Schistosoma mansoni venom allergen-like protein 4 (SmVAL4) is a novel lipid-binding SCP/TAPS protein that lacks the prototypical CAP motifs

Kelleher, A.; Darwiche, R.; Rezende, W.C.; Farias, L.P.; Leite, L.C.C.; Schneiter, R.; Asojo, O.A.

doi:10.1107/S1399004714013315

Figure 5
SmVAL4 binds chloresterol in vivo and in vitro. (a) Expression of SmVAL4 complements the sterol-export defect of yeast cells lacking their endogenous CAP proteins. Heme-deficient cells of the indicated genotype containing either an empty plasmid or a plasmid with SmVAL4 were radiolabeled with [¹⁴C]-cholesterol overnight, washed and diluted in fresh medium to allow export of cholesterol and cholesteryl acetate. Lipids were extracted from the cell pellet (P) and the culture supernatant (S) and separated by thin-layer chromatography. The positions of free cholesterol (FC), cholesteryl acetate (CA) and steryl esters (STE) are indicated on the right. The asterisk marks the position of an unidentified cholesterol derivative. (b) Quantification of the export of cholesteryl acetate in yeast cells lacking their endogenous CAP proteins. The export index indicates the relative percentage of cholesteryl acetate that is exported by the cells (the ratio between extracellular cholesteryl acetate and the sum of intracellular and extracellular cholesteryl acetate). Data represent the mean ± SD of two independent experiments. (c) SmVAL4 binds cholesterol in vitro. Sterol binding was assessed using increasing amounts of the purified protein and 50 pmol [³H]-cholesterol as the ligand. The protein was separated from unbound ligand by adsorption to an anion-exchange matrix and bound radioligand was quantified by scintillation counting. (d) Addition of an equimolar amount of unlabeled cholesterol (cold Chol) resulted in a corresponding reduction in binding of the radiolabeled ligand (hot Chol). (e) Addition of an excess of unlabeled cholesterol reduces binding of the radiolabeled ligand. Purified SmVAL4 (100 pmol) was incubated with 200 pmol [³H]-cholesterol as the ligand (hot Chol) and where indicated binding of the radioligand was in competition with 500 pmol unlabeled cholesterol (cold Chol).

BIOLOGICAL
CRYSTALLOGRAPHY

ISSN: 1399-0047

Volume 70| Part 8| July 2014| Pages 2186-2196

doi:10.1107/S1399004714013315

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