Purification and enzymatic characterization of a novel metalloprotease from Lachesis muta rhombeata snake venom.
Purification and enzymatic characterization of a novel metalloprotease from Lachesis muta rhombeata snake venom.
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DOI：
10.1186/s40409-018-0171-x
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Arantes EC
中科院分区：
文献类型：
--
作者：
Cordeiro FA;Coutinho BM;Wiezel GA;Bordon KCF;Bregge-Silva C;Rosa-Garzon NG;Cabral H;Ueberheide B;Arantes EC
菱形乳杆菌(Lachesis muta rhombeata，LMR)是拉丁美洲最大的毒蛇，其毒液主要含有丝氨酸和金属蛋白酶、L-氨基酸氧化酶和磷脂酶A2等酶组分。金属蛋白水解酶包括一大类锌依赖的蛋白水解酶，它们能裂解基底膜成分，如纤维连接蛋白、层粘连蛋白和IV型胶原。这些酶负责局部和全身的变化，包括出血、肌坏死和炎症。本研究旨在从LMR蛇毒(LmrV)中分离出第一个金属蛋白酶(LMR-MP)并对其进行酶学性质研究。通过两步层析纯化LMR-MP，并对其进行酶学鉴定。它对偶氮酶具有较强的蛋白水解性，在pH=7.0~9.0时活性最高。它被EDTA(一种去除锌的金属螯合剂)抑制，而PMSF、碘乙酸和胃抑素(分别是丝氨酸、半胱氨酸和天冬氨酸蛋白酶的抑制剂)对此没有影响。Ca~(2+)、Mg~(2+)、Ba~(2+)对酶有促进作用，而Al~(3+)、Cu~(2+)、Ni~(2+)、锌~(2+)对酶有抑制作用。此外，氯化锌对该酶有剂量依赖性的抑制作用。在血浆激肽释放酶(S-2302)、纤溶酶和链激酶型纤溶酶原(S-2251)和Xa因子(S-2222)的显色底物上，LMR-MP的活性也在S-2302上表现出最高的活性。在不同溶液(5 mM或50 mM碳酸氢铵，pH=7.8；0.1%三氟乙酸+ ，50%乙腈；磷酸盐缓冲液生理盐水，pH=7.4；50 mM醋酸钠，pH=4.0或醋酸铵pH=4.5)中的活性也进行了评价，结果表明，在酸性条件下，该酶的活性被消除。用MALDI-TOF测定了其分子质量(22,858Da)，并通过HCD和ETD碎片的高分辨质谱仪和数据库搜索密切相关物种的序列验证了其约90%的一级结构。它是一种新的酶，与属于P-I类的其他蛇毒金属蛋白酶(Svmps)有很高的同源性。该纯化工艺从LmrV中获得了一种新的高活性金属蛋白酶。这种新的分子可以帮助理解金属蛋白酶的作用机制，乳胶毒化，以及为其作为治疗工具的使用开辟新的视角。
Lachesis muta rhombeata (Lmr) is the largest venomous snake in Latin America and its venom contains mainly enzymatic components, such as serine and metalloproteases, L-amino acid oxidase and phospholipases A2. Metalloproteases comprise a large group of zinc-dependent proteases that cleave basement membrane components such as fibronectin, laminin and collagen type IV. These enzymes are responsible for local and systemic changes, including haemorrhage, myonecrosis and inflammation. This study aimed the isolation and enzymatic characterization of the first metalloprotease (Lmr-MP) from Lmr venom (LmrV). Lmr-MP was purified through two chromatographic steps and submitted to enzymatic characterization. It showed proteolytic activity on azocasein with maximum activity at pH 7.0–9.0. It was inhibited by EDTA (a metal chelator that removes zinc, which is essential for enzymatic activity) and no effect was observed with PMSF, iodoacetic acid or pepstatin (inhibitors of serine, cysteine and aspartyl proteases, respectively). Ca2+, Mg2+ and Ba2+ ions increased its activity, while Al3+, Cu2+, Ni2+ and Zn2+ inhibited it. Additionally, ZnCl2 showed a dose dependent inhibition of the enzyme. Lmr-MP activity was also evaluated upon chromogenic substrates for plasma kallikrein (S-2302), plasmin and streptokinase-activated plasminogen (S-2251) and Factor Xa (S-2222) showing the highest activity on S-2302. The activity in different solutions (5 mM or 50 mM ammonium bicarbonate, pH 7.8; 0.1% trifluoroacetic acid + 50% acetonitrile; phosphate buffer saline, pH 7.4; 50 mM sodium acetate, pH 4.0 or ammonium acetate pH 4.5) was also evaluated and the results showed that its activity was abolished at acidic pHs. Its molecular mass (22,858 Da) was determined by MALDI-TOF and about 90% of its primary structure was verified by high-resolution mass spectrometry using HCD and ETD fragmentations and database search against the sequence of closely related species. It is a novel enzyme which shared high identity with other snake venom metalloproteases (svMPs) belonging to the P-I group. The purification procedure achieved a novel pure highly active metalloprotease from LmrV. This new molecule can help to understand the metalloproteases mechanisms of action, the Lachesis envenoming, as well as to open new perspectives for its use as therapeutic tools.
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影响因子：
3.5
作者：
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通讯作者：
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作者：
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作者：
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DOI：
10.1186/s40409-016-0067-6
发表时间：
2016-01-01
影响因子：
2.4
作者：
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通讯作者：
Arantes, Eliane Candiani