Segment of glycosylation-deficient HGF alpha-chain

Abstract

The present invention provides a segment of glycosylation-deficient HGF having mutation(s) introduced into an amino acid sequence so as to prevent glycosylation at least one glycosylation site of a hepatocyte growth factor (HGF), and a method of producing the same. The segment of glycosylation-deficient HGF of the present invention has the same activity as that of a segment of glycosylated HGF, therefore, it is useful as an alternate for a segment of glycosylated HGF.

1 . A segment of glycosylation-deficient HGF, having a deficiency of at least one sugar chain at glycosylation site(s) of an HGF α chain, and selected from the following proteins (a) to (c), wherein the above-mentioned protein has an N terminal hairpin domain and the subsequent four kringle domains, has an antagonist activity against the action of HGF via a c-Met/HGF receptor, and has a neovascularization inhibitory activity: 
 (a) a protein having an amino acid sequence from 32nd to 494th in SEQ ID NO: 1,    (b) a protein having an amino acid sequence from 32nd to 494th of SEQ ID No. 1, wherein 1 to 30 amino acids are deleted, substituted, added or inserted in said amino acid sequence,    (c) a protein having a homology of at least 80% or higher with amino acids from 32nd to 494th of SEQ ID NO: 1.    
     
     
         2 . The segment of glycosylation-deficient HGF according to  claim 1 , wherein the amino acid(s) is/are substituted so as to prevent glycosylation of at least one glycosylation site of the protein.  
     
     
         3 . The segment of glycosylation-deficient HGF according to  claim 2 , wherein the substitution(s) of amino acid(s) is/are at least one of the following (a) to (d): 
 (a) Asn of at least one N-glycosylation site is substituted by another amino acid, wherein N-glycosylation site means a portion having consensus sequence represented by the amino acid sequence Asn-X-Ser or Asn-X-Thr wherein X represents an amino acid other than Pro,    (b) Ser or Thr of one N-glycosylation site, or Ser and/or Thr of two or more N-glycosylation sites, is/are substituted by another amino acid, wherein N-glycosylation site means a portion having consensus sequence represented by the amino acid sequence Asn-X-Ser or Asn-X-Thr wherein X represents an amino acid other than Pro,    (c) X of at least one N-glycosylation site is substituted by Pro, wherein N-glycosylation site means a portion having consensus sequence represented by the amino acid sequence Asn-X-Ser or Asn-X-Thr wherein X represents an amino acid other than Pro, and    (d) at least one of Ser and Thr which undergoes O-glycosylation is substituted by another amino acid.    
     
     
         4 . The segment of glycosylation-deficient HGF according to  claim 2 , wherein the substitution(s) of amino acid(s) is/are at least one of the following (a) to (d): 
 (a) amino acid(s) at 294th and/or 296th of an amino acid sequence of SEQ ID NO: 1 is/are substituted by another amino acid, and/or an amino acid at 295th thereof is substituted by Pro, leading thereby to no glycosylation at 294th,    (b) amino acid(s) at 402nd and/or 404th of an amino acid sequence of SEQ ID NO: 1 is/are substituted by another amino acid, and/or an amino acid at 403rd thereof is substituted by Pro, leading thereby to no glycosylation at 402nd, or    (c) an amino acid at 476th of an amino acid sequence of SEQ ID NO: 1 is substituted by another amino acid, leading thereby to no glycosylation at 476th.    
     
     
         5 . The segment of glycosylation-deficient HGF according to  claim 1 , wherein amino acids at 162nd to 166th of an amino acid sequence of SEQ ID NO: 1 are deleted.  
     
     
         6 . The segment of glycosylation-deficient HGF according to  claim 1 , wherein amino acids at 479th to 494th of an amino acid sequence of SEQ ID NO: 1 are deleted.  
     
     
         7 . A DNA comprising a base sequence coding the segment of glycosylation-deficient HGF according to  claim 1 .  
     
     
         8 . A vector containing the DNA according to  claim 7  incorporated therein.  
     
     
         9 . A method of producing the segment of glycosylation-deficient HGF according to  claim 1 , comprising introducing a vector containing a DNA comprising a base sequence coding the segment of glycosylation-deficient HGF according to  claim 1  into a cell, culturing the cell, allowing a segment of glycosylation-deficient HGF to be accumulated in the cell or to be secreted into culture solution of the cell, and collecting and purifying the segment of glycosylation-deficient HGF from the cell or culture solution of the cell.  
     
     
         10 . The method of producing the segment of glycosylation-deficient HGF according to  claim 9 , wherein the cell is a eukaryotic cell.  
     
     
         11 . The method of producing the segment of glycosylation-deficient HGF according to  claim 10 , wherein the eukaryotic cell is a yeast or insect cell.  
     
     
         12 . A method of producing the segment of glycosylation-deficient HGF according to  claim 1 , comprising introducing a vector containing a DNA comprising a base sequence coding the segment of glycosylation-deficient HGF according to  claim 1  into an insect individual, allowing a segment of glycosylation-deficient HGF to be accumulated in the insect individual, and collecting and purifying the segment of glycosylation-deficient HGF from the insect individual.  
     
     
         13 . A method of producing the segment of glycosylation-deficient HGF according to  claim 1 , comprising treating a segment of HGF having sugar chain(s) with an enzyme to remove the sugar chain(s) wholly or partially, and collecting and purifying the segment of glycosylation-deficient HGF from the enzymatic reaction mixture.  
     
     
         14 . A method of producing the segment of glycosylation-deficient HGF according to  claim 1 , comprising introducing a vector containing an incorporated DNA coding a segment of HGF having sugar chain(s) or a vector containing a DNA comprising a base sequence coding the segment of glycosylation-deficient HGF according to  claim 1  into a cell having no glycosylation ability, culturing the cell, allowing a segment of glycosylation-deficient HGF to be accumulated in the cell or to be secreted into culture solution of the cell, and collecting and purifying the segment of glycosylation-deficient HGF from the cell or culture solution of the cell.  
     
     
         15 . A method of producing the segment of glycosylation-deficient HGF according to  claim 1 , comprising synthesizing a segment of glycosylation-deficient HGF by cell-free protein synthesis system using a DNA having a base sequence coding a segment of HGF having sugar chain(s) or a DNA comprising a base sequence coding the segment of glycosylation-deficient HGF according to  claim 1  as a template, and collecting and purifying the segment of glycosylation-deficient HGF from the synthesis reaction mixture.  
     
     
         16 . A method of producing the segment of glycosylation-deficient HGF according to  claim 1 , comprising limited digestion of glycosylation-deficient HGF which lacks sugar chain(s) at at least one or all of glycosylation sites, by treatment with a protease or by chemical treatment of HGF, and collecting and purifying the segment of glycosylation-deficient HGF from the treatment solution.  
     
     
         17 . The method according to  claim 16 , wherein the protease is esterase.  
     
     
         18 . An agent comprising as an active ingredient the segment of glycosylation-deficient HGF according to  claim 1 .  
     
     
         19 . The agent according to  claim 18 , wherein the agent is a neovascularization inhibiting agent.  
     
     
         20 . The agent according to  claim 18 , wherein the agent is an antagonist against the action of HGF mediated via a c-Met/HGF receptor.  
     
     
         21 . The agent according to  claim 18 , wherein the agent is an agent suppressing invasion, growth or metastasis of tumor.  
     
     
         22 . The agent according to  claim 18 , wherein the agent is an apoptosis-inducing agent.  
     
     
         23 . The agent according  claim 20 , wherein the agent is an agent for prevention and/or treatment of an infectious disease.  
     
     
         24 . The agent according  claim 23 , wherein the infectious disease is an infectious disease caused by a malaria or  Listeria  parasite.  
     
     
         25 . A gene pharmaceutical composition, comprising the DNA according to  claim 7.