Dekhil Hafedh, Wisner Anne, Marrakchi Naziha, El Ayeb Mohamed, Bon Cassian, Karoui Habib
Laboratoire des Venins et Toxines, Institut Pasteur de Tunis, 13 place Pasteur, 1002 Tunis Belvédère, Tunisia.
Biochemistry. 2003 Sep 16;42(36):10609-18. doi: 10.1021/bi034790b.
The venoms of Viperidae snakes contain numerous serine proteinases that have been recognized to possess one or more of the essential activities of thrombin on fibrinogen and platelets. Among them, a platelet proaggregant protein, cerastocytin, has been isolated from the venom of the Tunisian viper Cerastes cerastes. Using the RACE-PCR technique, we isolated and identified the complete nucleotide sequence of a cDNA serine proteinase precursor. The recombinant protein was designated rCC-PPP (for C. cerastes platelet proaggregant protein), since its deduced amino acid sequence is more than 96% identical to the partial polypeptide sequences that have been determined for natural cerastocytin. The structure of the rCC-PPP cDNA is similar to that of snake venom serine proteinases. The expression of rCC-PPP in Escherichia coli system allowed, for the first time, the preparation and purification of an active protein from snake venom with platelet proaggregant and fibrinogenolytic activities. Purified rCC-PPP efficiently activates blood platelets at nanomolar (8 nM) concentrations, as do natural cerastocytin (5 nM) and thrombin (1 nM). It is able to clot purified fibrinogen and to hydrolyze alpha-chains. Thus, rCC-PPP could be therefore considered a cerastocytin isoform. By comparison with other snake venom serine proteinases, a Gly replaces the conserved Cys(42). This implies that rCC-PPP lacks the conserved Cys(42)-Cys(58) disulfide bridge. A structural analysis performed by molecular modeling indicated that the segment of residues Tyr(67)-Arg(80) of rCC-PPP corresponds to anion-binding exosite 1 of thrombin that is involved in its capacity to induce platelet aggregation. Furthermore, the surface of the rCC-PPP molecule is characterized by a hydrophobic pocket, comprising the 90 loop (Phe(90)-Val(99)), Tyr(172), and Trp(215) residues, which might be involved in the fibrinogen clotting activity of rCC-PPP.
蝰蛇科蛇类的毒液含有多种丝氨酸蛋白酶，这些酶已被确认对纤维蛋白原和血小板具有一种或多种凝血酶的基本活性。其中，一种血小板促聚集蛋白——角蝰细胞毒素，已从突尼斯角蝰（Cerastes cerastes）的毒液中分离出来。利用RACE-PCR技术，我们分离并鉴定了一种cDNA丝氨酸蛋白酶前体的完整核苷酸序列。该重组蛋白被命名为rCC-PPP（代表角蝰血小板促聚集蛋白），因为其推导的氨基酸序列与已确定的天然角蝰细胞毒素的部分多肽序列有超过96%的同一性。rCC-PPP cDNA的结构与蛇毒丝氨酸蛋白酶的结构相似。rCC-PPP在大肠杆菌系统中的表达首次实现了从蛇毒中制备和纯化具有血小板促聚集和纤维蛋白原溶解活性的活性蛋白。纯化后的rCC-PPP在纳摩尔（8 nM）浓度下能有效激活血小板，天然角蝰细胞毒素（5 nM）和凝血酶（1 nM）也有同样效果。它能够使纯化的纤维蛋白原凝固并水解α链。因此，rCC-PPP可被视为角蝰细胞毒素的一种同工型。与其他蛇毒丝氨酸蛋白酶相比，一个甘氨酸取代了保守的半胱氨酸（42）。这意味着rCC-PPP缺乏保守的半胱氨酸（42）-半胱氨酸（58）二硫键。通过分子建模进行的结构分析表明，rCC-PPP的酪氨酸（67）-精氨酸（80）残基片段对应于凝血酶的阴离子结合外位点1，该位点与其诱导血小板聚集的能力有关。此外，rCC-PPP分子表面有一个疏水口袋，由90环（苯丙氨酸（90）-缬氨酸（99））、酪氨酸（172）和色氨酸（215）残基组成，这可能与rCC-PPP的纤维蛋白原凝固活性有关。
Zotero 插件