Proteomic identification of the UDP-GlcNAc: PI α1-6 GlcNAc-transferase subunits of the glycosylphosphatidylinositol biosynthetic pathway of Trypanosoma brucei.

Zhe Ji,Michele Tinti,Michael A J Ferguson,Ziyin Li

doi:10.1371/journal.pone.0244699

Zhe Ji, Michele Tinti + Show 2 more

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https://doi.org/10.1371/journal.pone.0244699

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Abstract

The first step of glycosylphosphatidylinositol (GPI) anchor biosynthesis in all eukaryotes is the addition of N-acetylglucosamine (GlcNAc) to phosphatidylinositol (PI) which is catalysed by a UDP-GlcNAc: PI α1-6 GlcNAc-transferase, also known as GPI GnT. This enzyme has been shown to be a complex of seven subunits in mammalian cells and a similar complex of six homologous subunits has been postulated in yeast. Homologs of these mammalian and yeast subunits were identified in the Trypanosoma brucei predicted protein database. The putative catalytic subunit of the T. brucei complex, TbGPI3, was epitope tagged with three consecutive c-Myc sequences at its C-terminus. Immunoprecipitation of TbGPI3-3Myc followed by native polyacrylamide gel electrophoresis and anti-Myc Western blot showed that it is present in a ~240 kDa complex. Label-free quantitative proteomics were performed to compare anti-Myc pull-downs from lysates of TbGPI-3Myc expressing and wild type cell lines. TbGPI3-3Myc was the most highly enriched protein in the TbGPI3-3Myc lysate pull-down and the expected partner proteins TbGPI15, TbGPI19, TbGPI2, TbGPI1 and TbERI1 were also identified with significant enrichment. Our proteomics data also suggest that an Arv1-like protein (TbArv1) is a subunit of the T. brucei complex. Yeast and mammalian Arv1 have been previously implicated in GPI biosynthesis, but here we present the first experimental evidence for physical association of Arv1 with GPI biosynthetic machinery. A putative E2-ligase has also been tentatively identified as part of the T. brucei UDP-GlcNAc: PI α1-6 GlcNAc-transferase complex.

Highlights

Trypanosoma brucei is a protozoan pathogen that undergoes a complex life cycle between its tsetse fly vector and mammalian hosts
The proteomics data suggest that: (i) The T. brucei UDP-GlcNAc: PI α1–6 GlcNActransferase complex (GPI GnT) contains the expected subunits TbGPI3, TbGPI15, TbGPI19, TbGPI2, TbGPI1 and TbERI1. (ii) Like yeast, but unlike mammalian cells, DPM components are not subunits of the parasite complex. (iii) An Arv1-like protein (TbArv1) is a part of the parasite complex. (iv) A putative E2-ligase UbCE may be a part of the parasite complex
The limitations of this study are that it does not attempt to assess stoichiometry or topology of the T. brucei UDP-GlcNAc: PI α1–6 GlcNAc-transferase complex components and that it lacks an orthogonal confirmation of the presence of TbArv1 and TbUbCE in the complex

Summary

Introduction

Trypanosoma brucei is a protozoan pathogen that undergoes a complex life cycle between its tsetse fly vector and mammalian hosts. The parasite causes human African trypanosomiasis in humans and nagana in cattle in sub-Saharan Africa. The bloodstream form (BSF) of T. brucei produces a dense coat of GPI anchored variant surface protein (VSG) to protect it from the innate immune system and, through antigenic variation, the acquired immune response [1]. Other T. brucei surface molecules that have been.

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