Characterization of the substrate specificity of alpha1,3galactosyltransferase utilizing modified N-acetyllactosamine disaccharides.
alpha1,3galactosyltransferase (alpha1,3GalT) catalyzes the synthesis of a range of glycoconjugates containing the Galalpha1,3Gal epitope which is recognized by the naturally occurring human antibody, anti-Gal. This enzyme may be a useful synthetic tool to produce a range of compounds to further investigate the binding site of anti-Gal and other proteins with a Galalpha1,3Gal binding site. Thus, the enzyme has been probed with a series of type 2 disaccharide-C8(Galbeta1-4GlcNAc-C8) analogs. The enzyme tolerated acceptors with modifications at C2 and C3 of the N-acetylglucosamine residue, producing a family of compounds with a nonreducing alpha1,3 linked galactose. Compounds that did not serve as acceptors were evaluated as inhibitors. Interestingly, the type 1 disaccharide-C8, Galbeta1-3GlcNAc-C8, was a good inhibitor of the enzyme (Ki = 270 microM vs. Km = 190 microM for Galbeta1-4GlcNAc-C8). A potential photoprobe, based on a modified type 2 disaccharide (octyl 3-amino-3-deoxy-3-N-(2-diazo-3, 3, 3-trifluoropropionyl-beta-D-galactopyranosyl-(1, 4)-2-acetamindo-2-deoxy-beta-D-glycopyranoside, (DTFP-LacNAc-C8)), was evaluated as an inhibitor of alpha1,3GalT. alpha1,3GalT bound DTFP-LacNAc-C8 with an affinity (Ki = 300 microM) similar to that displayed by the enzyme for LacNAc-C8. Additional studies were done to determine the enzyme's ability to transfer a range of sugars from UDP-sugar donors. The results of these experiments demonstrated that alpha1,3GalT has a strict specificity for UDP-Gal. Finally, inactivation studies with various amino acid modifiers were done to obtain information on the importance of different types of amino acids for alpha1,3GalT activity.
Stults
C L
CL
Department of Chemistry and Biochemistry, San Francisco State University 1600 Holloway Avenue, San Francisco, CA 94132, USA.
Macher
B A
BA
Bhatti
R
R
Srivastava
O P
OP
Hindsgaul
O
O
D000606:Amino Sugars / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001665:Binding Sites; D019556:COS Cells; D002240:Carbohydrate Sequence; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism*; D000939:Epitopes / Q000737:chemistry / Q000378:metabolism; D005700:Galactosyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006001:Glycoconjugates / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology; D006801:Humans; D051379:Mice; D015335:Molecular Probes; D008969:Molecular Sequence Data; D010777:Photochemistry; D013379:Substrate Specificity; D014312:Trisaccharides / Q000096:biosynthesis / Q000737:chemistry
D000606:Amino Sugars; D000906:Antibodies; D004187:Disaccharides; D000939:Epitopes; D006001:Glycoconjugates; D015335:Molecular Probes; D014312:Trisaccharides; C000458:N-acetyllactosamine; D005700:Galactosyltransferases