五步蛇蛇毒金属蛋白酶cDNA的克隆和序列分析

抽提五步蛇毒腺总RNA,通过反转录PCR(RT-PCR)扩增出五步蛇毒腺中一种低分子量金属蛋白酶(aculysinl)的cDNA,克隆到pGMT-vector并测定了全序列.推导其编码的蛋白质序列,发现aculysinl是以酶原形式合成的分泌蛋白,酶原包括信号肽、前肽、金属蛋白酶成熟肽和间隔肽4个部分.金属蛋白酶成熟肽与其它蛇毒金属蛋白酶相比,蛋白质一级结构具有一定的同源性,有一个保守的Zn2+结合位点:HEXXHXXGXXH.Aculysinl含有6个半胱氨酸,推测形成3对链内二硫键.五步蛇低分子量金属蛋白酶cDNA的克隆,为研究蛇毒金属蛋白酶结构与功能的关系,以及开发治疗血栓药物打下了良好的基础     

蛇毒中的金属蛋白水解酶

从很早以前 ,人们就进行了探索蛇毒的奥秘。由于近代生物化学和分子生物学的快速进步 ,这为揭示蛇毒的本质及其作用机制 ,提供了有力条件。迄今为止 ,蛇毒被誉为自然界最集中的酶原之一 ,其中有功能独特的酶、毒性蛋白质和活性短肽等 ,目前已经在蛇毒中分离出至少 1 50种蛋白水解酶 ,这些蛋白酶可分为丝氨酸蛋白酶和金属蛋白酶 ,这里我们只讨论后者。蛇毒金属蛋白酶具有使纤维蛋白溶解的作用 ,其中有的酶具有底物专一性 ,只作用于凝血因子 X、血小板膜受体或 von Willebrand因子。很多蛇毒金属蛋白酶由多个结构域组成 ,如蛋白水解酶活性结…     

蛇毒丝氨酸蛋白酶多样性──底物专一性免疫化学及序列比较研究

本文报道烙铁头（Ｔｒｉｍｅｒｅｓｕｒｕｓｍｕｃｒｏｓｑｕａｍａｔｕｓ）蛇毒纤维蛋白原溶酶（ＴＭＶＦｇ），眼镜王蛇（Ｏｐｈｉｏｐｈａｇｕｓｈａｎｎａｈ）蛇毒纤维蛋白原溶酶（ｏｈＳ１），竹叶青（Ｔｒｉｍｅｒｅｓｕｒｕｓｓｔｅｊｎｅｇｅｒｉ）蛇毒专一纤溶酶原激活剂（ｓｖ－ｐＡ）对５种小分子多肽底物的底物专一性，及这些蛇毒丝氨酸蛋白酶对各种凝血因子（第Ｘ因子、凝血酶原、纤溶酶原、蛋白Ｃ）的作用，并和其它蛇毒丝氨酸蛋白酶如矛头蝮（Ｂｏｔｈｒｏｐｓａｔｒｏｘ）蛇毒凝血酶样酶（Ｂａｔｒｏｘｏｂｉｎ）、铜头蝮（Ａｇｋｉｓｔｒｏｄｏｎｃｏｎｔｏｒｔｒｉｘｃｏｎｔｏｒｔｒｉｘ）蛇毒蛋白Ｃ激活剂ＡＣＣ－Ｃ、蝰蛇（Ｖｉｐｅｒａｒｕｓｓｅｌｌｉ）毒第Ⅴ因子激活剂ＲＶＶ－Ｖ进行比较研究。通过酶标偶联免疫反应研究了抗ｓｖ－ＰＡ抗体与各种丝氨酸蛋白酶的免疫交叉反应，并对蛇毒丝氨酸蛋白酶及相应功能的哺乳动物蛋白酶进行了序列比较分析。从底物专一性多样性及已知序列结构分化上对这一类蛇毒丝氨酸蛋白酶的结构与功能进行了探讨和研究。     

蛇毒丝氨酸蛋白酶

蛇毒是含有许多种生物活性蛋白质和多肽的复杂混合物 ,在蛇毒中蛋白酶占据着独特的位置 ,特别是蝰亚科和蝮亚科。目前可将蛇毒中蛋白水解酶主要分成两大类 ,其中一类是可被ＰＭＳＦ或ＤＦＰ抑制其活性的丝氨酸蛋白酶类。它们的蛋白质序列与胰蛋白酶、激肽释放酶比较显示较大同源性 ,均具有一个共同活性中心和相同的酶催化机制。但由于活性中心外的序列差异导致底物专一性差异和生物活性差异。另一类是可被金属螯合剂ＥＤＴＡ抑制其活性的金属蛋白酶类。一般说来 ,参加它们催化作用活性中心的金属离子是锌 ,隶属于一个新的金属蛋白酶亚科 (ｍ…     

菜花烙铁头蛇毒中一种具有激肽释放酶与纤维蛋白原溶酶活性的Jerdonase

通过DEAESephadexA 5 0阴离子交换、超细SephadexG 10 0分子筛和反相高效液相C4 色谱层析 ,从菜花烙铁头蛇毒冻干粉中纯化出一种具有激肽释放酶活性和α纤维蛋白原溶酶活性的丝氨酸蛋白酶 ,命名为Jerdonase。在 12 .5 %胶浓度的SDS还原电泳条件下 ,该酶分子量大约为 5 5kD ,在非还原电泳条件下 ,分子量大约为 5 3kD。此酶是一种糖蛋白 ,含有约 35 .8%的中性糖。它的N末端氨基酸序列为IIGGDEENINEHPFLVALYDA ,其序列和蛇毒中其他丝氨酸蛋白酶具有非常高的序列相似性。Jerdonase能够催化BAEE、S 2 2 38和S 2 30 2的水解 ,其水解活性可被PMSF抑制 ,但是EDTA对此没有影响。Jerdonase能优先水解人纤维蛋白原的Aα链 ,同时伴随有微弱的Bβ链水解活性。另外 ,此酶能够水解牛低分子量的激肽原 ,释放舒缓激肽。总之 ,所有的结果表明Jerdonase是一个具有多功能活性的蛇毒丝氨酸蛋白酶     

竹叶青蛇毒凝血酶样酶氨基酸序列报道

蛇毒凝血酶样酶可作为蛋白酶结构与功能研究的良好模型,并已广泛用于各种血栓疾病的诊断和治疗,因而测定其一级结构具有重要意义．利用逆转录与聚合酶链反应相结合的ＲＴ－ＰＣＲ法,扩增出竹叶青蛇毒凝血酶样酶（ＴＳＶ－ＴＥＬ１）的ｃＤＮＡ;将扩增的ｃＤＮＡ片段克隆入ｐＧＥＭ－Ｔ载体中,经末端终止法测定核苷酸序列,推导出竹叶青蛇毒凝血酶样酶的全序列．竹叶青蛇毒凝血酶样酶由２３４个氨基酸组成并含有１个位于Ａｓｎ２０的Ｎ－型糖基结合位点．竹叶青蛇毒凝血酶样酶序列与其它蛇种来源凝血酶样酶具有较大相似性,其与黄绿烙铁头蛇毒凝血酶样酶序列相似度为８４％,与美洲矛头蝮蛇毒凝血酶样酶序列相似度为６８％,而与牛凝血酶Ｂ链序列相似度仅为２５％．     

中国长白山乌苏里蝮蛇金属蛋白酶解整合蛋白Ussurin基因克隆和序列分析

采用Clontech链转换建库试剂盒,建立了中国长白山乌苏里蝮蛇毒腺cDNA文库,从中克隆了金属蛋白酶/解整合蛋白Ussurin,并进行了序列分析。结果显示,Ussurin开框读码序列由1434bp组成,编码478个氨基酸。由核苷酸顺序推导的氨基酸序列可以看出,Ussurin最初的翻译产物是酶原前体;依次含有18氨基酸组成的信号肽,171氨基酸组成的酶原区和由289氨基酸组成的Ussurin（200氨基酸组成的金属蛋白酶结构域、16氨基酸组成的间隔区和73氨基酸组成的解整合蛋白结构域）。Ussurin的金属蛋白酶结构域含有3对二硫键;解整合蛋白结构域含有6对二硫键和特征性RGD(精氨酸甘氨酸天冬氨酸)结构。其基因序列和结构域组成与GenBank中蛇毒金属蛋白酶/解整合蛋白呈现高度同源性属于P-Ⅱ。氨基酸序列blast比对发现,酶原区和解整链蛋白结构域呈现极高的同源性,而金属蛋白酶结构域却出现了极高的变异,推测这些变异结构区是为了适应不同的底物、不同受体或同一受体的不同结构域。     

中国长白山乌苏里蝮蛇金属蛋白酶解整合蛋白Ussurin基因克隆和序列分析

采用Clontech链转换建库试剂盒 ,建立了中国长白山乌苏里蝮蛇毒腺cDNA文库 ,从中克隆了金属蛋白酶 解整合蛋白Ussurin ,并进行了序列分析。结果显示 ,Ussurin开框读码序列由 14 34bp组成 ,编码 4 78个氨基酸。由核苷酸顺序推导的氨基酸序列可以看出 ,Ussurin最初的翻译产物是酶原前体 ;依次含有 18氨基酸组成的信号肽 ,171氨基酸组成的酶原区和由 2 89氨基酸组成的Ussurin(2 0 0氨基酸组成的金属蛋白酶结构域、16氨基酸组成的间隔区和 73氨基酸组成的解整合蛋白结构域 )。Ussurin的金属蛋白酶结构域含有 3对二硫键 ;解整合蛋白结构域含有 6对二硫键和特征性RGD(精氨酸 甘氨酸 天冬氨酸 )结构。其基因序列和结构域组成与GenBank中蛇毒金属蛋白酶 解整合蛋白呈现高度同源性属于P Ⅱ。氨基酸序列blast比对发现 ,酶原区和解整链蛋白结构域呈现极高的同源性 ,而金属蛋白酶结构域却出现了极高的变异 ,推测这些变异结构区是为了适应不同的底物、不同受体或同一受体的不同结构域     

蛇毒丝氨蛋白酶多样性：底物专一性免疫化学及序列比较研究

本文报道烙铁头蛇毒纤维蛋白原溶酶,眼镜王蛇蛇毒纤维蛋白原溶酶,竹叶青蛇毒专一纤溶酶原激活剂对５种小分子多肽底物的底物专一性,及这些蛇毒丝氨酸蛋白酶对各种凝血因子（第Ｘ因子、凝血酶原、纤溶酶原、蛋白Ｃ）的作用,并和其它蛇毒丝氨酸蛋白酶如矛头蝮蛇毒凝血酶样酶、铜头蝮蛇毒蛋白Ｃ激活剂ＡＣＣ－Ｃ、蝰蛇毒第Ｖ因子激活剂ＲＶＶ－Ｖ进行比较研究。通过酶标偶联免疫反应研究了抗ｓｖ－ＰＡ抗体与各种丝氨酸蛋白酶的免疫     

一种新型的α-纤维蛋白原酶对血小板聚集的影响

SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测中国眼镜蛇毒蛋白酶水解纤维蛋白原的作用方式,发现该蛋白酶可迅速水解纤维蛋白原Aα链,对γ链作用较弱,对Bβ链无作用,是一种新型的α-纤维蛋白原酶.用比浊法测定血小板聚集率,证实中国眼镜蛇毒蛋白酶低剂量(0.025 mg/kg)、中剂量(0.05 mg/kg)以及高剂量(0.1 mg/kg)体内给药浓度依赖性地抑制ADP(10 .Μmol/L)和胶原(100 mg/L)诱导的兔血小板聚集.     

cDNA cloning and characterization of Agkistin,a new metalloproteinase from Agkistrodon halys

Agkistin was a new snake venom metalloproteinase (SVMP) gene which was cloned from Agkistrodon halys. Its deduced amino acid sequence has two additional cysteines (Cys407 and Cys426) in the disintegrin domain compared to other RGD containing SVMPs. The full-length gene (Agkistin) and its disintegrin region (named Agkistin-s) were expressed by baculovirus expression system (pFastBac-Htb vector) with His-tag, and their platelet aggregation-inhibition activity was evaluated. The expressed protein Agkistin can also induce apoptosis of HMEC cells in the basal medium after incubated at 37 degrees C for 20 h.     

Sequence of a cDNA clone encoding the zinc metalloproteinase hemorrhagic toxin e from Crotalus atrox: evidence for signal, zymogen, and disintegrin-like structures.

The sequence of two overlapping cDNA clones for the zinc metalloproteinase hemorrhagic toxin e (also known as atrolysin e, EC 3.4.24.44) from the venom gland of Crotalus atrox, the Western diamondback rattlesnake, is presented. The assembled cDNA sequence is 1975 nucleotides in length and encodes an open reading frame of 478 amino acids. The mature hemorrhagic toxin e protein as isolated from the crude venom has a molecular weight of approximately 24,000 and thus represents the processed product of this open reading frame. From the deduced amino acid sequence, it can be hypothesized that the enzyme is translated with a signal sequence of 18 amino acids, an amino-terminal propeptide of 169 amino acids, a central hemorrhagic proteinase domain of 202 amino acids, and a carboxy-terminal sequence of 89 amino acids. The propeptide has a short region similar to the region involved in the activation of matrix metalloproteinase zymogens. The proteinase domain is similar to other snake venom metalloproteinases, with over 57% identity to the low molecular weight proteinases HR2a and H2-proteinase from the Habu snake Trimeresurus flavoviridis. The carboxy-terminal region, which is not observed in the mature protein, strongly resembles the protein sequence immediately following the proteinase domain of HR1B (a high molecular weight hemorrhagic proteinase from the venom of T. flavoviridis) and the members of a different family of snake venom polypeptides known for their platelet aggregation inhibitory activity, the disintegrins. The cDNA sequence bears striking similarity to a previously reported sequence for a disintegrin cDNA. This report is evidence that this subfamily of venom metalloproteinases is synthesized in a proenzyme form which must be proteolytically activated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Jararhagin ECD-containing disintegrin domain: expression in escherichia coli and inhibition of the platelet-collagen interaction.

Jararhagin, a hemorrhagin from Bothrops jararaca venom, is a soluble snake venom component comprising metalloproteinase and disintegrin cysteine-rich domains and, therefore, is structurally closely related to the membrane-bound A Disintegrin And Metalloproteinase (ADAMs) protein family. Its hemorrhagic activity is associated with the effects of both metalloproteinase and disintegrin domains; the metalloproteinase enzymatically damages the endothelium and the disintegrin domain inhibits platelet-collagen interactions. The expression of whole jararhagin or its disintegrin domain has never been attempted before. The aim of this study was to investigate whether we could express the disintegrin domain of jararhagin and to verify whether this domain displays an inhibitory effect on the platelet-collagen interaction. Therefore, the cDNA fragment coding for the disintegrin plus cysteine-rich domains of jararhagin was cloned into the pET32a vector, used to transform the Escherichia coli AD494(DE3)pLysS strain. The thioredoxin-disintegrin fusion protein was recovered from the soluble extract of the cells, yielding up to 50 mg/liter culture. The fusion protein was isolated using polyhistidine binding resin which resulted in a main band of 45 kDa recognized by anti-native jararhagin antibodies. Antibodies raised in rabbits against the fusion protein had high enzyme-linked immunosorbent assay titers against native jararhagin and detected a band of 52 kDa on Western blots of whole B. jararaca venom demonstrating that these antibodies recognize the parent jararhagin molecule. Treatment of the fusion protein with enterokinase, followed by further capture of the enzyme, resulted in a band of 30 kDa, the expected size for jararhagin-C. Further purification of the cleaved disintegrin using FPLC Mono-Q columns resulted in one fraction capable of efficiently inhibiting collagen-induced platelet aggregation in a dose-dependent manner (IC(50) of 8.5 microg/ml).     

Complete amino acid sequence of kaouthiagin, a novel cobra venom metalloproteinase with two disintegrin-like sequences

The primary structure of kaouthiagin, a metalloproteinase from the venom of the cobra snake Naja kaouthia which specifically cleaves human von Willebrand factor (VWF), was determined by amino acid sequencing. Kaouthiagin is composed of 401 amino acid residues and one Asn-linked sugar chain. The sequence is highly similar to those of high-molecular mass snake venom metalloproteinases from viperid and crotalid venoms comprised of metalloproteinase, disintegrin-like, and Cys-rich domains. The metalloproteinase domain had a zinc-binding motif (HEXXHXXGXXH), which is highly conserved in the metzincin family. Kaouthiagin had an HDCD sequence in the disintegrin-like domain and uniquely had an RGD sequence in the Cys-rich domain. Metalloproteinase-inactivated kaouthiagin had no effect on VWF-induced platelet aggregation but still had an inhibitory effect on the collagen-induced platelet aggregation with an IC(50) of 0.2 microM, suggesting the presence of disintegrin-like activity in kaouthiagin. To examine the effects of these HDCD and RGD sequences, we prepared synthetic peptides cyclized by an S-S linkage. Both the synthetic cyclized peptides from the disintegrin-like domain and from the Cys-rich domain) had an inhibitory effect on collagen-induced platelet aggregation with IC(50) values of approximately 90 and approximately 4.5 microM, respectively. The linear peptide (RAAKHDCDLPELC) and the cyclized peptide had little effect on collagen-induced platelet aggregation. These results suggest that kaouthiagin not only inhibits VWF-induced platelet aggregation by cleaving VWF but also disturbs the agonist-induced platelet aggregation by both the disintegrin-like domain and the RGD sequence in the Cys-rich domain. Furthermore, our results imply that the corresponding part of the Cys-rich domain in other snake venom metalloproteinases also has a synergistic disturbing effect on platelet aggregation, serving as a second disintegrin-like domain. This is the first report of an elapid venom metalloproteinase with two disintegrin-like sequences.     

Primary structure and functional characterization of bilitoxin-1, a novel dimeric P-II snake venom metalloproteinase from Agkistrodon bilineatus venom

The amino acid sequence of the hemorrhagic toxin, bilitoxin-1, isolated from the venom of Agkistrodon bilineatus was determined by the Edman sequencing procedure of peptides derived from digests utilizing cyanogen bromide, clostripain, lysyl endopeptidase, and Staphylococcus aureus V8 protease. A molecular mass of 80,000 Da was observed in the nonreduced state and 48,000 Da was observed in the reduced state, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Each subunit consists of 291 amino acid residues and has a calculated molecular mass of 32,276 Da. The toxin contains fucose, galactosamine, glucosamine, galactose, mannose, and N-acetylneuraminic acid and three N-linked glycosylation consensus sites. Hydrazinolysis and ESI mass spectrometry revealed that asparagine was the carboxyl-terminal amino acid. The disintegrin-like domain of bilitoxin-1 lacks the RGD cell-binding sequence, which is substituted by the MGD sequence. Under certain conditions, the disintegrin domain is autoproteolytically processed from the native protein. Studies with the bilitoxin disintegrin demonstrated that it lacks platelet aggregation inhibitory activity, probably reflecting the substitution of RGD by MGD. The hemorrhagic activity of the asialobilitoxin-1 was only 25% of bilitoxin-1, while proteolytic activity was unaffected. The three-dimensional structure of this toxin was modeled and was shown to likely possess a structure similar to that of adamalysin II (Gomis-Rüth et al., EMBO J. 12, 151-157 (1993)) and the disintegrin kistrin (Adler et al., Biochemistry 32, 282-289 (1993)). In summary, here we report the first primary structure of a dimeric, P-II snake venom metalloproteinase and the biological role of bilitoxin-1 glycosylation and the disintegrin domain.     

A new gene structure of the disintegrin family: a subunit of dimeric disintegrin has a short coding region

Disintegrin is a potent platelet aggregation inhibitor isolated from various snake venoms. The cDNA of the snake venom disintegrin family precursor is well-known to encode pre-peptide, metalloprotease, spacer, and disintegrin domains. Recently, new types of disintegrins, dimeric disintegrins, have been isolated, and their amino acid sequences were determined to be approximately 65 amino acid residues in each subunit. We isolated a novel heterodimeric disintegrin, acostatin, from the venom of Agkistrodon contortrix contortrix, which consisted of 63 and 64 amino acid residues in the alpha chain and beta chain, and both chains had the Arg-Gly-Asp (RGD) sequence for binding platelet GPIIb/IIIa. The cDNA lengths of the alpha chain and the beta chain of acostatin were 902 bp and 2031 bp, respectively. The acostatin alpha chain precursor, surprisingly, has the only disintegrin domain alone and lacked almost all of the pre-peptide and metalloprotease domains. The precursor of the acostatin beta chain belongs to a well-known motif of disintegrin precursors. Furthermore, both precursors of alpha and beta chains of another heterodimeric disintegrin, piscivostatin, also have the same domain structures as those of acostatin subunits. These results indicate that the cDNAs of heterodimeric disintegrin subunits have quite a different length of coding region and their precursors have a novel domain structure of disintegrin-family proteins.     

两个菜花烙铁头蛇毒金属蛋白酶去整合素基因的克隆与序列分析(英文)

从菜花烙铁头蛇的毒腺中 ,利用RT PCR进行体外扩增 ,克隆到 2个金属蛋白酶 去整合素基因 ,命名为TJM 1、TJM 2 .TJM 1cDNA全长为 15 2 8bp ,编码 4 81个氨基酸 ;TJM 2cDNA全长为 15 78bp ,编码 4 84个氨基酸 .TJM 1和TJM 2都属于Ⅱ型蛇毒金属蛋白酶 ,由信号肽、前肽、金属蛋白酶、间隔肽和去整合素 5部分组成 .Ⅱ型蛇毒金属蛋白酶氨基酸序列的比较及进化分析显示 ,它可进一步分为两类 ,一类包括大多数Ⅱ型蛇毒金属蛋白酶 (其中含有TJM 1) ,而TJM 2和agkistin则组成了另一类 .并且TJM 2和agkistin的第 4 0 7位和第 4 2 6位残基都是半胱氨酸 ,而在其它Ⅱ型金属蛋白酶的相应位置 ,4 0 7位是丝氨酸 ,4 2 6位则缺失 .TJM 2和agkistin均有可与整合素α2 Ⅰ区域特异性结合的片段Q NRKRHDNAQ(残基 2 76～ 2 84 ) ,这个片段在其它Ⅱ型金属蛋白酶中并没有发现 .因此推断 ,TJM 2和agkistin可能属于一类新型的Ⅱ型蛇毒金属蛋白酶 .     

Inhibition of proliferation of ECV304 cells by a disintegrin from Chinese green tree viper

A novel disintegrin, stejnin, was purified from the Trimeresurus stejnegeri venom by gel filtration and reverse phase high performance liquid chromatography. The molecular weight of stejnin was determined to be 7428 Da by MALD-TOF MS analysis. The cDNA encoding the precursor of stejnin was cloned from the venom gland. From the deduced amino acid sequence, stejnin is composed of 71 amino acid residues contains the tripeptide sequence Arg-Gly-Asp (RGD), a well-known characteristic of the disintegrin family. Stejnin strongly inhibited ADP- and ristomycin-induced human platelet aggregation with IC50 of 45 and 50 nM, respectively. Stejnin also possessed potent inhibited cell proliferation of ECV304 cells.