Abstract
The COOH terminus of the externally disposed variant surface glycoprotein (VSG) of the eukaryotic pathogenic protozoan Trypanosoma brucei strain 427 variant MITat 1.4 (117) is covalently linked to a novel phosphatidylinositol-containing glycolipid. This conclusion is supported by analysis of the products of nitrous acid deamination or Staphylococcus aureus phosphatidylinositol-specific phospholipase C treatment of purified membrane-form VSG. Lysis of trypanosomes is accompanied by release of soluble VSG, catalyzed by activation of an endogenous phospholipase C. The only apparent difference between membrane-form VSG and soluble VSG is the removal of sn-1,2-dimyristylglycerol. The COOH-terminal glycopeptide derived by Pronase digestion of soluble VSG was characterized by chemical modification and digestion with alkaline phosphatase. The results are consistent with the single non-N-acetylated glucosamine residue being the reducing terminus of the oligosaccharide and in a glycosidic linkage to a myo-inositol monophosphate that is probably myo-inositol 1,2-cyclic monophosphate. A partial structure for the VSG COOH-terminal moiety is presented. This structure represents a new type of eukaryotic post-translational protein modification and membrane anchor. We discuss the relevance of this structure to observations that have been made with other eukaryotic membrane proteins.
Highlights
The COOH terminus of theexternally disposed var- variant surface glycoproteins (VSGs) mRNA translatiopnroductcsontain hydrophobic iant surface glycoprotein (VSG) of the eukaryotic CpOaOthH--terminal peptide extensions of 17 or 23 amino acids, ogenic protozoan Trypanosoma brucsetirain 427 var- depending on the variant, that are absent from mature VSG iant MITat 1.4 (117) is covalently linked to a novel (Boothroyd et al, 1980,1981; Cross 1984a)
The COOH terminus of theexternally disposed var- VSG mRNA translatiopnroductcsontain hydrophobic iant surface glycoprotein (VSG) of the eukaryotic CpOaOthH--terminal peptide extensions of 17 or 23 amino acids, ogenic protozoan Trypanosoma brucsetirain 427 var- depending on the variant, that are absent from mature VSG
The molecular basis of antigenic variation is the sequential expression of individual genes encoding immunologically distinct variant surface glycoproteins (VSGs’), which form a dense cell surface coat (Cross, 1984a; Turner, 1984)
Summary
Istry, Oxford University, South ParksRd., Oxford, United Kingdom. Materials-Lipid, sugar, and inositol standards and bovine intes-. Gas-LiquidChromatography-Gas-liquid chromatography was performed using a Varian 3700 gas chromatograph fitted with flameionization detectors Both sugar and inositol derivatives were separated on a glass column (2 m X 2 mm) packed with 5% SE-30 on Supelcoport 80/lOO mesh; Nz was used as carrier at 30 ml/min. Nitrous Acid Deamination ofsVSG-A 15-mg sample of purified intact sVSG 117 was deaminated in 1ml of 25 mM sodium acetate, pH 3.5,0.16 M NaN02 (5 h, 23 "C).To prevent possible base hydrolysis of the VSG polypeptide, NaBH4 reduction was omitted a t this stage and the neutralized reaction mixture was filtered through a Millex HV 0.45-pm membrane (Millipore Corp., Bedford, MA) and applied to a column (1.5 X 93.5 cm) of Bio-Gel P-10 (100-200 mesh) and eluted with 50 mM NH4HC03at 15ml/h. Glycoprotein Anchor and close to the column-included volume contained material that
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