Oligosaccharide preferences of beta 1,4-galactosyltransferase-I: Crystal structures of Met340His mutant of human beta 1,4-galactosyltransferase-I with a pentasaccharide and trisaccharides of the N-glycan moiety

beta-1,4-Galactosyltransferase-I (beta 4Gal-T1) transfers galactose from UDPgalactose to N-acetylglucosamine (GlcNAc) residues of the branched N-linked oligosaccharide chains of glycoproteins. In an N-linked biantennary oligosaccharide chain, one antenna is attached to the 3-hydroxyl-(1,3-arm), and the other to the 6-hydroxyl-(1,6-arm) group of mannose, which is beta-1,4-linked to an N-linked chitobiose, attached to the aspargine residue of a protein. For a better understanding of the branch specificity of beta 4Gal-T1 towards the GlcNAc residues of N-glycans, we have carried out kinetic and crystallographic studies with the wild-type human beta 4Gal-T1 (h-beta 4Gal-T1) and the mutant Met340His-beta 4Gal-T1 (h-M340H-beta 4Gal-T1) in complex with a GlcNAc-containing pentasaccharide and several GlcNAc-containing trisaccharides present in N-glycans. The oligosaccharides used were: pentasaccharide GlcNAc beta 1,2-Man alpha 1,6 (GlcNAc beta 1,2-Man alpha 1,3)Man; the 1,6-arm trisaccharide, GlcNAc beta 1,2-Man alpha 1,6-Man beta-OR (1,2-1,6-arm); the 1,3-arm trisaccharides, GlcNAc beta 1,2-Man alpha 1,3-Man beta-OR (1,2-1,3-arm) and GlcNAc beta 1,4-Man alpha 1,3-Man beta-OR (1,4-1,3-arm); and the trisaccharide GlcNA beta 1,4-GlcNAc beta 1,4-GlcNAc (chitotriose). With the wild-type h-beta 4Gal-T1, the K, of 1,2-1,6-arm is approximately tenfold lower than for 1,2-1,3arm and 1,4-1,3-arm, and 22-fold lower than for chitotriose. Crystal structures of h-M340H-P beta Gal-T1 in complex with the pentasaccharide and various trisaccharides at 1.9-2.0 angstrom resolution showed that beta 4Gal-T1 is in a closed conformation with the oligosaccharide bound to the enzyme, and the 1,2-1,6-arm trisaccharide makes the maximum number of interactions with the enzyme, which is in concurrence with the lowest K, for the trisaccharide. Present studies suggest that beta 4Gal-T1 interacts preferentially with the 1,2-1,6-arm trisaccharide rather than with the 1,2-1,3-arm or 1,4-1, 3-arm of a bi- or tri-antennary oligosaccharide chain of N-glycan. Published by Elsevier Ltd

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