| Blood group A and B gene-specified glycosyltransferases, respectively the alpha(1,3)N-acetylgalactosyltransferase and the alpha(1,3)galactosyltransferase are enzymes that synthesize the A and B antigens (glycoproteins and glycolipids) that are found on the surfaces of erythrocytes and other cells. The A enzyme uses UDP-GalNAc while the B enzyme uses UDP-Gal as the donors. The enzymes differ in only four amino acids. The minimum acceptor structure recognized by both the enzymes is alphaLFucp(1,2)betaDGal p-OR (R = H; for R = octyl, 1).; A series of analogs of this disaccharide, with modifications on the hydroxyl groups of the fucose moiety, were chemically synthesized to investigate the role of the hydroxyl groups in the recognition of the acceptor by these enzymes. Thus the Fuc 2, 3, 4-OH were replaced with H and OMe and the C-6' methyl with H (arabino analog). Evaluation of these analogs as acceptors show that modifications on the 3'-OH are tolerated by the enzymes. The 2'-OMe analog is not recognized by either as acceptor. The arabino analog is recognized by the A but not the B transferase. Acceptor recognition can thereby be directly ascribed to specific amino acid differences between the enzymes.; The acceptors recognized by the A transferase were used in the syntheses of modified blood group A antigens using UDP-GalNAc as donor. The arabino analog was used to assay a series of recombinant enzymes produced by sequentially changing the amino acids from the A to the B sequence. Although differential recognition of the acceptor by the mutants was observed, a single amino acid can not be definitely identified as being responsible for acceptor specificity.; Photoaffinity analogs of the acceptor 1 were synthesized to covalently label the A transferase in its active site by irradiation with UV light, isolate it by lectin affinity chromatography and subsequently establish the amino acid sequence of the active site. Thus, acceptor-based photoaffinity labeling of a glycosyltransferase was achieved although the low percentage of labeling prevented conventional sequencing analysis. Mass spectrometric analysis of the labeled fragments is currently being performed. |
