Snake Venomic of Crotalus durissus terrificus ... - ACS Publications

Snake Venomic of Crotalus durissus terrificussCorrelation with Pharmacological Activities ¨ hler,†,§ Jana Seifert,§ Martin von Bergen,§ Raghuvir K. Arni,| Dessislava Georgieva,†,‡ Michaela O Nicolay Genov,⊥ and Christian Betzel*,‡ Institute of Biochemistry and Molecular Biology, University of Hamburg, Laboratory of Structural Biology of Infection and Inflammation, c/o DESY, Notkestrasse 85, Building 22a, 22603 Hamburg, Germany, Department of Proteomics, Helmholtz-Centre for Environmental Research-UFZ, Permose Strasse 15, 04318 Leipzig, Germany, Department of Physics, IBILCE/UNESP, Cristo´vaõ Colombo 2265, CEP 15054-000, Saõ Jose´ do Rio Preto, SP Brazil, and Institute of Organic Chemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria Received November 13, 2009

The snake venomic of Crotalus durissus terrificus was analyzed by 2-D and 1-D electrophoresis and subsequent MS/MS and enzymatic assays. The venomic of the South American rattlesnake comprises toxins from seven protein families: phospholipases A2, serine proteinases, ecto-5′-nucleotidases, metalloproteinases, nerve growth factors, phosphodiesterases, and glutaminyl cyclase. The venom toxin composition correlates with the clinical manifestation of the crotalinae snake bites and explains pathological effects of the venom such as neurotoxicity, systemic myonecrosis, hemostatic disorders, myoglobinuria, and acute renal failure. The vast majority of toxins are potentially involved in neurotoxicity, myotoxicity, and coagulopathy. The predominant venom components are neurotoxic phospholipases A2 and serine proteinases. The venom is a rich source of 5′-nucleotidases (7.8% of the identified toxins) inducing hemostatic disorders. Analysis of the venom protein composition provided a catalogue for secreted toxins. The venomic composition of Crotalus d. terrificus and venom gland transcriptome of the synonymous subspecies Crotalus d. collilineatus show differences in the occurrence of protein families and in the abundance of toxins. Some of the venom components identified by the proteomic analysis were not reported in the transcriptome of the Crotalus d. collilineatus venom gland. Enzymatic activities of the Crotalus d. terrificus venom were determined and correlated with the proteomic composition. Keywords: snake venomic • Crotalus durissus terrificus • 2-D electrophoresis • electrospray mass spectrometry

Knowledge of the snake venom protein compositions is necessary for several reasons. On one side, snake envenoming is of public health significance in many countries of the South and Central America, Asia, Africa,1 and to a lesser extent in Europe, mainly in the South East and Central part of the continent. On the other side, snake venom is a rich source of biologically active compounds of therapeutical value. At present, it is not possible to predict how many pharmacologically active peptides and proteins are synthesized by venomous snakes influencing physiologically important systems in the living organisms as the blood coagulation cascade, hemostasis, tissue repair, and breathing. Venomic analysis is important for the basic research and better understanding of the ecological niche,

evolutionary relationships, for the preparation of more effective toxin-specific antivenoms, and for the design of new medicines regulating the blood pressure, with anticoagulant, anti-inflammatory, anticancer, or other activities for clinical use. The importance of snake venom protein compositions from medical point of view is discussed in details in a recently published review of Calvete.1 Analysis of the origin and evolution of snake venom proteome revealed that venom evolved once at the base of the advanced snake radiation and that the snake venom toxins evolved from recruitment of body proteins into the snakes.2,3 A single early origin of the venom system in snakes and lizards has also been suggested.4 During the last several years, the investigations of the groups of Calvete1,5-19 and Fox20-24 have contributed significantly to our understanding of the protein composition of snake venoms.

* To whom correspondence should be addressed. Tel.: +494089984744. Fax: +494089984747. E-mail: [email protected]. † These authors have contributed equally to this work. ‡ University of Hamburg. § Helmholtz-Centre for Environmental Research-UFZ. | IBILCE/UNESP. ⊥ Bulgarian Academy of Sciences.

Crotalus durissus is a rattlesnake found in all South American countries except Ecuador and Chile.25 Phylogeographical analysis showed that the colonization of the south part of the American continent by this species happened during the late Pliocene.25 The South American populations of C. durissus are phylogenetically closely related and eight subspecies are known.

1. Introduction

2302 Journal of Proteome Research 2010, 9, 2302–2316 Published on Web 03/07/2010

10.1021/pr901042p

 2010 American Chemical Society

Snake Venomic of Crotalus durissus terrificus However, the subspecific distinctions between C. d. cascavella, C. d. collilineatus, and C. d. terrificus were found to be insignificant, and the first two subspecies should be considered synonymous with the third.25 Crotalus durissus terrificus (Family: Viperidae; Subfamily: Crotalinae; Genus: Crotalus; Species: Crotalus durissus) is a snake of public health significance for South America. It is responsible for most of the lethal accidents due to snakebites in Brazil. The envenomation causes neurotoxic, myotoxic, and coagulopathic effects.26 C. d. terrificus is the most intensively studied subspecies of the genus Crotalus, in particular due to the presence in its venom of the neurotoxin crotoxin, the first animal venom toxin to be isolated.27 However, the venom components were subjected to phenomenological assays, and the global assessment of the venomic of this snake has not been published so far.

2. Materials and Methods 2.1. Collection of the Venom. Crude venom from C. d. terrificus, white in color, was obtained from a local serpentarium (San Maru Serpentarium, Brazil). The snakes were milked using a latex-covered specimen jar. The venom was filtered to remove potential mucosal contaminants. Polyethylene tubes, bottles, and pipettes were used to handle the sample to avoid a possible absorbance of snake venom components on glass materials. The lyophilized venom was stored at 4 °C. 2.2. 2-D Gel Electrophoresis and Electrospray Mass Spectrometry. Two-dimensional electrophoresis was performed as described previously.28 In brief, 500 µg of the total protein were mixed with 50 µL of a solution containing 8 M urea, 2% Triton X-100 (v/v), 2% CHAPS (w/v), 0.5% IPG (immobilized pH gradient) pH 3-10 nonlinear (NL) buffer (v/v) (GE Healthcare, Uppsala, Sweden), 65 mM dithiothreitol, and 0.01% bromphenol blue (w/v). The sample was agitated for 10 min at room temperature. Afterward rehydrating solution containing 8 M urea, 2% Triton X-100 (v/v), 0.5% IPG (immobilized pH gradient) pH 3-10 NL (v/v), 65 mM dithiothreitol, and 0.01% (w/v) bromphenol blue were added and the sample agitated for 5 min. To remove precipitates, the solution was centrifuged for 30 min at 13 000 rpm. The equilibration was performed with 400 µL of supernatant loaded on 18 cm Immobiline DryStrip pH 3-10 NL (GE Healthcare, Uppsala, Sweden). In the first dimension, proteins were separated by an IPGphore electrophoresis unit overnight (GE Healthcare, Uppsala, Sweden). After isoelectric focusing the strips were equilibrated for 15 min in equilibration buffer containing 0.05 M Tris/HCL pH 8.8, 30% glycerol (v/v), 6 M urea, 4% sodium dodecyl sulfate, and 2% dithioerythrithol. In a second equilibration step, the strips were incubated with 0.05 M Tris/HCL pH 8.8, 30% glycerol (v/v), 6 M urea, 4% sodium dodecylsulfate, and 2.5% iodoacetamide for 15 min. The prepared strips were stored at -20 °C until used in the second dimension. The second dimension was performed on a 12% polyacrylamide gel (160 × 160 × 1.0 mm3). The Protean II xi Cell-System (BioRad, Hercules, California) was used for running the gels. The following running conditions were used: 20 mA per gel for 10 min and 40 mA per gel for 4-5 h with cooling at 12 °C, respectively. Afterward, the gels were stained with CBB overnight and destained as described previously.29 Gels were scanned and imported into the Delta2D software package (Decodon, Greifswald, Germany).

research articles Protein bands of interest were cut from polyacrylamide gels and digested overnight using trypsin (Sigma, Munich, Germany) according to a protocol from Shevchenko modified in previous study.30 The cleaved peptides were eluted, concentrated by vacuum centrifugation and thereafter separated by RP nanoLC (LC1100 series, Agilent Technologies, Paolo Alto, California; column: Zorbax 300SB-C18, 3.5 µm, 150 × 0.075 mm; eluate: 0.1% formic acid in 0-60% acetonitrile). The peptides were analyzed by online MS/MS (LC/MSD TRAP XCT mass spectrometer, Agilent Technologies). Thereafter, a database search was conducted using the MS/ MS ion search (MASCOT, http://www.matrixscience.com) against all entries of NCBInr (GenBank; http://www.ncbi.nlm.nih.gov/ index.html) with subsequent parameters: trypsin digestion, up to one missed cleavage site, fixed modifications: carbamidomethyl (C), variable modifications: oxidation (M), peptide tol.: (1.2 Da, MS/MS tol.: (0.6 Da, peptide charge: +1, +2, and +3. Proteins with molecular masses less than 10 kDa were not identified. 2.3. LTQ-Orbitrap-MS Measurements. Proteins of interest were excised from the stained gels. The spots were subjected to in-gel trypsin digestion as previously described.30 Peptides were reconstituted in 0.1% formic acid, injected by an autosampler and concentrated on a trapping column (nanoAcquity UPLC column, C18, 180 µm × 2 cm, 5 µm, Waters, Eschborn, Germany) with water containing 0.1% formic acid at flow rate of 15 µL/min. After 4 min the peptides were eluted onto the separation column (nanoAcquity UPLC column, C18, 75 µm × 100 mm, 1.7 µm, Waters, Eschborn, Germany). Chromatography was performed using 0.1% formic acid in solvents A (100% water) and B (100% acetonitrile), with peptides eluted over 30 min with an 8-40% solvent B gradient using a nano-HPLC system (nanoAquity, Waters) coupled to an LTQOrbitrap mass spectrometer (Thermo Fisher Scientific). Continuous scanning of eluted peptide ions was carried out between 400-2000 m/z, automatically switching to MS/MS CID mode on ions exceeding an intensity of 2000. Raw MS/MS spectra were converted to mgf-files using TurboRaw2Mgf (ProQuantSuite v. 1.0.2708.25724). MS data were submitted to the in-house MASCOT server and searched against all Chordata entries in the National Centre for Biotechnology Information nucleotide database (NCBInr, January 2010; 2 001 908 protein entries) tolerating up to one tryptic missed cleavages, a mass tolerance of 10 ppm for precursor ions, 0.2 Da for MS/MS product ions allowing for methionine oxidation (dynamic modification), and cysteine carbamidomethylation (static modification). 2.4. Enzymatic Activities. Proteolytic activity was determined by the method of Johnson et al.31 The venom was assayed using 1.2% casein solution in Tris-HCl buffer, pH 7.4, at 37 °C. Undigested casein was precipitated with 0.5 M perchloric acid and centrifuged. Digested casein in the supernatant was determined by measuring the absorbance at 280 nm. Unit definition: One CTA unit liberates from cow casein 0.1 micro equivalents of tyrosine for 1 min at 37.5 °C. One CTA unit is equal to 0.096 proteolytic units as used by SIGMA Chemical Corporation, St. Louis, MO. Phospholipase A2 activity was determined using the Cayman Chemical Secretory PLA2 Assay kit (Ann Arbor, MI) containing a bee venom PLA2 as a standard. 1,2 - dithio analog of diheptanoyl phosphatidylcholine was used as a substrate. The release of free thiols upon the PLA2 catalyzed hydrolysis of the Journal of Proteome Research • Vol. 9, No. 5, 2010 2303

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Figure 1. 2-D gel pattern of the Crotalus durissus terrificus venom.

thioester bond at the sn-2 position was detected spectrophotometrically using 5,5′-dithiobis (2-nitrobenzoic acid). L-amino acid oxidase activity was determined by the method of Wellner et al.32 using L-phenylalanine as substrate. One unit of activity is the amount of enzyme required to give an absorbance of 0.030 at 300 nm. Alkaline phosphatase activity was measured by the method of Sulkowski et al.33 using p-nitrophenylphosphate as substrate. One unit of activity is defined as the amount of enzyme which liberates 1 µmole of p-nitrophenol per min. Acid phosphatase activity was determined by the method of Tu and Chua.34 o-Carboxyphenylphosphate (0.0036 M) was used as substrate and the initial rate of hydrolysis of the substrate at 25 °C was determined from the increase of the absorbance at 300 nm due to the liberation of salicylic acid. Venom concentration was adjusted to give a straight line at least for 5 min. One unit of acid phosphatase activity is equivalent to 1 µmole of the substrate hydrolyzed per min.

shown in Figure 2. The data are expressed as percentage of the protein sequences. The venom protein composition explains pharmaceutical effects and clinical manifestations of the South American rattlesnake envenomation. Neurotoxic pathological effects, systemic myonecrosis, coagulopathy, myoglo-

3. Results and Discussion 3.1. 2-D Gel Electrophoresis of the C. d. terrificus Venom and Protein Family Compositions. The venomic composition of C. d. terrificus was investigated by 2-D electrophoresis, and the separated protein bands were subjected to tryptic digestion. The venom components were identified by MS/MS and MASCOT search program (Figure 1 and Table 1). The oxidized methionine residues are indicated as Mox. The oxidation of Met is due probably to the procedure of harvesting and sample handling. The samples were dried in the presence of ambient air which is known to cause oxidation of methionine. The isolated toxins were assigned to the following protein families: phospholipases A2, serine proteinases, 5′-nucleotidases, metalloproteinases, nerve growth fators (NGFs), phosphodiesterases, and glutaminyl cyclase (Figure 1). The first two groups represent the major protein families containing the predominant number of toxins. The third one consists of eight representatives of 5′-nucleotidases, while the rest of the families have only a few members. The venomic of C. d. terrificus is 2304

Journal of Proteome Research • Vol. 9, No. 5, 2010

Figure 2. Proteome of the Crotalus durissus terrificus venom and transcriptome of the synonymous subspecie C. d. collilineatus venom gland. (A) Percent of toxin sequences found in the C. d. terrificus venom by proteome analysis (this work) and (B) percent of toxin coding sequences of the C. d. collilineatus venom gland by transcriptome analysis.66 PLA2, phospholipase A2.

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Snake Venomic of Crotalus durissus terrificus

Table 1. Assignment of the Proteins Isolated from the Spots of the 2-D Gel Electrophoresis of the Crotalus durissus terrificus Venom to Protein Families by MS/MS and MASCOT Database Search spot no.

protein

accession code

homology with a protein from

MASCOT score

matched peptides

peptide ion m/z

z

MS/MS derived sequence

672.3 860.0 765.3 523.3 672.3 430.2 860.0 713.2 423.2 521.6 732.4 523.3 672.4 430.2 860.0 423.2 730.5 521.8 358.2 524.3 426.2 732.5 523.3 672.3 476.4 860.0 423.1 521.6 732.5 523.1 672.3 430.3 859.9 426.2 732.4 523.3 859.9 521.6 524.2 523.2 430.7 476.3 860.0 645.1 521.6 785.9 425.7 732.5 523.2 672.3 859.9 521.7 785.9 425.8 732.5 523.2 419.7 630.7

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

K.CTGQDCYGGVAR.R K.ETPVLSNPGPYLEFR.D K.IIALGHSGFFEDQR.I R.VPTYVPLEK.E K.CTGQDCYGGVAR.R K.IINVGSEK.V K.ETPVLSNPGPYLEFR.D R.DEVEELQKHANK.L K.LTTLGVNK.I K.SSGNPILLNK.N R.VVSLNVLCTECR.V R.VPTYVPLEK.E K.CTGQDCYGGVAR.R K.IINVGSEK.V K.ETPVLSNPGPYLEFR.D K.LTTLGVNK.I K.YLGYLNVIFDDK.G K.SSGNPILLNK.N R.SPIDER.A R.HGQGMOXGELLQVSGIK.V K.VVYDLSR.K R.VVSLNVLCTECR.V R.VPTYVPLEK.E K.CTGQDCYGGVAR.R K.VGIIGYTTK.E K.ETPVLSNPGPYLEFR.D K.LTTLGVNK.I K.SSGNPILLNK.N R.VVSLNVLCTECR.V R.VPTYVPLEK.E K.CTGQDCYGGVAR.R K.IINVGSEK.V K.ETPVLSNPGPYLEFR.D K.VVYDLSR.K R.VVSLNVLCTECR.V R.VPTYVPLEK.E K.ETPVLSNPGPYLEFR.D K.SSGNPILLNK.N R.HGQGMGELLQVSGIK.V R.VPTYVPLEK.E K.IINVGSEK.V K.VGIIGYTTK.E K.ETPVLSNPGPYLEFR.D R.QVPVVQAYAFGK.Y K.SSGNPILLNK.N R.HGQGMOXGELLQVSGIK.V K.VVYDLSR.K R.VVSLNVLCTECR.V R.VPTYVPLEK.E K.CTGQDCYGGVAR.R K.ETPVLSNPGPYLEFR.D K.SSGNPILLNK.N R.HGQGMOXGELLQVSGIK.V K.VVYDLSR.K R.VVSLNVLCTECR.V R.VPTYVPLEK.E R.IQIHTAR.V K.TFLPIFVNPVN.-

1

ecto-5′-nucleotidase gi|211926756

Gloydius blomhoffi brevicaudus

157

4

2



302

8

3



452

11

4



267

7

5



225

6

6



195

4

7



271

9

8



265

7

9

phospho-diesterase

gi|109254994

126

2

10

phospho-diesterase

gi|109254994

120

2

419.8 630.6

2 2

R.IQIHTAR.V K.TFLPIFVNPVN.-

11

glutaminyl cyclase

gi|3868931

Sistrurus catenatus edwardsi Sistrurus catenatus edwardsi Bothrops jararaca

255

6

12

crotoxin basic chain 1

gi|171848868

Crotalus durissus terrificus

163

5

crotoxin basic chain 2 precursor

gi|129470

129

3


gi|171848868

Crotalus durissus terrificus Crotalus durissus terrificus

163

4

532.8 721.8 769.4 365.8 886.0 772.0 450.2 502.7 443.2 699.8 657.8 502.7 443.2 669.2 450.3 502.5 443.1 657.9

2 2 2 2 2 2 2 3 2 2 2 3 2 2 2 3 2 2

K.YFPPQLDGK.V K.LIFFDGEEAFVR.W R.NPVFPVYFLNTAR.W R.LEAIER.N R.NLNDLGLLNNYSSER.Q R.GVPILHLIPSPFPR.V -.HLLQFNK.M R.CCFVHDCCYGK.L K.SGYITCGK.G W.CEEQICECDR.V K.YGYMOXFYPDSR.C R.CCFVHDCCYGK.L K.SGYITCGK.G K.NEYMOXFYPDSR.C -.HLLQFNK.M R.CCFVHDCCYGK.L K.SGYITCGK.G K.YGYMOXFYPDSR.C

13

protein family

serine/threonine phosphatases serine/threonine phosphatases

serine/threonine phosphatases



serine/threonine phosphatases serine/threonine phosphatases


phospholipase A2

phospholipase A2 phospholipase A2

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Table 1. Continued spot no.

protein

crotoxin basic chain 2 precursor 14


16

serine proteinase precursor

17


18

halystase

19

pallase

20

21

22

accession code

gi|129470

Crotalus durissus terrificus gi|48429036 Crotalus durissus terrificus gi|123895843 Crotalus durissus durissus gi|123895843 Crotalus durissus durissus gi|3122187 Gloydius blomhoffi gi|3552036 Gloydius halys

matched peptides

peptide ion m/z

z

130

3

61

1

502.5 443.1 669.2 657.8

2 2 2 2

R.CCFVHDCCYGK.L K.SGYITCGK.G K.NEYMOXFYPDSR.C K.YGYMOXFYPDSR.C

219

4

199

3

107

2

188

3

596.4 537.8 584.8 559.9 596.4 537.7 559.8 444.7 559.9 756.9 388.7 559.8 388.7 739.9 559.8 717.4 696.0 1071.5 717.4 559.8

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

R.GDILVLLGVHR.L R.LKDVQTGVSK.D R.AAKPELPVTSR.T R.TLCAGILEGGK.G R.GDILVLLGVHR.L R.LKDVQTGVSK.D R.TLCAGILEGGK.G K.FFCLSSK.N R.TLCAGILEGGK.D M.VIGGDECNINEHR.F K.DIMOXLIR.L R.TLCAGILEGGK.D K.DIMOXLIR.L R.LDSPVSNSEHIAPL.S R.TLCAGILEGGK.D K.DDVLDKDIMOXLIK.L K.TLPDVPYCANIK.L K.LLDDAVCQPPYPELPATSR.T K.DDVLDKDIMOXLIK.L R.TLCAGILEGGK.D

749.8 845.5 781.5 560.00 781.5 717.6 696.0 1071.7 717.4 696.0 1071.6 749.8 559.8

2 2 2 2 2 2 2 2 2 2 2 2 2

-.VVGGDECNINEHR.F K.KNDEVLDKDIMOXLIK.L K.NDEVLDKDIMOXLIK.L R.TLCAGILEGGK.D K.KDDVLDKDIMOXLIK.L K.DDVLDKDIMOXLIK.L K.TLPDVPYCANIK.L K.LLDDAVCQPPYPELPATSR.T K.DDVLDKDIMOXLIK.L K.TLPDVPYCANIK.L K.LLDDAVCQPPYPELPATSR.T -.VVGGDECNINEHR.F R.TLCAGILEGGK.D

Gloydius ussuriensis

108

3

catroxase-2 precursor

gi|82244345

Crotalus atrox

168

3

serine proteinase isoform 3

gi|109254942 Sistrurus catenatus edwardsi gi|32469800 Agkistrodon contortrix contortrix

133

2

209

4

contortrixobin


protein family

phospholipase A2 phospholipase A2 peptidase S1

peptidase S1 serine protease serine protease peptidase S1 peptidase S1 serine protease peptidase S1

peptidase S1


gi|82244345

Crotalus atrox

192

4


gi|82244345

Crotalus atrox

142

3

contortrixobin

gi|32469800

Agkistrodon contortrix contortrix gi|123895843 Crotalus durissus durissus gi|109254944 Sistrurus catenatus edwardsi gi|123883733 Bothrops asper

127

2

124

2

596.3 559.8

2 R.GDILVLLGVHR.L 2 R.TLCAGILEGGK.G

120

3

156

3

gi|109254948 Sistrurus catenatus edwardsi gi|951152 Protobothrops mucrosquamatus gi|951152 Protobothrops mucrosquamatus gi|82244345 Crotalus atrox

114

2

652.9 753.9 510.2 644.8 603.8 832.1 588.4 886.0

2 2 2 2 2 3 2 2

100

2

445.9 559.7

2 K.FFCDSSK.T 2 R.TLCAGILEGGK.D

serine protease

67

1

559.8

2 R.TLCAGILEGGK.D

serine protease

143

3

126

2

717.4 696.1 714.6 717.4

2 2 3 2

560.2

2 R.TLCAGILEGGK.D

23

serine proteinase isoform 4

24

venom serine proteinase-like precursor serine proteinase isoform 6

26

preprotrimubin

27

preprotrimubin

28

catroxase-2 precursor serine proteinase isoform 3

gi|109254942 Sistrurus catenatus edwardsi

29

preprotrimubin

gi|951152

30

preprotrimubin

gi|951152

31

ancrod venombin-A protein C activator ACC-C crotoxin basic chain 1

gi|113827

2306

MASCOT score

calobin-1 precursor gi|13959630


33


Protobothrops mucrosquamatus Protobothrops mucrosquamatus Calloselasma rhodostoma

gi|171848868 Crotalus durissus terrificus


peptidase S1 peptidase S1 serine protease

serine protease K.NFQMOXQLGVHSK.K K.DDEKDKDIMOXLIR.L K.DKDIMOXLIR.L peptida se S1 K.NFQMOXQLGVHSK.K R.IMOXGWGTISPTK.E K.ETYPDVPHCANINILDHAVCR.A serine protease K.RDKDIMOXLIR.L R.ILCAGVLEGGIDTCHR.D

K.DDVLDKDIMOXLIK.L K.TLPDVPYCANIK.L K.LLDDAVCQPPYPELPATSR.T K.DDVLDKDIMOXLIK.L

peptidase S1 serine protease

76

1

559.4

2 R.TLCAGILEGGK.D

serine protease

63

1

559.7

2 R.TLCAGILEGGK.D

serine protease

78

1

756.9

2 -.VIGGDECNINEHR.F

peptidase S1

311

7

450.3 688.7 753.3 563.3 443.2 871.9 997.4

2 3 2 3 2 2 2

phospholipase A2

-.HLLQFNK.M K.DATDRCCFVHDCCYGK.L R.CCFVHDCCYGK.L K.CNTKWDIYPYSLK.S K.SGYITCGK.G K.GTWCEEQICECDR.V R.SLSTYKYGYMOXFYPDSR.C

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Snake Venomic of Crotalus durissus terrificus Table 1. Continued spot no.

protein

accession code

homology with MASCOT matched peptide a protein from score peptides ion m/z z


gi|129470


141

4

34



223

6

35



194

5

36

venom nerve growth factor; v-NGF precursor crotoxin basic chain 1

gi|82220600

97

3

107

3

85

3

184

5

Crotalus atrox

105

3


253

5

gi|48429036


172

7


gi|129470


150

5

phospholipase A2 F16


122

5

gyroxin-like B1_4 serine protease precursor metalloproteinase P-II

gi|164664996 Crotalus durissus terrificus gi|76365440 Crotalus durissus durissus gi|164664994 Crotalus durissus durissus

82

37 38 39

venom nerve growth factor; v-NGF precursor crotoxin basic chain 1

Crotalus durissus terrificus gi|171848868 Crotalus durissus terrificus gi|82220600 Crotalus durissus terrificus gi|171848868 Crotalus durissus terrificus

40


42

crotoxin acid chain gi|129456

47* crotoxin basic chain 1 precursor

49

50* gyroxin-like B1_3 serine protease precursor

gi|82244345


1

3 2 2 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2 2 3 2 2 2 2 2 3 2 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

78

1

799.3

2 K.NDDTCTGQSADCPR.N

metalloproteinase

203

5

446.2 947.5 428.7 885.9 839.0 502.5 443.2 871.8 409.7 657.8 502.5 443.2 409.7 669.3 519.7 780.1 798.9

2 3 2 2 3 3 2 2 2 2 3 2 2 2 2 3 2

serineprotease


gi|48429036

Crotalus durissus durissus

166

5


gi|129470


157

5

51

metalloproteinase P-II

gi|76365440

141

2

52


gi|76365440

Crotalus durissus durissus Crotalus durissus durissus

124

3

1170 1113.4 798.9

K.DATDRCCFVHDCCYGK.L R.CCFVHDCCYGK.L K.SGYITCGK.G K.GTWCKEQICECDR.V -.HLLQFNK.M K.MOXIKFETR.K R.CCFVHDCCYGK.L K.SGYITCGK.G R.VAAECLR.R K.YGYMOXFYPDSR.C -.HLLQFNK.M R.CCFVHDCCYGK.L K.GTWCEEQICECDR.V R.SLSTYKYGYMOXFYPDSR.C K.YGYMOXFYPDSR.C K.QYFFETK.C K.ALTMOXEGNQASWR.F R.IDSACVCVISR.K -.HLLQFNK.M R.SLSTYKYGYMOXFYPDSR.C K.YGYMOXFYPDSR.C K.QYFFETK.C K.ALTMOXEGNQASWR.F R.IDSACVCVISR.K -.HLLQFNK.M K.DATDRCCFVHDCCYGK.L R.CCFVHDCCYGK.L R.SLSTYKYGYMOXFYPDSR.C K.YGYMOXFYPDSR.C K.EKFFCPNKK.K K.FFCPNK.K K.DDVLDKDIMOXLIK. G.SLVEFETLMOXMK.I K.LTGCDPTTDVYTYR.Q K.AAAICFR.N R.NSMDTYDYK.Y K.YLQFSPENCQGESQPC.K.WDIYPYSLK.S K.SGYITCGK.G K.GTWCEEQICECDR.V R.VAAECLR.R R.RSLSTYK.Y K.YGYMOXFYPDSR.C R.CRGPSETC.K.SGYITCGK.G R.VAAECLR.R R.RSLSTYK.N K.NEYMOXFYPDSR.C R.CREPSETC.K.SGYITCGK.G R.VAAECLR.R R.RSLSTYK.N K.NGYMOXFYPDSR.C R.CRGPSETC.R.ILCAGVLEGGIDTCHR.D

protein family

688.7 753.3 443.2 871.9 450.3 470.8 753.3 443.2 818.5 657.7 450.3 753.3 874.3 997.5 657.8 481.7 692.4 640.8 450.3 665.3 657.8 481.7 706.4 640.4 450.3 688.7 753.3 665.6 657.8 599.3 406.7 717.2 672.4 831.4 404.7 568.3 972.4 592.8 443.2 871.8 409.7 427.7 657.8 483.7 443.2 409.7 427.7 669.3 519.7 443.2 409.7 427.7 633.3 483.7 885.9

R.VMGWGTIK.S K.SPQETLPDVPHCANINLLDYEVCR.T R.TAHPQFR.L R.ILCAGVLEGGIDTCHR.D K.VFDHLDWIQNIIAGSETVNCPS.R.CCFVHDCCYGK.L K.SGYITCGK.G K.GTWCEEQICECDR.V R.VAAECLR.R K.YGYMOXFYPDSR.C R.CCFVHDCCYGK.L K.SGYITCGK.G R.VAAECLR.R K.NEYMOXFYPDSR.C R.CREPSETC.R.IECDCGSIENPCCYATTCK.L K.NDDTCTGQSADCPR.N

2 R.IECDCGSIENPCCYATTCK.L 2 I.ECDCGSIENPCCYATTCK.L 2 K.NDDTCTGQSADCPR.N


phospholipase A2

NGF-beta phospholipase A2 NGF-beta phospholipase A2

peptidase S1 phospholipase A2

phospholipase A2

phospholipase A2

phospholipase A2

serine protease

phospholipase A2

phospholipase A2

metalloproteinase metalloproteinase


research articles

Georgieva et al.


protein

accession code


MASCOT score

matched peptides

peptide ion m/z

z


1170.0 1051.4 330.3 1119.0 798.5 855.9 443.2 409.7 427.7 669.3 519.7 443.2 871.8 409.7 427.7 649.8 483.7 443.2 409.7 427.7 633.3 483.7 443.2 871.8 409.7 427.7 657.8 443.2 409.7 427.7 669.3 519.7 443.2 409.7 427.7 633.3 443.2 871.8 409.7 427.7 657.8 443.2 409.7 427.7 625.3 443.2 409.7 427.7 669.3 443.2 871.8 409.7 427.7 649.8 483.7 443.2 409.7 427.7 625.3 483.7 443.2 409.7 427.7 669.3 443.2 871.8 409.7 427.7 649.8 483.7 443.2 409.7 427.7 625.3 483.7 443.2 409.7 427.7 669.3 425.2 752.7 443.3

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

R.IECDCGSIENPCCYATTCK.L K.LRPGSQCAEGMOXCCDQCR.F R.VSLVNK.N R.VSLVNKNDDTCTGQSADCPR.N K.NDDTCTGQSADCPR.N K.NDDTCTGQSADCPRN.V K.SGYITCGK.G R.VAAECLR.R R.RSLSTYK.N K.NEYMOXFYPDSR.C R.CREPSETC.K.SGYITCGK.G K.GTWCEEQICECDR.V R.VAAECLR.R R.RSLSTYK.Y K.YGYMFYPDSR.C R.CRGPSETC.K.SGYITCGK.G R.VAAECLR.R R.RSLSTYK.N K.NGYMOXFYPDSR.C R.CRGPSETC.K.SGYITCGK.G K.GTWCEEQICECDR.V R.VAAECLR.R R.RSLSTYK.Y K.YGYMOXFYPDSR.C K.SGYITCGK.G R.VAAECLR.R R.RSLSTYK.N K.NEYMOXFYPDSR.C R.CREPSETC.K.SGYITCGK.G R.VAAECLR.R R.RSLSTYK.N K.NGYMOXFYPDSR.C K.SGYITCGK.G K.GTWCEEQICECDR.V R.VAAECLR.R R.RSLSTYK.Y K.YGYMOXFYPDSR.C K.SGYITCGK.G R.VAAECLR.R R.RSLSTYK.N K.NGYMFYPDSR.C K.SGYITCGK.G R.VAAECLR.R R.RSLSTYK.N K.NEYMOXFYPDSR.C K.SGYITCGK.G K.GTWCEEQICECDR.V R.VAAECLR.R R.RSLSTYK.Y K.YGYMFYPDSR.C R.CRGPSETC.K.SGYITCGK.G R.VAAECLR.R R.RSLSTYK.N K.NGYMFYPDSR.C R.CRGPSETC.K.SGYITCGK.G R.VAAECLR.R R.RSLSTYK.N K.NEYMOXFYPDSR.C K.SGYITCGK.G K.GTWCEEQICECDR.V R.VAAECLR.R R.RSLSTYK.Y K.YGYMFYPDSR.C R.CRGPSETC.K.SGYITCGK.G R.VAAECLR.R R.RSLSTYK.N K.NGYMFYPDSR.C R.CRGPSETC.K.SGYITCGK.G R.VAAECLR.R R.RSLSTYK.N K.NEYMOXFYPDSR.C -.SLLQFNK.M R.CCFVHDCCYGK.L K.SGYITCGK.G

53


gi|76365440


300

6

56*


gi|129470


184

5


gi|48429036


177

6



144

5


gi|48429036


221

5


gi|129470


178

5



138

4


gi|48429036


145

5



129

4


gi|129470


120

4


gi|48429036


205

6



171

5


gi|129470


132

4


gi|48429036


197

6



143

5


gi|129470


130

4

chain A. crotoxin


99

3

57*

58*

59*

60*

61

2308


protein family

metalloproteinase

phospholipase A2

phospholipase A2

phospholipase A2

phospholipase A2

phospholipase A2


phospholipase A2

phospholipase A2

phospholipase A2


phospholipase A2


research articles

Snake Venomic of Crotalus durissus terrificus Table 1. Continued spot no.

protein

accession code


MASCOT score

matched peptides

peptide ion m/z

z


450.3 753.3 443.2 657.8 450.3 753.4 443.2 658.0 753.4 443.2 1009.5 753.3 443.2 669.0 450.3 753.3 443.2 753.3 443.1 657.9 688.3 753.3 593.0 443.4 888.3 699.7 325.7 658.0 483.7 688.3 753.3 443.4 325.7 669.8 450.3 753.4 443.2 879.8 699.8 657.9 450.3 753.4 442.7 879.9 699.8 657.8 753.4 442.7 1008.9 450.2 470.8 753.6 443.4 880.3 699.8 698.5 658.0 483.7 470.8 753.6 443.4 555.3 698.2 673.4 450.0 753.4 443.6 871.8 699.7 409.7 649.8 483.7 425.3 753.4 443.6 871.8 409.7 672.8 669.3 519.4

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 2 2 2 2 2 3 2 2 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 2 2

-.HLLQFNK.M R.CCFVHDCCYGK.L K.SGYITCGK.G K.YGYMOXFYPDSR.C -.HLLQFNK.M R.CCFVHDCCYGK.L K.SGYITCGK.G K.YGYMOXFYPDSR.C R.CCFVHDCCYGK.L K.SGYITCGK.G R.SLSTYKNEYMOXFYPDSR.C R.CCFVHDCCYGK.L K.SGYITCGK.G K.NEYMOXFYPDSR.C -.HLLQFNK.M R.CCFVHDCCYGK.L K.SGYITCGK.G R.CCFVHDCCYGK.L K.SGYITCGK.G K.YGYMOXFYPDSR.C K.DATDRCCFVHDCCYGK.L R.CCFVHDCCYGK.L K.WDIYPYSLK.S K.SGYITCGK.G K.GTWCEEQICECDR.V W.CEEQICECDR.V R.VAAECLRR.S K.YGYMOXFYPDSR.C R.CRGPSETC.K.DATDRCCFVHDCCYGK.L R.CCFVHDCCYGK.L K.SGYITCGK.G R.VAAECLRR.S K.NEYMOXFYPDSR.C -.HLLQFNK.M R.CCFVHDCCYGK.L K.SGYITCGK.G K.GTWCEEQICECDR.V W.CEEQICECDR.V K.YGYMOXFYPDSR.C -.HLLQFNK.M R.CCFVHDCCYGK.L K.SGYITCGK.G K.GTWCEEQICECDR.V W.CEEQICECDR.V K.YGYMOXFYPDSR.C R.CCFVHDCCYGK.L K.SGYITCGK.G R.SLSTYKNEYMOXFYPDSR.C -.HLLQFNK.M K.MOXIKFETR.K R.CCFVHDCCYGK.L K.SGYITCGK.G K.GTWCEEQICECDR.V W.CEEQICECDR.V R.SLSTYK.Y K.YGYMOXFYPDSR.C R.CRGPSETC.K.MOXIKFETR.K R.CCFVHDCCYGK.L K.SGYITCGK.G K.EQICECDR.V R.SLSTYK.N R.SLSTYKNEYMOXFYPDSR.C -.HLLQFNK.M R.CCFVHDCCYGK.L K.SGYITCGK.G K.GTWCEEQICECDR.V W.CEEQICECDR.V R.VAAECLR.R K.YGYMFYPDSR.C R.CRGPSETC.-.SLLQFNK.M R.CCFVHDCCYGK.L K.SGYITCGK.G K.GTWCKEQICECDR.V R.VAAECLR.R R.SLSTYKNEYMOXFYPDSR.C K.NEYMOXFYPDSR.C R.CREPSETC.-

62


gi|171848868


193

4

63


gi|171848868


147

4


gi|129470

84

3


gi|129470

116

3


gi|171848868

86

3

65


gi|48429036

165

3

66


gi|48429036

Crotalus durissus terrificus Crotalus durissus terrificus Crotalus durissus terrificus Crotalus durissus terrificus Crotalus durissus terrificus

264

9


gi|129470


203

5

67


gi|171848868


199

6

68


gi|171848868


174

6


gi|129470

133

3


gi|171848868

Crotalus durissus terrificus Crotalus durissus terrificus

307

9


gi|129470


224

6


gi|171848868


363

8

chain A, crotoxin

gi|171848867


335

8

64

69

70

protein family

phospholipase A2 phospholipase A2 phospholipase A2 phospholipase A2 phospholipase A2 phospholipase A2 phospholipase A2

phospholipase A2

phospholipase A2

phospholipase A2


phospholipase A2

phospholipase A2

phospholipase A2


research articles

Georgieva et al.


71

72

protein

accession code


gi|48429036


gi|129470


gi|129470


Crotalus durissus terrificus Crotalus durissus terrificus Crotalus durissus terrificus

MASCOT score

matched peptides

peptide ion m/z

z


106

3 2

2 2 2 2 2

R.CCFVHDCCYGK.L K.GTWCEEQICECDR.V K.YGYMOXFYPDSR.C R.CCFVHDCCYGK.L K.NEYMOXFYPDSR.C

phospholipase A2

100

753.2 879.7 657.9 753.2 669.2

64

2

887.9 669.2

2 2

K.GTWCKEQICECDR.V K.NEYMOXFYPDSR.C

phospholipase A2

binuria and acute renal failure are the major clinical features observed after accidents by C. d. terrificus.26,35 Phospholipases A2. The venom proteome analysis revealed high quantities and predominance of PLA2s, which are responsible for the neurotoxicity and failure of the neuromuscular junctions. Acidic and basic group II phospholipolytic enzymes represent 48.5% of the identified toxins (Figure 1, spots 12-14, 33-35, 37, 39, 42, 47, 50, 56-72). A number of crotoxin isoforms, the main neurotoxic component of the C. d. terrificus venom, were observed. Crotoxin is a noncovalent complex of a toxic basic phospholipase A2 (subunit B, crotactine) and a nonenzymatic subunit A (crotapotin), which consists of three polypeptides connected with disulfide bonds. The biological role of crotapotin is to avoid nonspecific binding of the toxic subunit and in this way potentiates the toxicity of PLA2.36 Crotoxin exists as a mixture of isoforms of the two subunits. The multiplicity and diversity of these isoforms result from post-translational modifications on a precursor.37 The MS/MS analysis revealed the presence of isoforms of the crotoxin basic subunits CB1 and CB2, their precursors, and the acidic subunit (Figure 1, Table 1). Crotoxin exerts neuropathological action by blocking the neuromuscular transmission38 and displays myotoxic, edema-inducing, bactericidal, and anticoagulant activities. Enzymatic activity is relevant for the lethal, myotoxic, and anticoagulant effects.39 This toxin also interferes with the activity of leukocytes and modulates immune and inflammatory responses.40 Zhang et al.41 reported the venom of C. d. terrificus to have analgesic activity. These authors found that the administration of crotoxin to cancer patients reduces the consumption of analgesics due to its action on the central nervous system. Both acidic and basic PLA2 isoforms are located mainly in the lower part of the 2-DE panel in the region of 11-15 kDa proteins (Figure 1). Their molecular masses correspond to those of monomeric enzymes. The number of basic neurotoxins is higher than that of their acidic counterparts. The proteins of the spots 12-14 and 33-35 show sequence homology with crotoxin isoforms CB1 and CB2.42,43 These toxins were classified as “oligomeric PLA2s” because their molecular masses are higher than that of the monomeric snake venom PLA2. The spots 47, 50, and 56-60 were measured again using a LTQ-Orbitrap-MS in order to obtain a higher resolution. The data obtained by this experiment approved the results from the ESI-MS measurements and revealed only 1-2 additional peptides. The LTQ-Orbitrap-MS results are shown in Table 1 and labeled by asterisks. The protein from spot 42 was identified as crotoxin acid chain A. Peptides isolated after trypsinolysis of the protein from this spot have sequences characteristic of the crotapotin precursor. However, it is not possible from the partial sequences of the isolated peptides to conclude categorically that proCA exists in the venom. We classify the protein from spot 2310


protein family

42 as an “isoform of the crotoxin acid chain”. Additional experiments by 1-D gel electrophoresis (please see paragraph 3.2) and analysis of the fractions of molecular masses 17-19 kDa using LTQ-Orbitrap-MS confirmed the results of the 2 DE. Serine Proteinases. Serine proteinases form the second big protein family of the C. d. terrificus venomic. This group of toxins with acidic isoelectric points and molecular masses in the region of 25 - 33 kDa constitutes 25.3% of the identified venom proteins. They are located in the upper left part of the 2-DE panel (Figure 1). Multiple isoforms of snake venom serine proteinases were isolated from 20 spots. On the basis of the sequence homology to halystase from the venom of Gloydius blomhoffii,44 pallase (Gloydius halys),45 calobin (Gloydius ussuriensis),46 catroxase-2 (Crotalus atrox),47 contortrixobin (Agkistrodon contortrix contortrix),48 trimubin (Protobothrops mucrosquamatus),49 ancrod (Malayan pitviper),50 and gyroxin (C. d. terrificus),51 these enzymes were classified as snake venom serine proteinases (SVSPs). The similar molecular masses also confirm this conclusion. In view of the large diversity of serine proteinases in the venom of the South American rattlesnake, it is surprising that only one representative of this protein family, gyroxin,51 has been investigated so far. Gyroxin induces hemotoxicity and a neurological syndrome called “barrel rotation”.52 Thus, the intravenous injection of this toxin into mice creates rotations around the long axis of the animal.51 Snake venom serine proteinases possess high substrate specificity and affect important physiological functions as blood coagulation and blood presure, fibrinolysis, platelet aggregation, the complement, and nervous systems.53 Some of them can activate Protein C which initiates anticoagulant activity.54 Most probably, venom serine proteinases are related to the hemostatic/clotting disturbances induced by the Crotalus d. terrificus snake bites. Envenomation by the South American rattlesnake degrades fibrinogen/fibrin and causes hypofibrinogenemia.55 The serine proteinases mentioned above cleave fibrinogen, that is, they act as fibrinogenases. Some of them hydrolyze preferentially the beta chain of fibrinogen (halystase), others hydrolyze the alpha chain of this protein (calobin, catroxase-2, trimubin). SVSPs are closely related proteins with a sequence homology among themselves of approximately 67%. In the same time, they show structural (only 40% sequence similarity) and functional differences to thrombin as regards the interactions with blood coagulation factors.53 SVSPs have anticoagulant activity because they induce the formation of friable clots which are readily degraded later on.53 The most popular representative of this protein family is ancrod from the Malayn pitviper which reduces the level of blood fibrinogen, prolongs blood clot formation and was used clinically as anticoagulant agent.50 Metalloproteinases. Four isoforms of class II metalloproteinases with acidic isoelectric points are grouped in the lower left part of the 2-DE panel (Figure 1, spots 49, 51, 52, 53). The

Snake Venomic of Crotalus durissus terrificus estimated molecular masses of 14-15 kDa are lower than those expected for this group of proteins. Class II snake venom metalloproteinases (SVMPs) have molecular masses of 30-60 kDa. They are composed of metalloproteinase domain and disintegrin domain.24 These snake venom components undergo processing to generate disintegrins.56 Most probably, the identified P-II SVMPs represent products of proteolytic processing of two-domain toxins. Metalloproteinases were not identified so far in the venom of C. d. terrificus. However, these enzymes exist in the venoms of other subspecies. For example, zinc-metalloproteinases type PII were found in the Crotalus durissus durissus and Crotalus durissus collilineatus venoms (http://www.uniprot.org). The presence of these enzymes in the C. d. terrificus venom can be explained by interbreeding between different subspecies and a subsequent gene flux. No proteins were identified in the spots 43, 44, 45, and 54 which are in the region of the group “disintegrin domains of metalloproteinases”. These spots were measured again using LTQ-Orbitrap-MS. However, no protein with a proper score was identified. The quality of the digestion and measurement procedures were proven by a BSA standard running in parallel. Metalloproteinases are responsible for the hemorrhagic activity of the snake venoms. These enzymes degrade basement membrane and weaken and disrupt the capillary wall, which further leads to bleeding.57 SVMPs exert also apoptotic and inflammatory effects.24 Despite of the presence of metalloproteinases, the C. d. terrificus venom lacks any hemorrhagic activity.58 It is known that some SVMPs are completely devoid of hemorrhagic activity.59 Moura-da-Silva et al.59 showed that the binding to collagen I and IV is a key factor for the hemorrhagic activity. A lack of affinity to collagen can explain the nonhemorrhagic nature of the C. d. terrificus venom metalloproteinases. 5′-Nucleotidases. A group of eight isoforms of 5′-nucleotidases (5′-NTs) was also identified (Figure 1, the right upper part of the 2-DE panel). These poorly investigated enzymes hydrolyze ADP to produce adenosine. 5′-NTs inhibit platelet aggregation via increased adenosine signaling.60 In this way they affect hemostasis and act as anticoagulants. 5′-NTs contribute significantly to the coagulopathy exerted by the Crotalus d. terrificus venom. Phosphodiesterases. The proteins in spots 9 and 10 (Figure 1, the right upper part of the 2-DE panel) were identified as phosphodiesterases (PHDEs). Snake venom phosphodiesterases are zinc metalloenzymes catalyzing R-β phosphoryl bond cleavage.61 PHDEs possess both exo- and endonuclease activities.62 Glutaminyl Cyclase. Glutaminyl cyclase (GC) was found in spot 11 (Figure 1, the upper right part of the 2-DE panel). This enzyme catalyzes the N-terminal glutamine cyclization of snake venom toxins protecting them from exopeptidase degradation.63 In this way GC contributes to the total venom toxicity. The absence of L-amino acid oxidases corresponds to the white color of the venom. The platelet activator convulxin, a C-type lectin which causes cardiovascular and respiratory disturbances,64 was not identified, but it exists in the venom of the South American rattlesnake. At the end of this work a paper of Calevete’s group on the snake venomics of the Crotalus durissus was “just accepted” for publication in the Journal of Proteome Research.65 The authors report partial venomic characterization of C. d. terrificus using reverse-phase HPLC, N-terminal sequences of isolated proteins, MS of proteins and tryptic peptides and

research articles

Figure 3. 1-D gel pattern of the Crotalus durissus terrificus venom. The left part of the figure shows molecular mass markers. The three bands in the region 17-19 kDa are labeled.

BLAST search. Comparison of our data with that published in65 is difficult due to the different approaches used, in accordance with the different purposes of the two groups. The authors mentioned above used HPLC which allowed the isolation and characterization of 18 compounds while we identified 93 toxins by 2-D electrophoresis and MS/MS analysis of the spots. We found a big group of 5′-nucleotidases as well as phosphodiesterases, nerve growth factors and glutaminyl cyclase in the venom of C. d. terrificus not identified by the other approach. There is a discrepancy also as regards the presence of LAAO, isolated by HPLC.65 We did not found LAAO in the white venom of the South American rattlesnake which was confirmed by enzymatic assays. Possible reasons for this could be the geographical origin of the snakes, diet etc. Of course, the venomic analysis performed by Calvete et al.65 is suitable for the comparative venomic and antivenomic characterization of South American rattlesnakes and allowed adequate conclusions about neurotoxicity as an adaptive trend along Crotalus dispersal in South America. 3.2. 1-D Gel Electrophoresis of the C. d. terrificus Venom. Trial to Identify Convulxin in the Venom. It is known that the venom of C. d. terrificus contains the toxin convulxin.64,66 However, the 2-D gel electrophoresis of the venom and the subsequent MS/MS analysis did not reveal the presence of this toxin. To prove the presence or absence of convulxin, the C. d. terrificus venom was subjected to 1-D gel electrophoresis (Figure 3). Three gel slices in the range 17-19 kDa, shown in Journal of Proteome Research • Vol. 9, No. 5, 2010 2311

research articles

Georgieva et al.

Table 2. Assignment of the Proteins Isolated from Bands in the Region 17-19 kDa of the 1-D Gel Electrophoresis (Shown in Figure 2) of the Crotalus durissus terrificus Venom bande

1

protein

3

2312


pI

MW

mascot matched peptide ion score peptides m/z z

Venom nerve growth factor; v-NGF; Precursor Crotoxin acid chain

gi|82220600

Crotalus durissus 8.90 27558 terrificus

171

4

gi|129456


130

4

Phospholipase A2 isoenzyme A; Crotoxin basic chain 1

gi|171848868 Crotalus durissus 8.74 14988 terrificus

104

7

Crotoxin basic chain 1; Phospholipase A2 F17 Crotoxin basic chain 1


64

4


64

4

Asrin

gi|111572527 Austrelaps 5.95 71183 superbus gi|224038264 Crotalus durissus 5.83 54916 collilineatus

49

2

48

6

Calobin I; Precursor Crotoxin basic chain 1; Phospholipase A2 F16

gi|13959630

Gloydius 6.38 29574 ussuriensis gi|239977495 Crotalus durissus 9.01 15120 terrificus

46

2

41

5

Acurhagin precursor Venom nerve growth factor; Short)vNGF; Precursor

gi|45331367

39

2

401

5

metalloprotease PII

2

accsession code

gi|82220600

Deinagkistrodon 5.03 70721 acutus Crotalus durissus 8.90 27558 terrificus

Venom nerve growth factor; Short)vNGF; Bj-NGF; Precursor Crotoxin acid chain

gi|82217029

Bothrops jararacussu

8.61 27601

291

5

gi|129456


101

4



88

7

Crotoxin basic chain 1; Phospholipase A2 F17 Crotoxin basic chain 1


74

4


69

4

metalloprotease PII

gi|224038264 Crotalus durissus 5.83 54916 collilineatus

59

3

Crotoxin basic chain 1; Phospholipase A2 F16


38

5

Venom nerve growth factor; v-NGF Precursor

gi|82220600

357

6



481.7 556.3 682.3 640.3 831.4 404.7 576.7 972.4 450.3 592.8 443.2 871.8 409.7 649.8 483.7 450.3 443.2 409.7 669.3 425.2 443.2 409.7 669.3 593.8 861.7 660.3 666.9 713.9 904.9 747.4 798.8 608.3 544.3 425.2 443.2 409.7 633.3 483.7 713.8 484.2 481.7 556.3 586.9 682.3 640.3 481.7 556.3 586.9 682.3 647.3 831.4 404.7 576.7 972.4 450.3 592.8 443.2 871.8 409.7 657.8 483.7 450.3 443.2 409.7 669.3 425.2 443.2 409.7 669.3 668.3 747.4 798.8 425.2 443.2 409.7 633.3 483.7 481.7 476.6 556.3 879.9 690.3 640.3

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 2 2 3 2 2 2 2 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 2 2 2 2


K.QYFFETK.C R.NPNPVPTGCR.G K.ALTMEGNQASWR.F R.IDSACVCVISR.K K.LTGCDPTTDVYTYR.Q K.AAAICFR.N R.NSMOXDTYDYK.Y K.YLQFSPENCQGESQPC.-.HLLQFNK.M K.WDIYPYSLK.S K.SGYITCGK.G K.GTWCEEQICECDR.V R.VAAECLR.R K.YGYMFYPDSR.C R.CRGPSETC.-.HLLQFNK.M K.SGYITCGK.G R.VAAECLR.R K.NEYMFYPDSR.C -.SLLQFNK.M K.SGYITCGK.G R.VAAECLR.R K.NEYMOXFYPDSR.C R.NDNAQLLTGIK.F R.AAKDDCDLPESCTGQSAECPTDR.F K.YEDTMQYELK.V K.VNGQPVVLHLEK.N K.DYSETHYSPDGR.K K.MOXCGVTQNWESNEPIK.K K.DLINVQPAAPNTLK.S K.NDDTCTGQSADCPR.N K.KVPNEDEQTR.V K.VPNEDEQTR.V -.SLLQFNK.M K.SGYITCGK.G R.VAAECLR.R K.NGYMOXFYPDSR.C R.CRGPSETC.K.DYSETHYSPDGR.E K.YENVEKEDEAPK.M K.QYFFETK.C R.NPNPVPTGCR.G R.HWNSYCTTTNTFVK.A K.ALTMEGNQASWR.F R.IDSACVCVISR.K K.QYFFETK.C R.NPNPVPTGCR.G R.HWNSYCTTTNTFVK.A K.ALTMEGNQASWR.F R.IDTACVCVISR.K K.LTGCDPTTDVYTYR.Q K.AAAICFR.N R.NSMOXDTYDYK.Y K.YLQFSPENCQGESQPC.-.HLLQFNK.M K.WDIYPYSLK.S K.SGYITCGK.G K.GTWCEEQICECDR.V R.VAAECLR.R K.YGYMOXFYPDSR.C R.CRGPSETC.-.HLLQFNK.M K.SGYITCGK.G R.VAAECLR.R K.NEYMOXFYPDSR.C -.SLLQFNK.M K.SGYITCGK.G R.VAAECLR.R K.NEYMOXFYPDSR.C K.YEDTMOXQYELK.V K.DLINVQPAAPNTLK.S K.NDDTCTGQSADCPR.N -.SLLQFNK.M K.SGYITCGK.G R.VAAECLR.R K.NGYMOXFYPDSR.C R.CRGPSETC.K.QYFFETK.C K.CRNPNPVPTGCR.G R.NPNPVPTGCR.G R.HWNSYCTTTNTFVK.A K.ALTMOXEGNQASWR.F R.IDSACVCVISR.K

research articles

Snake Venomic of Crotalus durissus terrificus Table 2. Continued

bande

protein

accsession code


pI

MW

mascot score

matched peptides

peptide ion m/z

z


481.7 476.6 556.3 879.9 690.3 647.3 831.4 404.7 576.7 972.4 450.3 592.8 443.2 871.3 409.7 487.8 657.8 483.7 450.3 443.2 409.7 487.8 669.3 425.2 443.2 409.7 487.8 625.3 483.7

2 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

K.QYFFETK.C K.CRNPNPVPTGCR.G R.NPNPVPTGCR.G R.HWNSYCTTTNTFVK.A K.ALTMOXEGNQASWR.F R.IDTACVCVISR.K K.LTGCDPTTDVYTYR.Q K.AAAICFR.N R.NSMOXDTYDYK.Y K.YLQFSPENCQGESQPC.-.HLLQFNK.M K.WDIYPYSLK.S K.SGYITCGK.G K.GTWCEEQICECDR.V R.VAAECLR.R R.VAAECLRR.S K.YGYMOXFYPDSR.C R.CRGPSETC.-.HLLQFNK.M K.SGYITCGK.G R.VAAECLR.R R.VAAECLRR.S K.NEYMOXFYPDSR.C -.SLLQFNK.M K.SGYITCGK.G R.VAAECLR.R R.VAAECLRR.S K.NGYMFYPDSR.C R.CRGPSETC.-

Venom nerve growth factor; v-NGF Bj-NGF; Precursor

gi|82217029

Bothrops jararacussu

8.61

27601

266

6

Crotoxin acid chain

gi|129456


4.21

16061

89

4


gi|171848868


8.74

14988

81

8

Crotoxin basic chain 1; Phospholipase A2 F17

gi|239977496


8.74

15157

58

5

Crotoxin basic chain 1;Phospholipase A2 F16

gi|239977495


9.01

15120

49

6

Figure 3, were cut, digested by trypsin and measured using a LTQ-Orbitrap-MS. The size range of the selected bands was chosen according to the theoretical molecular mass of both convulxin subunits (18.14 and 17.43 kDa). However, convulxin was not identified. The three bands contained nerve growth factor, crotoxin chain A, crotoxin basic chain 1, PLA2s (F16 and F17), asrin, metalloproteinase PII, serine proteinase calobin-1 and acurhagin precursor. The band compositions are shown in Table 2. It is known that the snake venom composition depends on several factors: diet, geographical factors, the age of the snake, etc. This can explain the absence or low quantity of convulxin in the investigated venom. 3.3. Proteomic of C. d. terrificus Venom versus Transcriptomic of C. d. collilineatus Venom Gland. The subspecies C. d. collilineatus is synonymous to C. d. terrificus25 and it is reasonable to compare data about the venom gland transcriptomic of the first subspecies66 with the venom proteomic of the second (this work). Comparison of both data sets showed similarities but also considerable differences in the distribution of proteins among the toxin families and in the nature of the toxins (Figure 2). The most abundant venom proteins according to the two approaches are neurotoxic PLA2s. Also, a low abundance of SVMPs and nerve growth factors was found by the venomic and venom gland transcriptomic analyses. The most drastic differences are about the presence and distribution of 5′-nucleotidases, vascular endothelial growth factors (VEGFs) and serine proteinases. 5′-nucleotidases represent 7.8% of the C. d. terrificus venom proteins (this work) while no such proteins have been found by the transcriptomic approach. We did not identify VEGFs which is in agreement with the data of Tokunaga et al.,67 who found that the C. d. terrificus venom lacks these proteins. On the contrary, transcriptomic analysis revealed five VEGF sequences.66 SVSPs are the second big protein family (this work) representing 25.3% of the venom proteins while according to the transcriptional profile of the venom gland they are only 2.5%.66 Venom

proteome includes also glutaminyl cyclase and phosphodiesterases not found by transcriptomic analysis. The transcripts encoding putative toxins not found by the proteomic approach include cardiotoxin, crotamin, C-type natriuretic peptide, convulxin and angiotensin-converting enzyme inhibitor.66 Peptides with a molecular mass below 10 kDa, like the first three toxins mentioned above, were not separated by the 2-DE. Possible reasons for the discrepancy of the data from the two approaches could be lack of transcripts encoding some of the venom proteins in the cDNA library, regulation on the translational level, preparation of the samples for analysis68 and the diet of the snakes or ontogenetic variations. Also, the transcriptomic analysis concerns the whole venom gland while the proteome represents only the venom proteins. Quantitative and qualitative differences in the data obtained by proteomic and transcriptomic analyses of snake venom toxins in one and the same subspecies have been reported also by other authors (ref 16 and references therein). 3.4. Enzymatic Activities of the C. d. terrificus Venom. Snake venom enzymes can induce pharmacological effects and contribute significantly to the total toxicity. We determined phospholipase A2, L-amino acid oxidase, protease, alkaline phosphatase and acidic phosphatase activities of the C. d. terrificus venom. The results are shown in Table 3. Neither LAAO nor acidic phosphatase activities were found. The PLA2 activity was similar to that of the Vipera a. ammodytes and Vipera a. meridionalis venoms69 and lower than the Daboia russelli siamensis phospholipolytic activity (Table 3). Phospholipases A2 hydrolyze natural biologically important phospholipids (phosphatidylcholine, choline plasmilogen, platelet aggregation factor etc.) including those in biological membranes which leads to their damage and changes in the permeability to ions and drugs (ref 70 and references therein). Products of PLA2 catalyzed reactions are lysophospholipids inducing tissue damage and arachidonic acidsa precursor of mediators of inflammation such as prostaglandins, leukotriens and thromJournal of Proteome Research • Vol. 9, No. 5, 2010 2313

research articles

Georgieva et al.

Table 3. Enzyme Activities of the Crotalus durissus terrificus Venom

Species

Crotalus durissus 5.90 terrificus Vipera ammodytes 4.30 ammodytesa Vipera ammodytes 6.75 meridionalisa 13.42 Daboia russelli siamensisa a

0.13

0

460

0

0.33

50

400

0

0.27

100

400

0

0.06

40

240

0

(5)

(6)

Data from ref 69.

boxanes.71 The venom of C. d. terrificus has a weak proteolytic/ caseinolytic activity which seems to be in contrast with the high content of serine proteinases. The low proteolytic activity toward casein can be explained with the high macromolecular substrate specificity of the serine proteinases hydrolyzing a few peptide bonds in their natural substrates. Furthermore, calobin does not show caseinolytic activity.46 The alkaline phosphatase activity of the Crotalus d. terrificus venom is higher than those of the other three venoms (Table 3). This type of activity is connected with dephosphorylation of natural substrates and contributes to the total toxicity. 3.5. Concluding Remarks. The venomic approach used in the present paper revealed that the venom of C. d. terrificus is a rich source of pharmaceutically important compounds with a potential to be used clinically as anticoagulants, for the treatment of thrombotic diseases, as tools in clinical assays, or as analgesics. It is surprising that the venom of the South American rattlesnake was intensively investigated during the last 70 years but only a few toxins, crotoxin, crotamine gyroxin, convulxin, and crotalphine, have been characterized. The venom proteome analysis revealed 93 different proteins with possible pharmacological activities. These toxins have molecular masses in the region 10-110 kDa. The number of biologically important compounds is higher, having in mind the presence of low molecular mass peptides. The data from the venomic analysis can be used for further studies on the structure, function, and pharmacological activity of individual toxins. 5′-Nucleotidases are of medical significance because these enzymes have a potential in the treatment of myocardial, renal, and intestinal ischemia or acute lung injury.60 The proteomic composition of the C. d. terrificus venom correlates with the effects of human envenoming by this snake.

Acknowledgment. This research was funded by grants from the Deutsche Forschungsgemeinschaft (project BE 1443-18-1 to C.B.), Fundaçaõ de Amparo a` Pesquisa do Estado de Saõ Paulo - FAPESP/Brazil (2007/54865-1 to R.K.A.), and the Bulgarian National Foundation for Scientific Research (Grant TK-B-1610/06 to N.G.). References (1) Calvete, J. J. Venomics: Digging into the evolution of venomous systems and learning to twist nature to fight pathology. J. Proteomics 2009, 72, 121–6. (2) Fry, B. G.; Wu ¨ ster, W. Assembling an arsenal: origin and evolution of the snake venom proteome inferred from phylogenetic analysis of toxin sequences. Mol. Biol. Evol. 2004, 21, 870–83. (3) Fry, B. G. From genome to “venome”: Molecular origin and evolution of the snake venom proteome inferred from phylogenetic

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Alkaline Acidic PLA2 Proteinase LAAO phosphatase phosphatase U/mg U/mg U/g U/g U/g


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