贻贝抗菌肽的分子设计、结构与功能研究
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摘要
抗菌肽是生物体在抵抗病原微生物的过程中自身产生的一类的肽类小分子,具有高效杀菌和不容易产生耐受性等优势,在新型生物抗生素的开发中具有广阔前景,海洋抗菌肽研究是当前热点之一。
为研究贻贝抗菌肽结构与功能的关系,同时对贻贝抗菌肽的人工设计与改造提供新的思路,本文以抗菌肽Mytilin-1(从厚壳贻贝血清中提取到)为研究对象,对其进行了空间结构模拟。为验证Mytilin-1的活性位点,我们对形成连接两段α-折叠的β-发夹结构所包含的肽段进行了截取和重新设计,并采用固相化学合成法,合成了两条新的序列相反的十肽:MDP-1和MDP-2。
实验表明,MDP-1和MDP-2均具有广谱抗菌活性,对革兰氏阳性菌、革兰氏阴性菌以及真菌均具有杀灭活性, MDP-2对于部分海洋弧菌类以及人类常见致病菌也有明显的的抑制作用。MDP-1和MDP-2均表现出很强的热稳定性,而且MDP-2在人血浆中经37℃孵育24hr后其抗菌活性未见明显下降。特殊的是,MDP-2的抗菌活性要比MDP-1高4到20倍。
为进一步了解MDP-1和MDP-2的空间结构和功能的关系,我们利用核磁共振技术NMR对其空间结构进行了解析,结果表明,MDP-1和MDP-2的空间结构与Mytilin-1的结构相比,其loop区域并未采取规则的β-发夹结构,而是采取了一种不规则的环结构(Loop)。MDP-1和MDP-2的氨基酸序列相反,其主链走向也有所差别,主要表现在loop的走向以及两条臂的走向有所不同;而氨基酸侧链,特别是MDP-1和MDP-2在两个Arg侧链的分布上存在明显不同,MDP-1的两个Arg侧链分布于loop的两侧,而MDP-2的两个Arg侧链位于loop的同一侧。这种分布特征有助于MDP-2正电荷的集中分布。鉴于此前的研究中,位于该loop上的两个Arg被认为是与其抗菌活性有直接联系,因此,在MDP-1和MDP-2中这两个Arg的分布的差异有可能是造成MDP-1和MDP-2的抗菌活性差异的原因。
研究结果为深入了解Mytilin的抗菌机制以及在此基础上开发具有应用价值的抗菌肽奠定了基础。
Antibacterial peptide is a class of small molecular peptides produced by the organism when it’s resisting pathogenic microbes. It has the advantages of high efficient sterilization and low risk of drug resistance, so it has the broad prospect in developing new organism antibiotics. Now the research on marine antibacterial peptides becomes one of the hot topics.
We studied the Mytilin-1 derived from serum of Mytilus coruscus and simulated its spatial structure for studying the relationship between spatial structure and antimicrobial activity and providing new ideas of manual designation and modification of Mytilin. For verification of the functional fragment of Mytilin-1, we synthesized two decapeptides corresponding to the main mimic structures connected theα-fold andβ-hairpin of mytilin-1 from Mytilus coruscus and evaluated their activities. The two decapeptides, which named MDP-1(Mytilin derived peptide-1) and MDP-2, were re-designed and synthesized by solid-phase peptide synthesis methods.
The antibacterial test of MDP-1 and MDP-2 shows that MDP-2 has stronger activity than MDP-1 despite the fact that both peptides can inhibit the growth of Gram-positive, Gram-negative bacteria and fungi. MDP-2 possesses the obvious inhibition to marine vibrio and pathogen. MDP-1 and MDP-2 have strong thermal stability, and their antibacterial activities didn’t drop obviously after culturing in the human plasm for 24h, 37℃.
For better understanding the relationship between spatial structure and function of MDP-1 and MDP-2, we analysis their spatial structure by 1H NMR。The result indicates that compared with structure of Mytilin-1, MDP-1 and MDP-2 have no regular structures ofβ-hairpin in loop area, and they have irregular loop structures instead. The amino acid sequences of MDP-1 and MDP-2 are opposite, which makes the trends of main chains are different. Meanwhile, the position of residues in the side chains are different, especially the position of Arg. The two Args of MDP-1 distribute on opposite side of the loop, but the two Args of MDP-2 distribute on same side, which help to make the positive charge more concentrate. According to the previous research, the two Arg are considered the functional part of antimicrobial activity. So the distribution difference of the two Arg may determine the intensity of antimicrobial activity.
The results lay a foundation for further research on the antibacterial mechanism of Mytilin and for further exploitation of antibacterial peptides with lower molecular mass and more stable structure.
为研究贻贝抗菌肽结构与功能的关系,同时对贻贝抗菌肽的人工设计与改造提供新的思路,本文以抗菌肽Mytilin-1(从厚壳贻贝血清中提取到)为研究对象,对其进行了空间结构模拟。为验证Mytilin-1的活性位点,我们对形成连接两段α-折叠的β-发夹结构所包含的肽段进行了截取和重新设计,并采用固相化学合成法,合成了两条新的序列相反的十肽:MDP-1和MDP-2。
实验表明,MDP-1和MDP-2均具有广谱抗菌活性,对革兰氏阳性菌、革兰氏阴性菌以及真菌均具有杀灭活性, MDP-2对于部分海洋弧菌类以及人类常见致病菌也有明显的的抑制作用。MDP-1和MDP-2均表现出很强的热稳定性,而且MDP-2在人血浆中经37℃孵育24hr后其抗菌活性未见明显下降。特殊的是,MDP-2的抗菌活性要比MDP-1高4到20倍。
为进一步了解MDP-1和MDP-2的空间结构和功能的关系,我们利用核磁共振技术NMR对其空间结构进行了解析,结果表明,MDP-1和MDP-2的空间结构与Mytilin-1的结构相比,其loop区域并未采取规则的β-发夹结构,而是采取了一种不规则的环结构(Loop)。MDP-1和MDP-2的氨基酸序列相反,其主链走向也有所差别,主要表现在loop的走向以及两条臂的走向有所不同;而氨基酸侧链,特别是MDP-1和MDP-2在两个Arg侧链的分布上存在明显不同,MDP-1的两个Arg侧链分布于loop的两侧,而MDP-2的两个Arg侧链位于loop的同一侧。这种分布特征有助于MDP-2正电荷的集中分布。鉴于此前的研究中,位于该loop上的两个Arg被认为是与其抗菌活性有直接联系,因此,在MDP-1和MDP-2中这两个Arg的分布的差异有可能是造成MDP-1和MDP-2的抗菌活性差异的原因。
研究结果为深入了解Mytilin的抗菌机制以及在此基础上开发具有应用价值的抗菌肽奠定了基础。
Antibacterial peptide is a class of small molecular peptides produced by the organism when it’s resisting pathogenic microbes. It has the advantages of high efficient sterilization and low risk of drug resistance, so it has the broad prospect in developing new organism antibiotics. Now the research on marine antibacterial peptides becomes one of the hot topics.
We studied the Mytilin-1 derived from serum of Mytilus coruscus and simulated its spatial structure for studying the relationship between spatial structure and antimicrobial activity and providing new ideas of manual designation and modification of Mytilin. For verification of the functional fragment of Mytilin-1, we synthesized two decapeptides corresponding to the main mimic structures connected theα-fold andβ-hairpin of mytilin-1 from Mytilus coruscus and evaluated their activities. The two decapeptides, which named MDP-1(Mytilin derived peptide-1) and MDP-2, were re-designed and synthesized by solid-phase peptide synthesis methods.
The antibacterial test of MDP-1 and MDP-2 shows that MDP-2 has stronger activity than MDP-1 despite the fact that both peptides can inhibit the growth of Gram-positive, Gram-negative bacteria and fungi. MDP-2 possesses the obvious inhibition to marine vibrio and pathogen. MDP-1 and MDP-2 have strong thermal stability, and their antibacterial activities didn’t drop obviously after culturing in the human plasm for 24h, 37℃.
For better understanding the relationship between spatial structure and function of MDP-1 and MDP-2, we analysis their spatial structure by 1H NMR。The result indicates that compared with structure of Mytilin-1, MDP-1 and MDP-2 have no regular structures ofβ-hairpin in loop area, and they have irregular loop structures instead. The amino acid sequences of MDP-1 and MDP-2 are opposite, which makes the trends of main chains are different. Meanwhile, the position of residues in the side chains are different, especially the position of Arg. The two Args of MDP-1 distribute on opposite side of the loop, but the two Args of MDP-2 distribute on same side, which help to make the positive charge more concentrate. According to the previous research, the two Arg are considered the functional part of antimicrobial activity. So the distribution difference of the two Arg may determine the intensity of antimicrobial activity.
The results lay a foundation for further research on the antibacterial mechanism of Mytilin and for further exploitation of antibacterial peptides with lower molecular mass and more stable structure.
引文
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[2] Hancock R. E. W.; Chapple, D. S. Peptide Antibiotics[J]. Antimicrobial Agents and Chemotherapy 1999, 43(6):7.
[3]陈建国;李国明.海洋生物抑菌物质的研究进展[J].国际检验医学杂志, 2006, 27(9):3.
[4]洪旭光;孙修勤;张进兴.海洋抗菌肽研究进展[J].高技术通讯, 2004, 14(2).
[5] Silphaduang U.; Noga, E. Antimicrobials peptide antibiotics in mast cells of fish[J]. Nature, 2001, 414:2.
[6]马海萍;梅雪婷;许实波.国外海洋抗菌活性物质的研究进展[J].中国海洋药物杂志, 2006, 25(3):4.
[7] Oren Z.; Shai, Y. A class of highly potent antibacterial peptides derived from pardaxin, a poreforming pep tide isolated from Moses sole fish[J]. European Journal of BioChemistry, 1996, 237:8.
[8] Park I.; Park, C.; Kim, M., et al. Parasin I,an antimicrobial peptide derived derived from histone H2A in the catfish,Parasilurus asotus[J]. FEBS Letters, 1998, 437(3):5.
[9] Lauth X.; Shike, H.; Burns, J., et al. Discovery and characterization of two isoforms of Moronecidin, a novel antimicrobial peptide from Hybrid Striped Bass[J]. The Journal of Biological Chemistry, 2002, 277:10.
[10] Nakamura T.; Furunaka, H.; Miyata, T., et al. Tachyplesin, a class of antimicrobial peptide from the hemocytes of the horseshoe crab (Tachypleus tridentatus). Isolation and chemical structure[J]. The Journal of Biological Chemistry, 1988, 263(32):5.
[11] Saito T.; Kawabata, S.; Shigenaga, T., et al. Innate immunity. Isolation of several cysteine-rich antimicrobial peptides from the blood of a mollusk[J]. The Journal of Biological Chemistry, 1995, 117(5).
[12] Schnapp D.; Kemp, G.; Smith, V., et al. Purification and characterization of a proline-rich antibacterial peptide, with sequence similarity to Bactenecin-7, from thehaemocytes of the shore crab, Carcinus Maenas[J]. European Journal of BioChemistry, 2004, 240(3):532.
[13] Destoumieux D.; Bulet, P.; Loew, D., et al. Penaeidins, a new family of antimicrobial peptides isolated from the shrimp Penaeu Vannamei (Decapoda)[J]. The Journal of Biological Chemistry, 1997, 272(45):9.
[14]洪旭光.皱纹盘鲍和栉孔扇贝抗菌肽的研究.中国海洋大学, 2008.
[15]送宏霞.紫贻贝_Mytilusedulis_抗菌肽的研究.中国海洋大学, 2007.
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