Biochemical and Biophysical Research Communications
Antifungal effect of CopA3 monomer peptide via membrane-active mechanism and stability to proteolysis of enantiomeric d-CopA3
Introduction
Infectious disease resulting in increased morbidity, mortality, and health-care costs is becoming a serious problem, mainly due to the emergence of antibiotic-resistant pathogens [1]. This clinical problem needs promising candidates for new antibiotics. Antimicrobial peptides produced by every living organism as a component of innate immunity, have received attention because they have many useful biological properties, including broad-spectrum antimicrobial activity, fast action and slow resistance development [2], [3]. Although the mechanism of action of antimicrobial peptides has not been shown clearly, it is believed that most antimicrobial peptides kill microbes by membrane permeabilization [4]. The membrane-permeabilizing action significantly hinders the development of resistance to antimicrobial peptides because it is difficult for a microbe to change the lipid composition of its membrane and this is in marked contrast to conventional antibiotics, which target specific molecules such as receptors and enzymes [5].
However, the use of antimicrobial peptides in vivo is mainly limited due to the loss of their activity in body fluids because of enzymatic degradation in the presence of proteases [6]. To overcome this limitation, d-amino acid enantiomers are expected to be resistant to proteolytic cleavage and several studies have reported. Synthesized d-enantiomers of naturally occurring membrane-active peptides such as cecropin A, magainin 2 and melittin exhibited antimicrobial activity similar to that of their natural form and were not sensitive to enzymatic degradation [7], [8], [9]. These results suggested that d-amino acid peptides would be very attractive candidates as a therapeutic agent.
Recently, we isolated coprisin (VTCDVLSFEAKGIAVNHSACALHCIALRKKGGSCQNGVCVCRN-NH2), a natural peptide consisting of 43-amino acids, from Dung beetle Copris tripartitus after it had been infected with pathogenic bacteria, and showed that coprisin exhibited antifungal activities by apoptotic mechanisms [10], [11]. In addition, we made a synthetic 9-mer analog peptide CopA3 (LLCIALRKK-NH2) by replacing the histidine residues of CopN5 (LHCIALRKK-NH2), which is the α-helical region in the natural peptide coprisin, with leucine to increase the hydrophobicity [10], [12]. The monomer and disulfide dimer of 9-mer CopA3 had antibacterial effects, and the antibiotic activity of CopA3 was higher than that of CopN5 [10], [13]. However, the antifungal activity and the mechanism of the CopA3 monomer are not known yet.
Therefore, in this study, the antifungal activity and the mechanism of the CopA3 monomer were investigated. In addition, enantiomeric d-CopA3 was synthesized, and its enzymatic degradation and antifungal activity in the presence of trypsin were investigated.
Section snippets
Solid-phase peptide synthesis
The peptide synthesis was done by Anygen Co. (Gwangju, Korea). Anygen Co. offers the following procedures for peptide synthesis. The assembly of the peptides was achieved with a 60 min cycle for each residue at ambient temperature using the following method: (1) 2-chlorotrityl (or 4-methylbenzhydrylamine amide) resin was charged to a reactor and then washed with DCM and DMF, respectively, and (2) a coupling step with vigorous shaking using a 0.14 mM solution of Fmoc-l-amino acids and Fmoc-l-amino
Synthesis of l- and d-CopA3
We focused on the short 9-mer CopA3 which is a functionally improved peptide over the 9-mer CopN5 representing the α-helical region of the natural peptide 43-mer coprisin. According to our previous study, CopA3 has not only cationicity but also an α-helical structure [10], [12]. In addition, a short peptide such as CopA3 has been specifically considered as a model for therapeutic agents because it can be produced at low cost and absorbed after oral administration without difficulty [24], [25].
Acknowledgment
This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ008158), Rural Development Administration, Republic of Korea.
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These authors contributed equally to this work and should be considered co-first authors.