Alpha 1,4-galactosyl transferase 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 acids 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 acids 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 - 9 . (Cancelled).  
     
     
         10 . A method for producing Gb3/CD77, comprising the steps of: 
 exposing the polypeptide according to  claim 1  or a transformed cell comprising a DNA encoding the polypeptide to lactosylceramide, to cause thereby enzymatic reaction; and    recovering Gb3/CD77.    
     
     
         11 . A method for producing a glycolipid represented by the following formula (10, comprising the steps of: 
 exposing the polypeptide according to  claim 1  or a transformed cell comprising a DNA encoding the polypeptide to galactosylceramide, to cause thereby enzymatic reaction; and    recovering the glycolipid represented by the following formula (1):      Galα1→4Gal-Cer  (1)    Wherein Gal represents a galactose residue, Cer represents a ceramide residue and α1→4 represents an α1-4 glycosidic linkage.    
     
     
         12 . An isolated DNA, which hybridizes under stringent conditions to the complement of SEQ ID NO:1, is about 1975 nucleotides, and which encodes a protein with an activity to transfer a galactose residue from a galactose donor to a C4 position of a galactose residue in lactosylceramide or galactosylceramide; and wherein the stringent conditions comprise washing in 2×SSC and 0.1% SDS at room temperature, and washing in 0.1×SSC and 0.1% SDS at a temperature of 50° or less.  
     
     
         13 . An isolated DNA, which encodes a polypeptide with an amino acid sequence in SEQ ID NO:2, and which is about 1975 nucleotides or less in length.  
     
     
         14 . The isolated DNA of  claim 12 , consisting essentially of nucleotides 269 to 1192 of SEQ ID NO:1.  
     
     
         15 . The isolated DNA of  claim 12 , consisting essentially of nucleotides 191 to 1192 of SEQ ID NO:1.  
     
     
         16 . The isolated DNA of  claim 12 , consisting essentially of nucleotides 134 to 1192 of SEQ ID NO:1.  
     
     
         17 . A vector comprising the isolated DNA of  claim 12 .  
     
     
         18 . A vector comprising the isolated DNA of  claim 13 .  
     
     
         19 . A vector comprising the isolated DNA of  claim 14 .  
     
     
         20 . A vector comprising the isolated DNA of  claim 15 .  
     
     
         21 . A vector comprising the isolated DNA of  claim 16 .  
     
     
         22 . A cell transformed with the isolated DNA of  claim 12 .  
     
     
         23 . A cell transformed with the isolated DNA of  claim 13 .  
     
     
         24 . A cell transformed with the isolated DNA of  claim 14 .  
     
     
         25 . A cell transformed with the isolated DNA of  claim 15 .  
     
     
         26 . A cell transformed with the isolated DNA of  claim 16 .  
     
     
         27 . A method of producing an α1,4-galactosyltransferase, comprising 
 culturing the cell of  claim 22  in a medium suitable for expressing the α1,4-galactosyltransferase; and    recovering the α1,4-galactosyltransferase from one or both of the medium and cell extract of the cell.    
     
     
         28 . A method of producing an α1,4-galactosyltransferase, comprising 
 culturing the cell of  claim 23  in a medium suitable for expressing the α1,4-galactosyltransferase; and    recovering the α1,4-galactosyltransferase from one or both of the medium and cell extract of the cell.    
     
     
         29 . A method of producing an α1,4-galactosyltransferase, comprising 
 culturing the cell of  claim 24  in a medium suitable for expressing the α1,4-galactosyltransferase; and    recovering the α1,4-galactosyltransferase from one or both of the medium and cell extract of the cell.    
     
     
         30 . A method of producing an α1,4-galactosyltransferase, comprising 
 culturing the cell of  claim 25  in a medium suitable for expressing the α1,4-galactosyltransferase; and    recovering the α1,4-galactosyltransferase from one or both of the medium and cell extract of the cell.    
     
     
         31 . A method of producing an α1,4-galactosyltransferase, comprising 
 culturing the cell of  claim 26  in a medium suitable for expressing the α1,4-galactosyltransferase; and    recovering the α1,4-galactosyltransferase from one or both of the medium and cell extract of the cell.