Identification of disulfide bonds among the nine core 2 N-acetylglucosaminyltransferase-M cysteines conserved in the mucin beta6-N-acetylglucosaminyltransferase family.
Bovine core 2 beta1,6-N-acetylglucosaminyltransferase-M (bC2GnT-M) catalyzes the formation of all mucin beta1,6-N-acetylglucosaminides, including core 2, core 4, and blood group I structures. These structures expand the complexity of mucin carbohydrate structure and thus the functional potential of mucins. The four known mucin beta1,6-N-acetylglucosaminyltransferases contain nine conserved cysteines. We determined the disulfide bond assignments of these cysteines in [(35)S]cysteine-labeled bC2GnT-M isolated from the serum-free conditioned medium of Chinese hamster ovary cells stably transfected with a pSecTag plasmid. This plasmid contains bC2GnT-M cDNA devoid of the 5'-sequence coding the cytoplasmic tail and transmembrane domain. The C18 reversed phase high performance liquid chromatographic profile of the tryptic peptides of reduced-alkylated (35)S-labeled C2GnT-M was established using microsequencing. Each cystine pair was identified by rechromatography of the C8 high performance liquid chromatographic radiolabeled tryptic peptides of alkylated bC2GnT-M on C18 column. Among the conserved cysteines in bC2GnT-M, the second (Cys(113)) was a free thiol, whereas the other eight cysteines formed four disulfide bridges, which included the first (Cys(73)) and sixth (Cys(230)), third (Cys(164)) and seventh (Cys(384)), fourth (Cys(185)) and fifth (Cys(212)), and eighth (Cys(393)) and ninth (Cys(425)) cysteine residues. This pattern of disulfide bond formation differs from that of mouse C2GnT-L, which may contribute to the difference in substrate specificity between these two enzymes. Molecular modeling using disulfide bond assignments and the fold recognition/threading method to search the Protein Data Bank found a match with aspartate aminotransferase structure. This structure is different from the two major protein folds proposed for glycosyltransferases.
Singh
Jaswant
J
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.
Khan
Gausal A
GA
Kinarsky
Leo
L
Cheng
Helen
H
Wilken
Jason
J
Choi
Kyung Hyun
KH
Bedows
Elliott
E
Sherman
Simon
S
Cheng
Pi-Wan
PW
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002241:Carbohydrates / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D006224:Cricetinae; D016895:Culture Media, Serum-Free; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D019992:Databases as Topic; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D016695:Glycosyltransferases / Q000378:metabolism; D051379:Mice; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009077:Mucins / Q000737:chemistry*; D017351:N-Acetylglucosaminyltransferases; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014357:Trypsin / Q000494:pharmacology
D002241:Carbohydrates; D016895:Culture Media, Serum-Free; D004220:Disulfides; D009077:Mucins; D010455:Peptides; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D016695:Glycosyltransferases; D017351:N-Acetylglucosaminyltransferases; C117361:beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-N-acetylglucosaminyltransferase 3; D014357:Trypsin; D003545:Cysteine