Exploring the Venom Proteome of the Western Diamondback Rattlesnake, Crotalus atrox, via Snake Venomics and Combinatorial Peptide Ligand Library Approaches Juan J. Calvete,*,† Elisa Fasoli,‡ Libia Sanz,† Egisto Boschetti,§ and Pier Giorgio Righetti‡ Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Cientı´ficas (CSIC), Jaume Roig 11, 46010 Valencia, Spain, Department of Chemistry, Materials and Chemical Engineering “Giulio Natta”, Politecnico di Milano, 20131 Milan, Italy, and Bio-Rad Laboratories, CEA-Saclay, 91181 Gif-sur-Yvette, France Received March 14, 2009
We report the proteomic characterization of the venom of the medically important North American western diamondback rattlesnake, Crotalus atrox, using two complementary approaches: snake venomics (to gain an insight of the overall venom proteome), and two solid-phase combinatorial peptide ligand libraries (CPLL), followed by 2D electrophoresis and mass spectrometric characterization of ingel digested protein bands (to capture and “amplify“ low-abundance proteins). The venomics approach revealed ∼24 distinct proteins belonging to 2 major protein families (snake venom metalloproteinases, SVMP, and serine proteinases), which represent 69.5% of the total venom proteins, 4 medium abundance families (medium-size disintegrin, PLA2, cysteine-rich secretory protein, and L-amino acid oxidase) amounting to 25.8% of the venom proteins, and 3 minor protein families (vasoactive peptides, endogenous inhibitor of SVMP, and C-type lectin-like). This toxin profile potentially explains the cytotoxic, myotoxic, hemotoxic, and hemorrhagic effects evoked by C. atrox envenomation. Further, our results showing that C. atrox exhibits a similar level of venom variation as Sistrurus miliarius points to a ”diversity gain” scenario in the lineage leading to the Sistrurus catenatus taxa. On the other hand, the two combinatorial hexapeptide libraries captured distinct sets of proteins. Although the CPLL-treated samples did not retain a representative venom proteome, protein spots barely, or not at all, detectable in the whole venom were enriched in the two CPLL-treated samples. The amplified low copy number C. atrox venom proteins comprised a C-type lectin-like protein, several PLA2 molecules, PIII-SVMP isoforms, glutaminyl cyclase isoforms, and a 2-cys peroxiredoxin highly conserved across the animal kingdom. Peroxiredoxin and glutaminyl cyclase may participate, respectively, in redox processes leading to the structural/functional diversification of toxins, and in the N-terminal pyrrolidone carboxylic acid formation required in the maturation of bioactive peptides such as bradykinin-potentiating peptides and endogenous inhibitors of metalloproteases. Our findings underscore the usefulness of combinatorial peptide libraries as powerful tools for mining below the tip of the iceberg, complementing thereby the data gained using the snake venomics protocol toward a complete visualization of the venom proteome. Keywords: Crotalus atrox • western diamond rattlesnake • snake venomics • venom proteome • viperid toxins • N-terminal sequencing • mass spectrometry • low-abundance proteins • combinatorial peptide ligand library • peroxiredoxin • glutaminyl cyclase
Introduction The pitvipers comprise the subfamily Crotalinae of the family Viperidae, found in Asia and the Americas. Currently, 208 species of pitvipers grouped into 28 genera are recognized (http://www.reptile-database.org). Pitvipers are distinguished by the presence of a heat-sensing pit organ located in an * Address correspondence to: Juan J. Calvete, Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Cientı´ficas (CSIC), Jaume Roig 11, 46010 Valencia, Spain. Phone: +34-96-339-1778. Fax: +34-96-3690800. E-mail:
[email protected]. † Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Cientı´ficas (CSIC). ‡ Politecnico di Milano. § Bio-Rad Laboratories. 10.1021/pr900249q CCC: $40.75
2009 American Chemical Society
indentation of the upper jaw, between the eye and the nostril on either side of the head. This infrared receptor is used to detect the distance and direction of warm-blooded prey.1,2 Pitvipers represent the only viperids found in the Americas. They appear to have dispersed into the New World as a single lineage from Asia during the late Oligocene or the early Miocene, between 22-24 Mya,3 at a time when eastern North America and Eurasia were widely separated across the Atlantic, whereas northeastern Asia and Alaska remained connected via the Behring land bridge. The colonization of the New Word was followed by rapid adaptive radiation in the middle-late Miocene and early Pliocene (16-3.6 Mya) giving rise to most of the approximately 126 currently recognized species included Journal of Proteome Research 2009, 8, 3055–3067 3055 Published on Web 04/17/2009
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in the 12 genera of New Word pitvipers. Within the North American group, the rattlesnakes (Sistrurus, Crotalus) form a clade, and Agkistrodon is their sister group.4 The genus Crotalus comprises 30-36 (http://www.reptile-database.org) species of terrestrial, live-bearers, pitvipers found only in the Americas, from southern Canada to northern Argentina.5 The genus name is derived from the Greek κFο´ τRλoν (krotalon), which means “clapper” or “castanet”, and refers to the rattle on the end of the tail that make this group so distinctive.5,6 The western diamondback rattlesnake, Crotalus atrox is the largest western rattlesnake, with adults commonly growing to more than 120 cm in length and reaching a body weight of up to 6.7 kg,5,6 and has the widest geographic range of all monotypic rattlesnake species. It lives in 10 southwestern U.S.A. states, from central and western Texas, through southern New Mexico and Arizona, and into southern California, and in most of the northern half of Mexico. Crotalus atrox is one of the more aggressive rattlesnake species found in North America and it is likely responsible for the majority of snakebite fatalities in northern Mexico and the second greatest number in the U.S.A. after Crotalus adamanteus.5 Across its expansive range, C. atrox takes up residence among communities of small mammals, usually hunting at night. It may be found in desert and semiarid ecosystems ranging from flat coastal plains to steep rocky canyons and hillsides. An ambush hunter, C. atrox typically sits near the trail of a mammal, waiting for it to pass by, then strikes at and releases the prey. The snake then follows the trail of the envenomated animal and swallows it whole. A comprehensive study showed that 94.8% by weight of C. atrox prey in Texas consisted of small mammals.7 Bird and lizards are also preyed upon, with lizards mostly being eaten by young snakes.7 Venom represents an adaptive trait that enabled advanced snakes to transition from a mechanical (constriction) to a chemical (venom) means of subduing and digesting prey. Snake venoms are deadly cocktails, each comprising unique mixtures of peptides and proteins naturally tailored by Natural Selection to act on vital systems of the prey or victim. Venom toxins likely evolved from proteins with a normal physiological function and appear to have been recruited into the venom proteome before the diversification of the advanced snakes, at the base of the Colubroid radiation.8–10 Although the venom of the diamondback is not particularly toxic (average venom yield, 175 - 600 mg; estimated human lethal dose, 100 mg),5 and venom toxicity decreases with age in this snake,11 the size of the snake allows a large capacity of venom which is released from its two prominent fangs. Like other American pit vipers, C. atrox’s venom is mainly cytotoxic, hemotoxic, myotoxic, and hemorrhagic, although smaller amounts of neurotoxins are also present.5 However, quantitative and qualitative intraspecific differences have been reported to occur with age and geographic origin.12–14 C. atrox bites are followed by intense pain at the site of envenomation, eventually radiating in all directions, swelling and edema, changes in pulse rate and blood pressure, nausea and vomiting.15 Adequate treatment of envenoming is critically dependent on the ability of antivenoms to neutralize the lethal toxins reversing thereby the signs of envenoming. A robust knowledge of venom composition and of the onset of ontogenetic, individual, and geographic venom variability may have applied importance for the treatment of bite victims and for the selection of specimens for the generation of improved antidotes.16 On the other hand, venoms represent a huge and essentially unexplored reservoir of bioactive components. The 3056
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high degree of target specificity makes toxins valuable scaffolds for drug development.17,18 Thus, understanding how toxins evolve may hold the key to understanding speciation at a molecular level and to learn how to use deadly toxins as therapeutic agents.19 We have developed proteomic protocols (“snake venomics” and “antivenomics”) for unravelling the composition, immunological profile, and evolution of snake venoms.16,20 A major conclusion drawn from snake venomics and transcriptomic analyses of viperid venom glands is that, despite venoms being complex mixture of proteins, venom proteins belong to only a few major families, including enzymes (serine proteinases, Zn2+-metalloproteases, L-amino acid oxidase, PLA2) and proteins without enzymatic activity (disintegrins, C-type lectins, natriuretic peptides, ohanin, myotoxins, CRISP toxins, nerve and vascular endothelium growth factors, cystatin and Kunitz-type protease inhibitors), though different venoms exhibit a distinct toxin family distribution profile.16,20,21 Abundant venom proteins may perform generic killing and digestive functions that are not prey specific, whereas lowabundance proteins may be more plastic either in evolutionary or ecological time scales. Low-abundance proteins may serve to “customize“ an individual venom to feeding on particular prey, may represent orphan molecules ”in search for a function”, or molecules involved in venom gland functioning. Hence, whereas abundant proteins are the primary targets for immunotherapy, minor components may represent scaffolds for biotechnological developments or may provide clues for understanding the biosynthesis of venom. Here, we have applied our snake venomics protocol to explore the toxin composition of C. atrox venom, and have used combinatorial hexapeptide ligand libraries to capture low-abundance venom proteins.22–25
Experimental Section Isolation of Venom Proteins. The venom of C. atrox was purchased from Latoxan (Valence, France). For reverse-phase HPLC separations, 2-5 mg of crude, lyophilized venom was 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 13 000g 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 SpeedVac (Savant). Given that the wavelength of absorbance for a peptide bond is 190-230 nm, protein detection at 215 nm allows to estimate the relative abundance (expressed as percentage of the total venom proteins) of the different protein families from the relation of the sum of the areas of the reversephase chromatographic peaks containing proteins from the same family to the total area of venom protein peaks in the reverse-phase chromatogram. In a strict sense, and according to the Lambert-Beer law, the calculated relative amounts correspond to the “% of total peptide bonds in the sample”, which is a good estimate of the % by weight (g/100 g) of a particular venom component. In those cases where an HPLC fraction shows the presence of several coeluting proteins, the relative abundance of each component was estimated by densitography.
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Exploring the Venom Proteome of Crotalus atrox 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 manufacturer’s instructions. Amino acid sequence similarity searches were performed against the available databanks using the BLAST program26 implemented in the WU-BLAST2 search engine at http://www.bork.emblheidelberg.de. The molecular masses of the purified proteins were determined by SDS-PAGE (on 12 or 15% polyacrylamide gels) and by electrospray ionization (ESI) mass spectrometry using an Applied Biosystems QTrap 2000 mass spectrometer27 operated in Enhanced Multiple Charge mode in the range m/z 600-1700. In-Gel Enzymatic Digestion and Mass Fingerprinting. Protein bands of interest were excised from Coomassie Brilliant Blue-stained SDS-PAGE gels and subjected to automated reduction with DTT and alkylation with iodoacetamide, and in-gel digestion with sequencing grade bovine pancreas trypsin (Roche) using a ProGest digestor (Genomic Solutions) following the manufacturer’s instructions. A volumeof 0.65 µL of the tryptic peptide mixtures (total volume of ∼ 20 µL) was spotted onto a 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 MALDITOF mass spectrometer, operated in delayed extraction and reflector modes. A tryptic peptide mixture of Cratylia floribunda seed lectin (Swiss-Prot accession code P81517) prepared and previously characterized in our laboratory was used as mass calibration standard (mass range, 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 electrospray ionization (ESI) mass spectrometric analysis using a QTrap mass spectrometer (Applied Biosystems)27 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-to-Q2 collision energy, 30-40 eV; Q3 entry barrier, 8 V; LIT (linear ion trap) 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 Swiss-Prot/TrEMBL database (UniProtKB/ Swiss-Prot Release 56.7 of 20-Jan-2009; http://us.expasy.org/ sprot/) plus the previously assigned peptide ion sequences from snake venomics projects carried out in our laboratory.16,20,21 MS/MS mass tolerance was set to (0.6 Da. Carbamidomethyl cysteine and oxidation of methionine were fixed and variable modifications, respectively. ProteoMiner Enrichment. We have used a combinatorial hexapeptide ligand library approach to capture low-abundance venom proteins.22–25 We have used two peptide libraries (ProteoMiner protein enrichment kit, hitherto termed Library1, and its carboxylated version, Library-2, not yet commercially available). The solid-phase combinatorial libraries consist of poly(hydroxymetacrilate) beads, each displaying the same hexapeptide. The ligand density is ca. 40-60 mmol/mL bead
volume, and the complete library may contain a set of 64 millions different hexapeptides.22 Peptide libraries and all materials for electrophoresis, such as gel plaques and reagents, were from Bio-Rad Laboratories (Hercules, CA). To capture low-abundance proteins, 2 g of lyophilized venom was dissolved in 40 mL of PBS (25 mM phosphate buffer, pH 7.2, containing 150 mM NaCl, and the complete protease inhibitor cocktail tablets from Roche Diagnostics, Basel, CH) by gently stirring, and incubated with 1 mL of Library-1 for 3 h at room temperature. Library-1 was then washed twice to remove excess soluble proteins not bound to the beads. The supernatant from Library-1 was then mixed with 1 mL of Library-2 for 3 h. After filtration, Library-2 was washed twice with PBS to remove nonadsorbed proteins. The captured protein species were separately eluted from the two libraries by boiling the beads in 5% SDS and 20 mM DTT. The elution step was repeated three times, each time with 0.5 mL of eluant. At the end of the procedure, all three eluates were pooled and each of the eluates from the two libraries was analyzed by SDSPAGE and by 2D electrophoresis. To this end, SDS had to be removed by precipitation with chloroform/methanol, followed by solubilization in 7 M urea, 2 M thiourea, 3% CHAPS, and 40 mM Tris (TUC buffer). 2D Electrophoresis. Whole venom and the eluates of Library-1 and Library-2 were solubilized in TUC buffer to a final concentration of 2 mg/mL. Disulfide bond reduction was performed at room temperature for 60 min by addition of TCEP [Tris(2-carboxyethyl)phosphine hydrochloride] at a final concentration of 5 mM. For alkylating sulfydryl groups, De-Streak [Bis-(2-hydroxyethyl)disulfide (HOCH2CH2)2S2)] was added to the solution at a final concentration of 150 mM (by dilution from the 8.175 M stock solution, Sigma-Aldrich), followed by addition of Ampholine (from a the 40% stock solution to a final concentration of 0.5%) and a trace amount of bromophenol blue. Seven-centimeter long IPG strips (Bio-Rad), pH range 3-10 L, were rehydrated in 150 µL of protein solution (containing a total of ca. 200 µg protein), for 4 h at room temperature. Isoelectric focusing (IEF) was carried out with a Protean IEF Cell (Bio-Rad) using a linear voltage gradient from 100 to 1000 V for 5 h, followed by 1000 V for 4 h, and an exponential gradient up to 5000 V, for a total of 25 kV/h. For the second dimension, the IPGs strips were equilibrated for 25 min in a solution containing 6 M urea, 2% SDS, 20% glycerol, and 375 mM Tris-HCl (pH 8.8) under gentle shaking. The IPG strips were then laid on 7.5-18% or on 7.5-22% acrylamide gradient SDS-PAGE gel slab with 0.5% agarose in the cathodic buffer (192 mM glycine, 0.1% SDS and Tris-HCl to pH 8.3). The electrophoretic run was performed at 5 mA/gel for 1 h, followed by 10 mA/gel for 1 h and 15 mA/gel until the dye front reached the gel bottom. Gels were incubated in a colloidal Coomassie Blue solution,28 and destaining was done in 7% acetic acid until clear background, followed by rinsing in deionized water.
Results and Discussion Characterization of the Venom Proteome of C. atrox Using the Venomics Approach. To characterize the complement of secreted proteins, pooled crude venom of C. atrox was fractionated by reverse-phase HPLC (Figure 1), followed by analysis of each chromatographic fraction by SDS-PAGE (Figure 1, inset), N-terminal sequencing, and MALDI-TOF mass spectrometry (Table 1). Protein fractions showing single electrophoretic band, molecular mass, and N-terminal sequence were straightforwardly assigned by BLAST analysis (http://www. Journal of Proteome Research • Vol. 8, No. 6, 2009 3057
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Calvete et al. affecting hemostasis, including hemorrhagic, fibrinolytic, activation of prothrombin and Factor X31–33 and serine proteinases interfere in platelet aggregation, blood coagulation and fibrinolysis.34
Figure 1. Reverse-phase HPLC separation of the C. atrox venom proteins. Two milligrams of C. atrox venom was applied to a Lichrosphere RP100 C18 column, which was then developed with the following chromatographic conditions: isocratically (5% B) for 10 min, followed by 5-15% B for 20 min, 15-45% B for 120 min, and 45-70% B for 20 min. Fractions were collected manually and characterized by N-terminal sequencing, mass spectrometry, and SDS-PAGE (Table 1). Inset, SDS-PAGE showing the protein composition of the reverse-phase HPLC separated venom protein fractions run under nonreduced (upper panels) and reduced (lower panel) conditions. Molecular mass markers (in kDa) are indicated at the left and at the right of the figure. Protein bands were excised and proteins were identified by tryptic peptide mass fingerprinting and CID-MS/MS of selected doubly or triply charged peptide ions. The results are listed in Table 1.
ncbi.nlm.nih.gov/BLAST) to a known protein family. 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 ingel tryptic digestion. 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 collision-induced dissociation tandem mass spectrometry. Product ion spectra were submitted through the online form of the search engine MASCOT (http://www.matrixscience.com) to identify snake venom proteins. The MS/MS data were also manually interpreted for de novo sequencing and the CID-MS/MS-deduced peptide ion sequences (Table 1) were submitted to BLAST similarity searches against a nonredundant database. The 30 fractions isolated by reverse-phase HPLC (Figure 1) comprised at least 24 different proteins (Table 1). Supporting the view that venom proteomes are mainly composed of proteins belonging to a few protein families,16,20,21 C. atrox proteins belong to 8 different groups of toxins (Figure 2, Table 2), distributed into 2 major protein families (snake venom metalloproteinases, SVMP, and serine proteinases) representing 69.5% of the total venom proteins, 4 medium abundance families (medium-size disintegrin, PLA2, cysteine-rich secretory protein, and L-amino acid oxidase) amounting to 25.8% of the venom proteins, and 3 minor protein families (vasoactive, bradykinin-influencing peptides; endogenous inhibitor of SVMP; and C-type lectin). This toxin profile fully explains the cytotoxic, myotoxic, hemotoxic, and hemorrhagic effects evoked by C. atrox envenomation. Thus, cytotoxic and myotoxic PLA2 molecules account for most of the muscle necrosis that results from envenenomation by crotaline snakes29,30 SVMPs, also called hemorrhagins, display a wide range of biological activities 3058
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The peptide TPPAGPDVGPR found in fraction Ca-2 (Figure 1, Table 1) is identical to a bradykinin-inhibitory peptide (BIP) reported in the venoms of three species of NewWorld pit vipers from the subfamily, the Mexican moccasin (Agkistrodon bilineatus), the prairie rattlesnake (Crotalus viridis viridis), and the South American bushmaster (Lachesis muta).35 BIPs are proteolytically released from larger (∼180-residue) precursors, which also encode an N-terminal bradykinin-potentiating peptide (BPP) and a C-terminal C-type natriuretic peptide (NAP)35 (e.g., UniProt entry B0VXV8 from the desert massasauga, Sistrurus catenatus edwardsi; Q90Y12 from the tropical rattlesnake, Crotalus durissus terrificus; and Q2PE51 from the South American rattlesnake, C.d. collineatus). The occurrence of BPP, BIP, and NAP in roughly equimolar amounts support the existence in C. atrox too of a common precursor of these three types of vasoactive peptides. The phylogenetic conservation in peptide primary structure is suggestive of a relevant biological role for BIP in the mode of action of New World snake venoms. A synthetic replicate of the BIP was found to antagonizes the vasodilatory actions of bradykinin at the B2 receptor in a rat-tail artery preparation.35 Although BIP appeared to be a less potent vasoconstrictor than safarotoxins,35 a group of 21-residue cardiotoxic peptides isolated from African snake venoms of genus Atractaspis that induce severe coronary vasoconstriction leading to bradycardia and vasospasm,36,37 BIP may disrupt the functioning of the cardiovascular system, supplementing the overall toxic effect of the snake venom. By contrast, the vasoactive peptide precursors found in Bothrops species, that is, Bothrops jararaca [Q6LEM5], Bothrops jararacussu [Q7T1M3], and Bothrops insularis [P68515], contain several N-terminal BPPs and the C-terminal NAP, but lack BIP, indicating a different evolutionary trend in this group of snakes, which have evolved hypotensive venoms. Mapping the different vasoactive peptide precursor structures onto a cladogram of Crotalinae suggests that duplications of the N-terminal region bearing the BPPs occurred in the common ancestor of Lachesis and Bothrops, and that BIP was subsequently lost in Bothrops (Figure 3). C. atrox is among the most thoroughly investigated crotalid species. The venom proteins identified in this work represent all the 24 (partial or full-length) C. atrox venom proteins present in the Serpentes Tox-Prot database (the UniProtKB/Swiss-Prot Toxin Annotation Program comprising 1168 annotated snake venom toxin entries from 158 species) (http://us.expasy.org/ sprot/tox-prot), with the exception of the PI-SVMP atroxase [Q91401] and the PIII-SVMP VAP-2A [A4PBQ9]. The latter entry has been only described at the transcript level, however. On the other hand, our proteomic analyses disclosed the presence of three novel venom components, a low molecular mass protein (Ca-12) and two PLA2 molecules (Ca-14/15 and Ca-17) (Figure 1, Table 1). These data suggest that the repertoire of C. atrox toxins deposited in the Tox-Prot database represent all major proteins secreted in the venom of the western diamond rattlesnake. However, to really understand how venoms work, quantitative data on the occurrence of individual toxins in a given venom are required. The proteomic data described in this paper provides an overall view of the relative abundance of the C. atrox venom toxins.
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Table 1. Assignment of the Reverse-Phase Isolated Fractions of C. atrox Venom (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 Bandsa peptide ion HPLC fraction Ca-
N-terminal sequencing
isotope-averaged molecular mass
m/z
z
385.2
1
MS/MS-derived sequence
1 2
TPPA TPPAGPDVGPR
1063.1*
3, 4
n.p.
m: 429.1
5
AGEECDCGSPANPCCDAAT
7505.4*
6
GEECDCGSPANPCCDAATCKLRP
7434.1*
ECDCGSPANPCCDAATCKLRPGA
7248.1*
977.8
2
GDWNDDTCTGQSADCPR
679.3 458.7 607.3
3 2 2
LRPGAQCADGLCCDQCR GTVCRPAR ZLWPRPQIPP
769.1
3
VSMVDRNDDTCTGQSADCPR
798.6 799.0 695.6
2 3 3
NDDTCTGQSADCPR GIECDCGSLENPCCYATTCK MRPGSQCAEGLCCDQCR
7
Blocked
1213.8*
8
CDCGSPANPCCDAATCKLRPGAQ
7061.8*
9
IECDCGSLENPCCYATTCKMRPG
7584.6*
10
GIECDCGSLENPCCYATTCKMRP
7641.3*
11
AKKRAGNGCFGLKLDRIGSMSGLGC
2537.6*b
12
Blocked
6 kDa
13
SLVELGKMILQETGK
13572*
14, 15
SVDFDSESPRKPEIQ
26679*
14, 15
NLLQFNKMIKIMTKK
TPPA
ZNW
14198*
446.7 501.7 735.2 555.7 697.8 723.9 658.9 769.4 642.8 617.3 704.6 438.3
2 2 2 2 2 2 3 2 2 2 3 2
FXXCPSR XGCE(210.1)WK (327.2)CEEEGGFCR MDGYTYSFK DGTDRCCFVHK TIICDVNNPCLK NPITSYGIYGCNCGVGSR MEWYPEAAANAER IGCAAAYCPSSK SLVQQAGCQDK QMQSDCPAICFCQNKII NLLQFNK
490.8 581.8 659.6 789.2 721.6 749.9
2 2 2 2 3 2
QICECDR TDIYSYSWK TDIYSYSWKR CCFVHDCCYEK NAFPFYTSYGCYCGWGGR VVGGDECNINEHR
565.3
2
FLVALYTFR
2 2 2 2
CCFVHDCCYGK DNIPSYDNK SLVQFETLIMK IMGWGTISPTK
protein/ protein family
N-terminal part of Ca-2 Bradykinin inhibitory peptide [∼P85025] Endogenous inhibitor of SVMPs Disintegrin crotatroxin [P68520] 1-72 Disintegrin crotatroxin [P68520] 2-72 Disintegrin crotatroxin [P68520] 4-72
Bradykinin-potentiating peptide [P0C7S6] Disintegrin crotatroxin [P68520] 5-70 Disintegrin atrolysin-e [P34182] 4-73 Disintegrin atrolysin-e [P34182] 3-73
C-type natriuretic peptide [∼Q90Y11] Unknown
K49-PLA2 [Q8UVZ7]
CRISP [Q7ZT99]
∼PLA2 [Q71QE8] C. viridis viridis
16-18
VVGGDECNINEHRSLVAIFVST
26900*
17 18
SLGQFETLIMKIAGR SLVQFETLIMKIAGR
13640* 13585*
19, 20
IIGGDECNRNEHRFLALVSSDG
25930*
753.3 533.3 654.9 596.2
162841
698.8 832.8 586.3
2 3 2
AAYPEYGLPATSR VILPDVPHCVNINLLNYSVCR GQENVWIGLR
621.3 639.8 736.9 772.7 639.9 753.3 475.8 776.8
2 2 2 2 3 2 2 3
517.2
2
DFSWEWTDR LWNDQVCESK YKPGCHLASFHR EFCVELVSLTGYR YGESLEIAEYISDYHK CCFVHDCCYGK PLA2 [P00624] YWLFPPK SAYGCYCGWGGHGLPQDATDR Serine proteinase [Q9PRW2/4, Q9PS55] YCYQEDR C-type lectin-like [∼ Q719L8]
20, 21
NNCPLDWLPMNGLCYKIFNQLK
SLVQFETLIMKIAGR
14.5 kDa1,9
21
VIGGDECNINEHRSL
27 kDa1,9
21-23
N.D.
301/ 169 kDa
Serine proteinase Catroxobin [Q7LZF5] PLA2 PLA2 [P00624]
Serine proteinase catroxase I [Q8QHK3]
Galactose-specific lectin [P21963]
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Table 1. Continued peptide ion HPLC fraction Ca-
22
N-terminal sequencing
VVGGDECNINEHRSL
isotope-averaged molecular mass
27 kDa1,9
m/z
z
789.6 749.9
2 2
GFCCPSGWSSYDR VVGGDECNINEHR
MS/MS-derived sequence
551.8 534.8
2 2
TLCAGIPEGGK VPNEDEQTR
23
V(V/I)GGDECNINEHRSLVAIFVSTE
27.6 kDa1,9
24
AHDRNPLEECFRETDY
56.8 kDa1,9
678.1
3
LNEFSQENENAWYFIK
23071*
612.2 619.2 519.3 532.3 485.6 557.8 748.9 777.9
2 2 2 2 2 2 3 2
DWYANLGPMR SAAQLYVESLR FWEDDGIR NPLEECFR VQVHFNAR VIEIQQNDR FDEIVGGMDQLPTSMYEAIK STGVVQDHSEINLR
53-56 kDa1,9
743.3 547.3 555.2 907.6 526.6
3 2 2 2 2
SHDNAQLLTSIAFDEQIIGR AYIGGICDPK TLNSFGEWR YVELFIVVDHGMYTK GNYYGYCR
110 kDa1
776.8 766.3 684.9 798.3 822.8 885.2 604.9 622.5
2 2 3 2 2 2 2 3
VCSNGHCVDVATAY VIGLAYVGSMCHPR ITVKPEAGYTLNAFGEWR HDNAQLLTAIDLDR MYEIVNTVNEIYR SGSQCGHGDCCEQCK FVELFLVVDK IENDADSTASISACNGLK
26 kDa1,9
860.6 662.1 548.2
2 2 2
IIVQSSADVTLDLFAK YEDAMQYEFK YNSDLNTIR
25 kDa1,9
637.1 701.9 803.8 612.2
2 2 2 2
YIELVVVADHR GASLCIMRPGLTK VHEIVNFINGFYR NTLNSFGEWR
25 kDa1,9
622.6 554.3 593.6 657.3 781.3 890.9 605.3
3 2 2 2 2 2 2
IENDADSTASISACNGLK YNSDLNIIR AYTSSMCNPR YVELVIVADHR VHELVNTINGFYR SHDHAQLLTAINFEGK NPDQQNLPQR
15 kDa1,9
804.9 612.2
2 2
VHEIVNFINGFYR NTLNSFGEWR
781.3 657.2
2 2
VHELVNTINGFYR YVELVIVADHR
555.2 777.8
2 2
TLNSFGEWR STGVVQDHSEINLR
23-25
26-30
28
NPEHQRYVELFIVVD
Blocked
Blocked
30
N.D.
42 kDa1,9
29, 30
N.D.
26 kDa
1,9
protein/ protein family
Serine proteinase catroxase 2 [Q8QHK2]
Serine proteinase catroxobin [Q7LZF5] L-amino acid oxidase [P56742]
PI-metalloprotease HT-e [P34182] 1-202
PIII-SVMP Catrocollastatin [Q90282]
PIII-SVMP VAP-1 [Q9DGB9]
PI-SVMP Atrolysin C [Q90392]
PI-SVMP Atrolysin B [Q90391, Q91401]
PI-SVMP Atrolysin D [P15167] PI-SVMP Atrolysin B [Q90391] fragment PIII-SVMP Atrolysin A [Q92043] PI-SVMP HT-e [P34182]
a X, Ile or Leu. Unless other stated, for N-terminal sequencing and MS/MS analyses, cysteine residues were pyridylethylated and carbamidomethylated, respectively. Molecular masses of the native components (*) were determined by electrospray-ionization mass spectrometry, MALDI-TOF, or by SDS-PAGE before (1) or after (9) sample reduction with β-mercaptoethanol; n.p., nonpeptidic material found; m, minor component; N.D., not determined. b Cysteines are engaged in disulfide linkage.
The absence of the DC-fragment, cat-C, which represents the C-terminal disintegrin-like and cysteine-rich domains from the proteolytically processed C. atrox PIII-SVMP catrocollastatin [Q90282],38,39 is worth mentioning. Venom-isolated DC-fragments have been reported to impair collagen-induced platelet aggregation in vitro by blocking the adhesive function of the 3060
Journal of Proteome Research • Vol. 8, No. 6, 2009
R2β1 platelet integrin.40,41 The low yield and uneven distribution of DC-fragments among viperid venoms, along with the striking observation that the disulfide bond pattern of the DC domains of full-length catrocollastatin as observed from the crystal structure42,43 is rather different from that determined by MS for cat-C isolated from a C. atrox venom pool;44 it has been
Exploring the Venom Proteome of Crotalus atrox
Figure 2. Overall protein composition of C. atrox venom. Relative abundance of different toxin families in the venom of C. atrox. BPP, bradykinin-potentiating peptide; BiP, bradykinin-inhibitor peptide; NAP, C-type natriuretic peptide; PLA2, phospholipase A2; CRISP, cysteine-rich secretory protein; C-type lectin, C-type lectinlike protein; LAO, L-amino acid oxidase; PI and PIII-SVMP, snake venom metalloproteinase from class I and III, respectively. Details of the individual proteins are shown in Table 1 and the percentages of the different toxin families in the venom are listed in Table 2. Table 2. Overview of the Relative Occurrence of Proteins (in Percentage of the total HPLC-Separated Proteins) of the Different Families in the Venom of C. atrox protein family
Vasoactive peptides: • Bradykinin-inhibitory peptide • Bradykinin-potentiating peptide • C-type natriuretic peptide Endogenous inhibitor of SVMPs Medium-size disintegrin PLA2 CRISP Serine proteinase C-type lectin-like: • Gal-specific lectin • Other C-type lectin-like L-amino acid oxidase Zn2+-metalloproteinases: • PI-SVMP • PIII-SVMP Other proteins:a • Peroxiredoxin • Glutaminyl cyclase a
% of total venom proteins
3.0 1.1 0.9 1.0 traces 6.2 7.3 4.3 19.8 1.7 1.6 0.1 8.0 49.7 22.4 27.3
