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

Zhe Ji, View ORCID ProfileMichele Tinti, View ORCID ProfileMichael A.J. Ferguson
doi: https://doi.org/10.1101/2020.12.16.423025
Zhe Ji
1The Wellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dundee, UK
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Michele Tinti
1The Wellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dundee, UK
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Michael A.J. Ferguson
1The Wellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dundee, UK
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  • ORCID record for Michael A.J. Ferguson
  • For correspondence: majferguson@dundee.ac.uk
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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. This enzyme has been shown to be a complex of at least seven subunits in mammalian cells and a similar complex of homologous subunits has been postulated in yeast. Homologs of most 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 partner proteins TbGPI15, TbGPI9, 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.

Graphical abstract First step of GPI anchor biosynthesis pathway in T.brucei BSF is catalysed by TbGPI3 complex.

Figure
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Posted December 16, 2020.
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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
bioRxiv 2020.12.16.423025; doi: https://doi.org/10.1101/2020.12.16.423025
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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
bioRxiv 2020.12.16.423025; doi: https://doi.org/10.1101/2020.12.16.423025

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