Local and systemic effects of BtaMP-1, a new weakly hemorrhagic Snake Venom Metalloproteinase purified from Bothriopsis taeniata Snake Venom

Highlights

• The purified BtaMP-1 is a new P-I class snake venom metalloproteinase from Bothriopsis taeniata snake venom.

• BtaMP-1 induces hemorrhage and local edema at relatively low doses.

• BtaMP-1 induce cytotoxicity on myoblast C2C12 cell line by selective degrading anchor proteins, producing cell detachment.

• BtaMP-1 induce lung hemorrhage and neutrophil infiltration and plasma fibrinogen depletion.

• BtaMP-1 induce in vivo selective tissue damage and affect hemostasis at relatively low doses and short time period.

Abstract

A new weak hemorrhagic metalloproteinase named BtaMP-1 was purified from Bothriopsis taeniata snake venom by molecular exclusion followed by anion exchange chromatographies. This protein showed a molecular mass of 25,968.16 Da and is composed of 218 amino acid residues. The multiple alignments of its partial amino acid sequence showed high structural identity with other P-I class SVMP. BtaMP-1 showed caseinolytic activity that was enhanced by Ca²⁺ ion, completely inhibited by chelating and reducing agents and can be classified as an α-fibrinogenolytic enzyme. Locally, BtaMP-1 induces hemorrhage and edema, but not myotoxicity. These findings were confirmed by histological analysis of mouse gastrocnemius muscle. “In vitro” studies suggest that BtaMP-1 induce cytotoxicity in myoblast C2C12 but not in the myotubes cell line. BtaMP-1 induced systemic alterations in mice with one MHD and two hours exposure; histological analysis of lungs showed hemorrhagic areas, congestion, and increase the thickness of alveolar septum. Also, this protein induced mild effects on kidney and disruption of coagulation by depletion of fibrinogen plasma levels. This work provides insights into the importance of BtaMP-1 biological effects in envenomation by Bothropsis taeniata snake venom and providing further evidence to understand the role of P-I class SVMP in ophidian envenomation.

Introduction

Snake venom metalloproteinases (SVMPs) are one of the most abundant protein families in snake venoms, accounting for up to 35% of the total protein content [1]. SVMPs are classified into three classes based on their domain composition [2], namely, P-I class consisting of proteins containing only the metalloproteinase domain, P-II class that contains proteins with a disintegrin-like domain in the C-terminus of the metalloproteinase domain, and P-III class that consists of proteins with a cysteine-rich domain (CRD) in addition to the two previous domains. P-III class SVMPs are further classified into P-IIIa that contains single-chain proteins, P-IIIb that contains precursors for P-I or P-II class SVMPs, P-IIIc that contains homodimers of P-IIIa and P-IIId that contains proteins with a C-type lectin-like domain anchored to the main chain by disulfide bridges. SVMPs are Zn²⁺-dependent endopeptidases and show proteolytic activity towards a broad spectrum of substrates [3]. SVMPs are responsible for local and systemic hemorrhage as a consequence of the degradation of extracellular matrix proteins such as collagen and nidogen [4]. These toxins also interfere with blood coagulation and platelet aggregation [5] and stimulate edema formation and pro-inflammatory responses [6].

P-III class SVMPs are potent hemorrhagic factors responsible for hemorrhage and other systemic effects mediated by the CRD that provides the specificity required for extracellular matrix protein degradation and interaction with target tissues. Extensive glycosylation of these proteins prevents their sequestration by plasma proteins such as α-macroglobulin [[7], [8], [9]]. In contrast, P-I class SVMPs have weak hemorrhagic activity compared to P-III class SVMPs because they lack the disintegrin-like domain and CRD. These SVMPs exert primarily local effects since their lack of glycosylation results in inhibition by α₂-macroglobulin in vitro [4,8]. However, despite this potential inhibition, several reports have shown that P-I class SVMPs can affect a variety of organs systemically, albeit at high doses and after extended periods of exposure [[10], [11], [12]]. In addition, P-I class SVMPs, with or without hemorrhagic activity, show defibrinating activity at relatively low doses and after short periods of exposure; this effect is observed within 2 h of intraperitoneal (i.p.) or intravenous (i.v.) administration [11,13,14]. Locally, P-I class SVMPs induce pro-inflammatory responses, edema and hemorrhage (although weaker than P-III class SVMPs) [15,16]. Myotoxicity induced by these proteins is mild, although the damage produced by some P-I class SVMPs can be rapid and significant [17,18]. In addition to these activities, some P-I class SVMPs are cytotoxic to a variety of cell types, primarily through their proteolytic degradation of anchorage proteins [[19], [20], [21]].

SVMPs (primarily P-I and P-III classes) account for up to 75% of proteins in Bothrops snake venoms (and those of the related genera Bothriechis, Bothriopsis and Bothrocophias) [1]. Bothriopsis taeniata, also known as the forest pit viper, is a semi-arboreal species that is widely distributed in lowland rainforests of South America, from Ecuador to Brazil [22]. Bothriopsis taeniata venom shows considerable enzymatic activity towards a variety of substrates [23,24], is hemorrhagic, and causes neutrophil recruitment in the mouse peritoneal cavity. These effects are strongly inhibited by chelating agents such as EDTA, suggesting the involvement of SVMPs [24]. The SVMPs in this venom probably contribute to the clinical signs of envenomation by B. taeniata, especially the inflammatory effects [25].

To date, only one report has described the purification of a toxin from B. taeniata venom, in this case, a phospholipase A₂ (PLA₂) [26]. Considering that few toxins have been characterized from this venom, in this report we describe the purification and biochemical characterization of a P-I class SVMP (BtaMP-1) from this venom and provide evidence for the involvement of this enzyme in local (hemorrhage, inflammation) effects and systemic alterations, including altered coagulation and selective organ damage.