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

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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 [ 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.

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

Fingerprint

Mucin-6
Disulfides
Cysteine
Mucin-1
Mucins
N-Acetylglucosaminyltransferases
Plasmids
Glycosyltransferases
Peptides
Molecular modeling
Cystine
Serum-Free Culture Media
Liquids
Conditioned Culture Medium
Blood Group Antigens
Aspartate Aminotransferases
Substrate Specificity
Cricetulus
Sulfhydryl Compounds
N-acetyllactosaminide beta-1,6-N-acetylglucosaminyltransferase

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Identification of disulfide bonds among the nine core 2 N-acetylglucosaminyltransferase-M cysteines conserved in the mucin β6-N-acetylglucosaminyltransferase family. / Singh, Jaswant; Khan, Gausal A.; Kinarsky, Leo; Cheng, Helen; Wilken, Jason; Choi, Kyung Hyun; Bedows, Elliott; Sherman, Simon; Cheng, Pi-Wan.

In: Journal of Biological Chemistry, Vol. 279, No. 37, 10.09.2004, p. 38969-38977.

Research output: Contribution to journalArticle

Singh, Jaswant ; Khan, Gausal A. ; Kinarsky, Leo ; Cheng, Helen ; Wilken, Jason ; Choi, Kyung Hyun ; Bedows, Elliott ; Sherman, Simon ; Cheng, Pi-Wan. / Identification of disulfide bonds among the nine core 2 N-acetylglucosaminyltransferase-M cysteines conserved in the mucin β6-N-acetylglucosaminyltransferase family. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 37. pp. 38969-38977.
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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 [ 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.",
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AU - Khan, Gausal A.

AU - Kinarsky, Leo

AU - Cheng, Helen

AU - Wilken, Jason

AU - Choi, Kyung Hyun

AU - Bedows, Elliott

AU - Sherman, Simon

AU - Cheng, Pi-Wan

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