Visualisation of experimentally determined and predicted protein N-glycosylation and predicted glycosylphosphatidylinositol anchor addition in Trypanosoma brucei.

Abstract

Background: Trypanosoma brucei is a protozoan parasite and the etiological agent of human and animal African trypanosomiasis. The organism cycles between its mammalian host and tsetse vector. The host-dwelling bloodstream form of the parasite is covered with a monolayer of variant surface glycoprotein (VSG) that enables it to escape both the innate and adaptive immune systems. Within this coat reside lower-abundance surface glycoproteins that function as receptors and/or nutrient transporters. The glycosylation of the Trypanosoma brucei surface proteome is essential to evade the immune response and is mediated by three oligosaccharyltransferase genes; two of which, TbSTT3A and TbSTT3B, are expressed in the bloodstream form of the parasite. Methods: We processed a recent dataset of our laboratory to visualise putative glycosylation sites of the Trypanosoma brucei proteome. We provided a visualisation for the predictions of glycosylation carried by TbSTT3A and TbSTT3B, and we augmented the visualisation with predictions for Glycosylphosphatidylinositol anchoring sites, domains and topology of the Trypanosoma brucei proteome. Conclusions: We created a web service to explore the glycosylation sites of the Trypanosoma brucei oligosaccharyltransferases substrates, using data described in a recent publication of our laboratory. We also made a machine learning algorithm available as a web service, described in our recent publication, to distinguish between TbSTT3A and TbSTT3B substrates.

Generation of a bloodstream formTrypanosoma bruceidouble glycosyltransferase null mutant competent in receptor-mediated endocytosis of transferrin

“…Protein groups enriched in the TbGT10 −/− / TbGT8 −/− sample, relative to the wild type and TbGT10 −/− /TbGT8 Flox/− conditional null mutant grown under permissive conditions samples, are indicated in (Table 1). Among these, two (ESAG2 and CBP1B) had predicted N -terminal signal peptides and 6 and 7 predicted N -glycosylation sites, respectively [27] (S4 Fig). Both are known to resolve at high molecular weight by SDS-PAGE in WT BSF cell lysates [13,18].…”

“…From these data, we conclude that at least some glycoproteins are affected as predicted in (Fig 4A), collapsing from very high apparent MW to, in the case of ESAG2 and CBP1B, around 60 kDa apparent MW upon loss of both TbGT10 and TbGT8. Consistent with their appearance in the soluble fraction upon hypotonic lysis, ESAG2 is predicted to be a GPI-anchored protein [27] while CBP1B is a soluble lysosomal/endosmal serine protease [28].…”

“…Prediction of a Signal Peptide (SP) by SignalP analysis indicated. N-glycosylation site prediction was performed [27]. Expanded sequence analysis of ESAG2 and CBP1B (bold) are shown in S4 Fig.…”

Generation of a bloodstream form Trypanosoma brucei double glycosyltransferase null mutant competent in receptor-mediated endocytosis of transferrin

“…Protein groups enriched in the TbGT10 -/- / TbGT8 -/- sample, relative to the wild type and TbGT10 -/- /TbGT8 Flox/- conditional null mutant grown under permissive conditions samples, are indicated in ( Table 1 ). Among these, two (ESAG2 and CBP1B) had predicted N -terminal signal peptides and 6 and 7 predicted N -glycosylation sites, respectively [ 27 ] ( S4 Fig ), indicative of Golgi trafficking and elaboration by glycosyltransferases. Both are known to resolve at high molecular weight by SDS-PAGE in wild type BSF cell lysates [ 13 , 18 ].…”

“…From these data, we conclude that at least some glycoproteins are affected as predicted in ( Fig 4A ), collapsing from very high apparent MW to, in the case of ESAG2 and CBP1B, around 60 kDa apparent MW upon loss of both TbGT10 and TbGT8. Consistent with their appearance in the soluble fraction upon hypotonic lysis, ESAG2 is predicted to be a GPI-anchored protein [ 27 ] while CBP1B is a soluble lysosomal/endosomal serine protease [ 28 ].…”