GLYCOSYL-PHOSPHATIDYLINOSITOLS OF TRYPANOSOMA-CONGOLENSE - 2 COMMON PRECURSORS BUT A NEW PROTEIN-ANCHOR

The parasitic protozoan Trypanosoma congolense exhibits a dense surfac e 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 mem brane anchor. A detailed study of the structure of T. congolense strai n 423 (clone BENat 1.3) variant surface glycoprotein glycosyl-phosphat idylinositol membrane anchor was performed. Radioactively labelled cor e-glycan prepared by dephosphorylation, deamination and reduction was analysed by high-pH anion-exchange chromatography, size-exclusion and lectin affinity chromatography. Additionally the glycosyl-phosphatidyl inositol membrane anchor core-glycan was purified from a bulk preparat ion of variant surface glycoprotein and subjected to mass spectrometry and methylation analysis. Using these methods we could identify a nov el galactose-beta 1 ,6-N-acetyl-glucosamine-beta 1,4-branch modifying the mannose adjacent to the glucosamine of the mannose-alpha 1,2-manno se-alpha 1,6-mannose-alpha 1,4 glucosamine core-glycan of the variant surface glycoprotein glycosyl-phosphatidylinositol membrane anchor. Fu rthermore the biosynthetic pathway leading to this novel structure was investigated. Two putative glycosyl-phosphatidylinositol anchor precu rsors were identified having structures identical to the previously ch aracterized Trypanosoma brucei brucei glycolipids P2 and P3 (also desi gnated glycolipid A and C) consistent with a trimannosyl core and a di myristoyl-glycerol. Both glycosyl-phosphatidylinositol anchor precurso rs of T. congolense do not possess the side-branch modification found on the mature protein membrane anchor, implying that the sugar side-ch ain is added to the anchor during its passage through the Golgi-appara tus. (C) 1996 Academic Press Limited