Elsevier

Colloids and Surfaces B: Biointerfaces

Volume 109, 1 September 2013, Pages 129-135
Colloids and Surfaces B: Biointerfaces

Surface activity and structures of two fragments of the human antimicrobial LL-37

https://doi.org/10.1016/j.colsurfb.2013.03.030Get rights and content

Highlights

  • Two fragments (LL-32 and LL-20) of the antimicrobial peptide LL-37 have been investigated in bulk and confined at the air/water interface.

  • LL-32 exhibits an increased antimicrobial activity compared to LL-37, while LL-20 is almost not active.

  • Both peptides are unstructured in bulk.

  • LL-32 transforms into an α-helix lying flat at the water surface but LL-20 forms a partly unstructured intermediate.

  • The ability of LL-32 to form an α-helical structure is in good agreement with its high antimicrobial activity.

Abstract

Two fragments of the antimicrobial peptide LL-37 (LL-32 and LL-20) have been characterized in adsorption layers at the air/buffer interface by infrared reflection absorption spectroscopy (IRRAS) and X-ray reflectivity (XR) measurements. As shown in previous work, LL-32 exhibits an increased antimicrobial activity compared to LL-37, while LL-20 is almost not active. It is shown in this work that the peptides differ drastically in their surface activity (equilibrium adsorption pressure) and their secondary structure, when they are adsorbed to the air/buffer interface. As concluded from the CD spectra, both peptides are unstructured in bulk. That means that the adsorption of the peptides to the air/buffer interface is connected to a secondary structure change. While LL-32 transforms into an α-helix lying flat at the buffer surface, with a helix diameter of 17 Å, LL-20 adopts a partly unstructured conformation. The dichroic ratio of LL-20 is reduced and the electron density profile shows the formation of a second layer. The ability of LL-32 to form a complete α-helical structure at the interface is in good agreement with its higher antimicrobial activity.

Introduction

Antimicrobial peptides (AMPs) are part of the immune defense system, and they can be found in every organism (some reviews can be found in [1], [2], [3]). More than thousand AMPs are yet identified and published in databases [4], [5]. Beyond their antibacterial, antifungal and antiviral activities, they are also involved in immunomodulatory activities and inflammatory processes [6], [7] and some are even active against cancer cells [8], [9]. This brings them into the focus of interest as an alternative to traditional antibiotics and even anti-cancer agents. AMPs are amphipathic, many of them are cationic, and a large percentage can adopt an alpha-helical conformation at the membrane [10]. But their sequences and their activities differ markedly [4]. Several modes of interaction between AMPs and the membrane are proposed, including a ‘carpet model’, where the peptides interact primarily with the lipid head groups, or the formation of ‘barrel-stave’ or ‘toroidal pores’, where the peptides penetrate the lipid bilayer [4], [7]. What they have in common is the fact that the cell membrane is their target. Due to the different composition of eukaryotic compared to vertebrate host cell membranes, most of the AMPs can distinguish between the target membranes [11]. One class of AMPs is the class of cathelicidins. They contain a highly conserved N-terminal domain called cathelin and a C-terminal domain that comprises an antimicrobial peptide [12]. In humans, only one peptide from the cathelicidin class is found, namely LL-37. It is cytotoxic against Gram-positive and Gram-negative bacteria [13], [14] as well as against tumor cells [15], but it is also hemolytic [13], [16]. It can inhibit the growth [17] and the adhesion of Candida albicans to cell surfaces [18]. LL-37 is released from its precursor hCAP18 by proteases and stored in the intracellular granules of neutrophilic granulocytes. It is more than an AMP, since it is involved in signaling, wound healing and up-regulated in inflammations. Furthermore, LL-37 can bind and neutralize lipopolysaccharides (LPS) from Gram-negative bacteria (for a review see [19]). LL-37 adopts mainly α-helical conformation when in contact with a membrane-mimetic environment. As resolved by NMR, a bend between Gly-14 and Glu-15 was found [20], but also a kink at Lys12 was proposed, which breaks the structure into two helices with an angle of 120° [21]. The antimicrobial core was identified between the residues 17–29 [22]. The residues 1–13 are involved in the hemolytic, but not in the antimicrobial activity of the peptide [16], [17]. The residues 32–37 are unstructured [20] and play a role only in the proteolytic resistance and the hemolytic activity [16]. The fragment LL-377–27 exhibits a good activity against microbes, but not against erythrocytes [23]. An orientation of the helix parallel to the membrane [24], [25] and a disruption of the membrane in a detergent-like carpet-mechanism was proposed for LL-37 [16], but even a formation of transmembrane pores has been reported [26], [27]. The mechanism of action was also explained by the fluctuations of the membrane due to the formation of voltage-dependent transient lesions [12].

Nevertheless, for the interaction of the peptide with a cell, the membrane surface plays the role of a target [28]. It can be regarded as an interface between a hydrophilic (membrane surface) and a hydrophobic (membrane core) environment. The hydrophobic effect is one of the main motifs inducing an ordered structure in a peptide [28], so the air/buffer interface is the simplest model to investigate the surface activity of peptides. Furthermore, it allows the investigation of the secondary structure induced by a hydrophilic/hydrophobic interface, and was successfully applied in the characterization of peptides [29], [30], [31], [32], [33]. The adsorption of peptides to the air/buffer interface reveals insight into the influence of hydrophobic interactions and a confinement on the peptide structure, while experiments with lipid monolayers will examine the influence of lipid charges and packing parameters. Moreover, because of the presence of LL-37 in the lung, on the skin, in sweat, and wound fluids (for review see [34]), the air/buffer interface receives even a biological relevance.

In our study, two fragments of LL-37, named LL-20 and LL-32, have been used. Both fragments lack the unstructured C-terminal part of the peptide. The antibacterial activity of LL-32 is increased compared to LL-37 [12], [14]. On the contrary, LL-20 exhibits a reduced antibacterial activity [35] compared to LL-37, probably because it lacks the assumed antimicrobial active core LL-3718–29 [20]. LL-32 carries a net charge of +6 (like LL-37), while LL-20 carries a smaller net charge of +4. In Fig. 1, the helical wheel projections of the two peptides are shown. The formation of an α-helix leads to the amphipathic structure of the peptide. It was already shown, that not only the antibacterial activity, but also the mode of action can be different for different fragments of LL-37 [36]. It is not clear, which parameters influence the antibacterial activity of LL-37 and its derivatives. On the one hand, it was considered for LL-37 that the interaction with the membrane is not mandatory based on electrostatics [37] and that the helical content correlates with the antibacterial activity [38]. On the other hand, no structure parameters could be defined, that explain the strong interaction of LL-32 with the membrane [12]. Our goal was to find decisive differences between the two peptide fragments. Therefore, we compare the structure of the peptides in aqueous solution with the structure formed at the air/liquid interface. The secondary structure elements in bulk were measured by circular dichroism (CD) experiments, since the optical activity of peptides depends on the arrangements of peptide bonds in different structures (α-helices, β-sheets etc.) [39]. When the peptide is adsorbed to the air/buffer interface, surface sensitive infrared reflection absorption spectroscopy (IRRAS) can obtain the secondary structure of the peptide at the buffer surface [40]. Additionally, X-ray reflectivity (XR) experiments were performed to obtain more subtle details of the peptide adsorption film [41].

Section snippets

Peptides

The two fragments of LL-37, namely LL-20 and LL-32, were synthesized with C-terminal amidation by the solid-phase peptide synthesis technique on an automatic peptide synthesizer (model 433 A; Applied Biosystems) on Rink amide resin according to the fastmoc synthesis protocol of the manufacturer, including the removal of the N-terminal Fmoc-group. The peptide was cleaved from the resin and deprotected and with 90% trifluoroacetic acid (TFA), 5% anisole, 2% thioanisole, 3% dithiothreitol for 3 h

Peptides in bulk

To determine the secondary structure of the peptides in bulk, CD experiments were performed (Fig. 2). Due to the strong absorption of HEPES and NaCl, the CD experiments were carried out in pure water. Both peptides exhibit a broad minimum at 198 nm and a tiny minimum at 230 nm. The spectra are typical for an unstructured conformation [45] and comparable to those of LL-37 [38]. Using the AGADIR algorithm [42], a theoretical helix content was calculated which amounts to 0.9% for LL-32 and to 0.3%

Conclusion

Two fragments (LL-32 and LL-20) of the antimicrobial peptide LL-37 were characterized in bulk and adsorbed at the air/buffer interface by CD and IRRA spectroscopy, respectively. XR was used to determine the electron density profiles perpendicular to the buffer surface. LL-32 and LL-20 are in water in an unstructured conformation. Theoretical calculations using AGADIR showed that the increase of the ionic strength does not change the secondary structure in bulk, but increases drastically the

Acknowledgements

We thank Rainer Bartels for peptide synthesis and HASYLAB at DESY, Hamburg, for beamtime and support. The financial support from the Deutsche Forschungsgemeinschaft (BR 1378-11-1) is gratefully acknowledged.

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