Elsevier

Peptides

Volume 27, Issue 12, December 2006, Pages 3100-3106
Peptides

The antimicrobial peptide cathelicidin interacts with airway mucus

https://doi.org/10.1016/j.peptides.2006.07.018Get rights and content

Abstract

Antimicrobial peptides (AMPs) and mucins are components of airway secretions and both contribute to the innate host defense system. At neutral pH, AMPs are positively charged, mucins negatively. It was the aim of the study to test whether these opposite charges result in interactions between AMPs and mucins. We measured binding of mucins isolated from porcine gastric mucosa to the cathelicidin LL-37 coated to multiwell plates and found that LL-37 electrostatically interacts with mucins. Circular dichroism spectra of the peptide revealed the induction of α-helical conformation by mucins. Addition of mucins to solutions of LL-37 significantly decreased the antimicrobial activity of the peptide against Pseudomonas aeruginosa and Streptococcus pneumoniae. We then tested whether LL-37 is bound to mucins in airway secretions from human subjects and found that a significant proportion of the peptide and its propeptide are bound to high molecular weight components. Together these data show that cationic AMPs interact with anionic mucins in airway secretions. Functions of AMPs are modulated by this interaction.

Introduction

The respiratory tract is shielded by a multi-component host defense system that involves structural, physical and functional mechanisms. Airway epithelial and host defense cells secrete a variety of factors that directly kill pathogens or modulate the inflammatory response. Classical components of the airway surface fluid (ASF) that have antimicrobial activity are lysozyme, lactoferrin, secretory phospholipase A2, and secretory leukocyte protease inhibitor (SLPI) [16]. Other substances, such as complement, surfactant proteins, and Clara-cell proteins (CC10, CCSP) likely contribute to host defense [21]. Cationic polypeptides have been identified as an essential part of the antimicrobial activity of human airway fluid [9].

Antimicrobial peptides (AMPs) are effector molecules of the innate immune system of the lung [23]. AMPs have a broad antimicrobial spectrum and lyse microbial cells by interaction with biomembranes. Besides their direct antimicrobial function, they have multiple roles as mediators of inflammation with impact on epithelial and inflammatory cells influencing diverse processes such as cytokine release, cell proliferation, angiogenesis, wound healing, chemotaxis, adaptive immunity, and protease–antiprotease balance. The defensins and the cathelicidins are the principal families of AMPs expressed in the lung [6]. The only human cathelicidin LL-37/hCAP-18 is produced by airway epithelial cells, type II pneumocytes, neutrophils, and macrophages [1], [3]. Besides its direct antimicrobial activity it has been proposed that LL-37 modulates angiogenesis [14], epithelial inflammation [19], and the activation of immune cells such as dendritic cells or monocytes [22]. One of the prominent physical features of AMPs is their strong positive charge at neutral pH. LL-37 has a positive charge of 5.8 at neutral pH. It has been demonstrated that LL-37 interacts with negatively charged macromolecules such as DNA [20] or glycosaminoglycans [11] and that these interactions have functional consequences.

Mucins are macromolecules that determine the biological and physical properties of mucus [15]. Products encoded by five secreted mucin genes are expressed in the human airways: MUC2, MUC5AC, MUC5B, MUC7, and MUC8. The mucin core protein is heavily glycosylated via O-glycosidic bounds. These sugar side chains contain sialic acid and N-acetylgalactosamine-6-sulfate that determine the strong negative charge of the molecules. Mucin glycoproteins have a host defense function and are part of the mucociliary clearance of the respiratory tract. Inhaled microorganisms are thought to be trapped within mucus and transported out of the lung.

Mucins are negatively charged, AMPs positively. Based on these opposite charges it is tempting to speculate that AMPs and mucins interact with each other. It was the aim of the study to investigate whether LL-37 interacts with mucins and whether this interaction modulates the antimicrobial activity of the AMP.

Section snippets

Patient materials

Spontaneous sputum was collected from five cystic fibrosis patients with severe lung disease (mean FEV1 43.0 ± 5.8% pred.). All patients (age 29.8 ± 2.8) were chronically infected with Pseudomonas aeruginosa and in a clinically stable condition. The samples were diluted 1:16 in phosphate buffer (10 mM KH2PO4/K2HPO4, pH 7.2), incubated with 50 μl DNAse (QIAGEN, Hilden, Germany) for 1 h at RT and vortexed several times during the procedure. Bronchoalveolar lavage fluid (BALF) was used from patients that

LL-37 binds to mucins

First we investigated whether the LL-37 binds to mucins. We determined the amounts of mucin bound to immobilized LL-37 using a lectin based assay. A native, uncoated plate was used as control. The results showed that precoating with LL-37 results in significantly increased levels of bound mucins (Fig. 1A Fig. 1). Washing with increasing concentrations of NaCl resulted in decreased amounts of bound mucins indicating that the nature of the binding between LL-37 and mucin is based on electrostatic

Discussion

The main finding of the present study is that LL-37 binds to mucins and that this interaction inhibits the antimicrobial activity of the peptide. It has been demonstrated before that cationic antimicrobial peptides interact with negatively charged macromolecules. DNA in airway secretions inhibits the antimicrobial activity of LL-37 [20]. Glycosaminoglycan-rich biological fluids also decrease the antimicrobial activity of this peptide [4]. Here we show that LL-37 binds to mucins and that this

Conclusions

Together these data show that cationic AMPs interact with anionic mucins in airway secretions and that their functions are modulated by this interaction. The decrease in antimicrobial activity is likely relevant for human diseases.

Acknowledgements

This study was supported by grants of the Deutsche Forschungsgemeinschaft (Ba 1641/5-1, 6-1), the German Ministry for Education and Sciences via CAPNETZ (01KI0432), and the Cystic Fibrosis Foundation (BALS03G0) to Robert Bals and Deutsche Forschungsgemeinschaft (SFB 630 Agents against Infectious Disease) and the Fonds der Chemischen Industrie (fellowship of Tanja Gulder and supplies).

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