title: TbGT8 is a bifunctional glycosyltransferase that elaborates N-linked glycans on a protein phosphatase AcP115 and a GPI-anchor modifying glycan in Trypanosoma brucei.
abstract: The procyclic form of Trypanosoma brucei expresses procyclin surface glycoproteins with unusual glycosylphosphatidylinositol-anchor side chain structures that contain branched N-acetyllactosamine and lacto-N-biose units. The glycosyltransferase TbGT8 is involved in the synthesis of the branched side chain through its UDP-GlcNAc: βGal β1-3N-acetylglucosaminyltransferase activity. Here, we explored the role of TbGT8 in the mammalian bloodstream form of the parasite with a tetracycline-inducible conditional null T. brucei mutant for TbGT8. Under non-permissive conditions, the mutant showed significantly reduced binding to tomato lectin, which recognizes poly-N-acetyllactosamine-containing glycans. Lectin pull-down assays revealed differences between the wild type and TbGT8 null-mutant T. brucei, notably the absence of a broad protein band with an approximate molecular weight of 110 kDa in the mutant lysate. Proteomic analysis revealed that the band contained several glycoproteins, including the acidic ecto-protein phosphatase AcP115, a stage-specific glycoprotein in the bloodstream form of T. brucei. Western blotting with an anti-AcP115 antibody revealed that AcP115 was approximately 10kDa smaller in the mutant. Enzymatic de-N-glycosylation demonstrated that the underlying protein cores were the same, suggesting that the 10-kDa difference was due to differences in N-linked glycans. Immunofluorescence microscopy revealed the colocalization of hemagglutinin epitope-tagged TbGT8 and the Golgi-associated protein GRASP. These data suggest that TbGT8 is involved in the construction of complex poly-N-acetyllactosamine-containing type N-linked and GPI-linked glycans in the Golgi of the bloodstream and procyclic parasite forms, respectively.
authors: Laboratory of Biochemistry, College of Pharmaceutical Sciences, Matsuyama University, Matsuyama, Ehime 790-8578, Japan. Electronic address: mnakanis@cc.matsuyama-u.ac.jp.
authors: Masayuki
authors: M
authors: Nakanishi
authors: Laboratory of Biochemistry, College of Pharmaceutical Sciences, Matsuyama University, Matsuyama, Ehime 790-8578, Japan.
authors: Moe
authors: M
authors: Karasudani
authors: Laboratory of Biochemistry, College of Pharmaceutical Sciences, Matsuyama University, Matsuyama, Ehime 790-8578, Japan.
authors: Takahiro
authors: T
authors: Shiraishi
authors: Laboratory of Biochemistry, College of Pharmaceutical Sciences, Matsuyama University, Matsuyama, Ehime 790-8578, Japan.
authors: Kazunori
authors: K
authors: Hashida
authors: Laboratory of Biochemistry, College of Pharmaceutical Sciences, Matsuyama University, Matsuyama, Ehime 790-8578, Japan.
authors: Mami
authors: M
authors: Hino
authors: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom.
authors: Michael A J
authors: MA
authors: Ferguson
authors: Laboratory of Biochemistry, College of Pharmaceutical Sciences, Matsuyama University, Matsuyama, Ehime 790-8578, Japan.
authors: Hiroshi
authors: H
authors: Nomoto
mesh_terms: D015153:Blotting, Western; D006031:Glycosylation; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D037121:Plant Lectins / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011134:Polysaccharides / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics
chemical_list: D037121:Plant Lectins; D011134:Polysaccharides; D015800:Protozoan Proteins; C034981:tomato lectin; C037199:poly-N-acetyllactosamine; D016695:Glycosyltransferases; D010749:Phosphoprotein Phosphatases; C406810:Try-AcP115 protein, Trypanosoma brucei
keywords: GPI-anchor; Glycosyltransferase; N-linked glycan; Tomato lectin; Trypanosoma brucei