A conserved flagellar pocket exposed high mannose moiety is used by African trypanosomes as a host cytokine binding molecule.
Trypanosomes use antigenic variation of their variant-specific surface glycoprotein (VSG) coat as defense against the host immune system. However, in order to sustain their growth, they need to expose conserved epitopes, allowing host macromolecule binding and receptor-mediated endocytosis. Here we show that Trypanosoma brucei uses the conserved chitobiose-oligomannose (GlcNAc(2)-Man(5-9)) moieties of its VSG as a binding ligand for tumor necrosis factor (TNF), a host cytokine with lectin-like properties. As endocytosis in trypanosomes is restricted to the flagellar pocket, we show that soluble flagellar pocket extracts, and in particular soluble VSG, inhibit the binding of (125)I-TNF to trypanosomes. The interaction between TNF and VSG is confirmed by affinity chromatography, biosensor, and dot-blot affinity measurements, and soluble VSG inhibition of TNF-mediated trypanolysis. In all approaches, removal of N-linked carbohydrates abrogates the TNF-VSG interaction. In addition, synthetic high mannose oligosaccharides can block TNF-VSG interactions, and a VSG glycopeptide carrying the GlcNAc(2)-Man(5-9) moiety is shown to inhibit TNF-mediated trypanosome killing in mixed parasite/macrophage cell cultures. Together, these results support the observation that TNF plays a role in growth control of trypanosomes and, moreover, suggest that, by the use of conserved VSG carbohydrates as lectin-binding epitopes, trypanosomes can limit the necessity to express large numbers of invariant surface exposed receptors.
Magez
S
S
Department of Immunology, Parasitology and Ultrastructure, Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint Genesius Rode, Belgium. stemagez@vub.ac.be
Radwanska
M
M
Stijlemans
B
B
Xong
H V
HV
Pays
E
E
De Baetselier
P
P
D000818:Animals; D001665:Binding Sites; D015374:Biosensing Techniques; D015153:Blotting, Western; D002240:Carbohydrate Sequence; D002845:Chromatography; D018920:Coculture Techniques; D016207:Cytokines / Q000378:metabolism*; D004187:Disaccharides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis; D005407:Flagella / Q000737:chemistry*; D006031:Glycosylation; D015151:Immunoblotting; D007700:Kinetics; D008024:Ligands; D008264:Macrophages; D008358:Mannose / Q000737:chemistry*; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D011485:Protein Binding; D013997:Time Factors; D014346:Trypanosoma brucei brucei; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000737:chemistry*
D016207:Cytokines; D004187:Disaccharides; D008024:Ligands; D014409:Tumor Necrosis Factor-alpha; D014643:Variant Surface Glycoproteins, Trypanosoma; C032438:chitobiose; D008358:Mannose