Glycosyl-phosphatidylinositols of : Two Common Precursors but a New Protein-anchor

The parasitic protozoan Trypanosoma congolense exhibits a dense surface coat which is pivotal for immunoevasion of the parasite. This dense surface coat is made of a single protein species, the variant surface glycoprotein, which is present in a high copy number. The protein is anchored to the plasma membrane by a glycosyl-phosphatidylinositol membrane anchor. A detailed study of the structure of T. congolense strain 423 (clone BENat 1.3) variant surface glycoprotein glycosyl-phosphatidylinositol membrane anchor was performed. Radioactively labelled core-glycan prepared by dephosphorylation, deamination and reduction was analysed by high- pH anion-exchange chromatography, size-exclusion and lectin affinity chromatography. Additionally the glycosyl-phosphatidylinositol mem brane anchor core-glycan was purified from a bulk preparation of variant surface glycoprotein and subjected to mass spectrometry and methylation analysis. Using these methods we could identify a novel galactose- β1,6- N -acetyl-glucosamine-β1,4-branch modifying the mannose adjacent to the glucosamine of the mannose-α1,2-mannose-α1,6-mannose-α1,4- glucosamine core-glycan of the variant surface glycoprotein glycosyl-phos phatidylinositol membrane anchor. Furthermore the biosynthetic pathway leading to this novel structure was investigated. Two putative glycosyl-phosphatidylinositol anchor precursors were identified having structures identical to the previously characterized Trypanosoma brucei brucei glycolipids P2 and P3 (also designated glycolipid A and C) consistent with a trimannosyl core and a dimyristoyl-glycerol. Both glycosyl-phosphatidylinositol anchor precursors of T. congolense do not possess the side-branch modification found on the mature protein membrane anchor, implying that the sugar side-chain is added to the anchor during its passage through the Golgi-apparatus.