Studies on citrullinated LL-37: detection in human airways, antibacterial effects and biophysical properties

Salma Al-Adwani,Melanie D Balhuizen,Birgitta Agerberth,Maarten Coorens,Peter Bergman,Ingemar Qvarfordt,Ákos Végvári,Edwin J A Veldhuizen,Astrid Gräslund,Michael Landreh,Anders Lindén,Margaretha E Smith,Cecilia Wallin

doi:10.1038/s41598-020-59071-7

Abstract

Arginine residues of the antimicrobial peptide LL-37 can be citrullinated by peptidyl arginine deiminases, which reduce the positive charge of the peptide. Notably, citrullinated LL-37 has not yet been detected in human samples. In addition, functional and biophysical properties of citrullinated LL-37 are not fully explored. The aim of this study was to detect citrullinated LL-37 in human bronchoalveolar lavage (BAL) fluid and to determine antibacterial and biophysical properties of citrullinated LL-37. BAL fluid was obtained from healthy human volunteers after intra-bronchial exposure to lipopolysaccharide. Synthetic peptides were used for bacterial killing assays, transmission electron microscopy, isothermal titration calorimetry, mass-spectrometry and circular dichroism. Using targeted proteomics, we were able to detect both native and citrullinated LL-37 in BAL fluid. The citrullinated peptide did not kill Escherichia coli nor lysed human red blood cells. Both peptides had similar α-helical secondary structures but citrullinated LL-37 was more stable at higher temperatures, as shown by circular dichroism. In conclusion, citrullinated LL-37 is present in the human airways and citrullination impaired bacterial killing, indicating that a net positive charge is important for antibacterial and membrane lysing effects. It is possible that citrullination serves as a homeostatic regulator of AMP-function by alteration of key functions.

Highlights

Arginine residues of the antimicrobial peptide LL-37 can be citrullinated by peptidyl arginine deiminases, which reduce the positive charge of the peptide
PAD2 and PAD4 are both expressed in neutrophils, where PAD2 is located in the cytosol and PAD4 is primarily located in the nucleus[17]
In vitro studies have demonstrated that recombinant PAD2 and PAD4 causes different degrees of citrullination for arginine residues in LL-37, which results in a reduced overall peptide charge and abrogation of its LPS neutralizing activity[15,19]

Summary

Introduction

Arginine residues of the antimicrobial peptide LL-37 can be citrullinated by peptidyl arginine deiminases, which reduce the positive charge of the peptide. Under inflammatory conditions, calcium-dependent peptidyl arginine deiminase enzymes (PADs) are expressed in the same location as the human cathelicidin LL-3715 These enzymes catalyse citrullination, a post-translational modification (PTM) in which positively-charged arginine residues are converted into citrulline, which reduces the charge of targeted proteins or peptides[12]. In vitro studies have demonstrated that recombinant PAD2 and PAD4 causes different degrees of citrullination for arginine residues in LL-37, which results in a reduced overall peptide charge and abrogation of its LPS neutralizing activity[15,19]. We determined the antimicrobial and biophysical properties of the synthetic citrullinated peptide by bacterial killing assays, transmission electron microscopy (TEM), isothermal titration calorimetry (ITC), mass spectrometry (MS) and circular dichroism (CD) and compared these properties with the native LL-37 peptide

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