A Single Point Mutation Reverses the Donor Specificity of Human Blood Group B-synthesizing Galactosyltransferase

Abstract

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.

High Resolution Structures of the Human ABO(H) Blood Group Enzymes in Complex with Donor Analogs Reveal That the Enzymes Utilize Multiple Donor Conformations to Bind Substrates in a Stepwise Manner

“…UDP-glucose (UDP-Glc) is a C4 epimer of UDP-Gal that binds GTB with comparable affinity (31), but whereas STD NMR experiments have shown the bound conformations of these nucleotide sugars to be comparable (32), UDP-Glc yields much lower enzymatic activity, 0% compared with UDP-GalNAc for GTA, which has a k cat of 17.5 s Ϫ1 , and 0.02% compared with UDP-Gal for GTB, which has a k cat of 5.1 s Ϫ1 (26,40). Here we report the structures to high resolution of GTA, GTB, AABB, ABBA, and ABBB grown in complex with UDP-C-Gal, UDP-Gal, and UDP-Glc with either synthetic H antigen 3 , providing fresh insight into the glycosyl transfer reaction.…”

The Influence of an Intramolecular Hydrogen Bond in Differential Recognition of Inhibitory Acceptor Analogs by Human ABO(H) Blood Group A and B Glycosyltransferases

“…Of the four amino acid differences between GTA and GTB, residue 266 has been shown to dominate the selection between UDP sugar donors (4), with GTA having Leu and GTB having Met. Small differences between the binding of H-disaccharide acceptor to GTA and GTB have been noted in the past (8) and attributed to Leu/Met-266; however, the positions of both the native (4) and now the 3-deoxy acceptor analogs in GTA and GTB are nearly superimposable. The large difference in position observed for the 3-amino analog emphasizes the influence that Leu/Met-266 can have on acceptor binding.…”

B-Transferase with a Pro234Ser Substitution Acquires AB-Transferase Activity

“…12) In that study, Marcus et al explained that a possible mechanism of this mismatch between the substrate specificity of the cloned enzyme (A) and the phenotype of red blood cells of individuals with this mutation (AB) might be due to the difference in length and form (briefly, a truncated and soluble form) of the recombinant P234S mutant. This hypothesis is consistent with our result for full-length B-transferase/P234S, in that the transfectants exhibited an AB phenotype.…”

“…6) The effects of amino acid mutations of the 234th and 266th residues were analyzed as to the substrate specificity and activity of the purified recombinant proteins, 5,12) but the effect on phenotypes has not been analyzed. In the present study, A/B/H-antigens on transfectants, in which B-transferase/P234S cDNA or A-transferase/L266M was introduced, were analyzed with other transfectants carrying cDNA, A-transferase, B-transferase, and A-transferase loss-of-function mutants (A-transferase/delG; typical O-genotype).…”