Looped Host Defense Peptide CLP-19 Binds to Microtubules and Inhibits Surface Expression of TLR4 on Mouse Macrophages

The looped host defense peptide CLP-19 is derived from a highly functional core region of the Limulus anti-LPS factor and exerts robust anti-LPS activity by directly interacting with LPS in the extracellular space. We previously showed that prophylactic administration of CLP-19 even 20 h prior to LPS challenge might significantly increase the survival rate in a lethal endotoxin shock mouse model. Such an effect may be associated with immune regulation of CLP-19. To investigate the underlying mechanisms, peptide affinity chromatography, immunofluorescence, and Western blotting procedures were used to identify α- and β-tubulin as direct and specific binding partners of CLP-19 in the mouse macrophage cell line RAW 264.7. Bioinformatic analysis using the AutoDock Vina molecular docking and PyMOL molecular graphics system predicted that CLP-19 would bind to the functional residues of both α- and β-tubulin and would be located within the groove of microtubules. Tubulin polymerization assay revealed that CLP-19 might induce polymerization of microtubules and prevent depolymerization. The immunoregulatory effect of CLP-19 involving microtubules was investigated by flow cytometry, immunofluorescence, and Western blotting, which showed that CLP-19 prophylactic treatment of RAW 264.7 cells significantly inhibited LPS-induced surface expression of TLR4. Taken together, these results suggest that CLP-19 binding to microtubules disrupts the dynamic equilibrium of microtubules, reducing the efficacy of microtubule-dependent vesicular transport that would otherwise translocate TLR4 from the endoplasmic reticulum to the cell surface.