Oligosaccharide Preferences of b1,4-Galactosyltransferase-I: Crystal Structures of Met340His Mutant of Human b1,4-Galactosyltransferase-I with a Pentasaccharide and Trisaccharides of the N-Glycan Moiety Velavan Ramasamy 1 , Boopathy Ramakrishnan 1,2 Elizabeth Boeggeman 1,2 , Daniel M. Ratner 3 , Peter H. Seeberger 3,4 and Pradman K. Qasba 1 * 1 Structural Glycobiology Section, Laboratory of Exper- imental and Computational Biology, Center for Cancer Research, National Cancer Institute at Frederick Frederick, MD 21702, USA 2 Basic Research Program SAIC-Frederick, Inc.; Laboratory of Experimental and Computational Biology, Center for Cancer Research, National Cancer Institute at Frederick Frederick, MD 21702, USA 3 Department of Chemistry Massachusetts Institute of Technology, Cambridge, MA 02139, USA 4 Laboratorium fu ¨ r Organische Chemie, ETH Ho ¨nggerberg/HCI F 315, Wolfgang-Pauli-Strasse 10, CH-8093, Zu ¨ rich Switzerland b-1,4-Galactosyltransferase-I (b4Gal-T1) transfers galactose from UDP- galactose to N-acetylglucosamine (GlcNAc) residues of the branched N-linked oligosaccharide chains of glycoproteins. In an N-linked bianten- nary 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 b-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 b4Gal-T1 towards the GlcNAc residues of N-glycans, we have carried out kinetic and crystallographic studies with the wild-type human b4Gal-T1 (h-b4Gal-T1) and the mutant Met340His-b4Gal-T1 (h-M340H-b4Gal-T1) in complex with a GlcNAc-containing pentasaccharide and several GlcNAc-containing trisaccharides present in N-glycans. The oligosaccharides used were: pentasaccharide GlcNAcb1,2-Mana1,6 (GlcNAcb1,2-Mana1,3)Man; the 1,6-arm trisaccharide, GlcNAcb1,2-Mana1,6-Manb-OR (1,2-1,6-arm); the 1,3-arm trisaccharides, GlcNAcb1,2-Mana1,3-Manb-OR (1,2-1,3-arm) and GlcNAcb1,4-Mana1,3-Manb-OR (1,4-1,3-arm); and the trisaccharide GlcNAcb1,4-GlcNAcb1,4-GlcNAc (chitotriose). With the wild-type h-b4Gal- 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 struc- tures of h-M340H-b4Gal-T1 in complex with the pentasaccharide and various trisaccharides at 1.9–2.0 A ˚ resolution showed that b4Gal-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 b4Gal-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. Keywords: b-1,4-galactosyltransferase-I; conformational change; N-glycan trisaccharides; human-b4Gal-T1-oligosaccharide crystal structures; b4Gal- T1 oligosaccharide preferences *Corresponding author 0022-2836/$ - see front matter Published by Elsevier Ltd. Abbreviations used: ER, endoplasmic reticulum; GT, glycosyltransferase; b4Gal-T, b1,4galactosyltransferase; b4Gal- T1, b1,4-Galactosyltransferase-I; b-b4Gal-T1, bovine b4Gal-T1; h-b4Gal-T1, human b4Gal-T1; UDP-Gal, UDP-a- galactose; UDP-H, UDP-hexanolamine; GlcNAc, N-acetylglucosamine; GlcNAc-T, N-acetylglucosaminyltransferase; OR, KO-CH 2 -CH 2 -CH 2 -CHZCH 2 ; PDB, RCSB protein data base. E-mail address of the corresponding author:qasba@helix.nih.gov doi:10.1016/j.jmb.2005.07.050 J. Mol. Biol. (2005) 353, 53–67