Tumor necrosis factor receptor associated factor 6 (TRAF6)

The invention provides methods and compositions relating to a novel tumor necrosis factor receptor associated factor number six (TRAF6) protein, which transcriptionally activates Nuclear Factor .kappa.B. The invention provides isolated TRAF6 hybridization probes and primers capable of hybridizing with the disclosed TRAF6 gene, nucleic acids encoding the subject TRAF6 proteins, methods of making the subject TRAF6 proteins, and methods of using the subject compositions in diagnosis and drug screening.

INTRODUCTION 
1. Field of the Invention 
The field of this invention is a class of human proteins involved in 
transcription and immuno-regulation. 
2. Background 
Nuclear factor .kappa.B (NF-.kappa.B) is a homo- or heterodimer of members 
of the Rel family of transcriptional activators that is involved in the 
inducible expression of a wide variety of important cellular genes 
including numerous cytokines, cytokine receptors, major histocompatibility 
antigens, serum amyloid A protein, etc. as well as many viral genes 
including genes of HIV, SV40, cytomegalovirus, etc. 
Several tumor necrosis factor receptor-associated factor (TRAF) proteins 
have been identified and shown to be involved in the signaling of various 
cellular responses including cytotoxicity, anti-viral activity, 
immuno-regulatory activities and the transcriptional regulation of a 
number of genes. 
Accordingly, the ability to exogenously modulate the activity of 
NF-.kappa.B and/or TRAF proteins would yield therapeutic application for 
numerous clinical indications. In addition, components of such pathways 
would provide valuable target reagents for automated, cost-effective, high 
throughput drug screening assays and hence would have immediate 
application in domestic and international pharmaceutical and biotechnology 
drug development programs. The present invention provides novel TRAF 
proteins which regulate NF-.kappa.B expression, their use in drug screens, 
and nucleic acids encoding the same. 
RELEVANT LITERATURE 
Rothe et al. (1994) Cell 78, 681-692 report that NF-.kappa.B expression can 
be mediated by the 75-80 kDa TNF receptor (TNF-R2), that a short region of 
the 78 amino acids at the C-terminus of the cytoplasmic domain of TNF-R2 
is required for signaling NF-.kappa.B activation, and that this region 
binds to closely related putative effectors, TRAF1 and TRAF2, see also, 
Hsu et al. (1995) Cell 81, 495 and Rothe et al., pending U.S. patent 
application Ser. No. 08/446,915. A third distinct TRAF, TRAF3, has been 
reported by Huet al. (1994) J. Biol Chem 269, 30069; Cheng et al. (1995) 
Science 267, 1494-1498; Mosialos et al. (1995) Cell 80, 389; and Sato et 
al. (1995) FEBS Letters, 358, 113. Rothe et al. (1995) Science 269, 
1424-1427 report TRAF2- (but not TRAF1- or 3-) mediated activation of 
NF-.kappa.B by TNF-R2 and CD40. 
SUMMARY OF THE INVENTION 
The invention provides methods and compositions relating to a novel tumor 
necrosis factor receptor associated factor number six (TRAF6) protein and 
gene. The subject TRAF6 proteins are encoded by cDNAs which hybridizes 
with SEQ ID NO:01 under high stringency conditions; specifically bind a 
natural intracellular TRAF6 binding target and comprise the amino sequence 
of SEQ ID NO:2 or fragment thereof sufficient to specifically bind a 
natural intracellular TRAF6 binding target. 
The invention also provides isolated TRAF6 hybridization probes and primers 
capable of hybridizing with the disclosed TRAF6 cDNA, nucleic acids 
encoding the subject TRAF6 proteins, methods of making the subject TRAF6 
proteins, and methods of using the subject compositions in diagnosis (e.g. 
genetic hybridization screens for TRAF6 gene mutations), and in the 
biopharmaceutical industry (e.g. reagents for screening chemical libraries 
for lead compounds for a pharmacological agent useful in the diagnosis or 
treatment of disease associated with immune regulation).

DETAILED DESCRIPTION OF THE INVENTION 
The nucleotide sequence of a natural cDNA encoding human TRAF6 is shown as 
SEQ ID NO:1 and the full conceptual translate shown as SEQ ID NO:2. The 
TRAF6 proteins of the invention include incomplete translates of SEQ ID 
NO:1 and deletion mutants of SEQ ID NO:2, which translates and deletions 
mutants have TRAF6-specific activity. 
TRAF6-specific activity or function may be determined by convenient in 
vitro, cell-based, or in vivo assays. Preferred proteins are capable of 
modulating NF-.kappa.B activation. Such activity or function may be 
demonstrated in cell culture (e.g. cell transfections) or in animals (e.g. 
in vivo gene therapy, transgenics). TRAF6 specific function can also be 
demonstrated by specific binding to a TRAF6 specific binding target, 
including natural binding targets such as TRAF6 (dimerization) and 
nonnatural targets such as TRAF6-specific antibodies. Finally, specific 
function can be assayed immunologically by the ability of the subject 
protein to elicit a TRAF6 specific antibody in a rodent or rabbit. 
TRAF6-specificity of the binding agent may be shown at a populational 
level by binding equilibrium constants (usually at least about 10.sup.7 
M.sup.-1, preferably at least about 10.sup.8 M.sup.-1, more preferably at 
least about 10.sup.9 M.sup.-1). A wide variety of cell-based and cell-free 
assays may be used to demonstrate TRAF6-specific binding; preferred are 
rapid in vitro, cell-free assays such as mediating or inhibiting 
TRAF6-protein (e.g. TRAF6--TRAF6, TRAF2 or TRAF3) binding, immunoassays, 
etc. In any event, TRAF6 specificity necessarily distinguishes the subject 
TRAF6 protein from TRAF1-5. 
The claimed TRAF6 proteins are isolated or pure and are typically 
recombinantly produced. An "isolated" protein for example, is 
unaccompanied by at least some of the material with which it is associated 
in its natural state, preferably constituting at least about 0.5%, and 
more preferably at least about 5% by weight of the total protein in a 
given sample and a pure protein constitutes at least about 90%, and 
preferably at least about 99% by weight of the total protein in a given 
sample. A wide variety of molecular and biochemical methods are available 
for generating, expressing and purifying the subject compositions, see 
e.g. Molecular Cloning, A Laboratory Manual (Sambrook, et al. Cold Spring 
Harbor Laboratory), Current Protocols in Molecular Biology (Eds. Ausubel, 
et al., Greene Publ. Assoc., Wiley-Interscience, NY) or that are otherwise 
known in the art. 
The invention provides TRAF6-specific binding agents, methods of 
identifying and making such agents, and their use in diagnosis, therapy 
and pharmaceutical development. For example, TRAF6-specific agents are 
useful in a variety of diagnostic and therapeutic applications, especially 
where disease or disease prognosis is associated with improper utilization 
of a pathway involving TRAF6, e.g. NF-.kappa.B activation. Novel 
TRAF6-specific binding agents include TRAF6-specific antibodies and other 
natural intracellular binding agents identified with assays such as one- 
and two-hybrid screens, non-natural intracellular binding agents 
identified in screens of chemical libraries such as describe below, etc. 
The invention also provides nucleic acids encoding the subject proteins, 
which nucleic acids may be part of TRAF6-expression vectors and may be 
incorporated into recombinant cells for expression and screening, 
transgenic animals for functional studies (e.g. the efficacy of candidate 
drugs for disease associated with TRAF6-mediated signal transduction), 
etc., and nucleic acid hybridization probes and replication/amplification 
primers having a TRAF6 cDNA specific sequence contained in SEQ ID NO:1 and 
sufficient to effect specific hybridization thereto (i.e. will 
specifically hybridize with TRAF6 cDNA (SEQ ID NO:1) in the presence of 
TRAF1-5 cDNA). Demonstrating specific hybridization generally requires 
high-stringency conditions, for example, hybridizing in a buffer 
comprising 30% formamide in 5.times.SSPE (0.18M NaCl, 0.01M NaPO.sub.4, pH 
7.7, 0.001M EDTA) buffer at a temperature of 42.degree. C. and remaining 
bound when subject to washing at 42.degree. C. with 0.2.times.SSPE; 
preferably hybridizing in a buffer comprising 50% formamide in 
5.times.SSPE buffer at a temperature of 42.degree. C. and remaining bound 
when subject to washing at 42.degree. C. with 0.2.times.SSPE buffer at 
42.degree. C. In any event, TRAF6 specific hybridization probes and 
primers necessarily distinguish TRAF6 cDNA from cDNA's encoding TRAF1-5. 
TRAF6 cDNA homologs can also be characterized by BLASTX (Altschul et al. 
(1990) Basic Local Alignment Search Tool, J Mol Biol 215, 403-410) 
probability scores. Using this nucleic acid sequence search program 
BLASTX, complete coding region TRAF6 cDNA homologs provide a Probability 
P(N) score of less than 1.0 e.sup.-200. 
The subject nucleic acids are isolated, i.e. unaccompanied by at least some 
of the material with which it is associated in its natural state, 
preferably constituting at least about 0.5%, preferably at least about 5% 
by weight of total nucleic acid present in a given fraction. The subject 
nucleic acids find a wide variety of applications including use as 
translatable transcripts, hybridization probes, PCR primers, diagnostic 
nucleic acids, etc.; use in detecting the presence of TRAF6 genes and gene 
transcripts and in detecting or amplifying nucleic acids encoding 
additional TRAF6 homologs and structural analogs. When used as expression 
constructs, the nucleic acids are usually recombinant, meaning they 
comprise a sequence joined to a nucleotide other than that which it is 
joined to on a natural chromosome. The subject nucleic acids may be 
contained within vectors, cells or organisms. 
In diagnosis, TRAF6 hybridization probes and/or primers find use in 
identifying wild-type and mutant TRAF6 alleles in clinical and laboratory 
samples. Mutant alleles are used to generate allele-specific 
oligonucleotide (ASO) probes for high-throughput clinical diagnoses. 
The invention provides efficient methods of identifying agents, compounds 
or lead compounds for agents active at the level of a TRAF6 modulatable 
cellular function. Generally, these screening methods involve assaying for 
compounds which modulate a TRAF6 interaction with a natural TRAF6 binding 
target. The methods are amenable to automated, cost-effective high 
throughput screening of chemical libraries for lead compounds. Identified 
reagents find use in the pharmaceutical industries for animal and human 
trials; for example, the reagents may be derivatized and rescreened in in 
vitro and in vivo assays to optimize activity and minimize toxicity for 
pharmaceutical development. Target indications may include infection, 
genetic disease, cell growth and regulatory disfunction, such as 
neoplasia, inflammation, hypersensitivity, etc. 
A wide variety of assays for binding agents are provided including 
protein--protein binding assays, immunoassays, cell based assays, etc. A 
preferred assay is a high-through put in vitro binding assay. Here, the 
TRAF6 compositions may be part of a fusion product with another peptide or 
polypeptide, e.g. a polypeptide that is capable of providing or enhancing 
protein--protein binding, stability under assay conditions, or a tag for 
detection or anchoring, etc. The assay mixtures comprise a natural 
intracellular TRAF6 binding target such as TRAF2, TRAF3 or TRAF6. While 
native binding targets may be used, it is frequently preferred to use 
portions (e.g. peptides, nucleic acid fragments) thereof so long as the 
portion provides binding affinity and avidity to the subject TRAF6 
conveniently measurable in the assay. The assay mixture also comprises a 
candidate pharmacological agent. Candidate agents encompass numerous 
chemical classes, though typically they are organic compounds; preferably 
small organic compounds and are obtained from a wide variety of sources 
including libraries of synthetic or natural compounds. A variety of other 
reagents may also be included in the mixture. These include reagents like 
salts, buffers, neutral proteins, e.g. albumin, detergents, etc. which may 
be used to facilitate optimal binding and/or reduce non-specific or 
background interactions, etc. Also, reagents that otherwise improve the 
efficiency of the assay, such as protease inhibitors, nuclease inhibitors, 
antimicrobial agents, etc. may be used. 
The resultant mixture is incubated under conditions whereby, but for the 
presence of the candidate pharmacological agent, the TRAF6 specifically 
binds the cellular binding target, portion or analog with a reference 
binding affinity. The mixture components can be added in any order that 
provides for the requisite bindings and incubations may be performed at 
any temperature which facilitates optimal binding, typically between 
4.degree. and 40.degree. C., more commonly between 15.degree. and 
40.degree. C. Incubation periods are likewise selected for optimal binding 
but also minimized to facilitate rapid, high-throughput screening, and are 
typically between 1 and 10 hours, preferably less than 5 hours, more 
preferably less than 2 hours. 
After incubation, the agent-biased binding between the TRAF6 and one or 
more binding targets is detected by any convenient way. For cell-free 
binding type assays, a separation step is often used to separate bound 
from unbound components. Separation may be effected by precipitation (e.g. 
TCA precipitation, immunoprecipitation, etc.), immobilization (e.g on a 
solid substrate), etc., followed by washing by, for examples, membrane 
filtration (e.g. Whatman's P-81 ion exchange paper, Polyfiltronic's 
hydrophobic GFC membrane, etc.), gel chromatography (e.g. gel filtration, 
affinity, etc.). In addition, one of the components usually comprises or 
is coupled to a label. A wide variety of labels may be 
employed--essentially any label that provides for detection of bound 
protein. The label may provide for direct detection as radioactivity, 
luminescence, optical or electron density, etc. or indirect detection such 
as an epitope tag, an enzyme, etc. A variety of methods may be used to 
detect the label depending on the nature of the label and other assay 
components. For example, the label may be detected bound to the solid 
substrate or a portion of the bound complex containing the label may be 
separated from the solid substrate, and thereafter the label detected. 
Labels may be directly detected through optical or electron density, 
radiative emissions, nonradiative energy transfers, etc. or indirectly 
detected with antibody conjugates, etc. For example, in the case of 
radioactive labels, emissions may be detected directly, e.g. with particle 
counters or indirectly, e.g. with scintillation cocktails and counters. 
A difference in the binding affinity of the TRAF6 protein to the target in 
the absence of the agent as compared with the binding affinity in the 
presence of the agent indicates that the agent modulates the binding of 
the TRAF6 protein to the TRAF6 binding target. Analogously, in the 
cell-based transcription assay also described below, a difference in the 
TRAF6 transcriptional induction in the presence and absence of an agent 
indicates the agent modulates TRAF6-induced transcription. A difference, 
as used herein, is statistically significant and preferably represents at 
least a 50%, more preferably at least a 90% difference. 
The following experiments and examples are offered by way of illustration 
and not by way of limitation. 
EXPERIMENTAL 
The human TRAF6 cDNA of SEQ ID NO:1 was cloned from human spleen cell and 
human umbilical vein endothelial cell cDNA libraries by high stringency 
hybridization: hybridization in 40% formamide, 5% Dextran sulfate, 0.5% 
SDS, 5.times.SSPE at 42.degree. C. followed by washes in 2.times.SSPE, 
0.1% SDS at 25.degree. C. and in 0.1.times.SSPE, 0.1% SDS at 42.degree. 
C.; using TRAF oligonucleotide probes. 
The resultant cDNA (SEQ ID NO:1) encodes human TRAF6 (SEQ ID NO:2). In 
cotransfection experiments, TRAF6 was shown to activate an 
NF-.kappa.B-dependent reporter gene, see FIG. 1. 
A yeast two-hybrid system was used to identify TRAF2, TRAF3 and TRAF6 as an 
intracellular binding targets of the TRAF6 protein of SEQ ID NO:2. A 
transfection based co-immunoprecipitation assay was also used to identify 
intracellular binding targets of the TRAF6 protein of SEQ ID NO:2. 
Briefly, FLAG-tagged TRAF1, 2, 3, 4 and 6 were cotransfected with 
HA-tagged TRAF6. Lysates were immunoprecipitated with anti-HA antibody and 
protein A glass beads. Western blot analysis using an anti-FLAG antibody 
revealed TRAF6--TRAF6 and TRAF6-TRAF3 complexes. 
Deletion mutagenesis of TRAF6 indicate that residues 115-522 are sufficient 
to mediate activation of NF-.kappa.B, see FIG. 2. In contrast, continuing 
the 5' deletion to the second Zn finger domain abolished activity; as did 
5' deletions through all five Zn finger domains, and deletions through the 
C domain. Similarly, an internal delection of the Zn finger domains 
abolished activity. 
EXAMPLES 
1. Protocol for High-Throughput Human TRAF6--TRAF6 Binding Assay. 
A. Reagents: 
Neutralite Avidin: 20 .mu.g/ml in PBS. 
Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hour at room temperature. 
Assay Buffer: 100 mM KCl, 20 mM HEPES pH 7.6, 0.25 mM EDTA, 1% glycerol, 
0.5% NP-40, 50 mM .beta.-mercaptoethanol, 1 mg/ml BSA, cocktail of 
protease inhibitors. 
.sup.33 P human TRAF6 (SEQUENCE ID NO:2, residues 1-522) 10.times.stock: 
10.sup.-8 -10.sup.-6 M unlabeled human TRAF6 supplemented with 
200,000-250,000 cpm of labeled human TRAF6 (Beckman counter). Place in the 
4.degree. C. microfridge during screening. 
Protease inhibitor cocktail (1000X): 10 mg Trypsin Inhibitor (BMB #109894), 
10 mg Aprotinin (BMB #236624), 25 mg Benzamidine (Sigma #B-6506), 25 mg 
Leupeptin (BMB #1017128), 10 mg APMSF (BMB #917575), and 2 mM NaVo.sub.3 
(Sigma #S-6508) in 10 ml of PBS. 
.DELTA.TRAF6: 10.sup.-8 -10.sup.-5 M biotinylated truncated .DELTA.TRAF6 
(SEQUENCE ID NO:2, residues 115-522) in PBS. 
B. Preparation of assay plates: 
Coat with 120 .mu.l of stock N-Avidin per well overnight at 4.degree. C. 
Wash 2 times with 200 .mu.l PBS. 
Block with 150 .mu.l of blocking buffer. 
Wash 2 times with 200 .mu.l PBS. 
C. Assay: 
Add 40 .mu.l assay buffer/well. 
Add 10 .mu.l compound or extract. 
Add 10 .mu.l .sup.33 P-human TRAF6 (20,000-25,000 cpm/0.1-10 
pmoles/well=10.sup.-9 -10.sup.-7 M final concentration). 
Shake at 25.degree. C. for 15 minutes. 
Incubate additional 45 minutes at 25.degree. C. 
Add 40 .mu.l biotinylated truncated .DELTA.TRAF6 (0.1-10 pmoles/40 .mu.l in 
assay buffer) 
Incubate 1 hour at room temperature. 
Stop the reaction by washing 4 times with 200 .mu.l PBS. 
Add 150 .mu.l scintillation cocktail. 
Count in Topcount. 
D. Controls for all assays (located on each plate): 
a. Non-specific binding 
b. Soluble (non-biotinylated truncated .DELTA.TRAF6) at 80% inhibition. 
2. Protocol for NF-.kappa.B-Dependent Reporter Gene Assay 
293 cells are transiently co-transfected with an E-selectin-luciferase 
reporter gene plasmid (Schindler et al. (1994) Mol Cell Biol 14, 5820) and 
TRAF6 expression vectors containing the TRAF6 coding region (see, SEQUENCE 
ID NO:1) produced as described for TRAF1, 2 and 3 in Rothe et al. (1995) 
Science 269, 1424. 
Control cells are transiently co-transfected with a CMV promoter luciferase 
reporter gene plasmid and/or TRAF2 expression vectors as described supra. 
The transfected cells are incubated 24 hours in the presence of the 
candidate compound or extract and then the cells harvested and luciferase 
activities determined and normalized on the basis of .beta.-galactosidase 
expression, as described in FIG. 3B of Rothe et al. (1995) Science 269, 
1424. 
All publications and patent applications cited in this specification are 
herein incorporated by reference as if each individual publication or 
patent application were specifically and individually indicated to be 
incorporated by reference. Although the foregoing invention has been 
described in some detail by way of illustration and example for purposes 
of clarity of understanding, it will be readily apparent to those of 
ordinary skill in the art in light of the teachings of this invention that 
certain changes and modifications may be made thereto without departing 
from the spirit or scope of the appended claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 2 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2248 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 230..1795 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GGCACGAGCCGCAGCTGGGGCTTGGCCTGCGGGCGGCCAGCGAAGGTGGCGAAGGCTCCC60 
ACTGGATCCAGAGTTTGCCGTCCAAGCAGCCTCGTCTCGGCGCGCAGTGTCTGTGTCCGT120 
CCTCTACCAGCGCCTTGGCTGAGCGGAGTCGTGCGGTTGGTGGGGGAGCCCTGCCCTCCT180 
GGTTCGGCCTCCCCGCGCACTAGAACGAGCAAGTGATAATCAAGTTACTATGAGT235 
MetSer 
CTGCTAAACTGTGAAAACAGCTGTGGATCCAGCCAGTCTGAAAGTGAC283 
LeuLeuAsnCysGluAsnSerCysGlySerSerGlnSerGluSerAsp 
51015 
TGCTGTGTGGCCATGGCCAGCTCCTGTAGCGCTGTAACAAAAGATGAT331 
CysCysValAlaMetAlaSerSerCysSerAlaValThrLysAspAsp 
202530 
AGTGTGGGTGGAACTGCCAGCACGGGGAACCTCTCCAGCTCATTTATG379 
SerValGlyGlyThrAlaSerThrGlyAsnLeuSerSerSerPheMet 
35404550 
GAGGAGATCCAGGGATATGATGTAGAGTTTGACCCACCCCTGGAAAGC427 
GluGluIleGlnGlyTyrAspValGluPheAspProProLeuGluSer 
556065 
AAGTATGAATGCCCCATCTGCTTGATGGCATTACGAGAAGCAGTGCAA475 
LysTyrGluCysProIleCysLeuMetAlaLeuArgGluAlaValGln 
707580 
ACGCCATGCGGCCATAGGTTCTGCAAAGCCTGCATCATAAAATCAATA523 
ThrProCysGlyHisArgPheCysLysAlaCysIleIleLysSerIle 
859095 
AGGGATGCAGGTCACAAATGTCCAGTTGACAATGAAATACTGCTGGAA571 
ArgAspAlaGlyHisLysCysProValAspAsnGluIleLeuLeuGlu 
100105110 
AATCAACTATTTCCAGACAATTTTGCAAAACGTGAGATTCTTTCTCTG619 
AsnGlnLeuPheProAspAsnPheAlaLysArgGluIleLeuSerLeu 
115120125130 
ATGGTGAAATGTCCAAATGAAGGTTGTTTGCACAAGATGGAACTGAGA667 
MetValLysCysProAsnGluGlyCysLeuHisLysMetGluLeuArg 
135140145 
CATCTTGAGGATCATCAAGCACATTGTGAGTTTGCTCTTATGGATTGT715 
HisLeuGluAspHisGlnAlaHisCysGluPheAlaLeuMetAspCys 
150155160 
CCCCAATGCCAGCGTCCCTTCCAAAAATTCCATATTAATATTCACATT763 
ProGlnCysGlnArgProPheGlnLysPheHisIleAsnIleHisIle 
165170175 
CTGAAGGATTGTCCAAGGAGACAGGTTTCTTGTGACAACTGTGCTGCA811 
LeuLysAspCysProArgArgGlnValSerCysAspAsnCysAlaAla 
180185190 
TCAATGGCATTTGAAGATAAAGAGATCCATGACCAGAACTGTCCTTTG859 
SerMetAlaPheGluAspLysGluIleHisAspGlnAsnCysProLeu 
195200205210 
GCAAATGTCATCTGTGAATACTGCAATACTATACTCATCAGAGAACAG907 
AlaAsnValIleCysGluTyrCysAsnThrIleLeuIleArgGluGln 
215220225 
ATGCCTAATCATTATGATCTAGACTGCCCTACAGCCCCAATTCCATGC955 
MetProAsnHisTyrAspLeuAspCysProThrAlaProIleProCys 
230235240 
ACATTCAGTACTTTTGGTTGCCATGAAAAGATGCAGAGGAATCACTTG1003 
ThrPheSerThrPheGlyCysHisGluLysMetGlnArgAsnHisLeu 
245250255 
GCACGCCACCTACAAGAGAACACCCAGTCACACATGAGAATGTTGGCC1051 
AlaArgHisLeuGlnGluAsnThrGlnSerHisMetArgMetLeuAla 
260265270 
CAGGCTGTTCATAGTTTGAGCGTTATACCCGACTCTGGGTATATCTCA1099 
GlnAlaValHisSerLeuSerValIleProAspSerGlyTyrIleSer 
275280285290 
GAGGTCCGGAATTTCCAGGAAACTATTCACCAGTTAGAGGGTCGCCTT1147 
GluValArgAsnPheGlnGluThrIleHisGlnLeuGluGlyArgLeu 
295300305 
GTAAGACAAGACCATCAAATCCGGGAGCTGACTGCTAAAATGGAAACT1195 
ValArgGlnAspHisGlnIleArgGluLeuThrAlaLysMetGluThr 
310315320 
CAGAGTATGTATGTAAGTGAGCTCAAACGAACCATTCGAACCCTTGAG1243 
GlnSerMetTyrValSerGluLeuLysArgThrIleArgThrLeuGlu 
325330335 
GACAAAGTTGCTGAAATCGAAGCACAGCAGTGCAATGGAATTTATATT1291 
AspLysValAlaGluIleGluAlaGlnGlnCysAsnGlyIleTyrIle 
340345350 
TGGAAGATTGGCAACTTTGGAATGCATTTGAAATGTCAAGAAGAGGAG1339 
TrpLysIleGlyAsnPheGlyMetHisLeuLysCysGlnGluGluGlu 
355360365370 
AAACCTGTTGTGATTCATAGCCCTGGATTCTACACTGGCAAACCCGGG1387 
LysProValValIleHisSerProGlyPheTyrThrGlyLysProGly 
375380385 
TACAAACTGTGCATGCGCTTGCACCTTCAGTTACCGACTGCTCAGCGC1435 
TyrLysLeuCysMetArgLeuHisLeuGlnLeuProThrAlaGlnArg 
390395400 
TGTGCAAACTATATATCCCTTTTTGTCCACACAATGCAAGGAGAATAT1483 
CysAlaAsnTyrIleSerLeuPheValHisThrMetGlnGlyGluTyr 
405410415 
GACAGCCACCTCCCTTGGCCCTTCCAGGGTACAATACGCCTTACAATT1531 
AspSerHisLeuProTrpProPheGlnGlyThrIleArgLeuThrIle 
420425430 
CTTGATCAGTCTGAAGCACCTGTAAGGCAAAACCACGAAGAGATAATG1579 
LeuAspGlnSerGluAlaProValArgGlnAsnHisGluGluIleMet 
435440445450 
GATGCCAAACCAGAGCTGCTTGCTTTCCAGCGACCCACAATCCCACGG1627 
AspAlaLysProGluLeuLeuAlaPheGlnArgProThrIleProArg 
455460465 
AACCCAAAAGGTTTTGGCTATGTAACTTTTATGCATCTGGAAGCCCTA1675 
AsnProLysGlyPheGlyTyrValThrPheMetHisLeuGluAlaLeu 
470475480 
AGACAAAGAACTTTCATTAAGGATGACACATTATTAGTGCGCTGTGAG1723 
ArgGlnArgThrPheIleLysAspAspThrLeuLeuValArgCysGlu 
485490495 
GTCTCCACCCGCTTTGACATGGGTAGCCTTCGGAGGGAGGGTTTTCAG1771 
ValSerThrArgPheAspMetGlySerLeuArgArgGluGlyPheGln 
500505510 
CCACGAAGTACTGATGCAGGGGTATAGCTTGCCCTCACTTGCTCAAAAACAACT1825 
ProArgSerThrAspAlaGlyVal 
515520 
ACCTGGAGAAAACAGTGCCTTTCCTTGCCCTGTTCTCAATAACATGCAAACAAACAAGCC1885 
ACGGGAAATATGTAATATCTACTAGTGAGTGTTGTTAGAGAGGTCACTTACTATTTCTTC1945 
CTGTTACAAATGATCTGAGGCAGTTTTTTCCTGGGAATCCACACGTTCCATGCTTTTTCA2005 
GAAATGTTAGGCCTGAAGTGCCTGTGGCATGTTGCAGCAGCTATTTTGCCAGTTAGTATA2065 
CCTCTTTGTTGTACTTTCTTGGGCTTTTGCTCTGGTGTATTTTATTGTCAGAAAGTCCAG2125 
ACTCAAGAGTACTAAACTTTTAATAATAATGGATTTTCCTTAAAACTTCAGTCTTTTTGT2185 
AGTATTATATGTAATATATTAAAAGTGAAAATCACTACCGCCTTGAAAAAAAAAAAAAAA2245 
AAA2248 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 522 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetSerLeuLeuAsnCysGluAsnSerCysGlySerSerGlnSerGlu 
151015 
SerAspCysCysValAlaMetAlaSerSerCysSerAlaValThrLys 
202530 
AspAspSerValGlyGlyThrAlaSerThrGlyAsnLeuSerSerSer 
354045 
PheMetGluGluIleGlnGlyTyrAspValGluPheAspProProLeu 
505560 
GluSerLysTyrGluCysProIleCysLeuMetAlaLeuArgGluAla 
65707580 
ValGlnThrProCysGlyHisArgPheCysLysAlaCysIleIleLys 
859095 
SerIleArgAspAlaGlyHisLysCysProValAspAsnGluIleLeu 
100105110 
LeuGluAsnGlnLeuPheProAspAsnPheAlaLysArgGluIleLeu 
115120125 
SerLeuMetValLysCysProAsnGluGlyCysLeuHisLysMetGlu 
130135140 
LeuArgHisLeuGluAspHisGlnAlaHisCysGluPheAlaLeuMet 
145150155160 
AspCysProGlnCysGlnArgProPheGlnLysPheHisIleAsnIle 
165170175 
HisIleLeuLysAspCysProArgArgGlnValSerCysAspAsnCys 
180185190 
AlaAlaSerMetAlaPheGluAspLysGluIleHisAspGlnAsnCys 
195200205 
ProLeuAlaAsnValIleCysGluTyrCysAsnThrIleLeuIleArg 
210215220 
GluGlnMetProAsnHisTyrAspLeuAspCysProThrAlaProIle 
225230235240 
ProCysThrPheSerThrPheGlyCysHisGluLysMetGlnArgAsn 
245250255 
HisLeuAlaArgHisLeuGlnGluAsnThrGlnSerHisMetArgMet 
260265270 
LeuAlaGlnAlaValHisSerLeuSerValIleProAspSerGlyTyr 
275280285 
IleSerGluValArgAsnPheGlnGluThrIleHisGlnLeuGluGly 
290295300 
ArgLeuValArgGlnAspHisGlnIleArgGluLeuThrAlaLysMet 
305310315320 
GluThrGlnSerMetTyrValSerGluLeuLysArgThrIleArgThr 
325330335 
LeuGluAspLysValAlaGluIleGluAlaGlnGlnCysAsnGlyIle 
340345350 
TyrIleTrpLysIleGlyAsnPheGlyMetHisLeuLysCysGlnGlu 
355360365 
GluGluLysProValValIleHisSerProGlyPheTyrThrGlyLys 
370375380 
ProGlyTyrLysLeuCysMetArgLeuHisLeuGlnLeuProThrAla 
385390395400 
GlnArgCysAlaAsnTyrIleSerLeuPheValHisThrMetGlnGly 
405410415 
GluTyrAspSerHisLeuProTrpProPheGlnGlyThrIleArgLeu 
420425430 
ThrIleLeuAspGlnSerGluAlaProValArgGlnAsnHisGluGlu 
435440445 
IleMetAspAlaLysProGluLeuLeuAlaPheGlnArgProThrIle 
450455460 
ProArgAsnProLysGlyPheGlyTyrValThrPheMetHisLeuGlu 
465470475480 
AlaLeuArgGlnArgThrPheIleLysAspAspThrLeuLeuValArg 
485490495 
CysGluValSerThrArgPheAspMetGlySerLeuArgArgGluGly 
500505510 
PheGlnProArgSerThrAspAlaGlyVal 
515520 
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