Effects of C-terminal amidation and heptapeptide ring on the biological activities and advanced structure of amurin-9KY, a novel antimicrobial peptide identified from the brown frog, Rana kunyuensis

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/H 2 O (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.

Bioinformatic Analysis of 1000 Amphibian Antimicrobial Peptides Uncovers Multiple Length-Dependent Correlations for Peptide Design and Prediction

“…In the APD database, 35% of amphibian AMPs are C-terminally amidated to increase the peptide net charge by +1 (for accuracy, this modification is considered in the APD when calculating the net charge of peptides). C-terminal amidation can be essential for the antimicrobial activity of amphibian peptides [ 133 , 149 ]. A plausible reason is stabilization of the helical structure to allow for the formation of one additional hydrogen bond.…”

“…It is likely that the Rana box is broken in reduced physiological conditions. Then, the free cysteines can enhance anti-oxidant ability of the peptide [ 149 ] or perform other functions yet to be elucidated.…”

Multidrug resistance crisis during COVID-19 pandemic: Role of anti-microbial peptides as next-generation therapeutics

“…Further, they penetrate the cell membrane resulting into bacterial death [137] . In last 5 years, there are many reports that have shown the effectiveness of AMPs against MDR pathogens, while parallel efforts have also been made to synthesize AMPs with enhanced therapeutic potential and minimal side effects [142] , [143] , [144] , [145] , [146] , [147] , [148] , [149] , [150] .…”

Engineering Approaches for the Development of Antimicrobial Peptide-Based Antibiotics

“…Usually, the N-terminal acetylation of AMPs increases the helicity of peptides and prevents enzymatic degradation, and its C-terminal amidation enhances structural stability and antimicrobial activity ( Figure 2 ) [ 37 , 38 , 39 , 40 ]. In a report by Alvares et al, the L1A peptide adopts a more helical conformation when its N-terminus is acetylated [ 37 ], and Li et al designed an L163 analog by amino-terminal acetylation, which exhibited higher stability against trypsin degradation [ 41 ].…”