title: 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 Glcalpha1-3Manalpha1-2Manalpha1-2Manalpha1-3(Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAc, Glcalpha1-3Manalpha1-2Manalpha1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAc, and Glcalpha1-3Manalpha1-2Manalpha1-2Manalpha1-3(Galbeta1-4GlcNAcbeta1-2Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAc. The possibility that these structures might arise from Glc1Man9GlcNAc2 by unusually rapid alpha-mannosidase processing was ruled out using a mixture of alpha-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(4-3)GlcNAc2 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.
authors.affiliation: Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, The Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, United Kingdom.
authors.forename: Deuan C
authors.initials: DC
authors.lastname: Jones
authors.forename: Angela
authors.initials: A
authors.lastname: Mehlert
authors.forename: M Lucia S
authors.initials: ML
authors.lastname: Güther
authors.forename: Michael A J
authors.initials: MA
authors.lastname: Ferguson
mesh_terms: D000818:Animals; D015139:Blotting, Southern; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology; D017353:Gene Deletion; D006020:Glycopeptides / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry; D006031:Glycosylation; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008745:Methylation; D008954:Models, Biological; D009154:Mutation; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry; D020033:Protein Isoforms; D021241:Spectrometry, Mass, Electrospray Ionization; D014346:Trypanosoma brucei brucei / Q000235:genetics*; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000737:chemistry; D000520:alpha-Glucosidases / Q000235:genetics*
chemical_list: D006020:Glycopeptides; D006023:Glycoproteins; D011134:Polysaccharides; D020033:Protein Isoforms; D014643:Variant Surface Glycoproteins, Trypanosoma; D004247:DNA; C034251:4-nitrophenyl-alpha-glucosidase; D006026:Glycoside Hydrolases; D000520:alpha-Glucosidases