Journal: Investigative Ophthalmology & Visual Science
Article Title: Antimicrobial Peptides Derived from the Immune Defense Protein CAP37 Inhibit TLR4 Activation by S100A9
doi: 10.1167/iovs.61.4.16
Figure Lengend Snippet: CAP37 and four CAP37-derived peptides directly bind TLR4/MD-2 in vitro. ELISA plates were coated with the indicated proteins and peptides. ( A , C ) His-tagged TLR4/MD-2 (at 0 or 10 nM) was added to the coated wells and binding was quantified using anti-His tag antibody. Data are mean ± SEM of OD values from three independent experiments, after values with 0-nM TLR4/MD-2 were removed as background. A one-way ANOVA, using Dunnett's multiple comparisons test, compared the binding of each protein ( A ) or peptide (B) to the binding of BSA. Significances of * P < 0.05, ** P < 0.01, and *** P < 0.001 are shown. ( B ) Dose-dependent binding of TLR4/MD-2 to indicated proteins. Data (mean ± SEM ) are fitted to a curve of nonlinear regression. A two-way ANOVA was used with Dunnett's multiple comparisons test to compare the binding of each protein to that of BSA at the same concentration of TLR4/MD-2. LPS shows a significant binding of ** P < 0.01 to TLR4/MD-2 at 50 nM and **** P < 0.0001 at all higher doses. CAP37 shows significance with ** P < 0.01 at 10 nM and **** P < 0.0001 at all higher doses of TLR4/MD-2. S100A8 showed significant binding to TLR4/MD-2 at 75 nM ( *** P < 0.001) and at 100 nM ( **** P < 0.0001). Binding of NE and CG was not significant. ( D ) Dose-dependent binding of TLR4/MD-2 to peptides. Results are plotted and analyzed as described in ( B ). At all tested doses of TLR4/MD-2, the binding of peptide 120-146WH 5R-MP showed maximum significance of **** P < 0.0001. Peptides 20-44, 20-44 5R-MP, and 120-146WH significantly bind TLR4/MD-2 at 25 nM and reach maximum significance ( **** P < 0.0001) at 50 nM. Binding of peptides 95-122 and 95-122 5R-MP was not significant at any tested dose.
Article Snippet: Recombinant human S100 calcium binding proteins S100A8 (untagged, pro-800) and S100A9 (fused to a C-terminus 8-His tag, pro-814) were purchased from Prospec Protein Specialists (Ness Ziona, Israel).
Techniques: Derivative Assay, In Vitro, Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay
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