Oligosaccharide Preferences of β1,4-Galactosyltransferase-I: Crystal Structures of Met340His Mutant of Human β1,4-Galactosyltransferase-I with a Pentasaccharide and Trisaccharides of the N-Glycan Moiety

β-1,4-Galactosyltransferase-I (β4Gal-T1) transfers galactose from UDP-galactose 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 β-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 β4Gal-T1 towards the GlcNAc residues of N-glycans, we have carried out kinetic and crystallographic studies with the wild-type human β4Gal-T1 (h-β4Gal-T1) and the mutant Met340His-β4Gal-T1 (h-M340H-β4Gal-T1) in complex with a GlcNAc-containing pentasaccharide and several GlcNAc-containing trisaccharides present in N-glycans. The oligosaccharides used were: pentasaccharide GlcNAcβ1,2-Manα1,6 (GlcNAcβ1,2-Manα1,3)Man; the 1,6-arm trisaccharide, GlcNAcβ1,2-Manα1,6-Manβ-OR (1,2-1,6-arm); the 1,3-arm trisaccharides, GlcNAcβ1,2-Manα1,3-Manβ-OR (1,2-1,3-arm) and GlcNAcβ1,4-Manα1,3-Manβ-OR (1,4-1,3-arm); and the trisaccharide GlcNAcβ1,4-GlcNAcβ1,4-GlcNAc (chitotriose). With the wild-type h-β4Gal-T1, the Km of 1,2-1,6-arm is approximately tenfold lower than for 1,2-1,3-arm and 1,4-1,3-arm, and 22-fold lower than for chitotriose. Crystal structures of h-M340H-β4Gal-T1 in complex with the pentasaccharide and various trisaccharides at 1.9–2.0 Å resolution showed that β4Gal-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 Km for the trisaccharide. Present studies suggest that β4Gal-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.