Copper regulates the interactions of antimicrobial piscidin peptides from fish mast cells with formyl peptide receptors and heparin

Phagocytic cells in fish secrete antimicrobial peptides (AMPs) such as piscidins, glycosaminoglycans such as heparin, and copper ions as first-line immune defenses. Recently, we established that Cu2+-coordination by piscidins 1 (P1) and 3 (P3) enhances their antibacterial activity against membranes and DNA. Interestingly, we noted that physicochemical similarities exist between both piscidins and other AMPs that interact with heparin and induce immune-cell chemotaxis through formyl peptide receptors (FPRs) involved in innate immunity. Thus, we postulated that P1 and P3 interact with heparin and FPRs but that these interactions distinctively depend on Cu2+ Here, we investigate the interactome potentiated by piscidins, heparin, FPR, and Cu2+ Utilizing FPR-transfected cells and neutrophils, we demonstrate that both piscidins exclusively use FPR1 and FPR2 to induce chemotaxis, and that Cu2+ reduces their chemotaxis induction. P1 is more effective at activating FPR1 than P3 and other known AMP ligands. Furthermore, the expression of Fpr2 on the surface of neutrophils is downregulated by both peptides. Using surface plasmon resonance, we show that piscidin-heparin interactions are Cu2+-dependent and reduced at the acidic pH of phagosomes. Although heparin decreases the antimicrobial activity of P3-Cu, it does not affect bacterial killing by P1-Cu and piscidin-mediated FPR1 aggregation. Copper's effects on modulating the micromolar-range interactions of both piscidins with FPR and heparin suggests that the interactome of these distinct immune agents plays an important role in innate immunity. The interactions between diverse host-defense molecules uncovered here may help inform the design of novel therapeutics to treat immune-related diseases. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

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