Identification of disulfide bonds among the nine core 2 N-acetylglucosaminyltransferase-M cysteines conserved in the mucin β6-N-acetylglucosaminyltransferase family

Jaswant Singh, Gausal A. Khan, Leo Kinarsky, Helen Cheng, Jason Wilken, Kyung Hyun Choi, Elliott Bedows, Simon Sherman, Pi Wan Cheng

Research output: Contribution to journalArticle

6 Scopus citations

Abstract

Bovine core 2 β1,6-N-acetylglucosaminyltransferase-M (bC2GnT-M) catalyzes the formation of all mucin β1,6-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 β1,6-N-acetylglucosaminyltransferases contain nine conserved cysteines. We determined the disulfide bond assignments of these cysteines in [35S]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 35S-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 (Cys113) was a free thiol, whereas the other eight cysteines formed four disulfide bridges, which included the first (Cys73) and sixth (Cys230), third (Cys164) and seventh (Cys 384), fourth (Cys185) and fifth (Cys212), and eighth (Cys393) and ninth (Cys425) 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.

Original languageEnglish (US)
Pages (from-to)38969-38977
Number of pages9
JournalJournal of Biological Chemistry
Volume279
Issue number37
DOIs
StatePublished - Sep 10 2004

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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