title: Oligosaccharide preferences of beta1,4-galactosyltransferase-I: crystal structures of Met340His mutant of human beta1,4-galactosyltransferase-I with a pentasaccharide and trisaccharides of the N-glycan moiety.
abstract: beta-1,4-Galactosyltransferase-I (beta4Gal-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 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 beta4Gal-T1 towards the GlcNAc residues of N-glycans, we have carried out kinetic and crystallographic studies with the wild-type human beta4Gal-T1 (h-beta4Gal-T1) and the mutant Met340His-beta4Gal-T1 (h-M340H-beta4Gal-T1) in complex with a GlcNAc-containing pentasaccharide and several GlcNAc-containing trisaccharides present in N-glycans. The oligosaccharides used were: pentasaccharide GlcNAcbeta1,2-Manalpha1,6 (GlcNAcbeta1,2-Manalpha1,3)Man; the 1,6-arm trisaccharide, GlcNAcbeta1,2-Manalpha1,6-Manbeta-OR (1,2-1,6-arm); the 1,3-arm trisaccharides, GlcNAcbeta1,2-Manalpha1,3-Manbeta-OR (1,2-1,3-arm) and GlcNAcbeta1,4-Manalpha1,3-Manbeta-OR (1,4-1,3-arm); and the trisaccharide GlcNAcbeta1,4-GlcNAcbeta1,4-GlcNAc (chitotriose). With the wild-type h-beta4Gal-T1, the K(m) 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-beta4Gal-T1 in complex with the pentasaccharide and various trisaccharides at 1.9-2.0A resolution showed that beta4Gal-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(m) for the trisaccharide. Present studies suggest that beta4Gal-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.
authors.affiliation: Laboratory of Experimental and Computational Biology, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702, USA.
authors.forename: Velavan
authors.initials: V
authors.lastname: Ramasamy
authors.forename: Boopathy
authors.initials: B
authors.lastname: Ramakrishnan
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Boeggeman
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Ratner
authors.forename: Peter H
authors.initials: PH
authors.lastname: Seeberger
authors.forename: Pradman K
authors.initials: PK
authors.lastname: Qasba
mesh_terms: D002236:Carbohydrate Conformation; D002384:Catalysis; D018360:Crystallography, X-Ray; D005700:Galactosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008715:Methionine / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D009844:Oligosaccharides; D008715:Methionine; D005700:Galactosyltransferases; C493310:beta1,4-galactosyltransferase, human