Highly conserved cysteines of mouse core 2 beta1,6-N-acetylglucosaminyltransferase I form a network of disulfide bonds and include a thiol that affects enzyme activity.
Core 2 beta1,6-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 (Cys217) is a free thiol, and eight are engaged in disulfide bonds, with pairs formed between Cys59-Cys413, Cys100-Cys172, Cys151-Cys199, and Cys372-Cys381. The only non-conserved residue within the beta1,6-N-acetylglucosaminyltransferase family, Cys235, is also a free thiol in the presence of dithiothreitol; however, in the absence of reductant, Cys235 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 Cys217 --> Ser mutant enzyme was insensitive to thiol reactants and displayed kinetic properties virtually identical to those of the wild-type enzyme, thereby showing that Cys217, 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 beta-glucosyltransferase fold.
Yen
Ten-Yang
TY
Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California 94132, USA.
Macher
Bruce A
BA
Bryson
Steve
S
Chang
Xiaoqing
X
Tvaroska
Igor
I
Tse
Roderick
R
Takeshita
Sawako
S
Lew
April M
AM
Datti
Alessandro
A
D000595:Amino Acid Sequence; D000818:Animals; D017122:Bacteriophage T4 / Q000201:enzymology; D001665:Binding Sites; D002412:Cations; D002448:Cell Adhesion; D002918:Chymotrypsin / Q000494:pharmacology; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005964:Glucosyltransferases / Q000737:chemistry; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry* / Q000378:metabolism*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry; D011134:Polysaccharides; D017510:Protein Folding; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000737:chemistry; D019163:Reducing Agents / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D012697:Serine Endopeptidases / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
D002412:Cations; D004220:Disulfides; D011134:Polysaccharides; D020033:Protein Isoforms; D011994:Recombinant Proteins; D019163:Reducing Agents; D013438:Sulfhydryl Compounds; D012694:Serine; D005964:Glucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C053024:beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-acetylglucosaminyl transferase; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C028344:glutamyl endopeptidase; D014357:Trypsin; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D003545:Cysteine; D004229:Dithiothreitol