New Insights on Moojase, a Thrombin-Like Serine Protease from Bothrops moojeni Snake Venom.

Fernanda Amorim,Loic Quinton,Suely Sampaio,Marco Sartim,Danilo Menaldo,Edwin De Pauw,Sante Carone,Thiago Silva

doi:10.3390/toxins10120500

Fernanda Amorim, Loic Quinton + Show 6 more

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https://doi.org/10.3390/toxins10120500

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Journal: Toxins	Publication Date: Nov 28, 2018
Citations: 17	License type: CC BY 4.0

Affiliation: Universidade de São Paulo, University of Liège

Abstract

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 Bothrops moojeni 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.

Highlights

Bothrops moojeni snake venom is a complex mixture of components, and -omics approaches have shown it to possess a great variety of different classes of toxins [1]
The serine protease active fraction CM10 was submitted to the chromatographic step with C18 reversed-phase chromatography (Figure 1B), resulting in a major fraction identified as the active serine protease
(Moojase at 20 μg/mL with fibrinogen at 3 mg/mL for 2 h at 37 ◦ C), our results showed that the serine protease mainly generated fibrinopeptide A (Figure 6C)

Summary

Introduction

Bothrops moojeni snake venom is a complex mixture of components, and -omics approaches have shown it to possess a great variety of different classes of toxins [1]. SVSPs have been shown to have a functional heterogeneity in vivo These toxins can affect several different pathways of the blood coagulation cascade, interfering with platelet aggregation, coagulation, blood pressure, complement system, and blood fibrinogen levels [5,6,7]. These various actions led to the categorization of SVSPs into different functional subtypes: thrombin-like enzymes [8], kallikrein-like [9], plasminogen activators [10], platelet aggregation inhibitors [11], protein-C activators [12], complement convertases [13], Factor-V activators [14], specific serpin inactivators [15], and prothrombin activators [16]. Several SVSPs have been described in the literature and in spite of their high versatility, the pharmacological targets and in vivo effects of most of them are still unclear [17]

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