Abstract
Rana kunyuensis is a species of brown frog that lives exclusively on Kunyu Mountain, Yantai, China. In the current study, a 279-bp cDNA sequence encoding a novel antimicrobial peptide (AMP), designated as amurin-9KY, was cloned from synthesized double-strand skin cDNA of R. kunyuensis. The amurin-9KY precursor was composed of 62 amino acid (aa) residues, whereas the mature peptide was composed of 14 aa and contained two cysteines forming a C-terminal heptapeptide ring (Rana box domain) and an amidated C-terminus. These structural characters represent a novel amphibian AMP family. Although amurin-9KY exhibited high similarity to the already identified amurin-9AM from R. amurensis, little is known about the structures and activities of amurin-9 family AMPs so far. Therefore, amurin-9KY and its three derivatives (amurin-9KY1–3) were designed and synthesized. The structures and activities were examined to evaluate the influence of C-terminal amidation and the heptapeptide ring on the activities and structure of amurin-9KY. Results indicated that C-terminal amidation was essential for antimicrobial activity, whereas both C-terminal amidation and the heptapeptide ring played roles in the low hemolytic activity. Circular dichroism (CD) spectra showed that the four peptides adopted an α-helical conformation in THF/H2O (v/v 1:1) solution, but a random coil in aqueous solution. Elimination of the C-terminal heptapeptide ring generated two free cysteine residues with unpaired thiol groups, which greatly increased the concentration-dependent anti-oxidant activity. Scanning electron microscopy (SEM) was also performed to determine the possible bactericidal mechanisms.
Highlights
Antimicrobial peptides (AMPs), which are small, cationic and amphipathic peptides widely distributed throughout organisms, are evolutionarily ancient weapons against environmental pathogens (Zasloff, 2002)
We identified a novel AMP family member, named amurin-9KY, from the skin secretions of R. kunyuensis
Among the AMP families identified from ranid species, the C-terminal heptapeptide ring (Rana box domain, two cysteine residues connected by a disulfide bridge) is a common structural feature, which broadly exists in families such as brevinin-1, brevinin-2, esculentin-1, esculentin-2, ranatuerin-1, ranalexin, japonicin-1, nigrocin-2, odorranain-C, odorranain-D, odorranain-F, odorranain-G, odorranain-H, odorranain-P1 and hainanenin (Conlon, 2004; Duda et al, 2002; He et al, 2012; Li et al, 2007; Matutte et al, 2000)
Summary
Antimicrobial peptides (AMPs), which are small, cationic and amphipathic peptides widely distributed throughout organisms, are evolutionarily ancient weapons against environmental pathogens (Zasloff, 2002). They are important components of innate immune systems and play key roles in the anti-infective immune responses of organisms (Radek & Gallo, 2007). AMPs possess strong and diverse antimicrobial activities against bacteria, fungi, viruses and even. The activities and specificities of AMPs can be affected by various factors such as molecular weight, sequence, charge, conformation, hydrophobicity and amphipathicity (Brogden, 2005). Most AMPs are thought to function by forming pores in the membranes of target microorganisms, leading to disruption of cellular integrity (Nicolas, 2009)
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