Local and systemic effects of BtaMP-1, a new weakly hemorrhagic Snake Venom Metalloproteinase purified from Bothriopsis taeniata Snake Venom
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.
Section snippets
Reagents and venom
Chromatographic columns, Sephadex G-75, and DEAE 8HR AP minicolumns were purchased from Waters (USA). All substrates, reagents and kits used were of analytical grade from commercial sources (Sigma-Aldrich and BioRad). Bothriopsis taeniata venom was donated by Prof. Dr. Ronald Navarro Oviedo from the Nacional University of São Augustín, Arequipa, Peru.
Animals
Male Swiss mice (20 ± 2 g) obtained from the Multidisciplinary Center for Biological Investigation (CEMIB)/UNICAMP were housed 6/cage on a wood
SVMP purification and biochemical characterization
A combination of conventional molecular exclusion chromatography (Fig. 1A) and anion exchange chromatography coupled to HPLC (Fig. 1B) resulted in the purification of a SVMP with weak hemorrhagic activity, referred to as BtaMP-1. The purification strategy yielded a homogeneous, single-chain protein with a relative molecular weight of 25 kDa (data not shown). Further confirmation of the purity of BtaMP-1 was provided by ESI-MS/MS that yielded a molecular mass of 25,968.16 Da, in agreement with
Conclusion
The results of this investigation show that BtaMP-1 from B. taeniata venom is a new member of P-I class SVMPs. This protein is highly proteolytic but is devoid of esterase and PLA2 activities, has weak hemorrhagic activity, induces local inflammation and causes limited hemorrhage in mice. BtaMP-1 produces cytotoxicity mainly by cell detachment and can exert effects on selected organs in vivo, in addition to consuming fibrinogen and causing coagulopathy. Together, these findings suggest that
Declaration of competing interest
The authors report no conflict of interest.
Acknowledgments
The author's thanks to Mr. Paulo A. Baldasso for technical assistance, to Prof. Dr. Stephen Hyslop for reviewing and correcting the English language, and the Mass Spectrometry Laboratory at Brazilian Biosciences National Laboratory, CNPEM/ABTLUS, Campinas, Brazil, for its support with the MS analyses.
Funding
This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001, and was part of the doctoral thesis of Frank Denis Torres Huaco.
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