The final step in the enzymatic synthesis of the ABO(H) blood group A and B antigens is catalyzed by two closely related glycosyltransferases, an ␣-(133)-N-acetylgalactosaminyltransferase (GTA) and an ␣-(133)-galactosyltransferase (GTB). Of their 354 amino acid residues, GTA and GTB differ by only four "critical" residues. High resolution structures for GTB and the GTA/GTB chimeric enzymes GTB/G176R and GTB/G176R/ G235S bound to a Glycosyltransferases synthesize carbohydrate moieties of glycoconjugates by catalyzing the sequential addition of monosaccharides from specific donors to specific acceptors. The ubiquitous presence of glycolipids and glycoproteins in all living systems underlines the importance of the glycosyltransferases superfamily, and the DNA of all domains of life encode for a large number of these enzymes (1). To date, crystal structures of glycosyltransferases have displayed a high degree of structural similarity even when there is low sequence homology (2-4). As such, glycosyltransferases provide an excellent example of the preferential conservation of structural phenotype over the conservation of sequence identity (2), which indicates that the mechanism of glycosylation, although not yet fully understood, has been conserved.

A single HT29 human colon adenocarcinoma cell was introduced into a fused-silica capillary and lysed, and the protein content was fluorescently labeled with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde. The labeled proteins were separated by capillary electrophoresis in a submicellar buffer and detected by laser-induced fluorescence in a postcolumn sheath-flow cuvette. Several dozen components were resolved. A number of experiments were done to verify that these components were proteins. Most components of the single-cell electropherogram had the same mobility as components present in the 30-100 kDa fraction of a protein extract prepared from the cell culture. One component was identified as a approximately 100 kDa protein by co-injecting the sample with purified protein obtained from an SDS-PAGE gel. Protein expression varied significantly between cells, but the average expression was consistent with that observed from a protein extract prepared from 10(6) cells.

Blood group A and B antigens are carbohydrate structures that are synthesized by glycosyltransferase enzymes. The final step in B antigen synthesis is carried out by an ␣1-3 galactosyltransferase (GTB) that transfers galactose from UDP-Gal to type 1 or type 2, ␣Fuc132␤Gal-R (H)-terminating acceptors. Similarly the A antigen is produced by an ␣1-3 N-acetylgalactosaminyltransferase that transfers N-acetylgalactosamine from UDP-GalNAc to H-acceptors. Human ␣1-3 N-acetylgalactosaminyltransferase and GTB are highly homologous enzymes differing in only four of 354 amino acids (R176G, G235S, L266M, and G268A). Single crystal x-ray diffraction studies have shown that the latter two of these amino acids are responsible for the difference in donor specificity, while the other residues have roles in acceptor binding and turnover. Recently a novel cis-AB allele was discovered that produced A and B cell surface structures. It had codons corresponding to GTB with a single point mutation that replaced the conserved amino acid proline 234 with serine. Active enzyme expressed from a synthetic gene corresponding to GTB with a P234S mutation shows a dramatic and complete reversal of donor specificity. Although this enzyme contains all four "critical" amino acids associated with the production of blood group B antigen, it preferentially utilizes the blood group A donor UDP-GalNAc and shows only marginal transfer of UDP-Gal. The crystal structure of the mutant reveals the basis for the shift in donor specificity.

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