哈尔滨地区黑斑蛙（Rana nigromaculata）夏季皮肤抗菌肽多样性的研究
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摘要
抗菌肽具有广谱杀菌、抑制病毒、抑制肿瘤细胞等作用,是两栖类动物天然免疫的主要成分。不同的环境中,抗原组成不同,两栖类机体会选择性地表达不同的抗菌肽谱。因而抗原是抗菌肽进化的主要压力。动物体如何根据环境抗原来变化表达谱呢?目前关于这个问题尚无研究可鉴。针对这个问题,我们2009年6月份在哈尔滨市郊采集黑斑蛙(Rana nigromaculata)作为研究对象,一组在野外捕获后立即处死,采集皮肤样品,另一组在实验室内用清水饲养3周后再处死,采集皮肤样品。通过比较两种情况下抗菌肽谱的异同点,尤其是抗菌肽的种类、理化性质、选择模式等,来阐释不同环境下抗菌肽的表达模式。结果显示,黑斑蛙共表达10个家族的22种抗菌肽,由65个基因编码的71种cDNA组成。其中有7个家族的17种抗菌肽(62种cDNA,分属于esculentin-2家族、nigrocin-2家族、odorranoin-S1家族、temporin-1家族、pelophylaxin-2家族、odorranain-P1家族,odorranain-B1家族)是实验室组和野外组所共同表达的,而有2个家族的4种抗菌肽(8种cDNA分属brevinin-1家族和pelphylaxin-3家族)为野外组所特有,1个家族的1种抗菌肽(3种cDNA属于nigromaculatin家族)为实验室组所特有。两组共有的家族和野外组特有的家族中,都属于碱性抗菌肽,除了nigrocin-2家族和odorranoin-S1家族以自由卷曲结构为主以外,其余家族的抗菌肽均形成轴向和断面两个维度上具有两亲性的α-螺旋结构。实验室组特有的抗菌肽nigromaculatin家族属于自由卷曲的酸性抗菌肽。temporin-1家族的cDNA达22种,且总表达量最高,为较强烈的负选择抗菌肽,野外组表达量稍高于实验室组。esculentin-2家族的cDNA有16种,但总表达量不高,亦为负选择或近于中性选择的抗菌肽。pelphylaxin-3家族包含6种不同的cDNA,在野外组总表达量仅次于temporin-1家族,均为正选择抗菌肽。odorranain-B1家族、brevinin-1、nigromaculatin家族的表达量均很低,而且都属于负选择抗菌肽。统计表明,随着与祖先基因的遗传距离的增加,抗菌肽表现出从负选择逐渐过渡到正选择的倾向。上述结果提示,(1)黑斑蛙基因组上至少具有65个基因编码71种抗菌肽。(2)在不同环境中黑斑蛙抗菌肽表达谱中有稳定的组分和环境特异性组分。稳定组分中的抗菌肽基因比较古老而保守,环境特异性组分的抗菌肽基因通常出现较晚,且以正选择模式进化。(3)环境抗原较少时(如实验室组),抗菌肽的表达从种类和丰度上均明显减少,即抗菌肽的表达具有明显的经济性原则。
Antimicrobial peptides (AMPs) are an important component of innate immunity of amphibians due to their functions of broad spectrum of bactericide, viral and tumor resistance. Amphibians use different AMP profile selectively to cope with different pathogenic community in different environment. This indicates an essential role of pathogenic community in the evolution of AMPs and raises a question how amphibians choose AMP profiles in coping with different pathogenic community. There has not been definite answer to this question up to date. We analyzed AMP profiles of black-spotted frog (Rana nigromaculata) in June 2009 in Harbin, China. Adult frogs were captured in the wild and divided into two groups randomly. One group was killed in situ immediately and skin samples were collected and preserved in liquid nitrogen. The other group was transferred into the lab and sampled skins after being raised in clear water for three weeks. Profiles, physical and chemical properties and mode of selection of AMPs were compared between the two groups. Seventy one cDNAs from 65 genes encoding 22 species of AMPs belonging to 10 families were cloned in total. Seven families containing 17 species of AMPs encoded by 62 cDNAs belonging to esculentin-2, nigrocin-2, odorranoin-S1, temporin-1, pelophylaxin-2, odorranain-P1 and odorranain-B1 were shared by the two groups. Two families of 4 species of AMPs encoded by 8 cDNAs belonging to brevinin-1 and pelphylaxin-3 were unique to the wild group. One species of AMP encoded by 3 cDNAs that was identified in ranids for the first time and named nigromaculatin was only seen in the lab group. All shared AMPs were alkaline and cationic peptides with typical a-helix structure except for family nigrocin-2 and odorranoin-S 1 with random coil structure. Family nigromaculatin was acidic and randomly coiled AMP. Family temporin-1 contained 22 cDNAs evolving under negative selection and revealed the highest expression, a little higher in the wild group relative to the lab group. Family esculentin-2 contained 16 cDNAs evolving under negative selection as well or near neutral selection. But their expression was moderate. Family pelphylaxin-3 containing 6 cDNAs under positive selection was the second abundant AMPs in the wild group relative to family temporin-1. Family odorranain-B1, brevinin-1 and nigromaculatin were all under negative selection but with low expression. Analysis also indicated that the selection of AMPs was in a trend of changing from negative selection to positive selection with increment of genetic distance relative to their ancestral genes. These results suggested that (1) the black-spotted frog has at least 65 gene loci encoding AMPs; (2) there are stable components and environmental specific components in AMP profiles when the black-spotted frog is exposed to different environment. The genes of stable AMP components are primordial and conservative. Whereas, the genes of environmental specific AMP components are usually recent and evolving in positive selection; (3) In case of simplification and reduction of pathogenic community such as lab group was, AMP expression may reduce in both AMP species and their abundance, indicating the expression of AMPs follow economic principle.
Antimicrobial peptides (AMPs) are an important component of innate immunity of amphibians due to their functions of broad spectrum of bactericide, viral and tumor resistance. Amphibians use different AMP profile selectively to cope with different pathogenic community in different environment. This indicates an essential role of pathogenic community in the evolution of AMPs and raises a question how amphibians choose AMP profiles in coping with different pathogenic community. There has not been definite answer to this question up to date. We analyzed AMP profiles of black-spotted frog (Rana nigromaculata) in June 2009 in Harbin, China. Adult frogs were captured in the wild and divided into two groups randomly. One group was killed in situ immediately and skin samples were collected and preserved in liquid nitrogen. The other group was transferred into the lab and sampled skins after being raised in clear water for three weeks. Profiles, physical and chemical properties and mode of selection of AMPs were compared between the two groups. Seventy one cDNAs from 65 genes encoding 22 species of AMPs belonging to 10 families were cloned in total. Seven families containing 17 species of AMPs encoded by 62 cDNAs belonging to esculentin-2, nigrocin-2, odorranoin-S1, temporin-1, pelophylaxin-2, odorranain-P1 and odorranain-B1 were shared by the two groups. Two families of 4 species of AMPs encoded by 8 cDNAs belonging to brevinin-1 and pelphylaxin-3 were unique to the wild group. One species of AMP encoded by 3 cDNAs that was identified in ranids for the first time and named nigromaculatin was only seen in the lab group. All shared AMPs were alkaline and cationic peptides with typical a-helix structure except for family nigrocin-2 and odorranoin-S 1 with random coil structure. Family nigromaculatin was acidic and randomly coiled AMP. Family temporin-1 contained 22 cDNAs evolving under negative selection and revealed the highest expression, a little higher in the wild group relative to the lab group. Family esculentin-2 contained 16 cDNAs evolving under negative selection as well or near neutral selection. But their expression was moderate. Family pelphylaxin-3 containing 6 cDNAs under positive selection was the second abundant AMPs in the wild group relative to family temporin-1. Family odorranain-B1, brevinin-1 and nigromaculatin were all under negative selection but with low expression. Analysis also indicated that the selection of AMPs was in a trend of changing from negative selection to positive selection with increment of genetic distance relative to their ancestral genes. These results suggested that (1) the black-spotted frog has at least 65 gene loci encoding AMPs; (2) there are stable components and environmental specific components in AMP profiles when the black-spotted frog is exposed to different environment. The genes of stable AMP components are primordial and conservative. Whereas, the genes of environmental specific AMP components are usually recent and evolving in positive selection; (3) In case of simplification and reduction of pathogenic community such as lab group was, AMP expression may reduce in both AMP species and their abundance, indicating the expression of AMPs follow economic principle.
引文
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[3]Sarah R, Dennison, Leslie HG, Morton, Andrea J, Shorrocks, Fredenrick H, David AP. A study on the interactions of Aurein 2.5 with bacterial membranes. Colloids and Surfaces B:Biointerfaces,2009,68:225～230
[4]Giacometti A, Cirioni O, Barchiesi F, Caselli F, Scalise G. In-vitro activity of polycationic peptides against Cryptosporidium parvum, Pneumocystis carinii and yeast clinical isolates. Journal of Antimicrobial Chemotherapy,1999,44:403～406
[5]姚玉淑,秦志辉.抗菌肽的研究现状和应用前景.中国基层医药,2005,12(7)：934～935
[6]Olson L,Soto AM. An antimicrobial peptide with low hemolytic activity from the skin of the paradoxical frog. Biochemcal Biophysical Research Communications,2001,288:2001～2005
[7]周霞,诸葛洪祥.抗菌肽的分子生物学研究进展.国外医学.流行病学.传染病学分册,2000,39：310～313
[8]Kyungik L, Song YS, Kyoungho K, Shin SL, Kyung Sh, Yangmee K. Antibiotic activity and structural analysis of the scorpion-derived antimicrobial peptide IsCT and it s analogs. Biochemcal Biophysical Research Communications,2004,323:712-719
[9]庞英明,段金廒,屈贤铭.昆虫抗菌肽结构与功能关系及其在分子设计中的应用.生命科学,2001,13：209～213
[10]Zhang L, Benz R, Hancock REW. Influence of proline residues on the antibacterial and synergistic activities of antimicrobial peptides. Biochemistry,1999,38:8102～8110
[11]Yonezawa A, Kuwahara J, Fujii N, Fujii N, Sugiura Y. Binding of tachyplesin I to DNA revealed by footprinting analysis:significant contribution of secondary structure to DNA binding and implication for biological action. Biochemistry,1992,31:2998～3004
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[13]Jenssen H, Hamill P, Hancock REW. Peptide antimicrobial agents. Clinical Microbiology Reviews,2006, 19(3):491～511
[14]Epand RM, Vogel HJ. Diversity of Antimicrobial peptides and their mechanisms of action. Biochemica et Biophysica Acta-Bimembranes,1999,1462(1-2):11～28
[15]Zhang LJ, Rozek A, Hancock REW. Interaction of cationic antimicrobial peptides with model membranes. Biological Chemistry,2001,276:35714～35722
[16]Dekker N, Cox RC, Kramer RA. Substrate specificity of the integral membrane protease Omp T determined by spatially addressed peptide libraries. Biochemistry,2001,40 (6):1694～1701
[17]Park Y, Lee DG, Jang SH. A Leu2 Lys2 rich antimicrobial peptide:activity and mechanism. Biochemica et Biophysica Acta,2003,1645(2):172～182
[18]Yang D, Chertov O, Oppenheim JJ. The role of mammalian antimicrobial peptides and proteins in awakening of innate host defenses and adaptive immunity. Cell Molecular Life Science,2001,58(7):978～989
[19]Zhou CC, Qi XB, Li P, Chen WN. High potency and broad-spectrum antimicrobial peptides synthesized via ring-opening polymerization of a-aminoacid-n-carboxyan-hydrides. Biomacromolecules,2010,11(1):60～67
[20]Mauel MJ. Miller DL, Frazier KS, Hines ME. Bacterial pathogens isolated from cultured bullfrogs (Rana
castesbeiana). Journal of Veterinary Diagnostic Investigation,2002,14:431～433
[211 Fehri LF, Sirand-Pugnet P, Gourgues G, Jan G, Wroblewski H, Blanchard A. Resistance to antimicrobial peptides
and stress response in Mycoplasma pulmonis. Antimicrobial Agents and Chemotherapy,2005,49(10): 4154-4165
[22]Chen Y, Xu X, Hong S. RGD-Tachyplesin inhibits tumor growth. Cancer Research,2001,61(6):2434～2438
[23]Mai JC, Mi Z, Kim SH, A proapoptotic peptide for the treatment of solid tumors. Cancer Research,2001,61 (21):7709～7712
[24]Ibrahim HR, Sugimoto Y, Aoki T. Ovotransferrin antimicrobial peptide (OTAP-92) kills bacteria through a membrane damage mechanism. Biochemica et Biophysica Acta,2000,1523(223):196～205
[25]Bechinger B. Structure and functions of channel-forming peptides:magainins, cecropins, melittin and alamethicin. Journal of Membrane Biology,1997,156 (3):197～11
[26]申吉泓,杨宇如,唐孝达.抗菌肽作用机制研究现状.国外医学肿瘤学分册,2003,30(5)：347～350
[27]Aboudy Y, Mendelson E, Shalit I, Bessalle R, Fridkin M. Activity of synthetic amphiphilic peptides and magainin-2 against herpes simplex virus types 1 and 2. International Journal of Peptide and Protein Research, 1994,43(6):573～582
[28]Waehinger M, Kleinsehlnidt A, Winder D. Antimicrobial peptides Melittin and Ceo pin inhibit replication of human immunodefieiency virus I by suppressing viral gene expression. Journal of General Virology,1998, 79:731-740
[29]Andreu D, Rivas L. Animal antimicrobial peptides:an overview. BioPolymers,1999,47:413-433
[30]王亚洲,徐文方.酶抑制剂类抗病毒药物的研究进展.中国新药杂志,2005,14(10)：1151～1155
[31]韩彤彤.东北林蛙皮肤抗菌肽抗病毒活性的初步研究.东北林业大学硕士学位论文,2006
[32]Lee WH, Li Y, Lai R, Li S, Zhang Y, Wang W. Variety of antimicrobial peptides in the Bombina maxima toad and evidence of their rapid diversification. European Journal of Immunology,2005,35:1220～1229
[33]Liu L, Zhao C, Heng HH, Ganz T. The Human β-defensin-1 and α-defensins are encoded by adjacent genes:two peptide families with differing disulfide topology share a common ancestry. Genomics,1997,43:316～320
[34]朱倩.东北林蛙皮肤抗菌肽多样性研究.东北林业大学硕士论文,2007
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