키조개 Atrina pectinata로부터 분리한 항균 펩타이드 및 그 유도체의 작용기작

Abstract

Mechanism of Antimicrobial Peptide and Its Analogs Derived from the Comb Pen Shell, Atrina pectinata

Sung-Youl Hong

Department of Biotechnology, Graduate School. Pukyong National University

Abstract

A 5.6 kDa antimicrobial peptide was purified from the acidified gill extract of the comb pen shell, Atrina pectinata, by cation-exchange and C18 reversed-phase high performance liquid chromatography. Comparison of the amino acid sequences and molecular weight of this peptide with those of other known antimicrobial polypeptides revealed that this antimicrobial peptide has high sequence homology with that of cgMolluscidin and was designated as apMolluscidin. apMolluscidin containing several dibasic residue repeats like Lys-Lys or diamino acid repeats like Lys-Gly. apMolluscidin showed potent antimicrobial activity against both Gram-positive bacteria (minimal effective concentrations [MECs]; 0.8-19.0 μg/ml) and Gram-negative bacteria (MECs; 1.0-4.0 μg/ml) without hemolytic activity. However, apMolluscidin did not show any significant activity against Candida albicans. The secondary structure prediction suggested that apMolluscidin might form an ordered amphipathic structure containing two α-helical regions. apMolluscidin did not show membrane permeabilization or leakage abilities. The full-length apMolluscidin cDNA contained 812-bp, including a 5’-untranslated region (UTR) of 82-bp, a 3’-UTR of 547-bp, and an open reading frame of 183-bp encoding 60 amino acids containing Met. The mRNA study of apMolluscidin suggests that it is expressed as a mature form in gills mainly. Two novel antimicrobial peptide analogs consisting of 13 amino acid residues in amphipathic α-helical structure were designed based on the central region of the parental sequence of apMolluscidin. To develop the lead peptides for antimicrobial applications, the net charge and the pI values were increased by substituting amino acids and amidation at the C-terminus. The experimental results showed that ap1 analog exhibited significant antimicrobial activity against all of the bacteria and Candida albicans tested, with some degree of hemolytic activity. In addition, bactericidal test, membrane permeabilization assay, DNA binding study, and chromogenic bacteriolytic plate assay indicated that ap1 acts directly on the bacterial membrane and killed bacteria with a bactericidal procedure via membrane permeabilizing function. AP1 analog also acted directly on the intracellular components such as DNA for its antimicrobial function. These results suggest AP1 has potential for the development of novel antimicrobial agents.