African trypanosomes are parasitic protozoa which are enveloped by a surface coat consisting of a matrix of identical glycoprotein molecules. Variations in the composition of these variant surface glycoproteins (VSGs) allow the parasite to escape the host's immune system and render effective immunoprophylaxis improbable. However, underlying the surface coat, all variant antigen types contain common membrane components, some of which can activate complement by the alternative pathway, leading to lysis of uncoated trypanosomes. Hence, stimulation of VSG release in vivo should be a potential form of chemotherapy, and we have therefore investigated the mode of attachment of VSG to the plasma membrane. Biochemical characterization of VSGs from several species has been performed on material purified after release from the cell surface following rupture of the trypanosome. We demonstrate here that VSGs of Trypanosoma brucei when bound to the membrane exist in a form which differs both biochemically and immunochemically from VSGs purified in the conventional manner. After rupture of the cell, membrane-form VSG (mfVSG) is enzymatically transformed into the commonly isolated water-soluble released form (sVSG). In conditions in which this modification does not take place, purified VSGs have amphiphilic properties and behave as integral membrane proteins by the criterion of charge-shift electrophoresis. The difference between the two forms lies in the C-terminal domain, which is phosphorylated in both forms. This domain in sVSGs contains an immunogenic oligosaccharide known as the cross-reacting determinant (CRD), attached to the C-terminal amino acid. Recognition of this determinant by anti-CRD antibodies is impaired in the membrane form.

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The survival of Trypanosoma cruzi, the causative agent of Chagas' disease, depends vitally on proteins and glycoconjugates that mediate the parasite/host interaction. Since most of these molecules are attached to the membrane by glycosylphosphatidylinositol (GPI), alternative means of chemotherapeutic intervention might emerge from GPI biosynthesis studies. The structure of the major 1G7 antigen GPI has been fully characterized by us (Güther, M. L. S., Cardoso de Almeida, M. L., Yoshida, N., and Ferguson, M. A. J.(1992) J. Biol. Chem. 267, 6820-6828; Heise, N., Cardoso de Almeida, M. L., and Ferguson, M. A. J.(1995) Mol. Biochem. Parasitol. 70, 71-84), and based on its properties we now report the complete precursor glycolipids predicted to be transferred to the nascent protein. Migrating closely to Trypanosoma brucei glycolipid A on TLC, such species, named glycolipids A-like 1 and A-like 2, were labeled with tritiated palmitic acid, myo-inositol, glucosamine, and mannose, but surprisingly only the less polar glycolipid A-like 1 incorporated ethanolamine. The predicted products following nitrous acid deamination and digestion with phospholipases A2, C, and D confirmed their GPI nature. Evidence that they may represent the anchor transferred to the 1G7 antigen came from the following analyses: (i) alpha-mannosidase treatments indicated that only one mannose was amenable to removal; (ii) their lipid moiety was identified as sn-1-alkyl-2-acylglycerol due to their sensitivity to phospholipase A2 (PLA2), mild base and by direct high performance TLC analysis of the corresponding benzoylated diradylglycerol components; and (iii) both glycolipids incorporated 3H-fatty acid only in the sn-2- and not in the sn-1-alkyl position as previously found in the GPI of the mature 1G7 antigen. Based on the differential [3H]ethanolamine incorporation pattern and the recent report that an aminoethylphosphonic acid (AEP) replaces ethanolamine phosphate (EtNH2-PO4) in the GPI in epimastigote sialoglycoproteins (Previato, J. O., Jones, C., Xavier, M. T., Wait, R., Travassos, L. R., Parodi, A. J., and Mendonça-Previato, L.(1995) J. Biol. Chem. 270, 7241-7250) it is proposed that glycolipid A-like 2 contains AEP and A-like 1 EtNH2-PO4. In the in vitro cell-free system both glycolipids were synthesized simultaneously and do not seem to bear a precursor/product relationship. Among the various components synthesized in vitro a glycolipid C-like corresponding to a form of glycolipid A-like 1 acylated on the inositol was also characterized. Phenylmethylsulfonyl fluoride, an inhibitor known to block the addition of ethanolamine phosphate in T. brucei but not in mammalian cells, also inhibits the synthesis of glycolipids A-like and C-like in T. cruzi, indicating that the putative trypanosome EtNH2-PO4/AEP transferase(s) might represent a potential target for chemotherapy.

The variant surface glycoprotein (VSG) ofthe African trypanosomes is the major membrane protein of the plasma membrane of the bloodstream stage of the parasite. It is anchored in the plasma membrane by a glycolipid covalently bound to the C-terminal amino acid of the protein. The by phase-separation in Triton X-114 (TX-114) and chromatography on DEAE-cellulose and Mono Q (Pharmacia) were performed as described (7). Phospholipase from T. brucei ILTat 1.25 was obtained by solubilization of VSG-depleted membranes in n-octyl glucoside, followed by covalent chromatography on Affi-Gel 501 (Bio-Rad), or by TX-114 extraction from VSG-depleted membranes, followed by phaseseparation at 37°C (J.W. and C.B., unpublished method). Purified MITat 1.6 mfVSG was radioiodinated on an affinity column of antibody as described (14), and ap63 was radioiodinated on living L. major promastigotes and then purified to homogeneity (3, 7). Phospholipase C, type III, from Bacillus cereus was obtained from Sigma. Antibody to the CRD of T. brucei was prepared and affinity-purified as described (5, 9). Staphylococcus aureus protein A conjugated to horseradish peroxidase was from Miles-Yeda (Rehovot, Israel). Endoglycosidase F was a gift from J. Kaufman (Basel Institute of Immunology).Gel Electrophoresis. NaDodSO4/polyacrylamide gel electrophoresis was done according to the method of Laemmli (15). Gels were fixed and stained with 0.1% Coomassie blue or were prepared for 3H fluorography by immersion in Amplify (Amersham). For immunoblotting, samples were fractionated by gel electrophoresis and were electrophoretically transferred to nitrocellulose paper, which then was stained with Ponceau S and photographed. After destaining, the nitrocellulose was saturated with 1% gelatin in TBS (10 mM Tris HCl/150 mM NaCl, pH 7.4) and was then incubated overnight with anti-CRD (2 ,tg/ml) prior to detection with S.aureus protein A-horseradish peroxidase.Phospholipase Digestions. Removal of [3H]myristate from ap63 was monitored by incubation of 2 ,ug of the labeled ap63 in 160 ,ul of TBS with 0.5 mM dithiothreitol and 0.05% Triton X-100 (TX-100) for 1 hr at 30°C in the presence (0.45 ,ul/ml) or absence of affinity-purified lipase. After digestion, samples were heated and reduced prior to analysis by electrophoresis in a 7.5-15% polyacrylamide gradient gel. To monitor conversion of the amphiphilic forms of p63 and VSG Abbreviations: TX-114, Triton X-114; TX-100, Triton X-100; VSG, variant surface glycoprotein; mfVSG and sVSG, membrane-form and soluble VSG, respectively; CRD, crossreacting determinant of VSG; ap63 and hp63, aipphiphilic and hydrophilic forms, respectively, of the p63 antigen of Leishmania. 5988The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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