Deletion of the Glucosidase II Gene in Trypanosoma brucei Reveals Novel N-Glycosylation Mechanisms in the Biosynthesis of Variant Surface Glycoprotein*

Abstract

The trypanosomatids are generally aberrant in their protein N-glycosylation pathways. However, protein N-glycosylation in the African trypanosome Trypanosoma brucei, etiological agent of human African sleeping sickness, is not well understood. Here, we describe the creation of a bloodstream-form T. brucei mutant that is deficient in the endoplasmic reticulum enzyme glucosidase II. Characterization of the variant surface glycoprotein, the main glycoprotein synthesized by the parasite with two N-glycosylation sites, revealed unexpected changes in the N-glycosylation of this molecule. Structural characterization by mass spectrometry, nuclear magnetic resonance spectroscopy, and chemical and enzymatic treatments revealed that one of the two glycosylation sites was occupied by conventional oligomannose structures, whereas the other accumulated unusual structures in the form of Glcα1–3Manα1–2Manα1–2Manα1–3(Manα1–6)Manβ1–4GlcNAcβ1–4GlcNAc, Glcα1–3Manα1–2Manα1–2Manα1–3(GlcNAcβ1–2Manα1–6)Manβ1–4GlcNAcβ1–4GlcNAc, and Glcα1–3Manα1–2Manα1–2Manα1–3(Galβ1–4GlcNAcβ1–2Manα1–6)Manβ1–4GlcNAcβ1–4GlcNAc. The possibility that these structures might arise from Glc₁Man₉GlcNAc₂ by unusually rapid α-mannosidase processing was ruled out using a mixture of α-mannosidase inhibitors. The results suggest that bloodstream-form T. brucei can transfer both Man₉GlcNAc₂ and Man₅GlcNAc₂ to the variant surface glycoprotein in a site-specific manner and that, unlike organisms that transfer exclusively Glc₃Man₉GlcNAc₂, the T. brucei UDP-Glc: glycoprotein glucosyltransferase and glucosidase II enzymes can use Man₅GlcNAc₂ and Glc₁Man₅GlcNAc₂, respectively, as their substrates. The ability to transfer Man₅GlcNAc₂ structures to N-glycosylation sites destined to become Man_4–3GlcNAc₂ or complex structures may have evolved as a mechanism to conserve dolichol-phosphate-mannose donors for glycosylphosphatidylinositol anchor biosynthesis and points to fundamental differences in the specificities of host and parasite glycosyltransferases that initiate the synthesis of complex N-glycans.