pleurocidin and Hemolysis

Pleurocidin (Ple) is a peptide derived from the winter flounder. In our previous study, we reported the antifungal effect of Ple and its mode of action. To develop novel antifungal peptides useful as therapeutic agents, two analogs, with amino acid substitutions, were designed to decrease the net hydrophobicity by Arg (R) or Ser (S)-substitution at the hydrophobic face of Ple without changing the amphipathic structure. By substituting Ser, the hydrophobicity of the peptide (anal-S) was decreased, and by substituting Arg, though the hydrophobicity of the peptide (anal-R) was decreased, the cationicity of this peptide was increased. CD measurements showed the substitution of Arg or Ser decrease the alpha-helical conformation of analog peptides. Studies with analog peptides have shown decreases in hydrophobicity and alpha-helicity do not affect antifungal activity but decrease hemolytic activity. These results suggest that highly hydrophobic and alpha-helical natures are not desirable in the design of antimicrobial peptides.

Topics: Amino Acid Sequence; Amino Acid Substitution; Antifungal Agents; Antimicrobial Cationic Peptides; Arginine; Cell Membrane; Cells, Cultured; Erythrocytes; Fish Proteins; Fungi; Hemolysis; Humans; Liposomes; Melitten; Molecular Sequence Data; Peptides; Permeability; Protein Structure, Secondary; Serine

Pleurocidin (Ple), a 25-residue alpha-helical antimicrobial peptide, isolated from skin mucosa of the winter flounder, shows potent bacterial cell selectivity. In this study, the effect of two glycine residues in positions 13 and 17 of Ple on structure and bacterial cell selectivity was investigated by Gly-->Ala substitution. Ala-substitution (Gly(13, 17)-->Ala, Gly13-->Ala and Gly17-->Ala) in positions 13 and 17 of Ple did not induce a significant change in antibacterial activity, but increased hemolytic activity. Both Gly(13, 17)-->Ala and Gly17-->Ala substitution did not cause a remarkable change in alpha-helical content in SDS micelles, while Gly(13, 17)-->Ala substitution caused a drastic increase in alpha-helical content. These results suggest that the hinge region from Gly13 to Gly17 of Ple is assumed to provide its conformational flexibility and bacterial cell selectivity.

Topics: Amino Acid Sequence; Amino Acid Substitution; Anti-Bacterial Agents; Bacteria; Circular Dichroism; Erythrocytes; Fish Proteins; Glycine; Hemolysis; Humans; Molecular Sequence Data; Proteins; Structure-Activity Relationship

A 25-mer cationic peptide pleurocidin, isolated from the winter flounder, has broad antibacterial activity. To clarify the structure-activity relationship, its properties and biological activity were examined. CD measurements showed that pleurocidin took an alpha-helical structure in the presence of DOPC/DOPG (3:1, anionic) vesicles. Very weak hemolytic activity of pleurocidin was observed and its antibacterial activity was moderate. Tryptophan fluorescence shift measurements showed that pleurocidin interacted weakly with a neutral phospholipid, but strongly with an acidic phospholipid. The peptide exhibited weak dye-leakage activity for DOPC (neutral) vesicles and moderate activity for acidic vesicles. From experiments on dye-leakage activity and membrane translocation of the peptide, it seemed likely that pleurocidin, like magainin 2, forms pores in the lipid membrane. A study of amino acid substitution in pleurocidin revealed that alpha-helicity, rather than hydrophobicity, affects the properties and activity of the peptide.

Topics: Animals; Anti-Bacterial Agents; Circular Dichroism; Fish Proteins; Gram-Negative Bacteria; Gram-Positive Bacteria; Hemolysis; Humans; Membranes, Artificial; Microbial Sensitivity Tests; Peptides; Phospholipids; Protein Transport; Proteins; Rabbits; Spectrometry, Fluorescence; Tryptophan