Abstract
The transferrin receptor of bloodstream form Trypanosoma brucei is a heterodimer encoded by expression site associated genes 6 and 7. This low-abundance glycoprotein with a single glycosylphosphatidylinositol membrane anchor and eight potential N-glycosylation sites is located in the flagellar pocket. The receptor is essential for the parasite, providing its only source of iron by scavenging host transferrin from the bloodstream. Here, we demonstrate that both receptor subunits contain endoglycosidase H-sensitive and endoglycosidase H-resistant N-glycans. Lectin blotting of the purified receptor and structural analysis of the released N-glycans revealed oligomannose and paucimannose structures but, contrary to previous suggestions, no poly-N-acetyllactosamine structures were found. Overlay experiments suggest that the receptor can bind to other trypanosome glycoproteins, which may explain this discrepancy. Nevertheless, these data suggest that a current model, in which poly-N-acetyllactosamine glycans are directly involved in receptor-mediated endocytosis in bloodstream form Trypanosoma brucei, should be revised. Sequential endoglycosidase H and peptide-N-glycosidase F treatment, followed by tryptic peptide analysis, allowed the mapping of oligomannose and paucimannose structures to four of the receptor N-glycosylation sites. These results are discussed with respect to the current model for protein N-glycosylation in the parasite. Finally, the glycosylation data allowed the creation of a molecular model for the parasite transferrin receptor. This model, when placed in the context of a model for the dense variant surface glycoprotein coat in which it is embedded, suggests that receptor N-glycosylation may play an important role in providing sufficient space for the approach and binding of transferrin to the receptor, without significantly disrupting the continuity of the protective variant surface glycoprotein coat.
Highlights
The tsetse-transmitted Trypanosoma brucei group of parasites cause human African trypanosomiasis and nagana in cattle and constitute a serious health problem for people and livestock in 36 countries of sub-Saharan Africa
Other less abundant glycoproteins are arranged either apparently randomly within the variant surface glycoproteins (VSGs) coat, like the invariant glycoproteins ISG65 and ISG75 [8,9], while others have specific surface locations, like Fla1 which locates to the flagellar adhesion zone [10] and the transferrin receptor which locates to the flagellar pocket [11]
While this life cycle stage shares some glycoproteins with the bloodstream form, like p67, tGLP1 and Fla1, others are clearly bloodstream form specific, like ISG65, ISG75, TbBMAP1 and the expression site associated gene (ESAG) 6 and ESAG7 subunits of the heterodimeric T. brucei transferrin receptor (TfR)
Summary
The tsetse-transmitted Trypanosoma brucei group of parasites cause human African trypanosomiasis and nagana in cattle and constitute a serious health problem for people and livestock in 36 countries of sub-Saharan Africa. The surface of the procyclic form parasite is dominated by 36106 copies of the GPI-anchored and Nglycosylated procyclin glycoproteins [4,16,17], about 16106 free GPI glycolipids [18,19] and a high-molecular weight glycoconjugate complex [20,21]. While this life cycle stage shares some glycoproteins with the bloodstream form, like p67, tGLP1 and Fla, others are clearly bloodstream form specific, like ISG65, ISG75, TbBMAP1 and the expression site associated gene (ESAG) 6 and ESAG7 subunits of the heterodimeric T. brucei transferrin receptor (TfR)
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