Methods for production of growth-promoting proteins and peptides for kidney epithelial cells

Abstract

Novel growth peptides derived from protein factors having molecular weights of about 22 and 45 kDa stimulate mitogenic activity of epithelial, but not fibroblastic cells, in particular, kidney epithelial cells. A source of the factors is scrape-wounded kidney epithelial cells in culture. Synthetic peptides having sixteen amino acids or less, in particular a hexapeptide, YPQGNH (SEQ ID NO: 2) maintain the mitogenic activity. The peptide AQPYPQGNHEASYG (14-Ser) (SEQ ID NO: 15) is effective in reversing acute renal failure in animals. The growth-promoting characteristics of the 22 and 45 kDa proteins and the peptides are useful in treating and diagnosing patients with kidney disease. Nucleotide sequences that encode the factor are useful to develop probes to locate similar factors, to identify genetic disorders involving the factor, and to produce the factor by genetic recombinant methods. The nucleotide sequences and fragments thereof, are also useful for diagnosis and treatment of kidney disorders.

We claim:  
     
         1 . A protein having the following characteristics: 
 (a) an estimated molecular weight of about 45 kDa, said estimate obtained by electrophoresing the HPLC-purified protein on an SDS-polyacrylamid gel;    (b) capability of stimulating mitogenic activity when in contact with cultured cells; and    (c) released by BSC-1 cells in culture by wounding.    
     
     
         2 . The protein of  claim 1  having a partial amino acid sequence at the amino terminal end of NH 2 -alanine-gluatamine-proline-tyrosine-proline-glutamine-glycine-asparagine-histidine-glutamic acid-X-alanine/serine-tyrosine-glycine-COOH (SEQ ID NO: 1).  
     
     
         3 . A protein having the following characteristics: 
 (a) an estimated molecular weight of about 22 kDa, said estimate obtained by electorphoresing the HPLC-purified protein on an SDS-polyacrylamide gel;    (b) capability of stimulating mitogenic activity when in contact with cultured cells; and    (c) released by BSC-1 cells in culture by wounding.    
     
     
         4 . The protein of  claim 3  having a partial amino acid sequence at the amino terminal end of NH 2 -alanine-glutamine-proline-tyrosine-proline-glutamine-glycine-asparagine-histidine-glutamic acid-alanine-thereonine-serine-serine-serine-phenylalinine-COOH (SEQ ID NO: 4).  
     
     
         5 . A peptide comprising an amino acid sequence of HN 2 -tyrosine-proline-glutaimine-glycine-asparagine-histidine-COOH (SEQ ID NO: 2) having a length of from 7 to 16 amino acids.  
     
     
         6 . A composition comprising the protein of  claim 1 .  
     
     
         7 . A method for producing a protein having the following characteristics: 
 (a) an estimated molecular weight of about 45 kDa, said estimate obtained by electrophoresing the HPLC-purified polypeptide on an SDS-polyacrylamide gel;    (b) capability of stimulating mitogenic activity when in contact with cultured cells; and    (c) released by BSC-1 cells in culture by scrape-wounding said method comprising:    (i) culturing kidney epithelial cells in media;    (ii) wounding cells in culture; and    (iii) obtaining the protein from the conditioned media.    
     
     
         8 . An antibody to the protein of  claim 1 .  
     
     
         9 . An antibody to the peptide AQPYPQGNHEASYG (SEQ ID NO: 15).  
     
     
         10 . An antibody to a peptide having an amino acid sequence selected from the group consisting of:  
       
         
           
                 
                 
                 
                 
               
                     
                     
                 
                     
                   AQPYPQGNHEATSSSF; 
                   (SEQ ID NO: 4) 
                     
                 
                     
                     
                 
                     
                   AQPYPQGNHEATSSS; 
                   (SEQ ID NO: 5) 
                 
                     
                     
                 
                     
                   AQPYPQGNHEA; 
                   (SEQ ID NO: 6) 
                 
                     
                     
                 
                     
                   AQPYPQGNHEAT; 
                   (SEQ ID NO: 7) 
                 
                     
                     
                 
                     
                   AQPYPQGNHEATS; 
                   (SEQ ID NO: 8) 
                 
                     
                     
                 
                     
                   AQPYPQGNHEATSS; 
                   (SEQ ID NO: 9) 
                 
                     
                     
                 
                     
                   AQPYPQGNHEATSY; 
                   (SEQ ID NO: 10) 
                 
                     
                     
                 
                     
                   AQPYPQGNHEAAYG; 
                   (SEQ ID NO: 11) 
                 
                     
                     
                 
                     
                   AQPYPQGNHEAAY; 
                   (SEQ ID NO: 12) 
                 
                     
                     
                 
                     
                   AQPYPQGNHEAA; 
                   (SEQ ID NO: 13) 
                 
                     
                     
                 
                     
                   AQPYPQGNHE; 
                   (SEQ ID NO: 14) 
                 
                     
                     
                 
                     
                   AQPYPQGNHEASYG; 
                   (SEQ ID NO: 15) 
                 
                     
                     
                 
                     
                   AQPYPQGNHEASY; 
                   (SEQ ID NO: 16) 
                 
                     
                     
                 
                     
                   AQPYPQGNHEAS; 
                   (SEQ ID NO: 17) 
                 
                     
                     
                 
                     
                   QPYPQGNHEA; 
                   (SEQ ID NO: 18) 
                 
                     
                     
                 
                     
                   AQPYPQGNH; 
                   (SEQ ID NO: 19) 
                 
                     
                     
                 
                     
                   QPYPQGNHE; 
                   (SEQ ID NO: 20) 
                 
                     
                     
                 
                     
                   PYPQGNHEA; 
                   (SEQ ID NO: 21) 
                 
                     
                     
                 
                     
                   QPYPQGNH; 
                   (SEQ ID NO: 22) 
                 
                     
                     
                 
                     
                   PYPQGNHE; 
                   (SEQ ID NO: 23) 
                 
                     
                     
                 
                     
                   YPQGNHEA; 
                   (SEQ ID NO: 24) 
                 
                     
                     
                 
                     
                   PYPQGNH; and 
                   (SEQ ID NO: 25) 
                 
                     
                     
                 
                     
                   YPQGNHE; 
                   (SEQ ID NO: 26) 
                 
                     
                     
                 
                     
                   YPQGNHEATSSSF; 
                   (SEQ ID NO: 27) 
                 
                     
                     
                 
                     
                   YPQGNHEATSSS; 
                   (SEQ ID NO: 28) 
                 
                     
                     
                 
                     
                   YPQGNHEATSS; 
                   (SEQ D NO: 29) 
                 
                     
                     
                 
                     
                   YPQGNHEATS; and 
                   (SEQ ID NO: 30) 
                 
                     
                     
                 
                     
                   YPQGNHEAT. 
                   (SEQ ID NO: 31) 
                 
                     
                     
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         11 . The antibody of  claim 8 , wherein the antibody is a monoclonal antibody.  
     
     
         12 . The antibody of  claim 10 , wherein the antibody is a monoclonal antibody.  
     
     
         13 . The method of making the protein of  claim 1 , said method comprising: 
 (a) obtaining a nucleotide sequence encoding the protein or peptide; and    (b) using the nucleotide sequence in a genetic expression system to make the protein or peptide.    
     
     
         14 . A diagnostic kit to measure the quantity of a WGF protein or a mitogenic peptide therefrom, in a biological sample, said kit comprising in separate containers: 
 (a) an antibody to EGF or to a mitogenic peptide therefrom; and    (b) a means for detecting a specific complex between the WGF protein or a mitogenic peptide and the antibody.    
     
     
         15 . The method of  claim 14 , wherein the mitogenic peptide comprises the sequence tyrosine-proline-glutamine-glycine-asparagine-histidine (SEQ ID NO: 2).  
     
     
         16 . A method of treating a person with acute renal failure, said method comprising: 
 (a) preparing a pharmacologically effective amount of native WGF protein or WGF-derived peptide in a suitable diluent; and    (b) administering the preparation to the person.    
     
     
         17 . The method of  claim 16 , wherein the WGF is ligated to acytolytic ligand for treatment of kidney cancer.  
     
     
         18 . The method of claim  25 , wherein the cytolytic ligand comprises a toxin fro treatment of kidney cancer.