Human β‐defensin‐3 structure motifs that are important in CXCR4 antagonism

Previously, we reported that human β‐defensin (hBD)‐3 can both antagonize CXCR4 function on T cells and promote receptor internalization in the absence of activation. In the present study, we explored the important structural elements of hBD‐3 that are involved in blocking CXCR4 activation by its natural ligand, stromal‐derived factor 1α (SDF‐1α; CXCL12). Results from site‐directed mutagenesis studies suggest that the ability of hBD‐3 to inhibit SDF‐1α–CXCR4 interaction, as assayed either by blocking SDF‐1 binding to CXCR4 or antagonizing SDF‐1‐induced Ca²⁺ mobilization, is correlated with the presence of hBD‐3 cysteine residues, specific surface‐distributed cationic residues, and the electrostatic properties and availability of both hBD‐3 termini. Specifically, hBD‐3 activity against CXCR4 is reduced by: (a) replacing all six cysteines; (b) replacing the cationic residues with acidic ones in the N‐terminus and C‐ terminus; (c) removal of the first 10 N‐terminal residues; and (d) replacing the surface‐exposed basic residues Lys8, Lys32 and Arg36 with neutral ones. The hBD‐3–CXCR4 interaction has potentially wide‐ranging implications for HIV‐related biology, as well as for a host of CXCR4‐dependent activities, including hematopoiesis, neurogenesis, angiogenesis, carcinogenesis, and immune cell trafficking. CXCR4 is highly expressed on T cells, monocytes, and epithelial cells. Therefore, understanding the structure–function relationship between hBD‐3 and CXCR4 that accounts for the antagonistic interaction between the two molecules may provide new insights into HIV/highly active antiretroviral therapy‐related pathology, as well as novel insights into the interaction between innate and adaptive immunity at mucosal sites.