The lipid-linked oligosaccharide donor specificities of Trypanosoma brucei oligosaccharyltransferases

We recently presented a model for site-specific protein N-glycosylation in Trypanosoma brucei whereby the TbSTT3A oligosaccharyltransferase (OST) first selectively transfers biantennary Man5GlcNAc2 from the lipid-linked oligosaccharide (LLO) donor Man5GlcNAc2-PP-Dol to N-glycosylation sequons in acidic to neutral peptide sequences and TbSTT3B selectively transfers triantennary Man9GlcNAc2 to any remaining sequons. In this paper, we investigate the specificities of the two OSTs for their preferred LLO donors by glycotyping the variant surface glycoprotein (VSG) synthesized by bloodstream-form T. brucei TbALG12 null mutants. The TbALG12 gene encodes the α1-6-mannosyltransferase that converts Man7GlcNAc2-PP-Dol to Man8GlcNAc2-PP-Dol. The VSG synthesized by the TbALG12 null mutant in the presence and the absence of α-mannosidase inhibitors was characterized by electrospray mass spectrometry both intact and as pronase glycopetides. The results show that TbSTT3A is able to transfer Man7GlcNAc2 as well as Man5GlcNAc2 to its preferred acidic glycosylation site at Asn263 and that, in the absence of Man9GlcNAc2-PP-Dol, TbSTT3B transfers both Man7GlcNAc2 and Man5GlcNAc2 to the remaining site at Asn428, albeit with low efficiency. These data suggest that the preferences of TbSTT3A and TbSTT3B for their LLO donors are based on the c-branch of the Man9GlcNAc2 oligosaccharide, such that the presence of the c-branch prevents recognition and/or transfer by TbSTT3A, whereas the presence of the c-branch enhances recognition and/or transfer by TbSTT3B

Identification of a glycosylphosphatidylinositol anchor-modifying β1-3 galactosyltransferase in <i>Trypanosoma brucei</i>

Trypanosoma brucei is the causative agent of human African sleeping sickness and the cattle disease nagana. Trypanosoma brucei is dependent on glycoproteins for its survival and infectivity throughout its life cycle. Here we report the functional characterization of TbGT3, a glycosyltransferase expressed in the bloodstream and procyclic form of the parasite. Bloodstream and procyclic form TbGT3 conditional null mutants were created and both exhibited normal growth under permissive and nonpermissive conditions. Under nonpermissive conditions, the normal glycosylation of the major glycoprotein of bloodstream form T. brucei, the variant surface glycoprotein and the absence of major alterations in lectin binding to other glycoproteins suggested that the major function of TbGT3 occurs in the procyclic form of the parasite. Consistent with this, the major surface glycoprotein of the procyclic form, procyclin, exhibited a marked reduction in molecular weight due to changes in glycosylphosphatidylinositol (GPI) anchor side chains. Structural analysis of the mutant procyclin GPI anchors indicated that TbGT3 encodes a UDP-Gal: β-GlcNAc-GPI β1-3 Gal transferase. Despite the alterations in GPI anchor side chains, TbGT3 conditional null mutants remained infectious to tsetse flies under nonpermissive conditions

Modeling of the N-Glycosylated Transferrin Receptor Suggests How Transferrin Binding Can Occur within the Surface Coat of Trypanosoma brucei

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