Highly Conserved Cysteines of Mouse Core 2 β1,6-N-Acetylglucosaminyltransferase I Form a Network of Disulfide Bonds and Include a Thiol That Affects Enzyme Activity

• Published in: The Journal of biological chemistry Vol. 278; no. 46; p. 45864
• Main Authors: Ten-Yang Yen, Bruce A. Macher, Steve Bryson, Xiaoqing Chang, Igor TvaroÅ¡ka, Roderick Tse, Sawako Takeshita, April M. Lew, Alessandro Datti
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 14.11.2003
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M303851200

Core 2 β1,6- N -acetylglucosaminyltransferase I (C2GnT-I) plays a pivotal role in the biosynthesis of mucin-type O -glycans that serve as ligands in cell adhesion. To elucidate the three-dimensional structure of the enzyme for use in computer-aided design of therapeutically relevant enzyme inhibitors, we investigated the participation of cysteine residues in disulfide linkages in a purified murine recombinant enzyme. The pattern of free and disulfide-bonded Cys residues was determined by liquid chromatography/electrospray ionization tandem mass spectrometry in the absence and presence of dithiothreitol. Of nine highly conserved Cys residues, under both conditions, one (Cys 217 ) is a free thiol, and eight are engaged in disulfide bonds, with pairs formed between Cys 59 –Cys 413 , Cys 100 –Cys 172 , Cys 151 –Cys 199 , and Cys 372 –Cys 381 . The only non-conserved residue within the β1,6- N -acetylglucosaminyltransferase family, Cys 235 , is also a free thiol in the presence of dithiothreitol; however, in the absence of reductant, Cys 235 forms an intermolecular disulfide linkage. Biochemical studies performed with thiolreactive agents demonstrated that at least one free cysteine affects enzyme activity and is proximal to the UDP-GlcNAc binding site. A Cys 217 → Ser mutant enzyme was insensitive to thiol reactants and displayed kinetic properties virtually identical to those of the wild-type enzyme, thereby showing that Cys 217 , although not required for activity per se , represents the only thiol that causes enzyme inactivation when modified. Based on the pattern of free and disulfide-linked Cys residues, and a method of fold recognition/threading and homology modeling, we have computed a three-dimensional model for this enzyme that was refined using the T4 bacteriophage β-glucosyltransferase fold.