SmSPO-1 assembles into a nine-subunit oligomer with potential To inhibit Toll-like receptor signaling - PubMed (original) (raw)

The Schistosoma mansoni protein Sm16/SmSLP/SmSPO-1 assembles into a nine-subunit oligomer with potential To inhibit Toll-like receptor signaling

Kristoffer Brännström et al. Infect Immun. 2009 Mar.

Abstract

The Sm16/SmSLP/SmSPO-1 (Sm16) protein is secreted by the parasite Schistosoma mansoni during skin penetration and has been ascribed immunosuppressive activities. Here we describe the strategy behind the design of a modified Sm16 protein with a decreased aggregation propensity, thus facilitating the expression and purification of an Sm16 protein that is soluble in physiological buffers. The Stokes radii and sedimentation coefficients of recombinant and native proteins indicate that Sm16 is an approximately nine-subunit oligomer. Analysis of truncated Sm16 derivatives showed that both oligomerization and binding to the plasma membrane of human cells depend on multiple C-terminal regions. For analysis of immunomodulatory activities, Sm16 was expressed in Pichia pastoris to facilitate the preparation of a pyrogen/endotoxin-free purified protein. Recombinant Sm16 was found to have no effect on T-lymphocyte activation, cell proliferation, or the basal level of cytokine production by whole human blood or monocytic cells. However, Sm16 exerts potent inhibition of the cytokine response to the Toll-like receptor (TLR) ligands lipopolysaccharide (LPS) and poly(I:C) while being less efficient at inhibiting the response to the TLR ligand peptidoglycan or a synthetic lipopeptide. Since Sm16 specifically inhibits the degradation of the IRAK1 signaling protein in LPS-stimulated monocytes, our findings indicate that inhibition is exerted proximal to the TLR complex.

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Figures

FIG. 1.

Construction of the engineered version of Sm16, Sm16(23-117)AA, with improved solubility and protein expression. (A) Schematic representation of recombinant Sm16 derivatives with successive truncations at the C terminus. Each truncated derivative is defined according to the amino acid sequence deduced from the cDNA sequence (numbers in parentheses). The location of hydrophobic residues Ile-92 and Leu-93 is indicated by a dashed line. The signal peptide of Sm16 consists of the first 22 residues, which are absent in the secreted form of the protein termed Sm16(23-117). All Sm16 derivatives contain an N-terminal His tag and an eight-residue C-terminal Flag tag. (B) Anti-Sm16 antibodies were used to detect recombinant Sm16 by immunoblot analysis of crude lysates of E. coli transformed with pET3d plasmid derivatives directing the expression of the indicated Sm16 proteins. (C) Coomassie brilliant blue-stained SDS-PAGE (upper panel) and immunoblot analysis with anti-Sm16 (lower panel) of crude lysates of E. coli transformed with pET3d derivatives directing the expression of the indicated Sm16 proteins. The abundantly expressed Sm16(23-117)AA protein was modified for improved solubility by replacing Ile-92 and Leu-93 with Ala, which also greatly improved expression. (D) Far-UV circular-dichroism (CD) spectra of Sm16(23-117)AA expressed in E. coli as a His-tagged protein and purified by metal ion affinity. Proteins were dissolved at 0.2 mg/ml in 2 mM sodium phosphate buffer and scanned at 20°C. The data indicate that ∼70% of the protein has an α-helical conformation. The values to the left of panels B and C are molecular sizes in kilodaltons.

FIG. 2.

Hydrodynamic parameters and molecular masses of recombinant Sm16(23-117)AA and native Sm16 in crude cercarial extract. (A) The Sm16(23-117)AA protein was expressed in E. coli with an N-terminal His tag and an eight-residue C-terminal Flag tag. The metal ion affinity-purified protein was analyzed by SDS-PAGE in the presence (+) or absence (−) of the reducing agent 2-mercaptoethanol. Proteins were visualized by Coomassie blue staining, and arrows indicate the positions of standards with the indicated molecular weights (103). (B) Sephadex 200 gel filtration chromatography of purified Sm16(23-117)AA. Fractions were analyzed by SDS-PAGE, and proteins were visualized and quantitated by Coomassie blue staining, followed by scanning of gels. Open and filled symbols indicate the elution peaks of protein standards with known Stokes radii and Sm16, respectively. (C) Sucrose gradient sedimentation of purified Sm16(23-117)AA. Fractions were analyzed by SDS-PAGE as described for panel B. Open and filled symbols indicate the positions of protein standards with known Stokes radii and Sm16, respectively. (D) Crude cercarial extract was separated by SDS-PAGE as described for panel A, and Sm16 was detected by immunoblotting. (E) Crude cercarial extract was gel filtered together with protein standards as described for panel B. The Sm16 content of each fraction was quantified by immunoblotting and a comparison of signal intensity with a serial dilution of cercarial extract. (F) Crude cercarial extract was sedimented on a sucrose gradient together with protein standards as described for panel C. Fractions were analyzed as described for panel E. The data plotted are representative of at least three independent analyses, and the interexperimental variations in the estimations of Stokes radii and sedimentation coefficients were <7%. The values to the left of panels A and D are molecular sizes in kilodaltons. a.u., arbitrary units; Seph, Sephadex.

FIG. 3.

Identification of two oligomeric forms in a preparation of the C-terminally truncated Sm16(23-98)AA protein. (A) The Sm16(23-98)AA protein contains Ala substitutions for Ile-92 and Leu-93 to prevent aggregation. The metal ion affinity-purified protein was separated by Sephadex 200 gel filtration, and fractions were analyzed and quantified as described in the legend to Fig. 2B. Open and filled symbols indicate the elution peaks of protein standards with known Stokes radii and Sm16(23-98)AA, respectively. The estimated Stokes radii of the two peaks are indicated. Peak fractions corresponding to Stokes radii of 5.5 and 3.1 nm, respectively, were pooled and analyzed by SDS-PAGE as described in the legend to Fig. 2A (insert). (B and C) Sedimentation properties of pooled peak fractions (insert in panel A) of Sm16(23-98)AA species with Stokes radii of 5.5 nm (panel B) and 3.1 nm (panel C) were analyzed as described in the legend to Fig. 2C. (D and E) Heparin binding of pooled peak fractions (insert in panel A) of Sm16(23-98)AA species with Stokes radii of 5.5 nm (panel D) and 3.1 nm (panel E). Proteins diluted in phosphate-buffered saline (0.15 M NaCl) were passed over a column containing heparin coupled to Sepharose. Columns were eluted with a linear gradient of 0.15 to 1.0 M NaCl. Proteins in the fractions were analyzed and quantitated as described in the legend to Fig. 2B. The data plotted are representative of at least two independent analyses, and the experimental errors in the estimations of Stokes radii and sedimentation coefficients were <7%. a.u., arbitrary units; Seph, Sephadex. The values to the left of the inserts in panel A are molecular sizes in kilodaltons.

FIG. 4.

Importance of C-terminal regions for Sm16 oligomerization and cell surface binding. (A) The fraction of approximately nine-subunit oligomers, defined as a >4.5-nm Stokes radius, in metal ion affinity-purified preparations of the truncated Sm16 derivatives depicted in Fig. 1A was determined by Sephadex 200 gel filtration. Where indicated by the superscript AA, amino acid residues Ile-92 and Leu-93 were replaced with Ala to prevent protein aggregation. (B) Cell surface binding of approximately nine-subunit oligomers isolated by Sephadex 200 gel filtration of the indicated Sm16 derivative. The Sm16(23-70) protein does not show detectable affinity for cells (data not shown) but was not included in the present analysis since it does not form detectable amounts of approximately nine-subunit oligomers (panel A). Binding to live K562 erythroleukemia cells was analyzed as described in Materials and Methods. Background binding, i.e., anti-Sm16 and fluorescein-conjugated swine anti-rabbit immunoglobulin, was ∼12% (dotted line). The presented data are representative of at least three independent experiments. a.u., arbitrary units.

FIG. 5.

Effect of P. pastoris_-expressed Sm16 on cytokine expression and proliferation of peripheral blood mononuclear cells stimulated by an antibody directed to the T-lymphocyte antigen/CD3 complex. Density gradient-purified blood mononuclear cells were cultured in medium alone or stimulated with the anti-CD3 antibody OKT3 (50 ng/ml) as indicated. IL-1β (A) or IL-2 (B) mRNA expression levels determined by quantitative RT-PCR after 18 h of incubation in the absence (open bars) or presence (filled symbols) of Sm16(23-117)AA_P (50 μg/ml) are shown. (C) Cell proliferation as determination by [3H]thymidine uptake during the last 6 h of a 78-h culture period in the presence of the indicated concentration of Sm16(23-117)AA_P_. The data plotted are representative of at least three independent experiments.

FIG. 6.

Effect of _P. pastoris_-expressed Sm16 proteins on LPS-induced production of proinflammatory cytokines in whole blood. Human blood was preincubated for 10 min with graded concentrations of P. pastoris_-expressed Sm16(23-70)P (triangles) or Sm16(23-117)AA_P (squares). Cells were then cultured for 6 h in the absence (open symbols) or presence (filled symbols) of LPS (50 ng/ml), followed by analysis of IL-6 (A), TNF-α (B), and IL-1β (C) in the supernatants. The data plotted are representative of at least 10 independent analyses performed in triplicate. Blood cells from four healthy donors were analyzed and found to be indistinguishable with regard to the effect of purified Sm16 derivatives.

FIG. 7.

Effect of Sm16(23-117)AA_P_ on cytokine production in response to various TLR ligands. Human blood was preincubated for 10 min with a buffer control (open bars) or P. pastoris_-expressed Sm16(23-117)AA_P (20 μg/ml) (filled bars). The buffer control, LPS (50 ng/ml), poly(I:C) (20 μg/ml), PG (5 μg/ml), or Pam3CSK4 (5 μg/ml) was then added as indicated for 6 h of incubation, followed by analysis of either IL-6 (upper panel) or IL-1RA (lower panels) in the supernatants. The data plotted are representative of at least four independent analyses performed in triplicate with blood from three donors.

FIG. 8.

Effect of Sm16(23-117)AA_P_ on the clonal monocytic cell line Mono-Mac-6. (A) Mono-Mac-6 cells seeded at 0.5 × 105/ml and cultured for 4 days with either a buffer control or LPS (2 ng/ml), as indicated, in the absence (open bars) or presence (filled bars) of Sm16(23-117)AA_P_ (20 μg/ml). Cell proliferation was analyzed by determination of [3H]thymidine uptake during the last 24 h of culture. (B) Mono-Mac-6 cells were cultured for 5 h in the absence (open bars) or presence (filled bars) of Sm16(23-117)AA_P_ (20 μg/ml). TLR4 surface expression was then analyzed by flow cytometry and expressed as the mean fluorescence signal.

FIG. 9.

Effect of Sm16(23-117)AA_P_ on cytokine production by Mono-Mac-6 cells in response to various TLR ligands. Mono-Mac-6 cells were preincubated for 10 min with a buffer control (open bars) or 20 μg/ml Sm16(23-117)AA_P_ (filled bars). The buffer control, LPS (50 ng/ml), poly(I:C) (20 μg/ml), or PG (5 μg/ml) was then added as indicated for 8 h of incubation, followed by quantification of the proinflammatory cytokine IL-6 or the anti-inflammatory cytokines IL-1RA and IL-10 in the supernatants. The data plotted are representative of at least four independent analyses performed in triplicate.

FIG. 10.

Effect of Sm16(23-117)AA_P_ on TLR ligand-induced intracellular signaling events. Mono-Mac-6 cells were preincubated for 10 min in the absence or presence of Sm16(23-117)AA_P_ (20 μg/ml) as indicated. A buffer control (Co), LPS (50 ng/ml), or PG (5 μg/ml) was then added as indicated. Cells were incubated at 37°C for 0.5 h (A), 1 h (B, top), or 8 h (B, bottom) and processed for immunoblot analysis. Filters were probed with either IκB-α (A) or anti-IRAK1 (B). Anti-α-tubulin was used as a control for equal loading. The presented data are representative of at least three independent analyses.

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The Schistosoma mansoni protein Sm16/SmSLP/SmSPO-1 assembles into a nine-subunit oligomer with potential To inhibit Toll-like receptor signaling - PubMed (original) (raw)