Enzymatic synthesis of blood group A and B trisaccharide analogues

Glycosyltransferases A and B utilize the donor substrates UDP-GalNAc and UD P-Gal, respectively, in the biosynthesis of the human blood group A and B t risaccharide antigens from the O(H)-acceptor substrates. These enzymes were cloned as synthetic genes and expressed in Escherichia coli, thereby gener ating large quantities of enzyme for donor specificity evaluations. The ami no acid sequence of glycosyltransferase A only differs from glycosyltransfe rase B by four amino acids, and alteration of these four amino acid residue s (Arg-176-->Gly, Gly-235-->Ser, Leu-266-->Met and Gly-268-->Ala) can chang e the donor substrate specificity from UDP-GalNAc to UDP-Gal. Crossovers in donor substrate specificity have been observed, i.e., the A transferase ca n 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 tra nsferase can utilize UDP-GlcNAc donor substrates synthesizing the blood gro up A trisaccharide analog alpha-D-Glcp-NAc-(1 --> 3)-[alpha-L-Fucp-(1 --> 2 )]-beta-D-Galp-O-(CH2)(7)CH3 (4). Recombinant blood group B was shown to us e UDP-GlcNAc donor substrates synthesizing blood group B trisaccharide anal og alpha-D-Glcp-(1 --> 3)-[alpha-L-Fucp-(1 --> 2)]-beta-D-Galp-O-(CH2)(7)CH 3 (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 t he rate of transfer with UDP-GlcNAc by the A enzyme was 0.4% that of UDP-Ga lNAc and the rate of transfer with UDP-Glc by the B enzyme was 0.01% that o f UDP-Gal, these cloned enzymes could be used for the enzymatic synthesis o f blood group A and B trisaccharide analogs 4 and 5. Crown in right of Cana da (C) 2000 Published by Elsevier Science Ltd. All rights reserved.