A Novel Plasminogen Activator from Snake Venom
A Novel Plasminogen Activator from Snake Venom
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DOI：
10.1074/jbc.270.17.10246
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发表时间：
1995-04
期刊：
影响因子：
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通讯作者：
Yun Zhang;A. Wisner;Y. Xiong;C. Bon
中科院分区：
文献类型：
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作者：
Yun Zhang;A. Wisner;Y. Xiong;C. Bon
从竹叶青蛇（Trimeresurus stejnegeri）毒液中鉴定并纯化出一种新型纤溶酶原激活剂（TSV - PA），达到均一性。它是一种单链糖蛋白，表观分子量为33000，等电点为pH 5.2。它通过一种酶促反应特异性激活纤溶酶原。TSV - PA对人天然谷氨酰纤溶酶原的激活是由于该分子在精氨酸 - 缬氨酸肽键处发生单一裂解。纯化的TSV - PA能催化几种三肽对硝基苯胺底物的水解，但它不激活也不降解凝血酶原、因子X或蛋白C，在无纤溶酶原存在时不使纤维蛋白原凝固，也不显示纤维蛋白（原）溶解活性。TSV - PA的活性易被苯甲基磺酰氟和对硝基苯基 - 对胍基苯甲酸酯抑制。根据TSV - PA的N端和内部肽段序列设计的寡核苷酸引物，用于通过聚合酶链反应扩增cDNA片段。这使得从由毒腺制备的cDNA文库中克隆出编码TSV - PA的全长cDNA。推导的TSV - PA完整氨基酸序列表明，成熟的TSV - PA蛋白由234个氨基酸组成，并在天冬酰胺处含有一个潜在的N - 糖基化位点。TSV - PA的序列与其他蛇毒蛋白酶具有高度的序列同一性：与铜头蝮蛇（Agkistrodon contortrix contortrix）毒液中的蛋白C激活剂有66%的同一性，与巴曲酶有63%的同一性，与蝰蛇（Russell's viper）毒液中的因子V激活剂有60%的同一性。另一方面，TSV - PA与尿激酶型纤溶酶原激活剂（u - PA）和组织型纤溶酶原激活剂（t - PA）的催化结构域仅有21 - 23%的序列相似性。此外，TSV - PA缺乏已被证明负责t - PA（KHRR）和u - PA（RRHR）与纤溶酶原激活剂抑制剂1型相互作用的序列位点。
A novel plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) has been identified and purified to homogeneity. It is a single chain glycoprotein with an apparent molecular weight of 33,000 and an isoelectric point of pH 5.2. It specifically activates plasminogen through an enzymatic reaction. The activation of human native Glu-plasminogen by TSV-PA is due to a single cleavage of the molecule at the peptide bond Arg-Val. Purified TSV-PA, which catalyzes the hydrolysis of several tripeptide p-nitroanilide substrates, does not activate nor degrade prothrombin, factor X, or protein C and does not clot fibrinogen nor show fibrino(geno)lytic activity in the absence of plasminogen. The activity of TSV-PA was readily inhibited by phenylmethanesulfonyl fluoride and by p-nitrophenyl- p-guanidinobenzoate. Oligonucleotide primers designed on the basis of the N-terminal and the internal peptide sequences of TSV-PA were used for the amplification of cDNA fragments by polymerase chain reaction. This allowed the cloning of a full-length cDNA encoding TSV-PA from a cDNA library prepared from the venom glands. The deduced complete amino acid sequence of TSV-PA indicates that the mature TSV-PA protein is composed of 234 amino acids and contains a single potential N-glycosylation site at Asn. The sequence of TSV-PA exhibits a high degree of sequence identity with other snake venom proteases: 66% with the protein C activator from Agkistrodon contortrix contortrix venom, 63% with batroxobin, and 60% with the factor V activator from Russell's viper venom. On the other hand, TSV-PA shows only 21-23% sequence similarity with the catalytic domains of u-PA and t-PA. Furthermore, TSV-PA lacks the sequence site that has been demonstrated to be responsible for the interaction of t-PA (KHRR) and u-PA (RRHR) with plasminogen activator inhibitor type 1.