High-resolution crystal structures of the human ABO blood group glycosyltransferase enzymes, unliganded and in complex with substrate molecules, reveal the basis for their substrate specificity and suggest amino acid residues important in catalysis. The human ABO(H) blood group glycosyltransferases differ in only 4 amino acid residues and are therefore two of the most homologous, naturally occurring glycosyltransferases known that utilize different naturally occurring donor molecules. An N-acetylgalactosaminyltransferase (GTA) uses a UDP-GalNAc donor to convert the H-antigen to the A-antigen, while a galactosyltransferase (GTB) uses a UDP-Gal donor to convert the H-antigen to B antigen. The resulting A and B blood group antigens differ from each other only in the substitution on the terminal saccharide residue of an acetamido for an hydroxyl group yet the potentially catastrophic effects of a mismatched blood transfusion makes them a paradigm for specificity in biosynthetic and immune recognition. The crystal structures of the catalytic domains of the cloned blood-group A and B enzymes have been determined to 1.80 and 1.65 A resolution, respectively, and those of the catalytic domains of the A and B enzymes in complex with the H antigen disaccharide and UDP to 1.35 and 1.32 A resolution, respectively. Glycosyltransferases that retain the stereochemistry of the donor sugar, such as GTA and GTB, have been postulated to function via a double-inversion mechanism, including a nucleophilic attack on the donor sugar anomeric carbon. The current structures support the double inversion mechanism and reveal, remarkably, that only two of the amino acids differing between GTA and GTB are positioned to select between the two donors and thus contribute to the stringent stereo- and regioselectivity in this biosynthesis. In addition, the structures of GTA and GTB in complex with H antigen alone have been determined to 1.58 and 1.46 A resolution, respectively, and show that, contrary to expectations, the acceptor substrate can bind in the absence of the donor substrate. The DxD motif in GTA and GTB coordinates a manganese ion, which in turn interacts with the UDP-sugar donor. Comparison of the GTA and GTB structures to the other two retaining glycosyltransferases (bovine alpha1-3galactosyltransferase, N. meningitidis LgtC) structures determined to date reveals that the coordination between the aspartate residues and manganese ion are the same. Interestingly, this coordination pattern is different from those observed in inverting glycosyltransferases, suggesting that these patterns may be characteristic of inverting versus retaining glycosyltranserases. The structure of an antibody fragment specific for the human blood group A trisaccharide antigen has also been determined to near-atomic resolution. This structure shows a pronounced pocket at the antigen-binding site, which is formed by four of the six complementarity determining regions, and is of the appropriate size and shape to accommodate the terminal N-acetylgalactosamine residue of the A trisaccharide antigen. A model of the Fv-trisaccharide complex shows several protein-carbohydrate interactions that would allow specific recognition of the blood group A antigen acetamido group.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/6188 |
| Date | January 2002 |
| Creators | Patenaude, Sonia I. |
| Contributors | Evans, Steve, |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Detected Language | English |
| Type | Thesis |
| Format | 159 p. |
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