Cloning of two novel P-III class metalloproteinases from Trimeresurus stejnegeri venom gland

Hemorrhagic toxins are widely distributed in viperid and crotalid snake venoms. Envenomation of Trimeresurus stejnegeri, a member of Crotalidae family, caused potent systemic and local hemorrhage. Up to now, there is no report on hemorrhage toxins from th

A new protein structure of P-II class snake venom metalloproteinases: it comprises metalloproteinase and disintegrin domains

A new metalloproteinase-disintegrin, named Jerdonitin, was purified from Trimeresurus jerdonii venom with a molecular weight of 36 kDa on SDS-PAGE. It dose-dependently inhibited ADP-induced human platelet aggregation with IC50 of 120 nM. cDNA cloning and sequencing revealed that Jerdonitin belonged to the class II of snake venom metalloproteinases (SVMPs) (P-II class). Different from other P-II class SVMPs, metalloproteinase and disintegrin domains of its natural protein were not separated, confirmed by internal peptide sequencing. 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 cannot be separated by posttranslationally processing. In summary, comparison of the amino 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. (C) 2003 Elsevier Inc. All rights reserved.

AN ACTIVATOR OF BLOOD-COAGULATION FACTOR-X FROM THE VENOM OF BUNGARUS-FASCIATUS

A specific activator of blood coagulation factor X was purified from the venom of Bungarus fasciatus by gel filtration and by ion-exchange chromatography on a Mono-Q column (FPLC). It consisted of a single polypeptide chain, with a mel. wt of 70,000 in reducing and non-reducing conditions. The enzyme had an amidolytic activity towards the chromogenic substrates S-2266 and S-2302 but it did not hydrolyse S-2238, S2251 or S-2222, which are specific substrates for thrombin, plasmin and factor Xa, respectively. The enzyme activated factor X in vitro and the effect was Ca2+ dependent with a Hill coefficient of 7.9. As with physiological activators, the venom activator cleaves the heavy chain of factor X, producing the activated factor Xa alpha. The purified factor X activator from B. fasciatus venom did not activate prothrombin, nor did it cleave or clot purified fibrinogen. The amidolytic activity and the factor X activation activity of the factor X activator from B. fasciatus venom were readily inhibited by serine protease inhibitors such as diisopropyl fluorophosphate (DFP), phenylmethanesulfonyl fluoride (PMSF), benzamidine and by soybean trypsin inhibitor but not by EDTA. These observations suggest that the factor X activator from B. fasciatus venom is a serine protease. It therefore differs from those of activators obtained from Vipera russelli and Bothrops atrox venoms, which are metalloproteinases.

A novel high molecular weight metalloproteinase cleaves fragment F1 of activated human prothrombin

A hemorrhagic proteinase, jerdohagin, was purified from Trimeresurus jerdonii venom by gel filtration and ion-exchange chromatographies. It was a single chain polypeptide with an apparent molecular weight of 96 kDa as estimated by SDS-PAGE under the non-reducing and reducing conditions. Internal peptide sequencing indicated that it consisted of metalloproteinase, disintegrin-like and cysteine-rich domains and belonged to the class III snake venom metalloproteinases (class P-III SVMPs). Like other typical metalloproteinases, hemorrhagic activities of jerdohagin were completely inhibited by EDTA, but not by PMSF. Jerdohagin preferentially degraded a-chain of human fibrinogen. Interestingly, jerdohagin did not activate human prothrombin, whereas it cleaved human prothrombin and fragment F1 of activated human prothrombin. (C) 2004 Elsevier Ltd. All rights reserved.

Expression, purification and characterization of recombinant Jerdonitin, a P-II class snake venom metalloproteinase comprising metalloproteinase and disintegrin domains

Jerdonitin is a P-II class snake venom metalloproteinase comprising metalloproteinase and disintegrin domains. In this study, we established a high-level expression system in Pichia pastoris and developed a purification strategy for the recombinant Jerdonitin. This recombinant Jerdonitin degraded fibrinogen at a level of activity comparable with its wild type. The effects of recombinant Jerdonitin on inhibiting ADP-induced human platelet aggregation were in a dose-dependent manner with an IC50 of 248 nM. In addition, we reported here that Jerdonitin can significantly inhibit the growth of several cell lines, including human liver cancer cells (Bel7402), human leukemia cells (K562) and human gastric carcinoma cells (BGC823). This study offers recombinant Jerdonitin that will be valuable for further functional and structural studies of Jerdonitin. (C) 2009 Elsevier Ltd. All rights reserved.

竹叶青蛇毒金属蛋白酶结构与功能的研究

出血性的蝰科蛇毒中含有丰富的蛇毒金属蛋白酶，本论文就竹叶青（Trimeresurus stejnegeri）蛇毒中的蛇毒金属蛋白酶的结构与功能进行研究。我们用生物化学手段从竹叶青（T.stejnegeri）的粗毒中分离纯化得到一个二聚体的P-IIIb亚型蛇毒金属蛋白酶，命名为TSV-DM。同时，用分子生物学方法从竹叶青（T.stejnegeri）的毒腺cDNA文库中克隆得到3个P-III型的蛇毒金属蛋白酶的cDNAs序列， 其中一个编码TSV-DM蛋白前体，另两个编码P-IIIc亚型的出血性蛇毒金属蛋白酶前体，分别命名为stejnihagin-A 和 stejnihagin-B。 经过阴离子层析和肝素亲和层析两步层析方法，我们从竹叶青（T. stejnegeri）蛇毒中分离纯化得到TSV-DM蛋白质，非还原条件下SDS-PAGE电泳表观分子量约110 kDa，还原条件下约为55 kDa。活性检测表明，TSV-DM降解牛纤维蛋白原Aα链快于Bβ链，且呈剂量依赖关系。但不降解明胶，不诱导出血，不具有促凝或者抗凝活性，以及不诱导或者抑制血小板聚集。蛋白质N-末端测序表明TSV-DM的成熟蛋白N-末端封闭。利用肽指纹图谱确证了TSV-DM的编码cDNA。TSV-DM的cDNA序列编码622个氨基酸残基的蛋白前体，包括信号肽、前肽、金属蛋白酶区域、间隔区、去整合素样区域和富含半胱氨酸区域。TSV-DM与其他P-III型蛇毒金属蛋白酶的一级结构序列比对发现TSV-DM和诱导血管内皮细胞凋亡的P-IIIb亚型蛇毒金属蛋白酶具有高度的同源性。但是用人脐带静脉血管内皮细胞系ECV304细胞作为靶细胞检测TSV-DM的诱导血管内皮细胞凋亡活性发现，TSV-DM只能抑制ECV304细胞的增殖和诱导细胞形态从多角形的内皮细胞向成纤维细胞样的梭形状改变。电泳检测抽提的片断化DNA以及流式细胞仪检测TSV-DM处理的ECV304细胞的DNA含量变化均表明TSV-DM不能诱导ECV304细胞的凋亡。 Stejnihagin-A 和stejnihagin-B是用PCR方法从竹叶青（T.stejnegeri）毒腺cDNA文库中克隆得到的两个P-III型蛇毒金属蛋白酶前体的cDNAs。这两个cDNA序列均编码600个氨基酸残基的蛋白前体，包括信号肽、前肽、金属蛋白酶区域、间隔区、去整合素样区域和富含半胱氨酸区域。推导成熟肽的氨基酸序列分析结果表明，stejnihagin-A 和stejnihagin-B不仅在一级结构序列上和来源于黄绿烙铁头（Trimeresurus flavoviridis）的HR1b具有高度同源性，高达79%， 而且在他们的金属蛋白酶区域的第100个氨基酸残基位置上均有一保守的半胱氨酸残基。结合序列比对和进化树的分析，我们推测stejnihagin-A、stejnihagin-B和HR1b有可能组成一个新的P-III型蛇毒金属蛋白酶亚型，命名为P-IIIc亚型。

菜花烙铁头蛇毒金属蛋白酶的研究

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。