Structural Basis for the Inactivity of Human Blood Group O2 Glycosyltransferase

Abstract

The human ABO(H) blood group antigens are carbohydrate structures generated by glycosyltransferase enzymes. Glycosyltransferase A (GTA) uses UDP-GalNAc as a donor to transfer a monosaccharide residue to Fuc␣1-2Gal␤-R (H)-terminating acceptors. Similarly, glycosyltransferase B (GTB) catalyzes the transfer of a monosaccharide residue from UDP-Gal to the same acceptors. These are highly homologous enzymes differing in only four of 354 amino acids, Arg/Gly-176, Gly/Ser-235, Leu/Met-266, and Gly/Ala-268. Blood group O usually stems from the expression of truncated inactive forms of GTA or GTB. Recently, an O 2 enzyme was discovered that was a fulllength form of GTA with three mutations, P74S, R176G, and G268R. We showed previously that the R176G mutation increased catalytic activity with minor effects on substrate binding. Enzyme kinetics and high resolution structural studies of mutant enzymes based on the O 2 blood group transferase reveal that whereas the P74S mutation in the stem region of the protein does not appear to play a role in enzyme inactivation, the G268R mutation completely blocks the donor GalNAc-binding site leaving the acceptor binding site unaffected.

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.…”

“…For example, AAAA refers to GTA, BBBB refers to GTB, and AABB refers to the chimera with the first two critical residues of GTA and the last two critical residues of GTB. Previous findings implicate the first amino acid in enzyme turnover (12,26), the second and third in acceptor recognition (14,27), and the third and fourth in donor selection (14,17,25,28,29).…”

“…1A, GTA catalyzes the transfer of an N-acetylgalactosaminyl (GalNAc) residue from UDP-GalNAc to HA to form the A antigen, and GTB catalyzes the transfer of a galactosyl (Gal) residue from UDP-Gal to HA to form the B antigen (14,15,20,(22)(23)(24)(25). Blood group O individuals generally express truncated or mutated forms of GTA and GTB (16,25,26), so biosynthesis terminates at the H antigen.…”

ABO(H) Blood Group A and B Glycosyltransferases Recognize Substrate via Specific Conformational Changes

“…Crystallization-All proteins were crystallized using conditions different from those reported previously (10,19,33,35,38,39). Whereas the first crystals of GTB were grown from relatively low protein concentrations (ϳ8 -15 mg/ml) and as a mercury derivative, the crystals in this paper were initially generated from higher protein concentrations (ϳ60 -75 mg/ml).…”

Preliminary joint neutron time-of-flight and X-ray crystallographic study of human ABO(H) blood group A glycosyltransferase

“…The glycosyltransferase GTA transfers the N-acetylgalactosaminyl monosaccharide residue from UDP to Gal O3 of the H antigen [minimal epitope Fuc-(1!2)Gal] with retention of the axial stereochemistry to generate the human blood group A antigen and is a model enzyme for studying the retaining glycosyltransfer mechanism (Alfaro et al, 2008;Lee et al, 2005;Letts et al, 2007;Nguyen et al, 2003;Patenaude et al, 2002;Persson et al, 2007;Schuman et al, 2007Schuman et al, , 2010Seto et al, 1999). Evaluation of hydrogenbonding partners and amino-acid protonation states is critical for understanding the catalytic mechanisms of GTA and indeed of all retaining glycosyltransferases.…”