Integrative characterization of the venom of the coral snake Micrurus dumerilii (Elapidae) from Colombia: Proteome, toxicity, and cross-neutralization by antivenom Paola Rey-Su´arez, Vitelbina N´un˜ ez, Juli´an Fern´andez, Bruno Lomonte PII: DOI: Reference:

S1874-3919(16)30029-X doi: 10.1016/j.jprot.2016.02.006 JPROT 2412

To appear in:

Journal of Proteomics

Received date: Revised date: Accepted date:

24 January 2016 10 February 2016 11 February 2016

Please cite this article as: Rey-Su´arez Paola, N´ un ˜ ez Vitelbina, Fern´andez Juli´ an, Lomonte Bruno, Integrative characterization of the venom of the coral snake Micrurus dumerilii (Elapidae) from Colombia: Proteome, toxicity, and cross-neutralization by antivenom, Journal of Proteomics (2016), doi: 10.1016/j.jprot.2016.02.006

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ACCEPTED MANUSCRIPT Integrative characterization of the venom of the coral snake Micrurus dumerilii (Elapidae) from Colombia: proteome, toxicity,

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and cross-neutralization by antivenom

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Paola Rey-Suárez‡1, Vitelbina Núñez 1,2, Julián Fernández3, Bruno Lomonte 3

Programa de Ofidismo y Escorpionismo, Universidad de Antioquia, Medellín, Colombia

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica,

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Escuela de Microbiología, Universidad de Antioquia, Medellín, Colombia

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San José, Costa Rica

Keywords: Micrurus dumerilii, coral snake, venom, elapid toxins, proteomics, venomics

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Address correspondence to: Paola Rey Suárez, MSc Programa de Ofidismo/Escorpionismo Universidad de Antioquia Medellín, Colombia E-mail: [email protected] Tel. (+57) 42196649

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ABSTRACT In Colombia, nearly 2.8% of the 4200 snakebite accidents recorded annually are

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inflicted by coral snakes (genus Micrurus). M. dumerilii has a broad distribution in this

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country, especially in densely populated areas. The proteomic profile of its venom was here studied by a bottom-up approach combining RP-HPLC, SDS-PAGE and MALDI-

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TOF/TOF. Venom proteins were assigned to eleven families, the most abundant being

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phospholipases A2 (PLA2; 52.0%) and three-finger toxins (3FTx; 28.1%). This compositional profile shows that M. dumerilii venom belongs to the 'PLA2-rich' phenotype,

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in the recently proposed dichotomy for Micrurus venoms. Enzymatic and toxic venom activities correlated with protein family abundances. Whole venom induced a conspicuous

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myotoxic, cytotoxic and anticoagulant effect, and was mildly edematogenic and proteolytic,

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whereas it lacked hemorrhagic activity. Some 3FTxs and PLA2s reproduced the lethal

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effect of venom. A coral snake antivenom to M. nigrocinctus demonstrated significant cross-recognition of M. dumerilii venom proteins, and accordingly, ability to neutralize its lethal effect. The combined compositional, functional, and immunological data here reported for M. dumerilii venom may contribute to a better understanding of these envenomings, and support the possible use of anti-M. nigrocinctus coral snake antivenom in their treatment. (191 words)

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BIOLOGICAL SIGNIFICANCE Coral snakes represent a highly diversified group of elapids in the New World, with

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nearly 70 species within the genus Micrurus. Owing to their scarce yields, the biochemical

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composition and toxic activities of coral snake venoms have been less well characterized than those of viperid species. In this work, an integrative view of the venom of Micrurus

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dumerilii, a medically relevant coral snake from Colombia, was obtained by a combined

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proteomic, functional, and immunological approach. The venom contains proteins from at least eleven families, with a predominance of phospholipases A2 (PLA2), followed by three-

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finger toxins (3FTx). According to its compositional profile, M. dumerilii venom can be grouped with those of several Micrurus species from North and Central America that

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present a PLA2-predominant phenotype, to date it is the most southerly coral snake species

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to do so. Other coral snake species that a 'PLA2-rich' venom, M. dumerilii venom contains

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both components that form MitTx, a pain-inducing heterodimeric complex recently characterized from the venom of M. tener, also present in M. mosquitensis and M. nigrocinctus venoms. In addition to a lethal three-finger toxin, PLA2s participate in the toxicity of M. dumerilii venom, some of them displaying ability to induce cytolysis, muscle necrosis, and lethality to mice. An antivenom to M. nigrocinctus demonstrated significant cross-recognition of M. dumerilii venom proteins, and accordingly, ability to neutralize its lethal effect, being of potential therapeutic usefulness in these envenomings.

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INTRODUCTION In the New World, the family Elapidae includes coral snakes, classified within three

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genera: Leptomicrurus, Micruroides, and Micrurus. The latter is the most abundant and

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diverse, comprising about 70 species, distributed from the southern United States to northeastern Argentina, in a wide range of climates and habitats [1, 2]. Like their Old

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World counterparts, these elapids have a proteroglyphous dentition connected to

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specialized venom glands that produce a variety of toxins, allowing them to rapidly subdue prey. With few exceptions, coral snakes have crepuscular and semifossorial habits, and

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have a restricted diet that mainly includes small snakes and lizards [2]. Out of the 80,000-129,000 snakebite envenoming cases estimated to occur annually

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in the American continent, coral snakes cause fewer than 5% [3-5]. However, neurotoxicity

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and potential lethality in envenomings by coral snakes underscore their medical relevance.

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Clinical features of these envenomings include palpebral ptosis, ophthalmoplegia, sialorrhea, and paralysis of the jaw, larynx, neck, and limb muscles. In severe cases, respiratory and cardiac arrest may occur, causing death [6-10]. Specific therapy for coral snake envenomings is based on the intravenous administration of antivenoms of heterologous origin which, however, vary in their paraspecific neutralizing ability due to antigenic differences among toxins from diverse species [4, 11-18]. In addition, it has been noted that the ability of antivenoms to inmunorecognize venom proteins correlates with their molecular mass, with weakest recognition towards low molecular weight proteins such as the three-finger toxins [19]. According to reports by the epidemiological surveillance system of Colombia [20], in 2014 coral snakes were responsible for at least 116 human envenoming cases, representing 2.8% of all snakebites recorded. Although it is not possible to ascribe these

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accidents to particular Micrurus species, it is likely that M. mipartitus and M. dumerilii represent the main species involved, on the basis of their broad distribution in all of the

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Colombian territory, especially in the densely populated areas.

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In previous studies, we analyzed the venom proteome of M. mipartitus from Colombia [21], and characterized Mipartoxin-I, its major neurotoxic component [22].

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However, the venom of M. dumerilii still remained largely unexplored, despite the medical

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relevance of this species.

M. dumerilii is a tricolored coral snake with broad red rings, bordered by narrow

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white rings with a medium black ring between them [1, 2]. It is a medium sized snake (adults ranging from 50 to 70 cm in length) found from the western lowlands of the Andes

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in Colombia, northern Ecuador, and interior valleys of the Colombian Andes, including the

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Western slopes of the Cordillera Oriental and Caribbean lowlands of Colombia eastward to

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Venezuela [1] (Fig.1). Studies on the protein composition, and the enzymatic and toxic activities of M. dumerilii venom have been scarce. Serafim et al. [23] demonstrated that this venom induces neuromuscular blockade in rat and avian preparations mainly due to postsynaptic action, and that it inhibits the twitch-tension elicited by indirect stimulation and the response to acetylcholine, without producing blockade of the response to KCl in chicks. In other studies, the venom of M. carinicauda (also classified as M. dumerilii carinicauda) was shown to induce prominent local myonecrosis after intramuscular injection in mice, but no hemorrhage or edema, at sublethal doses [24]. Tan and Ponnudurai [25] compared the enzymatic activities of several Micrurus venoms, and reported the presence of low proteolytic and phosphodiesterase activities in M. dumerilii venom, together with high phosphomonoesterase, L-amino acid oxidase, phospholipase A2, nucleotidase, and hyaluronidase activities. Only one toxin from this venom has been

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isolated and characterized, a phospholipase A2 that displays presynaptic neurotoxicity in vertebrate nerve-muscle preparations [26, 27].

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Aiming to expand knowledge on the medically relevant coral snakes of Colombia,

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in the present work we have determined the proteomic profile of M. dumerilii venom, in combination with the screening of its enzymatic and toxic activities, as well as its cross-

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recognition and neutralization by a coral snake antivenom raised against the venom of M.

2. MATERIALS AND METHODS

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2.1. Venom

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nigrocinctus from Costa Rica.

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The venom was a pool from 15 adult M. dumerilii kept at the Serpentarium of

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Universidad de Antioquia, Colombia. Venom was obtained by manual extraction, lyophilized, and stored at -20°C. Other venoms included for comparison in some assays were provided by the Serpentarium of Instituto Clodomiro Picado.

2.2 RP-HPLC and SDS-PAGE Two mg of venom were dissolved in 200 μL of 0.1% trifluoroacetic acid (TFA; solution A), centrifuged for 5 min at 5,000×g to remove debris, and separated by reversephase HPLC on a C18 column (250 × 4.6 mm, 5 μm particle size; Discovery Bio, Supelco) using an Agilent 1220 chromatograph with monitoring at 215 nm. Elution was performed at 1 mL/min by applying the following gradient toward solution B (acetonitrile, containing 0.1% TFA): 5% B for 5min, 5–15% B over 10 min, 15–45% B over 60 min, and 45–70% B over 12 min, as described [21]. Fractions were collected manually, dried by vacuum

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centrifugation, redissolved in water, and further separated on 12% SDS-polyacrylamide gels under reducing conditions (5% 2-mercaptoethanol, at 100 °C for 5 min), in a Mini-

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Protean electrophoresis system (Bio-Rad) run at 150 v. Proteins were visualized by

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2.3 Mass spectrometry and protein identification

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Coomassie blue R-250 staining.

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Protein bands were excised from gels and subjected to reduction with dithiothreitol and alkylation with iodoacetamide, followed by overnight in-gel digestion with sequencing

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grade bovine trypsin, as described [21]. The resulting peptides were analyzed by MALDITOF-TOF mass spectrometry on an Applied Biosystems 4800-Plus instrument (ABSciex).

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Mixtures of 0.5 µL of α-cyano-4-hydroxycinnamic acid and 0.5 µL of each sample were

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spotted onto an Opti-TOF 384 plate, dried, and analyzed in positive reflector mode. Spectra

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were acquired after 500 shots at a laser intensity of 3000, using as external standards CalMix-5 (ABSciex) spotted on the same plate. Up to 10 precursor ions from each MS spectrum were selected for automated collision-induced dissociation MS/MS spectra acquisition at 2KV, in positive mode (500 shots/spectrum, laser intensity of 3800). The resulting spectra were searched using ProteinPilot v.4 and the Paragon® algorithm (ABSciex) against the UniProt/SwissProt database for Serpentes (20150217), at a confidence level of ≥95%, for protein identification or protein family assignment by similarity. The relative abundance of each protein (expressed as % of total venom proteins) was estimated by integration of the chromatographic peak areas at 215 nm, using ChemStation C.01.06 (Agilent Technologies). When a peak from HPLC contained two or more SDS-PAGE bands, their relative distributions were estimated by densitometry using ImageLab (Bio-Rad), as previously described [28].

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2.4 Biological activities in vitro

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2.4.1 Coagulant and anticoagulant activity The coagulant effect of M. dumerilii venom was determined by recording the

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clotting times after addition of 20 μg (in 50 μL of phosphate-buffered saline; PBS; 0.12 M

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NaCl, 40 mM sodium phosphate, pH 7.2) to 200 μL of citrated human plasma, incubated at

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37°C [29]. Addition of PBS alone to plasma aliquots was used as a negative control, and all assays were performed in triplicates. The anticoagulant activity was tested by preincubating

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10 μg of venom (in 10 μL PBS) with 500 μL of citrated human plasma for 10 min at 37°C, in triplicates. Then, clotting times were recorded after adding 100 μL of 0.25 M CaCl 2.

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2.4.2 Proteolytic activity

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Plasma aliquots preincubated with PBS were used as controls.

Proteolysis was determined using azocasein as substrate [30]. Twenty µg of M. dumerilii venom (in 20 μL of 25 mM Tris, 0.15 M NaCl, 5 mM CaCl2, pH 7.4) were added to 100 μL of azocasein (10 mg/mL, dissolved in the same buffer), and incubated for 90 min at 37°C. The reaction was stopped by adding 200 μL of 5% trichloroacetic acid. After centrifugation, 100 μL of each supernatant were transferred to 96-well microplates, mixed with 100 μL of 0.5 M NaOH, and absorbances were recorded at 450 nm using a Multiskan (Thermo) reader. Activity was expressed as the increase of absorbance at 450 nm in comparison to the substrate alone.

2.4.3 L-amino acid oxidase activity

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L-amino acid oxidase activity was measured in triplicate using microplates, where 20 μg of M. dumerilii venom (in 10 μL of water) was added to 90 μL of a reaction mixture

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containing 250 μM L-Leucine, 2 mM o-phenylenediamine, and 0.8 U/mL horseradish peroxidase, in 50 mM Tris, pH 8.0 buffer. After incubation at 37°C for 60 min, the reaction

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was stopped with 50 μL of 2 M H2SO4, and absorbances were recorded at 492 nm [31].

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2.4.4 Cytotoxicity in C2C12 myotubes

The cytolytic activity of M. dumerilii venom was assayed on the myogenic C2C12

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cell line of murine origin (ATCC CRL-1772), differentiated to the myotube stage, as previously described [32]. Venom (40 μg) in 150 µL of assay medium (Dulbecco's

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modified Eagle Medium supplemented with 1% fetal bovine serum) was added to myotubes

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in 96-well plates. After 3 h of incubation at 37°C, supernatants were collected and the

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activity of lactate dehydrogenase (LDH) released from damaged cells was determined using a kinetic UV assay (LDH, Biocon). Controls for 0 and 100% cytotoxicity consisted of culture medium alone, and medium containing 0.1% Triton X-100, respectively. Experiments were carried out in triplicates.

2.4.5 Phospholipase A2 activity Phospholipase A2 (PLA2) activity of M. dumerilii venom was tested on the synthetic monodisperse substrate 4-nitro-3-octanoyloxy-benzoic acid (4-NOBA; [33]) and on micellar phosphatidylcholine (PC) by the phenol red colorimetric method [34]. In the first method, variable amounts of venoms (in 25 μL of 10 mM Tris, 10 mM CaCl2, 100 mM NaCl, pH 8.0 buffer) were added to microplate wells, mixed with 25 μL of substrate (1 mg/mL in acetonitrile) and 250 μL of the same buffer. After incubation for 60 min at 37°C,

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absorbances were recorded at 405 nm in a microplate reader and activity was expressed as the absorbance change in comparison to the substrate alone. In the second method, 20 μL

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containing various amounts of venom were added to 1 mL of substrate (0.25 % w/v sn-3phosphatidylcholine, 0.4 % v/v Triton X-100, 0.004% w/v phenol red) in a thermoregulated

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cuvette at 30 oC. After a stabilization period of 20 s, the decrease in absorbance at 558 nm

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was monitored continuously for 1 min. One unit of PLA2 activity was defined as the change

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of 0.001 in absorbance per min. Assays were performed in triplicate.

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2.5 Biological activities in vivo

All in vivo experiments were performed in Swiss-Webster mice (18–20 g body

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weight, for venom lethality), or in CD-1 mice (16–18 g for screening lethality of venom

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fractions, or 18–20 g for evaluating local tissue damaging effects). Assays followed

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protocols approved by the Institutional Committee for the Use and Care Laboratory Animals (CICUA, University of Costa Rica; 82–08, 132–13), and the Ethics Committee for Animal Testing (CEEA, University of Antioquia; Resolución Rectoral 18084).

2.5.1 Lethality

Varying doses of M. dumerilii venom, dissolved in 200 µL PBS, were injected into groups of four mice, by the intraperitoneal route. A control group received PBS alone. Animals were observed up to 48 h after injection to record deaths. The median lethal dose (LD50) was calculated by the Spearman–Karber method [35], using Toxicalc software [36].

2.5.2 Myotoxicity

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A group of four mice received an intramuscular injection containing 20 μg of M. dumerilii venom (in 50 μL PBS) into the gastrocnemius. Control mice received an injection

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of PBS. Blood was collected after 3 h from the tip of the tail into heparinized capillaries

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and the activity of creatine kinase (CK) in plasma was determined using an UV kinetic assay (CK-Nac, Biocon) [37]. Myotoxicity was also assessed by histological evaluation.

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Six h after injection, mice were sacrificed by inhalation of carbon dioxide and samples of

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gastrocnemius muscle were obtained for histological processing of formalin-fixed sections,

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hematoxylin-eosin staining, and microscopic evaluation.

2.5.3 Edematogenic activity

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A group of four mice received a subcutaneous injection of M. dumerilii venom (20

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µg in 50 µL PBS) in the left footpad. The right footpad, injected with 50 μL of PBS alone,

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was used as a negative control. Edema was estimated at different time points (0.5, 1, 3, 6 and 24 h) by measuring the footpad thickness with a low-pressure spring caliper (Ultra-Cal III Sylvac) as described [38].

2.5.4 Hemorrhagic activity

Groups of four mice received an intradermal injection of M. dumerilii venom (20 μg in 100 μL of PBS) in the abdominal skin. After 2 h, animals were euthanized by carbon dioxide inhalation, and their skins were dissected to measure the hemorrhagic areas [39].

2.6 Functional characterization of the main venom fractions Lethal, myotoxic and cytotoxic activities were assayed for the most abundant M. dumerilii venom HPLC fractions (5, 17, 19, 26, 28 and 29; Table 1). Additionally, fractions

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7 and 21 were included in the lethality assay only, due to the low amounts available. Protein concentrations were adjusted by measuring the absorbance at 280 nm on a

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NanoDrop reader (Thermo). In order to assess lethality, 20 μg of each fraction, dissolved in

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100 µL PBS, were injected into groups of three mice by the intravenous route, and deaths were scored after 48 h. For assessing myotoxic activity, the assay was performed as

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described in section 2.5.2, injecting 5 μg of each fraction intramuscularly, in three mice.

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Cytotoxic activity was determined as described in section 2.4.4, adding 20 μg of each

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fraction to myotubes, in triplicates.

2.7 Immunorecognition of M. dumerilii venom and its fractions by anti-M. nigrocinctus

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equine antivenom

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Cross-recognition of M. dumerilii venom, or its HPLC-resolved fractions, by an

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equine monospecific antivenom to M. nigrocinctus (SAC-ICP; Instituto Clodomiro Picado, University of Costa Rica; batch 520, expiry date March 2016) was tested by enzymeimmunoassay (ELISA). Microplates (Nunc) were coated with 1 µg/well of M. dumerilii venom or fractions, diluted in 100 µL of 0.1 M Tris, 0.15 M NaCl, pH 9.0 buffer, overnight at room temperature. Plates were washed five times with PBS, blocked for 60 min with 100 µL/well of PBS containing 2% bovine serum albumin (BSA), and decanted. Then, 100 µL of antivenom (serially diluted 1:500 to 1:64.000 for the whole venom assay, or 1:1000 for the fraction recognition assay), or an identical preparation of immunoglobulins from a nonimmunized horse as a control, in PBS-BSA, were added and incubated for 2 hr. Plates were washed five times with PBS, followed by the addition of 100 µL of anti-equine immunoglobulins-alkaline phosphatase conjugate (Sigma), diluted 1:4000 in PBS-BSA, and incubated for 2 hr. Finally, five washings were performed with PBS, and color was

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developed with 1 mg/ml p-nitrophenylphosphate in diethanolamine buffer, pH 9.8. Absorbances of triplicate wells were recorded at 405 nm. For comparative assessment of

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the immunorecognition of the homologous antigen, M. nigrocinctus venom was included in

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these assays.

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2.8 Neutralization of M. dumerilii venom by anti-M. nigrocinctus equine antivenom

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The ability of the equine SAC-ICP antivenom against M. nigrocinctus to neutralize the lethal activity of M. dumerilii venom was assessed by preincubation-type experiments.

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Groups of four mice (16-18 g) were injected i.p. with 500 μL of a solution that contained 60 µg of venom (equivalent to 3×LD50), which had been previously incubated for 30 min at

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37°C with antivenom, at a ratio of 200 μg venom/mL antivenom. A control group of mice

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recorded after 48 h.

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received the same dose of venom, incubated with PBS instead of antivenom. Deaths were

3. RESULTS AND DISCUSSION 3.1 Proteome of M. dumerilii venom Traditionally, studies on coral snake venoms have been limited by the low amounts obtained, and the difficulties of maintaining these snakes in captivity. Owing to the increasing sensitivity of current –omics analytical platforms, studies of the protein composition of coral snake venoms have become more feasible. However, knowledge on the venoms of the wide diversity of coral snakes, with nearly 70 species within the genus Micrurus, is only partial. Micrurus species with venom proteomes and/or transcriptomes that have been characterized include M. surinamensis [40], M. corallinus [41], M.

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altirostris [41], M. nigrocinctus [19], M. mipartitus [21], M. multifasciatus (also described as M. mipartitus from Costa Rica; [21]), M. frontalis [18], M. ibiboboca [18], M.

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lemniscatus [18], M. fulvius [42, 43], M. tener [44], M. mosquitensis [45], and M. alleni

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[45]. In the present work, we focused on the venom of M. dumerilii, a medically relevant coral snake species in Colombia, the overall venom characteristics of which were largely

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unknown.

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M. dumerilii venom was initially separated into 38 fractions by RP-HPLC. These were further resolved into a total of 62 electrophoretic bands (Fig.2). As expected from the

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chromatographic protocol used, the peaks eluting first (1-4) did not show protein bands by electrophoresis. These fractions could contain small peptides or non-proteinaceous

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components, for example adenosine, a nucleoside which has been detected in high amounts

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in the venoms of M. nigrocinctus, M. corallinus [46], and M. alleni [45], among coral

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snakes. However, the possible presence of adenosine in these first peaks of M. dumerilii venom was ruled out by mass spectrometry, as these did not present the parent/daughter ions of m/z 268+1/136+1 characteristic of this nucleoside. Further characterization of the early eluting peaks, which accounted for 2.8% of the total venom chromatographic area, was not pursued. However, it is interesting to point out that differences regarding the presence of adenosine among the different species of Micrurus exist, since its possible biological roles are still incompletely understood [47]. On the other hand, venom fractions 5 to 38 presented protein bands in SDS-PAGE (Fig.2) and all of the 62 bands obtained after this separation step could be assigned to protein families by MALDI-TOF/TOF analysis of their tryptic peptides (Table 1). Estimation of protein abundances by integration of peak areas, in combination with densitometry of electrophoretic bands, provided a general view of the proteome

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composition of M. dumerilii venom, as represented in Fig.3. Proteins belonging to 11 families were identified, with phospholipases A2 (PLA2) being clearly predominant

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(52.0%), followed by three-finger toxins (3FTx; 28.1%). Altogether, these two protein

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types account for 80% of the venom proteins, the rest being distributed, in descending proportions, by Kunitz-type trypsin inhibitor proteins (Kun), L-amino acid oxidases (LAO),

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serine proteases (SP), metalloproteinases (SVMP), C-type lectin/lectin-like proteins, and

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traces of phosphodiesterase (PDE), 5'-nucleotidase (5'nuc), phospholipase B (PLB), and hyaluronidase (Hya) proteins (Fig.3).

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A recent study revealed that Micrurus venoms studied to date show a compositional divergence, presenting either a PLA2-predominant or a 3FTx-predominant phenotype [45].

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The compositional data here obtained on M. dumerilii venom indicates that it presents a

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PLA2-predominant phenotype, resembling in this regard the venoms of M. fulvius, M. tener,

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M. nigrocinctus, and M. mosquitensis. This finding extends the range of coral snake species thus far observed to display the PLA2-predominant venom phenotype, from North and Central America, into the northern region of South America (Colombia), making M. dumerilii the geographically southern-most coral snake species displaying this characteristic.

A further observation of interest on M. dumerilii venom is the presence of both components that form the MitTx heterodimer, a complex originally discovered in the venom of M. tener, consisting of a Kunitz-type serine protease inhibitor (Mit-α) and a catalytically-inactive PLA2 homologue (Mit-β), involved in pain induction through its binding to acid-sensing receptors [48]. Since this kind of proteins has also been detected in the venoms of M. mosquitensis and M. nigrocinctus [45], both corresponding to the PLA2rich phenotype, the present findings point to a possible association between these two

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characteristics. An exception, however, may be the venom of M. fulvius, since in spite of expressing a PLA2-rich phenotype [43], a transcriptomic analysis of the venom gland did

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not find evidence of components related to those forming the MitTx heterodimer [42].

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Further studies are needed to determine the distribution pattern of this particular type of

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toxin across the venoms of Micrurus.

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3.2 Functional characteristics of M. dumerilii venom

The predominant effect displayed by Micrurus venoms is neurotoxicity, commonly

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associated with the presence of 3FTx and PLA2 proteins. The former often act as α– neurotoxins, binding to the nicotinic acetylcholine receptor to postsynaptically block

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transmission at the neuromuscular junction [49, 50]. A postsynaptic effect has been

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demonstrated for the crude venoms in some coral snake species such as M. lemniscatus

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[51], M. pyrrhocryptus [52], M. mipartitus [53], and M. dissoleucus [53]. On the other hand, a number of elapid venom PLA2s are known to act as β-neurotoxins, binding to undefined presynaptic sites at the neuromuscular junction, and interfering with neurotransmitter release by a mechanism that depends on their hydrolytic action on membrane phospholipids [54-56]. Presynaptic neurotoxicity has been reported for other coral snake venoms such as M. corallinus [57], M. altirostris [58], M. laticollaris [59] and M. dumerilii [23]. The median lethal dose (LD50) of M. dumerilii venom in mice was estimated at 1.18 g/g body weight (95% confidence limits: 0.75- 1.90 g/g), by the i.p route. In comparison with some other Micrurus venoms, for example those that display a 3FTx-rich phenotype, this lethal potency can be considered lower. The predominance of PLA2s in M. dumerilii venom suggested that this group of proteins could play a relevant role in its toxic activities.

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Indeed, a screening for lethality by i.v. injection of the most abundant venom fractions showed that peaks 21 and 26, corresponding to PLA2s (Table 1), were lethal to mice.

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However, lethal activity was also recorded for fraction 7, corresponding to a 3FTx (Table

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1), showing that both toxin groups contribute to the toxicity of this venom. Experimental studies have shown an extended spectrum of toxic activities for

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Micrurus venoms. These include myotoxicity [60, 61], cardiotoxicity [62], intravascular

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hemolysis [63], edema [64], nephrotoxicity [61], hemostatic alterations [65, 66] and, rarely, hemorrhagic activity [64], varying among species. The venom of M. dumerilii caused

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prominent myotoxicity when injected into the gastrocnemius of mice, inducing a marked increase in the plasma levels of creatine kinase (Fig.4) and a clear histological pattern of

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muscle fiber necrosis with only mild neutrophil infiltration (Fig.5). Similar effects have

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been recorded after injection of M. nigrocinctus venom [24, 60]. This is in contrast to the

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lack of myotoxicity described for M. mipartitus venom, tested under identical conditions [21]. A previous study had reported the ability of M. dumerilii (described as M. carinicauda) venom to increase plasma CK levels in mice [24], in agreement with present results. Additionally, Serafim et al. [23] described changes in the sarcolemma when this venom was added to preparations of isolated phrenic nerve-diaphragm muscle and chick biventer cervicis. However, in spite of the clear myotoxic activity of a number of coral snake venoms at the experimental level, clinical reports on myotoxicity in coral snakebite envenomings are lacking. It is possible that muscle necrosis is overlooked in humans owing to the low amounts of venom injected by these snakes, hence the lack of macroscopically prominent local effects. Another contributing factor is probably that specific laboratory tests such as creatine kinase determination and urinalysis are not performed on a routine basis [24].

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Knowing that the enzymatic activity of PLA2s is one of the mechanisms described for the induction of myotoxicity by snake venoms [67, 68], this activity was evaluated

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using two types of substrates: 4-NOBA and PC. Whole venom (Fig.6), as well as fractions

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identified as PLA2s (Table 2), demonstrated hydrolytic activity on both substrates. M. dumerilii venom had higher activity on the monodisperse 4-NOBA substrate than M.

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nigrocinctus venom, included as a reference (Fig.6A), but lower activity on the micellar PC

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substrate (Fig.6B), indicating that variations in substrate preference among the multiple PLA2 isoforms present in both venoms exist. Additionally, this venom demonstrated a high

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anticoagulant activity in vitro, increasing the coagulation time six-fold over control plasma, being higher than the activity described for M. mipartitus venom [22].

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The venom of M. dumerilii displayed cytotoxic activity, as evidenced by its activity

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on the murine myotubes of the C2C12 cell line, similar to the venom of M. nigrocinctus

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(Fig.7A), used as a control [69]. PLA2 fractions F17 and F19 were able to reproduce this effect although with a moderate potency (Fig.7B), suggesting the possibility of synergisms among venom components. Notably, however, other abundant PLA2 fractions such as F26 or F29, were completely inactive in this assay, demonstrating that cytotoxicity is a specific effect induced by particular PLA2 isoforms, rather than a general consequence of the catalytic activity of these phospholipolytic enzymes. Venoms from some elapids distributed in the Old World contain a particular type of 3FTxs known as cardiotoxins/cytotoxins, which exert rapid cytolytic effects on a variety of cultured cells [70-72]. However, none of the M. dumerilii major venom fractions corresponding to 3FTxs induced cytotoxicity, suggesting the probable lack of such toxin subtype in its composition, in agreement with the proteomic identification results (Table 1).

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PLA2s are venom components involved in the induction of edema [67]. Despite the high proportion of these enzymes in M. dumerilii venom, only moderate edema

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formation was recorded in the mouse footpad assay (Fig.8). Similar findings were reported

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for other venoms within this genus, such as M. altirostris, M. baliocoryphus, M. surinamensis, M. fulvius, and M. nigrocinctus [61]. These observations agree with the fact

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that edema is not a clinically relevant sign in envenomings caused by coral snakes [5, 8].

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A direct hemolytic activity upon erythrocytes of particular animal species such as dogs and mice, both in vitro and in vivo, has been observed for the venom of M. fulvius, and

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attributed to specific PLA2 isoforms [63]. In the case of M. dumerilii venom, signs of hemolytic activity such as a hematocrit decrease or reddish urine, were not observed in

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mouse experiments, reinforcing the notion that this is a very particular effect so far only

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described for M. fulvius, among coral snake venoms studied to date.

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M. dumerilii venom presented very low proteolytic activity upon azocasein in vitro (Fig.9), in agreement with its low metalloproteinase and serine proteinase content (1.8% and 1.9%, respectively; Fig.3). This conclusion is further supported by its lack of hemorrhagic effect in the mouse skin assay, and lack of coagulant activity in vitro (results not shown). L-amino acid oxidase is another enzyme generally present in low proportions in Micrurus venoms, and corresponded to 3.1% in the case of M. dumerilii. In agreement with its proteomic identification, the activity of this enzyme was demonstrated by a functional assay (Fig.10). Proteins of four families were detected in very low amounts (~0.1% or less) in the venom of M. dumerilii, posing a challenge to the study of their roles: hyaluronidase (Hya), phospholipase B (PLB), phoshodiesterase (PDE) and 5’-nucleotidase (5’nuc). The first enzyme is generally assumed to contribute to the spread of venom components in tissues

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by facilitating diffusion via the hydrolysis of long-chain tissue glycosaminoglycans [73, 74]. Hyaluronidase has also been detected in low amounts in the venom of M. alleni [45],

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and its transcripts were demonstrated in the venom gland of M. fulvius [42]. The function of phosphodiesterases in the venom is not clear, but they have been suggested to act

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together with 5’-nucleotidases in the generation of purine nucleosides, which, due their

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bioactivities, could contribute to the overall pathophysiology of envenoming [47, 73, 75].

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The presence of phosphodiesterases, or their RNA transcripts, has also been documented for the venoms of M. nigrocinctus [19], M. fulvius [42], and M. mosquitensis [45], among

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coral snakes. The possible roles of phospholipase B in envenomings are largely unknown. Bernheimer et al. [76] reported that a phospholipase B from the elapid Pseudechis colletti

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induced the conspicuous excretion of reddish urine in mice, later determined to correspond

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to myoglobinuria. However, in the case of M. dumerilii venom no macroscopic signs of

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myoglobinuria or hemoglobinuria were evidenced.

3.3 Cross-immunorecognition of M. dumerilii venom, its components, and its neutralization in mice.

The equine anti-coral snake antivenom raised against M. nigrocinctus venom in Costa Rica (SAC-ICP) was tested for cross-recognition and cross-neutralization of the venom of M. dumerilii. By ELISA, antibodies in this antivenom showed strong binding to the crude venom, generating a titration curve only slightly lower in comparison to the homologous venom (Fig.11). Analysis of the binding specificity of these antibodies was performed using the HPLC-resolved venom fractions in an ELISA assay, showing that protein fractions were recognized, albeit to variable degrees (Fig.12). Low-molecular mass components such as 3FTxs and Kunitz-type inhibitors resulted in weaker antibody binding

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signals, in comparison with larger proteins (Fig.12). This finding is in line with reports for other elapid venoms [19, 77, 78]. In contrast, fractions corresponding to PLA2s resulted in

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intense cross-recognition (Fig.12), indicating a considerable conservation of epitopes

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between these venom enzymes in M. dumerilii and M. nigrocinctus. In agreement with the ELISA results, the equine SAC-ICP antivenom neutralized the lethal effect of M. dumerilii

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venom in mice, with a median effective dose (ED50) of 200 μg venom/mL antivenom. Since

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this potency is similar to that standardized for the neutralization of the homologous venom, these results predict that the anti-M. nigrocinctus coral snake antivenom prepared in Costa

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Rica (SAC-ICP) should be useful in the treatment of cases of snakebite inflicted by M. dumerilii. This is in contrast to the lack of cross-recognition and neutralization by this

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antivenom toward some other coral snake venoms, such as that of M. mipartitus [21].

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4. CONCLUDING REMARKS

Data gathered through proteomic and functional analyses on the venom of a poorly known coral snake species, M. dumerilii, indicate that it is composed of proteins belonging to at least 11 families, with a marked predominance of PLA2s (~52%). 3FTxs also comprise a considerable portion (~28%) of the proteome of this venom. These compositional data indicate that the venom of M. dumerilii belongs to the group of Micrurus species that exhibit a PLA2-rich phenotype, as opposed to a 3FTx-predominant phenotype [45]. Interestingly, the lethal potency of this venom in mice appears to be lower than some venoms from coral snakes expressing the 3FTx-rich phenotype, and at least two of its PLA2s were found to exert lethal activity in mice. In addition to lethality, M. dumerilii venom exerts myotoxicity in vivo and cytotoxicity against myogenic cells in vitro, effects that could also be reproduced by some of its most abundant PLA2s. Finally, a significant

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degree of immunological cross-recognition of crude M. dumerilii venom, and its fractions, was demonstrated using an antivenom (SAC-ICP) raised against venom of M. nigrocinctus.

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These findings agree with the cross-neutralization of lethal activity observed, and suggest

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the possible usefulness of this therapeutic immunobiological in envenomings by M.

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dumerilii.

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Acknowledgements

Thanks are due to Colciencias (111556933661 and 617), Comité para el Desarrollo

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de la Investigacion and Sostenibilidad 2014-2015 (University of Antioquia), and Vicerrectoría de Investigación, University of Costa Rica (741-B3-760) for partial financial

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support of this study. This work was performed in partial fullfilment of the Ph.D. degree of

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Paola Rey-Suárez at the University of Antioquia, Colombia.

Disclosure of potential conflicts of interest Julián Fernández and Bruno Lomonte work at Instituto Clodomiro Picado, University of Costa Rica, where the therapeutic SAC-ICP anticoral snake antivenom used in this study is produced. The authors declare no other potential conflicts of interest regarding this manuscript.

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Figure 1

Figure 1: Micrurus dumerilii in South America (shaded area; adapted from [1]. Snake photo provided by Alejandro Ramirez.

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Figure 2

Figure 2: Separation of Micrurus dumerilii venom proteins by reverse-phase HPLC (A), followed by SDS-PAGE (12%) of fractions under reducing conditions. (B). Two mg of venom were applied to a C18 column (4.6 x 250 mm) and eluted with an acetonitrile gradient, shown as a dotted line in (A).

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Figure 3

Figure 3: Composition of Micrurus dumerilii venom proteome according to protein families, expressed as percentages of the total protein content. Abbreviations: phospholipase A2 (PLA2), three finger toxin (3FTx), Kunitz-type inhibitor (Kun), C-type lectin/lectin-like (C-lect), metalloproteinase (SVMP), L-amino acid oxidase (LAO), hyaluronidase (Hya), phospholipase B (PLB), serine proteinase (SP), phoshodiesterase (PDE), 5’-nucleotidase (5’nuc), and peptidic or non-proteic component (NP).

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Figure 4

Figure 4: Myotoxic activity of the venom of Micrurus dumerilii, compared to the venoms of M. mipartitus (Colombia) and M. nigrocinctus (Costa Rica). Twenty μg of each venom were injected intramuscularly in mice. Phosphate-buffered saline (PBS, pH 7.2) alone was injected in a control group. Plasma creatine kinase (CK) activity values were determined 3 h after venom injection. Bars represent the mean ± SD of three mice per group.

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Figure 5

Figure 5: Muscular necrosis in mice injected with Micrurus dumerilii venom. (A) Skeletal muscle from a mouse injected with phosphate-buffered saline, showing normal appearance. (B) Widespread necrosis of muscle fibers 6 h after the injection of 20 g of venom. Some edema and only a mild neutrophilic infiltration are observed. Hematoxilin/eosin stain (200X magnification).

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Figure 6

Figure 6: Phospholipase A2 activity of the venom of Micrurus dumerilii, compared to the venom of M. nigrocinctus, on the synthetic monodisperse substrate 4-nitro-3-octanoyloxybenzoic acid (A), and on micellar phosphatidylcholine (B), as described in Methods. Each point represents the mean ± SD (n=3).

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Figure 7

Figure 7: Cytotoxicity of Micrurus dumerilii and M. nigrocinctus venoms (A; 20 g/well) or the major M. dumerilii phospholipases A2 (HPLC fraction numbering as in Table 1) (B; 20 g/well) on murine C2C12 myotubes. Cytotoxicity was determined by the release of lactic dehydrogenase to the cell culture supernatants, as described in Methods. Triton X100 (TX-100) was used as a reference for 100% cytotoxicity. Bars represent the mean ± SD (n=3).

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Figure 8

Figure 8: Time course of the edema induced in the mouse footpad assay by Micrurus dumerilii venom (20 g). Phosphate-buffered saline (PBS) alone was injected as a control. Each point represents the mean ± SD of three mice per group.

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Figure 9

Figure 9: Proteolytic activity of Micrurus dumerilii venom compared to those of M. mipartitus and Bothrops asper, on an azocasein substrate (20 g/reaction well). Each bar represents the mean ± SD (n=3).

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Figure 10

Figure 10: L-amino acid oxidase activity of Micrurus dumerilii and M. mipartitus venoms (20 g/reaction). Bars represent the mean ± SD (n=3).

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Figure 11

Figure 11. Immunorecognition of Micrurus dumerilii venom by an equine antivenom produced against M. nigrocinctus. Venoms from both species were adsorbed onto microplates and bound antibodies were detected by an anti-horse IgG/alkaline phosphatase conjugate, as described in Methods. Normal horse serum was used as a negative control. Each point represents the mean ± SD (n=3).

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Figure 12

Figure 12. ELISA-based immunoprofiling of the equine anti-Micrurus nigrocinctus antivenom (SAC-ICP), against all RP-HPLC fractions of M. dumerilii venom bound to microwells. Normal horse serum was used as a negative control. Each bar represents the mean ± SD (n=3). For identification of venom fractions refer to Table 1

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Table 1: Assignment of the RP-HPLC fractions of Micrurus dumerilii venom to protein families by MALDI-TOF-TOF of

MS/MS-derived sequence

Conf

RI

Peptide ion

Sc

Protein family; ~ related protein *

(%)

m/z

SC

Mass ▼ (kDa),(Da)

z

1-4

2.4

5

5.4

10

1706,7

1

FSPGXBTSBTCPAGBK

6

0.8

10 (6572)

1655,6

1

ZBRPEYCNXPPDK

7

3.4

10

1036,4

1

WGCAASCPK

8

4.2

10

961,4 2324,9

1 1

1375,5

NU

%

Non proteic 93,2

6

3FTx; ~U3FVH8

99

12

Kunitz inhibitor 1a M.fulvius;~U3FAD2

98,1

7

Long chain neurotoxin 1c M.fulvius; ~U3FYB1

TFWNTHR HCASGETXCY(B cm)TFWNTHK

99 99

10 9

Alpha-neurotoxin homolog M.corallinus ~P58370

1

ENXCFTMFSAK

99

15

3FTx M.corallinus ~C6JUP4

99

17

Kunitz inhibitor 7 M.fulvius ~U3F5B9

PT ED

MA

Peak

PT

selected peptide ions from in-gel trypsin-digested protein bands.

1.7

9

2985,1

1

YYNPSTHSCXBFVYGGCEGNENNFK

10

0.8

10

1468,5 2985,1

1 1

YCYXPADPGPCR YYNPSTHSCXBFVYGGCEGNENNFK

99 99

9 14

Kunitz inhibitor 7 Micrurus fulvius; ~U3F5B9

11

0.8

9

1687,5

1

GPYNVCCSTDXCNK

99

16

3FTX M.laticollaris ~K9MCX0

12a

0.5

14

1373,4 1447,5 2983,1

1 1 1

CBDFVCNCDR HWXSFTNYGCY HWXSFTNYGCYCGYGGSGTPVDEXDK

99 99 99

7 10 31

Phospholipase A2 13 M.fulvius ~U3FYP8

12b

0.2

10

1310,4

1

(184.2)EFGCAASCPK

Man

Man

3FTX OH37 O.hannah ~B53B59

13a

0.6

13

1447,5 2983,1 1373,4

1 1 1

HWXSFTNYGCY HWXSFTNYGCYCGYGGSGTPVDEXDK CBDFVCNCDR

99 99 97.1

9 35 6

Phospholipase A2 13 M.fulvius ~U3FYP8

13b

0.6

10

2659.9

1

(433.2)FVTCPEGENHCYTTAXTAR

Man

Man

3FTX M.corallinus ~C6JUP5

14

2.0

14 (13411)

1373,4 2312,7

1

CBDFVCNCDR TNYGCYCGYGGSGTPVDEXDK

99 99

11 16 11

Phospholipase A2 13 M.fulvius ~U3FYP8

AC

CE

9

ACCEPTED MANUSCRIPT 46

99 99

1.9

14 (13411)

2125,6

1

YGCYCGYGGSGTPVDEXDR

16a

0.7

14

1373,4 2125,7 3084,1

1

CBDFVCNCDR YGCYCGYGGSGTPVDEXDR (Hcm)(Wox)XSFTNYGCYCGYGGSGTPVDEXDR

2983,1

1

HWXSFTNYGCYCGYGGSGTPVDEXDK

SC

15

PT

HWXSFTNYGCY HWXSFTNYGCYCGYGGSGTPVDEXDK

39

99

16

Phospholipase A2 24 M.fulvius ~U3FYP5

99 99 97

9 22 9

Phospholipase A2 24 M.fulvius ~U3FYP5

99

22

Phospholipase A2 13 M.fulvius ~U3FYP8

99

12

Phospholipase A2 24 M.fulvius ~U3FYP5

99

21

Phospholipase A2 24 M.fulvius ~U3FYP5

RI

1447,5 2983,0

0.2

10

2125,7

1

YGCYCGYGGSGTPVDEXDR

17

7.6

13 (13400)

2125,7

1

YGCYCGYGGSGTPVDEXDR

18a

1.3

14 (13357)

2983,1 1447,5

1 1

HWXSFTNYGCYCGYGGSGTPVDEXDK HWXSFTNYGCY

99 94,3

19 6

Phospholipase A2 13 M.fulvius ~U3FYP8

2125,7

1

YGCYCGYGGSGTPVDEXDK

99

17

Phospholipase A2 24 M.fulvius ~U3FYP5

2870,9 2855,0

1 1

SA(Wox)DFTNYGCYCGAGGSGTPVDEXDK SAWDFTNYGCYCGAGGSGTPVDEXDK

99 99

10 13

Basic phospholipase A2 M.corallinus ~B8AXW7

PT ED

MA

NU

16b

0.8

10 (7059)

3032,1

1

VTBYCSHACAXPASYEFVHCCBTDK

96,5

8

3FTx-3 M.corallinus ~C6JUP3

19

5.2

14

1447,5 2983,1

1 1

HWXSFTNYGCY HWXSFTNYGCYCGYGGSGTPVDEXDK

99 99

13 26

Phospholipase A2 13 M.fulvius ~U3FYP8

2855,0

1

SAWDFTNYGCYCGAGGSGTPVDEXDR

99,0

16

Basic phospholipase A2 M.corallinus ~B8AXW7

2840,9

1

AWDYN(Nda)YGCYCGBGGSGTPVDDXDR

99

14

PLA2-Sut-22 S.fasciata ~R4G7M2

2125,7

1

YGCYCGYGGSGTPVDEXDR

99

12

Phospholipase A2 24 M.fulvius ~U3FYP5

1041,3

1

AFVCNCDR

94,5

9

Phospholipase A2 3b M.fulvius ~U3FYP1

1.0

14 (13177)

AC

20a

CE

18b

20b

1.1

10 (7159)

1645,6 3280,2

1 1

PAFCYEDPPFFBK XTCVHFF(Yao)GBCDVNBNHFTTMSECNR

99 99

16 13

Kunitz-type neurotoxin MitTx-alpha M.tener tener ~G9I929

21a

4.3

14

2841,0

1

AWDYN(Nda)YGCYCGBGGSGTPVDDXDR

99

14

PLA2-Sut-22 S.fasciata ~R4G7M2

21b

1.6

11

1645,6 3280,2

1 1

PAFCYEDPPFFBK XTCVHFF(Yao)GBCDVNBNHFTTMSECNR

99 99

19 14

Kunitz-type neurotoxin MitTx-alpha M.tener tener ~G9I929

21c

1.0

9

1645,6 3280,2

1 1

PAFCYEDPPFFBK XTCVHFF(Yao)GBCDVNBNHFTTMSECNR

99 99

20 17

Kunitz-type neurotoxin MitTx-alpha M.tener tener ~G9I929

ACCEPTED MANUSCRIPT 47

2.1

14

1660,7

1

MTFFTPGFGWTBXK

99

12

3FTx-2 M.corallinus ~C6JUP2

22b

1.9

10

1645,5

1

PAFCYEDPPFFBK

99

10

Kunitz-type neurotoxin MitTx-alpha M.tener tener ~G9I929

23

2.4

13

1660,7

1

MTFFTPGFGWTBXK

99

9

3FTx-2 M.corallinus ~C6JUP2

24a

0.6

15

2883,0

1

(S fo)AWDFTNYGCYCGAGGSGTPVDEXDR

99

23

Basic phospholipase A2 M. corallinus ~B8AXW7

24b

0.3

12

1646,6

1

PAFCYEDPPFFBK

99

12

Kunitz-type neurotoxin MitTx-alpha M.tener tener ~G9I929

1660,7

1

MTFFTPGFGWTBXK

99

13

3FTx-2 M.corallinus ~C6JUP2

2202,7 1996,6

1 1

VHDDCYGEAEBV(Hox)GCWPK WTXYSYDCS(Nda)GBXTCK

99 99

11 15

Phospholipase A2 1d M.fulvius ~U3FYP6

2829,0

1

DGXDYD(daN)YGCYCGSGGSGTPVDDXDR

99

11

Phospholipase A2 isoform 6 P. nigriceps ~H8PG87

99

11

Phospholipase A2 22 M.fulvius ~U3F588

13.5

13

da

RI

SC NU

13 (13303)

MA

26

4.3

1996,6

1

WTXYSYDCS( N)GBXTCK

2843,0

1

DGXD(aoY)DNYGCYCGSGGSGTPVDDXDR

99

15

Phospholipase A2 isoform 6 P.nigriceps ~H8PG87

1810,7

1

TVENVGVSBVAPDNPER

99

13

Thaicobrin N.kaouthia ~P82885

PT ED

25

PT

22a

2.4

13 (13146)

2453,8 2306,8

1 1

FAWYGCYCGSGGSGTPVDEXDR AWYGCYCGSGGSGTPVDEXDR

99 99

11 17

Phospholipase A2 18 M.fulvius ~U3FYP7

28

8.3

10

1322,5

1

XCDDSSXPFXR

99

13

3FTX 6 M.fulvius ~U3EPK7

29

5.8

13

1350,5 2028,7 2528,8 1447,5

1 1 1 1

VHGCBPXVMFY (Vam)HGCBPXVMFYSFECR CTNDRV(Wky)A(Ddh)FVDYGCYCVAR (Pam)XVMFYSFECR

99 99 99 97,3

13 21 15 11

Basic phospholipase A2 homolog Tx-beta M.tener tener ~G9I930

30

0.3

13

1350,5 2028,7 2528,8 1084,3 1525,6

1

VHGCBPXVMFY (Vam)HGCBPXVMFYSFECR CTNDRV(Wky)A(Ddh)FVDYGCYCVAR NFVCNCDR (Tfo)ATXCXXTATYNR

99 99 99 99 96,2

15 20 19 12 11

Basic phospholipase A2 homolog Tx-beta M.tener tener ~G9I930

31a

0.1

15

1005,4 1915,7

1

XWEWTDR WNDTPCESXFAFXCR

89,6 42,3

8 5

C-type lectin 1a M.fulvius ~U3EPK2

31b

0.4

9

1763,6

1

AESADXAEYXYDYXK

99

14

C-type lectin 1a M.fulvius ~U3EPK2

32

0.6

20

1763,6

1

AESADXAEYXYDYXK

99

24

C-type lectin M.corallinus ~C6JUN9

AC

CE

27

ACCEPTED MANUSCRIPT 48

45

2114,9 2998,9 1416,6

1

XDFNG(Nda)TXGXAHXGSXCSPK YFVEVGEECDCGSPBDCBSACCDAR YXEFYVVVDNR

33b

0.1

35

1544,6 1450,4 1720,6 1911,5 2114,8 1416,6 2052,7 1011,4 1184,4

1

BYXEFYVVVDNR DDCDXPEXCTGR AABDDCDXPEXCTGR NGHPCBNNBGYCY(Nda)GK XDFNG(Nda)TXGXAHXGSXCSPK YXEFYVVVDNR TBPAYBFSSCSVBEHBR FYVVVDNR DMCFTXNBR

1297,4

1

SAECPTDSFBR

1944,8

1

XGVHNVHVHYEDEBXR

SC NU

MA

PT ED

99 99 99

7 12 15

Snake venom metalloproteinase 1 M.fulvius ~U3EPC7

99 99 99 99 99 99 99 97,9 89,4

16 15 12 23 21 14 15 9 9

Snake venom metalloproteinase 1 M.fulvius ~U3EPC7

99

14

Snake venom metalloproteinase-Ech-32 E.curta ~R4FJM6

99

12

Serine protease harobin H.hardwickii ~B5MCS0

PT

0.8

RI

33a

33c

0.07

32

1416,6

1

YXEFYVVVDNR

99

14

Snake venom metalloproteinase 4 M.fulvius ~U3FWL3

33d

0.1

30

2450.3

1

GTSMVAVTMAHEMGHNXGXNHDK

99 99 99 99 99

12 18 15 14 15

Snake venom metalloproteinase 1 M.fulvius ~U3EPC7

Snake venom metalloproteinase 1 M.fulvius ~U3EPC7

34a

0.07

0.02

20

130

XDFNG(N )TXGXAHXGSXCSPK

1544.9

BYXEFYVVVDNR

2053.1

TBPAYBFSSCSVBEHBR

1416.8

YXEFYVVVDNR

1416.8

CE

2115.2

1

AC

33e

da

YXEFYVVVDNR

99.0

11

1720.9

AABDDCDXPEXCTGR

99.0

13

1324.6

DPDYG(Mox)VEPGTK

99.0

12

1450.7

DDCDXPEXCTGR

99.0

14

1308.6

DPDYGMVEPGTK

99.0

15

1184.6

DMCFTXNBR

99.0

13

1200.6

D(Mox)CFTXNBR

73

7

1297.6

1

SAECPTDSFBR

99

12

Stephensease-1 H.stephensii ~B5KFV4

1498.9 2106.1 1243.7

1

VMEVXBWXDXPR RPDFSTXYXEEPDTTGHK SPNNXWVEER

99 99 99

10 9 12

Phosphodiesterase 1 M.fulvius~U3FAB3

ACCEPTED MANUSCRIPT 49

75

2421.3 2408.2

1

FHECNXGNXXCDAVVYNNXR HPDDNEWNHVSMCXV(Nda)GGGXR

98.3 96.8

8 8

Ecto-5'-nucleotidase 1 M.fulvius ~U3FYP9

34c

0.02

70

2421.3 1255.8 1389.8 1231.7 1449.8 1026.5 906.5

1

FHECNXGNXXCDAVVYNNXR FPXXSANXRPK XTXXHTNDVHAR EVVHFMNSXR VVSXNVXCTECR EDCYGGVAR VFPAMEGR

99 99 99 99 99 97.6 69.6

13 10 10 12 11 9 7

Ecto-5'-nucleotidase 1 M.fulvius ~U3FYP9

34d

0.3

45

1879.1

1

HXNFHXAXTGX(Edh)XWTK

99

17

Snake venom metalloproteinase 1 M.fulviu s~U3EPC7

34e

0.6

35

2115.1 1450.7 1416.8 1184.6 2053.0 1645.9

1

XDFNGN(da)TXGXAHXGSXCSPK DDCDXPEXCTGR YXEFYVVVDNR DMCFTXNBR TBPAYBFSSCSVBEHBR VYEMVNXXNB(Mox)YR

99 99 99 99 99 97.6

17 13 13 11 14 11

Snake venom metalloproteinase 1 M.fulvius ~U3EPC7

2172.1 1945.0

1

GDSGGPXXCDGBXBGXVSWGR XGVHNVHVHYEDEBXR

99 99

14 22

Snake venom serine protease BmSP B.multicinctus~A8BL57

1297.6

1

SAECPTDSFBR

99

12

SVMP-Ech-32 E.curta~R4FJM6

SC NU

MA

PT ED

35a

0.7

32

2172.1 1945.0

1

GDSGGPXXCDGBXBGXVSWGR XGVHNVHVHYEDEBXR

99 99

19 24

Snake venom serine protease BmSP B.multicinctus~A8BL57

35b

0.1

25

1856.0 1152.6

1

AC

CE

PT

0.01

RI

34b

TXBBXEVPXXGXDTCR XPFYADWXK

99 99

17 13

Prostasin-like protein M.fulvius~U3FBP8

35c

0.08

12

1152.6

1

XPFYADWXK

99

15

Prostasin-like protein M.fulvius ~U3FBP8

1416.7

1

YXEFYVVVDNR

99

11

MTP4 M.fulvius ~U3FWL3

2172.1

1

GDSGGPXXCDGBXBGXVSWGR

99

12

Snake venom serine protease B.multicinctus ~A8BL57

1484.8 1833.9 2275.2

1

EADYEEFXEXAR EFVBEDENAWYYXK XHFAGEYTANDHGWXDSTXK

99 99 99

13 17 17

L-amino acid oxidase 1a M.fulvius ~U3FYB2

36a

0.4

70

ACCEPTED MANUSCRIPT 50

99 99 99 99

27 13 23 19

L-amino acid oxidase 1a M.fulvius ~U3FYB2

99

19

L-amino acid oxidase B variant 1 E.coloratus ~A0A0A1WCY6

99

16

L-amino acid oxidase E.curta ~R4FJP5

99 99

15 16

Prostasin-like protein M.fulvius ~U3FBP8

2275.2 1131.5 1637.8 1484.7

1

XHFAGEYTANDHGWXDSTXK SDDXFSYER NDXEGWHVNXGPMR EADYEEFXEXAR

1310.7

1

RFDEXVGG(Mox)DR

2292.2

1

(Pox)E(Nda)TXSYVTADYVXVCSTSR

PT

58

10

RI

0.7

97.8

NU

36b

YAMGSXTSFVPYBFBHYFETVAAPVG(Bfo)

SC

3066.7

0.2

25

1152.6 1856.0

1

XPFYADWXK TXBBXEVPXXGXDTCR

37a

0.09

200

3082.6 2275.2 3066.6 1484.8

1

YA(Mox)GSXTSFVPYBFBHYFETVAAPVG(Kfo) XHFAGEYTANDHGWXDSTXK YAMGSXTSFVPYBFBHYFETVAAPVG(Kfo) EADYEEFXEXAR

99 99 97.5 96.3

11 8 9 8

L-amino acid oxidase 1a M.fulvius ~U3FYB2

1310.7

1

RFDEXVGG(Mox)DR

99

12

L-amino acid oxidase B variant 1 E.coloratus ~A0A0A1WCY6

1131.5 2275.1

1

SDDXFSYER XHFAGEYTANDHGWXDSTXK

98.9 7.4

8 5

L-amino acid oxidase E.curta ~R4FJP5

CE

PT ED

MA

36c

0.5

73

1484.7 1833.9 2275.2

1

EADYEEFXEXAR EFVBEDENAWYYXK XHFAGEYTANDHGWXDSTXK

99 99 99

14 9 16

L-amino acid oxidase 1a M.fulvius ~U3FYB2

37c

1.2

58

1637.9 1833.9 1484.8 2275.2 3082.7

1

NDXEGWHVNXGPMR EFVBEDENAWYYXK EADYEEFXEXAR XHFAGEYTANDHGWXDSTXK YA(Mox)GSXTSFVPYBFBHYFETVAAPVG(Kfo)

99 99 99 99 99

17 21 17 25 10

L-amino acid oxidase 1a M.fulvius ~U3FYB2

1310.7

1

RFDEXVGG(Mox)DR

99

9

L-amino acid oxidase B variant 1 E.coloratus ~A0A0A1WCY6

2235.3

1

HVVVVGAGMAGXSAAYVXAGAGHK

99

18

L-amino-acid oxidase O.scutellatus ~B4JHE3

2275.2 1833.9

1

XHFAGEYTANDHGWXDSTXK EFVBEDENAWYYXK

99 99

8 9

L-amino acid oxidase 1a M.fulvius

37d

0.08

42

AC

37b

ACCEPTED MANUSCRIPT 51

99 99 99 99

11 15 11 20

L-amino acid oxidase 1a M.fulvius ~U3FYB2

99 99

14 20

L-amino acid oxidase E.curta ~R4FJP5

99

16

L-amino acid oxidase B 1 E.coloratus ~A0A0A1WCY6

98

7

Hyaluronidase M.lebetina ~W8EEP5

99 99 99

15 13 12

Phospholipase B 1 M.fulvius ~U3FA79

99 99

14 17

Prostasin-like protein M.fulvius ~U3FBP8

1007.6 1963.2 1131.6 2275.2

1

FFBPXDXK TSGDXVXNDXSXXHBXPK SDDXFSYER XHFAGEYTANDHGWXDSTXK

1216.6 2292.2

1

FWEADGXHGGK (Pox)E(Nda)TXSYVTADYVXVCSTSR

1310.7

1

RFDEXVGG(Mox)DR

PT

42

11

RI

0.1

95.5

NU

37e

YAMGSXTSFVPYBFBHYFETVAAPVG(Kfo)

SC

3066.7

0.05

65

1243.7

1

NDBXXWXWR

38b

0.11

35

3079.8 1432.7 1460.8

1

SXEDGAXYXXEBVPNXVEYSDBTTXXR FGXDFSYEMAPR BVVPESXFAWER

38c

0.6

25

1152.7 1856.1

1

XPFYADWXB TXBBXEVPXXGXDTCR

PT ED

MA

38a

AC

CE

Cysteine residues are carbamidomethylated. X: Leu/Ile; B: Lys/Gln; ox: oxidized; da: deamidated; fo: formyl; dh: dehydrated; cm: carbamidomethyl; ao Amino;am Amidated; kyHydroxykynurenin ▼: reduced SDS-PAGE mass estimations, in kDa; in parentheses, the mass(es) observed by MALDI-TOF in selected peaks are indicated Confidence (Conf) and Score (Sc) values are calculated by the Paragon® algorithm of ProteinPilot®.

ACCEPTED MANUSCRIPT 52

Table 2: Functional characterization of selected HPLC-fractions of Micrurus dumerilii venom. Protein family

Lethality to mice†

Myotoxicity‡

F5

6776

3FTx

-

-

F7

n.t.

3FTx

+

n.t.

F17

13436

PLA2

-

F19

13485

PLA2

-

F21

n.t.

PLA2

+

F26

13289

PLA2

+

F28

7537

3FTx

-

F29

13840

PLA2

-/-

-

n.t.

n.t.

-

+/+

+ (41%)

+

+/+

+ (54%)

n.t.

n.t.

n.t.

+

+/+

-

-

-/-

-

MA -

-

+/-

-

RI

PT

Cytotoxicity§

NU

PLA2 activity (NOBA/PC)**

SC

Isotopeaveraged Mass*

determined by nano-ESI-MS in a QTrap 3200 mass spectrometer (ABsciex) operated in

D

*

Fraction

TE

positive enhanced multi-charge mode. tested by the intravenous injection of 20 g in mice of 16-18 g body weight.

‡

tested by the intramuscular injection of 5 g in mice of 18-20 g body weight followed by

AC CE P

†

determination of plasma creatine kinase activity. **

tested by incubation of 1 g or 4g of fractions with the substrates 4-nitro-3-octanoyloxybenzoic acid (NOBA) or micellar phosphatidylcholine (PC), respectively, as described in Methods.

§

tested on murine C2C12 myotube cultures by adding 20 g in 150 L/well, and determining the release of lactate dehydrogenase to supernatants, as described in Methods. In positive samples, % of cell death (after 3 h of toxin exposure) is indicated.

n.t.: not tested

ACCEPTED MANUSCRIPT 53

AC CE P

TE

D

MA

NU

SC

RI

PT

Graphical abstract

ACCEPTED MANUSCRIPT 54

HIGHLIGHTS

The venom proteome of Micrurus dumerilii from Colombia was determined



Phospholipases A2 are the predominant proteins, among 11 families



The venom induces a conspicuous myotoxic effect, reproduced by some PLA2s



Two PLA2s and a three-finger toxin reproduce the lethal venom effect



Anti-Micrurus nigrocinctus antivenom cross-neutralizes M. dumerilii venom

AC CE P

TE

D

MA

NU

SC

RI

PT

