Novel isoforms of human pregnancy-associated protein-E

Abstract

The invention provides isolated nucleic acids that encode three novel isoforms of human pregnancy associated plasma protein E, hPAPP-E, and fragments thereof, vectors for propagating and expressing PAPP-E nucleic acids, host cells comprising the nucleic acids and vectors of the present invention, proteins, protein fragments, and protein fusions of the novel PAPP-E isoforms, and antibodies thereto. The invention further provides transgenic cells and non-human organisms comprising human PAPP-E isoform nucleic acids, and transgenic cells and non-human organisms with targeted disruption of the endogenous orthologue of the human PAPP-E gene. The invention further provides pharmaceutical formulations of the nucleic acids, proteins, and antibodies of the present invention, and diagnostic, investigational, and therapeutic methods based on the PAPP-E nucleic acids, proteins, and antibodies of the present invention.

What is claimed is:  
     
         1 . An isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of: 
 (i) the nucleotide sequence of SEQ ID NO:1, (ii) the complement of the nucleotide sequence of SEQ ID NO:1; (iii) the nucleotide sequence of SEQ ID NO:2, (iv) a degenerate variant of the nucleotide sequence of SEQ ID NO:2, or (v) the complement (iii) or (iv), (vi) a nucleotide sequence that encodes a polypeptide having the sequence of SEQ ID NO:3, (vii) a nucleotide seuqence that encodes a polypeptide having the sequence of SEQ ID NO:3 with conservative amino acid substitutions, and (viii) a nucleotide sequence that is the complement of (vii) or (viii).    
     
     
         2 . An isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of: 
 (i) the nucleotide sequence of SEQ ID NO:4, (ii) the complement of the nucleotide sequence of SEQ ID NO:4, (iii) a nucleotide sequence that encodes a polypeptide having the sequence of SEQ ID NO:7, (iv) a nucleotide sequence that encodes a polypeptide having the sequence of SEQ ID NO:7 with conservative amino acid substitutions, and (iii) the complement of (i) or (ii),    wherein said isolated nucleic acid is no more than 50 kb in length.    
     
     
         3 . An isolated nucleic acid comprising a sequence that hybridizes under high stringency conditions to a hybridization probe the nucleotide sequence of which (i) encodes a polypeptide having the sequence of SEQ ID NO:7, (ii) encodes a polypeptide having the sequence of SEQ ID NO:7 with conservative amino acid substitutions, or (iii) is the complement of (i) or (ii), 
 wherein said isolated nucleic acid is no more than 50 kb in length.    
     
     
         4 . The isolated nucleic acid of  claim 3 , wherein said nucleic acid, or the complement of said nucleic acid, encodes a polypeptide having metalloproteinase activity.  
     
     
         5 . The isolated nucleic acid of  claim 3 , wherein said nucleic acid, or the complement of said nucleic acid, is expressed in placental tissue.  
     
     
         6 . An isolated nucleic acid comprising at least 17 nucleotides of (i) SEQ ID NO:4 or (ii) the complement of SEQ ID NO:4, 
 wherein said isolated nucleic acid is no more than 50 kb in length.    
     
     
         7 . An isolated nucleic acid comprising (i) a nucleotide sequence that encodes a peptide of at least 8 contiguous amino acids of SEQ ID NO:7, or (ii) the complement of a nucleotide sequence that encodes a peptide of at least 8 contiguous amino acids of SEQ ID NO:7, 
 wherein said isolated nucleic is no more than 50 kb in length.    
     
     
         8 . An isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of: 
 (i) the nucleotide sequence of SEQ ID NO:8, (ii) the complement of the nucleotide sequence of SEQ ID NO:8, (iii) the nucleotide sequence of SEQ ID NO:9, (iv) a degenerate variant of the nucleotide sequence of SEQ ID NO:9, or (v) the complement (iii) or (iv), (vi) a nucleotide sequence that encodes a polypeptide having the sequence of SEQ ID NO:10, (vii) a nucleotide sequence that encodes a polypeptide having the sequence of SEQ ID NO:10 with conservative amino acid substitutions, and (viii) a nucleotide sequence that is the complement of (vi) or (vii).    
     
     
         9 . An isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of: 
 (i) the nucleotide sequence of SEQ ID NO:11,    (ii) a degenerate variant of SEQ ID NO:11, (iii) the complement of (i) or (ii), (iv) a nucleotide sequence that encodes SEQ ID NO:12, (v) a nucleotide sequence that encodes SEQ ID NO:12 with conservative substititions, (vi) the complement of (iv) or (v),    wherein said isolated nucleic acid is no more than 50 kb in length.    
     
     
         10 . An isolated nucleic acid comprising a nucleotide sequence (i) that encodes a polypeptide having the sequence of SEQ ID NO:14, (ii) that encodes a polypeptide having the sequence of SEQ ID NO:14 with conservative amino acid substitutions, or (iii) the complement of a nucleotide sequence that encodes a polypeptide with the amino acid sequence of SEQ ID NO:14, 
 wherein wherein said isolated nucleic acid is no more than 50 kb in length.    
     
     
         11 . An isolated nucleic acid comprising a sequence that hybridizes under high stringency conditions to a probe the nucleotide sequence of which consists of SEQ ID NO:11 or the complement of SEQ ID NO:11, 
 wherein said isolated nucleic acid is no more than 50 kb in length.    
     
     
         12 . The isolated nucleic acid of  claim 11 , wherein said nucleic acid, or the complement of said nucleic acid, encodes a polypeptide having metalloproteinase activity.  
     
     
         13 . The isolated nucleic acid of  claim 11 , wherein said nucleic acid, or the complement of said nucleic acid, is expressed in placental tissue.  
     
     
         14 . An isolated nucleic acid comprising at least 17 nucleotides of (i) SEQ ID NO:11, (ii) a degenerate variant of SEQ ID NO:11, or (iii) the complement of (i) or (ii), 
 wherein said isolated nucleic acid is no more than 50 kb in length.    
     
     
         15 . An isolated nucleic acid comprising a nucleotide sequence that (i) encodes a polypeptide having the sequence of at least 8 contiguous amino acids of SEQ ID NO:12, (ii) encodes a polypeptide having the sequence of at least 8 contiguous amino acids of SEQ ID NO:12 with conservative amino acid substitutions, or (iii) is the complement of (i) or (ii).  
     
     
         16 . An isolated nucleic acid comprising a nucleotide sequence at least 90% identical to SEQ ID NO:11.  
     
     
         17 . An isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of: 
 (i) the nucleotide sequence of SEQ ID NO:15, (ii) a degenerate variant of SEQ ID NO:15, (iii) the complement of (i) or (ii), (iv) a nucleotide sequence that encodes a polypeptide having the sequence of SEQ ID NO:16, (v) a nucleotide sequence that encodes a polypeptide having the sequence of SEQ ID NO:16 with conservative amino acid substitutions, and (vi) the complement of (iv) or (v).    
     
     
         18 . An isolated nucleic acid comprising a sequence selected from the group consisting of: 
 (i) the nucleotide sequence of SEQ ID NO:17, (ii) a degenerate variant of SEQ ID NO:17, (iii) the complement of (i) or (ii), (iv) a nucleotide sequence that encodes SEQ ID NO:18, (v) a nucleotide sequence that encodes SEQ ID NO:18 with conservative amino acid substititions, and (vi) the complement of (i) or (ii).    
     
     
         19 . The isolated nucleic acid of  claim 18  wherein said nucleic acid, or the complement of said nucleic acid, encodes a polypeptide having metalloproteinase activity.  
     
     
         20 . The isolated nucleic acid of  claim 18  wherein said nucleic acid, or the complement of said nucleic acid, is expressed in placental tissue.  
     
     
         21 . An isolated nucleic acid comprising a nucleotide sequence of no more than one fragment of SEQ ID NOs:19 to 41 or the complement of SEQ ID NOs: 19 to 41, wherein said fragment comprises at least 17 contiguous nucleotides of said SEQ ID NO: or its complement and hybridizes under high stringency conditions to a nucleic acid expressed in human placenta.  
     
     
         22 . The isolated nucleic acid of claim  68 , wherein said fragment comprises at least 50 contiguous nucleotides of said SEQ ID NO: or its complement.  
     
     
         23 . An isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO:65 or its complement, 
 wherein said isolated nucleic acid is no more than 50 kb in length.    
     
     
         24 . The isolated nucleic acid of any of claims  1 - 23 , wherein said isolated nucleic acid is detectably labeled.  
     
     
         25 . The isolated nucleic acid of any one of claims  1 - 23 , wherein said nucleic acid is attached to a substrate.  
     
     
         26 . A microarray, comprising: a plurality of nucleic acids addressably disposed upon a substrate, 
 wherein at least one of said nucleic acids is the isolated nucleic acid of any one of 1-23.    
     
     
         27 . A recombinant nucleic acid vector, comprising: the isolated nucleic acid of any one of claims  1 - 23 .  
     
     
         28 . The vector of  claim 27 , wherein said isolated nucleic acid is operably linked to genetic elements capable of driving expression of said nucleic acid in a host cell.  
     
     
         29 . A host cell comprising the isolated nucleic acid of any one of claims  1 - 23  or the vector of either of claims  27 - 28 .  
     
     
         30 . An isolated polypeptide, said polypeptide comprising the amino acid sequence of SEQ ID NO:3, SEQ ID NO:10, or SEQ ID NO:16.  
     
     
         31 . An isolated polypeptide comprising a sequence of at least 8 contiguous amino acids of SEQ ID NO:7, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:18.  
     
     
         32 . An antibody, said antibody selected from the group consisting of: 
 (i) antibodies capable of binding specifically to polypeptides comprising an amino acid sequence as provided in SEQ ID NO:7, and the binding of which can be competitively inhibited by a polypeptide the sequence of which is given in SEQ ID NO:7;    (ii) antibodies capable of binding specifically to polypeptides comprising an amino acid sequence as provided in SEQ ID NO:14, and the binding of which can be competitively inhibited by a polypeptide the sequence of which is given in SEQ ID NO:14; and    (iii) antibodies capable of binding specifically to polypeptides comprising an amino acid sequence as provided in SEQ ID NO:18, and the binding of which can be competitively inhibited by a polypeptide the amino acid sequence of which is given in SEQ ID NO:18.    
     
     
         33 . The antibody of  claim 32 , which antibody is detectably labeled.  
     
     
         34 . A pharmaceutical composition comprising: 
 the nucleic acid of any one of claims  1 - 23 , the polypeptide of any one of claims  30 - 31 , or the antibody of  claim 32;  and    a pharmaceutically acceptable excipient.    
     
     
         35 . A method of antenatal diagnosis of dysgenetic pregnancies, comprising: 
 quantifying levels of PAPP-E protein circulating in maternal serum during first or second trimester of pregnancy, and comparing to a standard, decreased levels of circulating PAPP-E as compared to standard indicating increased likelihood of a dysgenetic pregnancy.    
     
     
         36 . A method of preventing or abrogating pregnancy, comprising: 
 vaccinating a human subject with the nucleic acid of any one of claims  1 - 23  or the polypeptide of any one of claims  30 - 31 , or administering an effective amount of the antibody of  claim 32.