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张芬, 郭志来, 陈燕, 李莉, 于海宁, 王义鹏. 2019: C-末端酰胺化和七肽环对新型崑嵛林蛙(Rana kunyuensis)抗菌肽amurin-9KY生物学活性和高级结构的影响. 动物学研究, 40(3): 198-204. DOI: 10.24272/j.issn.2095-8137.2018.070
引用本文: 张芬, 郭志来, 陈燕, 李莉, 于海宁, 王义鹏. 2019: C-末端酰胺化和七肽环对新型崑嵛林蛙(Rana kunyuensis)抗菌肽amurin-9KY生物学活性和高级结构的影响. 动物学研究, 40(3): 198-204. DOI: 10.24272/j.issn.2095-8137.2018.070
Fen Zhang, Zhi-Lai Guo, Yan Chen, Li Li, Hai-Ning Yu, Yi-Peng Wang. 2019: 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. Zoological Research, 40(3): 198-204. DOI: 10.24272/j.issn.2095-8137.2018.070
Citation: Fen Zhang, Zhi-Lai Guo, Yan Chen, Li Li, Hai-Ning Yu, Yi-Peng Wang. 2019: 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. Zoological Research, 40(3): 198-204. DOI: 10.24272/j.issn.2095-8137.2018.070

C-末端酰胺化和七肽环对新型崑嵛林蛙(Rana kunyuensis)抗菌肽amurin-9KY生物学活性和高级结构的影响

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

  • 摘要: 崑嵛林蛙(Rana kunyuensis)作为林蛙属的一种,仅自然生活于中国山东省烟台市昆嵛山境内。在本研究中,从合成的崑嵛林蛙皮肤cDNA文库中克隆获得一条279 bp的cDNA序列,该序列编码一种新型抗菌肽分子,命名为amurin-9KY。Amurin-9KY前体由62个氨基酸残基组成,其中成熟肽由14个氨基酸残基组成,包括由2个半胱氨酸残基形成的分子内七肽环和酰胺化的C-末端。这些结构特点表明amurin-9KY是一种新型两栖类抗菌肽家族成员之一。尽管amurin-9KY和之前从黑龙江林蛙(Rana amurensis)中发现的amurin-9AM具有较高的相似性,但是到目前为止对于amurin-9家族抗菌肽的结构和功能仍然未知。在本论文中,我们设计合成了amurin-9KY和3个改造体多肽amurin-9KY1-3,研究了C-末端酰胺化和七肽环对amurin-9KY结构和功能的影响。结果表明C-末端酰胺化对amurin-9KY的抗菌活性十分重要,而C-末端酰胺化和七肽环对amurin-9KY的溶血活性同时具有影响。圆二色谱(Circular dichroism, CD)结果表明上述4种多肽在TFE/H2O (v/v 1:1)溶液中均为α-螺旋型结构,而在水溶液中为无规则结构。打断七肽环的二硫键使2个半胱氨酸的巯基游离能够显著增加amurin-9KY的抗氧化活性。扫描电子显微镜(Scanning electron microscopy, SEM)同时也被用于研究amurin-9KY的抗菌机理。

     

    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.

     

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