Snake Venomics of Central American Pitvipers: Clues for

of K49 PLA2 molecules from a large variety of pitvipers from the genera Bothrops, Agkistrodon, Bothriechis, Crotalus, Trimeresurus, and Protobothr...
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Snake Venomics of Central American Pitvipers: Clues for Rationalizing the Distinct Envenomation Profiles of Atropoides nummifer and Atropoides picadoi Yamileth Angulo,† José Escolano,‡ Bruno Lomonte,† José María Gutiérrez,† Libia Sanz,‡ and Juan J. Calvete*,‡ Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica, and Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas (CSIC), Jaume Roig 11, 46010 Valencia, Spain Received September 20, 2007; Accepted October 29, 2007

We report the proteomic characterization of the Central American pitvipers Atropoides nummifer and Atropoides picadoi. The crude venoms were fractionated by reverse-phase high-performance liquid chromatography (HPLC), followed by analysis of each chromatographic fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequencing, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass fingerprinting, and collision-induced dissociation-tandem mass spectrometry (CID-MS/MS) of tryptic peptides. Each venom contained a number of bradykinin-potentiating peptides and around 25–27 proteins of molecular masses in the range of 7–112 kDa, belonging to only nine different toxin families (disintegrin, DC fragment, snake venom vascular endothelial growth factor, phospholipases A2, serine protease, cysteine-rich secretory proteins, C-type lectins, L-amino acid oxidase, and Zn2+-dependent metalloproteases), albeit distinctly distributed among the two Atropoides species. In addition, A. nummifer expresses low amounts of a three-finger toxin not detected in the venom of A. picadoi. The major toxins of A. nummifer belong to the PLA2 (relative abundance, 36.5%) and the serine proteinase (22%) families, whereas the most abundant A. picadoi toxins are Zn2+-dependent metalloproteinases (66.4%). We estimate that the similarity of venom proteins between the two Atropoides taxa may be around 14–16%. The high degree of differentiation in the venom proteome among congeneric taxa emphasizes unique aspects of venom composition of related species of Atropoides snakes and points to a strong role for adaptive diversification via natural selection as a cause of this distinctiveness. On the other hand, their distinct venom toxin compositions provide clues for rationalizing the low hemorrhagic, coagulant, and defibrinating activities and the high myotoxic and proteolytic effects evoked by A. nummifer snakebite in comparison to other crotaline snake venoms and the high hemorrhagic activity of A. picadoi. Keywords: Atropoides nummifer • Atropoides mexicanus • Atropoides picadoi • Bitis caudalis • jumping pitviper • Picadoi’s pitviper • snake venom protein families • proteomics • viperid toxins • snake venomics • N-terminal sequencing • mass spectrometry • three-finger toxin

Introduction Pitvipers (Crotalinae) are the most widely distributed subfamily of the venomous snake family Viperidae, with major radiations of its over 190 species (∼75% of viperid species allocated in 29 genera) (http://www.reptile-database.org) in both the Old and New Worlds.1 This diverse radiation has received substantial taxonomic and phylogenetic attention over the last several decades, yet the classification of the lineages within the subfamily Crotalinae remains controversial,2,3 par* To whom correspondence should be addressed: Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas (CSIC), Jaime Roig 11, 46010 Valencia, Spain. Telephone: +34-96-339-1778. Fax: +3496-369-0800. E-mail: [email protected]. † Universidad de Costa Rica. ‡ Consejo Superior de Investigaciones Científicas (CSIC).

708 Journal of Proteome Research 2008, 7, 708–719 Published on Web 12/28/2007

ticularly at the intergeneric level.1 Within Neotropical pitvipers, the Middle American lineage comprise genera Atropoides, Cerrophidion, and Porthidium, which form a clade inferred to be the sister group to a clade comprising the South American genera Bothrocophias, Bothrops, and Bothriopsis.2,3 Atropoides is a genus of thick-bodied (greatest recorded length of 120 cm) pit vipers feeding on small creatures, such as frogs, rodents, and lizards. Of the three currently recognized species of Atropoides, Atropoides olmec (Tuxtlan jumping pitviper) occupies the smallest range, occurring only in the Mexican Sierra de las Tuxtlas.1,4 The range of Atropoides picadoi (Picadoi’s jumping pitviper) is more extensive. It is found in the mountains of Costa Rica and western Panama at 50–1500 m altitude, including the Cordillera de Tilarán, the Cordillera Central, and the Cordillera de Talamanca.1 Atropoides num10.1021/pr700610z CCC: $40.75

 2008 American Chemical Society

Proteomics of Atropoides Snake Venoms mifer (Mexican jumping pitviper). inhabits the largest range, from San Luis Potosí in eastern Mexico to central Panama and can be found in various types of forest, including cloud forest and rain forest, at 40–1600 m altitude. The common names alludes to the ability that these snakes have to launch themselves at an attacker during a strike. Unlike most vipers, members of this genus will strike and then hold on and chew. However, the effects of the venom include only transient pain and mild swelling. A. nummifer possesses one of the least toxic pitviper venoms tested thus far in Central and South America.5 A. nummifer venom shows relatively low hemorrhagic, coagulant, and defibrinating activities6 but displays higher myotoxic and proteolytic effects than other crotaline snake venoms.7,8 In contrast, of 10 different Costa Rican pit viper venoms tested on mice, that of A. picadoi was the most hemorrhagic.9,10 The distinct signs and symptoms of envenomation by A. nummifer and A. picadoi snakebites suggests that venoms from these species may contain different toxin repertoires. Venoms represent the critical innovation in ophidian evolution that allowed advanced snakes to transition from a mechanical (constriction) to a chemical (venom) means of subduing and digesting prey larger than themselves, and as such, venom proteins have multiple functions, including immobilizing, paralyzing, killing, and digesting prey. Venom toxins likely evolved from proteins with normal physiological functions and appear to have been recruited into the venom proteome before the diversification of the advanced snakes, at the base of the Colubroid radiation.11–14 Given the central role that diet has played in the adaptive radiation of snakes,15 venom thus represents a key adaptation that has played an important function in the diversification of these animals. On the other hand, venom composition may retain information on its evolutionary history and may thus have a potential taxonomical value.16 However, despite the fact that characterization of the protein content of snake venoms may have a number of potential benefits for basic research, clinical diagnosis, development of new research tools and drugs of potential clinical use and for antivenom production strategies,17 only a single venom toxin sequence from any Atropoides species (PLA2 myotoxin II of A. nummifer, P82950)18 is annotated in the SwissProt/TrEMBL nonredundant database (Release 37 of July 2007). To address the need for detailed proteomic studies of snake venoms, we have initiated a snake venomics project whose long-term goal is the in-depth analysis of viperid venom proteomes. To date, we have reported the protein composition of the venoms from the North American rattlesnakes Sistrurus miliarius barbouri19,20 and Sitrurus catenatus (subspecies catenatus, tergeminus, and edwasdsii),20 the Tunisian vipers Cerastes cerastes, Cerastes vipera, and Macrovipera lebetina,21 and the Afrotropical species Bitis arietans (Ghana),22 Bitis gabonica gabonica,23 Bitis gabonica rhinoceros, Bitis nasicornis, and Bitis caudalis.16 Here, we report the proteomic characterization of the venoms of A. nummifer and A. picadoi.

Experimental Section Isolation of Venom Proteins. Crude venoms of A. nummifer (also denominated A. mexicanus; common name, jumping viper) and A. picadoi (Picadoi’s pitviper) were pooled from at least 20 specimens collected in Costa Rica and kept at the serpentarium of the Instituto Clodomiro Picado, University of Costa Rica in San José. For reverse-phase high-performance liquid chromatography (HPLC) separations, 2–5 mg of crude,

research articles lyophillized venom were dissolved in 100 µL of 0.05% trifluoroacetic acid (TFA) and 5% acetonitrile and insoluble material was removed by centrifugation in an Eppendorff centrifuge at 13000g for 10 min at room temperature. Proteins in the soluble material were separated using an ETTAN LC HPLC system (Amersham Biosciences) and a Lichrosphere RP100 C18 column (250 × 4 mm, 5 µm particle size) eluted at 1 mL/min with a linear gradient of 0.1% TFA in water (solution A) and acetonitrile (solution B) (5% B for 10 min, followed by 5–15% B over 20 min, 15–45% B over 120 min, and 45–70% B over 20 min). Protein detection was at 215 nm, and peaks were collected manually and dried in a Speed-Vac (Savant). The relative abundances (percent of the total venom proteins) of the different protein families in the venoms were estimated from the relation of the sum of the areas of the reverse-phase chromatographic peaks containing proteins from the same family to the total area of venom protein peaks. Characterization of HPLC-Isolated Proteins. Isolated protein fractions were subjected to N-terminal sequence analysis (using a Procise instrument, Applied Biosystems, Foster City, CA), following the instructions of the manufacturer. Amino acid sequence similarity searches were performed against the available databanks using the BLAST program24 implemented in the WU-BLAST2 search engine at http://www.bork.embl-heidelberg.de. The molecular masses of the purified proteins were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; on 12–15% polyacrylamide gels) and by electrospray ionization (ESI) mass spectrometry using an Applied Biosystems QTrap 2000 mass spectrometer25 operated in enhanced multiple charge mode in the range of m/z 600–1700. In-Gel Enzymatic Digestion and Mass Fingerprinting. Protein bands of interest were excised from a Coomassie Brilliant Blue-stained SDS-PAGE and subjected to automated reduction with dithiothreitol (DTT) and alkylation with iodoacetamide and in-gel digestion with sequencing-grade bovine pancreas trypsin (Roche) using a ProGest digestor (Genomic Solutions), following the instructions of the manufacturer. A total of 0.65 µL of the tryptic peptide mixtures (total volume of ∼20 µL) was spotted onto a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) sample holder, mixed with an equal volume of a saturated solution of R-cyano-4-hydroxycinnamic acid (Sigma) in 50% acetonitrile containing 0.1% TFA, dried, and analyzed with an Applied Biosystems Voyager-DE Pro MALDI-TOF mass spectrometer, operated in delayed extraction and reflector modes. A tryptic peptide mixture of Cratylia floribunda seed lectin (SwissProt accession code P81517) prepared and previously characterized in our laboratory was used as the mass calibration standard (mass range of 450–3300 Da). Collision-Induced Dissociation-Tandem Mass Spectrometry (CID-MS/MS). For peptide sequencing, the protein digest mixture was loaded in a nanospray capillary column and subjected to ESI mass spectrometric analysis using a QTrap mass spectrometer (Applied Biosystems)25 equipped with a nanospray source (Protana, Denmark). Doubly or triply charged ions of selected peptides from the MALDI-TOF mass fingerprint spectra were analyzed in enhanced resolution MS mode, and the monoisotopic ions were fragmented using the enhanced product ion tool with Q0 trapping. Enhanced resolution was performed at 250 amu/s across the entire mass range. Settings for MS/MS experiments were as follows: Q1, unit resolution; Q1-Q2 collision energy, 30–40 eV; Q3 entry barrier, 8 V; linear Journal of Proteome Research • Vol. 7, No. 2, 2008 709

research articles ion trap (LIT) Q3 fill time, 250 ms; and Q3 scan rate, 1000 amu/s. CID spectra were interpreted manually or using a licensed version of the MASCOT program (http://www.matrixscience.com) against a private database containing 927 viperid protein sequences deposited in the SwissProt/TrEMBL database (Knowledgebase Release 12 of July 2007; http://us.expasy.org/sprot/; 212 in SwissProt, 715 in TrEMBL) plus the previously assigned peptide ion sequences from snake venomics projects carried out in our laboratory.16,19–23 MS/MS mass tolerance was set to (0.6 Da. Carbamidomethyl cysteine and oxidation of methione were fixed and variable modifications, respectively. Variation in Venom Composition between Atropoides Taxa. We used similarity coefficients to estimate the similarity of venom proteins between taxa. These coefficients are similar to the bandsharing coefficients used to compare individual genetic profiles based on multilocus DNA fingerprints.26 We defined the protein similarity coefficient (PSC) between two species “a” and “b” in the following way: PSCab ) [2 × (number of proteins shared between a and b)/(total number of distinct proteins in a + total number of distinct proteins in b)] × 100. We judged two proteins (listed in Tables 1-3) as being different when they met one or more of these criteria: (1) had different N-terminal sequences and/or distinct internal peptides sequences (derived from MS/MS data) corresponding to homologous regions, (2) had different peptide mass fingerprints, (3) were of different sizes [judged by MALDI-TOF MS or SDSPAGE; For these comparisons, two proteins were judged to differ in size if they differed by more than our estimate of the 95% confidence interval for particular sizing techniques (0.01% for ESI-QTrap MS, 0.4% for MALDI-TOF MS-derived masses, and 1.4 kDa for SDS-PAGE-determined masses)], or (4) eluted in different reverse-phase HPLC peaks. We emphasize that these measures will give only minimum estimates of the similarities between the venom profiles. We suspect that a number of the proteins that we judge to be the same using the above criteria would be found to differ at one or more of these criteria if more complete information were available.

Results and Discussion Characterization of the Venom Proteomes of A. nummifer and A. picadoi. The crude venoms of A. nummifer and A. picadoi were fractionated by reverse-phase HPLC (Figures 1 and 3), followed by analysis of each chromatographic fraction by SDS-PAGE (Figures 2 and 4), N-terminal sequencing, and MALDI-TOF mass spectrometry (Tables 1 and 2). Protein fractions showing single electrophoretic band, molecular mass, and N-terminal sequence were straightforwardly assigned by BLAST analysis (http://www.ncbi.nlm.nih.gov/ BLAST) to a known protein family, indicating that, although a single toxin from A. nummifer (myotoxin II, P82950) is annotated in the public-accessible databases, representative members of most snake venom toxin families are present among the 927 viperid protein sequences deposited to date in the SwissProt/TrEMBL database (Knowledgebase Release 12 of July 2007; http://us.expasy.org/sprot/). Protein fractions showing heterogeneous or blocked N termini were analyzed by SDS-PAGE, and the bands of interest were subjected to automated reduction, carbamidomethylation, and in-gel tryptic digestion in a ProGest digestor (Genomic Solutions). The resulting tryptic peptides were then analyzed by MALDI-TOF mass fingerprinting followed by amino acid sequence determination of selected doubly and triply charged peptide ions by CID-MS/MS. Except for the protein eluting 710

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Angulo et al. in peak 22, which was identified as a PLA2 molecule highly related to myotoxin II,18 the peptide mass fingerprinting approach alone was unable to identify any protein in the databases. In addition, as expected from the rapid amino acid sequence divergence of venom proteins evolving under accelerated evolution,27–32 with a few exceptions, the product ion spectra did not match any known protein using the ProteinProspector (http://prospector.ucsf.edu) or the MASCOT (http://www.matrixscience.com) search programs. Furthermore, it was not too unusual that a product ion spectrum matched with high MASCOT score to a particular peptide sequence corresponds actually to a tryptic peptide of an homologue snake toxin containing one or more nearly isobaric amino acid substitutions. Hence, it is necessary to revise manually all of the CID-MS/MS spectra (to confirm the assigned peptide sequence or for performing de novo sequencing) and submit the deduced peptide ion sequences to BLAST similarity searches. Although the lack of any complete snake genome sequence is a serious drawback for the identification of venom proteins, high-quality MS/MS peptide ion fragmentation spectra usually yield sufficient amino acid sequence information derived from almost the complete series of sequence-specific b and/or y ions to unambiguously identify a homologue protein in the current databases. The outlined snake venomics approach allowed us to assign unambiguously all of the isolated venom toxins representing over 0.05% of the total venom proteins to known protein families (Tables 1 and 2). Supporting the view that venom proteomes are mainly composed of proteins belonging to a few protein families,12–14,16,19–23 the proteins found in the venoms of A. nummifer and A. picadoi cluster, respectively, into 11 and 10 different toxin families (bradykinin-potentiating peptides, disintegrin, DC fragment, snake venom vascular endothelial growth factor (svVEGF), PLA2, serine protease, cysteine-rich secretory proteins (CRISP), C-type lectins, L-amino acid oxidase (LAO), and Zn2+-dependent metalloproteases), albeit each species exhibiting distinct relative abundances, which are listed in Table 3. Large Venom Toxin Composition Variation between Atropoides Species: Occurrence of a Three-Finger Toxin in A. nummifer Venom. As judged from the data shown in Tables 1 and 2, the venom proteomes of A. nummifer and A. picadoi comprise a similar number, around 25–27, of distinct proteins (Figure 5). However, whereas the major toxins of A. nummifer belong to the PLA2 and the serine proteinase families, the most abundant A. picadoi toxins are Zn2+-dependent metalloproteinases (Table 3). Among the major A. nummifer PLA2 molecules, the protein eluting in peaks 22 and 23 (Table 1) exhibits similar chromatographic retention times, molecular mass, and N-terminal sequences as the PLA2 molecules from A. picadoi venom eluting in peaks 15 and 18 (Table 2). Similarly, the galactose-specific lectin found in each venom (An-27–30, Table 1 and Ap-21, Table 2) share chromatographic behavior, Nterminal sequence, molecular mass, and tryptic peptide ion sequences, and the same is true for the LAO proteins isolated in fractions An-35 (Figure 1 and Table 1) and Ap-27 (Figure 2 and Table 2) and the svVEGF (An-20 and Ap-14). On the other hand, the major PLA2 molecule eluted in peak An-21 (Table 1), which may correspond to the myotoxin I described by Rojas and co-workers,5 is uniquely expressed in A. nummifer venom. Similarly, among the major proteins found in Sistrurus venoms, PLA2 proteins appear to be exceptionally divergent at both the intra- and interspecific level,20 suggesting that they have been

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Proteomics of Atropoides Snake Venoms

Table 1. Assignment of the Reverse-Phase Fractions of A. nummifer (A. mexicanus) Venom, Isolated as in Figure 1, to Protein Families by N-Terminal Edman Sequencing, Mass Spectrometry, and Collision-Induced Fragmentation by nESI-MS/MS of Selected Peptide Ions from In-Gel-Digested Protein Bands (Separated by SDS-PAGE as in Figure 2)a HPLC fraction An-

N-terminal sequence

molecular mass

peptide m/z

ion z

MS/MS-derived sequence

1–4, 6, 8, 9, 13 5

np ND

370.6

2

(177.7)APAAPH

7

ND

348.8 532.4

2 2

PHHIPP TPPAGPDVGPR

10

ND

616.6

2

LGTPPAGPDVGPR

488.6

2

ZRFPQYR

684.3

3

508.2

2

LRPGAQCAEGL CCDQCR ARGDNPDDR

11

12

EAGEECDCGAPTNP CCDAAT

7710.3

PPPISPP

704.3

EECDCGAPTNPC CDAATCKL ECDCGAPTNPCCD AATCKLR

7452.3 7323.2

14

ND

508.2 630.2 397.2

2 2 2

15

blocked

622.8

2

ZKWDPPPISPP

16

6833*

712.3

17

LICEDCSLPNCD FLPGIL ND

24 062* 712.3

635.7

2 3

18 19 20

ND ND ND

24 390* 14 409* 38 kDa9/36 kDa1 13 kDa1, 8 kDa1

635.7 650.1 685.2 647.3 682.3 650.1 640.4

2 2 2 2 2 2 3

650.1

2

LYCFPSSPGDK (953.4)CDCGS PATCR LYCFPSSPGDK GXYGCNCGVGSR ZVMPFMEVYSR XDATCVCVXSR AXTMEGNQASWR GLYGCNCGVGSR NAIASYGLYGC NCGVGSR GXYGCNCGVGSR

640.4

3

482.6

2

569.1 925.4

2 2

509.9 702.4 751.8 460.2 491.3 479.6 669.9 874.8

2 2 2 2 2 2 2 2

21

22

23

SLYELGKMILQET GKNAIA

NLYQLWKMIL QETGKNA

SLIQFETLIMKIAGR

13 751.0

13 792.5

13 728*

bradykininpotentiating peptides bradykininpotentiating peptide bradykininpotentiating peptide Zn2+-metalloprotease fragment medium-size disintegrin bradykininpotentiating peptide medium-size disintegrin

LRPGAQCAEGL CCDQCR ARGDNPDDR PPGPVGPDR(223.0) PPGPPIPP

34 kDa9/16 kDa1

protein family

684.3

3

NAIASYGLYGC NCGVGSR NLYQLWK TDSYSYSWK NAAPSYGFY GCNCGVGR IYPKPLCK TIVCGKNNPCLK CCYKALTDCSPK MILQETGK DNLDTYNK YWFFPAK SLIQFETLIM(ox)K QXCECDKDAAXCXR

unknown bradykininpotentiating peptide bradykininpotentiating peptide three-finger toxin DC fragment

DC fragment PLA2 svVEGF

PLA2

PLA2 myotoxin I

PLA2 myotoxin II [P82950]

PLA2

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Table 1. Continued HPLC fraction An-

24 M m 25

N-terminal sequence

molecular mass

peptide ion m/z z

SVDFDSESPRKPEIQ VIGGDECNINEHRFL SLIQFETLIMKIAGQ VIGGDECNINEHRFL KNxGLDKDIMLIRLR

24 744* 38 kDa9/1 13 740* 28 kDa9/(14 + 17 kDa)1

VIGGDECNINEHRFL

30–33 kDa9/1

m 27 M m 28 m

SLIQFETLIMKIAGQ

13 740*

VIGGDECNINEHRSL

66 kDa9/33 kDa1

27–30

NNCPQDWLPMNGLCY

28 kDa /13 kDa

29

VIGGDECNINEHRSL

28 kDa9/1

30

V(V/I)GGDECNINEHRSL

28–34 kDa9/1

31

IIGGDECNINEHRFL

34 kDa9/1

32

IIGGDECNINEHRFL

29 kDa9/1

33–41

blocked

26 kDa9/1

34–41

ND

112 + 56 kDa /56 kDa

26 M

MS/MS-derived sequence

protein family

CRISP serine proteinase PLA2 two-chain serine proteinase 678.9 812.9 650.8 611.2 508.6 619.2

2 2 2 2 2 2

(203.2)XVLTAAHCNR CANINILDYIVCR GGDECNINEHR FLVALYDSHR (157.7)FPTXPER XNXNGYEVCR

serine proteinase

PLA2

V(I/F)GGDECNINEHR(S/F)L 26 kDa9/1 NNCPQDWLPMNGLCY 28 kDa9/13 kDa1

9

1

9

1

621.3

2

DFSWEWTDR

679.3 756.6 621.6 784.3 648.2 757.6 539.3 736.6 764.3 704.3 877.8 640.6 868.8 717.6 640.6 868.8 717.6 783.8 676.8 532.2

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

(316.2)VLTAAHCNR VIGGDECNINEHR DFSWEWTDR EFCVEXVSHAGYR XNXXDYQVCR VIGGDECNINEHR (145.1)YAGXPVCR PVEXTPVSXPSNPPXGSVCR IIGGDECNINEHR FXVAXHDYWSR TXCAGVXEGSTDTCDR XDXFDNEVCR XXCAGVXEGGTDTCNR (1003.3)PFQGXVSWGR XDXFDNEVCR XXCAGVXEGGTDTCNR (1003.3)PFQGXVSWGR XSHHDAQXXTAXVFDQNTXGR SMNXHXSXNDVEXWSNR NPXEECFR

serine proteinase Gal-specific lectin serine proteinase Gal-specific lectin serine proteinase

serine proteinase serine proteinase serine proteinase PI metalloprotease L-amino

acid

oxidase

37–40

ND

14–16 kDa1

40

ND

110 kDa9/42 kDa1

41

ND

78 kDa9/42 kDa1

758.3 641.3 634.8 684.0 783.8

2 2 2 3 3

676.8 900.3 636.1 502.4 900.3 636.1

3 2 2 2 2 2

ETDYEEFXEXAR SAGQXYQESXGK VGEVNQDPGXXK DCGDXVXNDXSXXHQXPK XSHHDAQXXTAXVFDQNTXGR PI metalloprotease fragment SMNXHXSXNDVEXWSNR (200.2)GQCAEGXCCEQCK PIII metalloprotease XYCFWSPGSR (289.2)PFQPVK (200.2)GQCAEGXCCEQCK PIII metalloprotease XYCFWSPGSR

a X, Ile or Leu; Z, pyrrolidone carboxylic acid; M(ox), methionine sulfoxide. Unless otherwise stated, for MS/MS analyses, cysteine residues were carbamidomethylated. Molecular masses of native proteins were determined by ESI ((0.02%) or MALDI-TOF (/) ((0.2%) mass spectrometry. Apparent molecular mass determined by SDS-PAGE of nonreduced (9) and reduced (1) samples. np ) nonpeptidic material found. M and m, denote mayor and minor products within the same HPLC fraction. The only previously reported venom protein from any Atropoides species is identified by its databank accession code.

the subject of strong balancing selection33 within and diversifying selection between taxa. Other studies have also shown that PLA2 genes show high levels of divergence between species and high levels of protein diversity at the population level.28,34–40 Serine proteinases and metalloproteinases have also diverged to a point where no structural similarity can be discerned between these toxins of A. nummifer and A. picadoi. In addition, A. nummifer venom departs from that of A. picadoi by the distinct occurrence in the former of a three-finger toxin (3FTx) (reverse-phase HPLC fraction An16 in Figures 1 and 2 and Table 1). Three-finger toxins appear to be widely distributed in Elapidae (including Hydrophiinae) venoms41 and typically block nicotinic acetylcholine receptors, resulting in postsynapyic neurotoxicity.42 Recently, 712

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three-finger toxins have also been described in Viperidae (Daboia russelli43 and Lachesis muta44) as well as Colubridae (Boiga dendrophila).45,46 These observations, along with our finding of a 3FTx in A. nummifer venom, indicate that this toxin family may be more widely distributed than previously thought and supports the proposal that venom toxins likely evolved from proteins with normal physiological functions recruited into the venom proteome before the diversification of the advanced snakes, at the base of the Colubroid radiation.11–14 The N-terminal sequence, molecular mass, and pharmacological activities of the Daboia russelli venom 3FTx indicate that it is a short-chain neurotoxin such as that found in Elapid venom.43 On the other hand, the colubrid 3FTx (termed denmotoxin) represents a bird-specific neu-

research articles

Proteomics of Atropoides Snake Venoms

Table 2. Assignment of the Reverse-Phase Fractions of A. picadoi Venom, Isolated as in Figure 3, to Protein Families by N-Terminal Edman Sequencing, Mass Spectrometry, and Collision-Induced Fragmentation by nESI-MS/MS of Selected Peptide Ions from In-Gel-Digested Protein Bands (Separated by SDS-PAGE as in Figure 4)a HPLC fraction An-

N-terminal sequence

molecular mass

peptide m/z

ion z

MS/MS-derived sequence

protein family

1, 2, 6 3 4 m 5 m 7 8 9 10 11 12 13 14

np ND

589.9

2

DTPPAGPDVGR

bradykinin-potentiating peptide

ND

489.9

2

ZQYNPYR

unknown

ND ND ND ND ND ECDCGAPTNPCCDAAT ND ND ND

395.9 634.9 625.9 537.9 555.3

2 2 2 2 2

FXQNXR VGEVNQDPGXXK (215.1)WPPGHHIPP (321.3)PGHHIPP XTAPFESS(277.1)

15

SLYQLWKMILQETGK

13 897.6

16

SLIQFETLIMKIAGR

13 806.6

17

NVDFDSESPRKPEIQ

24 787.1

18

SLIQFETLIMKIAGR

13 790.6

685.2 647.3 682.3 556.2 650.2 469.2 568.7 884.8 479.6 669.9 583.2 769.3 635.9 479.6 669.9 884.8

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

ZVMPFMEVYSR XDATCVCVXSR AXTMEGNQASWR NPNPVPTGCR GLYGCNCGVGSR SXYQXWK TDSYSYSWK TXCECDKDAAXCFR YWFFPAK SLIQFETLIM(ox)K NVDFDSESPR MEWYPEAAANAER KPEIQNEIVDLHNSLR YWFFPAK SLIQFETLIM(ox)K TXCECDKDAAXCFR

unknown LAO fragment bradykinin-potentiating peptide bradykinin-potentiating peptide unknown disintegrin (DC fragment) (DC fragment) svVEGF

19 20

VVGGDECNINEHRSL VVGGDECNINEHRSL

28 kDa9/1 39.2 Da9/1

21

VFGGDECNINEHRSL

29 kDa9/1

NNCPQDWLPMNGLCY

26 kDa9/13 kDa1

616.3 749.9 467.8 523.9 605.8 773.8 552.2 621.6 783.9 573.2 639.9 670.8 605.8 773.8 552.2 605.8 621.6 784.1 756.9 698.6 610.9 647.9 679.4 486.2 728.4 555.8 534.3 532.2 758.3 633.3 488.7 503.8 709.3 670.4

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

VKLNEDEQTR VVGGDECNINEHR XXCHXNR VXYQDAXPK INILDHAVCR VFGGDECNINEHR VLNEDEQTR DFSWEWTDR EFCVEXVSHAGYR NQPDHYQNK LWNDQVCESK NNCPQDWLPMNGLCYK INILDHAVCR VFGGDECNINEHR VLNEDEQTR INILDHAVCR DFSWEWTDR EFCVEXVSHAGYR VIGGDECNINEHR AAYPEYGXPATSR FXVAXYDSHR XNXXDYEVCR FXVAXYTFDXR (212.2)PNQDER SXPSSPPXVGSVCR TXDSFGEWR YNDGSDKXR NPXEECFR ETDYEEFXEXAR (145.2)VGPXQDHSPR GSNYGYCR TPFAAACXR HWADAEWFCAR YVEXVXVADYR

22

7341.2 23.5 kDa9/1 23.5 kDa9/1 26 kDa9/13 kDa1 18 kDa9, 10 kDa1

VFGGDECNINEHRSLVVLF 27.5 kDa9/1

NNCPQDWLPMNGLCY

19.5 kDa 26 kDa9/13 kDa1

23

VIGGDECNINEHRFL

30 kDa9/1

24

VVGGDECNINEHRFL

28 kDa9/1

25–27

TPEHQRYIELVIVVD

22.9 kDa/

27

ANDRNPLEECFRETD

56 kDa9/1

28

heterogeneous

58 kDa9/1 110 kDa9/16 kDa1

29

heterogeneous

68 kDa9/1

PLA2

PLA2

CRISP

PLA2

serine proteinase serine proteinase

serine proteinase

Gal-specific lectin

serine proteinase

serine proteinase Gal-specific lectin serine proteinase

serine proteinase

PI metalloproteinase L-amino

acid oxidase

PIII metalloproteinase C-type lectin-like PIII metalloproteinase

Journal of Proteome Research • Vol. 7, No. 2, 2008 713

research articles

Angulo et al.

Table 2. Continued HPLC fraction An-

N-terminal sequence

molecular mass

46 kDa9/1

30

31

heterogeneous

28 kDa9

heterogeneous

16 kDa1 14 kDa1 42 kDa9/1 16 kDa1 14 kDa1

peptide m/z

ion z

MS/MS-derived sequence

678.6 514.8 526.8 801.6 521.2 453.7 640.3 621.2 621.2 640.3 852.3 626.8 521.2 851.8

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

VAXVGXQXWSNR XPCAPEDVK GNYYGYCR MYEXANTVNDXYR YYAWIGLR TWEDAER DCPSDWSSYEGHCYR DCPSDESSYEGHCYR DCPSDESSYEGHCYR DCPSDWSSYEGHCYR SDVDYTXNSFAEWR (367.2)PDYCTGQSGDCPR YYAWIGLR SDVDYTXNSFAEWR

protein family

PIII metalloproteinase

C-type lectin-like

C-type lectin-like R subunit C-type lectin-like β subunit PIII metalloproteinase C-type lectin-like metalloproteinase fragment

a X, Ile or Leu; Z, pyrrolidone carboxylic acid; M(ox), methionine sulfoxide. Unless otherwise stated, for MS/MS analyses, cysteine residues were carbamidomethylated. Molecular masses of native proteins were determined by ESI ((0.02%) or MALDI-TOF (/) ((0.2%) mass spectrometry. Apparent molecular mass determined by SDS-PAGE of nonreduced (9) and reduced (1) samples. np, nonpeptidic material found. M and m, denote mayor and minor products within the same HPLC fraction.

Table 3. Overview of the Relative Occurrence of Proteins (in Percentage of the Total HPLC-Separated Proteins) of the Different Families in the Venoms of A. nummifer (A. mexicanus) and A. picadoi percent of total venom proteins protein family

A. nummifer

A. picadoi

bradykinin-potentiating peptides medium-size disintegrin three-finger toxin DC fragment sv-VEGF PLA2 serine proteinase CRISP C-type lectin-like L-amino acid oxidase Zn2+-metalloproteinases PI SVMP PIII SVMP

8.6 2.5