Comparative Analysis of the Venom Proteomes of Vipera ammodytes

Comparative Analysis of the Venom Proteomes of Vipera ammodytes ammodytes and Vipera ammodytes meridionalis Dessislava Georgieva,†,‡,# Michaela Risch,#,§ Anna Kardas,† Friedrich Buck,| Martin von Bergen,§ and Christian Betzel*,† Institute of Biochemistry and Molecular Biology, University of Hamburg, Martin-Luther-King Platz 6, 20146 Hamburg, Germany; Institute of Organic Chemistry, Bulgarian Academy of Sciences, “Akad. G. Bonchev”-strasse Bl.9, 1000 Sofia, Bulgaria; Helmholtz-Centre for Environmental Research-UFZ, Department of Proteomics, Permosestrasse 15, 04318 Leipzig, Germany; and Institute of Clinical Chemistry, University Medical Center HamburgsEppendorf, Martinistrasse 52, 20246 Hamburg, Germany Received June 15, 2007

The venom proteomics of Vipera ammodytes ammodytes and Vipera ammodytes meridionalis, snakes of public health significance and the most poisonous reptiles in Europe, were analyzed by FPLC, 2-D electrophoresis, sequence analysis, and MS/MS. FPLC analysis showed the presence of L-amino acid oxidase, monomeric and heterodimeric phospholipases A2, C-type lectin protein, and proteinases in the venom of V. a. ammodytes. Representatives of the same protein families were found in the venom of the other subspecies, V. a. meridionalis. N-terminally identical PLA2 neurotoxins were identified in both venoms. Difference in the PLA2 compositions of the venoms was also observed: a monomeric protein with phospholipase A2 activity, identical in the first 20 amino acid residues to the catalitically inactive acidic component of the heterodimeric PLA2 present in both venoms, was found only in that of V. a. meridionalis. Probably, this protein represents an intermediate form of the two components of the heterodimer. 2-D electrophoresis and MS/MS analysis showed that the two venoms shared a number of protein families: monomeric and heterodimeric Group II PLA2s, serine proteinases, Group I, II, and III metalloproteinases, L-amino acid oxidases (LAAOs), cysteine-rich secretory proteins, disintegrins, and growth factors. Totally, 38 venom components of the V. a. ammodytes, belonging to 9 protein families, and 67 components of the V. a. meridionalis venom belonging to 8 protein families were identified. The venom proteome of V. a. ammodytes shows larger diversity of proteins (139) in comparison to that of V. a. meridionalis (104 proteins). Most of the proteins are homologues of known representatives of the respective protein families. The protein compositions explain clinical effects of the V. ammodytes snakebites, such as difficulties in the breathing, paralysis, apoptosis, cloting disorders, hemorrhage, and tissue necrosis. The lists of secreted proteins by the two vipers can be used for further study of structure–function relationships in the toxins and for prediction and treatment of snakebite consequences. Keywords: Two-dimensional gel electrophoresis • mass spectrometry • N-terminal sequencing • snake venom • snake venom protein families

Introduction Two venomous Viperid snakes are widespread in East Europe: Vipera ammodytes ammodytes, inhabiting the west part of the region (Slovenia, Croatia, Serbia, West Bulgaria) and Vipera ammodytes meridionalis, a snake of public health significance in Greece, East Bulgaria, Roumania, Albania, and * Corresponding author: Prof. Dr. Ch. Betzel; University of Hamburg; Institute of Biochemistry and Molecular Biology; Martin-Luther-King-Platz 6; 20146 Hamburg, Germany. Tel.: +494089984744. Fax: +494089984747; E-mail: [email protected]. † University of Hamburg. ‡ Bulgarian Academy of Sciences. # These authors have contributed equally to this work. § Helmholtz-Centre for Environmental Research-UFZ. | University Medical Center HamburgsEppendorf.

866 Journal of Proteome Research 2008, 7, 866–886 Published on Web 02/08/2008

Turkey. Both snakes are the most frequent cause of snakebites and are responsible, in some cases, for deaths in the region. The venoms of these snakes cause a number of pathophysiological changes, such as local tissue damage, pain, paralysis, bleeding, fever, tachycardia, apoptosis, and other systemic symptoms. The V. ammodytes bite is a serious event that requires immediate hospital care.1 Venom proteomes of Viperid snakes inhabiting Africa and North America were recently analyzed and proteins belonging to different toxin families identified. Thus, the protein compositions of the venoms of Tunisian snakes Cerastes cerastes, Cerastes vipera and Macrovipera lebetina were determined using RP-HPLC, N-terminal sequence analysis, MALDI-TOF mass determination, and CID-MS/MS.2 The venom toxins of these snakes belong to a few protein families, and each venom 10.1021/pr070376c CCC: $40.75

 2008 American Chemical Society

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Vipera ammodytes Proteomics 2

has a distinct degree of protein composition complexity. The venom proteome of the East African Gaboon viper (Bitis gabonica gabonica) was analyzed by the same methods, and 35 proteins belonging to 12 toxin families have been identified.3 The same combination of analytical methods has been used for the determination of the venom protein compositions of three subspecies of Sisturus catenatus (S. c. catenatus, tergeminus, and edwardsii), and that of Sisturus miliarius barbouri.4 The venoms of the four Viperid snakes contain proteins from 11 families.4 The analysis showed that the protein family profile is not conserved in the different venoms. To date, little study has been done on the venom protein compositions of the European Viperid snakes. The major toxic component of the V. a. meridionalis venom, responsible for the neurotoxicity, is vipoxin, a heterodimeric complex between a basic, strongly toxic phospholipase A2 (PLA2) and an acidic, nontoxic, and catalytically inactive PLA2-like protein.5 In previous papers, we have described the high resolution X-ray structure of vipoxin6 and its separated components.7,8 No monomeric PLA2’s have been found in the venom of this snake so far. The existing information about the neurotoxicity of V. a. ammodytes is that the venom of this snake contains three basic monomeric PLA2’s (Ammodytoxins A, B, and C, Athxs) exhibiting presynaptic toxicity9 and a neutral PLA2 called ammodytin I2, which is not neurotoxic.10 It is the most enzymatically active phospholipase A2 in the venom of V. a. ammodytes.10 Membrane receptors for ammodytoxins were identified11 (and references therein). Recently, ammodytase, a 70 kDa venom metalloprotease, was isolated and characterized.12 There are 10 species of venomous snakes in Europe, but their venom proteomes are still unknown. Only a few toxins have been isolated and characterized. Here, we report on the venom proteomes of two Vipera ammodytes taxa: V. a. ammodytes and V. a. meridionalis. Novel proteins were identified for further structural and functional investigations. We describe the major protein families in the venoms of both reptiles in relation to the observed pathophysiological effects. The two taxa are similar in their venom proteomes. In addition, 2-D gel electrophoresis revealed some differences in the venom components. Detailed proteomic studies of snake venoms are important for basic research, development of new drugs, and for the clinical diagnosis and treatment of patients suffering of the snakebite consequences. The results can be taken into account for the preparation of neutralizing antivenom. At present, the only effective treatment of envenomation caused by V. ammodytes is intravenous administration of antisera prepared using the whole venom. However, this antivenom is not specific toward distinct proteins and contains a mixture of antibodies which can create undesired side effects. Knowledge of the venom components will allow the preparation of specific antivenom, using the major toxins as antigens. This will increase considerably its effectiveness and will decrease side effects.

Experimental Section Collection and Fractionation of Venoms. Venoms were collected from snakes originating from different parts of Bulgaria. Eight specimens of V. a. meridionalis were captured in the province Thrace, near the border with Greece, and the same number of specimens of V. a. ammodytes was captured in a region of northwest Bulgaria. Venom pooled from representatives of each subspecies was analyzed. 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 crude venoms were fractionated by FPLC. A total of 120 mg of the V. a. meridionalis venom was dissolved in 0.01 M sodium acetate buffer, pH 6.2, filtered, and applied on a cation exchange column Mono S 10/ 10. The chromatography was performed at pH 6.2 and a flow rate of 1.5 mL/min. The same procedure was repeated with equal amount of the venom from V. a. ammodytes, but the best separation of the protein components was obtained at pH 5.0. Fractions of 4 mL were collected and checked for PLA2 activity. Those containing phospholipase A2s were dialysed overnight against 0.01 M solution of Tris, pH 9.0, and rechromatographed at the same pH on an anion exchange column Mono Q 5/5. The isolated proteins were homogeneous by SDS-PAGE. The chemicals used for the fractionation of the venoms were delivered from Merck (Darmstadt, Germany). 2-D Gel Electrophoresis and Electrospray Mass Spectrometry. For two-dimensional electrophoresis, 500 µg of total protein was 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, a 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 was 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 mm). The Protean II xi Cell-System (Bio-Rad, Hercules, CA) was used to run 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.13 Gels were scanned and imported into the software Delta2D software package (Decodon, Greifswald, Germany). Protein bands of interest were cut from polyacrylamide gels and digested overnight using trypsin (Sigma, Munich, Germany). The cleaved peptides were eluted, concentrated by vacuum centrifugation, and thereafter separated by RP nanoLC (LC1100 series, Agilent Technologies, Paolo Alto, CA; 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). Journal of Proteome Research • Vol. 7, No. 3, 2008 867

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Figure 1. Fractionation of the V. a. ammodytes venom by FPLC on a Mono S 10/10 column at pH 5.0 and flow rate of 1.5 mL/min. The protein fraction which was not retained by the anion-exchanger contained L-amino acid oxidase. Peak I contained monomeric PLA2’s; peak II, ammodytin I2; peak III, anticoagulant factor X-binding protein; peak IV, thrombin-like serine protease; and peak V, the heterodimeric postsynaptic neurotoxin vipoxin.

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. Determination of Enzymatic Activities, Amino Acid Sequence Analysis, and SDS-PAGE. PLA2 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 analogue of diheptanoyl phosphatidylcholine was used as a substrate. The release of free thiols upon the PLA2catalyzed hydrolysis of the thioester bond at the sn-2 position was detected spectrophotometrically using 5,5′-dithiobis (2nitrobenzoic acid). L-Amino acid oxidase activity was determined by the method of Wellner et al.14 Proteolytic activity was determined as described elsewhere.15 The isolated proteins were N-terminaly sequenced using an automatic amino acid sequencer PROCISE from Applied Biosystems. SDS-PAGE was prepared according to Laemmli16 on a 12% (w/v) polyacrilamide gel under reducing and nonreducing conditions. A kit of molecular weight markers (Merck, Darmstadt, Germany) was run in the same gel for estimation the molecular masses of the separated proteins.

Results and Discussion Fractionation of the V. a. ammodytes venom by FPL Chromatography. Crude venom of V. a. ammodytes (120 mg) was fractionated by FPLC on a column Mono S 10/10, at pH 5.0 (Figure 1). The eluate in each tube was checked for phospholipase A2, LAAO, and proteolytic activities which are responsible for toxic effects of Viperid snake venoms: neurotoxicity/paralysis, edema, hematological disorders as hemolysis, blood coagulation disorders, hemorrhage, and apoptosis as well as other systemic symptoms. Part of the proteins were not 868

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retained by the cation-exchanger and were fast-eluted. They were collected as a separate fraction which possesses an L-amino acid oxidase activity. LAAOs are group of snake venom toxins involved in envenomation. There are data that these enzymes are responsible for tissue death, apoptosis, hemolysis, and hemorrhage.17 We purified the V. a. ammodytes enzyme to homogeneity. SDS-PAGE under reducing and nonreducing conditions showed the presence of a 55 kDa monomer. Peaks I–V were collected and dialyzed against 0.01 M Tris, pH 9.0, and each fraction was chromatographed on a column Mono Q. Most of the fractions showed phospholipase A2 and proteolytic activities. Peak I contained two monomeric PLA2’s of 14 kDa. The fractionation of peak I by FPLC on a Mono Q column as well as SDS-gel electrophoresis of both enzymes are shown in Figure 2. Probably, they are homologues of ammodytoxins.18 Several proteins were purified to homogeneity from the next fractions. Their purity and molecular masses were assessed by SDS-PAGE. N-terminal sequence analysis and searches in the protein database were used for the assignment of the isolated proteins to different snake venom protein families. A monomeric PLA2 with a high phospholipase activity was derived from peak II. The rechromatography of this enzyme on a Mono Q column was very similar to that shown in Figure 4 for the N-terminally identical protein from the venom of V. a. meridionalis. Molecular mass of approximately 14 kDa was determined by SDS-PAGE (Figure 2, inset). The sequence of 25 amino acid residues from the amino-terminus (Table 1) showed homology to ammodytin I2, a neutral PLA2 isolated from the venom of V. a. ammodytes originating from Slovenia.19 The most interesting observation was the presence in fraction V of a big quantity of vipoxin, a heterodimeric neurotoxin found only in the venom of the other subspecies, V. a. meridionalis. The toxin was purified to homogeneity by FPL rechromatography on a Mono Q column, and SDS-gel electrophoresis of the dissociated subunits is shown in Figure 2, inset. The molecular mass of the subunits was approximately 14 kDa. It was known that the V. a. ammodytes venom contains only

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Figure 2. Fractionation of two phospholipases A2 from peak I of Figure 1 by FPLC on a Mono Q 5/5 column at pH 9.0. (Inset) SDS-gel electrophoresis of V. a. ammodytes toxins after FPL chromatography and rechromatography on a Mono Q column: (1) phospholipase A2 from peak I-1; (2) phospholipase A2 from peak I-2; (3) homologue of ammodytin I2 isolated by FPL rechromatography of peak II from Figure 1 on a column Mono Q at pH 9.0; (4) dissociated vipoxin isolated after FPL rechromatography of peak V on a column Mono Q at pH 9.0; (5) homologue of the acidic subunit of vipoxin isolated by FPL chromatography and rechromatography of the V. a. meridionalis venom.

Figure 3. Fractionation of the V. .a meridionalis venom by FPLC on a Mono S column at pH 6.2 and flow rate of 1.5 mL/min. The protein fraction which was not retained on the column and fast-eluted contained L-amino acid oxidase. Peak I contained ammodytoxin I2 and peak II contained phospholipases A2s.

monomeric PLA2’s, ammodytoxins and ammodytins.10,11 The sequences of the first 20 amino acids of the heterodimeric PLA2, isolated from fraction V, were identical to those of the subunits A and B of vipoxin (Table 1). A heterodimeric protein with a molecular mass of 30.2 kDa was also isolated from fraction III. The N-terminal sequences of both subunits are shown in Table 1. Search in the PDB showed that it belongs to the family of snake venom anticoagulant factor X binding proteins (X-bp), interacting with the respective factor of the blood coagulation cascade. The mo-

lecular mass is also typical for this group of proteins. X-bps, together with factor IX binding proteins, belong to the C-type lectin superfamily.20 The N-terminal sequence of the A chain shows 55% identity to that of the factor X binding protein from Deinagkistrodon acutus, and is also homologous to the respective subunit of other factor IX/factor X binding snake venom proteins.20,21 A 27 kDa serine proteinase was isolated from fraction IV. The N-terminal sequence analysis showed that the enzyme belongs to the family of snake venom fibrin(ogen)olytic (thromJournal of Proteome Research • Vol. 7, No. 3, 2008 869

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Figure 4. The final purification step of ammodytin I2 from the venom of V. .a. meridionalis by FPLC on a Mono Q column. The major sharp peak contained the monomeric PLA2. Table 1. N-Terminal Sequences of Proteins Isolated from the Venoms of Vipera ammodytes ammodytes and Vipera ammodytes meridionalisa

a AmI2, ammodytin I2; Vipo B, B-chain of vipoxin; Vipo A, A-chain of vipoxin; VaX-bp A, chain A of the Factor X-binding protein isolated from the venom of V. a. ammodytes; DaX-bp A, chain A of the Factor X-binding protein from the venom of Deinagkistrodon acutus;20 VaX-bp B, chain B of the Factor X-binding protein from the venom of Vipera ammodytes ammodytes; DaX-bp B, chain B of the Factor X-binding protein from the venom of Deinagkistrodon acutus;20 Vaaserprot, serine proteinase isolated from the venom of V. a. ammodytes; Bothrombin, serine proteinase from Bothrops jararaca.22

bin-like) proteases. The N-terminal sequence is highly homologous to that of the serine proteinase bothrombin isolated from the venom of Bothrops jararaca.22 Fractionation of the V. a. meridionalis Venom by FPL Chromatography. Crude venom of V. a. meridionalis (120 mg) was fractionated on a column Mono S 10/10 by FPL chromatography (Figure 3). Part of the proteins were fast-eluted as in the case of V. a. ammodytes. This fraction contained L-amino acid oxidase. Venom PLA2s are the only well-characterized toxins in both subspecies so far. To compare in more detail the neurotoxins from this protein family, we have analyzed fractions showing phospholipase A2 activity. The other protein families were investigated by 2-DE and MS/MS. Determination 870


of the enzyme activity showed the presence of PLA2s in fractions I and II. The enzymes were further separated and purified on a column Mono Q. The chromatography of fraction I yielded one major peak and a small amount of contaminating products as it is shown in Figure 4. SDS-PAGE of the protein collected from the major fraction showed a single band at about 14 kDa. The N-terminal 25 amino acids of the protein from fraction I were identical to those of ammodytoxin I2, a component of the V. a. ammodytes venom. It was known so far that the venom of V. a. meridionalis contains only one heterodimeric neurotoxin, vipoxin23,24 (and citations therein). No venom monomeric PLA2s have been isolated from the subspecies meridionalis. The chromatographic profile of peak


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Figure 5. Fractionation of peak II from the venom of V. a. meridionalis, shown in Figure 3, by FPLC on a Mono Q column. The first fraction contained the heterodimeric neurotoxin vipoxin and the second contained catalytically active isoform of the acidic and enzymatically inactive PLA2-like protein of vipoxin, which is a natural inhibitor of the basic and toxic PLA2 subunit.

II is shown in Figure 5. Phospholipase A2 activity was found in both peaks. After rechromatography on a column Mono Q, the SDS-PAGE analysis showed a single band for each fraction at about 14 kDa. The N-terminal sequence analysis showed the presence of the heterodimeric phospholipase A2 vipoxin in the first fraction (Figure 5 and Table 1). A monomeric catalytically active PLA2 with a molecular mass of 14 kDa (Figure 2, band 5) was found in the second fraction. Surprisingly, the sequence of the N-terminal 20 residues of this protein was identical to that of the vipoxin acidic component (Vipo A, Table 1), which is devoid of phospholipase A2 activity. Most probably, the novel protein, which is highly homologous to the vipoxin acidic component and possesses PLA2 activity, represents an intermediate form of the two components of the dimer. This result supports the hypothesis about the evolution of the toxic enzyme into nontoxic inhibitor. Vipoxin is a stable complex between a basic strongly toxic PLA2 and an acidic nontoxic and catalytically inactive PLA2-like component.5 The complex can be dissociated in the presence of 6 M urea and at acidic pH’s. Free components of vipoxin have not been found so far in the V. a. meridionalis venom. The two components of the heterodimer are closely related proteins with 62% sequence identity.25 The acidic protein reduces the phospholipase A2 activity of the enzyme by up to 60%, and its toxicity,23 that is, the nontoxic component, is a natural inhibitor of the vipoxin basic PLA2. The heterodimeric complex is the first reported example of a high degree of structural homology between an enzyme and its natural inhibitor which plays a role in the stabilization of the toxin structure, preservation of the pharmacological activity (toxicity), and acts as chaperone during catalysis.24,26 It was supposed that the natural inhibitor is a product of divergent evolution of the unstable PLA2.26 2-D Gel Electrophoresis of the V. a. ammodytes Venom and Protein Family Compositions. The venom complexity of V. a. ammodytes was examined by 2-DE (Figure 6). The

Figure 6. 2-D gel pattern of the V. a. ammodytes venom. Fractionation was performed under the conditions described in the Experimental Section.

separated protein bands were subjected to tryptic digestion, and the venom components were identified by MS/MS and using MASCOT search program (Table 2). 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 major part of the isolated proteins were assigned to the following Journal of Proteome Research • Vol. 7, No. 3, 2008 871

872

gi|33355627


gi|15887054

acid oxidase

acid oxidase

L-amino

L-amino

okinaxobin-1 cerastocytin

triflin

triflin

okinaxobin-1 okinaxobin-1 venom serine proteinase 2C precursor mucofirase-1

30

49

51

55

56

60 61 67*

68

gi|602596

gi|129079 gi|129079 gi|82239725

gi|21305553

gi|21305553

gi|129079 gi|994878

gi|15887054

gi|763095

22b

gi:20530118

hemorrhagic metalloproteinase HR1a metalloproteinase

22a*

gi|83523626

acid oxidase

L-amino

06

gi|39841344

Group III SVMP

acid oxidase

L-amino

05

gi|15887054

gi|61104775

acid oxidase

L-amino

04

accession code

VLAIP-A

protein

spot no.

Protobothrops mucro-squamatus

Ovophis okinavensis Ovophis okinavensis Trimeresurus stejnegeri

Trimeresurus flavoviridis


Ovophis okinavensis Cerastes cerastes

Gloydius blomhoffi

Gloydius blomhoffi

Echis pyramidium


Echis ocellatus

Macrovipera lebetina

Viridovipera stejnegeri

Bothrops jararacussu

Gloydius blomhoffi

homology with a protein from:

63

75 71

158

164

70 44

183

137

48

93

107

200

151

156

Mascot score

1

1 1 1

2

3

1 1

6

3

2

1

2

3

5

4

5

matched peptides z

2

2 2 2 2 2 2 2

589.4 777.0 959.8 589.3 757.2 756.6 460.3 1123.7

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 2 2

959.8

475.7 955.5 567.5 569.2 583.5 567.5 665.2 569.2 583.0 663.0 526.7 757.1 711.2

567.5 569.2 583.2 462.9 894.9 676.7 569.5 583.4 462.7 834.7 567.7 665.2 569.4 894.8 447.1 578.5 718.3 692.7 718.3 745.9

peptide–ion m/z

R.KSAHIAPLSLPSSPPSVGSVCR.V

R.SVNPTASNMOXLK.M K.MOXEWYPEAAANAER.W R.KPEIQNEIIDLHNSLR.R R.SVNPTASNMOXLK.M -.VIGGDECNINEHR.F -.VIGGDECNINEHR.F K.SIIAGNTAAT.C

R.KPEIQNEIIDLHNSLR.R

K.LVIVVDHR.M K.CIDVNTAY.K.YPVKPSEEGK.S R.HDDIFAYEK.R R.IKFEPPLPPK.K K.YPVKPSEEGK.S K.EYLLK.E R.HDDIFAYEK.R R.IKFEPPLPPK.K K.FEPPLPPK.K K.FEPPLPPKK.A -.VIGGDECNINEHR.F -.VIGGAECNINEHR.S

K.YPVKPSEEGK.S R.HDDIFAYEK.R R.IKFEPPLPPK.K K.FEPPLPPK.K R.EEIQTFCYPSMOXIQK.W K.SAGQLYEESLQK.A K.HDDIFAYEK.R R.IKFEPPLPPK.K K.FEPPLPPK.K R.NDEEGWYANLGPMOXR.L K.YPVKPSEEGK.S K.EYLIK.E K.HDDIFAYEK.R R.EEIQTFCYPSMOXIQK.W K.VTLDLFGK.W R.KIPCAPQDVK.C R.LYCFDNLPEHK.N K.ASQLNLTPEQQR.Y R.LYCFDNLPEHK.N N.AQLLTAIDFNGTII.G

MS/MS derived sequence

Table 2. Assignment of the Spots of the 2D-Gel Electrophoresis of V. a. ammodytes to Protein Families by MS/MS and MASCOTa

acid oxidase

acid oxidase

acid oxidase

acid oxidase

thrombin-like proteinase

serine proteinase serine proteinase serine proteinase

CRISPs

serine proteinase thrombin-like platelet activator cysteine- rich secretory proteins (CRISPs)

L-amino

L-amino

metalloproteinase

metalloproteinase

metalloproteinase

L-amino

L-amino

acid oxidase

protein family L-amino

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disintegrin CC5

123

gi|18202444

gi|58177209

gi|58177209

vammin, chain A

vammin, chain A

gi|227635 gi|54036444

phospholipase A2 II vammin

122

120

gi|129481

gi|2914537

phospholipase A2 ammodytin L precursor

119

gi|6967298 gi|283845 gi|17375443 gi|283845 gi|227635 gi|33187126

vipoxin, chain B

phospholipase A2 phospholipase A2 Disintegrin EC6A phospholipase A2, acidic chain phospholipase A2 II ammodytin I2

112 117 118

gi|263157

gi|33187126 gi|50874562

nerve growth factor

111

gi|83523644 gi|1502368 gi|1408314

ammodytin I2 ammodytin L1

Group III SVMP lebetase Le3 phospholipase A2 acidic subunit

105 106 108

gi|547224 gi|299493

R-fibrinogenase A2 bilineobin

gi|461511 gi|83523632

gi|461511 gi|17933274

ancrod ussurin

ancrod Group III SVMP

gi|3552036

accession code

pallase

protein

104

96

spot no.

Table 2. (Continued)

Cerastes cerastes

Vipera aspis aspis

Vipera aspis aspis

Vipera ammodytes meridionalis Vipera aspis Vipera ammodytes ammodytes

Vipera ammodytes ammodytes Vipera ammodytes ammodytes

Vipera ammodytes ammodytes

Vipera ammodytes ammodytes Vipera aspis Echis carinatus sochureki Vipera aspis Vipera aspis Vipera ammodytes ammodytes

Daboia russelli

Echis ocellatus Macrovipera lebetina Vipera aspis

Calloselasma rhodostoma Echis ocellatus

Crotalus atrox Agkistrodon bilineatus

Agkistrodon bilineatus Gloydius ussuriensis

Gloydius halys


91

134

82

34 82

39

84 76

97

56 54 84 64 55 50

74

43 96 328

80 66

70 65

75 74

82

Mascot score

1

4

3

1 3

1

1 2

3

1 1 1 1 1 2

2

1 1 4

1 2

1 1

1 2

2

matched peptides

2 2 3 2 2 2 2 2 2 2

565.3 782.4 1053.5 565.3 782.4 465.3 658.7 565.2 522.0 989.6

2 2 2 2 2 2

740.9 804.2 857.7 746.1 804.2 584.7

2 3

2 2 2 1 2 2 2 2 2 2 2 2 2

748.6 819.7 1099.4 693.0 647.3 921.8 735.4 1080.6 735.3 695.9 857.7 921.7 746.1

696.2 1053.5

2 2 2 2 2 2

2 2 2 2 2 2 2

z

750.1 541.8 533.2 533.1 1050.6 735.0

756.8 444.5 750.2 749.1 444.5 749.1 756.8

peptide–ion m/z

K.HTVDLQIMOXR.V K.FTEHTACECRPR.R R.ETLVPILQEYPDEISDIFRPSCVAVLR.C K.HTVDIQIMOXR.V K.FTEHTACECRPR.R -.IVRPFLEVHER.S R.CSGCCTDESLK.C K.HTVDIQIMOXR.V K.FTEHTACECRPR.R R.GDDMOXNDYCTGISSDCPR.N

-.NLYQFGNMOXIFK.M R.ETLVPILQEYPDEISDIFRPSCVAVLR.C

R.ENGAIVCGSSTPCK.K R.ENGAIVCGSSTPCKK.Q R.VAAICFGENLNTYDK.K R.CCFVHSCCYAK.L R.ENGAIVCGSSTPCQK.Q K.LAIYYYSFK.K

R.CCFAQDCCYGR.V R.AAAICLGENVNTYDK.N K.NYEYYSISHCTEESEQC.K.CKNPNPVPSGCR.G R.INTACVCVISR.K R.VAAICFGENLNTYDKK.Y -.NLFQFGDMOXILQK.Y K.AMOXLDGLNDYCTGISSDCPR.N -.NLFQFGDMOXILQK.Y -.NLYQFGNMOXIFK.M R.VAAICFGENLNTYDK.K R.VAAICFGENLNTYDKK.Y R.CCFVHSCCYAK.L

-.VVGGDECNINEHR.S R.GESYFYCR.K R.HCVDVTTAY.R.HCVDVTTAY.K.SGNVNDYEVVNPQAVTGLPK.G -.NLFQFGDMOXILQK.T

M.VIGGDECNINEHR.F K.FFCLSSK.K -.VVGGDECNINEHR.S M.VIGGVECNINEHR.F K.FFCLSSK.T -.VPGGDECNINEHR.S -.VIGGDECNINEHR.F


disintegrin

VEGF

vascular endothelial growth factor (VEGF)

phospholipase A2

phospholipase A2 phospholipase A2 metalloproteinase phospholipase A2

nerve growth factor

metalloproteinase metalloproteinase phospholipase A2

R-fibrinogenase thrombin-like proteinase serine proteinase metalloproteinase

serine proteinase

protein family


research articles

Journal of Proteome Research • Vol. 7, No. 3, 2008 873

874


gi|33187132

gi|33187126

ammodytin I1 isoform 2

ammodytin I2

135

gi:50874500

Echis carinatus sochureki Cerastes vipera

gi|17375443 gi|77917362

ammodytin I2

Vipera aspis Macrovipera lebetina transmediterranea

gi|227635 gi|95007583

phospholipase A2 II MLD-containing dimeric disintegrins subunit ml-G2 precursor disintegrin EC6A disintegrin CV11 alpha precursor

134*

133

gi|50874302

ammodytin I1 (A)


Vipera ammodytes meridionalis Vipera ammodytes ammodytes

Vipera aspis aspis

Vipera aspis aspis

gi|50874288


Vipera aspis Vipera aspis aspis

ammodytin I1 (E)

gi|227635 gi|50874302

phospholipase A2 II ammodytin I1(A)


gi|33187126

gi|33187132

ammodytin I1

Ovophis okinavensis Cerastes cerastes Cerastes cerastes Vipera ammodytes ammodytes



ammodytin I2

gi|129079 gi|18202444 gi|18202444 gi|33187126

okinaxobin-1 disintegrin CC5 disintegrin CC5 ammodytin I2

125 129 131

132

gi|6967298

accession code

phospholipase A2

protein

124

spot no.


97

116

122 116

56 153

78

117

388

65 38

70

62 30 61 443

179

Mascot score

2

3

1

1 2

1 3

3

4

5

1 2

2

1 1 1 6

4

matched peptides

2 2 2 2 2

2 2 2 2 2 2

895.8 525.4 1081.4 1081.4 989.4 645.3 767.1 875.3 1060.0 767.1 921.8

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 2

z

767.2 939.1 696.3 768.8 939.1 767.2 858.2 922.1 952.3 806.8 767.2 880.4 874.6 939.2 768.4 874.6 939.2 696.1 968.6

858.2 921.8 952.2 807.4 757.3 989.9 989.5 767.2 1319.3 858.2 921.9 952.1 806.7

peptide–ion m/z

R.CCFVHDCCYGR.V R.VAAICFGENMOXNTYDK.K K.YMOXLYSLFDCKEESEQC.R.CCFVHDCCYGR.V R.VAAICFGENLNTYDKK.Y

M.NSANPCCDPITCKPR.K K.FLNPGTICK.R K.AMOXLDGLNDYCTGISSDCPR.N K.AKGDDMNDYCTGISSDCPR.N K.GDDMNDYCTGISSDCPR.N A.ICFGENLNTYD.K

R.VAAICFGENLNTYDK.K R.VAAICFGENLNTYDKK.Y K.YKNYPSSHCTETEQC.K.NYPSSHCTETEQC.-.VIGGDECNINEHR.F R.GDDMOXNDYCTGISSDCPR.N R.GDDMOXNDYCTGISSDCPR.N R.CCFVHDCCYGR.V K.LSIYSYSFENGDIVCGGDD PCLR.A R.VAAICFGENLNTYDK.K R.VAAICFGENLNTYDKK.Y K.YKNYPSSHCTETEQC.K.NYPSSHCTETEQC.R.CCFVHDCCYGR.V R.VAAICFGENMOXNTYDKK.Y -.NLYQFGNMOXIFK.M R.CYFVHDCCYGR.V R.VAAICFGENMOXNTYDKK.Y R.CCFVHDCCYGR.V R.VAAICFGENLNTYDK.K R.VAAICFGENLNTYDKK.Y K.YKNYPSSHCTETEQC.K.NYPSSHCTETEQC.R.CCFVHDCCYGR.V K.LSTYSYSFEKGDIVCGGDDPCLR.A R.VAAICFGENMOXNTYDK.K R.VAAICFGENMOXNTYDKK.Y R.CYFVHDCCYGR.V R.VAAICFGENMOXNTYDK.K R.VAAICFGENMOXNTYDKK.Y -.NLYQFGNMIFK.M -.MOXNSANPCCDPITCKPR.K


phospholipase A2

phospholipase A2

disintegrin

phospholipase A2

serine protease disintegrin disintegrin phospholipase A2

phospholipase A2

protein family


a

137

136

gi|237894

phospholipase A2–2

gi|1408314

gi|227636

gi|33187126

gi|237894

gi|50874266

phospholipase A2, subunit A

phospholipase A2 III

ammodytin I2

phospholipase A2–2

ammodytin I1 C)

gi|50874368

gi|227636

phospholipase A2 III

ammodytin I1 (C)

gi|50874368

ammodytin I1 (C)

gi|33187132

gi|50874302

ammodytin I1 (A)

ammodytin I1 soform 2

gi|33187132

accession code


protein

(*): De novo sequence analysis.

spot no.


Vipera aspis zinnikeri

Eristocophis macmahonii


Vipera aspis

Vipera aspis

Vipera aspis atra



Vipera aspis

Vipera aspis atra

Vipera aspis aspis



55

72

103

107

162

211

289

85

114

204

216

233

Mascot score

2

3

2

2

2

4

5

3

2

4

4

4

matched peptides

767.6 875.0 938.7 1059.5 768.4 875.0 938.7 1059.5 767.6 875.0 938.7 1059.5 653.6 478.5 767.6 874.7 938.8 767.8 875.1 939.0 678.5 1059.0 767.8 875.1 939.0 1059.0 735.6 819.7 653.7 717.8 767.8 806.7 767.8 874.9 938.1 767.8 874.9

peptide–ion m/z z

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 2 2 2 2


R.CCFVHDCCYGR.V R.VAAICFGENMOXNTYDK.K R.VAAICFGENMOXNTYDKK.Y K.YMOXLYSLFDCKEESEQC.R.CYFVHDCCYGR.V R.VAAICFGENMOXNTYDK.K R.VAAICFGENMOXNTYDKK.Y K.YMOXLYSLFDCKEESEQC.R.CCFVHDCCYGR.V R.VAAICFGENMOXNTYDK.K R.VAAICFGENMOXNTYDKK.Y K.YMOXFYSLLDCKEESEQC.-.HLSQFGDMOXINK.K -.HLSQFGDMOXINKK.T R.CCFVHDCCYGR.V R.VAAICFGENMOXNTYNK.K R.VAAICFGENMOXNTYNKK.Y R.CCFVHDCCYGR.V R.VAAICFGENMOXNTYDK.K R.VAAICFGENMOXNTYDKK.Y K.YMOXLYSLFDCK.E K.YMOXLYSLFDCKEESEQC.R.CCFVHDCCYGR.V R.VAAICFGENMOXNTYDK.K R.VAAICFGENMOXNTYDKK.Y K.YMOXFYSLLDCKEESEQC.-.NLFQFGDMOXILQK.T R.AAAICLGENVNTYDK.N -.HLSQFGDMOXINK.K -.HLSQFGDMOXINKK.T R.CCFVHDCCYGR.V K.NYPSSHCTETEQC.R.CCFVHDCCYGR.V R.VAAICFGENMOXNTYNK.K R.VAAICFGENMOXNTYNKK.Y R.CCFVHDCCYGR.V R.VAAICFGENMOXITYDK.Q

protein family

phospholipase A2

phospholipase A2


research articles


research articles

Georgieva et al. a

Table 3. Proteomic Compositions of the V. a. ammodytes and V. a. meridionalis Venoms Vaab

Vamb

monomeric phospholipase A2 monomeric phospholipase A2 monomeric phospholipase A2 monomeric phospholipase A2 monomeric phospholipase A2 monomeric phospholipase A2 monomeric phospholipase A2 monomeric phospholipase A2 neutral phospholipase A2 dimeric phospholipase A2 serine proteinase serine proteinase serine proteinase serine protease serine proteinase metalloproteinase metalloproteinase P-I metalloproteinase P-II metalloproteinase P-III metalloproteinase P-III hemorrhagic metalloproteinase HR1 a precursor L-amino acid oxidase platelet activator VEGFb VEGFb nerve growth factor CRISPb disintegrin disintegrin disintegrin disintegrin disintegrin disintegrin a Most of the proteins are homologues of the respective toxins. factor; CRISP ) cysteine-rich secretory protein.

monomeric monomeric monomeric monomeric monomeric monomeric monomeric

b

phospholipase A2 phospholipase A2 phospholipase A2

ammodytin I1 ammodytin I1 (A) ammodytin I1 (C) ammodytin I1 (D) ammodytin I1 (E) ammodytin I2 ammodytin I2(C) ammodytin I2 (E) ammodytin L(1′′) ammodytin L(3) phospholipase A2 phospholipase A2 vipoxin, chain B bothrombin bilineobin okinaxobin ussurin pallase mucofirase halystase R-fibrinogenase A2 metalloproteinase P-I lebetase Le3 VLAIP-A metalloproteinase P-III metalloproteinase precursor L-amino acid oxidase apoxin 1 cerastocytin VEGFb vammin, chain A nerve growth factor CRISPb disintegrin CC5 disintegrin CV11 disintegrin EC6A disintegrin EMF10B MLD-disintegrin RGD-disintegrin EO4A

Vaa ) V. a. ammodytes; Vam ) V. a. meridionalis; VEGF ) vascular endothelial growth

L-Amino acid oxidases, phospholipase A2s, metalloproteases, and serine proteases are the most abundant toxins in the venom of V. a. ammodytes. A variety of LAAOs were observed in the venomic profile (Figure 6, spots 4, 5, 6, 30, and 49). The first three spots of high intensity are grouped in the middle of the upper part of the panel. At least five different LAAOs were identified.

A diversity of Group II monomeric phospholipase A2s, probably differing from each other in their toxicity, was found Journal of Proteome Research • Vol. 7, No. 3, 2008

A2 A2 A2 A2

monomeric phospholipase A2 monomeric phospholipase A2 neutral phospholipase A2 dimeric phospholipase A2 serine proteinase serine proteinase serine proteinase serine proteinase serine proteinase metalloproteinase P-I metalloproteinase P-II metalloproteinase P-III metalloproteinase P-III L-amino acid oxidase L-amino acid oxidase VEGFb VEGFb nerve growth factor disintegrin -

protein families: L-amino acid oxidases, monomeric and dimeric PLA2s, serine/thrombin-like proteases, metalloproteases, disintegrins, nerve growth factors, vascular endothelial growth factors, and cysteine-rich secretory proteins (CRISPs). For a very few CRISPs, inhibitory effects on K+ channels were demonstrated.27 However, most venom CRISPs are of unknown activity.27 In total, 38 venom components of the V. a. ammodytes venom were identified. Most of the proteins are homologues of representatives of the families mentioned above (Table 3), and variations in their biochemical and physiological properties can be expected. This is an evidence for accelerated substitutions in the V. a. ammodytes venom toxins.

876

phospholipase phospholipase phospholipase phospholipase

Homology

in the V. a. ammodytes venom. A number of very intensive protein spots in the 2-D gel electrophoresis, fractions 108, 112, 117, 118, 119, 124, 131, 132, 133, 134, 135, 136, and 137 were identified as PLA2s. Part of them form a region in the lower right part of the panel (Figure 6). The large diversity of homologues reflects gene duplication28 (and references therein). Snake venom PLA2s have multiple functions such as neurotoxicity, cardiotoxicity, myotoxicity, antiplatelet, hypotensive, hemolytic, hemorrhagic, coagulant, anticoagulant, and edemainducing effects.29,30 These enzymes cause one of the most dangerous effects of the snake bite. 2-DE analysis confirmed the presence of the heterodimeric neurotoxin vipoxin which was found so far only in the venom of V. a. meridionalis.23,24 The analysis showed also a large number of metalloproteases (SVMP) in the venom of V. a. ammodytes (Figure 6, spots 11, 22, 104, 105, and 106). They are homologues of the three classes, P-I, P-II, and P-III zinc-chelating enzymes, produced by snake venoms. P-I metalloproteases contain only a protease domain; type P-II enzymes have a second disintegrin-like domain which facilitates the action by interacting with platelet receptors, and P-III are heterodimeric or multimeric metalloproteases, con-


research articles

Figure 7. 2-D gel pattern of the V. a. meridionalis venom. Fractionation was performed under the conditions described in the Experimental Section.

taining in addition a cysteine-rich domain.31 We identified homologues of lebetase Le3, type P-II metalloprotease and of VLAIP, an endothelial cell apoptosis-inducing heterodimeric glycosylated type P-III metalloprotease. Both enzymes are produced by Vipera lebetina.31 Snake venoms contain hemorrhagic and nonhemorrhagic metalloproteases. Two hemorrhagic proteins, VaH1 and VaH2,32 and the metalloprotease ammodytase with a very low hemorrhagic activity12 have been isolated from the V. a. ammodytes venom. The three enzymes are class III metalloproteases composed of protease, disintegrin-like and cysteine-rich domains. The V. a. ammodytes venom contains a number of serine proteases/thrombin-like enzymes (Tables 2 and 3). Five homologues of members of this protein family were observed in the 2-DE gel (Figure 6, spots 51, 60, 61, 67, 68, and 96). Some of them are located in the middle part of the panel. Snake venom serine proteases (SVSP) interfere with the hemostatic system, acting as activators of the fibrinolytic process, procoagulants, anticoagulants, or platelet-aggregating enzymes.33 Some SVSP have unique activities, for example, activation of platelets. A homologue of such a platelet-aggregating serine protease from C. cerastes, cerastocytin,34 was found in the venom of V. a. ammodytes (Table 3). Other group of pharmacologically active components of the V. a. ammodytes venom is that of disintegrins. They are located in the lowest line of spots in the 2-DE panel (Figure 6, spots 123, 125, and 129). This is a family of low molecular mass polypeptide inhibitors of integrins, cell surface glycoproteins which are transmembrane receptors that transduce signals from the extracellular matrix to the cell interior and vice versa. Thus, they mediate the extracellular matrix influence on cell growth and differentiation.35 Disintegrin family can be divided into two

subfamilies of monomeric and homo- or heterodimeric proteins.36 The 2-DE analysis revealed the presence of several homologues of the second subfamily in the V. a. ammodytes venom. Usually, the dimers were dissociated under the conditions used for the gel electrophoresis. Protein homologous to the 8 kDa subunit A of the heterodimeric disintegrin EC6 from the venom of E. carinatus, EC6A, was identified (Table 3). Two forms, monomeric (8 kDa) and a nondissociated dimeric (approximately 15–16 kDa) of a homologue of disintegrins were also observed (Table 3). Two homologues of vascular endothelial growth factors (VEGF) were identified in spots 120 and 122 in the left lower part of the panel (Figure 6). Snake venom VEGFs were isolated from Vipera aspis aspis (vammin) and from Daboia russelli russelli (VR-1).37 These proteins stimulate proliferation of vascular endothelial cells and induce strong hypotension on the arterial blood pressure. Spot 111 contains a homologue of a nerve growth factor. Two venom components in the spots 55 and 56 from the central part of the panel were identified as cysteine-rich secretory proteins (CRISPs). The spot 55 is one of the most intensive in the 2-DE gel (Figure 6). These proteins are homologous to triflin isolated from the venom of Japanese Mamushi snake (Agkistrodon blomhoffi). The function of snake venom CRISPs is not clear.27 2-D Gel Electrophoresis of the V. a. meridionalis Venom and Protein Family Composition: Comparison of the Proteomics of the Two Snake Venoms. The 2-DE pattern of the V. a. meridionalis venom is shown in Figure 7. The results of the MS/MS analyses of peptides obtained after hydrolysis of spots by trypsin were used for database search. The identified proteins were classified to the following protein families: Journal of Proteome Research • Vol. 7, No. 3, 2008 877

878

L-amino

07


gi|61104775

gi|83523626

VLAIP-A

Group III SVMP

11

gi|3552036

gi|143681919

gi|2994932 gi|1395962

pallase

pallase

serine protease kangshuanmei

bilineobin KN-BJ 2

27

28

gi|3552036

gi|763095 gi|83523644 gi|3552036

metalloprotease Group III SVMP pallase

19 24

18

gi|1502368 gi|31322303 gi|83523628

lebetase Le3 ussurin Group III SVMP

12

10

gi|1502368 gi|31322303 gi|83523636 gi|39841344

lebetase Le3 ussurin Group III SVMP L-amino acid oxidase

gi|5565692

gi|33355627

gi|15887054

accession code

09

apoxin

acid oxidase

L-amino

06

acid oxidase

protein

spot no.

Agkistrodon bilineatus Bothrops jararaca

Gloydius blomhoffi brevicaudus

Gloydius halys

Gloydius halys

Echis pyramidum Echis ocellatus Gloydius halys

Macrovipera lebetina Gloydius ussuriensis Echis ocellatus

Echis ocellatus


Macrovipera lebetina Gloydius ussuriensis Echis ocellatus Bothrops jararacussu

Crotalus atrox


Gloydius blomhoffi


85 70

85

125

83

40 41 118

60 60 53

74

92

49 49 34 129

107

169

222

Mascot score

1 3

2

3

2

1 1 3

1 1 2

2

3

1 1 1 3

5

5

8

matched peptides

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

603.4 745.4 573.3 444.9 603.4

z

834.2 567.2 665.3 569.2 583.3 527.0 438.7 895.7 834.9 567.3 665.3 569.5 438.7 665.3 1029.0 583.6 781.4 438.7 677.0 677.0 605.5 676.8 569.3 583.6 446.8 993.4 718.6 692.7 718.6 676.9 676.9 454.7 533.2 475.9 533.4 756.9 573.2 444.9 756.9 573.4 756.9 573.3 444.9 756.9

peptide–ion m/z


K.WDKDIMOXLIR.L -.VIGGDEHNINEHR.F K.EKFFCLSSK.N K.FFCLSSK.N K.WDKDIMOXLIR.L

R.NDKEGWYANLGPMOXR.L K.YPVKPSEEGK.S K.EYLLK.E R.HDDIFAYEK.R R.IKFEPPLPPK.K K.FEPPLPPKK.A K.STTDLPSR.F R.EEIQTFCYPSMOXIQK.W R.NDEEGWYANLGPMOXR.L K.YPVKPSEEGK.S K.EYLIK.E K.HDDIFAYEK.R K.STTDLPSR.F K.EYLLK.E R.EATVTYQTSANEMSSVTADYVIVCTTSR.A R.IKFEPPLPPK.K K.IFLTCK.K K.STTDLPSR.F R.NPQCILNQPLR.T R.NPQCILNKPLR.T K.CPLTLYQCR.A K.SAGQLYEESLQK.A K.HDDIFAYEK.R R.IKFEPPLPPK.K K.VTLDLFGK.W K.LTPGSQCADGECCDQCK.F R.LYCFDNLPEHK.N K.ASQLNLTPEQQR.Y R.LYCFDNLPEHK.N R.NPQCILNQPLR.T R.NPQCILNKPLR.T R.LFCEIVK.N R.HCVDVTTAY.K.LVIVVDHR.M R.HCVDVTTAY.M.VIGGDECNINEHR.F K.EKFFCLSSK.K K.FFCLSSK.K M.VIGGDECNINEHR.F K.EKFFCLSSK.K M.VIGGDECNINEHR.F K.EKFFCLSSK.K K.FFCLSSK.K -.VIGGDECNINEHR.F

Table 4. Assignment of the Spots of the 2D-Gel Electrophoresis of V. a. meridionalis to Protein Families by MS/MS and MASCOT

acid oxidase

acid oxidase

acid oxidase

serine proteinase

serine proteinase

peptidase M12B metalloproteinase serine proteinase

metalloproteinase serine proteinase metalloproteinase

metalloproteinase

L-amino

metalloproteinase

L-amino

L-amino

protein family


51

phospholipase A2, acidic subunit phospholipase A2, acidic subunit triflin

50 Vipera aspis


gi|48474867

Vipera aspis

Gloydius blomhoffi


Bothrops jararaca

Bothrops jararaca

Bothrops jararaca

Bothrops jararaca


Bothrops jararaca

Agkistrodon bilineatus Macrovipera lebetina

Agkistrodon bilineatus Gloydius halys

Agkistrodon bilineatus Agkistrodon bilineatus Gloydius ussuriensis

Gloydius halys


gi|1408314

gi|1408314

gi|75570145

L-amino

49

acid oxidase

gi|13959655

venom serine proteinase-like protein 2 precursor

45

gi|13959622

gi|13959622

KN-BJ 2

KN-BJ 2

40

gi|13959622

41

KN-BJ 2

gi|13959622

KN-BJ 2

gi|13959622

N-BJ 2

gi|13959655

gi|299493 gi|13959655

bilineobin venom serine proteinase-like protein 2 precursor

venom serine proteinase-like protein 2 precursor

gi|461511 gi|3552036

ancrod pallase

gi|461511 gi|299493 gi|17933274

ancrod bilineobin ussurin

accession code

gi|3552036

protein

pallase

39

38

37

32

31

spot no.


60

114

50

207

138

110

144

104

129

167

130

54 148

93 59

93 59 50

99

Mascot score

2

2

1

6

2

2

3

2

3

3

3

1 3

1 2

1 1 2

3

matched peptides

2 3 2

2 2 2 2 2 2 2 2

796.9 826.4 567.4 569.4 583.5 462.7 526.5 819.8

820.3 640.4 340.4

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

380.8 796.8 444.7 380.8 358.8 380.8 590.9 444.9 380.8 591.0 380.8 598.9 433.2

2

2 2 2 2 2 2

380.8 796.9 888.5 380.8 598.9 444.7

748.8

2 2 2 2 2 2 2 2 2 2 2 1

z

756.6 444.6 389.0 749.5 756.6 749.4 444.6 749.7 756.8 445.2 756.8 888.5

peptide–ion m/z

R.AAAICLGENVNTYDK.N R.KPEIQNEIIDLHNSLR.R K.TATPYK.S

R.CCFAQDCCYGR.V

R.TLCAGILQGGIDSCK.V R.NDKEGWYANLGPMR.L K.YPVKPSEEGK.S R.HDDIFAYEK.R R.IKFEPPLPPK.K K.FEPPLPPK.K K.FEPPLPPKK.A R.AAAICLGENVNTYDK.N

K.DIMLIR.L R.IMGWGTISTSK.V K.FFCLSSK.N K.DIMLIR.L R.IMGWGTISTSK.V K.DIMLIR.L R.IMOXGWGTISTSK.V R.IILGVHSK.N

K.DIMLIR.L R.TLCAGILQGGIDSCK.V K.FFCLSSK.N K.DIMLIR.L R.IMOXGWGTISTSK.V

K.DIMOXLIR.L R.TLCAGILQGGIDSCK.V K.FFCLSSK.N K.DIMLIR.L R.IMOXGWGTISTSK.V K.FFCLSSK.T

M.VIGGDECNINEHR.F K.FFCLSSK.K K.DIMOXLIR.L -.VVGGDECNINEHR.S -.VIGGDECNINEHR.F M.VIGGVECNINEHR.F K.FFCLSSK.T -.VVGGDECNINEHR.S M.VIGGDECNINEHR.F K.FFCLSSK.K -.VIGGDECNINEHR.F K.FFCLSSK.T


acid oxidase

phospholipase A2CRISP

phospholipase A2

L-amino

serine proteinase

serine proteinase

serine proteinase

serine proteinase

serine proteinase

serine proteinase

serine proteinase

serine proteinase

protein family


research articles


880


gi|3122187

gi|48093528

gi|33307101

pallase

halystase

serine proteinase BthaTl

serine protease KN13

55

Group III SVMP

Group III SVMP

67

68*

62 63 66

61

lebetase Le3 metalloproteinase/ disintegrin ussurin precursor lebetase Le3 metalloproteinase/ disintegrin ussurin precursor Group III SVMP ancrod phospholipase A2, acidic subunit

gi|83523634

gi|83523632

gi|83523636 gi|461511 gi|1408314

gi|1502368 gi|31322303

gi|1502368 gi|31322303

gi|48093528


60

gi|3552036

pallase

58

gi|3122187

halystase

gi|3552036

gi|48093528


pallase

gi|3552036

pallase

gi|3552036

57

56

gi|3552036

pallase

54

gi|48474867

triflin

53

gi|48474867

accession code

triflin

protein

52

spot no.


Echis ocellatus

Echis ocellatus

Echis ocellatus Agkistrodon bilineatus Vipera aspis

Macrovipera lebetina Gloydius ussuriensis

Macrovipera lebetina Gloydius ussuriensis

Bothrops alternatus

Gloydius halys

Gloydius halys

Gloydius blomhoffi

Bothrops alternatus

Gloydius halys


Bothrops alternatus

Gloydius blomhoffi

Gloydius halys

Gloydius halys




125

43 39 284

69 69

55 55

49

101

120

64

69

104

57

57

60

92

108

211

261

Mascot score

1

3

1 1 4

1 1

1 1

2

2

2

2

2

2

2

2

2

2

2

5

6

matched peptides

2 2 2 2 2 2 2 2 2 2

749.0 820.2 1099.9 619.3 541.5 533.5 513.2

2 2

2 3 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

z

605.9 749.9 735.6

676.8 676.8

959.9 692.5 659.4 581.1 769.5 1051.4 960.2 659.5 589.5 777.5 1051.3 756.8 388.8 756.9 388.8 763.8 388.8 756.9 388.8 562.1 388.8 756.9 388.8 756.9 388.8 763.9 388.8 756.9 388.9 756.9 388.8 756.9 388.8 676.9 676.9

peptide–ion m/z

R.CCFAQDCCYGR.V R.AAAICLGENVNTYDK.N K.NYEYYSISHCTEESEQC.K.RGESYFYCR.K R.GESYFYCR.K R.HCVDVTTAY.R.GESYFYCR.K

K.CPLTLYQCR.A -.VVGGDECNINEHR.S -.NLFQFGDMOXILQK.T

R.NPQCILNQPLR.T R.NPQCILNKPLR.T

R.KPEIQNEIIDLHNSLR.R R.KPEIQNEIIDLHNSLRR.S R.RSVNPTASNMLK.M R.SVNPTASNMLK.M K.MEWYPEAAANAER.W R.VIGGIKCGENIYMOXATYPAK.W R.KPEIQNEIIDLHNSLR.R R.RSVNPTASNMLK.M R.SVNPTASNMOXLK.M K.MOXEWYPEAAANAER.W R.VIGGIKCGENIYMOXATYPAK.W M.VIGGDECNINEHR.F K.DIMOXLIR.L M.VIGGDECNINEHR.F K.DIMOXLIR.L -.IIGGDECNINEHR.F K.DIMOXLIR.L -.VIGGDECDINEHR.F K.DIMOXLIR.L K.NHTQWNKDIMOXLIR.L K.DIMOXLIR.L M.VIGGDECNINEHR.F K.DIMOXLIR.L -.VIGGDECDINEHR.F K.DIMLIR.L -.IIGGDECNINEHR.F K.DIMOXLIR.L M.VIGGDECNINEHR.F K.DIMOXLIR.L M.VIGGDECNINEHR.F K.DIMOXLIR.L -.VIGGDECDINEHR.F K.DIMOXLIR.L R.NPQCILNQPLR.T R.NPQCILNKPLR.T


metalloproteinase

metalloproteinase

metalloproteinase serine proteinase phospholipase A2

metalloproteinase

metalloproteinase

serine roteinase

serineproteinase

serine proteinase

serine proteinase

serine proteinase

CRISP

CRISP

protein family


gi|37993393

phospholipase A2, acidic subunit

c-type lectin-2

81

gi|6647690 gi|58177209

acidic phospholipase A2 vammin, chain A

87

gi|58177209

vammin

gi|58177209

gi|283845 gi|2914537

phospholipase A2, acidic chain vipoxin, chain B

vammin

gi|6647690

gi|50874368

ammodytin I1 (C)

phospholipase A2

gi|33187132

gi|400714

phospholipase A2, RV-7


gi|1408314


gi|400499

86

85

84

82

gi|1408314

nerve growth factor

75

gi|83523632 gi|83523634 gi|3122187

Group III SVMP Group III SVMP halystase

70 71* 73

gi|83523632

accession code

Group III SVMP

protein

69

spot no.


Vipera palestinae Vipera aspis aspis

Vipera aspis aspis

Vipera aspis aspis

Vipera aspis Vipera ammodytes meridionalis

Vipera palestinae

Vipera aspis atra


Daboia russellii siamensis

Vipera aspis

Bitis gabonica

Vipera aspis

Daboia russellii russellii

Echis ocellatus Echis ocellatus Gloydius blomhoffi

Echis ocellatus


50 77

76

57

69 66

82

128

160

80

282

99

214

164

80

75

94

Mascot score

1 3

3

2

1 2

2

3

3

2

4

3

3

4

2 1 2

2

matched peptides

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

748.8 819.9 353.8 431.8 434.1 735.7 748.8 819.9 1099.9 819.9 1099.9 874.7 938.7 1059.6 874.7 938.7 874.8 938.7 735.2 867.0 584.5 564.5 782.4 697.5 564.8 782.5 875.3 697.4 564.8 782.2

z

541.4 533.2 541.4 513.2 444.9 388.7 533.4 481.9 693.4 549.4 647.4 735.4

peptide–ion m/z

K.HTVDIQIMOXR.V K.FTEHTACECRPR.R

K.HTVDIQIMOXR.V K.FTEHTACECRPR.R R.VAAICFGENMOXNTYDK.K -.IVRPFLEVHER.S

K.FTEHTACECRPR.R -.IVRPFLEVHER.S

K.LAIYYYSFK.K K.HTVDIQIMOXR.V

R.CCFAQDCCYGR.V R.AAAICLGENVNTYDK.N K.NYEYYSISHCTEESEQC.R.AAAICLGQNVNTYDK.N K.NYEYYSISHCTEESEQC.R.VAAICFGENMOXNTYDK.K R.VAAICFGENMOXNTYDKK.Y K.YMOXLYSLFDCKEESEQC.R.VAAICFGENMOXNTYDK.K R.VAAICFGENMOXNTYDKK.Y R.VAAICFGENMOXNTYDK.K R.VAAICFGENMOXNTYDKK.Y -.NLFQFGDMOXILQK.Y -.NLFQFAK.M

R.CCFAQDCCYGR.V R.AAAICLGENVNTYDK.N R.AFDEPK.R R.AFDEPKR.S K.NCFGLEK.E -.NLFQFGDMOXILQK.T

R.GESYFYCR.K R.HCVDVTTAY.R.GESYFYCR.K R.GESYFYCR.K K.FFCLSSK.N K.DIMOXLIR.L R.HCVDVTTAY.K.QYFFETK.C K.CKNPNPVPSGCR.G K.NPNPVPSGCR.G R.INTACVCVISR.K -.NLFQFGDMOXILQK.T


phospholipase A2 vascular endothelial growth factor

vascular endothelial growth factor

vascular endothelial growth factor

phospholipase A2

phospholipase A2

phospholipase A2

phospholipase A2

nerve growth factor

metalloproteinase metalloproteinase serine roteinase

metalloproteinase

protein family


research articles


882


93

92

gi|50874494

gi|129402

phospholipase A2

ammodytin I2(C) isoform

gi|50874466

ammodytin I2(D) isoform

gi|14917030

gi|14917030

neutral phospholipase A2


gi|18202444 gi|227635 gi|50874494

disintegrin CC5 phospholipase A2 II ammodytin I2(C) isoform

gi|18202444 gi|64445

disintegrin CC5 phospholipase A2

91

gi|50878135

ammodytin L(3)

90

gi|58177209

gi|2914537

vipoxin, chain B

vammin, chain A

gi|1408314

accession code


protein

89

88

spot no.


Vipera ammodytes meridionalis



Vipera ursinii


Cerastes cerastes Vipera aspis Vipera ammodytes meridionalis

Cerastes cerastes Vipera ammodytes

Vipera ammodytes montandoni

Vipera aspis aspis


Vipera aspis


4

4

250

4

4

5

1 1 5

1 3

4

3

3

2

matched peptides

254

120

193

254

62 48 279

84 80

152

61

113

133

Mascot score

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

819.9 434.3 584.5 594.8 697.4 564.8 782.3 692.4 746.2 735.3 404.8 989.9 692.4 746.2 404.8 990.3 675.7 494.3 767.3 395.3 857.9 948.3 494.3 767.3 395.3 857.9 806.9 767.3 395.3 857.9 802.9 435.8 767.3 948.3 803.4 767.4 857.9 615.3 807.1 767.4 857.9 615.3 802.4

z

735.78

peptide–ion m/z

R.VAAICFGENLNTYDK.K R.VAAICFGENLNTYDKK.Y K.NYPSSQCTETEQC.-

R.CCFVHDCCYGR.V R.VNGCDPK.L R.VAAICFGENLNTYDK.K K.YKNYPSSQCTETEQC.K.GKPQDATDR.C R.CCFVHDCCYGR.V R.VNGCDPK.L R.VAAICFGENLNTYDK.K K.NYPSSHCTETEQC.R.CCFVHDCCYGR.V R.VNGCDPK.L R.VAAICFGENLNTYDK.K K.NYPSSECTETEQC.-.NLYQFGK.M R.CCFVHDCCYGR.V K.YKDYPSSQCTETEQC.K.DYPSSQCTETEQC.R.CCFVHDCCYGR.V R.VAAICFGENLNTYDK.K R.VAAICFGENLNTYDKK.Y K.NYPSSHCTETEQC.R.CCFVHDCCYGR.V

R.CCFVHSCCYAK.L R.ENGAIVCGSSTSCK.K R.AAAICFR.E R.GDDMOXNDYCTGISSDCPR.N K.NPLTSYSFYGCHCGLGNK.G R.CCFVHSCCYAK.L R.AAAICFR.E R.GDDMOXNDYCTGISSDCPR.N -.NLYQFGNMIFK.M K.GKPQDATDR.C

K.HTVDIQIMOXR.V K.FTEHTACECRPR.R K.NPITSYSFYGCHCGLGNK.G

K.LAIYYYSFK.K R.VAANCFHQNK.N -.IVRPFLEVHER.S

R.AAAICLGENVNTYDK.N -.NLFQFAK.M

-.NLFQFGDMOXILQK.T


phospholipase A2

disintegrin phospholipase A2 phospholipase A2

disintegrin phospholipase A2

phospholipase A2

vascular endothelial growth factor (VEGF)

phospholipase A2

protein family


97

95

94

spot no.

gi|50874494

gi|50874466

gi|25453141


phospholipase A2

gi|129402

phospholipase A2


gi|50874466


gi|14917030


gi|129402


gi|50874494

gi|50874466



gi|14917030

phospholipase A2 isozyme PLA-1

gi|2914537

vipoxin, chain B

gi|50874494

gi|50874288

ammodytin I1(E) isoform


gi|50874430

accession code

ammodytin I2(A) variant

protein



Vipera ursinii



Vipera ursinii



Vipera ursinii

Vipera ammodytes



Vipera aspis aspis

Vipera aspis aspis


158

175

255

105

166

167

226

112

113

115

224

95

99

124

Mascot score

3

4

4

3

4

3

4

3

3

3

4

2

2

3

matched peptides

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

857.9 947.8 802.6 869.4 767.3 802.6 767.3 857.9 807.0 767.3 857.9 802.6 767.3 857.9 947.9 802.7 767.3 857.9 807.0 767.3 857.9 947.6 802.8 869.4 767.3 802.7 766.8 857.9 947.9 802.7 766.8 857.9 947.9 802.7 486.9 766.8 874.8

z

767.4 620.3 807.1 767.4 717.3 767.4 584.4 767.3

peptide–ion m/z

R.VAAICFGENLNTYDK.K K.YKNYPSSQCTETEQC.K.NYPSSQCTETEQC.R.CCFVHDCCYGR.V R.VAAICFGENLNTYDK.K K.YKNYPSSECTETEQC.K.NYPSSECTETEQC.K.GKPLDATDR.C R.CCFVHDCCYGR.V R.VAAICFGENMOXNTYDK.K

R.VAAICFGENLNTYDK.K K.YKNYPSSQCTETEQC.K.NYPSSQCTETEQC.R.CCFVHDCCYGR.V R.VAAICFGENLNTYDK.K K.NYPSSHCTETEQC.R.CCFVHDCCYGR.V R.VAAICFGENLNTYDK.K K.YKNYPSSECTETEQC.K.NYPSSECTETEQC.-.NLYQFGK.M R.CCFVHDCCYGR.V K.DYPSSQCTETEQC.R.CCFVHDCCYGR.V

R.CCFVHDCCYGR.V K.DYPSSQCTETEQC.R.CCFVHDCCYGR.V R.VAAICFGENLNTYDK.K K.NYPSSHCTETEQC.R.CCFVHDCCYGR.V R.VAAICFGENLNTYDK.K K.NYPSSECTETEQC.R.CCFVHDCCYGR.V

R.VAAICFGENLNTYDK.K K.YKNYPSSQCTETEQC.K.NYPSSQCTETEQC.-.NLYQFGK.M

R.CCFVHDCCYGR.V R.VAAICYGENLNTYDKK.Y K.NYPSSHCTETEQC.R.CCFVHDCCYGR.V K.GDIVCGGDDPCLR.A R.CCFVHDCCYGR.V K.LAIYYYSFK.K R.CCFVHDCCYGR.V


phospholipase A2

phospholipase A2

phospholipase A2

protein family


research articles


884


100

99

98

spot no.

gi|50874368

ammodytin I1 (C) isoform

gi|50874302

gi|50874302

ammodytin I1 (A) isoform


gi|33187132

gi|50874368

ammodytin I1 (C) isoform


gi|50874302


gi|50874384

gi|33187132

ammodytin I1isoform 2

ammodytin I1 (D) isoform

gi|50874384

accession code

ammodytin I1 (D) isoform

protein


Vipera aspis aspis

Vipera aspis atra

Vipera aspis aspis



Vipera aspis atra

Vipera aspis aspis




282

228

249

284

333

219

324

329

387

Mascot score

5

4

5

5

4

6

6

6

matched peptides

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

1314.8 875.2 939.3 678.4 1059.1 767.4 1314.6 875.2 939.3 678.4 1059.1 768.5 1314.6 875.2 939.3 678.4 1059.1 767.4 875.2 939.3 767.3 1314.9 874.8 938.8 1059.6 767.3 1314.9 874.8 938.4 1059.6 768.5 1314.9 874.8 938.8 1059.6 767.3 874.8 938.8 768.9 875.1 938.7 678.4 1059.1

z

767.4

peptide–ion m/z

R.CYFVHDCCYGR.V K.MOXGTYSYSFQNGDIVCGGDDPCLR.A R.VAAICFGENMOXNTYDK.K R.VAAICFGENMOXNTYDKK.Y K.YMOXLYSLFDCKEESEQC.R.CCFVHDCCYGR.V R.VAAICFGENMOXNTYDK.K R.VAAICFGENMOXNTYDKK. R.CYFVHDCCYGR.V R.VAAICFGENMOXNTYDK.K R.VAAICFGENMOXNTYDKK.Y K.YMOXLYSLFDCK.E K.YMOXLYSLFDCKEESEQC.-

K.MOXGTYSYSFENGDIVCGGDDPCLR.A R.VAAICFGENMOXNTYDK.K R.VAAICFGENMOXNTYDKK.Y K.YMOXLYSLFDCKEESEQC.R.CCFVHDCCYGR.V K.MOXGTYSYSFQNGDIVCGGDDPCLR.A R.VAAICFGENMOXNTYDK.K R.VAAICFGENMOXNTYDKK.Y K.YMOXLYSLFDCKEESEQC.-

K.MOXGTYSYSFENGDIVCGGDDPCLR.A R.VAAICFGENMOXNTYDK.K R.VAAICFGENMOXNTYDKK.Y K.YMOXLYSLFDCK.E K.YMOXLYSLFDCKEESEQC.R.CCFVHDCCYGR.V K.MOXGTYSYSFQNGDIVCGGDDPCLR.A R.VAAICFGENMOXNTYDK.K R.VAAICFGENMOXNTYDKK.Y K.YMOXLYSLFDCK.E K.YMOXLYSLFDCKEESEQC.R.CYFVHDCCYGR.V K.MOXGTYSYSFQNGDIVCGGDDPCLR.A R.VAAICFGENMOXNTYDK.K R.VAAICFGENMOXNTYDKK.Y K.YMOXLYSLFDCK.E K.YMOXLYSLFDCKEESEQC.R.CCFVHDCCYGR.V R.VAAICFGENMOXNTYDK.K R.VAAICFGENMOXNTYDKK.Y R.CCFVHDCCYGR.V

R.CCFVHDCCYGR.V


phosphorlipase A2 phosphorlipase A2

phospholipase A2

phospholipase A2

protein family


research articles

103

lebetase Le3 phospholipase A2

gi|1502368 gi|283845

Macrovipera lebetina Vipera aspis

90 54

1 1

2 2 2 2

111 gi|50874384 ammodytin I1 (D) isoform

Vipera Ammodytes meridionalis

4

875.4 938.9 1075.8 734.8

R.VAAICFGENMOXNTYDK.K R.VAAICFGENMOXNTYDKK.Y R.AVGDDMOXDDYCTGISSDCPR.N -.NLFQFGDMOXILQK.Y

phospholipase A2 3 187 Vipera aspis gi|1408314 101

phospholipase A2, acidic chain

219 gi|50874368 ammodytin I1 (C) isoform

Vipera aspis atra

4

2 2 2 2 2 2 2 2 2 2 2 3 282 gi|33187132 ammodytin I1 isoform 2


5

767.8 874.8 938.9 678.4 1059.1 767.8 875.1 938.9 735.9 748.1 820.0 877.4

R.CCFVHDCCYGR.V R.VAAICFGENMOXNTYDK.K R.VAAICFGENMOXNTYDKK.Y K.YMOXLYSLFDCK.E K.YMOXLYSLFDCKEESEQC.R.CCFVHDCCYGR.V R.VAAICFGENMOXNTYDK.K R.VAAICFGENMOXNTYDKK.Y -.NLFQFGDMOXILQK.T R.CCFAQDCCYGR.V R.AAAICLGENVNTYDK.N K.MOXGTYSYSFENGDIVCGGDDPCLR.A

protein family MS/MS derived sequence z peptide–ion m/z matched peptides Mascot score homology with a protein from: accession code protein spot no.


metalloproteinase phospholipase A2


L-amino acid oxidases (Figure 7, spots 6, 7, 10, 11, and 49), Group I, II, and III metalloproteinases (Figure 7, spots 9, 12, 18, 19, 60, 61, 62, 67, 68, 69, 70, 71, and 103), serine proteinases (Figure 7, spots 24, 27, 28, 31, 32, 37, 38, 39, 40, 41, 45, 54, 55, 56, 57, 58, 63, and 73), Group II phospholipase A2s (Figure 7, spots 50, 51, 66, 81, 82, 84, 85, 86, 88, 90, 91, 92, 93, 94, 95, 97, 98, 99, 100, 101, and 103), vascular endothelial growth factors (Figure 7, spots 84, 85, 86, 87, and 89), nerve growth factors (spot 75), disintegrin (spot 91), and cysteine-rich secretory proteins (CRISPs) (spots 51, 52, and 53). In total, 67 venom components belonging to 8 protein families were identified (Table 4). The proteomic composition explains the effects observed after snakebites by V. a. meridionalis. Evidently, PLA2s, which are one of the most abundant proteins in the venom, are responsible for the neurotoxic effects as difficulties in breathing and paralysis. Most probably, the clotting disorders are connected with the presence of thrombin-like and other proteinases. One interesting result is the observation in the V. a. meridionalis venom of homologues of the monomeric PLA2s ammodytin I2 and of ammodytin I1, which are neurotoxins typical of the other subspecies, V. a. ammodytes, also widespread in Europe. It is known that the neurotoxic component of the V. a. meridionalis venom is the heterodimeric vipoxin.5 In comparison to the closely related V. a. ammodytes, the composition of the V. a. meridionalis venom is less complex. 2-DE analysis revealed 139 protein components in the first case and 104 members of different protein families in the second. The number of the homologues of the most abundant protein families in the subspecies meridionalis is smaller than that in ammodytes. Visual inspection of the 2-D gels showed many similarities and some differences between the profiles of the two subspecies. There are regions within the gels which are quite similar. This is not surprising because the two snakes belong to the same genera. Additionally, comparison of the 2-D images demonstrates quantitave and qualitative differences. Some regions of the 2-D gel of the V. a. ammodytes venom have greater number and diversity of proteins in comparison to the respective regions in the gel of the V. a. meridionalis venom (Figures 6 and 7). Similarities and differences in the proteomic compositions of the two venoms are shown in Table 3.

Concluding Remarks Comparison of the V. a. ammodytes and V. a. meridionalis venom protein compositions revealed similarities and differences in their venom toxins. The venom PLA2 analysis showed that at present both subspecies produce considerable amounts of the dangerous neurotoxin vipoxin. This toxin is a promising candidate as an antigen for the preparation of antiserum against the neurological effects of Viper’s snakebite such as difficulties in breathing, cardiovascular problems, and paralysis. At present, the only treatment of the V. ammodytes’s snakebites is with an antiserum produced using the whole venom which is associated with side effects. Specific antivenom, prepared using the purified major neurotoxin(s), will be considerably more effective and will avoid the side effects. The identification of the other toxic agents creating hemorrhagic effects, apoptosis, and blood coagulation disorders, shed additional light on the toxicity of the venoms. This will allow prediction of the consequences of the snakebites and adequate medical treatment of patients. The isolation of a catalytically active homologue of the nontoxic and enzymatically inactive PLA2-like acidic compoJournal of Proteome Research • Vol. 7, No. 3, 2008 885

research articles nent of the vipoxin heterodimer demonstrates the unique evolution of the toxic and enzymatic functions into nontoxic and inhibitory ones. Our high resolution X-ray data6 showed that in the complex the acidic subunit blocks part of the toxicity site of the basic PLA2 which leads to considerable reduction of the neurotoxicity of the heterodimer. However, in the presence of a natural substrate, the complex dissociates and only the basic subunit, with several times higher toxicity, binds to the membrane.24 Most probably, the acidic protein is a product of evolution of the highly homologous basic subunit.

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PR070376C

Comparative Analysis of the Venom Proteomes of Vipera ammodytes

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