BthTX-II from Bothrops jararacussu venom has variants with different oligomeric assemblies: An example of snake venom phospholipases A2 versatility
Graphical abstract
Introduction
Snake venom phospholipases A2 (svPLA2s) are small (molecular mass around 14 kDa) and stable enzymes with seven disulphide bridges. They hydrolyse in an sn-2 reaction the ester bond of phospholipids in the bilipid interface of micelles, vesicles, and membranes, releasing lysophospholipids and fatty acid molecules, pro-inflammatory mediators, in a catalytic mechanism dependent on calcium ions [1], [2]. Their enzymatic activity is related to two regions: i) the catalytic network comprehended by residues His48, Tyr52, Tyr73 and Asp99 (numbering suggested by Renetseder et al. [3]); and ii) the calcium binding loop formed by Tyr28, Gly30, Gly32 and Asp49. In the catalysis, the calcium ion anchors the substrate through coordination of the phospholipid phosphate head, residues 28, 30 and 32 main chain oxygens and Asp49 side chain oxygens. His48 extracts a proton of a water molecule that promotes hydrolysis in the sn-2 ester bond [4], [5]. The relevance of the other residues of the catalytic network, Tyr52, Tyr73 and Asp99, is to maintain the His48 position [6]. Prior to catalysis, svPLA2 needs to be in an active monomeric state acquired through its interaction with the membrane in a process known as interfacial activation [7]. The PLA2 interface-binding surface region to the membrane (known as iFace), is distinct from the catalytic residues [7].
SvPLA2s are found in almost every venomous snake family (Viperidae, Elapidae and Colubridae) [8], with exception of African non-spitting cobras (subgenus: Uraeus) [9], [10], [11]. They can make up to 30–60% of the total venom in Viperidae snakes [12] and may be the most lethal fraction, as in the case of the Micrurus fulvius venom (family Elapidae) [13]. In Viperidae snakebites, some svPLA2s can cause quickly local myonecrosis, which may lead to permanent sequelae and disability if antivenom is administered belatedly [14]. These consequences may incapacitate rural workers, as these accidents are concentrated in the countryside with difficult access to health facilities [15].
Another challenge in the treatment of snakebites is the production of a polyvalent antivenom effective among different countries and different species, as the symptoms between snakes of the same family are not distinguishable and there is a high variability in snake venom composition within species as well as across different species [16]. This diversity is related to ontogeny [17], diet [18], seasonality [19], geographical location [20] and gender [21], [22]. In fact, the World Health Organization reclassified snakebites as a neglected tropical disease [23]. 1,800,000 to 2,700,000 ophidian accidents are estimated to occur each year, from which around 5% of the cases result in death, with the highest accidents frequency in Asia, Africa and Latin America [24], [25]. In Latin America, the majority of snakebite notification is related to snakes from the Bothrops genus (American lanceheads from the Viperidae family) [26].
The pharmacological activity of svPLA2s is intriguing, with the same scaffold, they are simultaneously neuro-, myo-, cyto- and cardiotoxic, anticoagulants and edematogenic [27], some of these are independent of catalysis [28], [29]. In addition, they present an antitumor effect, possibly inhibiting the growth factor of the vascular endothelium and its receptor system that are related to angiogenesis. Thus they can be both a potential anticancer agent and enable the discovery of new drugs [30], [31].
A protein with the same fold, known as PLA2-like protein, has a natural mutation in position 49 that prevents calcium coordination [32]. Even devoid of catalytic activity, this new group of proteins causes in in vivo experiments local myotoxicity, oedema, cytokine release, leukocyte recruitment, hyperalgesia and mechanical allodynia, analgesia and tumour growth inhibition [33]. In in vitro experiments, many toxic effects, such as cytotoxicity in different human cells, bactericidal, fungicidal and antiparasite action and liposome disruption have been observed [33].
PLA2-like proteins interact with the lipid bilayer and disorder the phospholipids affecting the integrity of the membrane, in a way that has not yet been fully elucidated [2]. Functional studies point to the importance of the C-terminal region [34], [35] and the dimeric assembly [36], [37] as responsible for the calcium-independent myotoxicity. In this C-terminal region and based on structural and functional studies of PLA2-like proteins with inhibitors [38], [39], two conserved clusters of residues were proposed as responsible for myotoxicity. While a cluster of basic residues would compose the iFace and would interact with the membrane, hydrophobic residues would disorder the membrane [40] in a process described by molecular dynamics calculations using normal mode analysis [41], [42]. The inhibitors aristolochic, caffeic [39], caftaric [43], rosmarinic [38], [44], chicoric acids [45], varespladib [46], [47], suramin [48], [49] and zinc [50] were identified to interact in these regions. Site-directed mutations confirm the importance of these positive and hydrophobic clusters, composed of Lys, Arg, Leu and Phe, since their point mutation to alanine causes the recombinant protein to lose part of its myotoxic activity [34].
Some svPLA2s have an overall high distribution of basic residues beyond the C-terminal region and have a dimeric assembly similar to other PLA2-like proteins [32]. It is the case of BthTX-II from Bothrops jararacussu [51], PrTX-III from Bothrops pirajai and MT-I from Bothrops asper, which do not need calcium ions to develop their toxicity in contrast to acid svPLA2s [52]. Their crystallographic structures present distorted calcium binding loop [52], [53], [54], [55] with two possibilities of dimers in their unit cell, a large one with the β-strand in the oligomeric contact [54] and a compact one with the calcium binding loop participating in a larger hydrophobic interface [52], [55]. The latter one is suggested by free energy analysis as the biological assembly [56]. Furthermore, phylogenetic studies show these three basic PLA2s having a higher similarity to PLA2-like proteins than other PLA2s and the compact dimer found in PLA2-like toxins is very similar to found in these basic PLA2s [52].
In spite of this observation, how exactly basic PLA2s develop their biological activity independently of calcium remains elusive. Herein, we characterized BthTX-II using mass spectrometry (MS), crystallography, small angle X-ray scattering (SAXS), dynamic light scattering (DLS) and enzymatic assays. The sample obtained by chromatographic purification contains at least three toxin variants revealed by intact mass MS data. Integrating structural and genetic information within SEQUENCE SLIDER software, we characterize two of these variants with their sequence varying in seven residues. Under physiological condition, these proteins are monomeric in a relaxed(R)-state, while in sodium citrate buffer in acid pH, they are mainly dimers in a Tense(T)-state, maintaining their enzymatic activity in both conditions. The knowledge of the BthTX-II action mechanism here obtained may aid the antivenom treatment with the structure-guided development of efficient inhibitors.
Section snippets
Protein purification
The lyophilized venom of Bothrops jararacussu was purchased from Centro de Extração de Toxinas Animais LTDA (Morungaba, SP, Brasil). BthTX-II, a basic PLA2, was isolated using gel filtration with 50 mM ammonium formate pH 3.5 and Superdex™ 75 10/300 GL column (GE™) followed by reverse-phase liquid chromatography with a gradient starting at 0.1% trifluoroacetic acid and ending at 0.1% trifluoroacetic acid and 80% acetonitrile in a μRPC C2/C18 ST 4.6/100 column (GE™) using AKTA purifier UPC-900
Primary structure characterization of BthTX-II variants using mass spectrometry
BthTX-II was isolated directly from lyophilized B. jararacussu snake venom after purification through size exclusion chromatographic step followed by a reverse-phase. The intact mass of BthTX-II was determined by MS as at least three variants, whose masses correspond to 13,712.30, 13,740.47 and 13,755.64 Da (Fig. 1). These variants are slightly different from the theoretical molecular mass of BthTX-II (13,995.14 Da) calculated from the sequence available in UniProt code P45881 [71]. Moreover,
Discussion
BthTX-II is a basic PLA2 from B. jararacussu first isolated in 1988 [57] by gel filtration followed by ion-exchange chromatography, characterized by a molecular mass of 13,976 Da and had its sequence revealed by Edman degradation [71]. Here, the ion-exchange chromatography second purification step was substituted by a reverse-phase and the sequencing, by MS. The sample was globally characterized by intact mass MS obtaining three BthTX-II variants (Fig. 1). The sequences of two of these were
Conclusion
We characterized new BthTX-II variants from Bothrops jararacussu venom with a broad combination of biophysics, biochemistry and bioinformatics techniques. The protein sample, under acid conditions, is predominantly dimeric with the calcium binding loop distorted in a T-state. We raise the hypothesis that in this condition BthTX-II can develop its myotoxic activity by a mechanism independent of catalysis similar to PLA2-like proteins [52] by the interaction of positive and hydrophobic residues
CRediT authorship contribution statement
RJB: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Supervision, Project administration, Validation and Roles/Writing – original draft. GHMS: Investigation and Validation. HBC. Investigation. DCP: Data curation, Investigation and Methodology. MON Investigation and Supervision. IU: Supervision and Validation. MRMF: Project administration, Resources, Funding acquisition, Supervision and Validation. All authors: Writing – review & editing.
Declaration of competing interest
None.
Acknowledgements
The authors thank the technical support of the MS facility in LNBIO (Campinas, Brazil), MX2 beamline in LNLS (Campinas, Brazil) and X6a beamline in NSLS-I (Upton, United States of America). The authors express their gratitude for the valuable discussions with Dr. Juan J. Calvete and Dr. Fábio Florença Cardoso. R.J.B. received a fellowship from FAPESP [grant number 16/24191-8], MRMF received a fellowship from CNPq [302883/2017-7] and IU was supported by PGC [PGC2018-101370-BI00
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