Antimicrobial Peptide Arenicin-1 Derivative Ar-1-(C/A) as Complement System Modulator

Abstract

Antimicrobial peptides (AMPs) are not only cytotoxic towards host pathogens or cancer cells but also are able to act as immunomodulators. It was shown that some human and non-human AMPs can interact with complement proteins and thereby modulate complement activity. Thus, AMPs could be considered as the base for complement-targeted therapeutics development. Arenicins from the sea polychaete Arenicola marina, the classical example of peptides with a β-hairpin structure stabilized by a disulfide bond, were shown earlier to be among the most prospective regulators. Here, we investigate the link between arenicins' structure and their antimicrobial, hemolytic and complement-modulating activities using the derivative Ar-1-(C/A) without a disulfide bond. Despite the absence of this bond, the peptide retains all important functional activities and also appears less hemolytic in comparison with the natural forms. These findings could help to investigate new complement drugs for regulation using arenicin derivatives.

Keywords: antimicrobial peptide; arenicin; complement regulation; complement system.

Figure 1

Fragment of the mass spectrum of Ar-1(C/A) after deconvolution for neutral molecule. The calculated monoisotopic mass of the peptide is 2694.525, the experimentally determined m/z value is 2694.519.

Figure 2

Circular dichroism spectra of Ar-1-(C/A) variant, Ar-1 and Ar-2 in (A) aqueous solution and in (B) complex with anionic SDS micelles (P:D = 1:350). Experimental data points are shown with symbols and fitted curves calculated with BeStSel are pictured as lines. Data for Ar-1 are taken from Panteleev et al. [23] and data for Ar-2 are from Ovchinnikova et al. [24].

Figure 3

Results of radial diffusion antimicrobial assay against (A,C) L. monocytogenes EGD, (B,D) E. coli ML-35p. Ar-1, Ar-2 and Ar-1-(C/A) were added at concentrations 64, 32, 16, 8 and 4 μM. As a negative control (w/o peptide), deionized water was used.

Figure 4

The action of arenicins on complement activation, expressed in H and E coefficients. Data are represented as mean ± standard deviation (n = 5). * p < 0.05; ** < 0.01; # p < 0.001 (H- and E-values vs. zero). (A) Alterations in lysis of antibody-sensitized sheep erythrocytes (E^sh) (CP model); (B) alterations in lysis of rabbit erythrocytes (E^rab) (AP model); (C) C3a accumulation in the model with E^sh; (D) C3a accumulation in the model with E^rab.

Figure 5

Correlation between H- and E-indexes. These coefficients were used for evaluation of the hemolytic activity of complement (H) and of complement-dependent C3a accumulation (E). (A) Correlation between the indexes in the E^sh CP model. Pearson correlation coefficient values were calculated as 0.92 for Ar-1, 0.96 for Ar-1-(C/A) and 0.99 for Ar-2. (B) Correlation between the indexes in the E^rab AP model. Pearson correlation coefficient values were calculated as 0.93 for Ar-1, 0.68 for Ar-1-(C/A) and 0.89 for Ar-2.

Figure 6

The primary structure of arenicins. Residues in red differ Ar-2 and Ar-1-(C/A) from Ar-1. Underlined residues in blue form a heparin-binding motif.