The lipid-linked oligosaccharide donor specificities of Trypanosoma brucei oligosaccharyltransferases

Abstract

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 1-6- 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 -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. © 2012 The Author(s).