Interaction between TNF and BmooMP-Alpha-I, a Zinc Metalloprotease Derived from Bothrops moojeni Snake Venom, Promotes Direct Proteolysis of This Cytokine: Molecular Modeling and Docking at a Glance

Abstract

Tumor necrosis factor (TNF) is a major cytokine in inflammatory processes and its deregulation plays a pivotal role in several diseases. Here, we report that a zinc metalloprotease extracted from Bothrops moojeni venom (BmooMP-alpha-I) inhibits TNF directly by promoting its degradation. This inhibition was demonstrated by both in vitro and in vivo assays, using known TLR ligands. These findings are supported by molecular docking results, which reveal interaction between BmooMP-alpha-I and TNF. The major cluster of interaction between BmooMP-alpha-I and TNF was confirmed by the structural alignment presenting Ligand Root Mean Square Deviation LRMS = 1.05 Å and Interactive Root Mean Square Deviation IRMS = 1.01 Å, this result being compatible with an accurate complex. Additionally, we demonstrated that the effect of this metalloprotease on TNF is independent of cell cytotoxicity and it does not affect other TLR-triggered cytokines, such as IL-12. Together, these results indicate that this zinc metalloprotease is a potential tool to be further investigated for the treatment of inflammatory disorders involving TNF deregulation.

Keywords: BmooMP-alpha-I; TACE; TNF; zinc metalloprotease.

Figure 1

Purification of BmooMP-alpha-I from Bothrops moojeni snake venom. (A) Separation on DEAE-Sephacel: crude venom (400 mg) was applied on the column (1.7 × 15 cm) and elution was carried out at 20 mL/h flow rate with ammonium bicarbonate (AMBIC) gradient buffer, pH 7.8, from 50 mM to 0.60 M; (B) Separation on Sephadex G-75: the active fraction (E2) was applied on the column (1.0 × 100 cm) and elutiom with 50 mM ammonium bicarbonate buffer at pH 7.8 was achieved at a flow rate of 20 mL/h; (C) Separation on Benzamidine Sepharose the fraction concentrate (E2G2) was applied on the column (20 × 15 cm) and elution was carried out at 40 mL/h flow rate with 50 mM glycine at pH 3.0. Pooled fractions are indicated by the closed circle; (D) SDS-PAGE in 12% (w/v). Lanes: 1–standard proteins; 2–non-reduced crude venom B. moojeni; 3–non-reduced BmooMP-alpha-I; (E) 2D electrophoresis of BmooMP-alpha-I solubilized in isoelectric focalization (IEF) solution were resolved by IEF capillary gel and then in 12% SDS-PAGE.

Figure 2

In vitro model for assessment of TNF production. (A) Levels of TNF determined after preincubation with LPS and treated BmooMP-alpha-I; (B) Levels of IL-12 p40 determined after preincubation with LPS and treated BmooMP-alpha-I; (C) Levels of TNF determined after preincubation with FSL-1 and treated BmooMP-alpha-I; (D) Levels of IL-12 p40 determined after preincubation with LPS and treated BmooMP-alpha-I. Macrophages were cultured in 96-well plates and after 24 h they were activated with Toll-like receptor (TLR) agonists: LPS (1 µg/mL); FSL-1 (1 µg/mL) or maintained with RPMI medium (control) at 37 °C and 5% CO₂. Cells were then treated with BmooMP-alpha-I (12 to 3.0 µg/mL) or maintained with RPMI medium (control) for additional 24 h at 37 °C and 5% CO₂. Levels of TNF and IL-12 were determined by ELISA kit according to the manufacturer’s instructions. Results are expressed as mean ± SD and compared to untreated controls by using Two-way Anova and Bonferroni multiple comparison post-test. ** p <0.01 and *** p <0.001; ns: no significant in relation to controls (RPMI medium).

Figure 3

In vivo model for assessment of TNF production. Mice were divided in groups (n = 10 per group) treated with the following conditions: PBS (control); PBS plus 100 µg of LPS (PBS + LPS); or BmooMP-alpha-I (50 µg) plus 100 µg of LPS (BmooMP-alpha-I + LPS). All animals received a final volume of 1 mL into their peritoneal cavity; the pretreatment was realized over 1 h and stimulated with LPS for 90 min. Serum TNF levels were measured by ELISA. The results are expressed as mean ± SD in triplicate for each experimental condition, and compared by using One-Way Anova and Tukey’s Multiple Comparison Test. * p <0.05 in relation to the group of control (mice that received injection with PBS, only).

Figure 4

Inhibitory effect of BmooMP-alpha-I on the TNF detection. (A) Determination of TNF levels; (B) Determination of IL-12 levels. The cytokines were detected after preincubation of capture antibody with 12 μg/mL BmooMP-alpha-I or 10 mM EDTA treated BmooMP-alpha-I (BmooMP-alpha-I(i)) for 45 min at 37 °C; (C) Determination of TNF levels (D) Determination of IL-12 levels. Now, the cytokines were detected after preincubation of recombinant TNF or IL-12 with 12 μg/mL BmooMP-alpha-I or 10 mM EDTA treated BmooMP-alpha-I (BmooMP-alpha-I(i)) for 45 min at 37 °C. Negative control was incubated only with sterile PBS. Levels of mouse recombinant TNF were quantified by ELISA kit according to the manufacturer’s instructions (DY 410-R & D Systems). Bars represent means ± SD out of five analyses for each experimental condition. Comparisons were carried out by using One-Way Anova and Dunnett’s Multiple Comparison Test. *** p <0.001; ns: not significant in relation to the negative controls.

Figure 5

Direct proteolytic effect of BmooMP-alpha-I on TNF cytokine. (A) Silver stained 18% SDS-PAGE. Lanes: 1-Molecular weight standard proteins; 2-Mouse recombinant TNF without incubation with BmooMP-alpha-I enzyme (control); 3-4-Mouse recombinant TNF incubated with 12; 6; or 3 μg/mL of BmooMP-alpha-I, respectively, for 45 min at 37 °C; (B) Percentage of TNF degradation by BmooMP-alpha-I (12; 6; and 3 μg/mL), determined by band intensity of the reaction products estimated by Kodak 1D image software in relation to negative control versus relative molecular mass of TNF; (C) Western blot of TNF protein treated with BmooMP-alpha-I. Immunoreactive bands were developed with ECL (Electron Chemiluminescent) Western substrate and visualized with an enhanced chemiluminescence system. Lanes: 1-Mouse recombinant TNF incubated with PBS (control); 2-Mouse recombinant TNF incubated with 24 µg/mL of BmooMP-alpha-I for 45 min at 37 °C.

Figure 5

Direct proteolytic effect of BmooMP-alpha-I on TNF cytokine. (A) Silver stained 18% SDS-PAGE. Lanes: 1-Molecular weight standard proteins; 2-Mouse recombinant TNF without incubation with BmooMP-alpha-I enzyme (control); 3-4-Mouse recombinant TNF incubated with 12; 6; or 3 μg/mL of BmooMP-alpha-I, respectively, for 45 min at 37 °C; (B) Percentage of TNF degradation by BmooMP-alpha-I (12; 6; and 3 μg/mL), determined by band intensity of the reaction products estimated by Kodak 1D image software in relation to negative control versus relative molecular mass of TNF; (C) Western blot of TNF protein treated with BmooMP-alpha-I. Immunoreactive bands were developed with ECL (Electron Chemiluminescent) Western substrate and visualized with an enhanced chemiluminescence system. Lanes: 1-Mouse recombinant TNF incubated with PBS (control); 2-Mouse recombinant TNF incubated with 24 µg/mL of BmooMP-alpha-I for 45 min at 37 °C.

Figure 6

MetaPocket 2.0 Analysis. (A) 3D view of the binding cavities in the complex formed between the metalloprotease and TNF. In blue is the binding cavity 1. In green is the binding cavity 2. In white is the binding cavity 3. In purple is the conserved histidine domains. In red is the active site containing the conserved catalytic consense sequence HEXXHXXGXXH; (B) Table containing the amino acids sequence of each binding cavity.

Figure 7

Analysis of the interaction of molecular complexes by Discovery Studio 3.5.0. (A) Interaction between the complex residues of 3GBO—2 TNF; (B) Demonstration of interacting residues in zoom out view. BmooMP-alpha-I is represented in red, and TNF recombinant murine in green. In purple the Zinc binding site is demonstrated.

Figure 8

Ramachandran plot: (A) The graphic demonstrates the phi-psi torsion angles for all residues in the BmooP-alpha-I and TNF molecular complex. The coloring/shading on the plot represents the allowed phi-psi backbone conformational regions, where the darkest areas (in red) correspond to the most favorable combinations of phi-psi values; (B) Details of the residues number in each region of Ramachandran plot and G-score.

Figure 9

Analysis of the potential cleavage sites in TNF by BmooP-alpha-I metalloprotease using PROSPER by homology with other metalloproteases. (A) Predicted cleavage sites P1 in TNF that can be cleaved and the main points of cleavage by metalloproteases that are indicated as a red line in the TNF respective regions; (B) Predicted cleavage sites P1 in TNF that can cleaved by metalloprotease in yellow and the predicted amino acids that interact with BmooMP-alpha-I, highlighted in red and white. The most reliable prediction site is highlighted in red and black.