Structure-activity studies on parasin I, a histone H2A-Derived Antimicrobial Peptide

Abstract

In response to epidermal injury, $\It{Parasilurus asotus}$, a catfish, secreted a strong antimicrobial peptide into the epithelial mucosal layer. The molecular mass of the antimicrobial peptide, named parasin I, was 2000.4 Da as determined by matrix associated laser desorption ionization mass spectrometry. The complete amino acid sequence of parasin I, which was determined by automated Edman degradation, was Lys-Gly-Arg-Gly -Lys-Gln-Gly-Gly-Lys-Val-Arg-Ala-Lys-Ala-Lys-Thr-Arg-Ser-Ser. Seventeen of the 19 residues in parasin I were identical to the N-terminal of histone H2A which implies that parasin I was cleaved off from the N-terminal of catfish histone H2A. Parasin I showed strong antimicrobial activity against a wide spectrum of microorganisms, without any hemolytic activity. Circular dichroism spectra of parasin I indicated a structural content of 11% $\alpha$-helix, 33% $\beta$ -sheet, and 56% random coils. Parasin I formed an amphipathic $\alpha$-helical structure (residues 9-17) flanked by two random coil regions (residues 1-8 and 18-19) in helix-promoting environments. Deletion of the lysine residue at the N-terminal [Pa(2-19)] resulted in loss of antimicrobial activity, but did not affect the $\alpha$-helical content of the peptide. The antimicrobial activity was recovered when the lysine residue was substituted with another basic residue, arginine $([R^1]Pa)$, but not with polar, neutral, or acidic residues. Progressive deletions from the C-terminal [Pa(1-17), Pa(1-15)] slightly increased the antimicrobial activity $(1-4 \mu g/ml)$ without affecting the a-helical content of the peptide. However, further deletion [Pa(1-14)] resulted in nearly complete loss of antimicrobial activity and a-helical structure. Confocal microscopic analysis and membrane permeabilization assays showed that parasin I and its analogs with comparable antimicrobial activities localized to the cell membrane and subsequently permeabilized the outer and cytoplasmic membranes. [Pa(...