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

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

Elimination of GPI2 suppresses glycosylphosphatidylinositol GlcNAc transferase activity and alters GPI glycan modification in Trypanosoma brucei

“…7 B ), reflecting its association with the GPI GlcNAc transferase complex. In a previous report ( 39 ), a GPI GlcNAc transferase complex isolated from T. brucei bloodstream by pull-down of cMyc-tagged TbGPI3 was seen to run closer to the 242-kDa marker. The reason for the difference on native PAGE between our data and the published report is not clear but could be due to heterogeneity of the complexes revealed by using different baits for pull-down (TbGPI3 versus TbGPI1) or differences in the architecture of the complexes in bloodstream versus procyclic trypanosomes.…”

“…The core subunits of the GPI GlcNAc transferase complex have been identified in T. brucei by bioinformatics ( 38 ) and quantitative proteomics ( 39 ): TbGPI1 (Tb927.3.4570), TbGPI2 (Tb927.10.6140), TbGPI3 (Tb927.2.1780), TbGPI15 (Tb927.5.3680), TbGPI19 (Tb927.10.10110), and TbERI1 (Tb927.4.780). TbDPM2 (Tb927.9.6440) is also listed in the T. brucei genome, but this may be a misannotation as the trypanosome dolichol phosphate mannose synthase, like its Saccharomyces cerevisiae counterpart, comprises a single protein, TbDPM1 ( 40 ).…”

Elimination of GPI2 suppresses glycosylphosphatidylinositol GlcNAc transferase activity and alters GPI glycan modification inTrypanosoma brucei

“…7B), reflecting its association with the GPI GlcNAc transferase complex. In a previous report (39), a GPI GlcNAc transferase complex isolated from T. brucei bloodstream by pull-down of cMyc-tagged TbGPI3 was seen to run closer to the 242 kDa marker. The reason for the difference on native PAGE between our data and the published report is not clear, but could be due to heterogeneity of the complexes revealed by using different baits for pull-down (TbGPI3 versus TbGPI1), or differences in the architecture of the complexes in bloodstream versus procyclic trypanosomes.…”

“…The GPI GlcNAc transferase complex was immunoprecipitated from parasites expressing myc-tagged TbGPI1 as previously described (39). Briefly, 2 × 10 8 trypanosomes were harvested by centrifugation, washed twice with PBS and lysed for 30 min on ice in 500 µl lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% digitonin, pH 7.4).…”

“…The core subunits of the GPI GlcNAc transferase complex have been identified in T. brucei by bioinformatics (38) and quantitative proteomics (39): TbGPI1 (Tb927.3.4570), TbGPI2 (Tb927.10.6140), TbGPI3 (Tb927.2.1780), TbGPI15 (Tb927.5.3680), TbGPI19 (Tb927.10.10110), and TbERI1 (Tb927.4.780) (TbDPM2 (Tb927.9.6440) is also listed in the T. brucei genome but this may be a mis-annotation as the trypanosome dolichol phosphate mannose synthase, like its yeast counterpart, comprises a single protein, TbDPM1 (40)). To explore the role of TbGPI2, we deleted the gene in T. brucei procyclic forms and characterized the knockout cells (TbGPI2-KO) using a variety of biochemical readouts.…”