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J. Biol. Chem., Vol. 282, Issue 24, 17890-17899, June 15, 2007

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A -1,2-Xylosyltransferase from Cryptococcus neoformans Defines a New Family of Glycosyltransferases^*

J. Stacey Klutts, Steven B. Levery^¶, and Tamara L. Doering¹

From the Departments of Molecular Microbiology and Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri 63110 and the ^¶Department of Chemistry, University of New Hampshire, Durham, New Hampshire 03824

Cryptococcus neoformans is an opportunistic fungal pathogen characterized by a prominent polysaccharide capsule that envelops the cell. Although this capsule is dispensable for in vitro growth, its presence is essential for virulence. The capsule is primarily made of two xylose-containing polysaccharides, glucuronoxylomannan and galactoxylomannan. There are likely to be multiple xylosyltransferases (XTs) involved in capsule synthesis, and the activities of these enzymes are potentially important for cryptococcal virulence. A -1,2-xylosyltransferase with specificity appropriate for capsule synthesis was purified 3000-fold from C. neoformans, and the corresponding gene was identified and cloned. This sequence conferred XT activity when expressed in Saccharomyces cerevisiae, which lacks endogenous XT activity. The gene, termed CXT1 for cryptococcal xylosyltransferase 1, encodes a 79-kDa type II membrane protein with an N-linked glycosylation site and two DXD motifs. These latter motifs are believed to coordinate divalent cation binding in the activity of glycosyltransferases. Site-directed mutagenesis of one DXD motif abolished Cxt1p activity, even though this activity does not depend on the addition of a divalent cation. This may indicate a novel catalytic mechanism for glycosyl transfer. Five homologs of Cxt1p were found in the genome sequence of C. neoformans and 34 within the sequences of other fungi, although none were found in other organisms. Many of the homologous proteins are similar in size to Cxt1p, and all are conserved with respect to the essential DXD motif. These proteins represent a new family of glycosyltransferases, found exclusively within the fungal kingdom.

Received for publication, March 6, 2007 , and in revised form, April 11, 2007.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) 905015.

^* This work was supported in part by National Institutes of Health Awards GM R01 071007 (to T. L. D.), GM F32 072341 (to J. S. K.), and R21 RR20355 (to S. B. L.), and a William Keck Foundation Postdoctoral Fellowship (to J. S. K.). 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 supplemental Table 1 and Fig. 1.

¹ To whom correspondence should be addressed: Dept. of Molecular Microbiology, Washington University School of Medicine, 660 South Euclid Ave., Campus Box 8230, St. Louis, MO 63110-1093. Tel.: 314-747-5597; Fax: 314-362-1232; E-mail: doering{at}wustl.edu.

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				S. A. Castle, E. A. Owuor, S. H. Thompson, M. R. Garnsey, J. S. Klutts, T. L. Doering, and S. B. Levery {beta}1,2-Xylosyltransferase Cxt1p Is Solely Responsible for Xylose Incorporation into Cryptococcus neoformans Glycosphingolipids Eukaryot. Cell, September 1, 2008; 7(9): 1611 - 1615. [Abstract] [Full Text] [PDF]

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