Fibrinogen-clotting enzyme, pictobin, from Bothrops pictus snake venom. Structural and functional characterization
Introduction
Snake venoms, particularly those of the Viperidae family (pit vipers and true vipers) contain high levels of serine proteinases (SVSPs) and metalloproteinases (SVMPs) among other toxins, which disrupt normal physiological processes such as coagulation and hemostasis. They may cause consumption of clotting factors and hypofibrinogenemia in human victims or experimental animals [1,2]. A group of SVSPs known as fibrinogen-clotting (thrombin-like enzymes, SV-TLEs) have been investigated by their potential therapeutic application (defibrination), as diagnostic reagents and as appropriate models to study mechanism of coagulation in vitro [3,4]. SVSPs are widely distributed among the Viperid venoms and especially abundant in Bothrops venoms [5]. They belong to the trypsin S1A family of PA(S) clan, which are synthesized as zymogens (~260 aa) [[4], [5], [6]]. In spite of 57–85% amino acid sequence identity among SVSPs, they are quite specific toward a given macromolecular substrate [6]. In general, they are single-chain glycoproteins showing typical chymotrypsin-like fold with the catalytic site cleft residing between two β-barrels, with the exception of their additional sixth disulfide linkage and C-terminal extension, which are unique features conserved in venom serine peptidases [7]. Importantly, most SVSPs are glycoproteins, bearing a variable number of N-linked glycosylation sites in non-homologous positions in their sequence, resulting in comparatively high molecular masses of even up to 67-kDa. The function of carbohydrate moieties was revealed for some of these enzymes. Among these functions, the carbohydrate content plays an important role in the interaction with substrates and consequently to maintain and/or improve their catalytic activity [3,6]. These enzymes display quite diverse substrate specificity, causing their versatility in pharmacological action on hemostatic system [3,[6], [7], [8], [9], [10]]. Thus, SVSPs are classified according to their activity into nine basic group: prothrombin activators, Factor X, VII, or V activators, plasminogen activators, serine proteinase inhibitors (serpins), platelet aggregation inducers, fibrin(ogen)olytic enzymes and thrombin-like enzymes (SV-TLEs) [2,4].
Most of coagulant SV-TLEs cause clotting of fibrinogen (Fg). Their molecular mechanism is partially similar to the one thrombin. Depending on the rates and cleavage specificity, with which they cleave off the fibrinopeptide from Fg, either A (FpA) or B (FpB), or both of them, these enzymes are classified into three groups termed venombin A, B or AB [2]. Furthermore, they have significant sequence similarities with thrombin, but SV-TLEs differ considerably in several functional properties such as: 1) a distinct preference to release FPA or FPB or both, 2) the absence of allosteric modulators, 3) the insensitivity to thrombin inhibitors (antithrombin III, hirudin and heparin) for most of them, and 4) they generally do not activate another coagulation factor, but some of them may induce platelet aggregation [[5], [6], [7], [8]]. Insights into the mechanism of action of serine proteinases showed that they employ a catalytic triad of serine (Ser195), histidine (His57) and aspartate (Asp102), with the residue numbering according to the α-chymotrypsinogen system [9], which is absolutely conserved among mammalian serine proteinases [[8], [9], [10]]. The anticoagulant action of SV-TLEs may occur in two forms: (i) by pseudo-procoagulant effect in which part of the coagulation cascade is catalyzed to produce unstable and poorly cross-linked clots, which are readily torn off from the site of formation, at even low shear forces, and (ii) by true anticoagulant effects where clot formation is directly impeded [11,12]. As a consequence of these differences, endogenous Fg decreases considerable and turns the blood uncoagulable. This hemostasis-blocking effect together with vessels wall degradation by venom proteinases lead to detrimental bleeding. Among the latter are metalloproteinases, which hydrolyze key components of the subendothelial basement membrane resulting in severe hemorrhage in the snakebite victim [2,9,10]. This persistent conversion of fibrinogen into fibrin and dissolution of friable and translucent thrombi results in consumptive coagulopathy and eventually leads to the inability to form stable thrombus. Other effects reported for SV-TLEs include activation of the complement system [13,14], alteration of the nervous and muscle systems [15,16].
The pit viper Bothrops pictus (Tschudi, 1845; Desert lancehead, Jergón de la costa) is one of the medically most important venomous snake in Peru. This species occurs on arid to semiarid costal foothills, river valleys and dry lower Andean slopes of the western Andes of Peru, from the central to northern Pacific coast between the Departments of Arequipa and the south of the Department of Ancash [17]. Due to its propensity to living near human settlements, it could be found in some of the arid valleys and low mountains close to Lima city. Thus, from January 1990 to April 1998, 23 cases of bothropic accidents were reported in the northern districts (Cono Norte) of the metropolitan area of Lima and assigned to B. pictus envenoming as the unique causative agent at the National Hospital Cayetano Heredia (Lima) [18]. Furthermore, from 2014 to date 100 cases of snakebites were registered in the Department of Lima, according to Centro Nacional de Epidemiología, Prevención y Control de Enfermedades – MINSA. Notably, due to rapid access to health services all patients received adequate treatment with anti-bothropic serum (Instituto Nacional de Salud, INS) within the first six hours. This data also supports the efficacy of the antivenom, raised at INS to neutralize the main toxic effects of Bothrops venoms.
Although B. pictus is medically highly relevant due to its geographical distribution, the pathophysiological mechanism of envenomation caused by B. pictus, is poorly understood as the venom compositions of this snake has been rarely investigated from the perspective of potential lead compound development [19,20]. In a preliminary study [21] we reported the purification of pictobin, a fibrinogen-clotting enzyme from B. pictus venom called pictobin (Mr = 52 kDa). Sequence analysis revealed that it is a SV-TLE. The present work aimed to characterize the structural and functional profile of pictobin in detail. Unprecedently, we demonstrate that a snake venom serine proteinase produces mitochondrial dysfunction by affecting mitochondrial dynamics and bioenergetics in culture cells. Taken together, this report highlights that glycosylated proteinases that target Fg and produced defibrinogenation in vivo, underlines the re-emerging interest in SVSPs as potential therapeutic leads in the development of novel anti-thrombotic and anti-cancer agent.
Section snippets
Venom, antivenoms and reagents
Bothrops pictus venom was collected from four adults specimens (3 males and 1 female) captured within the district of Pachacamac (southern of Lima, Perú), and kept in captivity at the Serpentarium “Oswaldo Meneses” - Museo de Historia Natural, Universidad Nacional Mayor de San Marcos (UNMSM), Lima, Perú. CM-Sephadex C-50, Sephadex G-100 and G-75, bovine thrombin, PNGase F, and o-glycosidase, human fibrinogen essentially plasminogen free, fibrinopeptides A and B, DL-BApNA, trypsin,
Purification of pictobin
Crude venom of B. pictus was separated into six peaks by CM-Sephadex C-50 column (Fig. 1A). Peak 4 containing Fg-clotting and amidolytic activity was collected for further purification. The desalted, lyophilized fraction was subjected to gel filtration chromatography on Sephadex G-100 column (Fig. 1B). Two major peaks (P4-I and P4-II) were obtained. The active fractions (P4-I) were concentrated and submitted to another gel filtration on a Sephadex G-75 column (Fig. 1C). The active compound
Discussion
The fibrino(geno)lytic enzymes including serine and metalloproteinases are widely distributed in several South American pit viper venoms. They lead to spectacular changes in physiological process in hemostasis, in the kallikrein-kinin system and in vascular integrity. Their principal targets are Fg, prothrombin and factor X [19,29,48]. This article reports the structural and functional properties of the Fg-clotting (SV-TLE), pictobin previously isolated from Peruvian B. pictus snake venom [21].
CRediT authorship contribution statement
Dan E. Vivas-Ruiz:Conceptualization, Methodology, Investigation, Writing - original draft, Resources, Visualization.Gustavo A. Sandoval:Investigation, Resources, Writing - original draft.Edgar Gonzalez-Kozlova:Investigation, Formal analysis, Visualization.Jacquelyne Zarria-Romero:Methodology, Resources, Investigation.Fanny Lazo:Investigation, Validation.Edith Rodríguez:Investigation, Validation.Henrique P.B. Magalhães:Investigation, Resources, Writing - review & editing.Carlos Chávez-Olortegui:
Acknowledgements
We thank S. Gontijo for her technical assistance. This work was supported by Convenio de Cooperación Bilateral CONCYTEC (Perú) – CNPq (Brazil), Grant 490269/2013-3, Fundação de Amparo y Pesquisa do Estado de Minas Gerais (FAPEMIG, Brazil, Grants APQ-01858-15, AUC-00022-16), Programa de Pos Graduação in Toxinology, Instituto Butantan, SP. Programa Nacional de Innovación para la Competitividad y Productividad – Innóvate Perú (Contrato N° 131- FINCyT-2013),Vicerrectorado de Investigación y
Declaration of competing interest
The authors declared there is no conflict of interest.
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2023, International Journal of Molecular Sciences
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