGGAAGTCAAG706 
GlnHisGlyIleAspAspSerSerAspAlaValGluValLys 
140145150 
GTCAAAGGGGTCGTCTTCCTCTACCGAGAGGGCTCTGCCCGC748 
ValLysGlyValValPheLeuTyrArgGluGlySerAlaArg 
155160165 
TATGCTTTCTCCTTCGCTGGAGCCCAGGAAGCCTGTGCTCGC790 
TyrAlaPheSerPheAlaGlyAlaGlnGluAlaCysAlaArg 
170175180 
ATCGGAGCCCGAATTGCCACCCCTGAGCAGCTGTATGCTGCC832 
IleGlyAlaArgIleAlaThrProGluGlnLeuTyrAlaAla 
185190 
TACCTCGGCGGCTATGAACAGTGTGATGCTGGCTGGCTGTCC874 
TyrLeuGlyGlyTyrGluGlnCysAspAlaGlyTrpLeuSer 
195200205 
GACCAAACCGTGAGGTACCCCATCCAGAACCCACGAGAAGCC916 
AspGlnThrValArgTyrProIleGlnAsnProArgGluAla 
210215220 
TGTTATGGAGACATGGATGGCTACCCTGGAGTGCGGAATTAC958 
CysTyrGlyAspMetAspGlyTyrProGlyValArgAsnTyr 
225230235 
GGAGTGGTGGGTCCTGATGATCTCTACGATGTCTACTGTTAT1000 
GlyValValGlyProAspAspLeuTyrAspValTyrCysTyr 
240245250 
GCCGAAGACCTAAATGGAGAACTGTTCCTAGGTGCCCCTCCC1042 
AlaGluAspLeuAsnGlyGluLeuPheLeuGlyAlaProPro 
255260 
GGCAAGCTGACGTGGGAGGAGGCTCGGGACTACTGTCTGGAA1084 
GlyLysLeuThrTrpGluGluAlaArgAspTyrCysLeuGlu 
265270275 
CGCGGTGCTCAGATCGCTAGCACGGGCCAGCTATACGCGGCA1126 
ArgGlyAlaGlnIleAlaSerThrGlyGlnLeuTyrAlaAla 
280285290 
TGGAATGGCGGCTTGGACAGATGTAGCCCTGGCTGGCTGGCT1168 
TrpAsnGlyGlyLeuAspArgCysSerProGlyTrpLeuAla 
295300305 
GATGGCAGTGTGCGGTACCCCATCATCACGCCCAGCCAACGC1210 
AspGlySerValArgTyrProIleIleThrProSerGlnArg 
310315320 
TGTGGGGGAGGCCTGCCAGGAGTCAAGACCCTCTTCCTCTTT1252 
CysGlyGlyGlyLeuProGlyValLysThrLeuPheLeuPhe 
325330 
CCCAACCAGACTGGCTTCCCCAGCAAGCAGAACCGCTTCAAT1294 
ProAsnGlnThrGlyPheProSerLysGlnAsnArgPheAsn 
335340345 
GTCTACTGCTTCCGAGACTCTGCCCATCCCTCTGCCTTCTCT1336 
ValTyrCysPheArgAspSerAlaHisProSerAlaPheSer 
350355360 
GAGCCTCCAGCCCAGCCTCTGATGGACTAGAGGCCATTGTCACAGTG1383 
GluProProAlaGlnProLeuMetAsp 
365370 
ACAGAGAAGCTGGAGGAACTGCAGTTGCCTCAGGAAGCTGTGGAGAGCGA1433 
GTCTCGTGGGGCGATCTACTCCATCCCCATCACAGAAGATGGGGGAGGAG1483 
GAAGCTCTACCCCAGAAGACCCAGCAGAGGCCCCCAG1520 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1519 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: 
(A) DESCRIPTION: DNA encoding a protein 
(v) FRAGMENT TYPE: entire sequence 
(vi) IMMEDIATE SOURCE: cat cortex 
(ix) FEATURE: 
(A) NAME/KEY: cat brain BEHAB 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
CGGCACGAGCTCGTGCCGA19 
ATTCGGCACAGAGGGACCGAGCGTGGACCCGGAGGAGAGCCCGGAGGAGA69 
GCCCGGAGGAGGCGCAAACTTGGCGGTGCGCACCCTAGCCCCGGCCCTCG119 
GCCTGCCGGAAGAAAACAAAGGCCCTGAGAGCTTAAGGAACTTGCAGCAA169 
GTTGACTAGCGCCCAGGTCTTGGTTCCGAGGAGGAATCCTGGTGGGGAGA219 
CAGGATCAGAAGCGAGGGTGTTAACAGTGAGTCCTTCCAGCAGCCTGAGC269 
ATGGCCCCACTGTTCCTGCCCCTGCTGATAGCCCTGGCCCTG311 
MetAlaProLeuPheLeuProLeuLeuIleAlaLeuAlaLeu 
510 
GCCCCGGGCCCCACGGCCTCAGCTGATGTCCTGGAAGGGGAC353 
AlaProGlyProThrAlaSerAlaAspValLeuGluGlyAsp 
152025 
AGCTCAGAGGACCGGGCCTTCCGCGTGCGCATCTCGGGCAAC395 
SerSerGluAspArgAlaPheArgValArgIleSerGlyAsn 
303540 
GCGCCGCTGCAGGGCGTGCTGGGCGGCGCCCTCACCATCTCG437 
AlaProLeuGlnGlyValLeuGlyGlyAlaLeuThrIleSer 
455055 
TGCCACGTTCACTACCTGCGGCCGCCGCCGGGCCGCCGGGCC479 
CysHisValHisTyrLeuArgProProProGlyArgArgAla 
606570 
GTGCTGGGCTCCCCGCGGGTCAAGTGGACCTTCCTGTCCGGG521 
ValLeuGlySerProArgValLysTrpThrPheLeuSerGly 
7580 
GGCCGGGAGGCCGAGGTGCTGGTGGCGCGGGGGCTGCGCGTC563 
GlyArgGluAlaGluValLeuValAlaArgGlyLeuArgVal 
859095 
AAGGTGAGCGAGGCCTACCGGTTCCGCGTGGCGCTGCCCGCC605 
LysValSerGluAlaTyrArgPheArgValAlaLeuProAla 
100105110 
TACCCGGCGTCCCTCACCGACGTCTCCCTGGCACTGAGCGAG647 
TyrProAlaSerLeuThrAspValSerLeuAlaLeuSerGlu 
115120125 
CTGCGGCCCAACGACTCTGGCATCTACCGCTGCGAGGTCCAG689 
LeuArgProAsnAspSerGlyIleTyrArgCysGluValGln 
130135140 
CACGGCATAGACGACAGCAGCGACGCCGTGGAGGTCAAGGTC731 
HisGlyIleAspAspSerSerAspAlaValGluValLysVal 
145150 
AAAGGGGTCGTCTTTCTCTACCGGGAGGGCTCTGCCCGCTAC773 
LysGlyValValPheLeuTyrArgGluGlySerAlaArgTyr 
155160165 
GCTTTCTCCTTCGCCCGGGCCCAGGAGGCCTGTGCCCGCATC815 
AlaPheSerPheAlaArgAlaGlnGluAlaCysAlaArgIle 
170175180 
GGAGCCCGCATCGCCACCCCGGAGCAGCTCTACGCTGCCTAC857 
GlyAlaArgIleAlaThrProGluGlnLeuTyrAlaAlaTyr 
185190195 
CTCGGGGGCTATGAGCAGTGCGATGCTGGCTGGCTGTCCGAC899 
LeuGlyGlyTyrGluGlnCysAspAlaGlyTrpLeuSerAsp 
200205210 
CAAACCGTGAGGTATCCCATCCAGACCCCACGGGAGGCCTGT941 
GlnThrValArgTyrProIleGlnThrProArgGluAlaCys 
215220 
TATGGAGACATGGATGGCTTCCCTGGGGTCCGGAACTATGGC983 
TyrGlyAspMetAspGlyPheProGlyValArgAsnTyrGly 
225230235 
CTGGTGGACCCGGATGACCTGTACGATATCTACTGCTATGCT1025 
LeuValAspProAspAspLeuTyrAspIleTyrCysTyrAla 
240245250 
GAAGACCTAAATGGAGAGCTGTTCCTGGGCGCCCCTCCAGAC1067 
GluAspLeuAsnGlyGluLeuPheLeuGlyAlaProProAsp 
255260265 
AACGTGACGCTGGAGGAGGCTACGGCATACTGCCGTGAGCGG1109 
AsnValThrLeuGluGluAlaThrAlaTyrCysArgGluArg 
270275280 
GGTGCAGAGATTGCTACCACGGGCCAGCTGTATGCAGCCTGG1151 
GlyAlaGluIleAlaThrThrGlyGlnLeuTyrAlaAlaTrp 
285290 
GATGGCGGCCTGGACCGCTGCAGCCCCGGCTGGCTGGCCGAT1193 
AspGlyGlyLeuAspArgCysSerProGlyTrpLeuAlaAsp 
295300305 
GGCAGCGTGCGCTACCCCATCGTCACGCCCAGCCAGCGCTGC1235 
GlySerValArgTyrProIleValThrProSerGlnArgCys 
310315320 
GGTGGGGGCCTGCCTGGCGTCAAGACTCTCTTCCTCTTCCCC1277 
GlyGlyGlyLeuProGlyValLysThrLeuPheLeuPhePro 
325330335 
AACCAGACCGGCTTCCCCAACAAGTACAGCCGCTTCAACGTC1319 
AsnGlnThrGlyPheProAsnLysTyrSerArgPheAsnVal 
340345350 
TACTGCTTCCGAGACTCTGGCCAGCCCTCCACCACCCCTGAG1361 
TyrCysPheArgAspSerGlyGlnProSerThrThrProGlu 
355360 
GCCTCTGACCAGCCTCTGACGGGCTGGAGGCCATTGTCACAG1403 
AlaSerAspGlnProLeuThrGlyTrpArgProLeuSerGln 
365370375 
TGACAGAGACCCTAGAGGAGCTCCACGTGCCGCGGGAAGCTGTGGAGAGC1453 
GAGTCCCGGGGAGCCATCTACTCCGTCCCCATTGTGGAGGATGGGGAGGT1503 
GCAAGGTCCCCCTCCA1519 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 329 residues 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: 
(A) DESCRIPTION: polypeptide 
(v) FRAGMENT TYPE: functional domains 
(ix) FEATURE: 
(A) NAME/KEY: rat aggrecan 
(x) PUBLICATION INFORMATION: 
(A) AUTHORS: Doege, K., Sasaki, M., Hori- 
gan, E., Hassell, J.R., and Yamada, Y. 
(B) TITLE: Complete primary structure of the rat 
cartilage proteoglycan core protein deduced from 
cDNA clones. 
(C) JOURNAL: J. Biol. Chem. 
(D) VOLUME: 262 
(F) PAGES: 17757-17767 
(G) DATE: 1987 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GluGluValProAspHisAspAsnSerLeuSerValSerIlePro 
51015 
GlnProSerProLeuLysAlaLeuLeuGlyThrSerLeuThrIle 
202530 
ProCysTyrPheIleAspProMetHisProValThrThrAlaPro 
354045 
SerThrAlaProLeuThrArgIleLysTrpSerArgValSerLys 
505560 
GluLysGluValValLeuLeuValAlaThrGluGlyGlnValArg 
657075 
ValAsnSerIleTyrGlnAspLysValSerLeuProAsnTyrPro 
808590 
AlaIleProSerAspAlaThrLeuGluIleGlnAsnLeuArgSer 
95100105 
AsnAspSerGlyIleTyrArgCysGluValMetHisGlyIleGlu 
110115120 
AspSerGluAlaThrLeuGluValIleValLysGlyIleValPhe 
125130135 
HisTyrArgAlaIleSerThrArgTyrThrLeuAspPheAspArg 
140145150 
AlaGlnArgAlaCysLeuGlnAsnSerAlaIleIleAlaThrPro 
155160165 
GluGlnLeuGlnAlaAlaTyrGluAspGlyPheHisGlnCysAsp 
170175180 
AlaGlyTrpLeuAlaAspGlnThrValArgTyrProIleHisThr 
185190195 
ProArgGluGlyCysTyrGlyAspLysAspGluPheProGlyVal 
200205210 
ArgThrTyrGlyIleArgAspThrAsnGluThrTyrAspValTyr 
215220225 
CysPheAlaGluGluMetGluGlyGluPheTyrAlaThrSerPro 
230235240 
GluLysPheThrPheGlnGluAlaAlaAsnGluCysArgThrVal 
245250255 
GlyAlaArgLeuAlaThrThrGlyGlnLeuTyrLeuAlaTrpGln 
260265270 
GlyGlyMetAspMetCysSerAlaGlyTrpLeuAlaAspArgSer 
275280285 
ValArgTyrProIleSerLysAlaArgProAsnCysGlyGlyAsn 
290295300 
LeuLeuGlyValArgThrValTyrLeuHisAlaAsnGlnThrGly 
305310315 
TyrProAspProSerSerArgTyrAspAlaIleCysTyrThr 
320325 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 333 residues 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: 
(A) DESCRIPTION: polypeptide 
(v) FRAGMENT TYPE: functional domains 
(ix) FEATURE: 
(A) NAME/KEY: rat neurocan 
(x) PUBLICATION INFORMATION: 
(A) AUTHORS: Rauch, U., Karthikeyan, L., 
Maurel, P., Margolis, R.U., and Margolis, 
R.K. 
(B) TITLE: Cloning and primary structure of neu- 
rocan, a developmentally regulated, aggregating 
chondroitin sulfate proteoglycan of brain. 
(C) JOURNAL: J. Biol. Chem. 
(D) VOLUME: 267 
(F) PAGES: 19536-19547 
(G) DATE: 1992 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
AspThrGlnAspThrThrThrThrGluLysGlyLeuHisMetLeu 
51015 
LysSerGlySerGlyProIleGlnAlaAlaLeuAlaGluLeuVal 
202530 
AlaLeuProCysPhePheThrLeuGlnProArgGlnSerProLeu 
354045 
GlyAspIleProArgIleLysTrpThrLysValGlnThrAlaSer 
505560 
GlyGlnArgGlnAspLeuProIleLeuValAlaLysAspAsnVal 
657075 
ValArgValAlaLysGlyTrpGlnGlyArgValSerLeuProAla 
808590 
TyrProArgHisArgAlaAsnAlaThrLeuLeuLeuGlyProLeu 
95100105 
ArgAlaSerAspSerGlyLeuTyrArgCysGlnValValLysGly 
110115120 
IleGluAspGluGlnAspLeuValThrLeuGluValThrGlyVal 
125130135 
ValPheHisTyrArgAlaAlaArgAspArgTyrAlaLeuThrPhe 
140145150 
AlaGluAlaGlnGluAlaCysHisLeuSerSerAlaThrIleAla 
155160165 
AlaProArgHisLeuAsnAlaAlaPheGluAspGlyPheAspAsn 
170175180 
CysAspAlaGlyTrpLeuSerAspArgThrValArgTyrProIle 
185190195 
ThrGlnSerArgProGlyCysTyrGlyAspArgSerSerLeuPro 
200205210 
GlyValArgSerTyrGlyArgArgAspProGlnGluLeuTyrAsp 
215220225 
ValTyrCysPheAlaArgGluLeuGlyGlyGluPheTyrValGly 
230235240 
ProAlaArgArgLeuThrLeuAlaGlyAlaArgAlaLeuCysGln 
245250255 
ArgGlnGlyAlaAlaLeuAlaSerValGlyGlnLeuHisLeuAla 
260265270 
TrpHisGluGlyLeuAspGlnCysAspProGlyTrpLeuAlaAsp 
275280285 
GlySerValArgTyrProIleGlnThrProArgArgArgCysGly 
290295300 
GlySerAlaProGlyValArgThrValTyrArgPheAlaAsnArg 
305310315 
ThrGlyPheProAlaProGlyAlaArgPheAspAlaTyrCysPhe 
320325330 
ArgAlaHis 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 328 residues 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: 
(A) DESCRIPTION: polypeptide 
(v) FRAGMENT TYPE: functional domains 
(ix) FEATURE: 
(A) NAME/KEY: human versican 
(x) PUBLICATION INFORMATION: 
(A) AUTHORS: Zimmermann, D.R., and Ruoslahti, E. 
(B) TITLE: Multiple domains of the large fibro- 
blast proteoglycan, versican. 
(C) JOURNAL: EMBO (Eur. Mol. Biol. Organ.) J. 
(D) VOLUME: 8 
(F) PAGES: 2975-2981 
(G) DATE: 1989 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
LeuHisLysValLysValGlyLysSerProProValArgGlySer 
51015 
LeuSerGlyLysValSerLeuProCysHisPheSerThrMetPro 
202530 
ThrLeuProProSerTyrAsnThrSerGluPheLeuArgIleLys 
354045 
TrpSerLysIleGluValAspLysAsnGlyLysAspLeuLysGlu 
505560 
ThrThrValLeuValAlaGlnAsnGlyAsnIleLysIleGlyGln 
657075 
AspTyrLysGlyArgValSerValProThrHisProGluAlaVal 
808590 
GlyAspAlaSerLeuThrValValLysLeuLeuAlaSerAspAla 
95100105 
GlyLeuTyrArgCysAspValMetTyrGlyIleGluAspThrGln 
110115120 
AspThrValSerLeuThrValAspGlyValValPheHisTyrArg 
125130135 
AlaAlaThrSerArgTyrThrLeuAsnPheGluAlaAlaGlnLys 
140145150 
AlaCysLeuAspValGlyAlaValIleAlaThrProGluGlnLeu 
155160165 
PheAlaAlaTyrGluAspGlyPheGluGlnCysAspAlaGlyTrp 
170175180 
LeuAlaAspGlnThrValArgTyrProIleArgAlaProArgVal 
185190195 
GlyCysTyrGlyAspLysMetGlyLysAlaGlyValArgThrTyr 
200205210 
GlyPheArgSerProGlnGluThrTyrAspValTyrCysTyrVal 
215220225 
AspHisLeuAspGlyAspPheHisLeuThrValProSerLysPhe 
230235240 
ThrPheGluGluAlaAlaLysGluCysGluAsnGlnAspAlaArg 
245250255 
LeuAlaThrValGlyGluLeuGlnAlaAlaTrpArgAsnGlyPhe 
260265270 
AspGlnCysAspTyrGlyTrpLeuSerAspAlaSerValArgHis 
275280285 
ProValThrValAlaArgAlaGlnCysGlyGlyGlyLeuLeuGly 
290295300 
ValArgThrLeuTyrArgPheGluAsnGlnThrGlyPheProPro 
305310315 
ProAspSerArgPheAspAlaTyrCysPheLysArgArg 
320325 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 326 residues 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: 
(A) DESCRIPTION: polypeptide 
(v) FRAGMENT TYPE: functional domains 
(ix) FEATURE: 
(A) NAME/KEY: rat link protein 
(x) PUBLICATION INFORMATION: 
(A) AUTHORS: Doege, K., Hassell, J.R., Ca- 
terson, B., and Yamada, Y. 
(B) TITLE: Link protein cDNA sequence reveals a 
tandemly repeated protein sequence. 
(C) JOURNAL: Proc. Natl. Acad. Sci. USA 
(D) VOLUME: 83 
(F) PAGES: 3761-3765 
(G) DATE: 1986 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
AspArgValIleHisIleGlnAlaGluAsnGlyProArgLeuLeu 
51015 
ValGluAlaGluGlnAlaLysValPheSerHisArgGlyGlyAsn 
202530 
ValThrLeuProCysLysPheTyrArgAspProThrAlaPheGly 
354045 
SerGlyIleHisLysIleArgIleLysTrpThrLysLeuThrSer 
505560 
AspTyrLeuArgGluValAspValPheValSerMetGlyTyrHis 
657075 
LysLysThrTyrGlyGlyTyrGlnGlyArgValPheLeuLysGly 
808590 
GlySerAspAsnAspAlaSerLeuIleIleThrAspLeuThrLeu 
95100105 
GluAspTyrGlyArgTyrLysCysGluValIleGluGlyLeuGlu 
110115120 
AspAspThrAlaValValAlaLeuGluLeuGlnGlyValValPhe 
125130135 
ProTyrPheProArgLeuGlyArgTyrAsnLeuAsnPheHisGlu 
140145150 
AlaArgGlnAlaCysLeuAspGlnAspAlaValIleAlaSerPhe 
155160165 
AspGlnLeuTyrAspAlaTrpArgGlyGlyLeuAspTrpCysAsn 
170175180 
AlaGlyTrpLeuSerAspGlySerValGlnTyrProIleThrLys 
185190195 
ProArgGluProCysGlyGlyGlnAsnThrValProGlyValArg 
200205210 
AsnTyrGlyPheTrpAspLysAspSerArgTyrAspValPheCys 
215220225 
PheThrSerAsnPheAsnGlyArgPheTyrTyrLeuIleHisPro 
230235240 
ThrLysLeuThrTyrAspGluAlaValGlnAlaCysLeuAsnAsp 
245250255 
GlyAlaGlnIleAlaLysValGlyGlnIlePheAlaAlaTrpLys 
260265270 
LeuLeuGlyTyrAspArgCysAspAlaGlyTrpLeuAlaAspGly 
275280285 
SerValArgTyrProIleSerArgProTrpArgArgCysSerPro 
290295300 
ThrGluAlaAlaValArgPheValGlyPheProAspLysLysHis 
305310315 
LysLeuTyrGlyValTyrCysPheArgAlaTyr 
320325 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 156 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: 
(A) DESCRIPTION: DNA encoding a polypeptide 
(v) FRAGMENT TYPE: partial sequence, PTR1 domain 
(vi) IMMEDIATE SOURCE: human brain 
(ix) FEATURE: 
(A) NAME/KEY: human BEHAB 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
GAGAGGGCTCTGCGCTATGCTTTCTCCTTTTCTGGGGCCCAG42 
GluArgAlaLeuArgTyrAlaPheSerPheSerGlyAlaGln 
510 
GAGGCTTGTGCCCGCATTGGAGCCCACATCGCCACCCCGGAG84 
GluAlaCysAlaArgIleGlyAlaHisIleAlaThrProGlu 
152025 
CAGCTCTATGCCGCCTACCTTGGGGGCTATGAGCAATGTGAT126 
GlnLeuTyrAlaAlaTyrLeuGlyGlyTyrGluGlnCysAsp 
303540 
GCTGGCTGGCTGTCGGATCAGACCGTGAGA156 
AlaGlyTrpLeuSerAspGlnThrValArg 
4550 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 371 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: 
(A) DESCRIPTION: polypeptide 
(v) FRAGMENT TYPE: entire sequence 
(vi) IMMEDIATE SOURCE: rat brain 
(ix) FEATURE: 
(A) NAME/KEY: rat BEHAB 
(D) OTHER INFORMATION: polypeptide encod- 
ed by (and set out under) SEQ ID NO 1 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
MetIleProLeuLeuLeuSerLeuLeuAlaAlaLeu 
510 
ValLeuThrGlnAlaProAlaAlaLeuAlaAspAspLeuLys 
152025 
GluAspSerSerGluAspArgAlaPheArgValArgIleGly 
303540 
AlaAlaGlnLeuArgGlyValLeuGlyGlyTrpValAlaIle 
4550 
ProCysHisValHisHisLeuArgProProProSerArgArg 
556065 
AlaAlaProGlyPheProArgValLysTrpThrPheLeuSer 
707580 
GlyAspArgGluValGluValLeuValAlaArgGlyLeuArg 
859095 
ValLysValAsnGluAlaTyrArgPheArgValAlaLeuPro 
100105110 
AlaTyrProAlaSerLeuThrAspValSerLeuValLeuSer 
115120 
GluLeuArgProAsnAspSerGlyValTyrArgCysGluVal 
125130135 
GlnHisGlyIleAspAspSerSerAspAlaValGluValLys 
140145150 
ValLysGlyValValPheLeuTyrArgGluGlySerAlaArg 
155160165 
TyrAlaPheSerPheAlaGlyAlaGlnGluAlaCysAlaArg 
170175180 
IleGlyAlaArgIleAlaThrProGluGlnLeuTyrAlaAla 
185190 
TyrLeuGlyGlyTyrGluGlnCysAspAlaGlyTrpLeuSer 
195200205 
AspGlnThrValArgTyrProIleGlnAsnProArgGluAla 
210215220 
CysTyrGlyAspMetAspGlyTyrProGlyValArgAsnTyr 
225230235 
GlyValValGlyProAspAspLeuTyrAspValTyrCysTyr 
240245250 
AlaGluAspLeuAsnGlyGluLeuPheLeuGlyAlaProPro 
255260 
GlyLysLeuThrTrpGluGluAlaArgAspTyrCysLeuGlu 
265270275 
ArgGlyAlaGlnIleAlaSerThrGlyGlnLeuTyrAlaAla 
280285290 
TrpAsnGlyGlyLeuAspArgCysSerProGlyTrpLeuAla 
295300305 
AspGlySerValArgTyrProIleIleThrProSerGlnArg 
310315320 
CysGlyGlyGlyLeuProGlyValLysThrLeuPheLeuPhe 
325330 
ProAsnGlnThrGlyPheProSerLysGlnAsnArgPheAsn 
335340345 
ValTyrCysPheArgAspSerAlaHisProSerAlaPheSer 
350355360 
GluProProAlaGlnProLeuMetAsp 
365370 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 378 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: 
(A) DESCRIPTION: polypeptide 
(v) FRAGMENT TYPE: entire sequence 
(vi) IMMEDIATE SOURCE: cat cortex 
(ix) FEATURE: 
(A) NAME/KEY: cat brain BEHAB 
(B) OTHER INFORMATION: polypeptide encod- 
ed by (and set out under) SEQ ID NO 2 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
MetAlaProLeuPheLeuProLeuLeuIleAlaLeuAlaLeu 
510 
AlaProGlyProThrAlaSerAlaAspValLeuGluGlyAsp 
152025 
SerSerGluAspArgAlaPheArgValArgIleSerGlyAsn 
303540 
AlaProLeuGlnGlyValLeuGlyGlyAlaLeuThrIleSer 
455055 
CysHisValHisTyrLeuArgProProProGlyArgArgAla 
606570 
ValLeuGlySerProArgValLysTrpThrPheLeuSerGly 
7580 
GlyArgGluAlaGluValLeuValAlaArgGlyLeuArgVal 
859095 
LysValSerGluAlaTyrArgPheArgValAlaLeuProAla 
100105110 
TyrProAlaSerLeuThrAspValSerLeuAlaLeuSerGlu 
115120125 
LeuArgProAsnAspSerGlyIleTyrArgCysGluValGln 
130135140 
HisGlyIleAspAspSerSerAspAlaValGluValLysVal 
145150 
LysGlyValValPheLeuTyrArgGluGlySerAlaArgTyr 
155160165 
AlaPheSerPheAlaArgAlaGlnGluAlaCysAlaArgIle 
170175180 
GlyAlaArgIleAlaThrProGluGlnLeuTyrAlaAlaTyr 
185190195 
LeuGlyGlyTyrGluGlnCysAspAlaGlyTrpLeuSerAsp 
200205210 
GlnThrValArgTyrProIleGlnThrProArgGluAlaCys 
215220 
TyrGlyAspMetAspGlyPheProGlyValArgAsnTyrGly 
225230235 
LeuValAspProAspAspLeuTyrAspIleTyrCysTyrAla 
240245250 
GluAspLeuAsnGlyGluLeuPheLeuGlyAlaProProAsp 
255260265 
AsnValThrLeuGluGluAlaThrAlaTyrCysArgGluArg 
270275280 
GlyAlaGluIleAlaThrThrGlyGlnLeuTyrAlaAlaTrp 
285290 
AspGlyGlyLeuAspArgCysSerProGlyTrpLeuAlaAsp 
295300305 
GlySerValArgTyrProIleValThrProSerGlnArgCys 
310315320 
GlyGlyGlyLeuProGlyValLysThrLeuPheLeuPhePro 
325330335 
AsnGlnThrGlyPheProAsnLysTyrSerArgPheAsnVal 
340345350 
TyrCysPheArgAspSerGlyGlnProSerThrThrProGlu 
355360 
AlaSerAspGlnProLeuThrGlyTrpArgProLeuSerGln 
365370375 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 52 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: 
(A) DESCRIPTION: polypeptide 
(v) FRAGMENT TYPE: partial sequence, PTR1 domain 
(vi) IMMEDIATE SOURCE: human brain 
(ix) FEATURE: 
(A) NAME/KEY: human BEHAB 
(D) OTHER INFORMATION: polypeptide encod- 
ed by (and set out under) SEQ ID NO 7 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
GluArgAlaLeuArgTyrAlaPheSerPheSerGlyAlaGln 
510 
GluAlaCysAlaArgIleGlyAlaHisIleAlaThrProGlu 
152025 
GlnLeuTyrAlaAlaTyrLeuGlyGlyTyrGluGlnCysAsp 
303540 
AlaGlyTrpLeuSerAspGlnThrValArg 
4550 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 residues 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: 
(A) DESCRIPTION: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: conserved PTR1 and PTR 2 region 
(D) OTHER INFORMATION: found in PTR family's HA- 
binding protein domain; two copies found in BEHAB; 
residue 7 is Ala or Ser; residue 9 is Gln or Gly; 
residue 10 is Thr or Ser 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
CysAspAlaGlyTrpLeuXaaAspXaaXaaValArgTyrProIle 
51015 
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