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WO1997010843A1 - Modified p53 constructs and uses therefor - Google Patents

Modified p53 constructs and uses therefor Download PDF

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Publication number
WO1997010843A1
WO1997010843A1 PCT/US1996/015188 US9615188W WO9710843A1 WO 1997010843 A1 WO1997010843 A1 WO 1997010843A1 US 9615188 W US9615188 W US 9615188W WO 9710843 A1 WO9710843 A1 WO 9710843A1
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ser
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arg
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PCT/US1996/015188
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French (fr)
Inventor
Thanos D. Halazonetis
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The Wistar Institute Of Anatomy And Biology
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Priority claimed from US08/697,221 external-priority patent/US5847083A/en
Application filed by The Wistar Institute Of Anatomy And Biology filed Critical The Wistar Institute Of Anatomy And Biology
Priority to AU72429/96A priority Critical patent/AU7242996A/en
Publication of WO1997010843A1 publication Critical patent/WO1997010843A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4746—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used p53
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates generally to the field of oncoproteins, and more specifically to p53.
  • Wild-type (wt) p53 is a sequence-specific DNA binding protein found in humans and other mammals, which has tumor suppressor function [See, e.g., Harris, Science. 262: 1980-1981 (1993)].
  • the wild-type p53 protein functions to regulate cell proliferation and cell death (also known as apoptosis) . It also participates in the response of the cell to DNA damaging agents [Harris (1993), cited above].
  • DNA damaging agents such as radiation and che otherapeutics commonly used for cancer treatment.
  • Fig. 3 illustrates the effects of a number of amino acid substitutions on DNA binding of the tumor- derived p53His273 mutant. Binding was assayed using a high affinity DNA site (oligonucleotide BC) by gel retardation shift analysis on native electrophoretic gels. The amino acids are abbreviated using the single letter code: His, H; Arg, R; Lys, K.
  • Fig. 4 illustrates the effect of the Thr284 to
  • the amino acids are abbreviated using the single letter code: Arg, R; Cys, C; Gin, Q; His, H; Lys, K.
  • Fig. 5 illustrates the results of an experiment relating to rescue of the transcriptional and tumor suppressor activities of tumor-derived p53 mutants.
  • Transcriptional activities from a reporter plasmid containing a high affinity p53 DNA site are presented as means ⁇ SE.
  • the activity of wild ⁇ type p53 was adjusted to 100%. No transcription was detected from a reporter lacking a p53 site.
  • Tumor suppressor activities in Saos-2 osteosarcoma cells are presented as means ⁇ SE of the number of tumor cell colonies per plate.
  • the amino acids are abbreviated using the single letter code: Arg, R; Cys, C; Gin, Q; His, H. threonine corresponding to amino acid residue 284 of the wild-type human p53 protein is changed to arginine.
  • the invention provides a method of enhancing the DNA-binding ability of a p53 construct having a p53 DNA binding domain comprising the step of modifying the codon encoding amino acid 284 to a codon encoding arginine.
  • the present invention provides a nucleic acid sequence encoding a protein of the invention.
  • These nucleic acids may be inserted into an appropriate vector for delivery to patients for gene therapy.
  • the nucleic acids may be inserted into a vector for in vitro expression of a protein of the invention, which is then introduced into patients.
  • Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.
  • Fig. 1 illustrates activation of DNA binding of common Class I mutants by antibody PAb421. Binding was assayed using a high affinity DNA site (oligonucleotide BC) by gel retardation shift analysis on native electrophoretic gels.
  • the amino acids are abbreviated using the single letter code: Cys, C; Gin, Q; His, H; Ser, S; Trp, W.
  • Fig. 2 illustrates activation of DNA binding of common Class I mutants by deletion of the p53 C-terminal 30 amino acids (residues 364-393) .
  • Binding was assayed using a high affinity DNA site (oligonucleotide BC) by gel retardation shift analysis on native electrophoretic gels in the presence of specific (S - oligonucleotide BC) or non-specific (NS - oligonucleotide TF3) unlabeled excess competitor DNA.
  • S - oligonucleotide BC specific - oligonucleotide BC
  • NS - oligonucleotide TF3 non-specific
  • the invention provides modified p53 protein constructs in which the amino acid residue corresponding to residue 284 of wild-type or native human p53 is modified from the native threonine to arginine.
  • the native Thr residue at position 284 may be substituted with Lys (K284) .
  • Lys284 was introduced into a p53His273 mutant, it was found to bind oligonucleotide BC somewhat better than the original p53His273 mutant, using the assay described in detail in Example 3. It will be understood that where reference is made to R284 in the following discussion, K284 may be substituted.
  • the modified p53 constructs of the invention may be derived from full-length p53.
  • p53Arg284 [SEQ ID NO: 3] and p53Lys284 [SEQ ID NO:4] are example ⁇ of such modified constructs.
  • the modified p53 constructs of the invention may contain a C-terminal p53 deletion.
  • a preferred deletion involves truncation of amino acid residues 364 - 393.
  • One example of such a truncated construct is p53Arg284 ⁇ 364-393 [SEQ ID NO: 17].
  • suitable deletions include truncation following amino acid residue 355, and deletions internal to this region (corresponding to residues 356-393 of SEQ ID N0:2) .
  • p53 protein constructs encompasses full-length and truncated p53 proteins containing a p53 DNA binding domain. Included in this Detailed Description of the Invention
  • the present invention provides modified p53 constructs containing arginine at the amino acid residue corresponding to residue 284 of wild-type human p53 [SEQ ID NO: 2].
  • the inventor has found that such a modification results in an increase in the DNA binding avidity of the p53 and more efficient tumor suppression than the corresponding unmodified construct.
  • the R284p53 [SEQ ID NO: 3] was found to bind DNA more avidly than wild-type p53 in vitro and to suppress colony growth of tumor cells about five- to six-fold more efficiently than wild-type p53 in tissue culture experiments.
  • the inventor demonstrates herein that the tumor suppressor function of common Class I tumor-derived p53 mutants can be restored and provides the means for pharmacological rescue of p53 function in cancer patients.
  • the inventor introduced a novel p53-DNA contact between a phosphate of the DNA backbone and p53. This was done by replacing Thr284 of wild-type human p53 with Arg. This substitution, in conjunction with the conformational switch that involves the C-terminus of p53 and allosterically regulates the activity of the p53 DNA binding domain, fully restored DNA binding of the tumor-derived p53 mutants. Furthermore, the transcriptional and tumor suppressing activities of these p53 mutants were also restored. 8
  • chimeric p53 proteins include proteins containing the N-terminal portion of p53 fused, optionally via a suitable linker, to a heterologous tetramerization domain.
  • a heterologous tetramerization domain includes any sequence of amino acids heterologous to p53 which forms stable homo-tetramers.
  • One particularly desirable tetramerization domain includes the tetrameric variant of the GCN4 LZ [Harbury et al,
  • GCN4 numbering follows Hinnenbusch et al, Proc. Natl. Acad. Sci. USA, 81:6442- 6446 (1984) and Ellenberger et al, Cell, 71: 1223-1237 (1992) .
  • Wild-type GCN4 is provided in SEQ ID NOS: 5 and 6.
  • the LZ variant has Ile at positions d of the coil and Leu at positions a [SEQ ID NO: 33], in contrast to the original zipper which has Leu and Val, respectively.
  • Suitable chimera include (from N-terminus to C-terminus) :
  • the above nucleotide sequences can be included within larger DNA or RNA fragments, or may be interrupted by introns.
  • nucleic acids encoding the modified proteins of the invention are present in the context of vectors suitable for amplification in prokaryotic or eukaryotic cells or for expression in cell-free extracts or lysates or in prokaryotic or eukaryotic cells.
  • vectors suitable for amplification in prokaryotic or eukaryotic cells or for expression in cell-free extracts or lysates or in prokaryotic or eukaryotic cells are known and many of these are commercially available.
  • plasmids with bacterial or yeast replication origins allow amplification in bacteria or yeast, respectively.
  • Such vectors allow the production of large quantities of nucleic acids encoding the proteins of the invention, which nucleic acids can be used for gene therapy or for expression of the modified p53 proteins of the invention.
  • expression vectors are known.
  • the vector pGEM4 (Promega, Madison, WI) is suitable for expression of the p53 proteins in cell-free lysates
  • the vector pSV2 [Mulligan et al, Proc. Natl. Acad. Sci. USA. 18:2072-2076 (1981)] is suitable for expression in mammalian cells.
  • Such vectors allow the production of the modified proteins of the invention in vitro for analysis of their functional properties or for delivery to patients.
  • one of skill in the art may readily select or construct another suitable expression vector.
  • nucleic acid sequences of the invention may be inserted into a vector capable of targeting and infecting a desired cell, either in vivo or ex vivo for which is fused via an Ile linker, to aa residues 352-393 of p53wt [SEQ ID NO: 2].
  • mutants include p53 having glutamine at residue 248 (p53Q248) [SEQ ID NO: 11], p53 having histidine at residue 273 (p53H273) [SEQ ID NO: 12], and p53 having cysteine at residue 273 (p53C273) [SEQ ID NO: 13].
  • Other p53 mutants which may be susceptible to this R284 mutation are known in the art.
  • Modifying the p53 protein construct according to the method of the invention involves altering the residue corresponding to aa residue 284 of human p53wt or of a p53 mutant containing the native Thr284 to Arg. This modification can be achieved by mutating the 284 codon using conventional site-directed mutagenesis techniques [R. Higuchi et al, in M. A. Innis et al, (eds.) , PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, pp. 177-183
  • the native codon 284 (ACA) is modified by site-directed mutagenesis to CGA or preferably CGT, which encodes Arg.
  • CGA native codon 284
  • CGT preferably CGT
  • the present invention further provides nucleic acid sequences encoding the modified p53 protein constructs of this invention.
  • the nucleic acid sequences of the invention 12 a pharmaceutically acceptable carrier, such as saline, and administered parenterally (or by other suitable means) in sufficient amounts to infect the desired cells and provide sufficient levels of p53 activity to arrest abnormal cellular proliferation.
  • a pharmaceutically acceptable carrier such as saline
  • Other pharmaceutically acceptable carriers are well known to those of skill in the art.
  • a suitable amount of the vector containing the chimeric nucleic acid sequences is between about 10 6 to 10° infectious particles per mL carrier. The delivery of the vector may be repeated as needed to sustain satisfactory levels of p53 activity, as determined by monitoring clinical symptoms.
  • this therapy may be combined with other therapies for the disease or condition being treated.
  • therapy involving the administration of a vector capable of expressing a modified p53 protein construct of the invention is well suited for use in conjunction with conventional cancer therapies, including surgery, radiation and chemotherapy.
  • nucleic acid sequences driving expression of a p53 protein of the invention may also be introduced as "naked DNA" by "carriers" other than viral vectors, such as liposomes, nucleic acid-coated gold beads or can simply be suspended in saline or the like and injected in situ [Fujiwara et al (1994) , cited above; Fynan et al , Proc. Natl. Acad. Sci.
  • a suitable amount of nucleic acid is between about 10 ⁇ g to about 1 mg per mL carrier.
  • a suitable amount of nucleic acid is between about 10 ⁇ g to about 1 mg per mL carrier.
  • many such viral vectors are useful for this purpose, e.g., adenoviruses, retroviruses and adeno-associated viruses (AAV) [Schreiber et al,
  • a recombinant viral vector e.g. an adenovirus
  • a recombinant viral vector comprises DNA of at least that portion of the viral genome which is capable of infecting the target cells operatively linked to the nucleic acid sequences of the invention.
  • infection is generally meant the process by which a virus transfers genetic material to its host or target cell.
  • the virus used in the construction of a vector of the invention is rendered replication-defective to remove the effects of viral replication on the target cells.
  • the replication-defective viral genome can be packaged by a helper virus in association with conventional techniques.
  • the vector(s) containing the nucleic acids encoding a protein of the invention is suspended in 14 effective to treat the conditions referred to below.
  • a preferred dose of a pharmaceutical composition containing a protein of this invention is generally effective above about 0.1 mg modified p53 protein, and preferably from about 1 mg to about 100 mg. Dosage units of such pharmaceutical compositions containing the proteins of this invention preferably contain about 1 mg to 5 g of the protein. These doses may be administered with a frequency necessary to achieve and maintain satisfactory p53 DNA binding and tumor suppressor activity levels. Although a preferred range has been described above, alternative doses for treatment of each type of tumor or other condition may be determined by those of skill in the art.
  • nucleic acids and proteins of the invention can be introduced into human patients for therapeutic benefits in conditions characterized by insufficient wild-type p53 activity.
  • the nucleic acids of the invention may be introduced into the patient in the form of a suitable viral vector (or by direct DNA delivery) to harness the patient's cellular machinery to express the proteins of the invention in vivo.
  • the proteins of the invention may be introduced into the patient in appropriate pharmaceutical formulations as described above.
  • compositions of thi ⁇ invention containing a protein of the invention or a nucleic acid or a viral vector which express a protein of the invention in vivo, may be employed to induce the cellular defense to DNA damaging agents.
  • DNA damaging agents include sunlight, UV irradiation, as well as radiation and chemotherapeutics used for cancer treatment.
  • UV irradiation examples include sunlight, UV irradiation, as well as radiation and chemotherapeutics used for cancer treatment.
  • modified p53 protein constructs of this invention may also be formulated into pharmaceutical compositions and administered using a therapeutic regimen compatible with the particular formulation.
  • compositions within the scope of the present invention include compositions containing a protein of the invention in an effective amount to have the desired physiological effect, e.g. to arrest the growth of cancer cells without causing unacceptable toxicity for the patient.
  • Suitable carriers for parenteral administration include aqueous solutions of the active compounds in water-soluble or water-dispersible form, e.g. saline.
  • suspensions of the active compounds may be administered in suitable conventional lipophilic carriers or in liposomes.
  • compositions may be supplemented by active pharmaceutical ingredients, where desired.
  • Optional antibacterial, antiseptic, and antioxidant agents in the compositions can perform their ordinary functions.
  • the pharmaceutical compositions of the invention may further contain any of a number of suitable viscosity enhancers, stabilizers, excipients and auxiliaries which facilitate processing of the active compounds into preparations that can be used pharmaceutically.
  • these preparations, as well as those preparations discussed below, are designed for parenteral administration.
  • compositions designed for oral or rectal administration are also considered to fall within the scope of the present invention.
  • suitable amount or “effective amount” means an amount which is 16
  • Plasmids of the pGEM series were used to generate in vitro translated p53 proteins, as previously described [T. Halazonetis and A. Kandil, EMBO J.. 12:5057-5064 (1993a); T. Halazonetis and A. Kandil, EMBO J. , 12:1021-1028 (1993b) ; J. L. Waterman et al, EMBO J. , 14:512-519 (1995)].
  • plasmid pGEMhump53wt (also termed pGEMhp53wtB) encodes full-length human wild-type p53.
  • This plasmid was prepared by PCR using a human p53 cDNA, which is readily available to those practicing the art. The PCR procedure was designed to incorporate unique restriction sites within the coding sequence of human p53: Kpn I at codon 218, Sst I at codon 299, Sst II at codon 333, Bst BI at codon 338 and Sal I immediately following the termination codon. An Msc I site at codon 138 was eliminated.
  • the proteins were derived from pGEMhump53wt by site-directed mutagenesis [Higuchi, in Innis et al, PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, pp. 177-183 (1990) ] of the codons indicated below.
  • In vitro amount of a composition of this invention patients may tolerate higher doses of such DNA damaging agents.
  • compositions of this invention are in inducing apoptosis of specific cells, such as proliferating lymphocytes.
  • a suitable amount of an appropriate pharmaceutical composition of this invention is administered to a subject to enhance the development of immune tolerance.
  • This method may employ both in vivo and ex vivo modes of administration.
  • this therapy is useful as the sole treatment or as an accessory treatment to prevent transplant rejection, or to treat autoimmune diseases, e.g. , systemic lupus erythrematosis, rheumatoid arthritis and the like.
  • the pharmaceutical compositions of this invention may also be employed to restore p53 function in tumor cells.
  • a suitable amount of the composition of this invention is administered systemically, or locally to the site of the tumor with or without concurrent administration of conventional cancer therapy (i.e. DNA damaging agents) .
  • compositions of this invention may be administered in methods to suppress cell proliferation in diseases other than cancers, which are characterized by aberrant cell proliferation.
  • diseases include psoriasis, atherosclerosis and arterial restenosis.
  • This method is conducted by administering a suitable amount of the selected composition systemically or locally to the patient.
  • These examples illustrate the preferred method for preparing exemplary modified p53 constructs of the invention and the biological activity of the modified p53 constructs. These examples are illustrative only and do not limit the scope of the invention. 18
  • the DNA binding activity of wild-type p53 is allosterically regulated by a basic region within the C- ter inal 30 amino acids of p53.
  • Monoclonal antibodies that mask this regulatory region, such as PAb421, or deletion of this region stimulate binding to DNA [T. Halanonetis et al, EMBJO J. , 12:1021-1028 (1993) ; T.R. Hupp et al, Cell. 21:875-886 (1992) ; J.L.F. Waterman et al, EMBO J. , ⁇ :512-519 (1995)].
  • some tumor-derived mutants have also been reported to bind DNA when allosterically activated by antibody PAb421 [T. Halazonetis and A.
  • Proteins corresponding to a) to h) each containing a deletion of the C-terminal 30 amino acid of human p53 ( ⁇ 364-393) , were also generated [SEQ ID NOS: 17-24]. These deletions permit in vitro DNA binding.
  • Plasmid pBC/TKseap has one copy of oligonucleotide BC [Halazonetis, EMBO J. , 12:1021-1028 (1993) cloned in the Eco RV site of pTKseap [Waterman, 1996] and expresses secreted alkaline phosphatase in a p53-responsive manner.
  • an Arg side chain introduced at position 284 could form electrostatic interactions with the phosphate oxygen atoms of DNA closest to its ⁇ -carbon and without violating bond lengths and angles. Modeling was performed with Quanta 4.1 (Molecular Simulations Inc., Burlington, MA) .
  • Example 1 All the proteins of Example 1 containing the 30 amino acid C-terminal deletion were expressed by in vitro translation and assayed for DNA binding using 0.2 ng 32 P- labeled DNA and, where indicated below, 100 ng unlabeled competitor DNA [J. L. F. Waterman et al, EMBO J. , 14: 512- 519 (1995)], The analysis was restricted to the C- terminally truncated proteins because full-length p53 translated in vitro is in a latent state and cannot bind DNA unless activated by a C-terminal truncation or by a monoclonal antibody (PAb421) that binds to the p53 C- terminus [Waterman et al, cited above].
  • PAb421 monoclonal antibody
  • Oligonucleotide BC which has the following sequence (top strand) is: [SEQ ID NO: 29] CC-GGGCA-TGTCC- GGGCA-TGTCC-GGGCATGT, and oligonucleotide structure of their DNA binding domain, have latent sequence-specific DNA binding activity, whereas Clas ⁇ II mutants, which have unfolded DNA binding domains [C. A. Finlay et al, Mol. Cell. Biol.
  • residues of the DNA binding domain of p53His273 were replaced with basic amino acids.
  • the substitutions targeted essentially all the residues close to the DNA backbone, except for those that already contact DNA or those that unequivocally stabilize the three-dimensional structure of p53 [Cho, cited above].
  • the targeted residues were: Glyll7, Thrll ⁇ , Alall9, Asn247, Thr284, Glu285 and Glu287.
  • Substitution of Thr284 with Arg enhanced binding of p53His273 to the high affinity DNA ⁇ ite, although binding wa ⁇ still dependent on allosteric activation by antibody PAb421 (Fig. 3) .
  • Substitution of Thr284 with Lys also enhanced binding of p53His273 to the high affinity DNA ⁇ ite, but less than sub ⁇ titution of 22
  • the Class I p53 mutants had either weak (p53Hi ⁇ 273) or no (p53Gln248 and p53Cys273) transcriptional activity. However, their transcriptional activity was enhanced to wild-type levels by the Thr284 to Arg substitution or, for p53Gln248, by combining the Thr284 to Arg substitution with C-terminal allosteric activation (Fig. 1) .
  • Tumor suppre ⁇ ing activity was tested in a colony formation assay, by cotransfecting Saos-2 osteosarcoma cells with 5 ⁇ g of pSV2hp53 expres ⁇ ion pla ⁇ mid directing p53 expre ⁇ ion, 0.5 ⁇ g of pSV7neo, a plasmid that confers neomycin/G418 resistance [K. Zhang et al, Proc. Natl. Acad. Sci. USA. 82:6281-6285 (1990)] and 24 ⁇ g of pBC12/PLseap [T. D. Halazonetis, Anticancer Res. , 11:285-292 (1992)], a carrier plasmid.
  • the transfected cell ⁇ were ⁇ elected for G418 re ⁇ i ⁇ tance, a neomycin relative. Two weeks later the colonies were stained with cry ⁇ tal violet and counted. High tumor suppressor activity corresponds to low colony formation.
  • the proteins containing the Arg284 modification ⁇ uppre ⁇ ed tumor colony formation more efficiently than the corresponding proteins without the Arg284 modification (Table 1) .
  • the magnitude of the effect is greater for the tumor-derived p53 mutants; however, even Ep21, which has the following sequence: [SEQ ID NO: 30] CCC-GAACA-TGTCC-CAACA-TGTTG-GGG, each contain a p53 binding site, which is underlined.
  • the BC oligonucleotide has a high affinity p53-binding site, while oligonucleotide Ep21 contains a lower affinity site, which is present in the regulatory sequences of the p21 gene [W. S.
  • Oligonucleotide Egadd45 has the sequence [SEQ ID NO: 31] ACA-GAACA-TGTCT-AAGCA-TGCTG-GGGA.
  • Oligonucleotide TF3 which contains three tandem repeats of [SEQ ID NO: 32] ATCACGTGAT, is a non-specific DNA [Halazonetis et al, EMBO J. , 11:1021-1028 (1993)].
  • Thr284 to Arg substitution enhanced binding of all p53 proteins examined (Fig. 4) .
  • oligonucleotides BC and Ep21 for wild-type p53 the effect is evident with oligonucleotides BC and Ep21, for p53Gln248 it is evident with oligonucleotide BC, for p53His273 and p53Cys273 it is evident with all oligonucleotides tested (Fig. 4) .
  • Example 3 Transcription and Tumor Suppres ⁇ ion A ⁇ ay ⁇
  • the proteins of Example 1 were examined for their transcriptional activity and tumor suppres ⁇ or activity. Wild-type p53 activate ⁇ transcription of target genes and suppresses tumor growth, whereas tumor- derived mutants lack both these activities [S.E. Kern et al, Science. 256: 827-830 (1992) ; C. A. Finlay et al, Cell, 5_7:1083-1093 (1989)].
  • the transcriptional activities of wild-type p53 and various p53 mutants were as ⁇ ayed with a p53-responsive reporter pla ⁇ mid in Saos-2 human o ⁇ teo ⁇ arcoma cells, which lack endogenous p53 [M.J.F.
  • transcriptional activity was determined by transfecting Saos-2 cell ⁇ with 2.5 ⁇ g pSV2hp53 expre ⁇ ion plasmid and 27.5 ⁇ g pBC/TKseap or pTKseap reporter pla ⁇ ids [Waterman et al, Cancer Res. , 24
  • MOLECULE TYPE DNA (genomic)
  • GGT TCT AAA TCA ACC AAC GAA AAT GTA TCT GCT TCC ACT TCT 879 Gly Ser Lys Ser Thr Asn Glu Asn Val Ser Ala Ser Thr Ser
  • MOLECULE TYPE DNA (genomic)
  • AAAAATTTCC GACTTTAAAT ACGGAAGATA AATACTCCAA CCTTTTTTTC 100
  • GAAAACTGTC AGTTTTTTGA AGAGTTATTT GTTTTGTTAC CAATTGCTAT 600
  • Lys Gin Arg Ser lie Pro Leu Ser Pro Ile Val Pro Glu Ser Ser
  • AAA CGT GCT AGA AAC ACT GAA GCC GCC AGG CGT TCT CGT GCG 1509 Lys Arg Ala Arg Asn Thr Glu Ala Ala Arg Arg Ser Arg Ala 235 240 245
  • MOLECULE TYPE protein Glu Ala Leu Glu Leu Lys Asp Ala Gin Ala Gly Lys Glu Pro Gly

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Abstract

A modified p53 protein or peptide having DNA binding in which amino acid residue 284 of a p53 protein or protein fragment is changed to Arginine or Lysine, is described. Also described are nucleotide sequences encoding the modified protein and vectors capable of expressing it.

Description

MODIFIED P53 CONSTRUCTS AND USES THEREFOR
Field of the Invention
The present invention relates generally to the field of oncoproteins, and more specifically to p53.
Background of the Invention
Wild-type (wt) p53 is a sequence-specific DNA binding protein found in humans and other mammals, which has tumor suppressor function [See, e.g., Harris, Science. 262: 1980-1981 (1993)]. The wild-type p53 protein functions to regulate cell proliferation and cell death (also known as apoptosis) . It also participates in the response of the cell to DNA damaging agents [Harris (1993), cited above]. In more than half of all human tumors p53 is inactivated by mutations and is therefore unable to arrest cell proliferation or induce apoptosis in response to DNA damaging agents, such as radiation and che otherapeutics commonly used for cancer treatment. The nucleotide and amino acid sequences of human p53 have been reported by Zakut-Houri et al, EMBO J. , 4.: 1251-1255 (1985) ; GenBank Code Hsp53]. The amino acid sequence of p53 is conserved across evolution [Soussi et al, Oncogene. 5_: 945-952 (1990) ], suggesting that its function is also conserved. The ability of p53 to bind DNA in a sequence-specific manner maps to amino acid residues 90-290 of human p53 [Halazonetis and Kandil, EMBO J.. 12: 5057-5064 (1993); Pavletich et al, Genes Dev.. 1_: 2556-2564 (1993) ; Wang et al, Genes Dev.. 1_: 2575-2586 (1993) ] and the tetramerization domain maps to amino acid residues 322-355 of human p53. Mutations of the p53 protein in most human tumors involve the sequence-specific DNA binding domain, so that the mutant proteins are unable to bind DNA [Bargonetti et al, Genes Dev. , 6 : 1886-1898 (1992)]. The loss of p53 function is critical for tumor development. using the single letter code: Cys, C; Gin, Q; His, H; Ser, S; Trp, W.
Fig. 3 illustrates the effects of a number of amino acid substitutions on DNA binding of the tumor- derived p53His273 mutant. Binding was assayed using a high affinity DNA site (oligonucleotide BC) by gel retardation shift analysis on native electrophoretic gels. The amino acids are abbreviated using the single letter code: His, H; Arg, R; Lys, K. Fig. 4 illustrates the effect of the Thr284 to
Arg substitution on binding of wild-type p53 and tumor- derived p53 mutants to a high affinity DNA site, and to the sites in the p21c-^ and gadd45 genes (oligonucleotides BC, Ep21 and Egadd45, respectively) . Binding was assayed by gel retardation shift analysis on native electrophoretic gels. Only the region of the gel corresponding to the p53-DNA complexes is shown. The amino acids are abbreviated using the single letter code: Arg, R; Cys, C; Gin, Q; His, H; Lys, K. Fig. 5 illustrates the results of an experiment relating to rescue of the transcriptional and tumor suppressor activities of tumor-derived p53 mutants. Transcriptional activities from a reporter plasmid containing a high affinity p53 DNA site (oligonucleotide BC) are presented as means ±SE. The activity of wild¬ type p53 was adjusted to 100%. No transcription was detected from a reporter lacking a p53 site. Tumor suppressor activities in Saos-2 osteosarcoma cells are presented as means ± SE of the number of tumor cell colonies per plate. The amino acids are abbreviated using the single letter code: Arg, R; Cys, C; Gin, Q; His, H. threonine corresponding to amino acid residue 284 of the wild-type human p53 protein is changed to arginine.
In another aspect, the invention provides a method of enhancing the DNA-binding ability of a p53 construct having a p53 DNA binding domain comprising the step of modifying the codon encoding amino acid 284 to a codon encoding arginine.
In yet another aspect, the present invention provides a nucleic acid sequence encoding a protein of the invention. These nucleic acids may be inserted into an appropriate vector for delivery to patients for gene therapy. Alternatively the nucleic acids may be inserted into a vector for in vitro expression of a protein of the invention, which is then introduced into patients. Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.
Brief Description of the Drawings
Fig. 1 illustrates activation of DNA binding of common Class I mutants by antibody PAb421. Binding was assayed using a high affinity DNA site (oligonucleotide BC) by gel retardation shift analysis on native electrophoretic gels. The amino acids are abbreviated using the single letter code: Cys, C; Gin, Q; His, H; Ser, S; Trp, W.
Fig. 2 illustrates activation of DNA binding of common Class I mutants by deletion of the p53 C-terminal 30 amino acids (residues 364-393) . Binding was assayed using a high affinity DNA site (oligonucleotide BC) by gel retardation shift analysis on native electrophoretic gels in the presence of specific (S - oligonucleotide BC) or non-specific (NS - oligonucleotide TF3) unlabeled excess competitor DNA. The amino acids are abbreviated Thus, the inventor has demonstrated that the tumor suppressor function of common Class I tumor-derived p53 mutants can be restored and that p53 function can be rescued in cancer patients.
I. p53 Mutant Proteins
Thus, in one aspect the invention provides modified p53 protein constructs in which the amino acid residue corresponding to residue 284 of wild-type or native human p53 is modified from the native threonine to arginine. In an alternate, and currently lesε preferred, embodiment, the native Thr residue at position 284 may be substituted with Lys (K284) . When Lys284 was introduced into a p53His273 mutant, it was found to bind oligonucleotide BC somewhat better than the original p53His273 mutant, using the assay described in detail in Example 3. It will be understood that where reference is made to R284 in the following discussion, K284 may be substituted.
The modified p53 constructs of the invention may be derived from full-length p53. p53Arg284 [SEQ ID NO: 3] and p53Lys284 [SEQ ID NO:4] are exampleε of such modified constructs. Alternatively, the modified p53 constructs of the invention may contain a C-terminal p53 deletion. Currently a preferred deletion involves truncation of amino acid residues 364 - 393. One example of such a truncated construct is p53Arg284Δ364-393 [SEQ ID NO: 17]. However, suitable deletions include truncation following amino acid residue 355, and deletions internal to this region (corresponding to residues 356-393 of SEQ ID N0:2) .
As used herein, "p53 protein constructs" encompasses full-length and truncated p53 proteins containing a p53 DNA binding domain. Included in this Detailed Description of the Invention
The present invention provides modified p53 constructs containing arginine at the amino acid residue corresponding to residue 284 of wild-type human p53 [SEQ ID NO: 2]. The inventor has found that such a modification results in an increase in the DNA binding avidity of the p53 and more efficient tumor suppression than the corresponding unmodified construct. For example, when wild-type p53 was so modified, the R284p53 [SEQ ID NO: 3] was found to bind DNA more avidly than wild-type p53 in vitro and to suppress colony growth of tumor cells about five- to six-fold more efficiently than wild-type p53 in tissue culture experiments. Particularly, the inventor demonstrates herein that the tumor suppressor function of common Class I tumor-derived p53 mutants can be restored and provides the means for pharmacological rescue of p53 function in cancer patients.
All references to human p53 residue numbers herein refer to the numbering scheme provided by
Zakut-Houri et al, (1985) [cited above], which is incorporated by reference, and reproduced in SEQ ID NOS: 1 and 2.
Without wishing to be bound by theory, to fully restore DNA binding to tumor-derived p53 mutants, such as Gln248, His273 and Cys273, the inventor introduced a novel p53-DNA contact between a phosphate of the DNA backbone and p53. This was done by replacing Thr284 of wild-type human p53 with Arg. This substitution, in conjunction with the conformational switch that involves the C-terminus of p53 and allosterically regulates the activity of the p53 DNA binding domain, fully restored DNA binding of the tumor-derived p53 mutants. Furthermore, the transcriptional and tumor suppressing activities of these p53 mutants were also restored. 8
(c) aa 1-325 of p53wt [SEQ ID NO: 2], fused via an Arg-Gly-Asn linker [SEQ ID NO: 8], to the heterologous sequence of (a) above;
(d) aa 1-325 of p53wt [SEQ ID NO: 2], fused via an Arg-Gly-Gly-Asn-Pro-Glu linker [SEQ ID NO: 9], to the heterologous sequence of (b) above;
(e) aa 1-323 of p53wt [SEQ ID NO: 2], fused via an Arg-Gly-Asn linker [SEQ ID NO: 8], to the heterologous sequence of (a) above; (f) aa 1-323 of p53wt [SEQ ID NO: 2], fused via an Arg-Gly-Gly-Asn-Pro-Glu linker [SEQ ID NO: 9], to the heterologous sequence of (b) above;
(g) aa 1-300 of p53wt [SEQ ID NO: 2], fused via a Gly-Gly-Asn-Gln-Ala linker [SEQ ID NO: 10], to the heterologous sequence of (b) above;
(h) aa 1-325 of p53wt [SEQ ID NO: 2], fused via an Arg-Gly-Asn linker [SEQ ID NO: 8], to the heterologous sequence of (a) above, fused via an Ile linker, to aa 352-393 of p53wt [SEQ ID NO: 2]; (i) aa 1-325 of p53wt [SEQ ID NO: 2], fused via an Arg-Gly-Gly-Asn-Pro-Glu linker [SEQ ID NO: 9], to the heterologous sequence of (b) above, which is fused via an Ile linker, to aa 352-393 of p53wt [SEQ ID NO: 2];
(j) aa 1-323 of p53wt [SEQ ID NO: 2], fused via an Arg-Gly-Asn linker [SEQ ID NO: 8], to the heterologous sequence of (a) above, which is fused via an Ile linker to aa 352-393 of p53wt [SEQ ID NO: 2] ;
(k) aa 1-325 of p53wt [SEQ ID NO: 2], fused via an Arg-Gly-Gly-Asn-Pro-Glu linker [SEQ ID NO: 9], to the heterologous sequence of (b) above, which is fused via an Ile linker, to aa residues 352-393 of p53wt [SEQ ID NO: 2] ; and
(1) aa 1-334 of p53wt [SEQ ID NO: 2], fused via an Asn linker, to the heterologous sequence of (a) above, definition are chimeric and mutant p53 proteins. Such proteins are known in the art.
Exemplary chimeric p53 proteins are described in detail in International Publication No. W096/16989, published June 6, 1996 and co-pending U.S. Patent
Application No. 08/347,792 and co-pending U.S. Patent application No. 08/431,357, which are incorporated by reference. For example, chimeric p53 proteins include proteins containing the N-terminal portion of p53 fused, optionally via a suitable linker, to a heterologous tetramerization domain. A heterologous tetramerization domain includes any sequence of amino acids heterologous to p53 which forms stable homo-tetramers. One particularly desirable tetramerization domain includes the tetrameric variant of the GCN4 LZ [Harbury et al,
Science, 262: 1401-1407 (1993)]. GCN4 numbering follows Hinnenbusch et al, Proc. Natl. Acad. Sci. USA, 81:6442- 6446 (1984) and Ellenberger et al, Cell, 71: 1223-1237 (1992) . Wild-type GCN4 is provided in SEQ ID NOS: 5 and 6. The LZ variant has Ile at positions d of the coil and Leu at positions a [SEQ ID NO: 33], in contrast to the original zipper which has Leu and Val, respectively. Suitable chimera include (from N-terminus to C-terminus) :
(a) aa 1-334 of p53wt [SEQ ID NO: 2], fused via an Asn linker, to a heterologous sequence spanning residues 249-281 of GCN4 containing isoleucines at positions d of the coiled coil and leucines at positions a [SEQ ID NO: 33] ;
(b) aa 1-334 of p53wt [SEQ ID NO: 2], fused via a Gly-Asn-Pro-Glu linker [SEQ ID NO: 7], to a heterologous sequence spanning residues 250-281 of GCN4 containing isoleucines at positions d of the coiled coil and leucines at positions a [SEQ ID NO: 33]; 10 include the complementary DNA sequence representing the non-coding strand, the messenger RNA sequence, the corresponding cDNA sequence and the RNA sequence complementary to the messenger RNA sequence. The above nucleotide sequences can be included within larger DNA or RNA fragments, or may be interrupted by introns.
In another embodiment the nucleic acids encoding the modified proteins of the invention are present in the context of vectors suitable for amplification in prokaryotic or eukaryotic cells or for expression in cell-free extracts or lysates or in prokaryotic or eukaryotic cells. Many such vectors are known and many of these are commercially available. For example, plasmids with bacterial or yeast replication origins allow amplification in bacteria or yeast, respectively. Such vectors allow the production of large quantities of nucleic acids encoding the proteins of the invention, which nucleic acids can be used for gene therapy or for expression of the modified p53 proteins of the invention. Similarly, expression vectors are known. For example, the vector pGEM4 (Promega, Madison, WI) is suitable for expression of the p53 proteins in cell-free lysates, while the vector pSV2 [Mulligan et al, Proc. Natl. Acad. Sci. USA. 18:2072-2076 (1981)] is suitable for expression in mammalian cells. Such vectors allow the production of the modified proteins of the invention in vitro for analysis of their functional properties or for delivery to patients. Alternatively, one of skill in the art may readily select or construct another suitable expression vector.
III. Gene Therapy
The nucleic acid sequences of the invention may be inserted into a vector capable of targeting and infecting a desired cell, either in vivo or ex vivo for which is fused via an Ile linker, to aa residues 352-393 of p53wt [SEQ ID NO: 2].
Also encompassed within the definition of "p53 protein constructs" are both naturally occurring and engineered mutant proteins. Exemplary mutants include p53 having glutamine at residue 248 (p53Q248) [SEQ ID NO: 11], p53 having histidine at residue 273 (p53H273) [SEQ ID NO: 12], and p53 having cysteine at residue 273 (p53C273) [SEQ ID NO: 13]. Other p53 mutants which may be susceptible to this R284 mutation are known in the art.
Modifying the p53 protein construct according to the method of the invention, involves altering the residue corresponding to aa residue 284 of human p53wt or of a p53 mutant containing the native Thr284 to Arg. This modification can be achieved by mutating the 284 codon using conventional site-directed mutagenesis techniques [R. Higuchi et al, in M. A. Innis et al, (eds.) , PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, pp. 177-183
(1990)]. For example, preferably, the native codon 284 (ACA) is modified by site-directed mutagenesis to CGA or preferably CGT, which encodes Arg. However, one of skill in the art can readily make alternative modifications resulting in an Arg codon at position 284.
Alternatively, conventional chemical synthesis techniques may be used to generate a p53 sequence containing this modification.
II. Nucleic Acid Sequences Encoding Modified p53 Proteins of the Invention
The present invention further provides nucleic acid sequences encoding the modified p53 protein constructs of this invention. In addition to the coding strand, the nucleic acid sequences of the invention 12 a pharmaceutically acceptable carrier, such as saline, and administered parenterally (or by other suitable means) in sufficient amounts to infect the desired cells and provide sufficient levels of p53 activity to arrest abnormal cellular proliferation. Other pharmaceutically acceptable carriers are well known to those of skill in the art. A suitable amount of the vector containing the chimeric nucleic acid sequences is between about 106 to 10° infectious particles per mL carrier. The delivery of the vector may be repeated as needed to sustain satisfactory levels of p53 activity, as determined by monitoring clinical symptoms.
As desired, this therapy may be combined with other therapies for the disease or condition being treated. For example, therapy involving the administration of a vector capable of expressing a modified p53 protein construct of the invention is well suited for use in conjunction with conventional cancer therapies, including surgery, radiation and chemotherapy. Alternatively, nucleic acid sequences driving expression of a p53 protein of the invention may also be introduced as "naked DNA" by "carriers" other than viral vectors, such as liposomes, nucleic acid-coated gold beads or can simply be suspended in saline or the like and injected in situ [Fujiwara et al (1994) , cited above; Fynan et al , Proc. Natl. Acad. Sci. USA, 90: 11478-11482 (1993) ; Cohen, Science. 259: 1691-1692 (1993) ; Wolff et al, Biotechniques, 11: 474-485 (1991)]. A suitable amount of nucleic acid is between about 10 μg to about 1 mg per mL carrier. However, one of skill in the art may modify the therapeutic dose as desired. gene therapy, and causing the encoded modified p53 protein construct of this invention to be expressed by that cell. Many such viral vectors are useful for this purpose, e.g., adenoviruses, retroviruses and adeno-associated viruses (AAV) [Schreiber et al,
Biotechniques. 14 : 818-823 (1993) ; Davidson et al, Nature Genetics, 3 : 219-223 (1993) ; Roessler et al, J. Clin. Invest. , 92: 1085-1092 (1993) ; S ythe et al, Ann. Thorac. Surg.. 5_7: 1395-1401 (1994) ; Kaplitt et al, Nature Genetics, 8 : 148-154 (1994)]. There has already been success using viral vectors driving expression of wild-type p53 [Fujiwara et al, Cancer Res. , 53 : 4129-4133 (1993) ; Fujiwara et al, Cancer Res. , 54 : 2287-2291 (1994) ; Friedmann, Cancer. 70(6 Suppl) : 1810-1817 (1992) ; Fujiwara et al, Curr. Opin. Oncol., 6 : 96-105 (1994)]. For use in gene therapy, these viral vectors containing nucleic acid sequences encoding a modified p53 protein construct of the invention, are prepared by one of skill in the art with resort to conventional techniques (see references mentioned above) . For example, a recombinant viral vector, e.g. an adenovirus, of the present invention comprises DNA of at least that portion of the viral genome which is capable of infecting the target cells operatively linked to the nucleic acid sequences of the invention. By "infection" is generally meant the process by which a virus transfers genetic material to its host or target cell. Preferably, the virus used in the construction of a vector of the invention is rendered replication-defective to remove the effects of viral replication on the target cells. In such cases, the replication-defective viral genome can be packaged by a helper virus in association with conventional techniques.
Briefly, the vector(s) containing the nucleic acids encoding a protein of the invention is suspended in 14 effective to treat the conditions referred to below. A preferred dose of a pharmaceutical composition containing a protein of this invention is generally effective above about 0.1 mg modified p53 protein, and preferably from about 1 mg to about 100 mg. Dosage units of such pharmaceutical compositions containing the proteins of this invention preferably contain about 1 mg to 5 g of the protein. These doses may be administered with a frequency necessary to achieve and maintain satisfactory p53 DNA binding and tumor suppressor activity levels. Although a preferred range has been described above, alternative doses for treatment of each type of tumor or other condition may be determined by those of skill in the art.
V. Therapeutic Indications
The nucleic acids and proteins of the invention can be introduced into human patients for therapeutic benefits in conditions characterized by insufficient wild-type p53 activity. As stated above, the nucleic acids of the invention may be introduced into the patient in the form of a suitable viral vector (or by direct DNA delivery) to harness the patient's cellular machinery to express the proteins of the invention in vivo. Alternatively, the proteins of the invention may be introduced into the patient in appropriate pharmaceutical formulations as described above.
As one example, the pharmaceutical compositions of thiε invention, containing a protein of the invention or a nucleic acid or a viral vector which express a protein of the invention in vivo, may be employed to induce the cellular defense to DNA damaging agents. Examples of DNA damaging agents include sunlight, UV irradiation, as well as radiation and chemotherapeutics used for cancer treatment. By administering a suitable IV. Pharmaceutical Compositions
The modified p53 protein constructs of this invention may also be formulated into pharmaceutical compositions and administered using a therapeutic regimen compatible with the particular formulation.
Pharmaceutical compositions within the scope of the present invention include compositions containing a protein of the invention in an effective amount to have the desired physiological effect, e.g. to arrest the growth of cancer cells without causing unacceptable toxicity for the patient.
Suitable carriers for parenteral administration include aqueous solutions of the active compounds in water-soluble or water-dispersible form, e.g. saline. Alternatively, suspensions of the active compounds may be administered in suitable conventional lipophilic carriers or in liposomes.
The compositions may be supplemented by active pharmaceutical ingredients, where desired. Optional antibacterial, antiseptic, and antioxidant agents in the compositions can perform their ordinary functions. The pharmaceutical compositions of the invention may further contain any of a number of suitable viscosity enhancers, stabilizers, excipients and auxiliaries which facilitate processing of the active compounds into preparations that can be used pharmaceutically. Preferably, these preparations, as well as those preparations discussed below, are designed for parenteral administration. However, compositions designed for oral or rectal administration are also considered to fall within the scope of the present invention.
Those of skill in the pharmaceutical art should be able to derive suitable dosages and schedules of administration. As used herein, the terms "suitable amount" or "effective amount" means an amount which is 16
Example 1 - p53 Protein Production
Plasmids of the pGEM series were used to generate in vitro translated p53 proteins, as previously described [T. Halazonetis and A. Kandil, EMBO J.. 12:5057-5064 (1993a); T. Halazonetis and A. Kandil, EMBO J. , 12:1021-1028 (1993b) ; J. L. Waterman et al, EMBO J. , 14:512-519 (1995)].
More specifically, plasmid pGEMhump53wt (also termed pGEMhp53wtB) encodes full-length human wild-type p53. This plasmid was prepared by PCR using a human p53 cDNA, which is readily available to those practicing the art. The PCR procedure was designed to incorporate unique restriction sites within the coding sequence of human p53: Kpn I at codon 218, Sst I at codon 299, Sst II at codon 333, Bst BI at codon 338 and Sal I immediately following the termination codon. An Msc I site at codon 138 was eliminated. These changes did not alter the sequence of the encoded p53, and were only performed to expedite construction of mutant proteins bearing altered tetramerization domains or point mutations associated with human cancer. The PCR product of the human p53 cDNA was digested with Neo I and Sal I and cloned in the vector pGEM4 [Promega, Madison, WI], which was linearized with Eco RI and Sal I. Synthetic oligonucleotides were used to bridge the Eco RI site of the vector and the Neo I site at the initiation codon of p53. Plasmid, pGEMhump53wt, was used to generate all the p53 mutants and modified p53 protein constructs described below, as well as for expression of wild-type p53 by in vitro translation [J.L.F. Waterman et al, EMBO J. , 14.: 512-519 (1995)]. The proteins were derived from pGEMhump53wt by site-directed mutagenesis [Higuchi, in Innis et al, PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, pp. 177-183 (1990) ] of the codons indicated below. In vitro amount of a composition of this invention, patients may tolerate higher doses of such DNA damaging agents.
Another therapeutic use of the compositions of this invention is in inducing apoptosis of specific cells, such as proliferating lymphocytes. According to this method of use, a suitable amount of an appropriate pharmaceutical composition of this invention is administered to a subject to enhance the development of immune tolerance. This method may employ both in vivo and ex vivo modes of administration. Preferably, this therapy is useful as the sole treatment or as an accessory treatment to prevent transplant rejection, or to treat autoimmune diseases, e.g. , systemic lupus erythrematosis, rheumatoid arthritis and the like. The pharmaceutical compositions of this invention may also be employed to restore p53 function in tumor cells. Desirably, a suitable amount of the composition of this invention is administered systemically, or locally to the site of the tumor with or without concurrent administration of conventional cancer therapy (i.e. DNA damaging agents) .
Additionally, the compositions of this invention may be administered in methods to suppress cell proliferation in diseases other than cancers, which are characterized by aberrant cell proliferation. Among such diseases are included psoriasis, atherosclerosis and arterial restenosis. This method is conducted by administering a suitable amount of the selected composition systemically or locally to the patient. These examples illustrate the preferred method for preparing exemplary modified p53 constructs of the invention and the biological activity of the modified p53 constructs. These examples are illustrative only and do not limit the scope of the invention. 18
Example 2 - DNA Binding Activity
The DNA binding activity of wild-type p53 is allosterically regulated by a basic region within the C- ter inal 30 amino acids of p53. Monoclonal antibodies that mask this regulatory region, such as PAb421, or deletion of this region stimulate binding to DNA [T. Halanonetis et al, EMBJO J. , 12:1021-1028 (1993) ; T.R. Hupp et al, Cell. 21:875-886 (1992) ; J.L.F. Waterman et al, EMBO J. , ϋ:512-519 (1995)]. Interestingly, some tumor-derived mutants have also been reported to bind DNA when allosterically activated by antibody PAb421 [T. Halazonetis and A. Kandil, EMBO J. , 12.: 5057-5064 (1993) ; T. Hupp et al, Nucl. Acids. Res.. 21:3167-3174 (1993); D. Niewolik et al, Oncogene, 10:881-890 (1995)]. The seven most common tumor-derived mutants: p53Hiεl75 [SEQ ID NO: 25], Gln248 [SEQ ID NO: 11], Trp248 [SEQ ID NO: 26], Ser249 [SEQ ID NO: 27], His273 [SEQ ID NO: 12], Trp282 [SEQ ID NO: 28] and Cys273 [SEQ ID NO: 13] [M. Hollstein et al, Science, 253:49-53 (1991)] were examined. The substitutions in these mutants target arginines 248 or 273 that contact DNA (Class I mutants) or arginines 175, 249 or 282 that stabilize the structure of the DNA binding domain (Class II mutants) [Y. Cho et al, Science. 265:346-355 (1994)]. Of the seven tumor- derived mutants, four recognized a high affinity p53 DNA site in the presence of PAb421 (Fig. 1) or when their C- ter inal 30 amino acids were deleted (Fig. 2) . Significantly, the mutants bound DNA in the presence of excess unlabeled non-specific DNA suggesting that they retain sequence specificity. Except for p53Trp248, allosteric activation enhanced DNA binding of all Class I mutants examined. DNA binding of Clasε II mutantε waε not activated, except for p53Trp282, which, like wild¬ type p53, bound DNA in the abεence and presence of PAb421. Thus, Class I mutants, which retain a native translated proteins were expressed using SP6 transcribed mRNA and rabbit reticulocyte lysates, as previously described [Halazonetis et al, Cell. 5_5:917-925 (1988)]. The following proteins were generated in this manner: a) Wild-type p53 (p53wt) [SEQ ID NO: 2] b) Wild-type p53 containing the Thr284 to Arg subεtitution (p53R284) [SEQ ID NO: 3] c) Tumor-derived mutant p53 glutamine 248 (p53Q248) [SEQ ID NO: 11] d) Tumor-derived mutant p53 glutamine 248 containing the Thr284 to Arg substitution (p53Q248R284) [SEQ ID NO: 14] e) Tumor-derived mutant p53 mutant histidine 273 (p53H273) [SEQ ID NO: 12] f) Tumor-derived mutant p53 histidine 273 containing the Thr 284 to Arg substitution (p53H273R284) [SEQ ID NO: 15] g) Tumor-derived mutant p53 cysteine 273 (p53C273) [SEQ ID NO: 13] h) Tumor-derived mutant p53 cysteine 273 containing the Thr 284 to Arg substitution (p53C273R284) [SEQ ID NO: 16] .
Proteins corresponding to a) to h) , each containing a deletion of the C-terminal 30 amino acid of human p53 (Δ364-393) , were also generated [SEQ ID NOS: 17-24]. These deletions permit in vitro DNA binding.
In addition, plasmid pSV2hp53wtB was used to express wild-type p53 in mammalian cells [M.J.F. Waterman et al, Cancer Res.. 56:158-163 (1996)]. Plasmid pBC/TKseap has one copy of oligonucleotide BC [Halazonetis, EMBO J. , 12:1021-1028 (1993) cloned in the Eco RV site of pTKseap [Waterman, 1996] and expresses secreted alkaline phosphatase in a p53-responsive manner. 20
Thr284 with Arg. All other substitutions either suppresεed or had no effect on p53His273 DNA binding (Fig. 3) .
The effects of the substitution of Thr284 with Arg can be rationalized using molecular modeling.
Specifically, using the coordinates of the wild-type p53 DNA binding domain bound to DNA [Cho, cited above] an Arg side chain introduced at position 284 could form electrostatic interactions with the phosphate oxygen atoms of DNA closest to its α-carbon and without violating bond lengths and angles. Modeling was performed with Quanta 4.1 (Molecular Simulations Inc., Burlington, MA) .
In the following experiments, the effect of the Thr284 to Arg substitution on binding to natural DNA sites was examined in the context of wild-type p53, of p53His273 and of the other Class I p53 mutants.
All the proteins of Example 1 containing the 30 amino acid C-terminal deletion were expressed by in vitro translation and assayed for DNA binding using 0.2 ng 32P- labeled DNA and, where indicated below, 100 ng unlabeled competitor DNA [J. L. F. Waterman et al, EMBO J. , 14: 512- 519 (1995)], The analysis was restricted to the C- terminally truncated proteins because full-length p53 translated in vitro is in a latent state and cannot bind DNA unless activated by a C-terminal truncation or by a monoclonal antibody (PAb421) that binds to the p53 C- terminus [Waterman et al, cited above].
For analysiε of DNA binding activity, these proteins were incubated with 32P-labeled oligonucleotides and subjected to electrophoresis as described [Halazonetis (1993a and 1993b) and Waterman (1995) , both cited above] . Oligonucleotide BC, which has the following sequence (top strand) is: [SEQ ID NO: 29] CC-GGGCA-TGTCC- GGGCA-TGTCC-GGGCATGT, and oligonucleotide structure of their DNA binding domain, have latent sequence-specific DNA binding activity, whereas Clasε II mutants, which have unfolded DNA binding domains [C. A. Finlay et al, Mol. Cell. Biol. , 8:531-539 (1988)], do not. Regarding the exceptions, we speculate that the large tryptophan side chain at position 248 precludes the p53Trp248 mutant from binding DNA due to steric interference with the DNA site. The ability of p53Trp282 to bind DNA may indicate that a small fraction of this mutant adopts the native fold.
Allosterically activated Class I p53 mutants compared favorably with wild-type p53 for binding to a high affinity DNA site. However, further experiments indicated that the mutants failed to recognize efficiently natural p53 sites, such as those present in the p2lcιp1 and gadd45 genes [W. S. El-Deiry et al, Cell. 25:817-825 (1993) , M.B. Kastan et al, Cell. 71:587-597 (1992)]. Since Class I p53 mutants apparently retain DNA binding sequence specificity, in an attempt to increaεe the affinity of Class I p53 mutantε for DNA, novel protein-DNA backbone contacts were introduced. Towards this goal, residues of the DNA binding domain of p53His273 were replaced with basic amino acids. The substitutions targeted essentially all the residues close to the DNA backbone, except for those that already contact DNA or those that unequivocally stabilize the three-dimensional structure of p53 [Cho, cited above]. The targeted residues were: Glyll7, Thrllδ, Alall9, Asn247, Thr284, Glu285 and Glu287. Substitution of Thr284 with Arg enhanced binding of p53His273 to the high affinity DNA εite, although binding waε still dependent on allosteric activation by antibody PAb421 (Fig. 3) . Substitution of Thr284 with Lys also enhanced binding of p53His273 to the high affinity DNA εite, but less than subεtitution of 22
56 -.158-163 (1996)]. The Class I p53 mutants had either weak (p53Hiε273) or no (p53Gln248 and p53Cys273) transcriptional activity. However, their transcriptional activity was enhanced to wild-type levels by the Thr284 to Arg substitution or, for p53Gln248, by combining the Thr284 to Arg substitution with C-terminal allosteric activation (Fig. 1) .
Tumor suppreεεing activity was tested in a colony formation assay, by cotransfecting Saos-2 osteosarcoma cells with 5 μg of pSV2hp53 expresεion plaεmid directing p53 expreεεion, 0.5 μg of pSV7neo, a plasmid that confers neomycin/G418 resistance [K. Zhang et al, Proc. Natl. Acad. Sci. USA. 82:6281-6285 (1990)] and 24 μg of pBC12/PLseap [T. D. Halazonetis, Anticancer Res. , 11:285-292 (1992)], a carrier plasmid. The transfected cellε were εelected for G418 reεiεtance, a neomycin relative. Two weeks later the colonies were stained with cryεtal violet and counted. High tumor suppressor activity corresponds to low colony formation.
Table 1
Tumor Colonies
Expressed Protein SE0 ID NO: (mean ± 1 S.E.) Human wild-type p53 2 11.3 ± 3.3 Human p53Δ364-393 17 17.7 ± 4.8 Human p53Arg284 3 2.0 ± 1.0
Human p53Arg284Δ364-393 18 4.7 ± 0.7
As illustrated in Table 1 above and in Fig. 5, the proteins containing the Arg284 modification εuppreεεed tumor colony formation more efficiently than the corresponding proteins without the Arg284 modification (Table 1) . The magnitude of the effect is greater for the tumor-derived p53 mutants; however, even Ep21, which has the following sequence: [SEQ ID NO: 30] CCC-GAACA-TGTCC-CAACA-TGTTG-GGG, each contain a p53 binding site, which is underlined. The BC oligonucleotide has a high affinity p53-binding site, while oligonucleotide Ep21 contains a lower affinity site, which is present in the regulatory sequences of the p21 gene [W. S. El-Deiry et al, Cell , 25:817-825 (1993)]. Oligonucleotide Egadd45 has the sequence [SEQ ID NO: 31] ACA-GAACA-TGTCT-AAGCA-TGCTG-GGGA. Oligonucleotide TF3 , which contains three tandem repeats of [SEQ ID NO: 32] ATCACGTGAT, is a non-specific DNA [Halazonetis et al, EMBO J. , 11:1021-1028 (1993)].
The Thr284 to Arg substitution enhanced binding of all p53 proteins examined (Fig. 4) . For wild-type p53 the effect is evident with oligonucleotides BC and Ep21, for p53Gln248 it is evident with oligonucleotide BC, for p53His273 and p53Cys273 it is evident with all oligonucleotides tested (Fig. 4) .
Example 3 - Transcription and Tumor Suppresεion Aεεayε The proteins of Example 1 were examined for their transcriptional activity and tumor suppresεor activity. Wild-type p53 activateε transcription of target genes and suppresses tumor growth, whereas tumor- derived mutants lack both these activities [S.E. Kern et al, Science. 256: 827-830 (1992) ; C. A. Finlay et al, Cell, 5_7:1083-1093 (1989)]. The transcriptional activities of wild-type p53 and various p53 mutants were asεayed with a p53-responsive reporter plaεmid in Saos-2 human oεteoεarcoma cells, which lack endogenous p53 [M.J.F. Waterman et al, Cancer Res. , 5_6:158-163 (1996)]. More particularly, transcriptional activity was determined by transfecting Saos-2 cellε with 2.5μg pSV2hp53 expreεεion plasmid and 27.5 μg pBC/TKseap or pTKseap reporter plaε ids [Waterman et al, Cancer Res. , 24
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(ii) TITLE OF INVENTION: Modified p53 Constructs and Uses
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(A) TELEPHONE: 215-540-9206
(B) TELEFAX: 215-540-5818
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1317 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear the tumor suppressor activity of wild-type p53 is enhanced by the Arg284 modification.
While the effect of the Arg284 modification seems to be greater for tumor-derived mutants, rather than wild-type p53, this is a reflection of the limitations of the asεayε uεed. In theεe aεsays, wild- type p53 demonstrates high activity. If the assays were adjusted so that wild-type p53 would have low activity, then the effect of the Arg284 modification would be as dramatic as observed with the tumor-derived p53 mutants.
Numerous modifications and variations of the present invention are included in the above-identified specification and are expected to be obvious to one of skill in the art. Such modifications and alterations to the compoεitions and processes of the preεent invention are believed to be encompaεεed in the scope of the claims appended hereto.
26
AAC AAG ATG TTT TGC CAA CTG GCC AAG ACC TGC CCT GTG CAG 267 Asn Lys Met Phe Cys Gin Leu Ala Lys Thr Cys Pro Val Gin
135 140
CTG TGG GTT GAT TCC ACA CCC CCG CCC GGC ACC CGC GTC CGC 609 Leu Trp Val Asp Ser Thr Pro Pro Pro Gly Thr Arg Val Arg 145 150 155
GCC ATG GCC ATC TAC AAG CAG TCA CAG CAC ATG ACG GAG GTT 651 Ala Met Ala Ile Tyr Lys Gin Ser Gin His Met Thr Glu Val 160 165 170
GTG AGG CGC TGC CCC CAC CAT GAG CGC TGC TCA GAT AGC GAT 693 Val Arg Arg Cys Pro His Hiε Glu Arg Cys Ser Asp Ser Asp 175 180 185
GGT CTG GCC CCT CCT CAG CAT CTT ATC CGA GTG GAA GGA AAT 735 Gly Leu Ala Pro Pro Gin His Leu Ile Arg Val Glu Gly Asn 190 195 200
TTG CGT GTG GAG TAT TTG GAT GAC AGA AAC ACT TTT CGA CAT 777 Leu Arg Val Glu Tyr Leu Asp Asp Arg Asn Thr Phe Arg His
205 210
AGT GTG GTG GTG CCC TAT GAG CCG CCT GAG GTT GGC TCT GAC 819 Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val Gly Ser Asp 215 220 225
TGT ACC ACC ATC CAC TAC AAC TAC ATG TGT AAC AGT TCC TGC 861 Cys Thr Thr Ile His Tyr Aεn Tyr Met Cys Asn Ser Ser Cys 230 235 240
ATG GGC GGC ATG AAC CGG AGA CCC ATC CTC ACC ATC ATC ACA 903 Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr 245 250 255
CTG GAA GAC TCC AGT GGT AAT CTA CTG GGA CGG AAC AGC TTT 945 Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe 260 265 270
GAG GTG CGT GTT TGT GCC TGT CCT GGG AGA GAC CGG CGC ACA 987 Glu Val Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr
275 280
GAG GAA GAG AAT CTC CGC AAG AAA GGG GAG CCT CAC CAC GAG 1029 Glu Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro Hiε Hiε Glu 285 290 295
CTG CCC CCA GGG AGC ACT AAG CGA GCA CTG CCC AAC AAC ACC 1071 Leu Pro Pro Gly Ser Thr Lys Arg Ala Leu Pro Aεn Aεn Thr 300 305 310 (ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 136..1314
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
GTCTAGAGCC ACCGTCCAGG GAGCAGGTAG CTGCTGGGCT CCGGGGACAC 50
TTTGCGTTCG GGCTGGGAGC GTGCTTTCCA CGACGGTGAC ACGCTTCCCT 100
GGATTGGCAG CCAGACTGCC TTCCGGGTCA CTGCC ATG GAG GAG CCG 147
Met Glu Glu Pro 1
CAG TCA GAT CCT AGC GTC GAG CCC CCT CTG AGT CAG GAA ACA 189 Gin Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gin Glu Thr 5 10 15
TTT TCA GAC CTA TGG AAA CTA CTT CCT GAA AAC AAC GTT CTG 231 Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu 20 25 30
TCC CCC TTG CCG TCC CAA GCA ATG GAT GAT TTG ATG CTG TCC 273 Ser Pro Leu Pro Ser Gin Ala Met Asp Asp Leu Met Leu Ser 35 40 45
CCG GAC GAT ATT GAA CAA TGG TTC ACT GAA GAC CCA GGT CCA 315 Pro Asp Asp Ile Glu Gin Trp Phe Thr Glu Asp Pro Gly Pro 50 55 60
GAT GAA GCT CCC AGA ATG CCA GAG GCT GCT CCC CCC GTG GCC 357 Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala
65 70
CCT GCA CCA GCA GCT CCT ACA CCG GCG GCC CCT GCA CCA GCC 399 Pro Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala 75 80 85
CCC TCC TGG CCC CTG TCA TCT TCT GTC CCT TCC CAG AAA ACC 441 Pro Ser Trp Pro Leu Ser Ser Ser Val Pro Ser Gin Lys Thr 90 95 100
TAC CAG GGC AGC TAC GGT TTC CGT CTG GGC TTC TTG CAT TCT 483 Tyr Gin Gly Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser 105 110 115
GGG ACA GCC AAG TCT GTA ACT TGC ACG TAC TCC CCT GCC CTC 525 Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu 120 125 130 28
Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser
80 85 90
Trp Pro Leu Ser Ser Ser Val Pro Ser Gin Lys Thr Tyr Gin Gly
95 100 105
Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lys
110 115 120
Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Aεn Lys Met Phe Cys
125 130 135
Gin Leu Ala Lys Thr Cys Pro Val Gin Leu Trp Val Asp Ser Thr
140 145 150
Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gin
155 160 165
Ser Gin His Met Thr Glu Val Val Arg Arg Cys Pro His Hiε Glu
170 175 180
Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gin His Leu Ile
185 190 195
Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Aεp Arg Asn
200 205 210
Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val
215 220 225
Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser
230 235 240
Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile
245 250 255
Thr Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe
260 265 270
Glu Val Arg Val Cys Ala Cys Pro Gly Arg Aεp Arg Arg Thr Glu
275 280 285
Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro
290 295 300
Pro Gly Ser Thr Lyε Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser
305 310 315
Pro Gin Pro Lys Lys Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu
320 325 330 AGC TCC TCT CCC CAG CCA AAG AAG AAA CCA CTG GAT GGA GAA 1113
Ser Ser Ser Pro Gin Pro Lys Lys Lyε Pro Leu Asp Gly Glu
315 320 325
TAT TTC ACC CTT CAG ATC CGT GGG CGT GAG CGC TTC GAG ATG 1155
Tyr Phe Thr Leu Gin Ile Arg Gly Arg Glu Arg Phe Glu Met
330 335 340
TTC CGA GAG CTG AAT GAG GCC TTG GAA CTC AAG GAT GCC CAG 1197
Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lyε Aεp Ala Gin
345 350
GCT GGG AAG GAG CCA GGG GGG AGC AGG GCT CAC TCC AGC CAC 1239
Ala Gly Lyε Glu Pro Gly Gly Ser Arg Ala His Ser Ser His 355 360 365
CTG AAG TCC AAA AAG GGT CAG TCT ACC TCC CGC CAT AAA AAA 1281
Leu Lys Ser Lys Lys Gly Gin Ser Thr Ser Arg His Lys Lyε 370 375 380
CTC ATG TTC AAG ACA GAA GGG CCT GAC TCA GAC TGA 1317
Leu Met Phe Lys Thr Glu Gly Pro Asp Ser Asp
385 390
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 393 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Glu Glu Pro Gin Ser Aεp Pro Ser Val Glu Pro Pro Leu Ser 1 5 10 15
Gin Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn
20 25 30
Val Leu Ser Pro Leu Pro Ser Gin Ala Met Asp Aεp Leu Met Leu
35 40 45
Ser Pro Asp Asp Ile Glu Gin Trp Phe Thr Glu Asp Pro Gly Pro
50 55 60
Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro
65 70 75 30
Ser Val Thr Cyε Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys
125 130 135
Gin Leu Ala Lys Thr Cys Pro Val Gin Leu Trp Val Asp Ser Thr
140 145 150
Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gin
155 160 165
Ser Gin His Met Thr Glu Val Val Arg Arg Cys Pro His His Glu
170 175 180
Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gin His Leu Ile
185 190 195
Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Aεp Arg Asn
200 205 210
Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val
215 220 225
Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser
230 235 240
Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile
245 250 255
Thr Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Aεn Ser Phe
260 265 270
Glu Val Arg Val Cyε Ala Cyε Pro Gly Arg Asp Arg Arg Arg Glu
275 280 285
Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro
290 295 300
Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser
305 310 315
Pro Gin Pro Lys Lys Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu
320 325 330
Gin Ile Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn
335 340 345
Glu Ala Leu Glu Leu Lys Asp Ala Gin Ala Gly Lys Glu Pro Gly
350 355 360
Gly Ser Arg Ala His Ser Ser His Leu Lys Ser Lyε Lys Gly Gin
365 370 375 Gin Ile Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn
335 340 345
Glu Ala Leu Glu Leu Lys Asp Ala Gin Ala Gly Lys Glu Pro Gly
350 355 360
Gly Ser Arg Ala His Ser Ser Hiε Leu Lys Ser Lys Lys Gly Gin
365 370 375
Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys Thr Glu Gly Pro
380 385 390
Asp Ser Asp
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 393 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Met Glu Glu Pro Gin Ser Asp Pro Ser Val Glu Pro Pro Leu Ser
1 5 10 15
Gin Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn
20 25 30
Val Leu Ser Pro Leu Pro Ser Gin Ala Met Asp Asp Leu Met Leu
35 40 45
Ser Pro Asp Asp Ile Glu Gin Trp Phe Thr Glu Asp Pro Gly Pro
50 55 60
Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro
65 70 75
Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser
80 85 90
Trp Pro Leu Ser Ser Ser Val Pro Ser Gin Lys Thr Tyr Gin Gly
95 100 105
Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lys
110 115 120 32
Ser Gin His Met Thr Glu Val Val Arg Arg Cys Pro Hiε His Glu
170 175 180
Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gin Hiε Leu Ile
185 190 195
Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Aεp Arg Aεn
200 205 210
Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val
215 220 225
Gly Ser Asp Cys Thr Thr Ile Hiε Tyr Aεn Tyr Met Cyε Asn Ser
230 235 240
Ser Cyε Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile
245 250 255
Thr Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe
260 265 270
Glu Val Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Lyε Glu
275 280 285
Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro Hiε His Glu Leu Pro
290 295 300
Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser
305 310 315
Pro Gin Pro Lys Lys Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu
320 325 330
Gin Ile Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Aεn
335 340 345
Glu Ala Leu Glu Leu Lys Asp Ala Gin Ala Gly Lys Glu Pro Gly
350 355 360
Gly Ser Arg Ala His Ser Ser His Leu Lys Ser Lyε Lys Gly Gin
365 370 375
Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys Thr Glu Gly Pro
380 385 390
Asp Ser Asp Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys Thr Glu Gly Pro
380 385 390
Asp Ser Asp
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 393 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Glu Glu Pro Gin Ser Asp Pro Ser Val Glu Pro Pro Leu Ser
1 5 10 15
Gin Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn
20 25 30
Val Leu Ser Pro Leu Pro Ser Gin Ala Met Asp Asp Leu Met Leu
35 40 45
Ser Pro Asp Asp Ile Glu Gin Trp Phe Thr Glu Aεp Pro Gly Pro
50 55 60
Aεp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro
65 70 75
Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser
80 85 90
Trp Pro Leu Ser Ser Ser Val Pro Ser Gin Lyε Thr Tyr Gin Gly
95 100 105
Ser Tyr Gly Phe Arg Leu Gly Phe Leu Hiε Ser Gly Thr Ala Lyε
110 115 120
Ser Val Thr Cyε Thr Tyr Ser Pro Ala Leu Asn Lyε Met Phe Cyε
125 130 135
Gin Leu Ala Lyε Thr Cyε Pro Val Gin Leu Trp Val Aεp Ser Thr
140 145 150
Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gin
155 160 165 34
AGT TTA TTT GCT TTA AAT CCA ATG GGT TTC TCA CCA TTG GAT 837 Ser Leu Phe Ala Leu Asn Pro Met Gly Phe Ser Pro Leu Asp 10 15 20
GGT TCT AAA TCA ACC AAC GAA AAT GTA TCT GCT TCC ACT TCT 879 Gly Ser Lys Ser Thr Asn Glu Asn Val Ser Ala Ser Thr Ser
25 30
ACT GCC AAA CCA ATG GTT GGC CAA TTG ATT TTT GAT AAA TTC 921 Thr Ala Lys Pro Met Val Gly Gin Leu Ile Phe Asp Lys Phe 35 40 45
ATC AAG ACT GAA GAG GAT CCA ATT ATC AAA CAG GAT ACC CCT 963 Ile Lys Thr Glu Glu Asp Pro Ile Ile Lys Gin Asp Thr Pro 50 55 60
TCG AAC CTT GAT TTT GAT TTT GCT CTT CCA CAA ACG GCA ACT 1005 Ser Asn Leu Asp Phe Asp Phe Ala Leu Pro Gin Thr Ala Thr 65 70 75
GCA CCT GAT GCC AAG ACC GTT TTG CCA ATT CCG GAG CTA GAT 1047 Ala Pro Aεp Ala Lyε Thr Val Leu Pro Ile Pro Glu Leu Aεp 80 85 90
GAC GCT GTA GTG GAA TCT TTC TTT TCG TCA AGC ACT GAT TCA 1089 Asp Ala Val Val Glu Ser Phe Phe Ser Ser Ser Thr Asp Ser
95 100
ACT CCA ATG TTT GAG TAT GAA AAC CTA GAA GAC AAC TCT AAA 1131 Thr Pro Met Phe Glu Tyr Glu Asn Leu Glu Asp Asn Ser Lys 105 110 115
GAA TGG ACA TCC TTG TTT GAC AAT GAC ATT CCA GTT ACC ACT 1173 Glu Trp Thr Ser Leu Phe Asp Asn Asp Ile Pro Val Thr Thr 120 125 130
GAC GAT GTT TCA TTG GCT GAT AAG GCA ATT GAA TCC ACT GAA 1215 Asp Asp Val Ser Leu Ala Asp Lys Ala Ile Glu Ser Thr Glu 135 140 145
GAA GTT TCT CTG GTA CCA TCC AAT CTG GAA GTC TCG ACA ACT 1257 Glu Val Ser Leu Val Pro Ser Asn Leu Glu Val Ser Thr Thr 150 155 160
TCA TTC TTA CCC ACT CCT GTT CTA GAA GAT GCT AAA CTG ACT 1299 Ser Phe Leu Pro Thr Pro Val Leu Glu Asp Ala Lys Leu Thr 165 170 175
CAA ACA AGA AAG GTT AAG AAA CCA AAT TCA GTC GTT AAG AAG 1341 Gin Thr Arg Lys Val Lys Lys Pro Asn Ser Val Val Lys Lys
180 185 (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1824 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 778..1620
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
ATCTTCGGGG ATATAAAGTG CATGAGCATA CATCTTGAAA AAAAAAGATG 50
AAAAATTTCC GACTTTAAAT ACGGAAGATA AATACTCCAA CCTTTTTTTC 100
CAATTCCGAA ATTTTAGTCT TCTTTAAAGA AGTTTCGGCT CGCTGTCTTA 150
CCTTTTAAAA TCTTCTACTT CTTGACAGTA CTTATCTTCT TATATAATAG 200
ATATACAAAA CAAAACAAAA CAAAAACTCA CAACACAGGT TACTCTCCCC 250
CCTAAATTCA AATTTTTTTT GCCCATCAGT TTCACTAGCG AATTATACAA 300
CTCACCAGCC ACACAGCTCA CTCATCTACT TCGCAATCAA AACAAAATAT 350
TTTATTTTAG TTCAGTTTAT TAAGTTATTA TCAGTATCGT ATTAAAAAAT 400
TAAAGATCAT TGAAAAATGG CTTGCTAAAC CGATTATATT TTGTTTTTAA 450
AGTAGATTAT TATTAGAAAA TTATTAAGAG AATTATGTGT TAAATTTATT 500
GAAAGAGAAA ATTTATTTTC CCTTATTAAT TAAAGTCCTT TACTTTTTTT 550
GAAAACTGTC AGTTTTTTGA AGAGTTATTT GTTTTGTTAC CAATTGCTAT 600
CATGTACCCG TAGAATTTTA TTCAAGATGT TTCCGTAACG GTTACCTTTC 650
TGTCAAATTA TCCAGGTTTA CTCGCCAATA AAAATTTCCC TATACTATCA 700
TTAATTAAAT CATTATTATT ACTAAAGTTT TGTTTACCAA TTTGTCTGCT 750
CAAGAAAATA AATTAAATAC AAATAAA ATG TCC GAA TAT CAG CCA 795
Met Ser Glu Tyr Gin Pro 1 5 36
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Met Ser Glu Tyr Gin Pro Ser Leu Phe Ala Leu Asn Pro Met Gly
1 5 10 15
Phe Ser Pro Leu Asp Gly Ser Lys Ser Thr Asn Glu Asn Val Ser
20 25 30
Ala Ser Thr Ser Thr Ala Lys Pro Met Val Gly Gin Leu Ile Phe
35 40 45
Asp Lys Phe Ile Lys Thr Glu Glu Asp Pro Ile Ile Lys Gin Asp
50 55 60
Thr Pro Ser Asn Leu Aεp Phe Asp Phe Ala Leu Pro Gin Thr Ala
65 70 75
Thr Ala Pro Asp Ala Lys Thr Val Leu Pro Ile Pro Glu Leu Asp
80 85 90
Asp Ala Val Val Glu Ser Phe Phe Ser Ser Ser Thr Asp Ser Thr
95 100 105
Pro Met Phe Glu Tyr Glu Asn Leu Glu Asp Asn Ser Lys Glu Trp
110 115 120
Thr Ser Leu Phe Asp Aεn Asp Ile Pro Val Thr Thr Aεp Aεp Val
125 130 135
Ser Leu Ala Asp Lys Ala Ile Glu Ser Thr Glu Glu Val Ser Leu
140 145 150
Val Pro Ser Asn Leu Glu Val Ser Thr Thr Ser Phe Leu Pro Thr
155 160 165
Pro Val Leu Glu Asp Ala Lys Leu Thr Gin Thr Arg Lys Val Lys
170 175 180
Lys Pro Asn Ser Val Val Lys Lyε Ser His His Val Gly Lys Asp
185 190 195
Asp Glu Ser Arg Leu Asp His Leu Gly Val Val Ala Tyr Asn Arg
200 205 210
Lys Gin Arg Ser lie Pro Leu Ser Pro Ile Val Pro Glu Ser Ser
215 220 225
Asp Pro Ala Ala Leu Lys Arg Ala Arg Asn Thr Glu Ala Ala Arg
230 235 240 TCA CAT CAT GTT GGA AAG GAT GAC GAA TCG AGA CTG GAT CAT 1383 Ser His His Val Gly Lys Asp Asp Glu Ser Arg Leu Asp His 190 195 200
CTA GGT GTT GTT GCT TAC AAC CGC AAA CAG CGT TCG ATT CCA 1425 Leu Gly Val Val Ala Tyr Asn Arg Lys Gin Arg Ser Ile Pro 205 210 215
CTT TCT CCA ATT GTG CCC GAA TCC AGT GAT CCT GCT GCT CTA 1467 Leu Ser Pro Ile Val Pro Glu Ser Ser Asp Pro Ala Ala Leu 220 225 230
AAA CGT GCT AGA AAC ACT GAA GCC GCC AGG CGT TCT CGT GCG 1509 Lys Arg Ala Arg Asn Thr Glu Ala Ala Arg Arg Ser Arg Ala 235 240 245
AGA AAG TTG CAA AGA ATG AAA CAA CTT GAA GAC AAG GTT GAA 1551 Arg Lys Leu Gin Arg Met Lys Gin Leu Glu Asp Lys Val Glu
250 255
GAA TTG CTT TCG AAA AAT TAT CAC TTG GAA AAT GAG GTT GCC 1593 Glu Leu Leu Ser Lys Asn Tyr His Leu Glu Asn Glu Val Ala 260 265 270
AGA TTA AAG AAA TTA GTT GGC GAA CGC TGATTTCATT 1630
Arg Leu Lys Lys Leu Val Gly Glu Arg 275 280
TACCTTTTAT TTTATATTTT TTATTTCATT CTCGTGTATA ACGAAATAGA 1680
TACATTCACT TAGATAAGAA TTTAATCTTT TTTATGCCAA TTTTCTTAAG 1730
TAGAATTTTA CACCACGCAT TTATAATCTG CCGTATGTTC TGGTATTTAC 1780
TGGTTAGGAA TAGATAAAAA AAACACTCAC GATGGGGGTC GAAC 1824
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 281 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein 38
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Arg Gly Gly Asn Pro Glu 1 5
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10
Gly Gly Asn Gin Ala
1 5
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 393 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
Met Glu Glu Pro Gin Ser Asp Pro Ser Val Glu Pro Pro Leu Ser 1 5 10 15
Gin Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn
20 25 30
Val Leu Ser Pro Leu Pro Ser Gin Ala Met Asp Asp Leu Met Leu
35 40 45
Ser Pro Asp Asp Ile Glu Gin Trp Phe Thr Glu Asp Pro Gly Pro
50 55 60
Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro
65 70 75 Arg Ser Arg Ala Arg Lys Leu Gin Arg Met Lys Gin Leu Glu Asp
245 250 255
Lys Val Glu Glu Leu Leu Ser Lys Asn Tyr His Leu Glu Asn Glu
260 265 270
Val Ala Arg Leu Lys Lys Leu Val Gly Glu Arg
275 280
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Gly Asn Pro Glu
1
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Arg Gly Asn 1
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide 40
Gin Ile Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn
335 340 345
Glu Ala Leu Glu Leu Lys Asp Ala Gin Ala Gly Lys Glu Pro Gly
350 355 360
Gly Ser Arg Ala His Ser Ser His Leu Lyε Ser Lyε Lyε Gly Gin
365 370 375
Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys Thr Glu Gly Pro
380 385 390
Asp Ser Asp
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 393 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Met Glu Glu Pro Gin Ser Asp Pro Ser Val Glu Pro Pro Leu Ser 1 5 10 15
Gin Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn
20 25 30
Val Leu Ser Pro Leu Pro Ser Gin Ala Met Asp Asp Leu Met Leu
35 40 45
Ser Pro Asp Asp Ile Glu Gin Trp Phe Thr Glu Asp Pro Gly Pro
50 55 60
Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro
65 70 75
Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser
80 85 90
Trp Pro Leu Ser Ser Ser Val Pro Ser Gin Lys Thr Tyr Gin Gly
95 100 105
Ser Tyr Gly Phe Arg Leu Gly Phe Leu Hiε Ser Gly Thr Ala Lys
110 115 120 Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser
80 85 90
Trp Pro Leu Ser Ser Ser Val Pro Ser Gin Lys Thr Tyr Gin Gly
95 100 105
Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lyε
110 115 120
Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys
125 130 135
Gin Leu Ala Lys Thr Cys Pro Val Gin Leu Trp Val Asp Ser Thr
140 145 150
Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gin
155 160 165
Ser Gin His Met Thr Glu Val Val Arg Arg Cys Pro His His Glu
170 175 180
Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gin His Leu Ile
185 190 195
Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Aεp Aεp Arg Aεn
200 205 210
Thr Phe Arg Hiε Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val
215 220 225
Gly Ser Aεp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser
230 235 240
Ser Cys Met Gly Gly Met Asn Gin Arg Pro Ile Leu Thr Ile Ile
245 250 255
Thr Leu Glu Asp Ser Ser Gly Aεn Leu Leu Gly Arg Aεn Ser Phe
260 265 270
Glu Val Arg Val Cyε Ala Cyε Pro Gly Arg Aεp Arg Arg Thr Glu
275 280 285
Glu Glu Asn Leu Arg Lyε Lys Gly Glu Pro His His Glu Leu Pro
290 295 300
Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser
305 310 315
Pro Gin Pro Lys Lyε Lyε Pro Leu Aεp Gly Glu Tyr Phe Thr Leu
320 325 330 42
Ser Thr Ser Arg His Lys Lyε Leu Met Phe Lyε Thr Glu Gly Pro
380 385 390
Asp Ser Asp
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 393 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
Met Glu Glu Pro Gin Ser Asp Pro Ser Val Glu Pro Pro Leu Ser 1 5 10 15
Gin Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn
20 25 30
Val Leu Ser Pro Leu Pro Ser Gin Ala Met Asp Asp L«ΪU Met Leu
35 40 45
Ser Pro Aεp Aεp Ile Glu Gin Trp Phe Thr Glu Asp Pro Gly Pro
50 55 60
Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro
65 70 75
Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser
80 85 90
Trp Pro Leu Ser Ser Ser Val Pro Ser Gin Lys Thr Tyr Gin Gly
95 100 105
Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lys
110 115 120
Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys
125 130 135
Gin Leu Ala Lys Thr Cys Pro Val Gin Leu Trp Val Asp Ser Thr
140 145 150
Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gin
155 160 165 Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys
125 130 135
Gin Leu Ala Lys Thr Cys Pro Val Gin Leu Trp Val Asp Ser Thr
140 145 150
Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gin
155 160 165
Ser Gin His Met Thr Glu Val Val Arg Arg Cys Pro His His Glu
170 175 180
Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gin His Leu Ile
185 190 195
Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp Arg Asn
200 205 210
Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val
215 220 225
Gly Ser Aεp Cyε Thr Thr Ile Hiε Tyr Aεn Tyr Met Cys Asn Ser
230 235 240
Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile
245 250 255
Thr Leu Glu Asp Ser Ser Gly Aεn Leu Leu Gly Arg Aεn Ser Phe
260 265 270
Glu Val Hiε Val Cyε Ala Cyε Pro Gly Arg Aεp Arg Arg Thr Glu
275 280 285
Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro
290 295 300
Pro Gly Ser Thr Lys Arg Ala Leu Pro Aεn Aεn Thr Ser Ser Ser
305 310 315
Pro Gin Pro Lys Lys Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu
320 325 330
Gin Ile Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn
335 340 345
Glu Ala Leu Glu Leu Lys Asp Ala Gin Ala Gly Lys Glu Pro Gly
350 355 360
Gly Ser Arg Ala His Ser Ser His Leu Lys Ser Lys Lys Gly Gin
365 370 375 44 (2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 393 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Met Glu Glu Pro Gin Ser Asp Pro Ser Val Glu Pro Pro Leu Ser 1 5 10 15
Gin Glu Thr Phe Ser Aεp Leu Trp Lyε Leu Leu Pro Glu Asn Asn
20 25 30
Val Leu Ser Pro Leu Pro Ser Gin Ala Met Asp Asp Leu Met Leu
35 40 45
Ser Pro Asp Asp Ile Glu Gin Trp Phe Thr Glu Asp Pro Gly Pro
50 55 60
Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro
65 70 75
Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser
80 85 90
Trp Pro Leu Ser Ser Ser Val Pro Ser Gin Lys Thr Tyr Gin Gly
95 100 105
Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lyε
110 115 120
Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys
125 130 135
Gin Leu Ala Lys Thr Cys Pro Val Gin Leu Trp Val Asp Ser Thr
140 145 150
Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gin
155 160 165
Ser Gin Hiε Met Thr Glu Val Val Arg Arg Cys Pro His His Glu
170 175 180
Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gin His Leu Ile
185 190 195 Ser Gin His Met Thr Glu Val Val Arg Arg Cyε Pro His His Glu
170 175 180
Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gin His Leu Ile
185 190 195
Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp Arg Asn
200 205 210
Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val
215 220 225
Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser
230 235 240
Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile
245 250 255
Thr Leu Glu Asp Ser Ser Gly Aεn Leu Leu Gly Arg Aεn Ser Phe
260 265 270
Glu Val Cyε Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu
275 280 285
Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro
290 295 300
Pro Gly Ser Thr Lys Arg Ala Leu Pro Aεn Aεn Thr Ser Ser Ser
305 310 315
Pro Gin Pro Lyε Lyε Lyε Pro Leu Aεp Gly Glu Tyr Phe Thr Leu
320 325 330
Gin Ile Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Aεn
335 340 345
Glu Ala Leu Glu Leu Lys Asp Ala Gin Ala Gly Lys Glu Pro Gly
350 355 360
Gly Ser Arg Ala His Ser Ser His Leu Lys Ser Lys Lys Gly Gin
365 370 375
Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys Thr Glu Gly Pro
380 385 390
Asp Ser Asp 46
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
Met Glu Glu Pro Gin Ser Asp Pro Ser Val Glu Pro Pro Leu Ser
1 5 10 15
Gin Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn
20 25 30
Val Leu Ser Pro Leu Pro Ser Gin Ala Met Asp Asp Leu Met Leu
35 40 45
Ser Pro Asp Aεp Ile Glu Gin Trp Phe Thr Glu Aεp Pro Gly Pro
50 55 60
Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro
65 70 75
Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser
80 85 90
Trp Pro Leu Ser Ser Ser Val Pro Ser Gin Lys Thr Tyr Gin Gly
95 100 105
Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lys
110 115 120
Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys
125 130 135
Gin Leu Ala Lys Thr Cys Pro Val Gin Leu Trp Val Asp Ser Thr
140 145 150
Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gin
155 160 165
Ser Gin His Met Thr Glu Val Val Arg Arg Cys Pro His His Glu
170 175 180
Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gin His Leu Ile
185 190 195
Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Aεp Aεp Arg Asn
200 205 210
Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val
215 220 225
Gly Ser Asp Cys Thr Thr Ile Hiε Tyr Asn Tyr Met Cys Asn Ser
230 235 240
Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile
245 250 255 Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp Arg Asn
200 205 210
Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val
215 220 225
Gly Ser Asp Cys Thr Thr Ile Hiε Tyr Aεn Tyr Met Cyε Asn Ser
230 235 240
Ser Cys Met Gly Gly Met Asn Gin Arg Pro Ile Leu Thr Ile Ile
245 250 255
Thr Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe
260 265 270
Glu Val Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Arg Glu
275 280 285
Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro
290 295 300
Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser
305 310 315
Pro Gin Pro Lys Lys Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu
320 325 330
Gin Ile Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn
335 340 345
Glu Ala Leu Glu Leu Lyε Asp Ala Gin Ala Gly Lys Glu Pro Gly
350 355 360
Gly Ser Arg Ala His Ser Ser His Leu Lys Ser Lys Lys Gly Gin
365 370 375
Ser Thr Ser Arg His Lyε Lyε Leu Met Phe Lyε Thr Glu Gly Pro
380 385 390
Asp Ser Asp
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 393 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein 48
Ser Pro Asp Asp Ile Glu Gin Trp Phe Thr Glu Asp Pro Gly Pro
50 55 60
Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro
65 70 75
Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser
80 85 90
Trp Pro Leu Ser Ser Ser Val Pro Ser Gin Lys Thr Tyr Gin Gly
95 100 105
Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lys
110 115 120
Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys
125 130 135
Gin Leu Ala Lys Thr Cyε Pro Val Gin Leu Trp Val Asp Ser Thr
140 145 150
Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gin
155 160 165
Ser Gin His Met Thr Glu Val Val Arg Arg Cys Pro His His Glu
170 175 180
Arg Cys Ser Asp Ser Aεp Gly Leu Ala Pro Pro Gin Hiε Leu Ile
185 190 195
Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp Arg Asn
200 205 210
Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val
215 220 225
Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser
230 235 240
Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile
245 250 255
Thr Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe
260 265 270
Glu Val Cys Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Arg Glu
275 280 285
Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro
290 295 300 Thr Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe
260 265 270
Glu Val His Val Cys Ala Cys Pro Gly Arg Aεp Arg Arg Arg Glu
275 280 285
Glu Glu Aεn Leu Arg Lyε Lyε Gly Glu Pro Hiε His Glu Leu Pro
290 295 300
Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser
305 310 315
Pro Gin Pro Lys Lys Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu
320 325 330
Gin Ile Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Aεn
335 340 345
Glu Ala Leu Glu Leu Lyε Aεp Ala Gin Ala Gly Lyε Glu Pro Gly
350 355 360
Gly Ser Arg Ala Hiε Ser Ser His Leu Lyε Ser Lyε Lyε Gly Gin
365 370 375
Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys Thr Glu Gly Pro
380 385 390
Asp Ser Asp
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 393 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Met Glu Glu Pro Gin Ser Asp Pro Ser Val Glu Pro Pro Leu Ser 1 5 10 15
Gin Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn
20 25 30
Val Leu Ser Pro Leu Pro Ser Gin Ala Met Asp Aεp Leu Met Leu
35 40 45 50
Trp Pro Leu Ser Ser Ser Val Pro Ser Gin Lys Thr Tyr Gin Gly
95 100 105
Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lys
110 115 120
Ser Val Thr Cyε Thr Tyr Ser Pro Ala Leu Aεn Lyε Met Phe Cyε
125 130 135
Gin Leu Ala Lyε Thr Cyε Pro Val Gin Leu Trp Val Asp Ser Thr
140 145 150
Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gin
155 160 165
Ser Gin His Met Thr Glu Val Val Arg Arg Cys Pro His His Glu
170 175 180
Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gin His Leu Ile
185 190 195
Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp Arg Asn
200 205 210
Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val
215 220 225
Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser
230 235 240
Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile
245 250 255
Thr Leu Glu Asp Ser Ser Gly Aεn Leu Leu Gly Arg Asn Ser Phe
260 265 270
Glu Val Arg Val Cys Ala Cyε Pro Gly Arg Asp Arg Arg Thr Glu
275 280 285
Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro
290 295 300
Pro Gly Ser Thr Lys Arg Ala Leu Pro Aεn Asn Thr Ser Ser Ser
305 310 315
Pro Gin Pro Lys Lyε Lyε Pro Leu Asp Gly Glu Tyr Phe Thr Leu
320 325 330
Gin Ile Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn
335 340 345 Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser
305 310 315
Pro Gin Pro Lyε Lys Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu
320 325 330
Gin Ile Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn
335 340 345
Glu Ala Leu Glu Leu Lys Asp Ala Gin Ala Gly Lys Glu Pro Gly
350 355 360
Gly Ser Arg Ala His Ser Ser His Leu Lys Ser Lys Lys Gly Gin
365 370 375
Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys Thr Glu Gly Pro
380 385 390
Asp Ser Asp
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 363 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
Met Glu Glu Pro Gin Ser Asp Pro Ser Val Glu Pro Pro Leu Ser 1 5 10 15
Gin Glu Thr Phe Ser Aεp Leu Trp Lyε Leu Leu Pro Glu Aεn Asn
20 25 30
Val Leu Ser Pro Leu Pro Ser Gin Ala Met Asp Aεp Leu Met Leu
35 40 45
Ser Pro Asp Asp Ile Glu Gin Trp Phe Thr Glu Asp Pro Gly Pro
50 55 60
Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro
65 70 75
Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser
80 85 90 52
Ser Gin His Met Thr Glu Val Val Arg Arg Cys Pro His His Glu
170 175 180
Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gin His Leu Ile
185 190 195
Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Aεp Arg Asn
200 205 210
Thr Phe Arg Hiε Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val
215 220 225
Gly Ser Aεp Cyε Thr Thr Ile Hiε Tyr Aεn Tyr Met Cyε Asn Ser
230 235 240
Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile
245 250 255
Thr Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe
260 265 270
Glu Val Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Arg Glu
275 280 285
Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro
290 295 300
Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser
305 310 315
Pro Gin Pro Lyε Lys Lys Pro Leu Aεp Gly Glu Tyr Phe Thr Leu
320 325 330
Gin Ile Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn
335 340 345
Glu Ala Leu Glu Leu Lyε Asp Ala Gin Ala Gly Lys Glu Pro Gly
350 355 360
Gly Ser Arg
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 363 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein Glu Ala Leu Glu Leu Lys Asp Ala Gin Ala Gly Lys Glu Pro Gly
350 355 360
Gly Ser Arg
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 363 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
Met Glu Glu Pro Gin Ser Asp Pro Ser Val Glu Pro Pro Leu Ser
1 5 10 15
Gin Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn
20 25 30
Val Leu Ser Pro Leu Pro Ser Gin Ala Met Asp Asp Leu Met Leu
35 40 45
Ser Pro Asp Asp Ile Glu Gin Trp Phe Thr Glu Asp Pro Gly Pro
50 55 60
Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro
65 70 75
Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser
80 85 90
Trp Pro Leu Ser Ser Ser Val Pro Ser Gin Lys Thr Tyr Gin Gly
95 100 105
Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lys
110 115 120
Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe Cyε
125 130 135
Gin Leu Ala Lys Thr Cys Pro Val Gin Leu Trp Val Asp Ser Thr
140 145 150
Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gin
155 160 165 54
Thr Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Aεn Ser Phe
260 265 270
Glu Val Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu
275 280 285
Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro
290 295 300
Pro Gly Ser Thr Lys Arg Ala Leu Pro Aεn Aεn Thr Ser Ser Ser
305 310 315
Pro Gin Pro Lyε Lyε Lyε Pro Leu Asp Gly Glu Tyr Phe Thr Leu
320 325 330
Gin Ile Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn
335 340 345
Glu Ala Leu Glu Leu Lys Asp Ala Gin Ala Gly Lys Glu Pro Gly
350 355 360
Gly Ser Arg
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 363 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
Met Glu Glu Pro Gin Ser Asp Pro Ser Val Glu Pro Pro Leu Ser 1 5 10 15
Gin Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn
20 25 30
Val Leu Ser Pro Leu Pro Ser Gin Ala Met Asp Asp Leu Met Leu
35 40 45
Ser Pro Asp Asp Ile Glu Gin Trp Phe Thr Glu Aεp Pro Gly Pro
50 55 60
Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro
65 70 75 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
Met Glu Glu Pro Gin Ser Asp Pro Ser Val Glu Pro Pro Leu Ser
1 5 10 15
Gin Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn
20 25 30
Val Leu Ser Pro Leu Pro Ser Gin Ala Met Asp Aεp Leu Met Leu
35 40 45
Ser Pro Aεp Aεp Ile Glu Gin Trp Phe Thr Glu Aεp Pro Gly Pro
50 55 60
Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro
65 70 75
Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser
80 85 90
Trp Pro Leu Ser Ser Ser Val Pro Ser Gin Lys Thr Tyr Gin Gly
95 100 105
Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lys
110 115 120
Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys
125 130 135
Gin Leu Ala Lys Thr Cyε Pro Val Gin Leu Trp Val Aεp Ser Thr
140 145 150
Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lyε Gin
155 160 165
Ser Gin Hiε Met Thr Glu Val Val Arg Arg Cyε Pro Hiε His Glu
170 175 180
Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gin His Leu Ile
185 190 195
Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp Arg Asn
200 205 210
Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val
215 220 225
Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Aεn Ser
230 235 240
Ser Cyε Met Gly Gly Met Asn Gin Arg Pro Ile Leu Thr Ile Ile
245 250 255 56
Gin Ile Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn
335 340 345
Glu Ala Leu Glu Leu Lys Asp Ala Gin Ala Gly Lys Glu Pro Gly
350 355 360
Gly Ser Arg
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 363 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
Met Glu Glu Pro Gin Ser Asp Pro Ser Val Glu Pro Pro Leu Ser 1 5 10 15
Gin Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn
20 25 30
Val Leu Ser Pro Leu Pro Ser Gin Ala Met Asp Aεp Leu Met Leu
35 40 45
Ser Pro Asp Asp Ile Glu Gin Trp Phe Thr Glu Aεp Pro Gly Pro
50 55 60
Aεp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro
65 70 75
Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser
80 85 90
Trp Pro Leu Ser Ser Ser Val Pro Ser Gin Lyε Thr Tyr Gin Gly
95 100 105
Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lys
110 115 120
Ser Val Thr Cyε Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys
125 130 135
Gin Leu Ala Lys Thr Cys Pro Val Gin Leu Trp Val Asp Ser Thr
140 145 150 Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser
80 85 90
Trp Pro Leu Ser Ser Ser Val Pro Ser Gin Lys Thr Tyr Gin Gly
95 100 105
Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lys
110 115 120
Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe Cyε
125 130 135
Gin Leu Ala Lys Thr Cyε Pro Val Gin Leu Trp Val Aεp Ser Thr
140 145 150
Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gin
155 160 165
Ser Gin His Met Thr Glu Val Val Arg Arg Cys Pro His His Glu
170 175 180
Arg Cys Ser Aεp Ser Aεp Gly Leu Ala Pro Pro Gin His Leu Ile
185 190 195
Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Aεp Asp Arg Asn
200 205 210
Thr Phe Arg Hiε Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val
215 220 225
Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser
230 235 240
Ser Cys Met Gly Gly Met Aεn Gin Arg Pro Ile Leu Thr Ile Ile
245 250 255
Thr Leu Glu Aεp Ser Ser Gly Aεn Leu Leu Gly Arg Aεn Ser Phe
260 265 270
Glu Val Arg Val Cyε Ala Cyε Pro Gly Arg Aεp Arg Arg Arg Glu
275 280 285
Glu Glu Aεn Leu Arg Lyε Lyε Gly Glu Pro His Hiε Glu Leu Pro
290 295 300
Pro Gly Ser Thr Lyε Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser
305 310 315
Pro Gin Pro Lyε Lyε Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu
320 325 330 58 (2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 363 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
Met Glu Glu Pro Gin Ser Asp Pro Ser Val Glu Pro Pro Leu Ser 1 5 10 15
Gin Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn
20 25 30
Val Leu Ser Pro Leu Pro Ser Gin Ala Met Asp Aεp Leu Met Leu
35 40 45
Ser Pro Aεp Aεp Ile Glu Gin Trp Phe Thr Glu Asp Pro Gly Pro
50 55 60
Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro
65 70 75
Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser
80 85 90
Trp Pro Leu Ser Ser Ser Val Pro Ser Gin Lys Thr Tyr Gin Gly
95 100 105
Ser Tyr Gly Phe Arg Leu Gly Phe Leu Hiε Ser Gly Thr Ala Lys
110 115 120
Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys
125 130 135
Gin Leu Ala Lyε Thr Cys Pro Val Gin Leu Trp Val Asp Ser Thr
140 145 150
Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gin
155 160 165
Ser Gin His Met Thr Glu Val Val Arg Arg Cys Pro His His Glu
170 175 180
Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gin His Leu Ile
185 190 195 Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gin
155 160 165
Ser Gin His Met Thr Glu Val Val Arg Arg Cys Pro His His Glu
170 175 180
Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gin His Leu Ile
185 190 195
Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Aεp Arg Asn
200 205 210
Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val
215 220 225
Gly Ser Asp Cyε Thr Thr Ile Hiε Tyr Aεn Tyr Met Cyε Aεn Ser
230 235 240
Ser Cyε Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile
245 250 255
Thr Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe
260 265 270
Glu Val Hiε Val Cyε Ala Cyε Pro Gly Arg Aεp Arg Arg Thr Glu
275 280 285
Glu Glu Asn Leu Arg Lys Lyε Gly Glu Pro His His Glu Leu Pro
290 295 300
Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser
305 310 315
Pro Gin Pro Lys Lys Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu
320 325 330
Gin Ile Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn
335 340 345
Glu Ala Leu Glu Leu Lys Asp Ala Gin Ala Gly Lys Glu Pro Gly
350 355 360
Gly Ser Arg 60
Gin Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn
20 25 30
Val Leu Ser Pro Leu Pro Ser Gin Ala Met Asp Asp Leu Met Leu
35 40 45
Ser Pro Asp Asp Ile Glu Gin Trp Phe Thr Glu Asp Pro Gly Pro
50 55 60
Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro
65 70 75
Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser
80 85 90
Trp Pro Leu Ser Ser Ser Val Pro Ser Gin Lys Thr Tyr Gin Gly
95 100 105
Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lys
110 115 120
Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys
125 130 135
Gin Leu Ala Lys Thr Cys Pro Val Gin Leu Trp Val Asp Ser Thr
140 145 150
Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gin
155 160 165
Ser Gin His Met Thr Glu Val Val Arg Arg Cys Pro His His Glu
170 175 180
Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gin His Leu Ile
185 190 195
Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp Arg Aεn
200 205 210
Thr Phe Arg Hiε Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val
215 220 225
Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser
230 235 240
Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile
245 250 255
Thr Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe
260 265 270 Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Aεp Aεp Arg Asn 200 205 210
Thr Phe Arg Hiε Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val 215 220 225
Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser 230 235 240
Ser Cys Met Gly Gly Met Aεn Arg Arg Pro Ile Leu Thr Ile Ile 245 250 255
Thr Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe 260 265 270
Glu Val His Val Cys Ala Cyε Pro Gly Arg Asp Arg Arg Arg Glu 275 280 285
Glu Glu Asn Leu Arg Lyε Lyε Gly Glu Pro Hiε Hiε Glu Leu Pro 290 295 300
Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn Aεn Thr Ser Ser Ser 305 310 315
Pro Gin Pro Lys Lyε Lyε Pro Leu Asp Gly Glu Tyr Phe Thr Leu 320 325 330
Gin lie Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn 335 340 345
Glu Ala Leu Glu Leu Lyε Aεp Ala Gin Ala Gly Lyε Glu Pro Gly 350 355 360
Gly Ser Arg
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 363 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Met Glu Glu Pro Gin Ser Asp Pro Ser Val Glu Pro Pro Leu Ser 1 5 10 15 62
Trp Pro Leu Ser Ser Ser Val Pro Ser Gin Lys Thr Tyr Gin Gly
95 100 105
Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lys
110 115 120
Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys
125 130 135
Gin Leu Ala Lys Thr Cys Pro Val Gin Leu Trp Val Asp Ser Thr
140 145 150
Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gin
155 160 165
Ser Gin His Met Thr Glu Val Val Arg Arg Cyε Pro Hiε Hiε Glu
170 175 180
Arg Cyε Ser Aεp Ser Aεp Gly Leu Ala Pro Pro Gin Hiε Leu Ile
185 190 195
Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp Arg Asn
200 205 210
Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val
215 220 225
Gly Ser Asp Cys Thr Thr Ile Hiε Tyr Aεn Tyr Met Cyε Aεn Ser
230 235 340
Ser Cyε Met Gly Gly Met Aεn Arg Arg Pro Ile Leu Thr Ile Ile
245 250 255
Thr Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe
260 265 270
Glu Val Cyε Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Arg Glu
275 280 285
Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro
290 295 300
Pro Gly Ser Thr Lys Arg Ala Leu Pro Aεn Aεn Thr Ser Ser Ser
305 310 315
Pro Gin Pro Lyε Lyε Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu
320 325 330
Gin Ile Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn
335 340 345 Glu Val Cys Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu
275 280 285
Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro Hiε Hiε Glu Leu Pro
290 295 300
Pro Gly Ser Thr Lyε Arg Ala Leu Pro Aεn Asn Thr Ser Ser Ser
305 310 315
Pro Gin Pro Lys Lys Lyε Pro Leu Aεp Gly Glu Tyr Phe Thr Leu
320 325 330
Gin Ile Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn
335 340 345
Glu Ala Leu Glu Leu Lys Asp Ala Gin Ala Gly Lys Glu Pro Gly
350 355 360
Gly Ser Arg
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 363 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
Met Glu Glu Pro Gin Ser Asp Pro Ser Val Glu Pro Pro Leu Ser 1 5 10 15
Gin Glu Thr Phe Ser Asp Leu Trp Lyε Leu Leu Pro Glu Asn Asn
20 25 30
Val Leu Ser Pro Leu Pro Ser Gin Ala Met Asp Asp Leu Met Leu
35 40 45
Ser Pro Asp Asp Ile Glu Gin Trp Phe Thr Glu Asp Pro Gly Pro
50 55 60
Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro
65 70 75
Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser
80 85 90 64
Ser Gin His Met Thr Glu Val Val Arg His Cys Pro His His Glu
170 175 180
Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gin His Leu Ile
185 190 195
Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp Arg Asn
200 205 210
Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val
215 220 225
Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser
230 235 240
Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile
245 250 255
Thr Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Aεn Ser Phe
260 265 270
Glu Val Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu
275 280 285
Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro
290 295 300
Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser
305 310 315
Pro Gin Pro Lys Lyε Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu
320 325 330
Gin Ile Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn
335 340 345
Glu Ala Leu Glu Leu Lys Asp Ala Gin Ala Gly Lys Glu Pro Gly
350 355 360
Gly Ser Arg Ala His Ser Ser His Leu Lys Ser Lys Lys Gly Gin
365 370 375
Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys Thr Glu Gly Pro
380 385 390
Asp Ser Asp Glu Ala Leu Glu Leu Lys Asp Ala Gin Ala Gly Lys Glu Pro Gly
350 355 360
Gly Ser Arg
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 393 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
Met Glu Glu Pro Gin Ser Asp Pro Ser Val Glu Pro Pro Leu Ser
1 5 10 15
Gin Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn
20 25 30
Val Leu Ser Pro Leu Pro Ser Gin Ala Met Asp Asp Leu Met Leu
35 40 45
Ser Pro Asp Asp Ile Glu Gin Trp Phe Thr Glu Aεp Pro Gly Pro
50 55 60
Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro
65 70 75
Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser
80 85 90
Trp Pro Leu Ser Ser Ser Val Pro Ser Gin Lys Thr Tyr Gin Gly
95 100 105
Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lys
110 115 120
Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys
125 130 135
Gin Leu Ala Lyε Thr Cys Pro Val Gin Leu Trp Val Asp Ser Thr
140 145 150
Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gin
155 160 165 66
Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp Arg Asn
200 205 210
Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val
215 220 225
Gly Ser Aεp Cyε Thr Thr Ile Hiε Tyr Asn Tyr Met Cys Asn Ser
230 235 240
Ser Cys Met Gly Gly Met Asn Trp Arg Pro Ile Leu Thr Ile Ile
245 250 255
Thr Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe
260 265 270
Glu Val Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu
275 280 285
Glu Glu Asn Leu Arg Lyε Lys Gly Glu Pro His His Glu Leu Pro
290 295 300
Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn Aεn Thr Ser Ser Ser
305 310 315
Pro Gin Pro Lyε Lyε Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu
320 325 330
Gin Ile Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn
335 340 345
Glu Ala Leu Glu Leu Lys Asp Ala Gin Ala Gly Lyε Glu Pro Gly
350 355 360
Gly Ser Arg Ala His Ser Ser His Leu Lys Ser Lys Lyε Gly Gin
365 370 375
Ser Thr Ser Arg His Lyε Lys Leu Met Phe Lys Thr Glu Gly Pro
380 385 390
Asp Ser Asp
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 393 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 393 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
Met Glu Glu Pro Gin Ser Asp Pro Ser Val Glu Pro Pro Leu Ser
1 5 10 15
Gin Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn
20 25 30
Val Leu Ser Pro Leu Pro Ser Gin Ala Met Asp Asp Leu Met Leu
35 40 45
Ser Pro Asp Aεp Ile Glu Gin Trp Phe Thr Glu Aεp Pro Gly Pro
50 55 60
Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro
65 70 75
Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser
80 85 90
Trp Pro Leu Ser Ser Ser Val Pro Ser Gin Lys Thr Tyr Gin Gly
95 100 105
Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lyε
110 115 120
Ser Val Thr Cyε Thr Tyr Ser Pro Ala Leu Aεn Lyε Met Phe Cyε
125 130 135
Gin Leu Ala Lyε Thr Cyε Pro Val Gin Leu Trp Val Aεp Ser Thr
140 145 150
Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lyε Gin
155 160 165
Ser Gin Hiε Met Thr Glu Val Val Arg Arg Cyε Pro His His Glu
170 175 180
Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gin His Leu Ile
185 190 195 68
Thr Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe
260 265 270
Glu Val Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu
275 280 285
Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro
290 295 300
Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser
305 310 315
Pro Gin Pro Lys Lys Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu
320 325 330
Gin Ile Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn
335 340 345
Glu Ala Leu Glu Leu Lys Asp Ala Gin Ala Gly Lyε Glu Pro Gly
350 355 360
Gly Ser Arg Ala Hiε Ser Ser His Leu Lyε Ser Lys Lys Gly Gin
365 370 375
Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys Thr Glu Gly Pro
380 385 390
Asp Ser Asp
(2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 393 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
( i) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
Met Glu Glu Pro Gin Ser Asp Pro Ser Val Glu Pro Pro Leu Ser 1 5 10 15
Gin Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn
20 25 30
Val Leu Ser Pro Leu Pro Ser Gin Ala Met Aεp Asp Leu Met Leu
35 40 45 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
Met Glu Glu Pro Gin Ser Asp Pro Ser Val Glu Pro Pro Leu Ser 1 5 10 15
Gin Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn
20 25 30
Val Leu Ser Pro Leu Pro Ser Gin Ala Met Aεp Asp Leu Met Leu
35 40 45
Ser Pro Aεp Asp Ile Glu Gin Trp Phe Thr Glu Asp Pro Gly Pro
50 55 60
Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro
65 70 75
Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser
80 85 90
Trp Pro Leu Ser Ser Ser Val Pro Ser Gin Lys Thr Tyr Gin Gly
95 100 105
Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lys
110 115 120
Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys
125 130 135
Gin Leu Ala Lys Thr Cys Pro Val Gin Leu Trp Val Asp Ser Thr
140 145 150
Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gin
155 160 165
Ser Gin His Met Thr Glu Val Val Arg Arg Cys Pro His His Glu
170 175 180
Arg Cyε Ser Aεp Ser Aεp Gly Leu Ala Pro Pro Gin His Leu Ile
185 190 195
Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp Arg Asn
200 205 210
Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val
215 220 225
Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser
230 235 240
Ser Cys Met Gly Gly Met Asn Arg Ser Pro Ile Leu Thr Ile Ile
245 250 255 70
Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser
305 310 315
Pro Gin Pro Lys Lys Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu
320 325 330
Gin Ile Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn
335 340 345
Glu Ala Leu Glu Leu Lys Asp Ala Gin Ala Gly Lys Glu Pro Gly
350 355 360
Gly Ser Arg Ala His Ser Ser His Leu Lyε Ser Lys Lys Gly Gin
365 370 375
Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys Thr Glu Gly Pro
380 385 390
Asp Ser Asp
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: CCGGGCATGT CCGGGCATGT CCGGGCATGT 30
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: CCCGAACATG TCCCAACATG TTGGGG 26 Ser Pro Asp Asp Ile Glu Gin Trp Phe Thr Glu Asp Pro Gly Pro
50 55 60
Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro
65 70 75
Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser
80 85 90
Trp Pro Leu Ser Ser Ser Val Pro Ser Gin Lyε Thr Tyr Gin Gly
95 100 105
Ser Tyr Gly Phe Arg Leu Gly Phe Leu Hiε Ser Gly Thr Ala Lyε
110 115 120
Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lyε Met Phe Cyε
125 130 135
Gin Leu Ala Lys Thr Cys Pro Val Gin Leu Trp Val Asp Ser Thr
140 145 150
Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gin
155 160 165
Ser Gin His Met Thr Glu Val Val Arg Arg Cys Pro His Hiε Glu
170 175 180
Arg Cyε Ser Aεp Ser Aεp Gly Leu Ala Pro Pro Gin Hiε Leu Ile
185 190 195
Arg Val Glu Gly Aεn Leu Arg Val Glu Tyr Leu Asp Asp Arg Asn
200 205 210
Thr Phe Arg Hiε Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val
215 220 225
Gly Ser Aεp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser
230 235 240
Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile
245 250 255
Thr Leu Glu Asp Ser Ser Gly Aεn Leu Leu Gly Arg Aεn Ser Phe
260 265 270
Glu Val Arg Val Cyε Ala Cyε Pro Gly Arg Aεp Trp Arg Thr Glu
275 280 285
Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro
290 295 300 72
Ala Ser Thr Ser Thr Ala Lys Pro Met Val Gly Gin Leu Ile Phe
35 40 45
Asp Lys Phe Ile Lyε Thr Glu Glu Asp Pro Ile Ile Lys Gin Asp
50 55 60
Thr Pro Ser Asn Leu Asp Phe Asp Phe Ala Leu Pro Gin Thr Ala
65 70 75
Thr Ala Pro Aεp Ala Lys Thr Val Leu Pro Ile Pro Glu Leu Asp
80 85 90
Asp Ala Val Val Glu Ser Phe Phe Ser Ser Ser Thr Asp Ser Thr
95 100 105
Pro Met Phe Glu Tyr Glu Asn Leu Glu Asp Asn Ser Lyε Glu Trp
110 115 120
Thr Ser Leu Phe Aεp Aεn Aεp Ile Pro Val Thr Thr Asp Asp Val
125 130 135
Ser Leu Ala Asp Lys Ala Ile Glu Ser Thr Glu Glu Val Ser Leu
140 145 150
Val Pro Ser Asn Leu Glu Val Ser Thr Thr Ser Phe Leu Pro Thr
155 160 165
Pro Val Leu Glu Aεp Ala Lys Leu Thr Gin Thr Arg Lys Val Lys
170 175 180
Lys Pro Asn Ser Val Val Lys Lys Ser Hiε Hiε Val Gly Lyε Aεp
185 190 195
Asp Glu Ser Arg Leu Aεp Hiε Leu Gly Val Val Ala Tyr Aεn Arg
200 205 210
Lyε Gin Arg Ser Ile Pro Leu Ser Pro Ile Val Pro Glu Ser Ser
215 220 225
Asp Pro Ala Ala Leu Lyε Arg Ala Arg Aεn Thr Glu Ala Ala Arg
230 235 240
Arg Ser Arg Ala Arg Lyε Leu Gin Arg Met Lyε Gin Ile Glu Aεp
245 250 255
Lyε Leu Glu Glu Ile Leu Ser Lys Leu Tyr Hiε Ile Glu Asn Glu
260 265 270
Leu Ala Arg Ile Lys Lys Leu Leu Gly Glu Arg
275 280 (2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: ACAGAACATG TCTAAGCATG CTGGGGA 27
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: ATCACGTGAT 10
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 281 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
Met Ser Glu Tyr Gin Pro Ser Leu Phe Ala Leu Asn Pro Met Gly 1 5 10 15
Phe Ser Pro Leu Asp Gly Ser Lys Ser Thr Aεn Glu Aεn Val Ser
20 25 30 74
7. The modified p53 construct according to claim 4 wherein the p53 amino acid sequence is a chimeric p53 protein.
8. The modified p53 construct according to claim 4 wherein the p53 amino acid sequence contains an engineered p53 DNA binding domain.
9. A pharmaceutical composition comprising a modified p53 protein construct according to claim 1 and a pharmaceutically acceptable carrier.
10. A method of enhancing the DNA-binding ability of a p53 construct having a p53 DNA binding domain comprising the step of: modifying the codon encoding the amino acid correεponding to residue 284 of wild-type p53 to a codon encoding arginine, whereby the resulting modified p53 construct is characterized by enhanced DNA-binding ability.
11. The method according to claim 10 wherein the p53 amino acid sequence iε a natural or engineered mutant p53.
12. The method according to claim 10 wherein the p53 amino acid εequence is a chimeric p53 protein.
13. A nucleotide sequence encoding a modified p53 protein construct having DNA binding activity comprising a p53 amino acid sequence in which the threonine corresponding to amino acid residue 284 of the wild-type p53 protein is changed to arginine.

Claims

What is claimed is:
1. A modified p53 protein construct having DNA binding ability comprising a p53 amino acid sequence in which the threonine corresponding to amino acid residue 284 of the wild-type human p53 protein is changed to arginine.
2. The modified p53 construct according to claim 1 wherein the p53 amino acid sequence iε full- length human wild-type human p53.
3. The modified p53 conεtruct according to claim 1 wherein the p53 amino acid εequence iε human wild-type p53 bearing a deletion of all or a fragment of the C-terminal reεidues 356 to 393.
4. The modified p53 construct according to claim 1 wherein the p53 amno acid sequence is a natural or engineered mutant p53 sequence.
5. The modified p53 construct according to claim 4 wherein the p53 amino acid sequence is a p53 mutant amino acid sequence selected from the group consisting of: a mutant p53 having glutamine at amino acid position 248, a mutant p53 having histidine at amino acid position 273, and a mutant p53 having cysteine at amino acid poεition 273.
6. The modified p53 conεtruct according to claim 5 wherein the p53 amino acid εequence iε deleted of all or a fragment of the C-terminal reεidues 356 to 393. 76
22. The method according to claim 19 wherein the condition is cancer.
14. A vector comprising a nucleotide sequence encoding a modified p53 protein construct having DNA binding activity comprising a p53 amino acid sequence in which the threonine corresponding to amino acid residue 284 of the wild-type p53 protein is changed to arginine.
15. A pharmaceutical composition comprising a nucleic acid sequence according to claim 13 and a pharmaceutically acceptable carrier.
16. A pharmaceutical composition comprising a vector according to claim 14 and a pharmaceutically acceptable carrier.
17. A method of treating a condition associated with deficient p53 activity comprising the step of administering a pharmaceutical composition according to claim 9.
18. A method of treating a condition asεociated with deficient p53 activity compriεing the step of adminiεtering a pharmaceutical compoεition according to claim 15.
19. A method of treating a condition associated with deficient p53 activity comprising the step of administering a pharmaceutical composition according to claim 16.
20. The method according to claim 17 wherein the condition is cancer.
21. The method according to claim 18 wherein the condition is cancer.
PCT/US1996/015188 1995-09-22 1996-09-20 Modified p53 constructs and uses therefor WO1997010843A1 (en)

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US480295P 1995-09-22 1995-09-22
US60/004,802 1995-09-22
US08/697,221 US5847083A (en) 1996-08-21 1996-08-21 Modified p53 constructs which enhance DNA binding
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000022115A3 (en) * 1998-10-13 2000-09-21 Univ Texas Assays for identifying functional alterations in the p53 tumor suppressor

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5362623A (en) * 1991-06-14 1994-11-08 The John Hopkins University Sequence specific DNA binding by p53
WO1995017213A1 (en) * 1993-12-21 1995-06-29 Sloan-Kettering Institute For Cancer Research P53-based polypeptide fragments, nucleic acid molecules encoding same, and uses thereof
US5573925A (en) * 1994-11-28 1996-11-12 The Wistar Institute Of Anatomy And Biology P53 proteins with altered tetramerization domains

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5362623A (en) * 1991-06-14 1994-11-08 The John Hopkins University Sequence specific DNA binding by p53
WO1995017213A1 (en) * 1993-12-21 1995-06-29 Sloan-Kettering Institute For Cancer Research P53-based polypeptide fragments, nucleic acid molecules encoding same, and uses thereof
US5573925A (en) * 1994-11-28 1996-11-12 The Wistar Institute Of Anatomy And Biology P53 proteins with altered tetramerization domains

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CELL, 27 November 1992, Vol. 71, HUPP et al., "Regulation of the Specific DNA Binding Function of p53", pages 875-886. *
SCIENCE, 05 July 1991, Vol. 253, HOLLSTEIN et al., "p53 Mutations in Human Cancers", pages 49-53. *
THE EMBO JOURNAL, 1993, Vol. 12, No. 13, HALAZONETIS et al., "Conformational Shifts Propagate from the Oligomerization Domain of p53 to its Tetrameric DNA Binding Domain and Restore DNA Binding to Select p53 Mutants", pages 5057-5064. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000022115A3 (en) * 1998-10-13 2000-09-21 Univ Texas Assays for identifying functional alterations in the p53 tumor suppressor
US6429298B1 (en) 1998-10-13 2002-08-06 Board Of Regents, The University Of Texas System Assays for identifying functional alterations in the p53 tumor suppressor

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