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J. Biol. Chem., Vol. 279, Issue 34, 35644-35655, August 20, 2004

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Five Genes Involved in Biosynthesis of the Pyruvylated Gal1,3-Epitope in Schizosaccharomyces pombe N-Linked Glycans^*

Ekaterina N. Andreishcheva¶, Jeremy P. Kunkel¶, Trent R. Gemmill, and Robert B. Trimble||**

From the Wadsworth Center, New York State Department of Health, Albany, New York 12201-0509 and the ^||Department of Biomedical Sciences, School of Public Health, State University of New York at Albany, Albany, New York 12201

The N-linked galactomannans of Schizosaccharomyces pombe have pyruvylated Gal1,3-(PvGal) caps on a portion of the Gal1,2-residues in their outer chains (Gemmill, T. R., and Trimble, R. B. (1998) Glycobiology 8, 1087–1095). PvGal biosynthesis was investigated by ethyl methanesulfonate mutagenesis of S. pombe, followed by the isolation of cells devoid of negatively charged N-glycans by Q-Sepharose exclusion and failure to bind human serum amyloid P component, which acts as a lectin for terminal PvGal residues. Mutant glycans were characterized by lectin binding, saccharide composition, exoglycosidase sensitivity, and NMR spectroscopy. Restoration of the cell surface negative charge by complementation with an S. pombe genomic library led to the identification of five genes involved in PvGal biosynthesis, which we designated pvg1–pvg5. Pvg1p may be a pyruvyltransferase, since NMR of pvg1^– mutant N-glycans revealed the absence of only the pyruvyl moiety. Pvg2p–Pvg5p are crucial for attachment of the Gal1,3-residue that becomes pyruvylated. Pvg3p is predicted to be a member of the 1,3-galactosyltransferase family, and Pvg3p-green fluorescent protein labeling was consistent with Golgi localization. Predicted Pvg1p and Pvg3p functions imply that Gal1,3-is added to the galactomannans and is then pyruvylated in situ, rather than by an en bloc addition of PvGal1,3-caps to the outer chain. Pvg4p-green fluorescent protein targeted to the nucleus, and its sequence contains a MADS-box DNA-binding and dimerization domain; however, it does not appear to solely control transcription of the other identified genes. Pvg2p and/or Pvg5p may contribute to an enzyme complex. Whereas a functional role for the PvGal epitope in S. pombe remains unclear, it is nonessential for either cell growth or mating under laboratory conditions.

Received for publication, March 31, 2004 , and in revised form, June 1, 2004.

^* This work was supported in part by National Institutes of Health, United States Public Health Service, Grant GM23900 (to R. B. T.). 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.

The on-line version of this article (available at http://www.jbc.org) contains one additional figure and table.

Present address: Laboratory of Bacterial Toxins, Center for Biologics Evaluation and Research, Federal Drug Administration, 8800 Rockville Pike, Bethesda, MD 20892.

^¶ These authors contributed equally to this work.

^** To whom correspondence should be addressed: Wadsworth Center, C-547, New York State Department of Health, P.O. Box 509, Albany, NY 12201-0509. Tel.: 518-474-5511; Fax: 518-402-5381; E-mail: trimble{at}wadsworth.org.

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