Chaperone Requirements for Biosynthesis of the Trypanosome Variant Surface Glycoprotein

Abstract

Background Trypanosoma brucei does not respond transcriptionally to several endoplasmic reticulum (ER) stress conditions, including tunicamycin or dithiothreitol, indicating the absence of a conventional unfolded protein response. This suggests divergent mechanisms for quality control (QC) of ER protein folding and export may be present in trypanosomes. As the variant surface glycoprotein (VSG) represents ∼90% of trypanosome plasma membrane protein, it is possible that VSG has evolved to fold efficiently to minimize ER folding burden.Methodology/Principal FindingsWe demonstrate the presence of a QC system by pharmacological inhibition of the trypanosome 26S proteasome. This indicates active proteasome-mediated VSG turnover as ∼2.5 fold more VSG is recovered from cell lysates following MG132 inhibition. An in silico scan of the trypanosome genome identified 28 open reading frames likely to encode polypeptides participating in ER nascent chain maturation. By RNA interference we monitored the importance of these gene products to proliferation, VSG abundance and cell morphology. 68% of the cohort were required for normal proliferation, and depletion of most of these factors resulted in increased VSG abundance, suggesting involvement in ERQC and degradation.Conclusions/SignificanceThe retention of genes for, and the involvement of many gene products in, VSG folding indicates a substantial complexity within the pathways required to perform this role. Counterintuitively, for a super-abundant antigen VSG is apparently made in excess. The biosynthetic excess VSG appears to be turned over efficiently by the proteasome, implying that considerable VSG is rejected by the trypanosome ERQC mechanism. Accordingly, the VSG polypeptide is not well optimized for folding, as only ∼30% attains the native state. Finally as much of the core ERQC system is functionally conserved in trypanosomes, the pathway has an ancient evolutionary origin, and was present in the last common eukaryotic ancestor.

Functional cooperation between BiP and calreticulin in the folding maturation of a glycoprotein in Trypanosoma cruzi

“…In summary, the activities of BiP and CRT are finely tuned to complement each other in order to nurture the complete folding process of TcrCATL, from the access of the growing chain to the ER to the final acquisition of the native structure. The present report provides further evidence on early evolutionary acquisition of the basic tenets of the QC mechanism of glycoprotein folding represent [37]. …”

Leishmania amazonensis Arginase Compartmentalization in the Glycosome Is Important for Parasite Infectivity

“…Treatment with a trypanosome proteasome inhibitor, MG-132 [24], led to ARG accumulation in the WT and both add-backs parasites, enabling ARG detection in arg − /+ arg ΔSKL western blot and confirming the cytosolic location of ARG by confocal immunolabeling.…”

Mosaic VSGs and the Scale of Trypanosoma brucei Antigenic Variation

“…Evidence for progressive mosaicism—the stepwise increase in complexity of an expressed mosaic ‘string’ within an infection, similar to that in Anaplasma marginale [42]—was limited: the sheer number of different variants present may have prevented detection of intermediate mosaics, but it is also possible that mosaicism is a rapid process, with multiple segments accumulating in a short period. A useful, novel mosaic is a VSG able both to form a functional coat and escape circulating immune responses: rapid SGC, allied with efficient selection—the death of individual trypanosomes that have activated a dysfunctional mosaic VSG or perhaps even a form of VSG quality control [43]—would enable a sublineage to efficiently explore the space of potential mosaics, favouring production of a variant that fulfils these criteria. In more natural hosts, where the greater number of switches arising from greater population size accelerates the kinetics of antigenic variation, the infection is likely to progress to this phase sooner, as the easily-activated VSG s are neutralised and unique variants remain to drive prolonged infection [25].…”