Purification and characterisation of membrane-form variant surface glycoproteins of Trypanosoma brucei.
The surface of Trypanosoma brucei contains 10 copies of the glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG; Cross 1975). On cell lysis the anchor is rapidly cleaved by an endogenous GPI-speci®c phospholipase C (GPI-PLC; Cardoso de Almeida and Turner 1983; BuÈ low and Overath 1986). Several methods have been described for puri®cation of membraneand soluble-form VSG, with yields ranging from 25% to 75% (Cross 1975, 1984; Gurnett et al. 1986; Schell and Overath 1990). This study describes the quantitative isolation of soluble-form VSG from dierent T. brucei variant clones by inhibition and reactivation of the endogenous GPI-PLC with p-chloromecuribenzenesulfonic acid (PCMBS) and dithiothreitol (DTT) during the puri®cation procedure. Bloodstream forms of T. brucei variant clone MITat 1.4 (117a) (Cross 1975; BuÈ low and Overath 1986) that had been grown in mice and puri®ed by diethylaminoethyl-cellulose chromatography (Lanham and Godfrey 1970) were suspended in lysis buer [50 mM Na-HEPES (pH 7.0), 2.5 mM ethylenediaminetetraacetic acid (EDTA), 2 mM ethylene glycol tetraacetic acid (EGTA), 200 lM Na-p-tosyl-L-lysine chloromethyl ketone (TLCK), 400 lM phenylmethylsulfonyl ̄uoride (PMSF), 10 lM leupeptin, 2 lM trans-epoxysuccinylL-leucylamido(4-guanidino)butane (E-64), 1 lM pepstatin A (Steverding et al. 1994)] containing 10 mM PCMBS so as to inactivate the endogenous GPI-PLC (Gurnett et al. 1986). After incubation on ice with occasional shaking until no intact cell could be observed microscopically, the suspension was centrifuged (114,000 g, 1 h). All of the VSG (Fig. 1a,b, lanes 1) remained in the particulate fraction since no VSG was detected in the soluble fraction as demonstrated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining or by immunoblotting (Fig. 1a,b, lanes 2). The particulate fraction was solubilized in PBS [137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4 1.4 mM KH2PO4, (pH 7.2)] in the presence of 0.2% Triton X-100 by sonication (3 ́ 2 min). Then, DTT and EDTA were added to give ®nal concentrations of 1 and 2.5 mM, respectively. After incubation for 15 min at 37 C and subsequent centrifugation (114,000 g, 1 h), all of the VSG appeared in the soluble fraction (Fig. 1a,b, lanes 3) due to cleavage of the GPI anchor by reactivated GPI-PLC (see below). Insoluble proteins remained associated with the particulate fraction (Fig. 1a,b, lanes 4). The purity of the isolated VSG was 92% as determined by densitometric scanning of a Coomassie-blue-stained gel (data not shown). The same puri®cation pattern was obtained using culture-adapted trypanosomes of the cell line TC 221 (Hirumi et al. 1980) and bloodstream forms of T. brucei rhodesiense clone MVAT4 (Alarcon et al. 1994; data not shown). Cleavage of the GPI anchor of VSG by GPI-PLC produces an epitope known as the cross-reacting determinant (CRD; Zamze et al. 1988). For con®rmation that the VSG was released from the particulate fraction on reactivation of GPI-PLC by DTT the presence of the CRD on the VSG was examined. As the source of antiCRD antibodies a polyvalent antiserum against hydrophilic gp63 (hgp63) from Leishmania mexicana was used (Bahr et al. 1993; Chaudhri et al. 1994). The VSG did not react with anti-CRD antibodies before DTT treatment (Fig. 2, upper panel, lane 1) but did so strongly thereafter (Fig. 2, upper panel, lane 2). Furthermore, after DTT treatment the VSG had a slightly greater apparent molecular mass (Fig. 2, lower panel, cf. lane 2 with lane 1) as previously reported for the soluble-form VSG, i.e., with a cleaved GPI anchor (Cardoso de Almeida and Turner 1983; Gurnett et al. 1986). These reParasitol Res (1998) 84: 524 ± 525 Ó Springer-Verlag 1998