Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Molecular Microbiology and Microbial Pathogenesis

Language

English (en)

Date of Award

January 2009

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Tamara Doering

Abstract

Cryptococcus neoformans is an environmental yeast and an opportunistic pathogen capable of causing a meningoencephalitis in human hosts. The organism produces an extensive polysaccharide capsule that is unique among pathogenic fungi and absolutely required for its virulence. Work in the Doering laboratory on the capsule and other glycoconjugates of C. neoformans has focused on the identification of glycosyltransferases, enzymes that catalyze the transfer of a sugar moiety from an active donor to a specific acceptor, creating a particular linkage. Previous work demonstrated that xylose residues, derived from the nucleotide sugar UDP-xylose, are necessary for cryptococcal virulence. An assay to detect xylosyltransferase activity was developed in the laboratory using a radiolabeled UDP-xylose donor, a dimannose acceptor, and protein fractions from C. neoformans as the source of enzymatic activity. Using this assay, several discrete xylosyltransferase activities have been detected, including one that depends on the presence of manganese cations as a cofactor. The identification and characterization of the protein responsible for this activity has been the focus of these dissertation studies. The product of the manganese-dependent xylosyltransferase activity was analyzed by mass spectrometry and NMR and found to be xylose-α-phosphate-6-mannose-α-1,3-mannose, indicating that the enzyme responsible is, unexpectedly, a xylosylphosphotransferase: Xpt1p). There are no reports in the literature of similar glycan structures, suggesting that Xpt1p is a novel enzyme capable of generating a unique sugar linkage. The locus encoding Xpt1p activity was identified based on limited homology to a known mammalian glycosylphosphotransferase and confirmed by activity studies of a deletion mutant. Xpt1p was subsequently shown to prefer the donor and acceptor molecules UDP-xylose and mannose, respectively. It was further found to play a role in the glycosylation of cellular proteins, in particular the synthesis of O-linked glycan structures, and has been suggested to exist in a multimeric protein complex. This thesis details these studies of Xpt1p and considers the future directions of this research. Altogether, this work has broadened our understanding of glycan synthesis in general and the synthesis of cryptococcal glycans in particular.

Comments

Permanent URL: http://dx.doi.org/10.7936/K72B8W1J

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