Biological and Proteomic Analysis of Venom from the Puerto Rican Racer (Alsophis Portoricensis: Dipsadidae)

Toxicon 55 (2010) 558–569

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Toxicon

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Biological and proteomic analysis of venom from the Puerto Rican Racer (Alsophis portoricensis: Dipsadidae)

Caroline L. Weldon, Stephen P. Mackessy*

School of Biological Sciences, University of Northern Colorado, 501 20th Street, CB 92, Greeley, CO 80639-0017, USA article info abstract

Article history: The Puerto Rican Racer Alsophis portoricensis is known to use venom to subdue lizard prey, Received 12 August 2009 and extensive damage to specific lizard body tissues has been well documented. The Received in revised form toxicity and biochemistry of the venom, however, has not been explored extensively. We 27 September 2009 employed biological assays and proteomic techniques to characterize venom from Accepted 2 October 2009 A. portoricensis anegadae collected from Guana Island, British Virgin Islands. High metal- Available online 14 October 2009 loproteinase and gelatinase, as well as low acetylcholinesterase and phosphodiesterase activities were detected, and the venom hydrolyzed the a-subunit of human fibrinogen Keywords: Biological roles very rapidly. SDS-PAGE analysis of venoms revealed up to 22 protein bands, with masses of w Colubrid 5–160 kDa; very little variation among individual snakes or within one snake between CRISP venom extractions was observed. Most bands were approximately 25–62 kD, but MALDI- Enzymes TOF analysis of crude venom indicated considerable complexity in the 1.5–13 kD mass a-Fibrinogenase range, including low intensity peaks in the 6.2–8.8 kD mass range (potential three-finger Gland histology toxins). MALDI-TOF/TOF MS analysis of tryptic peptides confirmed that a 25 kDa band was Hemorrhage a venom cysteine-rich secretory protein (CRiSP) with sequence homology with tigrin, Mass spectrometry a CRiSP from the natricine colubrid Rhabdophis tigrinus. The venom was quite toxic to NSA Metalloproteinase mice (Mus musculus:LD 2.1 mg/g), as well as to Anolis lizards (A. carolinensis: 3.8 mg/g). Toxin 50 ¼ Histology of the venom gland showed distinctive differences from the supralabial salivary glands (serous vs. mucosecretory), and like the Brown Treesnake (Boiga irregularis), another rear-fanged snake, serous secretory cells are arranged in densely packed secretory tubules, with little venom present in tubule lumina. These results clearly demonstrate that venom from A. portoricensis shares components with venoms of front-fanged snakes as well as with other rear-fanged species. Venom from A. portoricensis, in particular the prominent metalloproteinase activity, likely serves an important trophic function by facilitating prey handling and predigestion of prey. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction a function of phylogeny and diet, as well as the inﬂuence of several other factors (Mackessy, 2009). An important bio- A prominent evolutionary trend seen among the logical role of venom in snakes is the immobilization of advanced snakes is toward the production of toxic oral potentially dangerous, struggling prey, and by injecting secretions (venoms) and away from constriction as the venom which slows, paralyzes or kills the prey, the snake dominant method of subduing prey (Kochva, 1987). secures a meal and avoids injury. Venom of many species, Venoms vary in complexity and composition, in part as particularly viperids, also produces extensive tissue necrosis and likely acts to increase rates of digestion of large prey items by ‘‘predigesting’’ the prey (Thomas and * Corresponding author. Tel.: þ1 970 351 2429; fax: þ1 970 351 2335. Pough, 1979; Mackessy, 1988). Because many venomous E-mail address: [email protected] (S.P. Mackessy). snakes are capable of consuming prey items of much larger

0041-0101/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2009.10.010 C.L. Weldon, S.P. Mackessy / Toxicon 55 (2010) 558–569 559 size in proportion to their bodies than non-venomous commonly characterized by minor to significant bleeding snakes (Greene, 1997), the increased surface area available (Kuch and Mebs, 2002) due in large part to the presence of for gastric digestion caused by the venom breaking down hemorrhagic toxins (Takeya and Iwanaga, 1998), which can the prey internally as the snake digests the prey’s external be serine proteinases or metalloproteinases, although surface is selectively advantageous (Mackessy, 1988). hemorrhage is not necessarily due only to the proteinase The paraphyletic family ‘‘Colubridae’’ is the largest components. Metalloproteinases may also show muscle- family of modern snakes, containing at least 700 venomous damaging activity, and myotoxic snake venom metal- species world-wide (Cadle, 1994; Vidal, 2002). This clade of loproteinases have been isolated from the venoms of several advanced snakes has been the subject of numerous colubrid snakes (e.g., Philodryas olfersii: Prado-Franceschi morphological and molecular analyses, and the phyloge- et al., 1998; Rhabdophis tigrinus: Komori et al., 2006; netic hypotheses of recent work (Vidal et al., 2007; Quijada- P. patagoniensis: Peichoto et al., 2007). Mascarenãs and Wu¨ ster, 2009) will be followed here; The Puerto Rican racer (Alsophis portoricensis), now however, the term ‘‘colubrid’’ will be used generically and considered a member of the family Dipsadidae (Vidal et al., for convenience. These snakes vary greatly in size, distri- 2007), is a rear-fanged colubrid snake found on numerous bution and diet, and although various species are not islands in the Caribbean. Alsophis portoricensis are ground- closely related, many possess enlarged rear teeth and dwelling, diurnal snakes that have been observed enveno- a Duvernoy’s gland (Weinstein and Kardong, 1994), mating prey before consumption (Rodriguez-Robles and a homolog of the venom gland of front-fanged snakes. Thomas,1992). The diet of A. portoricensis primarily includes Colubrid snakes are generally considered harmless, but lizards (Anolis sp.) and Eleutherodactylus frogs, but they have there have been documented human fatalities from enve- also been observed preying on dead fish, bird eggs, iguanas, nomation by the Boomslang (Dispholidus typus)(Pope, other snakes, and rats (Rodriguez-Robles and Leal, 1993a, b; 1958) and species from three other genera of colubrids Norton, 1993). Although typically hesitant to bite when (Thelotornis, Rhabdophis and Philodryas)(FitzSimons and confronted, A. portoricensis has been documented to cause Smith, 1958; Mittleman and Goris, 1976; Kornalik and cases of human envenomation resulting in edema, paraes- Taborska, 1978; Ogawa and Sawai, 1986; Prado-Franceschi thesia and ecchymosis at the bite site (Heatwole and et al., 1996). It is probable that the ‘‘non-venomous’’ nature Banuchi, 1966; R. Platenberg and K. Lindsay, pers. comm.). of most colubrids is not due to a lack of enzymes or toxins Because venom composition appears to be related to prey in the venom, but instead is a result of evolution of venoms type and form (Mackessy,1988; Daltry et al.,1996; Gibbs and that differentially affect non-mammalian taxa (Mackessy Mackessy, 2009), the types of toxins utilized by particular et al., 2006; Pawlak et al., 2009). With the vast majority of snake species to facilitate prey handling may differ colubrid venoms still unstudied, there is opportunity to depending on the type of prey animal utilized. For example, discover novel biological compounds with possible medical venom from the Brown Treesnake (Boiga irregularis) has implications; however, one obstacle to the characterization been shown to possess taxon-specific toxins that affect birds of colubrid venoms is the exceedingly small quantities of and reptiles to a much greater extent than mammals, and raw material previously obtainable (Mackessy, 2002). Most this snakes feeds primarily on non-mammalian prey colubrids are of a small size and, therefore, yield small (Mackessy et al., 2006; Pawlak et al., 2009). Alsophis por- amounts of venom. While this previously hindered study, toricensis venom may also be more toxic to reptiles than to efficient extraction is now possible (Hill and Mackessy, mammals because reptiles make up the majority of the diet 1997, 2000). of this snake. Hegeman (1961) commented on the hemolytic Snake venoms are of considerable interest pharmaco- and proteolytic activities of A. portoricensis venom, but was logically because they contain compounds useful as probes unable to characterize it further due to a lack of both crude of ion channels and other physiological processes and as material and technical sophistication. The predatory use of leads for drug development. Colubrid snakes typically have venom by A. portoricensis has been documented, and enve- less complex venoms than those of front-fanged snakes nomated Anolis lizards appeared to suffer respiratory (Viperidae, Elapidae), but they share many of the same distress resulting from hemorrhage in the lungs (Prieto- components including metalloproteinases, serine prote- Hernandez,1985), perhaps due to the action of SVMPs. In the ases, phosphodiesterases, acetylcholinesterases, phospho- current study, biological assays and proteomic techniques lipases A2 (PLA2) and three-finger toxins (3FTXs) were employed to characterize more fully the venom of (Mackessy, 1998, 2002, 2009; Hill and Mackessy, 2000; Fry A. portoricensis and to provide a biochemical explanation for et al., 2003; Pawlak et al., 2006). Several of these (metal- the effects of envenomation previously described. We also loproteinase and acetylcholinesterase activities) have been hypothesized that like B. irregularis venom, A. portoricensis previously reported in the venom of the Puerto Rican Racer, venom would have taxon-specific effects, with greater Alsophis portoricensis (Hegeman, 1961). potency toward lizard prey. Metalloproteinases are the most abundant and diverse enzymatic proteins found in most viperid venoms (Fox and 2. Materials and methods Serrano, 2005; Pahari et al., 2007). Hemorrhagic metalloproteinases are responsible for the severe local inflam- 2.1. Reagents mation and tissue necrosis seen in human envenomations (Rucavado et al., 1995, 1999), and are biologically very Protein concentration reagents were purchased from important to the predigestion effects of these venoms on BioRad Inc. (San Diego, CA). Novex Mark 12 unstained prey (Mackessy, 1988, 1993b). Colubrid envenomations are molecular mass standards, MES running buffer, LDS sample 560 C.L. Weldon, S.P. Mackessy / Toxicon 55 (2010) 558–569 buffer and precast electrophoretic gels (Zoom, NuPAGE was assayed and evaluated at 405 nm (Mackessy, 1993a). Novex Bis-Tris, Novex Zymogram) were obtained from Ten mg of crude venom was used and specific activities were Invitrogen, Inc. (San Diego, CA). Phospholipase A2 assay kit reported as nmol product/min/mg protein. All enzyme was purchased from Cayman Chemical Co. (Ann Arbor, MI). assays were performed on venom from three individual Immobiline DryStrip immobilized pH gradient (IPG) strips, snakes, all done in triplicate. Negative controls were also buffers and an Ettan IPGphor isoelectric focusing unit were performed in triplicate. Positive controls included obtained from GE/Amersham Biosciences, Inc. (Piscataway, numerous rattlesnake venoms assayed previously (Mack- NJ). Acetyl-L-Lys-L-Leu-L-Asn-L-Lys-L-Arg-paranitroaniline essy, 2008). The metalloproteinase and phosphodiesterase was synthesized by EZ Biolab (Carmel, IN). A Trypsin Profile assays described above were also used to determine IGD kit and all other buffers and reagents (analytical grade) activities in fractions generated from SE-HPLC. were purchased from Sigma-Aldrich, Inc. (St. Louis, MO). Some colubrid snake venoms, including those of P. olfersii and P. patagoniensis, have been shown to degrade 2.2. Alsophis portoricensis venom fibrinogen, interfering with blood coagulation via hypofibrinogenemia (Assakura et al., 1994; Peichoto et al., 2007). Venom was extracted from A. portoricensis collected on Fibrinogenase activity was determined using 20 mg of crude Guana Island, British Virgin Islands and housed at the UNC venom incubated with human fibrinogen (final concen- Animal Facility in accordance with UNC-IACUC protocol No. tration 0.5 mg/mL) in a total volume of 200 mL for periods of 9204.1. Snakes were extracted no more frequently than 0, 1, 5, 10, 30, and 60 min (Ouyang and Huang, 1979). once every 6 weeks using the method of Hill and Mackessy Twenty mL of this reaction mixture was removed at each (1997) with ketamine-HCl (32 mg/kg) followed by pilo- time point and mixed with an equal volume of 4% SDS and carpine HCl (6 mg/kg), and venom was collected from the 5% 2-mercaptoethanol. Five mL aliquots of the resulting base of the enlarged rear maxillary teeth into micro- solutions were combined with 2 LDS buffer, electro- capillary tubes. The venom was lyophilized and stored phoresed on a 12% NuPAGE gel, stained with Coomassie frozen at 20 C until needed. Protein content of the Brilliant Blue, and destained. All gels were imaged using an venom was determined using bovine gamma globulin as Alpha Imager system. a standard and the method of Bradford (1976) as modified by BioRad Inc. These calculated protein concentrations, 2.4. One-dimensional gel electrophoresis performed in triplicate for each crude venom sample, were the basis of all other analyses and enzymatic activity Crude venom from two extractions of three different calculations. A. portoricensis was used to assess the number and relative molecular masses of venom components as well as to 2.3. Enzyme assays determine whether variation in venom composition exists among individuals and/or between extractions. NuPage Metalloproteinase activity of the crude venom was Bis-Tris gels (Invitrogen, Inc.) and MES running buffer were evaluated using azocasein as a substrate (Aird and da Silva, utilized for SDS-PAGE analysis, with samples and buffers 1991). The activity was expressed as DA342 nm/min per mg prepared according to the manufacturer as described venom protein. Phosphodiesterase activity was assayed previously (Mackessy et al., 2006). Thirty mg of crude using the methods of Bjork (1963) and Laskowski Sr., 1980 A. portoricensis venom was added to each lane. Other crude as modified by Mackessy (1998). Thymidine 50-mono- colubrid venoms (30 mg each) as well as 2.5 mg purified phospho-p-nitrophenyl ester substrate was incubated with denmotoxin (a 3FTx from Boiga dendrophila venom; Pawlak crude venom and specific activity was expressed as et al., 2006) were run for comparison, and 5 mL Mark 12 DA400 nm/min per mg protein. Acetylcholinesterase activity standards (Invitrogen, Inc.) was used to estimate masses. was determined using 10 mg crude venom incubated with The gel was then stained with Coomassie Brilliant Blue, acetylthiocholine iodide substrate (Ellman et al., 1961)in destained and imaged with an AlphaImager. a jacketed cuvette at 37 C. The absorbance at 412 nm was The number and relative size of components with recorded every minute for 15 min and the linear portion of endoproteinase (gelatinase) activity were determined the graph was used to calculate activity, reported as mmol using varying amounts of crude venom (0.001, 0.005, 0.01, product formed per minute per mg venom protein. Phos- 0.05, 0.1, 0.5, 1.0, and 5.0 mg per lane) run on a 10% gelatin pholipase A2 activity (Huang and Mackessy, 2004)was Zymogram gel (Munekiyo and Mackessy, 1998). The gel was evaluated using a commercially available kit (Cayman placed sequentially in renaturing and developing buffers Chemical Co.) as described by the manufacturer using (1 h each) and incubated at 37 C overnight with gentle 1.0 mg crude A. portoricensis venom (200 mL final total agitation. The gel was then stained with 0.1% Coomassie volume). Absorbance was measured at 414 nm every Brilliant Blue, destained, and imaged. Areas of gelatinase minute for 15 min (37 C) and activity was reported as mmol activity appeared clear on a blue field. product/min/mg protein. Activity toward paranitroaniline- derived (pNA) synthetic substrates for thrombin (BzPhe- 2.5. Two-dimensional gel electrophoresis ValArg-pNA), trypsin (BzArg-pNA), thrombin/trypsin (TosylGlyProArg-pNA), plasmin (ValLeuLys-pNA), arginine Seven cm IPG strips (GE/Amersham Biosciences, Inc.) of esterase (CBZ-L-Arg-pNA), kallikrein (ProPheArg-pNA), pH 3–11 were rehydrated with crude venom solutions from elastase (SuccAlaAlaAla-pNA), leucine aminopeptidase either A. portoricensis or Crotalus ruber (Red Diamondback (Leu-pNA), and cone snail CRiSP (AcLysLeuAsnLysArg-pNA) Rattlesnake) and subjected to isoelectric focusing using an C.L. Weldon, S.P. Mackessy / Toxicon 55 (2010) 558–569 561

Ettan IPGphor isoelectric focusing unit (GE Amersham venom were evaluated using both lizards (Anolis carolinensis) Biosciences, Inc.) as per manufacturer instructions. Briefly, and non-Swiss albino (NSA) mice (Mus musculus). Mice 160 mg of lyophilized venom from each snake was dissolved were bred in the UNC Animal Facility and lizards were in 125 mL of rehydration buffer (8 M urea, 2% CHAPS, 2.0% obtained from Animal Attractions (Greeley, CO); all proce- IPG buffer, trace bromphenol blue, and freshly added DTT dures were reviewed and approved by the UNC IACUC (18.2 mM)). These solutions were added to each strip in IPG (protocol No. 9401). Venom was introduced intraperito- strip holders, covered with mineral oil, and rehydrated for neally (IP) in sterile saline, with doses adjusted for the body 12 h at 22 C and 30 V on the IPGphor system. The strips mass of each animal. Three animals were used at each were then focused for 4.5 h at 22 C with a maximum of dosage in an effort to minimize animal use numbers and 5000 V (50 mA/strip). After focusing, the strips were were monitored for 24 h post injection. The 24 h LD50 (Reed reduced (4 mL of Tris/SDS equilibration buffer with 65 mM and Muench, 1938) was expressed in mg venom/g animal DTT) for 15 min and then alkylated (4 mL of Tris/SDS body mass. Necropsies were performed on animals that equilibration buffer with 135 mM iodoacetamide) for succumbed to the venom to analyze further the extent of 15 min. Next, the strips were equilibrated with 4 mL tissue damage. NuPAGE LDS sample buffer (1) for 10 min, placed in the IPG wells of ZOOM gels, overlain with 0.5% agarose, and run 2.9. Hemorrhagic assay according to the manufacturer’s instructions. After staining with Coomassie Brilliant Blue and destaining, the spots on The hemorrhagic activity of the crude venom was the two gels were visually compared with each other as evaluated using a method based on Bjarnason and Fox well as with the molecular mass standards. (1983). Lyophilized crude venom was dissolved in 25 mLof 0.9% sterile saline and injected intradermally (ID) into 2.6. MALDI-TOF mass spectrometry and trypsin digestion 4 week old female NSA mice. After a 2 h incubation period, the mice were euthanized using CO2. The skins were then A sample of lyophilized crude venom was analyzed using removed, pinned fur side down, photographed and a Bruker Ultraflex MALDI-TOF/TOF mass spectrometer hemorrhagic spots measured to the nearest mm. Dosages (Proteomics and Metabolomics Facility, CSU, Fort Collins, were given in duplicate at 0 (control), 1.0 and 5.0 mg. The CO). Approximately 1 mg of crude venom was spotted onto mice used in this assay (six total) were bred in the UNC sinapinic acid matrix. For identification of venom proteins, Animal Facility and all procedures were reviewed and bands of interest (50, 25 and 16 kDa) were excised from a 1D approved by the UNC IACUC (protocol No. 9401). SDS-NuPAGE gel of crude A. portoricensis venom and proteins were digested using a trypsin in-gel digest kit 2.10. Histology of the venom (Duvernoy’s) gland (Sigma-Aldrich, Inc.; product #PP0100). Digest samples were C18 ZipTip cleaned and then subjected to MALDI-TOF- One adult A. portoricensis was euthanized and the MS/MS analysis on an ABI 4800 mass spectrometer. Singly- glands were rapidly removed and preserved in buffered charged ions of selected peptides from the MALDI-TOF mass formalin as described previously (Mackessy and Baxter, fingerprint spectra were sequenced by collision-induced 2006). Preserved tissue was then serially dehydrated, dissociation tandem mass spectrometry. The spectra embedded in paraffin and 5 mm sagittal sections were generated were then analyzed using ABI ProteinPilot soft- obtained and stained with either hematoxylin/eosin or ware, and sequence data were compared against the BLASTP Periodic Acid-Schiff (Humason, 1978; sectioning and database (http://blast.ncbi.nlm.nih.gov/Blast.cgi, 15 January staining performed by Colorado Histo-Prep, Ft. Collins, CO). 2009) in order to identify venom proteins. Light micrographs were then recorded digitally using an Olympus CX41 microscope with a CCD camera, and images 2.7. Size exclusion (SE) HPLC were captured using SPOT Digital software (Diagnostics Instruments, Inc.) version 4.0.5. Images were optimized Five hundred mg of crude venom was dissolved in 200 mL using Adobe Photoshop Creative Suites 2.0. of 25 mM HEPES buffer, pH 6.8, containing 100 mM NaCl and 5.0 mM CaCl2, injected onto a TosoHaas TSK G2000 3. Results SWxl size exclusion column (7.8 mm ID 300 mm, 5.0 mm) equilibrated with the same buffer and run at a flow rate of 3.1. Snake sizes, venom yields and protein content 0.3 mL/min using a Waters HPLC system (Mackessy et al., 2006). The fractions were collected at 1 min intervals and Average snake length was 582 mm (SVL), and mass was absorbance was measured at 280 nm (Empower 2 soft- 68.9 g (N ¼ 33). Based on 30 extractions, venom yields for ware). Fractions from each peak were run on a 1D gel and each of six snakes averaged 160 mL (SD ¼70 mL). Dry mass compared with results for crude A. portoricensis venom. of venom averaged 5.9 mg (2.0 mg), and protein content Fractions were also assayed for acetylcholinesterase and of venoms averaged 89%. metalloproteinase activity as above. 3.2. Enzyme assays 2.8. Toxicity tests There was substantial metalloproteinase activity toward Because lizards make up a substantial part of Alsophis azocasein, with activities of the venoms of the three spec- diet, taxon-specificity and lethal toxicity of crude A. portoricensis imens averaging 1.25 (DA342 nm/min/mg venom protein). 562 C.L. Weldon, S.P. Mackessy / Toxicon 55 (2010) 558–569

Table 1 Speciﬁc activities of A. portoricensis venom toward various substrates. All values are mean values for three individual snakes, assayed in triplicate, standard deviation.

Assay Substrate Activity St. Dev. Units

Metalloproteinase Azocasein 1.254 0.086 DA342 nm/min/mg 0 Phosphodiesterase Thymidine 5 -monophospho-p-nitrophenyl ester 0.046 0.023 DA400 nm/min/mg Acetylcholinesterase Acetylthiocholine iodide 0.450 0.010 mmol product/min/mg

Phospholipase A2 1, 2-Dithiodiheptanoyl phosphatidylcholine 0.000 0.000 mmol product/min/mg Thrombin BzPheValArg-pNA 1.187 0.002 nmol/min/mg Plasmin ValLeuLys-pNA 0.251 0.0003 nmol/min/mg Elastase SuccAlaAlaAla-pNA 0.084 0.0002 nmol/min/mg Thrombin/trypsin TosylGlyProArg-pNA 37.85 0.011 nmol/min/mg Trypsin BzArg-pNA 0.028 0.0001 nmol/min/mg Kallikrein ProPheArg-pNA 0.527 0.001 nmol/min/mg Leucine aminopeptidase Leu-pNA 3.590 0.002 nmol/min/mg Cone snail CRiSP AcLysLeuAsnLysArg-pNA 1.940 0.001 nmol/min/mg Arginine esterase CBZ-L-Arg-pNA 0.419 0.0004 nmol/min/mg

Phosphodiesterase activity of A. portoricensis venom was 0.05 mg/lane, but activity was detectable at 0.005 mg/lane. 0.069 (DA400 nm/min/mg venom protein), relatively low The ﬁbrinogen digest revealed an almost immediate compared with several other colubrids (Hill and Mackessy, hydrolysis of the a-subunit of ﬁbrinogen (20 mg venom), 2000). The acetylcholinesterase assays revealed low and after 60 min there appeared to be some degradation of activity levels of 0.45 mmol product formed/min/mg venom the b-subunit as well (Fig. 2). protein. Low activity toward the thrombin/trypsin-like pNA substrate (TosylGlyProArg-pNA) was observed (37.8 nmol 3.3. One-dimensional gel electrophoresis product/min/mg protein). Assays for activity toward all other pNA substrates showed very low or no activity Approximately 20 protein bands were observed (Table 1), and there was no detectable PLA activity. The 2 between 10 and 80 kDa, with minimal variation in relative zymogram gel showed prominent gelatinase activity, as band intensity between venom extractions and among evidenced by the large cleared area in the gel at approxi- three different A. portoricensis individuals (Fig. 3, lanes mately 45 kDa (Fig. 1). The optimal amount of venom was 11–16). Prominent bands were found between 45 and 62 kDa, and one at 25 kDa, and three very faint, high molecular weight bands (w90–160 kDa) were also observed. There were several very faint, lower molecular mass bands (w5–9 kDa) also present (not visible in Fig. 3). Many of the other colubrid venoms compared also had prominent bands at 25 kDa and at 50–60 kDa (lanes 3–9), but A. portoricensis venom appeared to have greater

Fig. 2. Fibrinogen digest assay gel showing degradation of subunits after incubation with A. portoricensis venom for speciﬁc time intervals. Molecular mass standards appear in the far left lane. First lane after standards shows the three ﬁbrinogen subunits (a, b, g) intact at time 0. The following groups Fig. 1. Ten percent acrylamide zymogram gel image showing gelatinase of three lanes (venom from snake #86, #87 and #88) show rapid loss of activity of A. portoricensis venom. Activity of crude venom (0.05 mg/lane) a-subunit after incubation with venom (1, 5, 10, 30 and 60 min). Note the from each of three A. portoricensis (#86, #87, #88) is seen as clear regions of degradation products visible below the g-subunit band indicating partial the gel. Molecular mass standards appear in the left lane. degradation of the b-subunit by 60 min (arrow at right). C.L. Weldon, S.P. Mackessy / Toxicon 55 (2010) 558–569 563

a sequence of WAYQCAYDHSPRSSR (Fig. 6). A BLASTP search for this peptide revealed 100% sequence identity with residues 52–66 of tigrin (mature protein chain), a 25 kDa CRiSP isolated from Rhabdophis tigrinus venom (Yamazaki et al., 2002). The band at 16 kDa was identiﬁed as a hemoglobin blood contaminant; a peptide sequence derived from this protein matched a hemoglobin peptide only (data not shown). Attempts to obtain internal sequence of the putative metalloproteinase (w50 kDa) via MALDI-TOF-MS/MS were unsuccessful, perhaps due to the presence of numerous other proteins with similar masses.

3.6. Size exclusion HPLC

Five peaks were observed eluting between 19 and 47 min, and enzyme activities (metalloproteinase and Fig. 3. 1-D SDS PAGE comparing venoms of A. portoricensis to other colubrids. Molecular mass standards appear in lanes 1, 10, and 17. Samples: 2.5 mg purified denmotoxin (lane 2); crude venom (30 mg) from other colubrids: Hydrodynastes gigas (lane 3), Lioheterodon madagascariensis (lane 4), Leptodeira annulata (lane 5), Ahaetulla nasuta (lane 6), Boiga cyanea (lane 7), Boiga dendrophila (lane 8), Boiga irregularis (lane 9). Lanes 11–16: A. portoricensis, three individual snakes (A–C), first and second extractions (1 & 2) for each. Note the lack of variation between repeat extractions and between individuals. complexity in the 31–50 and 12–20 kDa range. Alsophis venoms appeared to lack detectable amounts of homologs of denmotoxin (lane 2), a taxon-specific, monomeric 3FTx, whereas homologs were present in all three Boiga venoms analyzed (lanes 7–9).

3.4. Two-dimensional gel electrophoresis

The 2D A. portoricensis venom gel had approximately 30 protein spots that spanned most of the 3–11 pH range, with the majority of the spots in the higher molecular mass, acidic region (Fig. 4A). Spots of the same molecular mass but of differing isoelectric points (likely isoforms of single proteins) were visible at w45, 58, and 62 kDa, as evidenced by a series of repeating spots (Fig. 4A, arrows). Discrete protein spots were visible at w35 and 50 kDa, but several were horizontally streaked, possibly due to incomplete focusing. Probable or conﬁrmed identity of several protein spots is indicated for Fig. 4A. The C. ruber gel (Fig. 4B) showed many more distinct proteins than the A. portoricensis gel, demonstrating the comparatively lower complexity of the proteome of A. portoricensis, typical of many colubrid snakes (Mackessy et al., 2006).

3.5. Mass spectrometry

MALDI-TOF spectrograms of the crude venom were generated using 1–14 and 15–70 kDa windows. The 1–14 kDa window revealed several species of low molec- Fig. 4. Two-dimensional SDS PAGE gels comparing complexity of A. portoricensis ular mass, including 3.56, 4.25, 11.62 and 12.59 kDa peaks venom (A) to that of Crotalus ruber (B); 160 mg venom each. Molecular mass as well as numerous lower intensity peaks (Fig. 5A). Four standards appear along the left of both gels. Top three arrows in (A) indicate major peaks at 17.47, 23.13, 25.18, and 50.36 kDa were possible protein isoforms in the acidic region between 50 and 60 kDa (likely visible in the 15–70 kDa scan (Fig. 5B). Following band 1 – metalloproteinases, 2 – acetylcholinesterases and 3 – phosphodiesterases); identiﬁed proteins are indicated by arrows 4 (CRiSP) and 5 (hemo- excision and trypsin digestion, MALDI-TOF-MS/MS spec- globin contaminant). Note the general lack of proteins in the lower trometry and analysis with ProteinPilot software revealed molecular mass region on the A. portoricensis gel compared with the same that a peptide derived from the 25 kDa protein band had region on the C. ruber gel. 564 C.L. Weldon, S.P. Mackessy / Toxicon 55 (2010) 558–569

Fig. 5. MALDI-TOF spectrograms of crude A. portoricensis venom generated using a Bruker Ultraﬂex mass spectrometer. (A) 1–14 kDa and (B) 15–70 kDa window scans. Note the numerous small mass peptides (w1400–9900 Da) in A which appear to be of very low abundance (based on comparison with 1D & 2D SDS-PAGE). phosphodiesterase) were detected in peak I fractions for animals of both species which received lethal doses (19–26 min) only (Fig. 7). Aliquots taken from the fractions revealed substantial hemorrhage at the injection site and that corresponded to each of the ﬁve peaks were electro- surrounding muscle tissue, as well as to the lungs. phoresed on a 1D gel. When compared to the 1D SDS-PAGE gel of crude venom, peak I encompassed the 45–58 kDa 3.8. Hemorrhagic assay bands, peak II (28–32 min) corresponded to the 25 kDa band (CRiSP), and the three peaks that eluted between 34 Hemorrhage was present in the subcutaneous tissue at and 47 min corresponded to the four lower molecular all ID dosages of crude venom (Fig. 8). The largest hemor- weight bands observed between 11 and 20 kDa. rhagic spot, present from the 5.0 mg venom assay, was 12 11 mm (Fig. 8C). 3.7. Lethal toxicity 3.9. Gland histology The venom was quite toxic to both mice and lizards, with an IP LD50 of 2.1 mg crude venom/gram body mass in mice Sagittal sections of the venom gland (Fig. 9)showedan and an IP LD50 for Anolis carolinensis of 3.8 mg/g. Necropsies overall pattern of organization similar to that of the Brown C.L. Weldon, S.P. Mackessy / Toxicon 55 (2010) 558–569 565

Fig. 6. MALDI-TOF MS/MS analysis of A. portoricensis crude venom. (A) Reducing SDS-PAGE image of A. portoricensis crude venom (50 mg/lane) from which the 25 and 16 kDa bands were excised and in-gel digested with trypsin. (B) Spectrogram and analysis using ProteinPilot software of a peptide derived from the 25 kDa protein from A. portoricensis venom.

Treesnake, Boiga irregularis (Zalisko and Kardong, 1992). The 4. Discussion Alsophis gland is composed of lobular subunits arranged into numerous secretory tubules which are lined by serous Many enzymes and toxins appear to be present in both secretorycells. The difference of these secretorycells from the front-fanged snakes and colubrids, and the biological roles (muco)polysaccharide-secreting cells of the supralabial sali- of venom in prey handling and digestion appear to be vary gland, which abuts the anterior end of the venom gland, universal (Mackessy, 1988, 1993a; Mackessy et al., 2006; was apparent by the differential staining of the glands (Figs. Pawlak et al., 2006, 2009; Gibbs and Mackessy, 2009). 9A,B,D). Mucosecretory cells of the salivary gland contained Despite the diminutive size of most venomous colubrid large diffuse cytoplasmic granules which were absent from snakes, they are still very capable predators, and many the (serous) cells of the venom gland. Most secretory tubules exclusively use a chemical means of dispatching prey. The were empty of venom. A large (often basal) storage lumen, relatively small size of the rear teeth is likely compensated common among front-fanged snakes, was absent in Alsophis, for by extreme maxillary rotation that allows for engage- as is typical of most colubrid venom glands. ment of these teeth into prey tissues. By ‘‘chewing’’ their

0. 051 0041. IIIkaeP 031.0 * 021.0 011.0 100.0 090.0 080.0 070.0 060.0 Abs (280 nm) (280 Abs 050.0 040.0 030.0 III VI 020.0 V 010.0 0.000

00.0 00.5 00.01 00.51 00.02 00.52 00.03 00.53 00.04 00.54 00.05 00.55 00.06 00.56 700.0 0 )nim(emiT

Fig. 7. HPLC size exclusion chromatogram of 500 mg crude venom. The ﬁrst peak in the chromatogram represents the metalloprotease and phosphodiesterase- containing peak (*), the second peak is the CRiSP peak, and the small peaks further downstream are the lower molecular mass toxins (w10–16 kDa). Flow rate was 0.3 mL/min, and absorbance was monitored at 280 nm. 566 C.L. Weldon, S.P. Mackessy / Toxicon 55 (2010) 558–569

Fig. 8. Hemorrhagic effects of A. portoricensis venom on NSA mice. Inner surface of mouse skin 2 h after intradermal injection with (A) 0.9% saline control, (B) 1 mg venom and (C) 5 mg venom dosages. Rule has 1 mm increments. prey as opposed to striking and releasing, colubrid snakes is likely that A. portoricensis venom, with high levels of are able to introduce venom into prey tissues at multiple metalloproteinase activity, has a similar role. The levels of sites, allowing the venom to produce diffused toxic effects. metalloproteinase activity detected were very high for Relatively long contact time appears to be necessary for a colubrid snake, and were comparable to those measured significant effects in humans (e.g., Kuch and Mebs, 2002), for venoms from some species of rattlesnakes (Bjarnason due in part to the intracellular storage of venom, and bites and Fox, 1994; Hill and Mackessy, 2000; Mackessy, 2008). from most colubrid snakes are not of clinical concern. Because metalloproteinases cause both localized and However, A. portoricensis occasionally are responsible for systemic hemorrhage and necrosis (Bjarnason and Fox, more serious human envenomations, and symptoms of 1995; Gutie´rrez and Rucavado, 2000), one or more metal- a recent extended bite included edema, generalized loproteinases are likely responsible for at least some of the arthralgia, paraesthesia in the bitten digit and minor observed damage in the mouse and lizard tissues. ecchymosis in the bitten limb (R. Platenberg and K. Lindsay, The severity of an envenomation depends on the nature of pers. comm.). Given the toxicity of A. portoricensis venom the venom components and, as demonstrated in this and toward both mice and lizards and anecdotal observations of other studies, colubrid snakes possess some venom human cases, an extended bite by a very large adult snake proteins in common with the venoms of front-fanged could easily result in clinically significant symptoms of snakes. Crude venom from A. portoricensis caused severe envenomation. localized ecchymosis, hemorrhage and necrosis in both Rattlesnakes use venom as a means of promoting mice and lizards, a finding consistent with the feeding digestion of large food items through ‘‘predigestion’’ of the behavior observations of Thomas 1985); hemorrhage was prey (e.g. Thomas and Pough, 1979; Mackessy, 1988), and it particularly pronounced in the lungs. C.L. Weldon, S.P. Mackessy / Toxicon 55 (2010) 558–569 567

Fig. 9. Structure of the venom (Duvernoy’s) gland of A. portoricensis. Overall organization is similar to that of Boiga irregularis, with the secretory epithelial tissue organized into lobules (L) containing secretory tubules (ST) and surrounded by connective tissue (CT), often also supporting blood vessels (BV). Secretory tubules join to form a larger lobular duct (LD), apparently ﬁlled with venom. Note differential staining of supralabial salivary gland (SG) compared to the venom gland (VG) in A, B & D; large (muco)polysaccharide-rich cytoplasmic granules are apparent in the SG, whereas the cells of the VG are primarily serous. A, B and C – hematoxylin/eosin stain; D – PASH stain. Scale bars ¼ 200 mm (A, B, D) and 50 mm (C).

However, the venom did not appear to show taxon- Ryan, 1997; Anderson and Dufton, 1998), A. portoricensis specific toxicity and was somewhat less toxic to Green apparently does not. The low acetylcholinesterase activity Anoles (Anolis carolinensis) than it was to inbred mice. was consistent with the low cholinesterase levels reported by Perhaps because envenomation is only one aspect of the Hegeman (1961). The combination of potent hemorrhagic, feeding sequence, the snake may rely more heavily on metalloproteinase, gelatinase and fibrinogenase activities mechanically subduing prey via constriction rather than (with low phosphodiesterase and acetylcholinesterase using chemical means (venom). It is also possible that activities) could certainly explain the pain and inflammation species of Anolis which are native to Guana Island associated with reported bites from A. portoricensis.By (including A. cristatellus and A. stratulus; Rodda et al., 2001) damaging structural proteins and interfering with the blood may be more susceptible to venom than A. carolinensis, and clot cascade, Alsophis venom is most likely used both to two different species of lizards have been shown to display immobilize and to digest prey. The snake’s relatively small different sensitivities to Boiga irregularis (also a colubrid size and hesitation to bite are the primary reasons it is not snake) venom (Mackessy et al., 2006). Alsophis venom considered more of a hazard to humans. possessed very high gelatinase activity, with quantities as One dimensional electrophoresis verified that a consis- low as 0.005 mg producing discernable activity on zymo- tent venom product was obtained from A. portoricensis gram gels. Gelatinase and collagenase activities of metal- specimens and that little variation exists in the venom loproteinases, which degrade structural proteins in the composition among individual snakes. This lack of varia- basement membrane, contributed to the hemorrhage and tion has been noted among venoms of captive Brown necrosis observed in envenomated prey tissues. The venom Treesnakes (Mackessy et al., 2006) and among venoms also contained a-fibrinogenase activity, with levels similar sampled at 6 month intervals from wild Prairie Rattle- to that seen in venoms from snakes in the genus Philodryas snakes (Crotalus viridis viridis) in Colorado (Mackessy, (Peichoto et al., 2007) and from many rattlesnakes (Mack- unpubl. data). When compared with several other colubrid essy, 1993a). By disrupting the blood clot cascade and species on 1D SDS-PAGE, A. portoricensis venom also inducing hypofibrinogenemia, fibrinogenases are likely appeared to have greater complexity in the higher molec- responsible for much of the bleeding associated with ular mass region. Only w20 proteins were visible following colubrid snake bites. 1D SDS-PAGE, but there were over 30 distinct proteins Although some species of colubrid snakes have high levels observed following 2D SDS-PAGE. As with most colubrid of acetylcholinesterase in their venoms (e.g., Broaders and venoms (Mackessy, 2002), the venom proteome was less 568 C.L. Weldon, S.P. Mackessy / Toxicon 55 (2010) 558–569 complex than that of front-fanged viperids such as C. ruber Acknowledgements (30 vs. w100), and the region of greatest complexity appeared to be restricted to the 45–60 kDa acidic region of Funding for this project was provided by a BioScience the gel. These proteins likely represent different isoforms of grant from the Colorado Office of Economic Development one or more phosphodiesterases, metalloproteinases and/ and International Trade. We thank Gad Perry and Robert or acetylcholinesterases in A. portoricensis venom, as indi- Powell for providing the Alsophis specimens used for this cated by SE-HPLC and SDS-PAGE of fractions. research. 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