Alpha 1,4-galactosyltransferase and dna encoding thereof

Abstract

The object of the present invention is to provide α1,4-galactosyltransferase to transfer a galactose residue to C4 position of galactose residue of lactosylceramide or galactosylceramide, and DNA coding for the enzyme. What is provided includes the following polypeptides (a) and (b), and DNAs encoding thereof: (a) a polypeptide consisting of an amino acid sequence represented by the amino acid Nos. 46-353 in SEQ ID NO: 2; or (b) a polypeptide which comprises an amino acid sequence including substitution, deletion, insertion or transposition of one or few amino acids in the amino acid sequence of (a) and which has an enzymatic activity to transfer a galactose residue from a galactose donor to C4 position of galactose residue of lactosylceramide or galactosylceramide which serves as an acceptor.

1 . A polypeptide of (a) or (b) below:
 (a) a polypeptide consisting of an amino acid sequence represented by the amino acid Nos. 46-353 in SEQ ID NO: 2; or   (b) a polypeptide which comprises an amino acid sequence including substitution, deletion, insertion or transposition of one or few amino acids in the amino acid sequence of (a) and which has an enzymatic activity to transfer a galactose residue from a galactose donor to C4 position of galactose residue of lactosylceramide or galactosylceramide which serves as an acceptor.   
     
     
         2 . A polypeptide of (a′) or (b′) below:
 (a′) a polypeptide consisting of an amino acid sequence represented by the amino acid Nos. 20-353 in SEQ ID NO:2; or   (b′) a polypeptide which comprises an amino acid sequence including substitution, deletion, insertion or transposition of one or few amino acids in the amino acid sequence of (a′) and which has an enzymatic activity to transfer a galactose residue from a galactose donor to C4 position of galactose residue of lactosylceramide or galactosylceramide which serves as an acceptor.   
     
     
         3 . A polypeptide of (a″) or (b″) below:
 (a″) a polypeptide consisting of an amino acid sequence represented by SEQ ID NO:2; or   (b″) a polypeptide which comprises an amino acid sequence including substitution, deletion, insertion or transposition of one or few amino acids in the amino acid sequence of (a″) and which has an enzymatic activity to transfer a galactose residue from a galactose donor to C4 position of galactose residue of lactosylceramide or galactosylceramide which serves as an acceptor.   
     
     
         4 . A DNA encoding the polypeptide according to any one of  claims 1  to  3 . 
     
     
         5 . The DNA according to  claim 4  represented by (a) or (b) below:
 (a) a DNA comprising a nucleotide sequence represented by nucleotide Nos. 269 to 1192 in SEQ ID NO:1; or   (b) a DNA hybridizable with a DNA comprising a nucleotide sequence represented by SEQ ID NO:1, a nucleotide sequence complimentary to SEQ ID NO:1, or a part of those sequences, under a stringent condition.   
     
     
         6 . The DNA according to  claim 5  encoding a polypeptide having an enzymatic activity to transfer a galactose residue from a galactose donor to C4 position of galactose residue of lactosylceramide or galactosylceramide which serves as an acceptor. 
     
     
         7 . A recombination vector containing the DNA as described in any one of  claims 4  to  6 . 
     
     
         8 . A transformed cell obtained by transfecting a host cell with the DNA according to any one of  claims 4  to  6 , or the recombination vector according to  claim 7 . 
     
     
         9 . A method for producing the polypeptide according to any one of  claims 1  to  3 , comprising the steps of:
 expressing the polypeptide according to  claims 1  to  3  by culturing the transformed cell according to  claim 8  in a medium suitable for expressing the polypeptide; and   recovering said polypeptide from the medium and/or a cell extract of the cultured transformed cell.   
     
     
         10 . A method for producing Gb3/CD77, comprising the steps of:
 exposing the polypeptide according to any one of  claims 1  to  3 , or a cultured product of the transformed cell according to  claim 8 , to lactosylceramide, to cause thereby enzymatic reaction; and   recovering Gb3/CD77.   
     
     
         11 . A method for producing a glycolipid represented by the following formula (I), comprising the steps of:
 exposing the polypeptide according to any one of  claims 1  to  3 , or a cultured product of the transformed cell according to  claim 8 , to galactosylceramide, to cause thereby enzymatic reaction; and   recovering the glycolipid represented by the following formula (I):
   Galα1→4Gal-Cer  (1) 
   
       wherein Gal represents a galactose residue, Cer represents a ceramide residue and α1→4 represents an α1-4 glycosidic linkage.