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Eukaryotic Cell doi:10.1128/EC.00061-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Dolichyl-phosphate-glucose is used to make O-glycans on glycoproteins of Trichomonas vaginalis

Kariona A. Grabiska, Sudip K. Ghosh, Ziqiang Guan, Jike Cui, Christian R. H. Raetz, Phillips W. Robbins, and John Samuelson^*

Department of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, Boston, Massachusetts 02118; Department of Biotechnology, Indian Institute of Technology-Kharagpur, Kharagpur 721302, India; Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

^* To whom correspondence should be addressed. Email: jsamuels{at}bu.edu.

	Abstract

Trichomonas vaginalis, the protist that causes vaginal itching, has a huge genome with numerous gene duplications. Recently we found that Trichomonas has numerous genes encoding putative dolichyl-phosphate-glucose (Dol-P-Glc) synthases (encoded by ALG5 genes) despite the fact that Trichomonas lacks the glycosyltransferases (encoded by ALG6, ALG8, and ALG10 genes) that use Dol-P-Glc to glucosylate dolichyl-PP-linked glycans. In addition, Trichomonas does not have a canonical DPM1 gene encoding a dolichyl-phosphate-mannose (Dol-P-Man) synthase. Here we show Trichomonas membranes have roughly 300 times the Dol-P-Glc synthase activity of Saccharomyces cerevisiae membranes and about 1/5^th the Dol-P-Man synthase activity of Saccharomyces. Endogenous Dol-P-hexoses of Trichomonas are relatively abundant and contain 16 isoprene units. Five paralogous Trichomonas ALG5 gene products have Dol-P-Glc synthase activity when expressed as recombinant proteins, and these Trichomonas Alg5s correct a carboxypeptidase N-glycosylation defect in a Saccharomyces alg5 mutant in vivo. A recombinant Trichomonas Dpm1, which is deeply divergent in its sequence, has Dol-P-Man synthase activity. When radiolabeled Dol-P-Glc is incubated with Trichomonas membranes, Glc is incorporated into reducing and non-reducing sugars of O-glycans of endogenous glycoproteins. To our knowledge, this is the first demonstration of Dol-P-Glc as a sugar donor for O-glycans on glycoproteins.