Venomic and Antivenomic Analyses of the Central ... - ACS Publications

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Venomic and Antivenomic Analyses of the Central American Coral Snake, Micrurus nigrocinctus (Elapidae) Julian Fernandez,† Alberto Alape-Giron,†,‡ Yamileth Angulo,†,‡ Libia Sanz,§ Jose María Gutierrez,† Juan J. Calvete,§ and Bruno Lomonte*,† †

Instituto Clodomiro Picado, Facultad de Microbiología, and ‡Departamento de Bioquímica, Escuela de Medicina, Universidad de Costa Rica, San Jose, Costa Rica § Instituto de Biomedicina de Valencia, CSIC, Jaume Roig 11, 46010 Valencia, Spain ABSTRACT: The proteome of the venom of Micrurus nigrocinctus (Central American coral snake) was analyzed by a “venomics” approach. Nearly 50 venom peaks were resolved by RPHPLC, revealing a complex protein composition. Comparative analyses of venoms from individual specimens revealed that such complexity is an intrinsic feature of this species, rather than the sum of variable individual patterns of simpler composition. Proteins related to eight distinct families were identified by MS/MS de novo peptide sequencing or N-terminal sequencing: phospholipase A2 (PLA2), three-finger toxin (3FTx), L-amino acid oxidase, C-type lectin/lectin-like, metalloproteinase, serine proteinase, ohanin, and nucleotidase. PLA2s and 3FTxs are predominant, representing 48 and 38% of the venom proteins, respectively. Within 3FTxs, several isoforms of short-chain R-neurotoxins as well as muscarinic-like toxins and proteins with similarity to long-chain κ-2 bungarotoxin were identified. PLA2s are also highly diverse, and a toxicity screening showed that they mainly exert myotoxicity, although some are lethal and may contribute to the known presynaptic neurotoxicity of this venom. An antivenomic characterization of a therapeutic monospecific M. nigrocinctus equine antivenom revealed differences in immunorecognition of venom proteins that correlate with their molecular mass, with the weakest recognition observed toward 3FTxs. KEYWORDS: Micrurus nigrocinctus, coral snake, venom, elapid toxins, proteomics, venomics, antivenomics, mass spectrometry

1. INTRODUCTION Snake venoms are secretions produced by specialized exocrine glands located in the upper jaw, injected into the prey through a fang-based delivery system which independently appeared in Viperidae, Elapidae and Atractaspididae.1 These venoms contain a complex mixture of proteins selectively expressed in the glands, which have specific activities that contribute to subdue, kill, and/ or digest the prey.2 Venom proteins were adapted to serve toxic functions from relatively few ancient and versatile scaffolds by changes in surface exposed residues.3 The corresponding genes evolved at an accelerated rate after multiple recruitment and amplification events, which occurred before and after the diversification of the advanced snakes.4,5 Several high-throughput approaches have been used to characterize the complete set of proteins in the venoms from many species of Viperidae.6-8 However, less information is available on the proteomes of Atractaspididae and Elapidae. Elapid venom proteomes characterized to date are those from Naja naja atra, N. n. kaouthia, N. nigricollis, N. katiensis, N. pallida, N. nubiae, N. mossambica, Pseudonaja textilis, Ophiophagus hannah, Bungarus fasciatus, Demansia vestigiata, Micrurus surinamensis, and M. pirrhocryptus.9-16 In addition, elapid venom gland transcriptomes have been characterized for Oxyuranus scutellatus, Bungarus flaviceps, and Micrurus corallinus.17-19 Coral snakes, the representatives of the family Elapidae in the Americas, include the genera Leptomicrurus, Micruroides, and r 2011 American Chemical Society

Micrurus, the latter being the most diverse with nearly sixty species.20 Micrurus nigrocinctus, commonly known as Central American coral snake, is distributed from southern Mexico to northern Colombia.21 In Costa Rica it inhabits the Pacific versant (Figure 1A), where it is found from sea level to 1500 m of altitude. It is a fossorial, small to moderate-sized snake (average 60-70 cm, maximum 110 cm) which feeds primarily, although not exclusively, on small colubrid snakes.22,23 The incidence of envenomings by M. nigrocinctus in Central America is low, probably below 1-2% of all snakebites. Nonetheless, they are of medical concern, since the risk of a fatal outcome in severe human envenomings is high if timely treatment is not provided.24,25 This species produces small amounts of venom, as reflected by an average yield of nearly 5 mg for specimens in captivity,26 but its venom induces potent and progressive neurotoxic effects which may lead to respiratory paralysis,25 a shared feature with many Micrurus venoms.27-29 Median lethal doses (LD50) of this venom in mice are within the range of 0.3-0.5 μg/g (intravenous), 0.4-1.2 μg/g (intraperitoneal), or 1.7-2.5 μg/g (subcutaneous).26,30-32 The biochemical composition and toxic activities of M. nigrocinctus venom are only partially known. Experimentally, this venom induces electrophysiological alterations in neuromuscular Received: October 29, 2010 Published: January 31, 2011 1816

dx.doi.org/10.1021/pr101091a | J. Proteome Res. 2011, 10, 1816–1827

Journal of Proteome Research

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In addition to these PLA2s, four partial41 and one complete amino acid sequence42 for short R-neurotoxins with nicotinic acetylcholine receptor-binding activity have been reported. Venomics, a proteomic strategy developed for the analysis of snake venoms,6,7 was applied in the present study to characterize the protein profile of M. nigrocinctus venom, aiming to identify its major components and their possible relationships to toxic activities. This information was combined with an antivenomic analysis, to characterize the immunoreactivity of a monospecific therapeutic antivenom produced against this medically relevant, widely distributed representative of the family Elapidae in Central America.

2. MATERIALS AND METHODS 2.1. Venom and its Fractionation by RP-HPLC

Figure 1. (A) Micrurus nigrocinctus and its geographic distribution in Costa Rica (gray area; images reproduced with permission from Instituto Nacional de Biodiversidad, INBio, Costa Rica). Dots labeled “a-f” indicate locations of individual specimens whose venoms are analyzed in Figure 4. (B) Overall composition of the venom according to protein families, expressed as percentages. Three-finger toxins (3FTx), phospholipases A2 (PLA2), L-amino acid oxidases (LAO), C-type lectins/ lectin-like (C-lect), metalloproteinases (SVMP), serine proteinases (SP), ohanin (OH), and nucleotidase (NU).

preparations that suggest the action of both post- and presynaptic toxins, with a predominance of the former.33 In addition, a local myotoxic action after intramuscular injection has been demonstrated in mice,29,34-36 as well as in neuromuscular preparations exposed to the crude venom.33 These neurotoxic and myotoxic actions are in agreement with similar observations performed with the venoms of other Micrurus species15,37,38 However, only few toxins from M. nigrocinctus have been purified and characterized, partly due to the limited availability of its venom. Mochca-Morales et al.39 reported the N-terminal amino acid sequences of three M. nigrocinctus venom phospholipases A2 (PLA2) which were toxic to mice. Alape-Giron et al.3 purified and determined the complete amino acid sequences of two basic PLA2s, nigroxins A and B, which induced myonecrosis in mice. The sequence of nigroxin A is identical in 27 out of the 28 N-terminal residues to one of the PLA2s (UniProt KB accession number P21792) reported by Mochca-Morales et al.39 Myotoxic activity was also documented for a purified acidic PLA2 of this venom,40 although no information on its sequence is available.

M. nigrocinctus venom was a pool from more than fifty specimens from Costa Rica, maintained at the serpentarium of Instituto Clodomiro Picado (ICP). The venom was lyophilized and stored at -20 °C. For reverse-phase (RP) HPLC separations, 2-3 mg of venom were dissolved in 200 μL of 5% acetonitrile containing 0.1% trifluoroacetic acid (TFA; solution A), centrifuged for 5 min at 13 000 rpm, and loaded on a C18 column (250 4.6 mm, 5 μm particle; Vydac) using an Agilent 1200 chromatograph. Elution was performed at 1 mL/min by applying a gradient toward solution B (95% acetonitrile, 0.1% TFA), as follows: 5% B for 5 min, 5-15% B over 10 min, 1545% B over 60 min, and 45-70% B over 12 min. Absorbance was monitored at 215 nm, and fractions were manually collected, and dried in a vacuum centrifuge (Savant) for subsequent characterization. The relative abundance of each protein (% of total venom proteins) was estimated by integration of the peak signal at 215 nm, using ChemStation B.04.01 (Agilent). If a fraction contained two or more SDS-PAGE-separated bands, their relative distribution was estimated by densitometry, using ImageJ 1.4 (http://rsb.info.nih.gov/ij/).

2.2. Characterization of the RP-HPLC Venom Fractions

Each venom fraction obtained after RP-HPLC was analyzed by SDS-PAGE under reducing (15% gels) and nonreducing (12% gels) conditions. Protein bands were excised from the Coomassie blue R-250-stained gels and subjected to reduction with dithiothreitol and alkylation with iodoacetamide, followed by in-gel digestion with sequencing grade bovine trypsin on an automated processor (ProGest, Digilab), according to the manufacturer. The resulting peptide mixtures were analyzed by MALDI-TOF-TOF mass spectrometry on an Applied Biosystems 4800-Plus instrument. Mixtures of 0.5 μL of R-cyano-4-hydroxycinnamic acid and 0.5 μL of each peptidic sample were spotted onto an Opti-TOF 384-well plate, dried, and analyzed in positive reflector mode. Spectra were acquired using a laser intensity of 3000 and 1625 shots per spectrum, using as internal standards CalMix-5 (ABSciex), prespotted on the same plate. Up to 100 precursor peaks 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 3000). The resulting spectra were analyzed using ProteinPilot 2.0.1 (Applied Biosystems) to identify proteins, at a confidence level of 99%. Tryptic digests not identified by MALDI-TOF/TOF were further subjected to nanoelectrospray ionization (nESI)-MS/MS analysis by direct infusion on a Q-Trap 3200 instrument (Applied Biosystems). Doubly- or 1817

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Table 1. Assignment of Proteins in RP-HPLC Separated Fractions of Micrurus nigrocinctus Venom to Protein Families peptide ion z

protein family; related protein*

%

mass (kDa)

m/z

3

7.9

7

2204.1

1

GCGCPTVBPGXHXSCCASDK

3FTx; M. nigrocinctus ∼ P80548

4

1.5

7

2203.9

1



1138.4

1

GVBVECCMR

3FTx; M. corallinus ∼ Q9PUB7

2056.8

1

HASDSQTTTCXSGXCYBK

fraction

peptide sequence

1, 2

unknown

5

1.6

7

2203.9

1



6

1.3

7

2203.9

1



7 8

2.2 4.1

7 7

2203.9 478.8

1 2

GCGCPTVBPGXHXSCCASDK NPTNXXER

3FTx; M. nigrocinctus ∼ P80548 3FTx; M. corallinus ∼ ACS74998

9

3.8

7

1310. 6

1

AXEFGCAASCPK

3FTx; Laticauda colubrina ∼ P0C8R6

10

0.5

14

1298.6

1

NXYQFBNMXK

PLA2; M. nigrocinctus ∼ P21792

10b

1.5

7

655.9

2

AXEFGCAASCPK


11

0.4

14

1235.6

1

NXXDFBNMXK

PLA2; M. nigrocinctus nigroxin B ∼ P81167

11b

1.3

7

1310.6

1

AXEFGCAASCPK


12

0.6

14

1298.6

1

NXYQFBNMXK


12b 13a

0.4 1.2

7 14

1310.6 1235.7

1 1

AXEFGCAASCPK NXXDFBNMXK

3FTx; Laticauda colubrina ∼ P0C8R6 PLA2; M. nigrocinctus nigroxin B ∼ P81167

1373.4

1

CBDFVCNCDR

13b

0.3

7

655.9

2

AXEFGCAASCPK

14

0.7

14

1235.7

1

NXXDFBNMXK

PLA2; M. nigrocinctus nigroxin B ∼ P81167

15a

2.9

14

1298.7

1

NXYQFBNMXK

PLA2; M. nigrocinctus P21792

15b

0.1

7

1373.6

1

CBDFVCNCDR

995.4 1801

1 2

WHMXVPGR (203.2)ETCADGQNXCFBR

MuTx; Naja kaouthia MuTx-like protein-2 ∼ P82463 MuTx; Dendroaspis angusticeps mt-7 - 3FEV

NXXDFBNMXK

PLA2; M. nigrocinctus nigroxin A or B ∼ P81166 or P81167

N-te

16

2.1

14

1235.7

1 N-te

17a

2.9

14

1298.7

1 N-te

17b

0.5

18a

4


NLYQFKNMIKCTNTR

NLIDFKNMIKCTNTR NXYQFBNMXK


NLYQFKNMIKCTNTR

7

1645.7

1

TRGCAATCPBAEYR

MuTx; Dendroaspis angusticeps mt-7 - 3FEV

14

1770.5

1

CCQVHDDCYGEAEK

PLA2; Oxyuranus scutellatus ∼ AAZ22633

1264.5 1264.6

1 1

NXQYXBNMXK NXYQXBNMXK

PLA2; M. nigrocinctus ∼ P21791 PLA2, M. nigrocinctus ∼ P21792

CBDFVCNCDR


1373.4 18b

1.3

7

844.5

2

GPYNVCCSTDXCNR

3FTx, M. frontalis ∼ P86421

19a

0.3

14

1373.4

1

CBDFVCNCDR


19b

0.1

7

20

4.1

21

1.5

14

844.5

2

GPYNVCCSTDXCNR

3FTx; Micrurus frontalis ∼ P86421

1881.8

1

BTYBYDCSEGBXTCK

PLA2; Bungarus flaviceps ∼ Q7T2Q5

TAAXCFAK


NLYQLKNMIKCTNTR APYNNBNFK

PLA2; M. nigrocinctus nigroxin A ∼ P81166

881.4

1

1095.5

N-te 1

1373.5

1

CBDFVCNCDR

22a

0.4

14

1373.5

1

CBDFVCNCDR


22b

1

10

854.4

2

(186.2)TXFCDNSNVPSXR

3FTx; M. corallinus ∼ ACS74996

23a

0.9

16

1770.6

1

CCQVHDDCYGEAEK

PLA2; Oxyuranus scutellatus ∼ AAZ22633

23b

1.1

14

1568.7

1

SPYNNNNYNXDXK

PLA2; Laticauda colubrina ∼ Q8UUI0

23c

1.9

7

854.4

2

(186.2)TXFCDNSNVPSXR

3FTx; M. corallinus ∼ ACS74996

24 25a

1.4 1.9

14 14

1373.6 1373.4

1 1

CBDFVCNCDR CBDFVCNCDR

PLA2; M. nigrocinctus nigroxin A or B ∼ P81166 or P81167 PLA2; M. nigrocinctus nigroxin A or B ∼ P81166 or P81167

25b

1.3

7

1373.4

26

4.1

14

1 N-te

1373.4

1

CBDFVCNCDR


NLIQFKNMIKCTNTR


CBDFVCNCDR 1818



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Table 1. Continued peptide ion fraction

%

mass (kDa)

m/z

27

3.6

14

1373.5 881.4

28

3

14

z

protein family; related protein*

peptide sequence 1 1

CBDFVCNCDR TAAXCFAK


1944.7

1

NXYQFBNMXQCTTBR


1373.4

1

CBDFVCNCDR


29a

0.5

17

1769.6

1

CCQVHDNCYGEAEK

PLA2; Naja sputatrix ∼ Q9I900

29b

2.1

14

1373.5

1

CBDFVCNCDR


30

5.3

31a 31b

0.6 0.4

14 11

31c

1.4

7

32a

2.7

11

32b

0.7

7

33

2

34a 34b

1373.4

1

CBDFVCNCDR


1937.7

1

BTYKcYDCSEGBXTCK

PLA2; Bungarus flaviceps ∼ Q7T2Q5

1937.7

1

BTYKcYDCSEGBXTCK

PLA2; Bungarus flaviceps, ∼ Q7T2Q5 unknown

625.8

3

(631.5)PGVAGCAVTCPR

3 FTx; M. corallinus ∼ ACS74994

848

2

TPQXCPEGQDVCYK

3 FTx; Bungarus multicinctus κ2 bungarotoxin, ∼ P15816

625.8

3

(631.5)PGVAGCAVTCPR

3 FTx; M. corallinus ∼ ACS74994

11

848

2

TPQXCPEGQDVCYK

3 FTx; Bungarus multicinctus κ2 bungarotoxin, ∼ P15816

0.6

14

1568.7

1

SPYNNNNYNXDXK

PLA2; Laticauda colubrina ∼ Q8UUI0

0.2

11

1810.8

1

TVENVGVSQVAPDNPER

OH; Naja kaouthia thaicobrin ∼ P82885

35 36

2.9 0.6

14

1810.9 1810.7

1 1

TVENVGVSQVAPDNPER TVENVGVSQVAPDNPER

OH; Naja kaouthia thaicobrin ∼ P82885 OH; Naja kaouthia thaicobrin ∼ P82885

37

0.3

38

0.4

864.3

1

GCAATCPK

MuTx; Dendroaspis angusticeps mt-7 - 3FEV

39

1.2

503.3

2

XWEWTDR

C-type lectin; M. corallinus ∼ ACS74933

40

0.6

958.5

2

WNDTPCESXFAFXCR


41

0.4

958.4

2

WNDTPCESXFAFXCR


42

1

unknown 20

43

0.5

65

44

2.4

43

45

0.4 43

N-te

DKYLQVKKYIETYVI

SVMP; Rhinocephalus nigrescens ∼ ABQ01139

708.8

2 N-te

YXEFYVVVDNR TPEQDRYLQVKKYI

SVMP; Hoplocephalus stephensii ∼ ABQ01135 SVMP; Naja atra ∼ ADF43026

1910.8

1

NGHPCQNNQGYCYNGK

SVMP; Naja kaouthia ∼ P82942

1910.6

1

NGHPCQNNQGYCYNGK

SVMP; Naja kaouthia ∼ P82942

649.4

2

SAECPTDSFQR

SVMP; Hoplocephalus stephensii ∼ ABQ01135

1269.5

1

ZVPVVQAYAFGK

NU; Gloydius blomhoffi ecto-50 -nucleotidase ∼ BAG82602

46

1.5

1484.7

1

EADYEEFXEXAR

LAO; Oxyuranus scutellatus ∼ Q4JHE3

47

0.8

1216.6

1

FWEADGXHGGK


48

0.5

1484.7 1484.5

1 1

EADYEEFXEXAR EADYEEFXEXAR


49

0.7

IVGGSDARSGQWPWQ

SP; Taeniopygia guttata serine proteinase ∼ XP002188242

N-te

* Abbreviations: 3FTx, three-finger toxin; PLA2, phospholipase A2; OH, ohanin-like; MuTx, muscarinic toxin; SVMP, metalloproteinase; NU, nucleotidase; LAO, L-amino acid oxidase; SP, serine proteinase; N-te, N-terminal sequence. Cysteine residues determined in MS/MS analyses are carbamidomethylated, unless otherwise stated. X, Leu/Ile; B, Lys/Gln; Z, pyrrolidone carboxylic acid; Kc, carbamylated Lys.

triply charged ions of peptides selected from the MALDI-TOF mass fingerprint spectra were analyzed in Enhanced Resolution mode (250 amu/s), and the monoisotopic ions were fragmented using the Enhanced Product Ion tool with Q0 trapping. Settings for MS/MS analyses were: Q1, unit resolution; collision energy, 25-40 eV; linear ion trap Q3 fill time, 250 ms; and Q3 scan rate, 1000 amu/s. Resulting CID spectra were interpreted with the aid of the BioAnalyst 1.5 manual sequencing tool, and the deduced sequences or complete spectra were submitted to BLAST (http://blast.ncbi.nlm.nih.gov), or MASCOT (http://www. matrixscience.com) databases, respectively, for protein identification. In addition to MS analyses, selected undigested fractions from the RP-HPLC separation were submitted to N-terminal amino acid sequencing on a Procise instrument (Applied Biosystems), according to manufacturer’s instructions.

2.3. Individual Variations

The chromatographic profiles of venom samples obtained from six M. nigrocinctus specimens from several locations of Costa Rica were compared. Three corresponded to individuals kept in captivity for over one year at the ICP serpentarium, whereas the other three corresponded to first venom extractions from newly arrived snakes. Venoms were collected in plastic vials, centrifuged, and stored at -20 °C. Two-hundred μL of each sample, diluted in 800-1000 μL of solution A, were analyzed by RP-HPLC as described in section 2.1. 2.4. Lethality and Myotoxicity of PLA2 Fractions

Major venom fractions containing PLA2s (peaks 15, 17, 18, 20, 22, 26, 27, 31, 33; numbering as in Table 1 and Figure 2) were screened for lethal and myotoxic activities. Fractions were vacuum-dried, redissolved in water, and adjusted for protein 1819



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Figure 2. Elution profile of Micrurus nigrocinctus venom proteins by RP-HPLC. Two to three milligrams of pooled venom were fractionated on a C18 column, as described in Materials and Methods (gradient line is omitted for clarity). Fractions were analyzed by SDS-PAGE under reducing conditions (top; mass markers indicated in kDa) and characterized by MALDI-TOF/TOF, nESI-MS/MS tryptic peptide MS/MS de novo sequencing or N-terminal sequencing, as summarized in Table 1. In SDS-PAGE under nonreducing conditions (not shown), fractions containing PLA2s produced very strong and concentration-dependent smears, which made interpretation of their electrophoretic patterns difficult, and were therefore omitted.

concentration using the Quickstart Bradford Protein Assay (BioRad). Five micrograms of protein from each fraction, in a volume of 100 μL, was administered to two mice (16-18 g body weight) by the intraperitoneal route, and deaths were scored after 48 h. Screening for myotoxic activity was performed by injecting 5 μg of protein from each fraction intramuscularly in two mice (1820 g). Two control mice received an identical injection of PBS. Blood was collected from the tail after 3 h, and plasma creatine kinase (CK) activity was determined by a kinetic UV assay (CKNac, Biocon). 2.5. Antivenomic Analyses

The ability of an equine monospecific antivenom to M. nigrocinctus (ICP, batch 448; expiry September 2012) to immunodeplete venom components was evaluated as described.43 In brief, 1 mg of crude venom was added to 1 mL of antivenom (containing ∼24 mg of caprylic acid-purified whole IgG), and incubated overnight on a rotary mixer, at room temperature. After centrifuging at 13 000 rpm for 15 min, the supernatant was incubated with 0.8 mL of Sepharose-Protein G beads (GE Healthcare) for 2 h at room temperature, with gentle rotation, in a spin column. After centrifuging at 4000 rpm for 1 min, 500 μL of the final supernatant was analyzed by RP-HPLC as described. As a control, a parallel sample in which antivenom was substituted by phosphate-buffered saline (PBS) was analyzed under otherwise identical conditions. Antibody recognition of M. nigrocinctus venom components was also evaluated by immunoblotting. RP-HPLC separated venom fractions were loaded on 5-20% gradient gels and separated by SDS-PAGE under reducing conditions, in duplicate gels. Proteins were transferred to nitrocellulose membranes using a Mini-Protean cell (Bio-Rad) at 100 mAmp for 2 h, and visualized by reversible Ponceau-S staining. After saturating the membranes in PBS containing 1%

bovine serum albumin (PBS-BSA) for 1 h, one set was incubated with a 1:3000 dilution of antivenom (in PBS-BSA), and the other with an equal dilution of nonimmune caprylic acid-purified horse IgG, as a control, for 2 h at room temperature. After five washings with PBS-BSA containing 0.1% Tween-20, bound antibodies were detected by a further 2 h incubation with antihorse IgGalkaline phosphatase conjugate (Sigma) diluted 1:6000 in PBSBSA. After thorough washing, color was developed simultaneously for both sets of membranes using BCIP/NBT (Chemicon) as chromogenic substrate.

3. RESULTS AND DISCUSSION 3.1. Venomics

The elution profile of M. nigrocinctus venom proteins separated by RP-HPLC resolved nearly 50 peaks, revealing a complex pattern (Figure 2). This complexity appears to be higher than that observed in chromatographic analyses of venoms from other coral snakes, such as M. alleni, M. multifasciatus,42 M. surinamensis,14 or other elapids.12,16 Unlike some snake venoms which present a protein distribution heavily biased toward one or few major components, for example in Naja naja siamensis44 or several Crotalus species,45,46 the protein constituents of M. nigrocinctus venom are more uniformly distributed. Most of the chromatographic fractions contain less than 2% of the total proteins (average 1.6%, median 1.2%), ranging from 0.1 to 7.9% (Figure 2). Proteins related to eight families were identified by MS/MS de novo peptide sequencing or N-terminal sequencing in M. nigrocinctus venom (Table 1): phospholipase A2 (PLA2), three-finger toxin (3FTx), L-amino acid oxidase (LAO), C-type lectin/lectinlike (C-lect), metalloproteinase (SVMP), serine proteinase (SP), ohanin (OH), and nucleotidase (NU). The two outstanding protein families are PLA2s and 3FTxs, which account for 48 and 1820



Figure 3. Diversity of three-finger toxins (3FTx) and phospholipases A2 (PLA2) in Micrurus nigrocinctus venom, as revealed by MALDI-TOF mass spectrometry. A number of proteins with masses compatible with short- and long-chain 3FTxs are evidenced in (A), whereas several proteins with masses typical of elapid PLA2s are shown in (B).

38% of the venom proteins, respectively (Figure 1B). Their prominence is a shared feature of Micrurus venoms studied by proteomic or transcriptomic approaches so far,14,18 although it is noteworthy that in M. nigrocinctus the PLA2s predominate over 3FTxs, in contrast to most elapids.10,16,19 Alongside their abundance, PLA2s and 3FTxs are highly diverse in M. nigrocinctus venom, as they were present in 22 and 18 chromatographic fractions, respectively (Table 1). MALDI-TOF analysis of the crude venom in MS linear mode confirmed the diversity of proteins in the range of masses corresponding to these two families (Figure 3). Such abundance and diversity suggests that PLA2s and 3FTxs are likely to play major biological roles in this venom. Although this diverse cocktail of venom proteins may be the consequence of neutral evolutionary processes, it may also reflect the acquisition of a versatile toxic arsenal needed to cope with a variable array of prey items characteristic of this species.22,47 Considering that the venom was obtained from a large number of specimens, it was of interest to determine whether the observed complexity is a consequence of intraspecific variations. Therefore, venom samples of six individual specimens from various locations of Costa Rica were analyzed by RP-HPLC. As shown in Figure 4, despite few minor differences in the region

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corresponding to PLA2s, there were no prominent qualitative changes in their overall chomatographic profiles, which mainly varied in the relative size of some peaks. For example, the conspicuous R-neurotoxin eluting at ∼20 min (Figure 4), which represents the most abundant component of the pooled venom (fraction 3 of Table 1; 7.9%), showed higher relative levels in specimens B, D, and E, than in specimens A, C, and F, but was present in all of them. Further, the chromatographic profiles of venom samples obtained from newly collected or from long-term captive specimens were similar. Overall, these analyses indicate that the complex protein composition observed in the pooled venom of M. nigrocinctus does not result from the sum of highly variable individual patterns of lower complexity, but is rather an intrinsic feature of this species. This conclusion is also in agreement with a previous evaluation of intraspecific variability in individual M. nigrocinctus venoms performed by electrophoresis.48 Among PLA2 components, the majority presented peptide fragments with sequences identical to either the previously characterized nigroxins A and B of M. nigrocinctus, which exert myotoxicity,3,41 or to the neurotoxic PLA2s reported for this species by Mochca-Morales et al.39 However, some of the PLA2s showed peptides related to enzymes from M. corallinus, or to Old World elapid species of the genera Naja, Bungarus, Laticauda, Oxyuranus, and Lapemis. Since presynaptic neurotoxicity is a well-known effect for many elapid PLA2s,49 a screening for lethal activity in mice was conducted for the major PLA2-containing fractions of M. nigrocinctus venom. The injection of 5 μg (0.3 μg/g) of most of these proteins did not cause death, except in the case of fraction 27 (intraperitoneal) and of fraction 26 (intramuscular). The lethal activity of these PLA2 fractions is suggestive of a possible presynaptic neurotoxic activity, which would agree with the observations by Goularte et al.,33 who recorded electrophysiological alterations compatible with a presynaptic effect in isolated neuromuscular preparations exposed to the crude venom. A similar effect has been described for a PLA2 of M. dumerilii carinicauda.50 On the other hand, the lack of lethal activity at this dose in most PLA2-containing fractions suggests that they might have activities other than presynaptic neurotoxicity. Along this line, a screening for myotoxic activity by intramuscular injection of 5 μg of these proteins confirmed such effect (Figure 5). Fractions 15, 17, 18, 20 22, 26, 27, and 31 caused plasma CK elevations of at least 5-fold above control levels. Furthermore, the myotoxic activity of peak 26 was remarkably potent, leading to a 125-fold CK increase over controls (Figure 5). A marked local necrosis of skeletal muscle tissue, and the presence of hyaline casts in renal tubules, probably corresponding to myoglobin, were confirmed for this potent PLA2 fraction (Figure 6). The adaptive role of myotoxicity in this and other elapid venoms could be associated with the digestion of muscle mass in the prey, since widespread myonecrosis is likely to contribute to a more effective digestion of muscle proteins by proteinases.51 The second most abundant venom proteins were 3FTxs, which eluted earlier than PLA2s in the RP-HPLC separation. The major 3FTx (fraction 3; 7.9%) showed identity to the short-chain R-neurotoxin (P80548) characterized by Rosso et al.,42 and similar proteins were detected in several other fractions (Table 1). Accordingly, Rosso et al.42 and Alape-Giron et al.41 have shown that multiple protein fractions from M. nigrocinctus venom specifically bind to the acetylcholine receptor of the electric organ of Torpedo sp. The observation of lack 1821



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Figure 4. Elution profile of venoms from individual Micrurus nigrocinctus specimens by RP-HPLC. Samples A-C correspond to captive individuals, whereas samples D-F correspond to first extractions from newly arrived snakes. Geographic origin of the specimens is indicated in the map shown in Figure 1. Chromatography conditions are identical to those of Figure 2.

of lethality in the majority of PLA2 fractions, together with the abundance of R-neurotoxins, suggests that the latter play the predominant role in the neurotoxic effect that leads to paralysis. In addition to short-chain R-neurotoxins, acting postsynaptically on nicotinic acetylcholine receptors,52 other proteins of the 3FTx family were identified. These include few proteins with structural similarity to muscarinic or muscarinic-like toxins (MuTx) such as mt-7 from Dendroaspis angusticeps or MuTxlike protein-2 from Naja kaouthia (Table 1), which target subtypes of muscarinic acetylcholine receptors, and represent about 1% of the venom. Furthermore, long-chain 3FTxs were

also recognized, including proteins with homology to κ-2 bungarotoxin from Bungarus multicinctus, which constitute 4.7% of the venom (Table 1). Long-chain 3FTxs are known to target neuronal nicotinic acetylcholine receptors,52 and their presence in M. nigrocinctus venom had been previously demonstrated by their antigenic cross-reactivity to the long-chain neurotoxin-I from N. oxiana and by N-terminal sequence similarity.41 Thus, the presence of MuTx-like and κ-neurotoxin-like proteins of the 3FTx family expands the range of possible biological and toxic activities exerted by this venom, which deserve further studies. The predominant effects induced by M. 1822



Figure 5. Increase of plasma creatine kinase (CK) in mice due to skeletal muscle necrosis induced by major PLA2-containing fractions from Micrurus nigrocinctus venom separated by RP-HPLC. Five micrograms of protein was injected by intramuscular route in two mice and, after 3 h, CK activity was determined and compared to mice that received an identical phosphatebuffered saline (PBS) injection. Bars represent mean ( SD, except for fractions 17 and 22, representing single mice (due to insufficient protein). Fraction numbers correspond to Figure 2 and Table 1.

Figure 6. Light micrographs of sections of (A) gastrocnemius muscle and (B) kidney of a mouse injected with 5 μg of PLA2 fraction 26 of Micrurus nigrocinctus venom. The mouse died 6 h after the injection and tissue samples were obtained immediately after death. Notice widespread myonecrosis in (A), with cells showing clumping of myofibrillar material as a consequence of hypercontraction (arrows). In (B), hyaline casts (arrows), probably containing myoglobin, are observed in renal tubules. A blood vessel (v) is also present. Magnification: 200 in A and 400 in B. Hematoxylin-eosin staining.

nigrocinctus venom, both in mice and in its natural prey (colubrid snakes), are neurotoxicity and myotoxicity.23,30,34

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It is suggested that neurotoxicity is predominantly mediated by R-neurotoxins, with a possible role of few presynaptically acting PLA2s, whereas myotoxicity is induced by PLA2s. At least two proteins related to thaicobrin/ohanin, first described in Naja and Ophiophagus species, respectively, were identified in M. nigrocinctus (fractions 35 and 36, Table 1). The highly homologous (93% identity) thaicobrin and ohanin define an independent family of toxins, of which only ohanin from O. hannah has been thoroughly characterized.53,54 Ohanin induces hypolocomotion and hyperalgesia in mice, by a mechanism unrelated to neuromuscular blockade.53 In contrast to ohanin, which constitutes only 0.1% of O. hannah venom, the related proteins in M. nigrocinctus represent 3.8%. It will be of interest to determine whether they exert similar actions as ohanin, which may reduce the mobility of envenomed prey. Recent reports of ohanin-related transcripts in the venom gland of two viperids, Lachesis muta55 and Crotalus durissus cumanensis,56 suggest that this recently recognized toxin family could be more widely distributed than previously thought, and perhaps not restricted to elapids, if protein expression of their transcripts is confirmed. Venoms of elapid snakes generally present a predominance of low molecular mass proteins, in contrast to viperid counterparts, where larger proteins are commonly found.9 This was indeed the case for M. nigrocinctus, which showed a low content of high molecular mass components. Among these, P-III metalloproteinases represent 4.3% of the venom. Proteins within this family are generally linked to disturbances of the hemostatic system, hemorrhage, tissue damage or digestive roles.57 The fact that M. nigrocinctus venom does not alter coagulation parameters in vivo, is devoid of hemorrhagic action,34 and has a very low proteolytic activity in vitro,58 would be in agreement with the low content of metalloproteinases in its proteome, suggesting a marginal role in these envenomings. Micrurus venoms, in general, display weak proteolytic activities.58-60 Metalloproteinases of the P-III class have been purified from other elapid venoms, mostly of the genus Naja.61-64 They act on various physiological substrates, such as the complement system, von Willebrand factor, and pro-TNFR. It is necessary to explore the biological activities of metalloproteinases in Micrurus venoms, although it is highly unlikely that they play a relevant role in the pathophysiology of envenomings by M. nigrocinctus. Other high molecular mass components of low abundance in M. nigrocinctus venom belong to C-type lectin/lectin-like, L-amino acid oxidase, nucleotidase, and serine proteinase families (Figure 1B). Accordingly, L-amino acid oxidase and 50 -nucleotidase activities have been reported in the crude venom of this species, as well as in other Micrurus venoms,59 although their precise functional roles are yet to be determined. C-type lectin/lectin-like components of snake venoms exert a number of activities, mostly related to the disruption of hemostasis by targeting particular plasma proteins or blood cells, especially platelets.65 The identification of proteins of this family in M. nigrocinctus, efficiently resolved by RP-HPLC, opens possibilities to further study their structural and functional properties. 3.2. Antivenomics

Within the framework of the venomic characterization of M. nigrocinctus, an evaluation of the equine monospecific coral snake antivenom produced at ICP, and therapeutically used in Central America, was performed through an “antivenomics” 1823



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Figure 7. Immunodepletion of Micrurus nigrocinctus venom components by the equine antivenom (anticoral snake) produced at Instituto Clodomiro Picado, University of Costa Rica. Venom was preincubated with (A) phosphate-buffered saline, or (B) coral snake antivenom. Then, both samples were incubated with Protein G-Sepharose beads to remove the equine IgG and its bound venom components. Proteins remaining in the supernatant were finally analyzed by RP-HPLC. For simplicity, only some of the fractions (numbered as in Figure 2 and Table 1) are labeled. In panel (B), arrows point to completely immunodepleted fractions. Peaks corresponding to phenol (*) present in the antivenom, and excess IgG, are indicated.

approach.7,66 When tested for its ability to immunodeplete the different venom components, this antivenom achieved only partial reductions of venom chromatographic peaks in most cases, especially for the various 3FTx- and PLA2-containing fractions (Figure 7). In contrast, peaks corresponding to fractions containing higher molecular mass proteins (fractions 35 and above) were highly or completely depleted. Due to the limitations of the technique regarding the amount of venom proteins needed for adequate detection during the analytical RP-HPLC step, in relation to the intrinsic dilution introduced by the antivenom per se, the highest ratio of venom/antivenom assayed corresponds to 1 mg/mL. This coral snake antivenom is routinely standardized to a neutralizing potency (effective dose 50% against lethality in mice) of at least 0.5 mg venom/mL antivenom, and the particular batch studied here had a potency of 0.7 mg/mL. It is possible that higher antivenom/venom ratios may lead to stronger immunodepletions than those recorded in Figure 7, but this could not be tested. In any case, under the conditions described, immunodepletions can be grouped into three regions of the venom chromatogram: the high molecular mass proteins showed the best depletions, followed by intermediate depletion of proteins eluting in the central PLA2 region, while the poorest depletions corresponded to proteins eluting in the 3FTx region (Figure 7). These findings evidence a relationship between molecular mass of the toxins and immunogenicity (as reflected by the specific antibody contents against each venom component leading to immunodepletion), which can be expected considering that high mass and structural complexity tend to enhance protein immunogenicity, and viceversa.67 This relationship between molecular mass of toxins and immunogenicity has also been described in viperid snake venoms.43,68 Efforts are needed to improve the immune

response to low molecular mass venom proteins, especially in the cases of venoms whose toxicity depends on such components, as in elapid species. The immunodepletion analysis was complemented by immunoblotting of all RP-HPLC separated venom fractions, probed with the same antivenom. In agreement with the former, immunoblotting revealed a weak recognition of the 3FTx components, a slightly higher recognition of PLA2s, and a stronger recognition of the high molecular mass proteins. An example of these results (covering the chromatographic region of 3FTxs and the first part of the PLA2 region) is shown in Figure 8. An unexpected, but reproducible finding was a faint recognition of few venom components, most notably some PLA2s (Figure 8) by the normal horse immunoglobulins used as a control. As a general rule, antivenoms prepared against venoms containing low molecular mass neurotoxins, such as those of elapid snakes or of some crotalids, achieve lower lethality-neutralizing potencies than those prepared against viperids.31,69,70 The antivenomic data here presented help to understand this pattern in the case of M. nigrocinctus, by disclosing the relatively low proportions of antibodies against the highly lethal 3FTxs, both in immunodepletion and immunoblotting assessments. In the latter, this conclusion can be clearly illustrated by comparing the high amount of proteins available on the nitrocellulose membrane (Figure 8A), to the weak intensities resulting from their probing with antivenom (Figure 8B). Although it would be highly valuable to find novel strategies to enhance the immune response toward neurotoxic 3FTxs, the antibody levels present in this antivenom have proven clinically effective for the treatment of coral snake envenomings in Central America.25 Further, the ability of this and related antivenoms to neutralize the myotoxic, PLA2, and acetylcholine receptor-binding activities of M. nigrocintus venom, in addition to its lethal effect, have been confirmed experimentally.28,30-33,71 1824



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digestion.23 Newly identified 3FTxs in this venom, related to MuTx/MuTx-like proteins or to long-chain κ-neurotoxins, should be further studied to determine their possible relevance in envenomings by this coral snake. Similarly, previously unknown proteins of other families such as the ohanin-like and C-type lectin/lectin-like venom components, offer interesting opportunities to study their structures and biological activities with current technologies and micromethods, in a taxonomic group of elapids that has traditionally received limited attention due to the scarcity of their venoms. Finally, the antivenomic characterization here presented helps to understand the current limitations of Central American coral snake antivenom regarding potency, by revealing the immunodominance of high molecular mass venom components, probably not involved in lethality, over smaller proteins with proven lethal effects such as the 3FTxs. Such disparate immunogenicity of M. nigrocinctus venom proteins can also be inferred by a previous study where 11 monoclonal antibodies against this venom were obtained, recognizing high molecular mass proteins or PLA2s, but none directed against a 3FTx.72 These findings altogether stress the relevance of devising new strategies to improve the antibody response toward poorly immunogenic, but clinically relevant, snake venom toxins.

’ AUTHOR INFORMATION Corresponding Author

*Dr. Bruno Lomonte, Instituto Clodomiro Picado, Universidad de Costa Rica, San Jose, Costa Rica. E-mail: bruno.lomonte@ ucr.ac.cr, Tel. (þ506) 2229-0344.

Figure 8. Immunoblotting analysis of equine coral snake antivenom (ICP) against proteins from selected Micrurus nigrocinctus venom fractions separated by RP-HPLC and SDS-PAGE on 5-20% gradient gels, under reducing conditions. Samples shown correspond to 3FTxs and PLA2s from the first third of the chromatogram (numbered as in Figure 2 and Table 1). Molecular mass standards are labeled at the left, in kDa. (A) Ponceau-S reversible stain of proteins before probing with the equine antivenom. The same membrane, after antivenom probing, is shown in (B). Note the stronger recognition of some PLA2s in lanes 12-17, in comparison to the weak immunostaining of 3FTxs in lanes 3-8 (C). Control nonimmune equine IgG was probed in a duplicate nitrocellulose membrane against these fractions. Note a faint staining of some venom fractions, especially PLA2s in lanes 15-17, by normal equine immunoglobulins in (C).

4. CONCLUDING REMARKS The venom of M. nigrocinctus is composed of proteins classified within eight structural families, of which PLA2s and 3FTxs are quantitatively predominant and highly diverse, contributing to its considerable complexity. The short-chain R-neurotoxins acting on the nicotinic acetylcholine receptor of neuromuscular junctions41,42 are likely to have a leading role in lethality induced by this venom, with a possible contribution by other types of 3FTxs and some PLA2s, but further studies remain to be done to clarify this point. Most of the PLA2 enzymes did not behave as lethal toxins when tested at doses high enough for neurotoxic PLA2s to express this effect, but mainly displayed myotoxic activity, and thus may be fulfilling a role in prey

’ ACKNOWLEDGMENT Financial support to this work was provided by CONARE, CRUSA-CSIC (project 2009CR0021), Vicerrectoría de Investigacion-Universidad de Costa Rica (UCR; 741-A7-611), Ministerio de Innovacion y Ciencia (Madrid, Spain; grants BFU2007-61563 and BFU2010-17373), PROMETEO/2010/ 005 from the Generalitat Valenciana, CYTED (206AC0281) and Acciones Integradas (CSIC-UCR 2006CR0010). Analyses performed at the Proteomics Laboratory of Instituto Clodomiro Picado were supported by CONARE and Vicerrectoría de Investigacion-UCR. Thanks are also due to our colleagues at the ICP Industrial Division and Serpentarium, especially to Mr. Danilo Chacon, in charge of the live collection of M. nigrocinctus, and Dr. Mahmood Sasa, for fruitful discussions. We also wish to acknowledge Dr Deckar Rojas and Eng. Leonardo Gabino (ABSciex) for excellent support on MS instrumentation. ’ REFERENCES (1) Vonk, F. J.; Admiraal, J. F.; Jackson, K.; Reshef, R.; de Bakker, M. A.; Vanderschoot, K.; van den Berge, I.; van Atten, M.; Burgerhout, E.; Beck, A.; Mirtschin, P. J.; Kochva, E.; Witte, F.; Fry, B. G.; Woods, A. E.; Richardson, M. K. Evolutionary origin and development of snake fangs. Nature 2008, 454, 630–633. (2) Fry, B. G.; Vidal, N.; Normamn, J. A.; Vonk, F. J.; Scheib, H.; Ramjan, S. F.; Kuruppu, S.; Fung, K.; Hedges, S. B.; Richardson, M. K.; Hodgson, W. C.; Ignjatovic, V.; Summerhayes, R.; Kochva, E. Early evolution of the venom system in lizards and snakes. Nature 2006, 439, 584–588. (3) Alape-Giron, A.; Persson, B.; Cederlun, E.; Flores-Díaz, M.; Gutierrez, J. M.; Thelestam, M.; Bergman, T.; J€ornvall, H. Elapid venom toxins: multiple recruitments of ancient scaffolds. Eur. J. Biochem. 1999, 259, 225–234. 1825


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