Abstract
The surface coat of Trypanosoma brucei is composed of 10(7) molecules of the variant surface glycoprotein (VSG). Each VSG molecule is tethered to the cell membrane by a glycolipid moiety which contains 1,2-dimyristoyl-sn-phosphatidylinositol (Ferguson, M. A. J., Low, M. G., and Cross, G. A. M. (1985) J. Biol. Chem. 260, 14547-14555). Following cell lysis, an endogenous phospholipase C cleaves dimyristoyl glycerol from the glycolipid, releasing soluble VSG. We have purified this enzyme, which we designate VSG lipase, by detergent extraction, (NH4)2SO4 fractionation, hydrophobic chromatography, and cation exchange chromatography. It is purified 2600-fold and is virtually homogeneous. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the apparent molecular mass is 37 kDa. In solutions containing the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), the Stokes radius (2.6 nm), S20,w (3.7 S), and v (0.77 cm3/g) of VSG lipase suggest a molecular mass for the native enzyme of about 47 kDa, part of which may be due to bound CHAPS. Therefore, it is probably monomeric. VSG lipase does not require Ca2+; it is stimulated by chelating agents or dithiothreitol, and it is inhibited by some sulfhydryl reagents. The purified enzyme appears to be highly specific. Under the conditions of our assay, it cleaves the VSG glycolipid, a biosynthetic precursor of the VSG glycolipid, and, to a much lesser extent, 1,2-dimyristoyl-sn-phosphatidylinositol. There was no apparent cleavage of other myristate-containing lipids of trypanosomes or 1-stearoyl-2-arachidonoyl-sn-phosphatidylinositol.
Highlights
The surface coat of Trypanosoma brucei is composed host’s immune response by antigenic variation
Cardoso de Almeida and Turner (6) resolved this paradox when they discovered that boiling trypanosomes in SDS allows isolation of an amphiphilic or membrane formof VSG
Lipase suggest a molecular mass for the native enzyme Recent structural studies in several laboratories have reof about 47 kDa, part of which may be due to bound vealed that membrane formof VSG (mfVSG) contains a glycolipid covalently linked to CHAPS
Summary
10% trichloroacetic acid, as described by Lowry et al (27) unless noted otherwise. SDS (0.5%) was included to avoid interference by Trypanosomes-T.brucei Isolated from the blood of Swiss mice or Wistar rats (parasitemia about 109/ml)by DEAE-cellulose chromatography (23) using 50 mM NaCl, 5 mM KCl, 55 mMD-ghCOSe, 50 mM Na-Bicine, pH 8.0 (BBS)
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