AbstractTrypanosoma brucei is a protozoan that causes Human African Trypanosomiasis and nagana in cattle. The bloodstream form (BSF) of T. brucei has a dense surface-coat of variant surface glycoprotein (VSG) that acts as the first line of defence against the host immune system while the main cell surface glycoproteins of the procyclic form (PCF) are the procyclins. VSGs and procyclins are attached to the cell membrane through glycosylphosphatidylinositol (GPI) anchors. Despite its importance, the GPI biosynthetic pathway in T. brucei is not fully elucidated.
In this thesis, we have applied epitope tagging, chemical crosslinking and quantitative proteomics methods to look for new components of protein complexes of the GPI pathway. Three components of the pathway were tagged and immunoprecipitated with or without chemical crosslinking and the results were as follows:
For TbGPI12, the GlcNAc-PI de-N-acetylase, we discovered that, contrary to expectations, it is not complexed with TbMTI but with a different potential partner protein. We also optimised methodologies in this part of the study.
For the TbGPI3 component of GPI GnT, we identified its expected partner proteins TbGPI15, TbGPI19, TbGPI2, TbGPI1 and TbERI1 and also identified two new components of the complex, namely an Arv1-like protein (TbArv1) and a putative E2-ligase.
For the GPI inositol deacylase (TbdeAc2) that catalyses inositol deacylation of GPI intermediates, we showed that it is present in a ~240 kDa complex and, using both crosslinking and stable isotope in cell culture (SILAC) methodology, identified partner protein(s) encoded by genes Tb927.11.11740 and/or Tb927.11.11750. Individual reverse pull-down experiments showed that only Tb927.11.11750 was directly associated with TbdeAc2. Our attempts to generate a null and conditional null mutant for Tb927.11.11750 were not successful. However, we considered the possibility that Tb927.11.11750 (which contains a lipase motif) encodes a GPI PLA2 enzyme; an activity adjacent to TbdeAc2 in the pathway. We therefore used immunoprecipitated Tb927.11.11750 in an in vitro assay for GPI PLA2 activity using a radiolabelled and purified glycolipid A’ substrate. However, no GPI-PLA2 activity could be detected under the conditions used. Taken together, we postulate that Tb927.11.11750 encodes an essential product for the survival of BSF T. brucei and hypothesise that the Tb927.11.11750 protein may be required for stabilisation and/or regulation of TbdeAc2.
|Date of Award
|China Scholarship Council
|Michael Ferguson (Supervisor)