Differential Recognition of the Type I and II H Antigen Acceptors by the Human ABO(H) Blood Group A and B Glycosyltransferases

James A Letts,Natisha L Rose,Ying R Fang,Christopher H Barry,Svetlana N Borisova,Nina O.L Seto,Monica M Palcic,Stephen V Evans

doi:10.1074/jbc.m507620200

James A Letts, Natisha L Rose + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m507620200

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Abstract

The human ABO(H) blood group A and B antigens are generated by the homologous glycosyltransferases A (GTA) and B (GTB), which add the monosaccharides GalNAc and Gal, respectively, to the cell-surface H antigens. In the first comprehensive structural study of the recognition by a glycosyltransferase of a panel of substrates corresponding to acceptor fragments, 14 high resolution crystal structures of GTA and GTB have been determined in the presence of oligosaccharides corresponding to different segments of the type I (alpha-l-Fucp-(1-->2)-beta-D-Galp-(1-->3)-beta-D-GlcNAcp-OR, where R is a glycoprotein or glycolipid in natural acceptors) and type II (alpha-l-Fucp-(1-->2)-beta-D-Galp-(1-->4)-beta-d-GlcNAcp-OR) H antigen trisaccharides. GTA and GTB differ in only four "critical" amino acid residues (Arg/Gly-176, Gly/Ser-235, Leu/Met-266, and Gly/Ala-268). As these enzymes both utilize the H antigen acceptors, the four critical residues had been thought to be involved strictly in donor recognition; however, we now report that acceptor binding and subsequent transfer are significantly influenced by two of these residues: Gly/Ser-235 and Leu/Met-266. Furthermore, these structures show that acceptor recognition is dominated by the central Gal residue despite the fact that the L-Fuc residue is required for efficient catalysis and give direct insight into the design of model inhibitors for GTA and GTB.

Highlights

Sequence homology among the hundreds of known GTs
The human ABO(H) blood group A and B antigens are synthesized by the specific GTs A (GTA) and B (GTB), which transfer GalNAc from UDP-GalNAc and Gal from UDP-Gal, respectively, to the H antigen disaccharide ␣-L-Fucp-(132)-␤-D-Galp-OR
The remaining two critical residues (Arg/Gly-176 and Gly/Ser-235) were found to have little effect on donor specificity. This pattern was understood upon the determination of the structures of GTA and GTB in the presence and absence of UDP and the H antigen disaccharide ␣-L-Fucp-(132)-␤-D-Galp-O(CH2)7CH3 (7), which showed that only Leu/Met-266 and Gly/Ala-268 are positioned to affect donor recognition

Summary

Introduction

Sequence homology among the hundreds of known GTs. There are currently 79 different families of GTs; but to date, only 20 contain structurally characterized examples (4). GTA and GTB are prototypic retaining GTs and are model probes of this class of enzyme They are the two most homologous, naturally occurring GTs known to transfer distinct naturally occurring donors and differ only in 4 of 354 amino acid residues: Arg/Gly-176, Gly/Ser-235, Leu/Met-266, and Gly/Ala-268 (5). The remaining two critical residues (Arg/Gly-176 and Gly/Ser-235) were found to have little effect on donor specificity This pattern was understood upon the determination of the structures of GTA and GTB in the presence and absence of UDP and the H antigen disaccharide ␣-L-Fucp-(132)-␤-D-Galp-O(CH2)7CH3 (7), which showed that only Leu/Met-266 and Gly/Ala-268 are positioned to affect donor recognition. Removing the ␣-L-Fuc residue (to form N-acetyllactosamine (LacNAc) in the case of the type II H antigen: ␤-D-Galp-(134)-D-GlcNAcp) results in a weak acceptor for GTA, indicating that the fucose residue is essential for efficient catalysis (10)

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