Leishmania and Trypanosoma surface glycoproteins have a common glycophospholipid membrane anchor.

Abstract

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501 (Bio-Rad), or by TX-114 extraction from VSG-depleted membranes, followed by phaseseparation at 37°C (J.W. and C.B., unpublished method).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.

Internal and Surface-Localized Major Surface Proteases ofLeishmaniaspp. and Their Differential Release from Promastigotes

“…Indeed, in L. mexicana, MSPs account for 1 and 0.1% of total proteins in promastigotes and amastigotes, respectively (1). Promastigote cell-associated MSP is predominantly attached to the cell surface by glycosylphosphatidylinositol anchors (4,5). However, our laboratory and others have observed that as much as one-third of the cell-associated MSP is located intracellularly, as determined by a combination of surface biotinylation, immunoelectron microscopy, and cytofluorometry (42,47).…”

Evidence for glycosyl-phosphatidylinositol anchoring of Toxoplasma gondii major surface antigens.

“…Biochemical analyses of the GPI-anchors have shown that they contain a phospholipid structure linked covalently to an oligosaccharidic structure containing inositol, glucosamine, and ethanolamine (8-10, 20, 25). In many cases, GPIanchored proteins are released from membranes by treatment with phosphatidylinositol-specific phospholipase C (Pl-PLC) (19); this treatment uncovers a common reactive determinant to soluble forms of GPI-anchored proteins (1,2,5,28).…”

“…The cross-reacting epitope has been found in other protozoa, such as Paramecium spp. (2) and Leishmania major (1) and is one of the principal criteria used in the identification of a GPI-anchor (28).…”

Major Surface Protease of Trypanosomatids: One Size Fits All?

“…As indicated above, most of the Leishmania MSPs are bound to the plasma membranes of promastigotes through a GPI anchor (9). Several groups, including ours, have reported that some MSPs of several Leishmania spp.…”