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J. Biol. Chem., Vol. 279, Issue 37, 38969-38977, September 10, 2004

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Identification of Disulfide Bonds among the Nine Core 2 N-Acetylglucosaminyltransferase-M Cysteines Conserved in the Mucin 6-N-Acetylglucosaminyltransferase Family^*

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

From the Department of Biochemistry and Molecular Biology and ^||Department of Obstetrics and Gynecology, College of Medicine, and the ^¶Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198

Bovine core 2 1,6-N-acetylglucosaminyltransferase-M (bC2GnT-M) catalyzes the formation of all mucin 1,6-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 1,6-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 (Cys³⁸⁴), 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.

Received for publication, January 30, 2004 , and in revised form, June 4, 2004.

^* The Bioinformatics Core Facility of the University of Nebraska Medical Center used in these studies was supported in part by National Institutes of Health (NIH) Cancer Center Support Grant P30 CA36727, NIH Grant RR15635 from the COBRE Program, NIH Grant P20 RR16469 from the BRIN Program, NCI, NIH, Training Grant CA09746 to the Eppley Institute Cancer Center, and the Nebraska Research Initiatives as well as NIH Grant HL48282 (to P.-W. C.), the University of Nebraska Medical Center Olson Center for Women's Health (to E. B.), and an Emley Fellowship (to J. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this work.

^** To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 985870 Nebraska Medical Center, Omaha, NE 68198-5870. Tel.: 402-559-5776; Fax: 402-559-6650; E-mail: pcheng{at}unmc.edu.

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