Abstract
LL-37, the only member of the mammalian cathelicidin in humans, plays an essential role in innate immunity by killing pathogens and regulating the inflammatory response. However, at an inflammatory focus, arginine residues in LL-37 can be converted to citrulline via a reaction catalyzed by peptidyl-arginine deiminases (PAD2 and PAD4), which are expressed in neutrophils and are highly active during the formation of neutrophil extracellular traps (NETs). Citrullination impairs the bactericidal activity of LL-37 and abrogates its immunomodulatory functions. Therefore, we hypothesized that citrullination-resistant LL-37 variants would retain the functionality of the native peptide in the presence of PADs. To test this hypothesis, we synthetized LL-37 in which arginine residues were substituted by homoarginine (hArg-LL-37). Bactericidal activity of hArg-LL-37 was comparable with that of native LL-37, but neither treatment with PAD4 nor exposure to NETs affected the antibacterial and immunomodulatory activities of hArg-LL-37. Importantly, the susceptibilities of LL-37 and hArg-LL-37 to degradation by proteases did not significantly differ. Collectively, we demonstrated that citrullination-resistant hArg-LL-37 is an attractive lead compound for the generation of new agents to treat bacterial infections and other inflammatory diseases associated with enhanced PAD activity. Moreover, our results provide a proof-of-concept for synthesis of therapeutic peptides using homoarginine.
Highlights
Antimicrobial peptides (AMPs) have a broad spectrum of antibacterial and immunomodulatory activities and constitute an ancient branch of the innate immune system [1,2]
Experiments using synthetic LL-37 revealed that post-translational modifications including proteolysis [23], non-enzymatic conversion of lysine residues to homocitrulline [24], and deimination of arginine residues [12,13,14] significantly influence the antimicrobial and immunomodulatory functions of LL-37
We used synthetic LL-37 in which arginine residues were substituted by citrullination-resistant homoarginine residues. hArg-LL-37 retained its biological activity upon PAD4 treatment
Summary
Antimicrobial peptides (AMPs) have a broad spectrum of antibacterial and immunomodulatory activities and constitute an ancient branch of the innate immune system [1,2]. LL-37 is released from an 18-kDa precursor protein (hCAP-18) by protease-3 in neutrophils [4] and by a serine protease belonging to the kallikrein family in keratinocytes [5] and contains 37 amino acids with two leucine residues at its N-terminus. This cationic and amphipathic peptide kills microbes by binding to and permeabilizing the negatively charged bacterial cell membrane [6,7]. Our findings indicate that hArg-LL-37 can be used as a therapeutic replacement for LL-37 in pathological inflammatory conditions such as psoriasis and rheumatoid arthritis, in which citrullination of the native peptide compromises its antibacterial and immunoregulatory activities [15,16]
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