Zoological Research ›› 2019, Vol. 40 ›› Issue (3): 198-204.doi: 10.24272/j.issn.2095-8137.2018.070

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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

Fen Zhang1,#, Zhi-Lai Guo3,#, Yan Chen1, Li Li3, Hai-Ning Yu2,*, Yi-Peng Wang1,*   

  1. 1 Department of Pharmaceutical Sciences, College of Pharmaceutical Sciences, Soochow University, Suzhou Jiangsu 215123, China
    2. Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian Liaoning 116023, China
    3. School of Life Sciences, Guizhou Normal University, Guiyang Guizhou 550001, China
  • Received:2018-06-20 Accepted:2018-07-30 Online:2019-05-18 Published:2019-04-19
  • Contact: Hai-Ning Yu, Yi-Peng Wang E-mail:yipengwang@suda.edu.cn; yuhaining@dlut.edu.cn
  • Supported by:
    This work was supported by grants from the National Natural Science Foundation of China (31772455), Natural Science Foundation of Jiangsu Province (BK20160336 and BK20171214), Natural Science Foundation of College in Jiangsu Province (16KJB350004), Suzhou Science and Technology Development Project (SYN201504 and SNG2017045)

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 a-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.

Key words: , Antimicrobial peptides, Rana kunyuensis, Amurin-9KY, Heptapeptide ring, C-terminal amidation, Structure activity relationship

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