Identification of disulfide bonds among the nine core 2 N-acetylglucosaminyltransferase-M cysteines conserved in the mucin beta6-N-acetylglucosaminyltransferase family

The Journal of Biological Chemistry
J SinghPi-Wan Cheng

Abstract

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 incl...Continue Reading

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Citations

Sep 19, 2012·The Journal of Biological Chemistry·Armen PetrosyanPi-Wan Cheng
Apr 1, 2008·The International Journal of Biochemistry & Cell Biology·Prakash RadhakrishnanPi-Wan Cheng

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