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

Abstract

Schistosomiasis is a parasitic disease that affects over 200 million people. Vaccine candidates have been identified, including Schistosoma mansoni venom allergen-like proteins (SmVALs) from the SCP/TAPS (sperm-coating protein/Tpx/antigen 5/pathogenesis related-1/Sc7) superfamily. The first SmVAL structure, SmVAL4, was refined to a resolution limit of 2.16 Å. SmVAL4 has a unique structure that could not be predicted from homologous structures, with longer loops and an unusual C-terminal extension. SmVAL4 has the characteristic α/β-sandwich and central SCP/TAPS cavity. Furthermore, SmVAL4 has only one of the signature CAP cavity tetrad amino-acid residues and is missing the histidines that coordinate divalent cations such as Zn(2+) in other SCP/TAPS proteins. SmVAL4 has a cavity between α-helices 1 and 4 that was observed to bind lipids in tablysin-15, suggesting the ability to bind lipids. Subsequently, SmVAL4 was shown to bind cholesterol in vitro. Additionally, SmVAL4 was shown to complement the in vivo sterol-export phenotype of yeast mutants lacking their endogenous CAP proteins. Expression of SmVAL4 in yeast cells lacking endogenous CAP function restores the block in sterol export. These studies suggest an evolutionarily conserved lipid-binding function shared by CAP proteins such as SmVAL4 and yeast CAP proteins such as Pry1.

Keywords: Ancylostoma secreted protein; CAP; Saccharomyces cerevisiae; Schistosoma mansoni; TAPs; sperm-coating protein; sterol binding; venom allergen-like protein; venom antigen 5.

Figure 1

The SmVAL4 monomer. (a) A Coomassie-stained SDS gel reveals the purity of the SmVAL4 sample and its monomeric mass of ∼20 kDa. Lane M contains molecular-weight marker (labeled in kDa). (b) SEC-MALS analysis reveals that SmVAL4 is an ∼20 kDa monomer in solution. (c) Fit of N-­glycosylated Asn118 and proximal residues into a 2F _o − F _c electron-density map (gray) calculated from the refined model of SmVAL4 and contoured at 1.2σ. NAG, N-acetyl-d-glucosamine. (d) Topology of the SmVAL4 structure.

Figure 2

Structural features of SmVAL4 and primary-sequence alignment with selected representative CAP proteins. The sequences were aligned with ClustalW2 and the secondary-structural features are illustrated with the coordinates of SmVAL4 and GLIPR2 using ESPript (Gouet et al., 2003 ▶). The different secondary-structure elements shown are α-helices (large squiggles labeled α), 3₁₀-helices (small squiggles labeled η), β-strands (arrows labeled β) and β-turns (labeled TT). Identical residues are shown in white on a red background and conserved residues are highlighted in yellow. The locations of the cysteine residues involved in disulfide bonds are numbered in green and the signature CRISP motifs are identified by red bars. The representative CAP structures are NaASP2 (PDB entry 1u53), tablysin-15 (PDB entry 3u3n), GAPR-1 (PDB entry 1smb), sGLIPR1 (PDB entry 3qnx) and vCRISP (PDB entry 1rc9).

Figure 3

Comparison of the SmVAL4 structure with representative CAP structures. The top row shows ribbon diagrams of SmVAL4 (PDB entry 4p27), NaASP2 (PDB entry 1u53), tablysin-15 (PDB entry 3u3n), GAPR-1 (PDB entry 1smb), sGLIPR1 (PDB entry 3q2u) and vCRISP (PDB entry 1rc9). The core α–β–α sandwich is formed by the three-stranded β-sheet between the labeled helices. The second row is another view of the proteins in which the central cavity is visible. The third row is from the same view as the second row and shows the differences in the charge distribution of these CAP proteins, colored blue for positively charged and red for negatively charged regions. The differences in the charge distribution in proximity to the central cavity is obvious in the structures. The Zn²⁺ complexed with sGLIPR1 and sitting in the central cavity is shown in magenta. The palmitate bound to the tablysin-15 structure is shown in a black stick representation.

Figure 4

The CAP cavity. (a) The superposed central cavity of CAPs reveals that key residues corresponding to the Zn²⁺-binding site superimpose well in representative CAP structures. CAP structures are colored as follows: SmVAL4, gray; NaASP2, green; tablysin-15, blue; GAPR-1, yellow; sGLIPR1, magenta; vCRISP, cyan. The numbers correspond to those for GLIPR1 and the Zn²⁺ ion is shown as a red sphere. (b) The same region and view for SmVAL4 alone reveals the absence of the His that coordinates Zn²⁺; numbering corresponds to that of SmVAL4. (c) The same region and view for sGLIPR1 alone; numbering corresponds to that of sGLIPR1.

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).

Figure 6

The calveolin-binding motif. (a) The conserved calveolin-binding motif (CBM) is evident in the alignment of the sequences of SmVAL4, GAPR1, Pry1 and Pry2. The secondary-structural elements shown are for SmVAL4 and GAPR1 (PDB entry 4aiw). The location of the CBM is identified with a blue line, while the CRISP1 motif is shown as a red line. The figure was generated using EsPript and ClustalW and structural elements are labeled as described in Fig. 2 ▶. (b) The superposed ribbon structures of SmVAL4 (gray) and GAPR1 (cyan) reveals the conformational flexibility of the CBM (shown in stick representation and identified with a blue arrow). The inositol hexakisphosphate (IP6) that was co-crystallized with GAPR1 is shown in a cyan stick representation. (c) Close-up of the superposed CBM showing the conformational difference of the loops.

Figure 7

The palmitate-binding motif in tablysin-15. (a) Mode of palmitate binding in tablysin-15; the ribbon is shown in gray and interacting residues and palmitate are shown as gray sticks. Also shown is the surface charge in the cavity. (b) Superposition of the plamitate-binding region of tablysin-15 (shown in cyan ribbon) with SmVAL4 (shown in gray) reveals that SmVAL4 can have the same palmitate-binding mode as tablysin-15.