Novel cystine knot protein and materials and methods for making it

Abstract

The present invention relates to polynucleotide and polypeptide molecules for zsig51, a novel member of the cystine knot family of polypeptides. The polypeptides comprise a sequence of amino acid residues that is at least 80% identical in amino acid sequence to residues 1 through 106 of SEQ ID NO:2. The invention further provides therapeutic and diagnostic methods utilizing the polynucleotides, polypeptides, and antagonists of the polypeptides.

We claim:  
     
         1 . An isolated polypeptide comprising at least 15 consecutive amino acid residues of SEQ ID NO:2.  
     
     
         2 . An isolated polypeptide that is at least 80% identical in amino acid sequence to residues 1 through 106 of SEQ ID NO:2, said polypeptide comprising cysteine residues at positions corresponding to residues 8, 34, 38, 66, 96, and 98 of SEQ ID NO:2; a glycine residue at a position corresponding to residue 36 of SEQ ID NO:2; and beta strand-like regions corresponding to residues 9-17, 29-34, 38-43, 59-64, 67-71, and 90-95 of SEQ ID NO:2.  
     
     
         3 . The isolated polypeptide according to  claim 2  further comprising cysteine residues at positions corresponding to residues 25, 65, 80, and 101 of SEQ ID NO:2.  
     
     
         4 . The isolated polypeptide according to  claim 2  wherein amino acid residues corresponding to residues 8, 11, 12, 14, 29, 30, 32, 34, 43, 44, 60, 63, 64, 65, 71, 74, 80, 90, 91, 93, and 94 of SEQ ID NO:2 are Cys, His, Pro, Asn, His, Val, Gln, Cys, Phe, Pro, Thr, Ser, Gln, Cys, Leu, Val, Cys, Ile, Phe, Ala, and Arg, respectively; and an amino acid residue corresponding to residue 75 of SEQ ID NO:2 is Lys or Arg.  
     
     
         5 . The isolated polypeptide according to  claim 2  wherein said polypeptide is at least 95% identical to residues 1 through 106 of SEQ ID NO:2.  
     
     
         6 . The isolated polypeptide according to  claim 2  comprising residue 1 through residue 106 of SEQ ID NO:2 or residue 1 through residue 106 of SEQ ID NO:29.  
     
     
         7 . The isolated polypeptide according to  claim 2 , covalently linked to an affinity tag.  
     
     
         8 . The isolated polypeptide according to  claim 2 , covalently linked to an immunoglobulin constant region.  
     
     
         9 . An isolated protein comprising a first polypeptide complexed to a second polypeptide, wherein said first polypeptide is at least 80% identical in amino acid sequence to residues 1 through 106 of SEQ ID NO:2 and comprises cysteine residues at positions corresponding to residues 8, 34, 38, 66, 96, and 98 of SEQ ID NO:2; a glycine residue at a position corresponding to residue 36 of SEQ ID NO:2; and beta strand-like regions corresponding to residues 9-17, 29-34, 38-43, 59-64, 67-71, and 90-95 of SEQ ID NO:2, and wherein said protein modulates cell proliferation, differentiation, or metabolism.  
     
     
         10 . The isolated protein according to  claim 9  wherein said first polypeptide further comprises cysteine residues at positions corresponding to residues 25, 65, 80, and 101 of SEQ ID NO:2.  
     
     
         11 . The isolated protein according to  claim 9  wherein amino acid residues of said first polypeptide corresponding to residues 8, 11, 12, 14, 29, 30, 32, 34, 43, 44, 60, 63, 64, 65, 71, 74, 80, 90, 91, 93, and 94 of SEQ ID NO:2 are Cys, His, Pro, Asn, His, Val, Gln, Cys, Phe, Pro, Thr, Ser, Gln, Cys, Leu, Val, Cys, Ile, Phe, Ala, and Arg, respectively; and an amino acid residue corresponding to residue 75 of SEQ ID NO:2 is Lys or Arg.  
     
     
         12 . The isolated protein according to  claim 9  wherein said first polypeptide is at least 95% identical to residues 1 through 106 of SEQ ID NO:2.  
     
     
         13 . The isolated protein according to  claim 9  wherein said protein is a heterodimer.  
     
     
         14 . The isolated protein according to  claim 13  wherein said second polypeptide is a glycoprotein hormone common alpha subunit.  
     
     
         15 . The isolated protein according to  claim 9  wherein said first polypeptide comprises residue 1 through residue 106 of SEQ ID NO:2 or residue 1 through residue 106 of SEQ ID NO:29.  
     
     
         16 . The isolated protein according to  claim 9  wherein said protein is a homodimer.  
     
     
         17 . The isolated protein according to  claim 16  wherein each of said first and second polypeptides comprises residue 1 through residue 106 of SEQ ID NO:2 or residue 1 through residue 106 of SEQ ID NO:29.  
     
     
         18 . An isolated polynucleotide encoding a polypeptide according to  claim 2 .  
     
     
         19 . The isolated polynucleotide according to  claim 18  comprising a sequence of nucleotides as shown in SEQ ID NO:4 from nucleotide 70 through nucleotide 387 or SEQ ID NO:30 from nucleotide 70 through nucleotide 387.  
     
     
         20 . The isolated polynucleotide according to  claim 18  comprising a sequence of nucleotides as shown in SEQ ID NO:1 from nucleotide 125 through nucleotide 442.  
     
     
         21 . The isolated polynucleotide according to  claim 18  wherein said polynucleotide is from 318 to 1000 nucleotides in length.  
     
     
         22 . The isolated polynucleotide according to  claim 18  wherein said polynucleotide is DNA.  
     
     
         23 . An expression vector comprising the following operably linked elements: 
 a transcription promoter;    a DNA segment encoding a polypeptide that is at least 90% identical in amino acid sequence to residues 1 through 106 of SEQ ID NO:2, said polypeptide comprising cysteine residues at positions corresponding to residues 8, 34, 38, 66, 96, and 98 of SEQ ID NO:2; a glycine residue at a position corresponding to residue 36 of SEQ ID NO:2; and beta strand-like regions corresponding to residues 9-17, 29-34, 38-43, 59-64, 67-71, and 90-95 of SEQ ID NO:2; and    a transcription terminator.    
     
     
         24 . The expression vector according to  claim 23  wherein said polypeptide further comprises cysteine residues at positions corresponding to residues 25, 65, 80, and 101 of SEQ ID NO:2.  
     
     
         25 . The expression vector according to  claim 23  wherein amino acid residues corresponding to residues 8, 11, 12, 14, 29, 30, 32, 34, 43, 44, 60, 63, 64, 65, 71, 74, 80, 90, 91, 93, and 94 of SEQ ID NO:2 are Cys, His, Pro, Asn, His, Val, Gln, Cys, Phe, Pro, Thr, Ser, Gln, Cys, Leu, Val, Cys, Ile, Phe, Ala, and Arg, respectively; and an amino acid residue corresponding to residue 75 of SEQ ID NO:2 is Lys or Arg.  
     
     
         26 . The expression vector according to  claim 23  wherein said polypeptide is at least 95% identical to residues 1 through 106 of SEQ ID NO:2.  
     
     
         27 . The expression vector according to  claim 23  wherein said polypeptide comprises residue 1 through residue 106 of SEQ ID NO:2 or residue 1 through residue 106 of SEQ ID NO:29.  
     
     
         28 . The expression vector according to  claim 23  wherein said DNA segment further encodes a secretory peptide operably linked to said polypeptide.  
     
     
         29 . The expression vector according to  claim 28  wherein said DNA segment encodes residue −23 through residue 106 of SEQ ID NO:2 or residue −23 through residue 106 of SEQ ID NO:29.  
     
     
         30 . A cultured cell into which has been introduced an expression vector according to  claim 23 , wherein said cell expresses the polypeptide encoded by the DNA segment.  
     
     
         31 . A pharmaceutical composition comprising a polypeptide according to  claim 2  in combination with a pharmaceutically acceptable vehicle.  
     
     
         32 . A method of producing a polypeptide comprising: 
 culturing a cell into which has been introduced an expression vector according to  claim 23 , whereby said cell expresses the polypeptide encoded by the DNA segment; and    recovering the expressed polypeptide.    
     
     
         33 . An antibody that specifically binds to an epitope of a polypeptide according to  claim 2 .  
     
     
         34 . A method for detecting a genetic abnormality in a patient, comprising: 
 obtaining a genetic sample from a patient;    incubating the genetic sample with a polynucleotide comprising at least 14 contiguous nucleotides of SEQ ID NO:1 or the complement of SEQ ID NO:1, under conditions wherein said polynucleotide will hybridize to complementary polynucleotide sequence, to produce a first reaction product;    comparing said first reaction product to a control reaction product, wherein a difference between said first reaction product and said control reaction product is indicative of a genetic abnormality in the patient.    
     
     
         35 . An oligonucleotide probe or primer comprising 14 contiguous nucleotides of a polynucleotide of SEQ ID NO:4 or a sequence complementary to SEQ ID NO:4.