toxins Article
Venom of the Coral Snake Micrurus clarki: Proteomic Profile, Toxicity, Immunological Cross-Neutralization, and Characterization of a Three-Finger Toxin Bruno Lomonte 1, *, Mahmood Sasa 1 , Paola Rey-Suárez 2 , Wendy Bryan 1 and José María Gutiérrez 1 1 2
*
Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José 11501, Costa Rica; [email protected] (M.S.); [email protected] (W.B.); [email protected] (J.M.G.) Programa de Ofidismo y Escorpionismo, Universidad de Antioquia, Medellín 050010, Colombia; [email protected] Correspondence: [email protected]; Tel.: +506-2511-7888
Academic Editor: Stephen P. Mackessy Received: 26 March 2016; Accepted: 2 May 2016; Published: 5 May 2016
Abstract: Micrurus clarki is an uncommon coral snake distributed from the Southeastern Pacific of Costa Rica to Western Colombia, for which no information on its venom could be found in the literature. Using a ‘venomics’ approach, proteins of at least nine families were identified, with a moderate predominance of three-finger toxins (3FTx; 48.2%) over phospholipase A2 (PLA2 ; 36.5%). Comparison of this venom profile with those of other Micrurus species suggests that it may represent a more balanced, ‘intermediate’ type within the dichotomy between 3FTx- and PLA2 -predominant venoms. M. clarki venom was strongly cross-recognized and, accordingly, efficiently neutralized by an equine therapeutic antivenom against M. nigrocinctus, revealing their high antigenic similarity. Lethal activity for mice could be reproduced by a PLA2 venom fraction, but, unexpectedly, not by fractions corresponding to 3FTxs. The most abundant venom component, hereby named clarkitoxin-I, was identified as a short-chain (type I) 3FTx, devoid of lethal effect in mice, whose target remains to be defined. Its amino acid sequence of 66 residues shows high similarity with predicted sequences of venom gland transcripts described for M. fulvius, M. browni, and M. diastema. Keywords: monadal coral snake; Elapidae; neutralization
venom;
toxin;
Micrurus;
antivenom;
proteome;
1. Introduction Coral snakes (Elapidae) comprise some 71 species that are widely distributed in tropical and subtropical regions of the New World, from Southeastern USA to Central Argentina [1–3]. Envenomings by these snakes are far less frequent than those inflicted by viperids, but are of medical concern [4,5]. Micrurus clarki (Figure 1A) is an uncommon, slender to medially robust coral snake with a reported maximum length of 920 mm, although most adults average between 400 and 600 mm. This species has a tricolored pattern with narrow yellow bands bordering the black bands followed by single red bands, and a characteristic black head cap that extends over most of the snout and parietals. M. clarki has a discontinuous distribution from the Tárcoles basin in Central Pacific Costa Rica southward to the Pacific lowlands of Western Panamá, where it inhabits lowlands of the Canal Zone, to Pacific Darien, and Western Colombia. Along this distribution, M. clarki inhabits tropical wet forest, but also transitional tropical wet-tropical dry forest, where it is mostly found at low elevations, with some Toxins 2016, 8, 138; doi:10.3390/toxins8050138
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with some exceptions reported up to 900 m above sea level [1,6,7]. Little is known about the natural exceptions reported up to 900 m above sea level [1,6,7]. Little is known about the natural history of this history of this coral snake. The species was named in honor of H.C. Clark, a medical doctor that coral snake. The species was named in honor of H.C. Clark, a medical doctor that directed the Gorgas directed the Gorgas Memorial Institute in Panamá, considered a pioneer in tropical medicine [8]. It is Memorial Institute in Panamá, considered a pioneer in tropical medicine [8]. It is considered mainly a considered mainly a terrestrial and primarily nocturnal species whose natural diet is known to terrestrial and primarily nocturnal species whose natural diet is known to include the marbled swamp include the marbled swamp eel Synbranchus marmoratus, and colubrid snakes [6]. eel Synbranchus marmoratus, and colubrid snakes [6].
Figure 1. (A) Micrurus clarki and (B) separation of its venom (2 mg) by RP-HPLC, followed by (C) Figure 1. (A) Micrurus clarki and (B) separation of its venom (2 mg) by RP‐HPLC, followed by (C) SDS-PAGE. Venom was fractionated on a C18 RP-HPLC column and eluted with an acetonitrile gradient SDS‐PAGE. was fractionated on further a C18 RP‐HPLC column and eluted an acetonitrile (dashed line)Venom at 1 mL/min. Fractions were separated by SDS-PAGE under with reducing conditions. gradient (dashed line) at 1 mL/min. Fractions were further separated by SDS‐PAGE under reducing Molecular weight markers (Mw) are indicated in kDa. Coomassie-stained bands were excised, in-gel conditions. Molecular weight markers (Mw) are indicated in kDa. bands were digested with trypsin, and subjected to MALDI-TOF/TOF analysis forCoomassie‐stained assignment to protein families, excised, in‐gel digested with trypsin, and subjected to MALDI‐TOF/TOF analysis for assignment to as shown in Table 1. protein families, as shown in Table 1.
Our search of the mainstream literature databases did not find any information on the venom of Our search of the mainstream literature databases did not find any information on the venom of M. clarki, whose general characteristics seem to be completely unknown. As part of an ongoing effort M. clarki, whose general characteristics seem to be completely unknown. As part of an ongoing effort to characterize the venom proteomes of all snake species from Costa Rica [9], in the present work we to characterize the venom proteomes of all snake species from Costa Rica [9], in the present work we report the composition, toxicity, immunological cross-recognition, and neutralization of the venom of report the composition, toxicity, immunological cross‐recognition, and neutralization of the venom M. clarki obtained from specimens collected in the Southeastern Pacific of this country. In addition, a of M. clarki obtained from specimens collected in the Southeastern Pacific of this country. In addition, three-finger toxin from this venom was isolated and characterized. a three‐finger toxin from this venom was isolated and characterized. 2. Results and Discussion 2. Results and Discussion 2.1. Proteomic Profile of Micrurus Clarki Venom 2.1. Proteomic Profile of Micrurus Clarki Venom The venom of M. clarki (1A) was initially separated into 33 fractions by RP-HPLC (Figure 1B), The venom M. clarki was bands initially separated into 33 fractions RP‐HPLC which were furtherof resolved into (1A) 42 protein after SDS-PAGE (Figure 1C). Afterby in-gel tryptic (Figure 1B), which were further resolved into 42 protein bands after SDS‐PAGE (Figure 1C). After in‐ digestion of these, followed by MALDI-TOF-TOF analysis, protein family assignments were obtained gel tryptic of these, to followed by 95.9% MALDI‐TOF‐TOF analysis, protein The family assignments for 34 of thedigestion bands, estimated represent of the total venom proteins. remaining eight were obtained for 34 of the bands, estimated to represent 95.9% of the total venom proteins. bands for which no identifications were obtained (indicated as “unknown” in Table 1 and FigureThe 2), remaining eight bands for which no identifications were obtained (indicated as ‘‘unknown’’ in Table altogether add to the remaining 4.1% of the venom proteins. 1 and Figure 2), altogether add to the remaining 4.1% of the venom proteins. Although not markedly predominant, the protein family with the highest abundance in M. clarki Although not markedly predominant, the protein family with the highest abundance in M. clarki venom corresponds to three-finger toxins (3FTx), which account for almost half of the protein content venom corresponds to three‐finger toxins (3FTx), which account for almost half of the protein content (48.2%), followed by phospholipase A2 (PLA2 ), representing roughly one-third (36.5%). (48.2%), followed by phospholipase A2 (PLA2), representing roughly one‐third (36.5%).
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Table 1. Assignment of the RP-HPLC/SDS-PAGE separated fractions of Micrurus clarki venom to protein families by MALDI-TOF-TOF of selected peptide ions from in-gel trypsin-digested protein bands. Peak
%
Peptide Ion
Mass, kDa İ (Da)
m/z
z
MS/MS-Derived Sequence *
Conf (%)
Sc **
Protein Family; ~ Related Protein and Species *** PNP; adenosine
1
1.3
-
-
-
-
-
-
2–6
1.1
-
-
-
-
-
-
PNP 3FTx; ~U3FVH8, 3FTx 4a Micrurus fulvius
7
3.2
10 İ (6757)
1707.0
1
FSPGIQTSQTCPAGQK
99.0
11
8
5.9
9 İ (6638, 7276)
976.5
1
MFIRTHR
76.6
5
1322.7
1
ICDDSSIPFLR
99.0
7
FYFAYQCTSK
man
man
99.0
11
99.0
11
-
-
9
0.2
9İ
1314.7
1
10
8.0
9 İ (6361, 6451)
2663.2
1
1375.6
1
DGFYSVTCTEKENLCFT MFSAR ENLCFTMFSAR
9İ
-
-
-
11 12
13
14
0.4 1.8
3.6
6.9
9 İ (7045, 7140, 7198)
9 İ (7071, 7201)
9 İ (6845)
1661.8
1
GAYNVCCSTDLCNK
99.0
15
1374.7
1
KCLEFIYGGCQ
99.0
12
1786.9
1
VCYTIFLVGPSYPpy EK
93.5
6
1687.8
1
GPYNVCCSTDLCNK
99
7
1310.7
1
AIEFGCAASCPK
99
12
3FTx; ~P01414 , toxin FS-2 Dendroaspis polylepis 3FTx; ~U3EPK7, 3FTx 6 Micrurus fulvius 3FTx; ~C6JUP1, 3FTx Micrurus corallinus 3FTx; ~C6JUP4, 3FTx Micrurus corallinus Unknown 3FTx; ~U3FAE1, 3FTx 3a Micrurus fulvius Kunitz; ~Q6ITB9, mulgin-3 Pseudechis australis 3FTx; ~AKN63197, 3FTx Micrurus diastema 3FTx; ~B3EWF8, mipartoxin-I Micrurus mipartitus 3FTx; ~AKN63198, 3FTx DK, Micrurus diastema
15a
0.4
12 İ
-
-
-
-
-
unknown
15b
2.5
9 İ (6843, 7311)
1314.7
1
FYFAYQCTSK
man
man
3FTx; ~C6JUP1, 3FTx Micrurus corallinus
16a
0.3
12 İ
-
-
-
-
-
unknown
16b
0.2
10 İ
1373.6
1
CKDFVCNCDR
99.0
7
PLA2 ; ~U3FYP8, PLA2 13 Micrurus fulvius
17a
1.7
13 İ
1373.6 1085.4
1 1
CKDFVCNCDR DFVCNCDR
99.0 96.8
14 6
PLA2 ; ~U3FYP8, PLA2 13 Micrurus fulvius
17b
1.2
9İ
-
-
-
-
-
unknown
2969.3
1
99.0
12
18
6.6
13 İ (13353)
1373.5
1
99.0
12
2983.3
1
98.5
6
HWVSFTNYGCYCGYGG SGTPVDELDK CKDFVCNCDR HWLSFTNYGCYCGYGG SGTPVDELDK
PLA2 ; ~P81167, nigroxin B Micrurus nigrocinctus PLA2 ; ~U3FYP8, PLA2 13 Micrurus fulvius
19
11.3
13 İ (13477)
1373.6
1
CKDFVCNCDR
99.0
9
PLA2 ; ~U3FYN8, PLA2 9 Micrurus fulvius
20
1.8
13 İ
2855.3
1
SAWDFTNYGCYCGAGG SGTPVDELDR
99.0
15
PLA2 ; ~Q8AXW7, PLA2 Micrurus corallinus
2841.3
1
AWDYNNda YGCYCGQG GSGTPVDDLDR
99
12
1335.7
1
VHDDCYAAAEK
99
18
1827.8
1
CCQVHDNCYNEAEK
99
22
1278.6
1
VHDDCYAAAEK
99.0
7
1373.5
1
CKDFVCNCDR
99.0
8
2883.2
1
Sf˝ AWDFTNYGCYCGAG GSGTPVDELDR
99.0
15
21
5.3
12 İ (13331)
22a
2.4
20 İ
22b
4.5
13 İ
PLA2 ; ~R4G7M2, Sut-22 Suta fasciata PLA2 ; ~ U3FYP1, PLA2 3b Micrurus fulvius PLA2 ; ~ A4FS04, natratoxin, Naja atra PLA2 ; ~U3FYP1, PLA2 3b Micrurus fulvius PLA2 ; ~U3FYP8, PLA2 13 Micrurus fulvius PLA2 ; ~Q8AXW7, PLA2 Micrurus corallinus
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Table 1. Cont. Peak
%
Peptide Ion
Mass, kDa İ (Da)
m/z
z
MS/MS-Derived Sequence *
Conf (%)
Sc **
Protein Family; ~ Related Protein and Species ***
VHDDCYAAAEK
99.0
8
PLA2 ; ~U3FYP1, PLA2 3b Micrurus fulvius
99.0
16
PLA2 ; ~P00606, PLA2 Bungarus multicinctus
23a
0.6
13 İ
1278.5
1
23b
2.1
11 İ
2883.3
1
24
16.1
10 İ (7537)
1478.7 1322.6 1108.6
1 1 1
RICDDSSIPFLR ICDDSSIPFLR KGCASSCPKN
99 99 99
12 15 13
3FTx; ~U3EPK7, 3FTx 10a Micrurus fulvius
25
0.9
10 İ
1322.6
1
ICDDSSIPFLR
99.0
9
3FTx; ~U3EPK7, 3FTx 6 Micrurus fulvius
26
1.1
22 İ
1183.7
1
NVWIGLNDPR
man
man
CTL; ~ACC67944, mannose-binding 1 Pseudechis porphyriacus
27
0.1
-
-
-
-
-
-
28a
0.2
38 İ
PWky IGYVNYGCYCGAGG SGTPVDELDR
1054.5
1
TYWHYER
99.0
11
1130.6
1
EVFDGHTIGR
man
man
unknown MP; ~U3FWL3, MTP4 Micrurus fulvius MP; ~P85314, MP Bothrops moojeni MP: ~R4G7J1, MP-Hop-13 Hoplocephalus bungaroides
28b
0.2
29 İ
1297.6
1
SAECPTDSFQR
99.0
7
29a
0.6
38 İ
-
-
-
-
-
unknown
29b
0.2
28 İ
1011.6
1
FYVVVDNR
90.9
9
MP; ~ABQ01135, stephensease-1 Hoplocephalus. stephensii
29c
0.2
25 İ
1011.6
1
FYVVVDNR
man
man
MP; ~ABQ01135, stephensease-1 Hoplocephalus. stephensii
30a
0.4
51 İ
995.6
1
Fdh YVVVDNR
man
man
MP; ~ABQ01135, stephensease-1 Hoplocephalus. stephensii
30b
0.4
46 İ
995.6
1
Fdh YVVVDNR
man
man
MP; ~ABQ01135, stephensease-1 Hoplocephalus. stephensii
30c
0.1
29 İ
2171.1
1
GDSGGPLICNGQIQGIVS WGR
99.0
7
SP; ~Q5MCS0, harobin Hydrophis hardwickii
30d
0.2
20 İ
1329.7
1
IHDIKWNFEK
99.0
13
GPX; ~XP_013914274, glutathione peroxidase 3 Thamnophis sirtalis
1637.8
1
NDLEGWHVNLGPMR
99.0
23
LAO; ~U3FYQ2, LAAO 1a Micrurus fulvius
1131.5 1576.9 1484.7
1 1 1
SDDIFSYER IQDNTENVRVAYRam EADYEEFLEIAR
99.0 99.0 99.0
13 13 19
-
1963.1
1
TSGDIVINDLSLIHQLPK
99.0
7
LAO; ~U3FYQ2, LAAO 1a Micrurus fulvius
2275.1
1
99.0
8
-
1484.7
1
IHFAGEYTANDHGWIDS TIK EADYEEFLEIAR
99.0
9
1310.7
1
RFDEIVGGM˝x DR
99.0
10
1460.8
1
QVVPESLFAWER
95.0
7
LAO; ~A0A0A1WCY6, B variant Echis coloratus PLB: ~V8ND68, PLB-like 1 Ophiophagus hannah
1484.7 1963.0
1 1
99.0 99.0
13 17
2275.0
1
99.0
20
1131.5 1576.8
1 1
EADYEEFLEIAR TSGDIVINDLSLIHQLPK IHFAGEYTANDHGWIDS TIK SDDIFSYER IQDNTENVRVAYRam
99.0 99.0
11 12
-
-
-
-
-
31a
31b
31c
31d
2.2
0.5
1.1
0.7
61 İ
50 İ
42 İ
20 İ
LAO; ~U3FYQ2, LAAO 1a Micrurus fulvius
unknown
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Table 1. Cont. Toxins 2016, 8, 138 Peak
%
5 of 15
Mass, kDa İ (Da)
31f 31e
0.4 0.2
14 ▼ 17 İ
32
0.9 0.4
14 İ
31f 32
23 ▼
Peptide Ion m/z 1637.8 z 1589.8 1 1484.7 1 1466.7 1833.8‐ 1 1637.8 1 1484.7 1152.6 1 -
-
MS/MS-Derived Conf (%) Sequence * NDLEGWHVNLGPMR 1 99.0 99.0 NDLEGWHVNLGPMdt R 1 EADYEEFLEIAR Epy ADYEEFLEIAR 99.099.0
‐
‐ EFVQEDENAWYYIK NDLEGWHVNLGPMR EADYEEFLEIAR 1 LPFYADWIK -
99.0 ‐ 99.0 99.099.0 -
Protein Family; ~ Related Protein and Species ***
Sc ** 12 9 1117
LAO; ~U3FYQ2, LAAO 1a unknown Micrurus fulvius SP; ~U3FBP8, prostasin‐ like protein Micrurus fulvius unknown
19‐ 12 1712 -
▼ reduced SDS‐PAGE apparent mass, in kDa. Selected peaks were analyzed by mass spectrometry, SP; ~U3FBP8, prostasin-like 0.9 23 İ 1152.6 1 LPFYADWIK 99.0 12 protein Micrurus fulvius and their masses (Da) are shown in parentheses; * Cysteine residues are carbamidomethylated. İ reduced SDS-PAGE apparent mass, in kDa. Selected peaks were analyzed by mass spectrometry, and their Possible, although unconfirmed/ambiguous amino acid modifications, suggested by the automated masses (Da) are shown in parentheses; * Cysteine residues are carbamidomethylated. Possible, although identification software are shown in superscript, with the following abbreviations: ox: oxidized; da: unconfirmed/ambiguous amino acid modifications, suggested by the automated identification software are shown in superscript, with the following abbreviations: ox: oxidized; da: deamidated; py: pyroglutamic; deamidated; py: pyroglutamic; fo: formyl; dh: dehydrated; am: amidated; ky: hydroxykynurenin; dt: fo: formyl; dh: dehydrated; am: amidated; ky: hydroxykynurenin; dt: dethiomethyl; ** Confidence (Conf) ® algorithm of dethiomethyl; ** Confidence (Conf) and score (Sc) values are calculated by the Paragon and score (Sc) values are calculated by the Paragon® algorithm of ProteinPilot® v.4.0. Few spectra that ® v.4.0. Few spectra by that were ***manually resolved are indicated by ‘‘man’‘; ProteinPilot were manually resolved are indicated “man’‘; Abbreviations: PNP: peptide or non-proteic; 3FTx: three-finger toxin; PLA A2 ; Kunitz: 3FTx: Kunitz-type serine proteinase inhibitor; CTL: C-typeA2; 2 : phospholipase *** Abbreviations: PNP: peptide or non‐proteic; three‐finger toxin; PLA 2: phospholipase lectin/lectin-like; MP: metalloproteinase; SP: serine proteinase; LAO: L-amino acid oxidase; PLB: phospholibase Kunitz: Kunitz‐type serine proteinase inhibitor; CTL: C‐type lectin/lectin‐like; MP: metalloproteinase; B; GPX: glutathione peroxidase. SP: serine proteinase; LAO: L‐amino acid oxidase; PLB: phospholibase B; GPX: glutathione peroxidase.
Figure 2. Composition of Micrurus clarki venom proteome according to protein families, expressed Figure 2. Composition of Micrurus clarki venom proteome according to protein families, expressed as as percentages of the total protein content. 3FTx: three-finger toxin; PLA2 : phospholipase A2 ; LAO: percentages of the total protein content. 3FTx: three‐finger toxin; PLA 2: phospholipase A 2; LAO: L‐amino L-amino acid oxidase; CTL: C-type lectin/lectin-like; MP: metalloproteinase, SP: serine proteinase; acid oxidase; CTL: C‐type lectin/lectin‐like; MP: metalloproteinase, SP: serine proteinase; KUN: Kunitz-type serine proteinase inhibitor; GPX: Glutathione peroxidase; PLB: phospholipase B; KUN: Kunitz‐type serine proteinase inhibitor; GPX: Glutathione peroxidase; PLB: phospholipase B; PNP: peptides and/or non-proteinaceous components; UNK: unknown/unidentified. PNP: peptides and/or non‐proteinaceous components; UNK: unknown/unidentified.
In addition addition toto abundant 3FTxs 2s, other proteins detected in proportions lower proportions In itsits abundant 3FTxs andand PLAPLA proteins detected in lower (within 2 s, other (within of 0.9%–3.8%) to the acid L‐amino acid oxidase, metalloproteinase, serine the rangethe of range 0.9%–3.8%) belong to belong the L-amino oxidase, metalloproteinase, serine proteinase, proteinase, Kunitz‐type serine proteinase inhibitor, and C‐type lectin/lectin‐like families, together Kunitz-type serine proteinase inhibitor, and C-type lectin/lectin-like families, together with traces with traces (~0.2%) of phospholipase B and glutathion peroxidase components (Figure 2). Altogether, (~0.2%) of phospholipase B and glutathion peroxidase components (Figure 2). Altogether, proteins in proteins in this venom belong to at least nine families. As expected, the rapidly eluting peaks in the this venom belong to at least nine families. As expected, the rapidly eluting peaks in the RP-HPLC RP‐HPLC separation of the venom (Figure 1B; peaks 1–6), did not show proteins by electrophoresis, separation of the venom (Figure 1B; peaks 1–6), did not show proteins by electrophoresis, and are and are considered to contain either small peptides or non‐proteinaceous compounds (PNP; 2.4%). considered to contain either small peptides or non-proteinaceous compounds (PNP; 2.4%). The most The most intense of these initial were by analyzed by and nESI‐MS, and clear evidence for intense of these initial peaks (1–3)peaks were (1–3) analyzed nESI-MS, clear evidence for adenosine adenosine being present in obtained peak 1 was obtained (Figure 3). Adenosine has found in several being present in peak 1 was (Figure 3). Adenosine has been found inbeen several venoms from venoms from Micrurus species, although not ubiquitously [10,11], and may have a contributing role Micrurus species, although not ubiquitously [10,11], and may have a contributing role in envenoming in envenoming strategies [12], a hypothesis which still remains to be studied in detail. strategies [12], a hypothesis which still remains to be studied in detail.
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Figure 3. Detection of adenosine in Micrurus clarki venom by nESI-MS/MS. (A) Fraction 1 from Figure 1 Figure 3. Detection of adenosine in Micrurus clarki venom by nESI‐MS/MS. (A) Fraction 1 from Figure 1 was directly infused into the nano-spray ion source of a QTrap 3200 mass spectrometrer, and scanned was directly infused into the nano‐spray ion source of a QTrap 3200 mass spectrometrer, and scanned in positive enhanced MS mode; and (B) the ion labeled at m/z 268.2 (dotted red circle in A) was selected in positive enhanced MS mode; and (B) the ion labeled at m/z 268.2 (dotted red circle in A) was for collision-induced dissociation, showing the characteristic fragment of m/z 136.1, which corresponds selected for collision‐induced dissociation, showing the characteristic fragment of m/z 136.1, which to the transition of adenosine to adenine. corresponds to the transition of adenosine to adenine.
2.2. Immunological Properties of Micrurus Clarki Venom 2.2. Immunological Properties of Micrurus Clarki Venom Since Since the the antivenom antivenom used used therapeutically therapeutically for for coral coral snake snake envenomings envenomings in in Central Central America America (SAC-ICP) is prepared by immunization of horses with the venom of a single species, M. nigrocinctus, (SAC‐ICP) is prepared by immunization of horses with the venom of a single species, M. nigrocinctus, it was of interest to characterize its immunological cross-recognition and cross-neutralization toward it was of interest to characterize its immunological cross‐recognition and cross‐neutralization toward the untested venom of M. clarki. As shown by ELISA titration curves, this antivenom strongly the untested venom of M. clarki. As shown by ELISA titration curves, this antivenom strongly cross‐ cross-recognized M. clarki whole venom, resulting in antibody binding signals virtually as high as recognized M. clarki whole venom, resulting in antibody binding signals virtually as high as those those obtained for the homologous nigrocinctus) venom (Figure Thisfinding findingevidences evidences the the high high obtained for the homologous (M. (M. nigrocinctus) venom (Figure 4). 4).This degree of antigenic conservation between venoms of these two coral snake species. Following this degree of antigenic conservation between venoms of these two coral snake species. Following this result, detailed cross-recognition analysis was was performed using using the RP-HPLC-separated venom result, aa more more detailed cross‐recognition analysis performed the RP‐HPLC‐separated fractions of M. clarki, revealing additional information. First, it evidenced that the antivenom contains venom fractions of M. clarki, revealing additional information. First, it evidenced that the antivenom antibodies against all of the venom fractions tested, since the binding signals recorded were, in all cases, contains antibodies against all of the venom fractions tested, since the binding signals recorded were, significantly higher than those of the negative control equine immunoglobulins (Figure 5). Second, it in all cases, significantly higher than those of the negative control equine immunoglobulins (Figure 5). showed that immunorecognition of smaller proteins, suchproteins, as 3FTxs such or Kunitz-type is Second, it showed that immunorecognition of smaller as 3FTxs components, or Kunitz‐type generally weaker in comparison to recognition of the larger PLA2 s or LAAOs2s or LAAOs (Figure 5). (Figure 5). The lower components, is generally weaker in comparison to recognition of the larger PLA titers of antibodies against low molecular mass proteins may reflect differences in their immunogenicity, The lower titers of antibodies against low molecular mass proteins may reflect differences in their and/or epitope availability in the immunoassay, and is in agreement with earlier studies performed immunogenicity, and/or epitope availability in the immunoassay, and is in agreement with earlier with other Micrurus venoms [13,14] or with venoms from other elapids [15,16]. studies performed with other Micrurus venoms [13,14] or with venoms from other elapids [15,16].
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Figure 4. Cross‐recognition of the venom of Micrurus clarki by an equine antivenom raised against M. Figure 4. Cross-recognition of the venom of Micrurus clarki by an equine antivenom raised against Figure 4. Cross‐recognition of the venom of Micrurus clarki by an equine antivenom raised against M. nigrocinctus venom (SAC‐ICP), as evaluated by ELISA. Crude venom from each of the two species M. nigrocinctus venom (SAC-ICP), as evaluated by ELISA. Crude venom from each of the two species nigrocinctus venom (SAC‐ICP), as evaluated by ELISA. Crude venom from each of the two species was coated onto microplates at 1 g/well, as described in Methods. Serial dilutions of the antivenom was coated onto microplates at 1 µg/well, as described in Methods. Serial dilutions of the antivenom was coated onto microplates at 1 g/well, as described in Methods. Serial dilutions of the antivenom were added and the binding of antibodies was detected by anti‐equine immunoglobulins conjugated were added and the binding of antibodies was detected by anti-equine immunoglobulins conjugated to were added and the binding of antibodies was detected by anti‐equine immunoglobulins conjugated to alkaline phosphatase, followed by color development using p‐nitrophenylphosphate substrate. A alkaline phosphatase, followed by color development using p-nitrophenylphosphate substrate. A mock to alkaline phosphatase, followed by color development using p‐nitrophenylphosphate substrate. A mock antivenom prepared using the plasma plasma of a a non-immunized normal, non‐immunized non‐immunized horse, was used as a a antivenom prepared using the plasma of a normal, horse, was usedwas as aused negative mock antivenom prepared using the of normal, horse, as negative control for background. Each point represents mean ± SD of triplicate wells. control for background. Each point represents mean ˘ SD of triplicate wells. negative control for background. Each point represents mean ± SD of triplicate wells.
Figure 5.5. 5. Immunorecognition Immunorecognition of of the RP-HPLC RP‐HPLC fractions fractions of of Micrurus Micrurus clarki clarki venom venom by by an an equine equine Figure Immunorecognition of the the Figure RP‐HPLC fractions of Micrurus clarki venom by an equine monospecific antivenom raised against M. nigrocinctus venom (SAC‐ICP), as evaluated by ELISA. monospecific antivenom raised against M. nigrocinctus venom (SAC-ICP), as evaluated by ELISA. monospecific antivenom raised against M. nigrocinctus venom (SAC‐ICP), as evaluated by ELISA. Venom fractions fractions (0.4 g/well) were coated onto microplates, and the binding binding of antibodies antibodies was Venom (0.4 µg/well) were coated ontoonto microplates, and the binding of antibodies was detected Venom fractions (0.4 g/well) were coated microplates, and the of was detected as described in Methods, using anti‐equine immunoglobulins conjugated to alkaline as described Methods,in using anti-equine conjugated toconjugated alkaline phosphatase, detected as in described Methods, using immunoglobulins anti‐equine immunoglobulins to alkaline phosphatase, followed by color color development using p‐nitrophenylphosphate p‐nitrophenylphosphate substrate. Mock followed by color development using p-nitrophenylphosphate substrate. Mock antivenom prepared phosphatase, followed by development using substrate. Mock antivenom prepared using the plasma of a normal, non‐immunized horse, was used as a negative using the plasma of a normal, non-immunized horse, was used as a negative control for background. antivenom prepared using the plasma of a normal, non‐immunized horse, was used as a negative control for background. Each bar represents mean ± SD of triplicate wells. All bars corresponding to Each bar represents mean ˘ SD of triplicate wells. All bars corresponding to SAC-ICP have values control for background. Each bar represents mean ± SD of triplicate wells. All bars corresponding to SAC‐ICP have values that are significantly higher (p
Venom of the Coral Snake Micrurus clarki: Proteomic Profile, Toxicity, Immunological Cross-Neutralization, and Characterization of a Three-Finger Toxin Bruno Lomonte 1, *, Mahmood Sasa 1 , Paola Rey-Suárez 2 , Wendy Bryan 1 and José María Gutiérrez 1 1 2
*
Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José 11501, Costa Rica; [email protected] (M.S.); [email protected] (W.B.); [email protected] (J.M.G.) Programa de Ofidismo y Escorpionismo, Universidad de Antioquia, Medellín 050010, Colombia; [email protected] Correspondence: [email protected]; Tel.: +506-2511-7888
Academic Editor: Stephen P. Mackessy Received: 26 March 2016; Accepted: 2 May 2016; Published: 5 May 2016
Abstract: Micrurus clarki is an uncommon coral snake distributed from the Southeastern Pacific of Costa Rica to Western Colombia, for which no information on its venom could be found in the literature. Using a ‘venomics’ approach, proteins of at least nine families were identified, with a moderate predominance of three-finger toxins (3FTx; 48.2%) over phospholipase A2 (PLA2 ; 36.5%). Comparison of this venom profile with those of other Micrurus species suggests that it may represent a more balanced, ‘intermediate’ type within the dichotomy between 3FTx- and PLA2 -predominant venoms. M. clarki venom was strongly cross-recognized and, accordingly, efficiently neutralized by an equine therapeutic antivenom against M. nigrocinctus, revealing their high antigenic similarity. Lethal activity for mice could be reproduced by a PLA2 venom fraction, but, unexpectedly, not by fractions corresponding to 3FTxs. The most abundant venom component, hereby named clarkitoxin-I, was identified as a short-chain (type I) 3FTx, devoid of lethal effect in mice, whose target remains to be defined. Its amino acid sequence of 66 residues shows high similarity with predicted sequences of venom gland transcripts described for M. fulvius, M. browni, and M. diastema. Keywords: monadal coral snake; Elapidae; neutralization
venom;
toxin;
Micrurus;
antivenom;
proteome;
1. Introduction Coral snakes (Elapidae) comprise some 71 species that are widely distributed in tropical and subtropical regions of the New World, from Southeastern USA to Central Argentina [1–3]. Envenomings by these snakes are far less frequent than those inflicted by viperids, but are of medical concern [4,5]. Micrurus clarki (Figure 1A) is an uncommon, slender to medially robust coral snake with a reported maximum length of 920 mm, although most adults average between 400 and 600 mm. This species has a tricolored pattern with narrow yellow bands bordering the black bands followed by single red bands, and a characteristic black head cap that extends over most of the snout and parietals. M. clarki has a discontinuous distribution from the Tárcoles basin in Central Pacific Costa Rica southward to the Pacific lowlands of Western Panamá, where it inhabits lowlands of the Canal Zone, to Pacific Darien, and Western Colombia. Along this distribution, M. clarki inhabits tropical wet forest, but also transitional tropical wet-tropical dry forest, where it is mostly found at low elevations, with some Toxins 2016, 8, 138; doi:10.3390/toxins8050138
www.mdpi.com/journal/toxins
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with some exceptions reported up to 900 m above sea level [1,6,7]. Little is known about the natural exceptions reported up to 900 m above sea level [1,6,7]. Little is known about the natural history of this history of this coral snake. The species was named in honor of H.C. Clark, a medical doctor that coral snake. The species was named in honor of H.C. Clark, a medical doctor that directed the Gorgas directed the Gorgas Memorial Institute in Panamá, considered a pioneer in tropical medicine [8]. It is Memorial Institute in Panamá, considered a pioneer in tropical medicine [8]. It is considered mainly a considered mainly a terrestrial and primarily nocturnal species whose natural diet is known to terrestrial and primarily nocturnal species whose natural diet is known to include the marbled swamp include the marbled swamp eel Synbranchus marmoratus, and colubrid snakes [6]. eel Synbranchus marmoratus, and colubrid snakes [6].
Figure 1. (A) Micrurus clarki and (B) separation of its venom (2 mg) by RP-HPLC, followed by (C) Figure 1. (A) Micrurus clarki and (B) separation of its venom (2 mg) by RP‐HPLC, followed by (C) SDS-PAGE. Venom was fractionated on a C18 RP-HPLC column and eluted with an acetonitrile gradient SDS‐PAGE. was fractionated on further a C18 RP‐HPLC column and eluted an acetonitrile (dashed line)Venom at 1 mL/min. Fractions were separated by SDS-PAGE under with reducing conditions. gradient (dashed line) at 1 mL/min. Fractions were further separated by SDS‐PAGE under reducing Molecular weight markers (Mw) are indicated in kDa. Coomassie-stained bands were excised, in-gel conditions. Molecular weight markers (Mw) are indicated in kDa. bands were digested with trypsin, and subjected to MALDI-TOF/TOF analysis forCoomassie‐stained assignment to protein families, excised, in‐gel digested with trypsin, and subjected to MALDI‐TOF/TOF analysis for assignment to as shown in Table 1. protein families, as shown in Table 1.
Our search of the mainstream literature databases did not find any information on the venom of Our search of the mainstream literature databases did not find any information on the venom of M. clarki, whose general characteristics seem to be completely unknown. As part of an ongoing effort M. clarki, whose general characteristics seem to be completely unknown. As part of an ongoing effort to characterize the venom proteomes of all snake species from Costa Rica [9], in the present work we to characterize the venom proteomes of all snake species from Costa Rica [9], in the present work we report the composition, toxicity, immunological cross-recognition, and neutralization of the venom of report the composition, toxicity, immunological cross‐recognition, and neutralization of the venom M. clarki obtained from specimens collected in the Southeastern Pacific of this country. In addition, a of M. clarki obtained from specimens collected in the Southeastern Pacific of this country. In addition, three-finger toxin from this venom was isolated and characterized. a three‐finger toxin from this venom was isolated and characterized. 2. Results and Discussion 2. Results and Discussion 2.1. Proteomic Profile of Micrurus Clarki Venom 2.1. Proteomic Profile of Micrurus Clarki Venom The venom of M. clarki (1A) was initially separated into 33 fractions by RP-HPLC (Figure 1B), The venom M. clarki was bands initially separated into 33 fractions RP‐HPLC which were furtherof resolved into (1A) 42 protein after SDS-PAGE (Figure 1C). Afterby in-gel tryptic (Figure 1B), which were further resolved into 42 protein bands after SDS‐PAGE (Figure 1C). After in‐ digestion of these, followed by MALDI-TOF-TOF analysis, protein family assignments were obtained gel tryptic of these, to followed by 95.9% MALDI‐TOF‐TOF analysis, protein The family assignments for 34 of thedigestion bands, estimated represent of the total venom proteins. remaining eight were obtained for 34 of the bands, estimated to represent 95.9% of the total venom proteins. bands for which no identifications were obtained (indicated as “unknown” in Table 1 and FigureThe 2), remaining eight bands for which no identifications were obtained (indicated as ‘‘unknown’’ in Table altogether add to the remaining 4.1% of the venom proteins. 1 and Figure 2), altogether add to the remaining 4.1% of the venom proteins. Although not markedly predominant, the protein family with the highest abundance in M. clarki Although not markedly predominant, the protein family with the highest abundance in M. clarki venom corresponds to three-finger toxins (3FTx), which account for almost half of the protein content venom corresponds to three‐finger toxins (3FTx), which account for almost half of the protein content (48.2%), followed by phospholipase A2 (PLA2 ), representing roughly one-third (36.5%). (48.2%), followed by phospholipase A2 (PLA2), representing roughly one‐third (36.5%).
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Table 1. Assignment of the RP-HPLC/SDS-PAGE separated fractions of Micrurus clarki venom to protein families by MALDI-TOF-TOF of selected peptide ions from in-gel trypsin-digested protein bands. Peak
%
Peptide Ion
Mass, kDa İ (Da)
m/z
z
MS/MS-Derived Sequence *
Conf (%)
Sc **
Protein Family; ~ Related Protein and Species *** PNP; adenosine
1
1.3
-
-
-
-
-
-
2–6
1.1
-
-
-
-
-
-
PNP 3FTx; ~U3FVH8, 3FTx 4a Micrurus fulvius
7
3.2
10 İ (6757)
1707.0
1
FSPGIQTSQTCPAGQK
99.0
11
8
5.9
9 İ (6638, 7276)
976.5
1
MFIRTHR
76.6
5
1322.7
1
ICDDSSIPFLR
99.0
7
FYFAYQCTSK
man
man
99.0
11
99.0
11
-
-
9
0.2
9İ
1314.7
1
10
8.0
9 İ (6361, 6451)
2663.2
1
1375.6
1
DGFYSVTCTEKENLCFT MFSAR ENLCFTMFSAR
9İ
-
-
-
11 12
13
14
0.4 1.8
3.6
6.9
9 İ (7045, 7140, 7198)
9 İ (7071, 7201)
9 İ (6845)
1661.8
1
GAYNVCCSTDLCNK
99.0
15
1374.7
1
KCLEFIYGGCQ
99.0
12
1786.9
1
VCYTIFLVGPSYPpy EK
93.5
6
1687.8
1
GPYNVCCSTDLCNK
99
7
1310.7
1
AIEFGCAASCPK
99
12
3FTx; ~P01414 , toxin FS-2 Dendroaspis polylepis 3FTx; ~U3EPK7, 3FTx 6 Micrurus fulvius 3FTx; ~C6JUP1, 3FTx Micrurus corallinus 3FTx; ~C6JUP4, 3FTx Micrurus corallinus Unknown 3FTx; ~U3FAE1, 3FTx 3a Micrurus fulvius Kunitz; ~Q6ITB9, mulgin-3 Pseudechis australis 3FTx; ~AKN63197, 3FTx Micrurus diastema 3FTx; ~B3EWF8, mipartoxin-I Micrurus mipartitus 3FTx; ~AKN63198, 3FTx DK, Micrurus diastema
15a
0.4
12 İ
-
-
-
-
-
unknown
15b
2.5
9 İ (6843, 7311)
1314.7
1
FYFAYQCTSK
man
man
3FTx; ~C6JUP1, 3FTx Micrurus corallinus
16a
0.3
12 İ
-
-
-
-
-
unknown
16b
0.2
10 İ
1373.6
1
CKDFVCNCDR
99.0
7
PLA2 ; ~U3FYP8, PLA2 13 Micrurus fulvius
17a
1.7
13 İ
1373.6 1085.4
1 1
CKDFVCNCDR DFVCNCDR
99.0 96.8
14 6
PLA2 ; ~U3FYP8, PLA2 13 Micrurus fulvius
17b
1.2
9İ
-
-
-
-
-
unknown
2969.3
1
99.0
12
18
6.6
13 İ (13353)
1373.5
1
99.0
12
2983.3
1
98.5
6
HWVSFTNYGCYCGYGG SGTPVDELDK CKDFVCNCDR HWLSFTNYGCYCGYGG SGTPVDELDK
PLA2 ; ~P81167, nigroxin B Micrurus nigrocinctus PLA2 ; ~U3FYP8, PLA2 13 Micrurus fulvius
19
11.3
13 İ (13477)
1373.6
1
CKDFVCNCDR
99.0
9
PLA2 ; ~U3FYN8, PLA2 9 Micrurus fulvius
20
1.8
13 İ
2855.3
1
SAWDFTNYGCYCGAGG SGTPVDELDR
99.0
15
PLA2 ; ~Q8AXW7, PLA2 Micrurus corallinus
2841.3
1
AWDYNNda YGCYCGQG GSGTPVDDLDR
99
12
1335.7
1
VHDDCYAAAEK
99
18
1827.8
1
CCQVHDNCYNEAEK
99
22
1278.6
1
VHDDCYAAAEK
99.0
7
1373.5
1
CKDFVCNCDR
99.0
8
2883.2
1
Sf˝ AWDFTNYGCYCGAG GSGTPVDELDR
99.0
15
21
5.3
12 İ (13331)
22a
2.4
20 İ
22b
4.5
13 İ
PLA2 ; ~R4G7M2, Sut-22 Suta fasciata PLA2 ; ~ U3FYP1, PLA2 3b Micrurus fulvius PLA2 ; ~ A4FS04, natratoxin, Naja atra PLA2 ; ~U3FYP1, PLA2 3b Micrurus fulvius PLA2 ; ~U3FYP8, PLA2 13 Micrurus fulvius PLA2 ; ~Q8AXW7, PLA2 Micrurus corallinus
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Table 1. Cont. Peak
%
Peptide Ion
Mass, kDa İ (Da)
m/z
z
MS/MS-Derived Sequence *
Conf (%)
Sc **
Protein Family; ~ Related Protein and Species ***
VHDDCYAAAEK
99.0
8
PLA2 ; ~U3FYP1, PLA2 3b Micrurus fulvius
99.0
16
PLA2 ; ~P00606, PLA2 Bungarus multicinctus
23a
0.6
13 İ
1278.5
1
23b
2.1
11 İ
2883.3
1
24
16.1
10 İ (7537)
1478.7 1322.6 1108.6
1 1 1
RICDDSSIPFLR ICDDSSIPFLR KGCASSCPKN
99 99 99
12 15 13
3FTx; ~U3EPK7, 3FTx 10a Micrurus fulvius
25
0.9
10 İ
1322.6
1
ICDDSSIPFLR
99.0
9
3FTx; ~U3EPK7, 3FTx 6 Micrurus fulvius
26
1.1
22 İ
1183.7
1
NVWIGLNDPR
man
man
CTL; ~ACC67944, mannose-binding 1 Pseudechis porphyriacus
27
0.1
-
-
-
-
-
-
28a
0.2
38 İ
PWky IGYVNYGCYCGAGG SGTPVDELDR
1054.5
1
TYWHYER
99.0
11
1130.6
1
EVFDGHTIGR
man
man
unknown MP; ~U3FWL3, MTP4 Micrurus fulvius MP; ~P85314, MP Bothrops moojeni MP: ~R4G7J1, MP-Hop-13 Hoplocephalus bungaroides
28b
0.2
29 İ
1297.6
1
SAECPTDSFQR
99.0
7
29a
0.6
38 İ
-
-
-
-
-
unknown
29b
0.2
28 İ
1011.6
1
FYVVVDNR
90.9
9
MP; ~ABQ01135, stephensease-1 Hoplocephalus. stephensii
29c
0.2
25 İ
1011.6
1
FYVVVDNR
man
man
MP; ~ABQ01135, stephensease-1 Hoplocephalus. stephensii
30a
0.4
51 İ
995.6
1
Fdh YVVVDNR
man
man
MP; ~ABQ01135, stephensease-1 Hoplocephalus. stephensii
30b
0.4
46 İ
995.6
1
Fdh YVVVDNR
man
man
MP; ~ABQ01135, stephensease-1 Hoplocephalus. stephensii
30c
0.1
29 İ
2171.1
1
GDSGGPLICNGQIQGIVS WGR
99.0
7
SP; ~Q5MCS0, harobin Hydrophis hardwickii
30d
0.2
20 İ
1329.7
1
IHDIKWNFEK
99.0
13
GPX; ~XP_013914274, glutathione peroxidase 3 Thamnophis sirtalis
1637.8
1
NDLEGWHVNLGPMR
99.0
23
LAO; ~U3FYQ2, LAAO 1a Micrurus fulvius
1131.5 1576.9 1484.7
1 1 1
SDDIFSYER IQDNTENVRVAYRam EADYEEFLEIAR
99.0 99.0 99.0
13 13 19
-
1963.1
1
TSGDIVINDLSLIHQLPK
99.0
7
LAO; ~U3FYQ2, LAAO 1a Micrurus fulvius
2275.1
1
99.0
8
-
1484.7
1
IHFAGEYTANDHGWIDS TIK EADYEEFLEIAR
99.0
9
1310.7
1
RFDEIVGGM˝x DR
99.0
10
1460.8
1
QVVPESLFAWER
95.0
7
LAO; ~A0A0A1WCY6, B variant Echis coloratus PLB: ~V8ND68, PLB-like 1 Ophiophagus hannah
1484.7 1963.0
1 1
99.0 99.0
13 17
2275.0
1
99.0
20
1131.5 1576.8
1 1
EADYEEFLEIAR TSGDIVINDLSLIHQLPK IHFAGEYTANDHGWIDS TIK SDDIFSYER IQDNTENVRVAYRam
99.0 99.0
11 12
-
-
-
-
-
31a
31b
31c
31d
2.2
0.5
1.1
0.7
61 İ
50 İ
42 İ
20 İ
LAO; ~U3FYQ2, LAAO 1a Micrurus fulvius
unknown
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Table 1. Cont. Toxins 2016, 8, 138 Peak
%
5 of 15
Mass, kDa İ (Da)
31f 31e
0.4 0.2
14 ▼ 17 İ
32
0.9 0.4
14 İ
31f 32
23 ▼
Peptide Ion m/z 1637.8 z 1589.8 1 1484.7 1 1466.7 1833.8‐ 1 1637.8 1 1484.7 1152.6 1 -
-
MS/MS-Derived Conf (%) Sequence * NDLEGWHVNLGPMR 1 99.0 99.0 NDLEGWHVNLGPMdt R 1 EADYEEFLEIAR Epy ADYEEFLEIAR 99.099.0
‐
‐ EFVQEDENAWYYIK NDLEGWHVNLGPMR EADYEEFLEIAR 1 LPFYADWIK -
99.0 ‐ 99.0 99.099.0 -
Protein Family; ~ Related Protein and Species ***
Sc ** 12 9 1117
LAO; ~U3FYQ2, LAAO 1a unknown Micrurus fulvius SP; ~U3FBP8, prostasin‐ like protein Micrurus fulvius unknown
19‐ 12 1712 -
▼ reduced SDS‐PAGE apparent mass, in kDa. Selected peaks were analyzed by mass spectrometry, SP; ~U3FBP8, prostasin-like 0.9 23 İ 1152.6 1 LPFYADWIK 99.0 12 protein Micrurus fulvius and their masses (Da) are shown in parentheses; * Cysteine residues are carbamidomethylated. İ reduced SDS-PAGE apparent mass, in kDa. Selected peaks were analyzed by mass spectrometry, and their Possible, although unconfirmed/ambiguous amino acid modifications, suggested by the automated masses (Da) are shown in parentheses; * Cysteine residues are carbamidomethylated. Possible, although identification software are shown in superscript, with the following abbreviations: ox: oxidized; da: unconfirmed/ambiguous amino acid modifications, suggested by the automated identification software are shown in superscript, with the following abbreviations: ox: oxidized; da: deamidated; py: pyroglutamic; deamidated; py: pyroglutamic; fo: formyl; dh: dehydrated; am: amidated; ky: hydroxykynurenin; dt: fo: formyl; dh: dehydrated; am: amidated; ky: hydroxykynurenin; dt: dethiomethyl; ** Confidence (Conf) ® algorithm of dethiomethyl; ** Confidence (Conf) and score (Sc) values are calculated by the Paragon and score (Sc) values are calculated by the Paragon® algorithm of ProteinPilot® v.4.0. Few spectra that ® v.4.0. Few spectra by that were ***manually resolved are indicated by ‘‘man’‘; ProteinPilot were manually resolved are indicated “man’‘; Abbreviations: PNP: peptide or non-proteic; 3FTx: three-finger toxin; PLA A2 ; Kunitz: 3FTx: Kunitz-type serine proteinase inhibitor; CTL: C-typeA2; 2 : phospholipase *** Abbreviations: PNP: peptide or non‐proteic; three‐finger toxin; PLA 2: phospholipase lectin/lectin-like; MP: metalloproteinase; SP: serine proteinase; LAO: L-amino acid oxidase; PLB: phospholibase Kunitz: Kunitz‐type serine proteinase inhibitor; CTL: C‐type lectin/lectin‐like; MP: metalloproteinase; B; GPX: glutathione peroxidase. SP: serine proteinase; LAO: L‐amino acid oxidase; PLB: phospholibase B; GPX: glutathione peroxidase.
Figure 2. Composition of Micrurus clarki venom proteome according to protein families, expressed Figure 2. Composition of Micrurus clarki venom proteome according to protein families, expressed as as percentages of the total protein content. 3FTx: three-finger toxin; PLA2 : phospholipase A2 ; LAO: percentages of the total protein content. 3FTx: three‐finger toxin; PLA 2: phospholipase A 2; LAO: L‐amino L-amino acid oxidase; CTL: C-type lectin/lectin-like; MP: metalloproteinase, SP: serine proteinase; acid oxidase; CTL: C‐type lectin/lectin‐like; MP: metalloproteinase, SP: serine proteinase; KUN: Kunitz-type serine proteinase inhibitor; GPX: Glutathione peroxidase; PLB: phospholipase B; KUN: Kunitz‐type serine proteinase inhibitor; GPX: Glutathione peroxidase; PLB: phospholipase B; PNP: peptides and/or non-proteinaceous components; UNK: unknown/unidentified. PNP: peptides and/or non‐proteinaceous components; UNK: unknown/unidentified.
In addition addition toto abundant 3FTxs 2s, other proteins detected in proportions lower proportions In itsits abundant 3FTxs andand PLAPLA proteins detected in lower (within 2 s, other (within of 0.9%–3.8%) to the acid L‐amino acid oxidase, metalloproteinase, serine the rangethe of range 0.9%–3.8%) belong to belong the L-amino oxidase, metalloproteinase, serine proteinase, proteinase, Kunitz‐type serine proteinase inhibitor, and C‐type lectin/lectin‐like families, together Kunitz-type serine proteinase inhibitor, and C-type lectin/lectin-like families, together with traces with traces (~0.2%) of phospholipase B and glutathion peroxidase components (Figure 2). Altogether, (~0.2%) of phospholipase B and glutathion peroxidase components (Figure 2). Altogether, proteins in proteins in this venom belong to at least nine families. As expected, the rapidly eluting peaks in the this venom belong to at least nine families. As expected, the rapidly eluting peaks in the RP-HPLC RP‐HPLC separation of the venom (Figure 1B; peaks 1–6), did not show proteins by electrophoresis, separation of the venom (Figure 1B; peaks 1–6), did not show proteins by electrophoresis, and are and are considered to contain either small peptides or non‐proteinaceous compounds (PNP; 2.4%). considered to contain either small peptides or non-proteinaceous compounds (PNP; 2.4%). The most The most intense of these initial were by analyzed by and nESI‐MS, and clear evidence for intense of these initial peaks (1–3)peaks were (1–3) analyzed nESI-MS, clear evidence for adenosine adenosine being present in obtained peak 1 was obtained (Figure 3). Adenosine has found in several being present in peak 1 was (Figure 3). Adenosine has been found inbeen several venoms from venoms from Micrurus species, although not ubiquitously [10,11], and may have a contributing role Micrurus species, although not ubiquitously [10,11], and may have a contributing role in envenoming in envenoming strategies [12], a hypothesis which still remains to be studied in detail. strategies [12], a hypothesis which still remains to be studied in detail.
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Figure 3. Detection of adenosine in Micrurus clarki venom by nESI-MS/MS. (A) Fraction 1 from Figure 1 Figure 3. Detection of adenosine in Micrurus clarki venom by nESI‐MS/MS. (A) Fraction 1 from Figure 1 was directly infused into the nano-spray ion source of a QTrap 3200 mass spectrometrer, and scanned was directly infused into the nano‐spray ion source of a QTrap 3200 mass spectrometrer, and scanned in positive enhanced MS mode; and (B) the ion labeled at m/z 268.2 (dotted red circle in A) was selected in positive enhanced MS mode; and (B) the ion labeled at m/z 268.2 (dotted red circle in A) was for collision-induced dissociation, showing the characteristic fragment of m/z 136.1, which corresponds selected for collision‐induced dissociation, showing the characteristic fragment of m/z 136.1, which to the transition of adenosine to adenine. corresponds to the transition of adenosine to adenine.
2.2. Immunological Properties of Micrurus Clarki Venom 2.2. Immunological Properties of Micrurus Clarki Venom Since Since the the antivenom antivenom used used therapeutically therapeutically for for coral coral snake snake envenomings envenomings in in Central Central America America (SAC-ICP) is prepared by immunization of horses with the venom of a single species, M. nigrocinctus, (SAC‐ICP) is prepared by immunization of horses with the venom of a single species, M. nigrocinctus, it was of interest to characterize its immunological cross-recognition and cross-neutralization toward it was of interest to characterize its immunological cross‐recognition and cross‐neutralization toward the untested venom of M. clarki. As shown by ELISA titration curves, this antivenom strongly the untested venom of M. clarki. As shown by ELISA titration curves, this antivenom strongly cross‐ cross-recognized M. clarki whole venom, resulting in antibody binding signals virtually as high as recognized M. clarki whole venom, resulting in antibody binding signals virtually as high as those those obtained for the homologous nigrocinctus) venom (Figure Thisfinding findingevidences evidences the the high high obtained for the homologous (M. (M. nigrocinctus) venom (Figure 4). 4).This degree of antigenic conservation between venoms of these two coral snake species. Following this degree of antigenic conservation between venoms of these two coral snake species. Following this result, detailed cross-recognition analysis was was performed using using the RP-HPLC-separated venom result, aa more more detailed cross‐recognition analysis performed the RP‐HPLC‐separated fractions of M. clarki, revealing additional information. First, it evidenced that the antivenom contains venom fractions of M. clarki, revealing additional information. First, it evidenced that the antivenom antibodies against all of the venom fractions tested, since the binding signals recorded were, in all cases, contains antibodies against all of the venom fractions tested, since the binding signals recorded were, significantly higher than those of the negative control equine immunoglobulins (Figure 5). Second, it in all cases, significantly higher than those of the negative control equine immunoglobulins (Figure 5). showed that immunorecognition of smaller proteins, suchproteins, as 3FTxs such or Kunitz-type is Second, it showed that immunorecognition of smaller as 3FTxs components, or Kunitz‐type generally weaker in comparison to recognition of the larger PLA2 s or LAAOs2s or LAAOs (Figure 5). (Figure 5). The lower components, is generally weaker in comparison to recognition of the larger PLA titers of antibodies against low molecular mass proteins may reflect differences in their immunogenicity, The lower titers of antibodies against low molecular mass proteins may reflect differences in their and/or epitope availability in the immunoassay, and is in agreement with earlier studies performed immunogenicity, and/or epitope availability in the immunoassay, and is in agreement with earlier with other Micrurus venoms [13,14] or with venoms from other elapids [15,16]. studies performed with other Micrurus venoms [13,14] or with venoms from other elapids [15,16].
Toxins 2016, 8, 138 Toxins 2016, 8, 138 Toxins 2016, 8, 138
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Figure 4. Cross‐recognition of the venom of Micrurus clarki by an equine antivenom raised against M. Figure 4. Cross-recognition of the venom of Micrurus clarki by an equine antivenom raised against Figure 4. Cross‐recognition of the venom of Micrurus clarki by an equine antivenom raised against M. nigrocinctus venom (SAC‐ICP), as evaluated by ELISA. Crude venom from each of the two species M. nigrocinctus venom (SAC-ICP), as evaluated by ELISA. Crude venom from each of the two species nigrocinctus venom (SAC‐ICP), as evaluated by ELISA. Crude venom from each of the two species was coated onto microplates at 1 g/well, as described in Methods. Serial dilutions of the antivenom was coated onto microplates at 1 µg/well, as described in Methods. Serial dilutions of the antivenom was coated onto microplates at 1 g/well, as described in Methods. Serial dilutions of the antivenom were added and the binding of antibodies was detected by anti‐equine immunoglobulins conjugated were added and the binding of antibodies was detected by anti-equine immunoglobulins conjugated to were added and the binding of antibodies was detected by anti‐equine immunoglobulins conjugated to alkaline phosphatase, followed by color development using p‐nitrophenylphosphate substrate. A alkaline phosphatase, followed by color development using p-nitrophenylphosphate substrate. A mock to alkaline phosphatase, followed by color development using p‐nitrophenylphosphate substrate. A mock antivenom prepared using the plasma plasma of a a non-immunized normal, non‐immunized non‐immunized horse, was used as a a antivenom prepared using the plasma of a normal, horse, was usedwas as aused negative mock antivenom prepared using the of normal, horse, as negative control for background. Each point represents mean ± SD of triplicate wells. control for background. Each point represents mean ˘ SD of triplicate wells. negative control for background. Each point represents mean ± SD of triplicate wells.
Figure 5.5. 5. Immunorecognition Immunorecognition of of the RP-HPLC RP‐HPLC fractions fractions of of Micrurus Micrurus clarki clarki venom venom by by an an equine equine Figure Immunorecognition of the the Figure RP‐HPLC fractions of Micrurus clarki venom by an equine monospecific antivenom raised against M. nigrocinctus venom (SAC‐ICP), as evaluated by ELISA. monospecific antivenom raised against M. nigrocinctus venom (SAC-ICP), as evaluated by ELISA. monospecific antivenom raised against M. nigrocinctus venom (SAC‐ICP), as evaluated by ELISA. Venom fractions fractions (0.4 g/well) were coated onto microplates, and the binding binding of antibodies antibodies was Venom (0.4 µg/well) were coated ontoonto microplates, and the binding of antibodies was detected Venom fractions (0.4 g/well) were coated microplates, and the of was detected as described in Methods, using anti‐equine immunoglobulins conjugated to alkaline as described Methods,in using anti-equine conjugated toconjugated alkaline phosphatase, detected as in described Methods, using immunoglobulins anti‐equine immunoglobulins to alkaline phosphatase, followed by color color development using p‐nitrophenylphosphate p‐nitrophenylphosphate substrate. Mock followed by color development using p-nitrophenylphosphate substrate. Mock antivenom prepared phosphatase, followed by development using substrate. Mock antivenom prepared using the plasma of a normal, non‐immunized horse, was used as a negative using the plasma of a normal, non-immunized horse, was used as a negative control for background. antivenom prepared using the plasma of a normal, non‐immunized horse, was used as a negative control for background. Each bar represents mean ± SD of triplicate wells. All bars corresponding to Each bar represents mean ˘ SD of triplicate wells. All bars corresponding to SAC-ICP have values control for background. Each bar represents mean ± SD of triplicate wells. All bars corresponding to SAC‐ICP have values that are significantly higher (p
