三种两栖类动物皮肤和脑中活性多肽结构、功能与多样性分析
文献类型：学位论文
| 作者 | 马玉芳 |
| 学位类别 | 博士 |
| 答辩日期 | 2009-06 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 赖仞 |
| 关键词 | 两栖动物 微蹼铃蟾 大蹼铃蟾 华西雨蛙 抗菌肽 活性多肽 |
| 其他题名 | Structure, Function and Molecular Diversity Analysis of Bioactive Peptides from Three Amphibians |
| 中文摘要 | 两栖动物是最原始的陆生脊椎动物，分布比较广泛。无尾目两栖动物现有3000 多种，它们皮肤裸露、光滑，为适应广泛的栖息地和生态条件，已进化出各种有效的皮肤防御系统。抗菌肽（Antimicrobial peptides，AMPs）作为两栖类先天防御系统的重要组成部分，在皮肤分泌液中含量异常丰富。我们以来源于云南省普洱市景东县的铃蟾科微蹼铃蟾（Bombina microdeladigitora）和楚雄州双柏县雨蛙科华西雨蛙（Hyla annectans）为实验材料，对其皮肤分泌液中抗菌肽的分子多样性并对其结构和功能进行研究。微蹼铃蟾皮肤抗菌肽多样性非常丰富，我们从单一个体中克隆得到了64 条编码不同抗菌肽的cDNA 序列，其中有两条序列只编码Maximin 一种抗菌肽，其余 62 条均编码Maximin 和Maximin H 两类抗菌肽。这64 条cDNA 序列共编码44 种Maximins 和30 种Maximin Hs，其中有32 种Maximins 和20 种Maximin Hs 为新鉴定的抗菌肽，其余和铃蟾属其它种中发现的抗菌肽具有相同的序列。除了皮肤外，两栖动物的脑也是抗菌肽的丰富资源库。我们分别从微蹼铃蟾和大蹼铃蟾（B. maxima）脑中得到了大量新的抗菌肽cDNA 序列。其中从微蹼铃蟾脑中克隆到21 条新的cDNA序列，共编码16 种Maximins 和10 种Maximin Hs，其中7 种Maximins 和4 种Maximin Hs 为新鉴定的抗菌肽。从大蹼铃蟾脑中克隆到39 条新的cDNA 序列，编码27 种Maximins 和20 种Maximin Hs，其中16 种 Maximins 和12 种Maximin Hs 为新鉴定的抗菌肽。在以上新鉴定的抗菌肽中，Maximins 均为阳离子抗菌肽，Maximin Hs 中除以前鉴定的Maximin H5 外，尚有十余种阴离子抗菌肽。抗菌肽碱基转换/颠换（R=s/v）分析表明，RMaximin<1 而RMaximin H>1，说明这两种抗菌肽碱基转换和颠换发生的几率并不相同，Maximin 间差异主要由碱基颠换引起，而Maximin H 则主要由碱基转换引起。种间进化分析表明，大蹼铃蟾和微蹼铃蟾的遗传距离较近，而它们与欧洲花铃蟾（B. variegata）的遗传距离均较远。种内各部分遗传距离差异较大。与信号肽和酸性间隔肽相比，成熟肽的遗传距离明显增大，其中Maximin 的进化速度比Maximin H 更快。抗菌肽Maximin 和Maximin H 种内、种间均存在正选择（ω>1），而信号肽和酸性间隔肽在分化过程中没有正选择（ω<1），说明Maximin 和Maximin H 经受着达尔文正选择驱动的快速进化，是抗菌肽多样性产生的根本原因。这与抗菌肽参与最终的生物防御功能，从而增加物种对环境的适应是一致的。功能研究发现，有些微蹼铃蟾Maximins 抗菌肽是多功能分子，不但对革兰氏阴性菌、革兰氏阳性菌和真菌起抗菌作用，而且还具有很强的抗氧化功能。脑中抗菌肽基因的大量表达也预示着抗菌肽可能在神经信号传导中起一定作用。用基因克隆方法我们从微蹼铃蟾皮肤得到大量缓激肽前体序列，由1-4 个拷贝的Bombinakinin 或1-4 个拷贝的Bombinakinin 和1 个拷贝的Bombinakinin-GAP 组成。这与大蹼铃蟾皮肤中缓激肽前体由1-8 个拷贝的Bombinakinin 或1-8 个拷贝的Bombinakinin 和1 个拷贝的Bombinakinin-GAP 组成有所不同。按同样方法，我们从大蹼铃蟾脑中也得到了三条缓激肽前体序列，其中两条含有6 个 Bombinakinin 拷贝，另一条含2 个Bombinakinin 拷贝。通过比较只含Bombinakinin 和同时含有Bombinakinin 和Bombinakinin-GAP 的前体cDNA 序列后发现， 前者序列中缺失了一段碱基序列TGCGGGTA， 从而导致移码突变， 终止了 Bombinakinin-GAP 的表达。通过生物化学的手段从微蹼铃蟾皮肤分泌液中分离到一种丝氨酸蛋白酶抑制剂BMSI1，与铃蟾属其它种中的胰蛋白酶抑制剂具有很高的相似性。根据已有铃蟾属丝氨酸蛋白酶抑制剂cDNA 序列设计引物，以皮肤cDNA 为模板，扩增丝氨酸蛋白酶抑制剂的基因序列，结果得到两条不同的序列。这两条前体序列与铃蟾属其它两栖动物皮肤中的丝氨酸蛋白酶抑制剂具有高度相似性（>70%），而且它们都含有10 个半胱氨酸残基。BMSI1 对五种丝氨酸蛋白水解发色底物的抑制活性测定表明，BMSI 1 能抑制胰蛋白酶和凝血酶的水解活性，其K(i)分别为0.02 μM 和0.15 μM。通过随机筛选cDNA 文库的方法，我们从大蹼铃蟾脑中得到了一条完整的 Somatostatin（SST）序列，根据该序列，我们在大蹼铃蟾和微蹼铃蟾脑cDNA 文库中筛选到两条变异体序列SST-L(Leu11-SST-14)和SST-R(Arg14-SST-14)。功能研究表明，这两种变异体具有和SST 相似的生物学功能，可抑制肿瘤细胞增殖、抑制细胞因子释放以及具有一定的镇痛作用。从大蹼铃蟾和微蹼铃蟾脑中得到了阿片肽前体POMC 和Proenkephalin 的 cDNA 序列，序列比对发现与东方铃蟾具有较高的同源性。从华西雨蛙皮肤cDNA 中克隆得到两类活性多肽，命名为Annins。其中一类为抗菌肽类似肽，共11 条序列，编码单一的成熟肽序列，其信号肽与雨蛙科信号肽具有很高的同源性，但酸性间隔肽和成熟肽相差较大。其成熟肽由15-17 个氨基酸残基组成，活性分析表明无抗菌和抗氧化作用，但在较高浓度时对部分细菌和多种细胞有促进生长作用，推测可能在使伤口快速愈合方面起重要作用；另一类编码具有2 个拷贝的成熟肽序列，成熟肽由5 个氨基酸残基组成，具有一定的镇痛活性，其镇痛机理可能是拮抗bradykinin 作用。 |
| 英文摘要 | Amphibians that are the most primitive terrestrial vertebrates have wide distribution throughout the world. Their skins are naked and smooth. In order to adapt a wide range of habitats and ecological conditions, they have developed a variety of skin effective defense system. As an important component of the body's innate defense system of the amphibians, antimicrobial peptides (AMPs) are abundant in the skin secretions. We selected Bombina microdeladigitora and Hyla annectans in Yunnan province to study the diversity, function and structure of AMPs in amphibian skin secretions. In this study, 64 novel cDNAs encoding 44 Maximins and 30 Maximin Hs were cloned from skin cDNA library of B. microdeladigitora, of which 32 Maximins and 20 Maximin Hs were novel and others were same as AMPs cloned from other Bombina species. Besides skin, we also found AMPs genes were abundantly expressed in brain of B. microdeladigitora and B. maxima. Twenty one novel cDNAs encoding 16 Maximins and 10 Maximin Hs and 39 novel cDNAs encoding 27 Maximins and 20 Maximin Hs were cloned in brain of B. microdeladigitora and B. maxima, respectively. Among the AMPs, all Maximins are cations and tens of Maximin Hs are anions including Maximin H5 identified before. The analysis of base transition/transversion showed RMaximin<1 while RMaximin H >1. These results indicated that the diversity of Maximins caused mainly by base transition, while Maximin Hs by transversion. The interspecies evolutionary distance analysis showed that B. maxima are close to B. microdeladigitora, while they are both far away from B. variegata. The intraspecific evolutionary distance is differing from domains. The distances between the mature peptides are larger than that between signal peptides and acidic spacer peptides. Our results also showed that the evolutionary rate was faster in Maximins than in Maximin Hs. Furthermore, there is a positive selection on Maximins and Maximin Hs intra- and inter-species (ω>1), while no positive selection is found in signal peptides and acidic spacer peptides. These results suggested that the rapid evolution of Darwin’s positive selection on Maximins and Maximin Hs is the primary cause of diversity in AMPs. This is consistent with the most important defensive functions of AMPs that is essential for amphibian survival in different conditions. Functional study found that AMPs in B. microdeladigitora not only have activites of antibacteria and anti-fungi, but also have a strong anti-oxidative function. In addition, the expression of AMP genes in brain tissue gives us information that AMPs may also play a role in neural transmission. A large number of cDNAs encoding bradykinin-related peptides were cloned from B. microdeladigitora. These sequences include 1-4 Bombinakinin tandem repeats or 1-4 Bombinakinin tandem repeats with a Bombinakinin-GAP, while previous reports show that 1-8 Bombinakinin tandem repeats or 1-8 Bombinakinin tandems repeats with a Bombinakinin-GAP in B. maxima. We also gained three cDNAs encode Bombinakinin tandems from the brain of B. maxima, two have 6 Bombinakinin tandems with or without a Bombinakinin-GAP, the other one has 2 Bombinakinin tandems. By comparing the cDNA sequences between Bombinakinin and Bombinakinin with a Bombinakinin-GAP, we found that the missing sequence TGCGGGTA lead to frameshift mutation, which brought the termination of coding sequence forward and caused the loss of Bombinakinin-GAP. Two serine protease inhibitors (named BMSI 1 and BMSI 2, respectively) were identified from the skin secretions of the toad, B. microdeladigitora. The cDNAs encoding BMSIs were cloned from the cDNA library prepared from the toad skin. The deduced complete amino acid sequences of BMSIs indicate that mature BMSI 1 and BMSI 2 are composed of 60 amino acids including 10 half-cystines to form 5 disulfide bridges. A FASTA search in the databanks revealed that BMSIs exhibit sequence similarity with other serine protease inhibitors from amphibians of the genus Bombina. BMSI 1 potently inhibited trypsin and thrombin with a K(i) value of 0.02 μM and 0.15 μM, respectively. Sequence analysis revealed that all serine protease inhibitors from five amphibians of the genus Bombina share highly conserved primary structures. By random screening of cDNA library of the brain of B. maxima, a complete Somatostatin（SST）sequence was obtained, then two variant sequences SST-L (Leu11-SST-14) and SST-R (Arg14-SST-14) were gained by means of gene clone from B. maxima and B. microdeladigitora. The two mutational proteins have similar function with SST that inhibit proliferation of some tumor cell lines and release of cytokines, and have analgesic activity. Moreover, the cDNA sequences of POMC and proenkephalin homologous with B. orientalis were screened in brains of B. maxima and B. microdeladigitora. Two types of active peptides called annin were found in skin of Hyla annectans,. One type of annin cDNAs encodes peptides with 15-17 amino acid residues. Their signal peptides are higher homologous to those from Hylidae, while the acidic spacer peptides and mature peptides are significantly difference. They share homologous with AMPs from other Hylidae species but have neither antibacterial nor anti-oxidative activity. MTT assay showed they could promote cancer cell and endothelial cell (HUVEC) growth at a high concentration. Another type of cDNAs encode pentapeptide with analgesic activity which might be caused by the antagonism of bradykinin. |
| 语种 | 中文 |
| 公开日期 | 2010-10-22 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6300]  |
| 专题 | 昆明动物研究所_动物毒素室 |
| 推荐引用方式 GB/T 7714 | 马玉芳. 三种两栖类动物皮肤和脑中活性多肽结构、功能与多样性分析[D]. 北京. 中国科学院研究生院. 2009. |
入库方式： OAI收割
来源：昆明动物研究所
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