Citrullination-Resistant LL-37 Is a Potent Antimicrobial Agent in the Inflammatory Environment High in Arginine Deiminase Activity

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.

The Citrullination-Neutrophil Extracellular Trap Axis in Chronic Diseases

“…Aberrant citrullination may either lead to the creation of novel epitopes which can become immunogenic, especially if the modified proteins are abundantly accumulated in tissues or it may cause a shift in the activity of a protein or an enzyme, possibly further causing other problems related to the up-or downregulation J Innate Immun 2022;14:393-417 DOI: 10.1159/000522331 of the affected molecule. For example, antibacterial activity of cathelicidin LL-37 can be reduced after citrullination [31].…”

Peptides for Health Benefits 2020

“…Besides its potent activity against several highly virulent and antibiotic resistant bacterial pathogens, cecropin KJ has demonstrated to be one of the few peptides active against esophageal cancer. In the study of Bryzek et al [ 4 ], arginine residues in human cathelicidin LL-37 are substituted by homoarginine citrulline to produce a new generation of agents to treat bacterial infections and other inflammatory diseases associated with peptidyl-arginine deiminases activity. Binder and Skerra [ 5 ] combine PASylation ® technology with enzymatic in situ N-acetylation by RimJ to obtain a long-acting version of immunostimulatory peptide thymosin α1 in Escherichia coli at high yield.…”