Trypanosoma brucei gambiense and T. b. rhodesiense: Concanavalin A Binding to the Membrane and Flagellar Pocket of Bloodstream and Procyclic Forms¹

Abstract

We have measured binding of fluorescein-conjugated succinyl-concanavalin A (Fl-s-Con A) to bloodstream and procyclic forms of Trypanosoma brucei gambiense and to bloodstream forms of T. b. rhodesiense by flow cytofluorimetry. Bloodstream forms bound an order of magnitude less lectin than procyclic forms. Trypsin-treating cells enhanced binding of Fl-s-Con A to bloodstream forms 3-16-fold depending on the strain and the length of trypsinization but had little effect on Fl-s-Con A binding by procyclics. The trypsinization protocol used did not remove major common glycoproteins detected on lectin blots of either life cycle form but removed greater than 95% of the variant specific glycoprotein and fragments derived from this protein of bloodstream forms. Microscopically detectable Fl-s-Con A binding to bloodstream forms was confined to the flagellar pocket. Trypsinized bloodstream forms and procyclics bound Fl-s-Con A in the flagellar pocket, on the flagellum, and on the cell surface. Lectin remained cell associated but appeared to redistribute towards the flagellum and pocket when cells that had bound lectin on ice were subsequently incubated at physiological temperatures. The Fl-s-Con A binding had specificity characteristic of the interaction between the lectin and oligosaccharides. These results are consistent with the hypothesis that the variant specific surface glycoprotein blocks binding of the lectin to surface glycoproteins of bloodstream forms and suggest that concanavalin A-binding glycoproteins are abundant in the flagellar pocket of both life cycle forms.

A MORN Repeat Protein Facilitates Protein Entry into the Flagellar Pocket of Trypanosoma brucei

“…This is supported by the observation that antibodies directed against the Cterminal domain of VSG (which is close to the outer leaflet of the plasma membrane) label the entire surface of fixed cells but do not label live cells (45). Trypsinization of live cells removes VSG and results in ConA labeling of the entire surface, probably due to recognition of other glycoproteins that were previously shielded (26,27). In live cells, the only ConA labeling is observed in and adjacent to the FP (26,27).…”

“…As an additional test of endocytic activity, uptake of fluorophore-conjugated concanavalin A (ConA) was assayed (26). ConA is a lectin (ϳ80 Å across) that can bind to glycoproteins in and adjacent to the FP (26,27).…”

“…ConA is a lectin (ϳ80 Å across) that can bind to glycoproteins in and adjacent to the FP (26,27). The glycoprotein-ConA complexes are then taken up by endocytosis.…”

“…Accordingly, ConA labels the entire surface of fixed T. brucei cells (44). However, surface labeling by ConA is not seen in experiments with live cells (26,27). Fixation presumably makes the glycan groups on VSG more accessible to ConA.…”

Membrane domains and flagellar pocket boundaries are influenced by the cytoskeleton in African trypanosomes

“…This region is characterized by a narrowing of the FP at its apical end until it is little larger in diameter than the flagellum. Again, the cytoskeleton appears central to this: in particular, an electron-dense structure called the ''collar'' can be seen on the cytoplasmic side of this region, and ablation of a protein that localizes to this structure disrupts FP morphogenesis (7).Because of its central importance to pathogenicity, the endocytic activity of T. brucei has long been studied by using well-defined markers for endocytosis and subcellular compartments (1,(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). These studies have established the speed, timing, and routes of uptake and traffic within the cell (1,16,18,20), but a number of critical questions remain unanswered.…”