菜花烙铁头蛇毒金属蛋白酶的研究
文献类型：学位论文
| 作者 | 陈润强 |
| 学位类别 | 博士 |
| 答辩日期 | 2005-06 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 熊郁良 ; 张云 |
| 关键词 | 菜花烙铁头蛇毒 蛇毒金属蛋白酶 去整合素 纤维蛋白原 凝血酶原 克隆表达 整合素 |
| 其他题名 | Studies on Snake Venom Metalloproteinases from Trimeresurus Jerdonii |
| 中文摘要 | 本论文结合生物化学与分子生物学手段对云南产菜花烙铁头蛇毒（几互刃eresrs户厂由刀打）的金属蛋白酶进行了系统深入的研究。从中分离纯化到两个新的蛇毒金属蛋白酶：二型蛇毒金属蛋白酶Jerdonit认和三型蛇毒金属蛋白酶jerdohagin。采用又卫PCR的方法得到了两个cDNA。通过蛋白质胰蛋白酶水解的内肚测序分析，其中一条为编码Jerdo址tin的，cDNA。序列分析发现，另外一条是一个含有RTS短链去整合素cDNA序列，命名为jerdos七atin，并对其进行了表达和活性鉴定。，Jerdonitin是一个表观分子量为为kDa的单链蛋白。它的c砚认全长1578bP，由金属蛋白酶、间隔区和去整合素区组成，说明它是二型蛇毒金属蛋白酶。但是与其它二型蛇毒金属蛋白酶不同的是，Jerdonitin的成熟蛋白由金属蛋白酶和去整合素结构域组成。与其它二型相比，Jerdonitin"多了两个半肤氨酸的（CysZ19andCys238）分别位于间隔区和去整合素区，Cys219可能和后面去整合素区的自由半眺氨酸残基Cys238形成一对二硫键，这对二硫键可能阻止了在后翻译加工过程中去整合素区的释放。通过Jerdonitin和其它二型蛇毒金属蛋白酶氨基酸序列比较和进化分析，结合天然蛋白结构数据，说明Jerdonitin是二型蛇毒金属蛋白酶的一个新亚型。和其它蛇毒金属蛋白酶一样，Jerdon九in是a一纤溶酶，并且它的活性都能被金属鳌合剂EDTA完全抑制，而不受丝氨酸蛋白酶抑制剂PMSF影响。像其独特的结构一样，Jerdonitin不仅具有纤维蛋白原降解的蛇毒金属蛋白酶活性，而且也有抑制ADP诱导的血小板聚集的非酶活性。Jerdohagin是一个表观分子量为96kDa单链蛋白。测定其内肤发现它有金属蛋白酶、去整合素样和富含半耽氨酸区组成，说明jerdohagig是三型蛇毒金属蛋白酶。像其它典型的蛇毒金属蛋白酶一样，jerdohagin的出血活性能完全被EDTA抑制，而不受R涯SF的影响。Jerdohagin是仪纤溶酶专一酶切人纤维蛋白原的。链。用氧化的胰岛素B链作底物，它可以水解仰r16一Leu17肤键。有趣的是，jerdohagin不激活人凝血酶原，但是它酶切人凝血酶原和凝血酶原激活后产生的激活片段F1。Jerdostotin的cDNA全长邹3bp，由信号肚、前肤和去整合素三个区组成，其与。btustotin和vieris七atin有较高的相似性（80％）。jerdostatin的序列是第一个报道的短链去整合素的cD以序列，它的产生机制和aCostatin一a有相似之处。值得注意的是，jerdostatin和obtus七atin八ipris，tatin不同的氨基酸残基（8／9）主要分布在含有整合素识别区的C末端。采用硫氧还蛋·白作为融合蛋白表达jerdostatin：，表达纯化后发现有两个j．erdostatin表达，，命名为：jerdo就。tin一1和'rjerdostotin一2。质谱分析显示它们的八个半肤氨酸都参与形成了四对二硫键，它俩可能是分别含有天然和非天然二硫键结构的多肚。但是jerdostatin一1的活性比rjerdostat还一2高两个数量级。和含有KTS的去整合素一样，重组jerdos七atin的异构体都选择性抑制整合素。lpl结合胶原IV，rjerdost就in一1的抑制活性分别是。b加stotin和viperistotill的1／1时和1／2500。氨基酸残基的组成不同尽管没有影响jerdost就in对整合素alpl的抑制选择性，但是它造成的化学环境不同可能导致抑制活性的差异。 |
| 英文摘要 | Snake venom metalloproteina'ses (SVMPs) from Trimeresurus jerdonii venom were studied systematically and comprehensively using biochemistry and molecular biology methods. Two novel SVMPs, Jerdonitin and jerdohagin, which belong to Class PII and PIE SVMPs, respectively, were purified. Two cDNA clones were obtained by RT-PCR from the venom gland mRNA of the pit viper Trimeresurus jerdonii. Trypsin-digested internal peptide sequencing -suggested that one of the cDNA clones encoded Jerdonitin. Sequence analysis indicated that the other cDNA clone encoded a novel RTS-containing short disintegrin, designated as jerdostatin. Moreover, jerdonitin was expressed and characterized.Jerdonitin has an apparent molecular weight of 36 kDa. The precursor cDNA of Jerdonitin contains 1578 bp ORE cDNA cloning and sequencing revealed that Jerdonitin belonged to Class PII SVMPs. Internal peptide sequencing indicated that Jerdonitin was different from other P-II class SVMPs in that the metalloproteinase and disintegrin domains of its mature protein were not separated. Compared to other P-II class SVMPs, Jerdonitin has two additional cysteines (Cys219 and Cys238) located in the spacer domain and disintegrin domain respectively .They probably form a disulfide bond and therefore the metalloproteinase and disintegrin domains can not be separated by posttranslationally processing. Comparison of the ammo acid sequences of Jerdonitin with those of other P-II class SVMPs by sequence alignment and phylogenetic analysis, in conjunction with natural protein structure data, suggested that it was a new type of P-II class SVMPs. Like other SVMPs, Jerdonitin is a-fibrinogeriases and its activity can be inhibited by EDTA, but not by PMSF. As indicated by its typical protein structure, Jerdonitin has not only the characteristics of a metalloproteinase, but also that of RGD-containing disintegrin, which inhibits ADP-induce platelet aggregation.Jerdohagin had an apparent molecular weight of 96 kDa. Internal peptide sequencing indicated that it is consisted of metalloproteinase, disintegrin-like and cysteine-rich domains and belongs to the class HI SVMPs. Like other typical metalloproteinases, hemorrhagic activities of jerdohagin are completely inhibited by EDTA, but not by PMSF. Jerdohagin preferentially degrades a-chain of human fibrinogen. Using the B-chain of oxidized insulin as substrate, it could hydrolyze the Tyr16-Leu17 peptide bond. Interestingly, jerdohagin does not activate human prothrombin, whereas it cleaves human prothrombin and activation fragment Fl of activated human prothrombin.The precursor cDNA of jerdostatin contains a 333 bp ORF encoding a67-residue pre-peptide and a 43-amino-acid disintegrin domain. The aminoacid sequence of jerdostatin displays 80% identity with that of theKTS-disintegrins obtustatin and viperistatin. The jerdostatin cDNA structurereported here represents the first complete ORF of a short disintegrin andpoints to a mechanism for the generation of jerdostatin from a short-codinggene. This generation mechanism is similar to that of acostatin-a. Noteworthy,the 8/9 different residues between jerdostatin and obtustatin/viperistatin aresegregated within the C-terminal half of the molecule, including theintegrin-recognition loop and the C-terminal tail. These two structural elementsform a continuous conformational functional epitope in the NMR structure ofobtustatin. Two recombinant jerdostatin conformers, rjerdostatin-1 andrjerdostatin-2, were expressed in E. coli as fusion proteins with thioredoxin.Mass spectrometric analysis of the fusion tag-free jerdostatin conformersshowed that in both cases their 8 cysteine residues were involved in theformation of 4 disulphide bonds, and may represent, respectively, native andnon-native disulphide bond conformers. However, rjerdostatin-1 was over twoorders of magnitude more potent than rjerdostatin-2. Like the KTS-disintegrins,the recombinant jerdostatin conformers selectively inhibits the binding of the aiPi integrin to immobilized collagen IV, though rjerdostatin-1 was about 100 and 2500 times less potent than the KTS-disintegrins obtustatin and viperistatin, respectively. The amino acid residues of jerdostatin which depart from the primary structures of its homolog KTS-disintegrins may create a distinct chemical environment responsible for the lower inhibitory activity of jerdostatin, though these substitutions do not affect the restricted inhibitory selectivity of j erdostatin towards integrin a1B1. |
| 语种 | 中文 |
| 公开日期 | 2010-10-15 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6178]  |
| 专题 | 昆明动物研究所_其他 昆明动物研究所_动物活性蛋白多肽组学 |
| 推荐引用方式 GB/T 7714 | 陈润强. 菜花烙铁头蛇毒金属蛋白酶的研究[D]. 北京. 中国科学院研究生院. 2005. |
入库方式： OAI收割
来源：昆明动物研究所
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