The complete primary structures of the Asn-linked oligosaccharides from the conserved glycosylation site of the type-I variant surface glycoproteins of Trypanosoma hrucei MITat 1.4 and MITat 1.6 were determined using a combination of exoglycosidase digestions, permethylation analysis, acetolysis and 'H NMR. Both variants contained almost exclusively oligomannose-type oligosaccharides, identical in structure to those of mammalian glycoproteins. The oligosaccharides ranged in size from (Man),(GlcNAc), to (Man),(GlcNAc),. The relative abundance of each component was similar in both variants. The major components were (Man),(GlcNAc)2 and (Mar~),(GlcNAc)~ with slightly less (Man)9(GlcNAc)2 and ( M~~) , ( G~C N A C )~ and much less ( M~~) , ( G I C N A C )~. Both variants also contained the same structural isomers. The close similarity of the oligomannose series indicates identical processing at the conserved site in both variants.The surface of the parasitic protozoan Trypanosomu hrucei is covered by a dense monolayer of variant surface glycoprotein (VSG) which forms a protective barrier against lysis by host serum components. Each trypanosome contains up to a thousand different VSG genes each of which encodes an immunologically distinct VSG molecule. Only one VSG gene is expressed at a time. A trypanosome population is able to evade the immune response of the host by a change in expression, in a small number of the population, to a different VSG variant [l, 21.The most fully characterised VSG variant to date is that from trypanosome subclone MITat 3.4. Its complete aminoacid sequence [3], details of secondary structure [4], cDNA sequence [5] and the primary structure of its glycosylphosphatidylinositol membrane anchor [6] have all been determined. The present analysis of the Asn-linked oligosaccharides completes the full primary structural characterisation of one VSG molecule and is the first report of the complete structure of Asn-linked oligosaccharides in T. brucei.VSGs can be placed into three groups according to aminoacid sequence homology in the COOH-terminal third of the molecule [7, 81. All VSGs belonging to the type-I subclass contain a conserved Asn-glycosylation site located approximately 50 residues from the mature COOH-terminus lying within the region of peptide homology. It is the only Asnglycosylation site present in both variants MITat 1.4 and MITat 1.6 and contains mannose and N-acetylglucosamine as its sole sugar constituents [7].Correspondence to S . E. Zamze, Oxford Glycobiology Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, EnglandAbbreviations. COSY, two-dimensional corrclation spectroscopy; GC/MS, gas chromatography/mass spectrometry; GlcNAcol, N-acetylglucosaminitol; VSG, variant surface glycoprotein.
MATERIALS AND METHODS
MaterialsBio-Gel P-4 (-400 mesh), Dowex AG-50x12, Dowex AG-3X4A and Chelex-100 were obtained from Bio-Rad Laboratories Ltd (Watford, UK). QAE-Sephadex A-25 was obtained from Pharmacia Ltd (Milton Keynes, UK). Aspergillus phoenicus ...