Ss 1,3-n-acetyl-d-galactosamine transferase protein, nucleic acid encoding the same and method of examining canceration using the same

Abstract

The N-acetyl-D-galactosamine transferase protein of the present invention is characterized by transferring N-acetyl-D-galactosamine to N-acetyl-D-glucosamine with β1,3 linkage, and it preferably has the amino acid sequence shown in SEQ ID NO: 2 or 4. The canceration assay according to the present invention uses a nucleic acid for measurement which hybridizes under stringent conditions to the nucleotide sequence shown in SEQ ID NO: 1 or 3 or a nucleotide sequence complementary to at least one of them.

1 . A β1,3-N-acetyl-D-galactosamine transferase protein which transfers N-acetyl-D-galactosamine to N-acetyl-D-glucosamine with β1,3 linkage.  
     
     
         2 . The glycosyltransferase protein according to  claim 1 , which has at least one of the following properties (a) to (c): 
 (a) acceptor substrate specificity 
 when using an oligosaccharide as an acceptor substrate, the protein shows transferase activity toward Bz-β-GlcNAc, GlcNAc-β1-4-GlcNAc-β-Bz, Gal-β1-3-(GlcNAc-β1-6) GalNAc-α-pNp, GlcNAc-β1-3-GalNAc-α-pNp and GlcNAc-β1-6-GalNAc-α-pNp (“GlcNAc” represents an N-acetyl-D-glucosamine residue, “GalNAc” represents an N-acetyl-D-galactosamine residue, “Bz” represents a benzyl group, “pNp” represents a p-nitrophenyl group, and “-” represents a glycosidic linkage. Numbers in these formulae each represent the carbon number in the sugar ring where a glycosidic linkage is present, and “α” and “β” represent anomers of the glycosidic linkage at the 1-position of the sugar ring. An anomer whose positional relationship with CH 2 OH or CH 3  at the 5-position is trans and cis is represented by “α” and “β”, respectively);  
   (b) reaction pH 
 the activity is lower in a pH range of 6.2 to 6.6 than in other pH ranges; or  
   (c) divalent ion requirement 
 although the activity is enhanced at least in the presence of Mn 2+ , Co 2+  or Mg 2+ , the Mn 2+ -induced enhancement of the activity is almost completely eliminated in the presence of Cu + .  
   
     
     
         3 . A glycosyltransferase protein which comprises the following polypeptide (A) or (B): 
 (A) a polypeptide which has the amino acid sequence shown in SEQ ID NO: 2 or 4; or    (B) a polypeptide which has an amino acid sequence with substitution, deletion or insertion of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or 4 and which transfers N-acetyl-D-galactosamine to N-acetyl-D-glucosamine with β1,3 linkage.    
     
     
         4 . The glycosyltransferase protein according to  claim 3 , wherein the polypeptide (A) consists of a polypeptide having an amino acid sequence covering amino acids 189 to 500 shown in SEQ ID NO: 2.  
     
     
         5 . The glycosyltransferase protein according to  claim 3 , wherein the polypeptide (A) consists of a polypeptide having an amino acid sequence covering amino acids 36 to 500 shown in SEQ ID NO: 2.  
     
     
         6 . The glycosyltransferase protein according to  claim 3 , which consists of a polypeptide having an amino acid sequence sharing at least more than 30% identity with an amino acid sequence covering amino acids 189 to 500 shown in SEQ ID NO: 2 or amino acids 35 to 504 shown in SEQ ID NO: 4.  
     
     
         7 . A nucleic acid consisting of a nucleotide sequence encoding the polypeptide according to  claim 3  or a nucleotide sequence complementary thereto.  
     
     
         8 . The nucleic acid according to  claim 7 , which consists of the nucleotide sequence shown in SEQ ID NO: 1 or 3 or a nucleotide sequence complementary to at least one of them.  
     
     
         9 . The nucleic acid according to  claim 7 , which consists of a nucleotide sequence covering nucleotides 565 to 1503 shown in SEQ ID NO: 1 or a nucleotide sequence complementary thereto.  
     
     
         10 . The nucleic acid according to  claim 7 , which consists of a nucleotide sequence covering nucleotides 106 to 1503 shown in SEQ ID NO: 1 or a nucleotide sequence complementary thereto.  
     
     
         11 . The nucleic acid according to  claim 7 , which consists of a nucleotide sequence covering nucleotides 103 to 1512 shown in SEQ ID NO: 3 or a nucleotide sequence complementary thereto.  
     
     
         12 . The nucleic acid according to  claim 7 , which is DNA.  
     
     
         13 . A vector carrying the nucleic acid according to  claim 7 .  
     
     
         14 . A transformant containing the vector according to  claim 13 .  
     
     
         15 . A method for producing a β1,3-N-acetyl-D-galactosamine transferase protein, which comprises growing the transformant according to  claim 14  to express the glycosyltransferase protein and collecting the glycosyltransferase protein from the transformant.  
     
     
         16 . An antibody recognizing the β1,3-N-acetyl-D-galactosamine transferase protein according to  claim 1 .  
     
     
         17 . An antibody recognizing the β1,3-N-acetyl-D-galactosamine transferase protein according to  claim 3 .  
     
     
         18 . A nucleic acid consisting of a nucleotide sequence encoding the polypeptide according to  claim 4  or a nucleotide sequence complementary thereto.  
     
     
         19 . A nucleic acid consisting of a nucleotide sequence encoding the polypeptide according to  claim 5  or a nucleotide sequence complementary thereto.  
     
     
         20 . A nucleic acid consisting of a nucleotide sequence encoding the polypeptide according to  claim 6  or a nucleotide sequence complementary thereto.