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 Glc␣1-3Man␣1-2Man␣1-2Man␣1-3(Man␣1-6)Man␤1-4GlcNAc␤1-4GlcNAc, Glc␣1-3Man␣1-2Man␣1-2Man␣1-3(GlcNAc␤1-2Man␣1-6)Man␤1-4GlcNAc␤1-4GlcNAc, and Glc␣1-3Man␣1-2Man␣1-2Man␣1-3(Gal␤1-4GlcNAc␤1-2Man␣1-6)Man␤1-4GlcNAc␤1-4GlcNAc. The possibility that these structures might arise from Glc 1 Man 9 GlcNAc 2 by unusually rapid ␣-mannosidase processing was ruled out using a mixture of ␣-mannosidase inhibitors. The results suggest that bloodstream-form T. brucei can transfer both Man 9 GlcNAc 2 and Man 5 GlcNAc 2 to the variant surface glycoprotein in a site-specific manner and that, unlike organisms that transfer exclusively Glc 3 Man 9 GlcNAc 2 , the T. brucei UDP-Glc: glycoprotein glucosyltransferase and glucosidase II enzymes can use Man 5 GlcNAc 2 and Glc 1 Man 5 GlcNAc 2 , respectively, as their substrates. The ability to transfer Man 5 GlcNAc 2 structures to N-glycosylation sites destined to become Man 4 -3 GlcNAc 2 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.The parasitic protozoan Trypanosoma brucei, transmitted by the tsetse fly, is the causative agent of nagana in cattle and African trypanosomiasis or sleeping sickness in humans. During the bloodstream stage of the life cycle, the cells are covered in a densely packed coat of variant surface glycoprotein (VSG) 3 The VSG coat serves as a physical barrier to components of the host complement system and undergoes antigenic variation (1). There are many VSG genes, and each encodes a GPIanchored glycoprotein with one to three N-glycosylation sites (2, 3). The cell line used in this study expresses VSG variant 221 (also known as MiTat1.2). VSG221 carries two occupied N-glycosylation sites, the glycan structures of which have been fully characterized (4). The Asn-428 site, 5 residues from the GPI attachment site, is occupied mostly by oligomannose structures (Man 7-9 GlcNAc 2 ), whereas the Asn-263 site is occupied by small biantennary structures rangin...

Polypeptide binding specificities of Saccharomyces cerevisiae oligosaccharyltransferase accessory proteins Ost3p and Ost6p

“…4,12 The various enzymes from a single organism can have different glycan and protein substrate specificities, such that gene duplication and divergence may have increased the catalytic range of efficient glycosylation. [13][14][15][16][17] Higher eukaryotes have evolved multiprotein complex OTases, in which subunits are involved in selection of mature glycan substrate and regulation of activity. [18][19][20] Duplication and divergence of multiprotein OTase subunits has also occurred, with genes encoding Stt3p and Ost3/6p proteins present in two copies in some organisms.…”

An antibody produced in tobacco expressing a hybrid β-1,4-galactosyltransferase is essentially devoid of plant carbohydrate epitopes

“…2 and Table 1). One major class of N-glycans consisted of high-Man oligosaccharides (Man [5][6][7][8][9] GlcNAc 2 ) totaling up to 34% of the total relative peak area. Another very abundant class of N-glycans was made up of hybrid oligosaccharides, of which the products at m͞z ϭ 1,460.4 and 1,622.5 were by far the most abundant components, together accounting for 27% of total N-glycans, as estimated by relative peak area.…”

“…In fact, it appears that the end product of ER-associated N-glycan synthesis may be identical in all eukaryotes examined up until now with the exception of trypanosomatids (7). It is beyond the ER and mainly in the Golgi that the wide diversity in eukaryotic N-glycan structures is created.…”

“…It is beyond the ER and mainly in the Golgi that the wide diversity in eukaryotic N-glycan structures is created. Through a series of trimming reactions by glycosidases in the ER and cis-Golgi compartments, the so-called high-Man N-glycans (Man [5][6][7][8][9] GlcNAc 2 ) are produced. The actual formation of complex-type N-glycans is initiated with the addition of the first GlcNAc onto Man 5 GlcNAc 2 .…”

N‐glycomic profiling of a glucosidase II mutant of Dictyostelium discoideum by ‘‘off‐line’’ liquid chromatography and mass spectrometry

“…The biological role of glucosidase II (EC 3.2.1.84) has been assessed with a range of mutant cell lines and organisms, including Arabidopsis thaliana, Candida albicans, Saccharomyces cerevisiae and Trypanosoma brucei knock-outs as well as a hypercellulolytic strain of Trichoderma reesei, a mouse lymphoma cell line and the Dicytostelium discoideum modA (M31) mutant (9)(10)(11)(12)(13)(14)(15) . A recurring feature of the plant and fungal mutants is the occurrence of cell wall phenotypes suggesting that glucosidase II is required for proper formation of cell walls; indeed in Dictyostelium, which also produces cellulose, stunted fruiting bodies are the obvious morphological phenotype of the modA strain.…”