TbGT8 is a bifunctional glycosyltransferase that elaborates N-linked glycans on a protein phosphatase AcP115 and a GPI-anchor modifying glycan in Trypanosoma brucei

Masayuki Nakanishi,Moe Karasudani,Takahiro Shiraishi,Kazunori Hashida,Mami Hino,Michael A.J Ferguson,Hiroshi Nomoto

doi:10.1016/j.parint.2014.01.007

Masayuki Nakanishi, Moe Karasudani + Show 5 more

Open Access

https://doi.org/10.1016/j.parint.2014.01.007

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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 110kDa 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.

Highlights

Glycans that are covalently attached to proteins can modulate protein properties such as folding, activity, stability, trafficking, and recognition [1]
Treatment of the lysates with peptide Nglycosidase (PNGase) F, which removes N-linked glycans from proteins, resulted in a near-complete loss of lectin binding. These results demonstrate that TbGT8 is involved in the synthesis of N-linked glycans of approximately 110 kDa glycoproteins and is functional even if an hemagglutinin epitope (HA)-tag is fused to its C-terminus
We revealed a role for TbGT8 in the enhancement of AcP115 N-linked glycans in the bloodstream form

Summary

Introduction

Glycans that are covalently attached to proteins can modulate protein properties such as folding, activity, stability, trafficking, and recognition [1]. These functions are known to be important to multicellular organisms, which require highly organized intercellular interactions, and the expression of incomplete or modified glycans often leads to embryonic death or congenital disease [2,3]. Our knowledge about glycan functions in unicellular parasites such as Trypanosoma brucei is less advanced, despite the fact that these organisms synthesize an impressive range of glycoconjugates [4]. The parasite has a complex life cycle between its mammalian hosts and tsetse fly vectors. The parasite glycans have been extensively analyzed in both the mammalian bloodstream and insect-dwelling procyclic forms of the parasite, and these studies have revealed the existence of asparagine (N)-linked oligomannose, paucimannose, and complex type glycans, as well as protein GPI-anchors bearing complex glycan side chains [5,6,7,8,9,10,11,12]

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