期刊
PARASITOLOGY INTERNATIONAL
卷 63, 期 3, 页码 513-518出版社
ELSEVIER IRELAND LTD
DOI: 10.1016/j.parint.2014.01.007
关键词
Glycosyltransferase; Trypanosoma brucei; N-linked glycan; GPI-anchor; Tomato lectin
类别
资金
- JSPS KAKENHI [22590385]
- MAJF [085622]
- Wellcome Trust [100476]
- Grants-in-Aid for Scientific Research [22590385] Funding Source: KAKEN
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: beta Gal beta 1-3N-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 10 kDa 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. (C) 2014 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY license
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