Enzymatic synthesis of blood group A and B trisaccharide analogues.
Glycosyltransferases A and B utilize the donor substrates UDP-GalNAc and UDP-Gal, respectively, in the biosynthesis of the human blood group A and B trisaccharide antigens from the O(H)-acceptor substrates. These enzymes were cloned as synthetic genes and expressed in Escherichia coli, thereby generating large quantities of enzyme for donor specificity evaluations. The amino acid sequence of glycosyltransferase A only differs from glycosyltransferase B by four amino acids, and alteration of these four amino acid residues (Arg-176-->Gly, Gly-235-->Ser, Leu-266-->Met and Gly-268-->Ala) can change the donor substrate specificity from UDP-GalNAc to UDP-Gal. Crossovers in donor substrate specificity have been observed, i.e., the A transferase can utilize UDP-Gal and B transferase can utilize UDP-GalNAc donor substrates. We now report a unique donor specificity for each enzyme type. Only A transferase can utilize UDP-GlcNAc donor substrates synthesizing the blood group A trisaccharide analog alpha-D-Glcp-NAc-(1-->3)-[alpha-L-Fucp-(1-->2)]-beta-D-Galp-O-(CH2 )7CH3 (4). Recombinant blood group B was shown to use UDP-Glc donor substrates synthesizing blood group B trisaccharide analog alpha-D-Glcp-(1-->3)-[alpha-L-Fucp-(1-->2)]-beta-D-Galp-O-(CH2) 7CH3 (5). In addition, a true hybrid enzyme was constructed (Gly-235-->Ser, Leu-266-->Met) that could utilize both UDP-GlcNAc and UDP-Glc. Although the rate of transfer with UDP-GlcNAc by the A enzyme was 0.4% that of UDP-GalNAc and the rate of transfer with UDP-Glc by the B enzyme was 0.01% that of UDP-Gal, these cloned enzymes could be used for the enzymatic synthesis of blood group A and B trisaccharide analogs 4 and 5.
Seto
N O
NO
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ont. nina.seto@nrc.ca
Compston
C A
CA
Szpacenko
A
A
Palcic
M M
MM
D000017:ABO Blood-Group System; D002240:Carbohydrate Sequence; D005700:Galactosyltransferases / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000378:metabolism*; D009844:Oligosaccharides / Q000096:biosynthesis*; D025942:Oligosaccharides, Branched-Chain; D013379:Substrate Specificity; D014312:Trisaccharides / Q000096:biosynthesis*; D014532:Uridine Diphosphate Glucose / Q000378:metabolism; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
D000017:ABO Blood-Group System; D009844:Oligosaccharides; D025942:Oligosaccharides, Branched-Chain; D014312:Trisaccharides; C101335:blood group A trisaccharide; C406478:blood group B trisaccharide; D014537:Uridine Diphosphate N-Acetylglucosamine; D005700:Galactosyltransferases; D017350:N-Acetylgalactosaminyltransferases; C003742:UDPgalactosamine-galactose acetylgalactosaminyltransferase; C023155:blood-group-substance alpha-D-galactosyltransferase; D014532:Uridine Diphosphate Glucose