Laboratório de Toxinologia, Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto 14040-903, Brazil.
Laboratory of Mass Spectrometry, Department of Chemistry, University of Liège, 4000 Liège, Belgium.
Toxins (Basel). 2018 Nov 28;10(12):500. doi: 10.3390/toxins10120500.
Snake venom serine proteases (SVSPs) are enzymes that are capable of interfering in various parts of the blood coagulation cascade, which makes them interesting candidates for the development of new therapeutic drugs. Herein, we isolated and characterized Moojase, a potent coagulant enzyme from snake venom. The toxin was isolated from the crude venom using a two-step chromatographic procedure. Moojase is a glycoprotein with N-linked glycans, molecular mass of 30.3 kDa and acidic character (pI 5.80⁻6.88). Sequencing of Moojase indicated that it is an isoform of Batroxobin. Moojase was able to clot platelet-poor plasma and fibrinogen solutions in a dose-dependent manner, indicating thrombin-like properties. Moojase also rapidly induced the proteolysis of the Aα chains of human fibrinogen, followed by the degradation of the Bβ chains after extended periods of incubation, and these effects were inhibited by PMSF, SDS and DTT, but not by benzamidine or EDTA. RP-HPLC analysis of its fibrinogenolysis confirmed the main generation of fibrinopeptide A. Moojase also induced the fibrinolysis of fibrin clots formed in vitro, and the aggregation of washed platelets, as well as significant amidolytic activity on substrates for thrombin, plasma kallikrein, factor Xia, and factor XIIa. Furthermore, thermofluor analyses and the esterase activity of Moojase demonstrated its very high stability at different pH buffers and temperatures. Thus, studies such as this for Moojase should increase knowledge on SVSPs, allowing their bioprospection as valuable prototypes in the development of new drugs, or as biotechnological tools.
蛇毒丝氨酸蛋白酶 (SVSPs) 是能够干扰血液凝固级联反应各个部分的酶，这使它们成为开发新治疗药物的有趣候选物。在此，我们从蛇毒液中分离并鉴定了 Moojase，一种有效的凝血酶。该毒素是通过两步色谱程序从粗毒液中分离得到的。Moojase 是一种带有 N-连接聚糖的糖蛋白，分子量为 30.3 kDa，呈酸性（pI 5.80⁻6.88）。Moojase 的测序表明它是 Batroxobin 的同工型。Moojase 能够以剂量依赖的方式凝结血小板贫血浆和纤维蛋白原溶液，表明具有类凝血酶特性。Moojase 还能迅速诱导人纤维蛋白原 Aα 链的蛋白水解，然后在延长孵育时间后降解 Bβ 链，这些作用被 PMSF、SDS 和 DTT 抑制，但被苯甲脒或 EDTA 抑制。其纤维蛋白原水解的 RP-HPLC 分析证实主要生成纤维蛋白肽 A。Moojase 还诱导体外形成的纤维蛋白凝块的纤维蛋白溶解，以及洗涤血小板的聚集，以及对凝血酶、血浆激肽释放酶、因子 Xia 和因子 XIIa 的底物具有显著的氨肽酶活性。此外，Moojase 的热荧光分析和酯酶活性表明其在不同的 pH 缓冲液和温度下具有非常高的稳定性。因此，对 Moojase 的此类研究应该增加对 SVSPs 的了解，允许将其生物勘探作为开发新药的有价值原型，或作为生物技术工具。
