Proteomic Interrogation of Venom Delivery in Marine Cone Snails

Proteomic Interrogation of Venom Delivery in Marine Cone Snails: Novel Insights into the Role of the Venom Bulb Helena Safavi-Hemami,† Neil D. Young,‡ Nicholas A. Williamson,§ and Anthony W. Purcell*,† Department of Biochemistry and Molecular Biology, The Bio21 Molecular Science and Biotechnology Institute, and Department of Veterinary Science, University of Melbourne, Victoria, Australia Received May 11, 2010

Cone snails of the genus Conus are predatory marine gastropods mainly found in the shallow waters of the tropics and warm temperate seas. To prey on other marine organisms including fish, cone snails have evolved complex venoms synthesized and delivered by a highly sophisticated venom apparatus. Upon prey discovery, the venom is perfused through a harpoon-like radula tooth and rapidly injected into the prey to cause paralysis. While the venom components of cone snails have been intensively characterized, the mechanism of venom translocation and loading prior to and during injection remains elusive. The involvement of the venom bulb, a muscular dilation of the venom gland has been suggested, however evidence is sparse. Here, we use a combination of proteomics, molecular biology, and morphological examination to elucidate the potential role of the venom bulb in venom translocation and delivery. Analysis of the venom bulb proteome clearly demonstrated a function of this organ in muscular movement and, more interestingly, in burst muscle contraction. Morphological examination revealed high structural similarities to the mantle muscle of squids, animals known for their rapid escape response. We sequenced and further characterized arginine kinase, a key protein of rapid muscular movement in invertebrates and show high concentrations of this enzyme in the bulb when compared to the venom gland and the foot muscle. Proteins characteristic for venom biosynthesis were low in abundance. On the basis of our findings, we suggest that the bulb of cone snails is a highly specialized organ of venom translocation. Delivery of venom is driven by burst contractions of the bulb rapidly forcing the venom through the radula tooth into the prey. Keywords: cone snails • conotoxins • venom bulb • venom gland;burst contraction • arginine kinase

Introduction Predatory marine cone snails (genus Conus) are among the most successful marine genera inhabiting the shallow waters of the tropics and temperate regions.1 Although cone snails move relatively slowly and are unable to swim, the ability to produce extremely potent venoms allows them to feed on a broad variety of marine organisms, including fish. Most of the biologically active venom components are relatively small, disulfide-rich peptides called conotoxins or conopeptides (for review, see ref 2). The venom is synthesized in the epithelial cells of the long, convoluted venom gland and packed into a variety of different granules before being secreted into the gland’s lumen.3 Upon prey discovery, the snail’s proboscis is extended and loaded with a highly specialized radula tooth that functions as both a harpoon and hypodermic needle.4 Morphological examinations and high-speed video recordings on the feeding mechanism of Conus catus and juveniles of Conus pennaceus showed that prior to injection the radula tooth is held tightly within a constricted proboscis.5,6 When the pro* To whom correspondence should be addressed. E-mail: apurcell@ unimelb.edu.au, Phone: +613 83442288, Fax: +613 93481421. † Department of Biochemistry and Molecular Biology. ‡ Department of Veterinary Science. § The Bio21 Molecular Science and Biotechnology Institute.

5610 Journal of Proteome Research 2010, 9, 5610–5619 Published on Web 09/06/2010

boscis touches the prey its muscles relax and the radula tooth is propelled into the prey by a ballistic mechanism presumably generated through pressurized liquid at the base of the tooth.5,6 The tooth does not leave the venom apparatus and is instead held tightly at the tip of the proboscis by a ring of muscles. By an unknown mechanism, venom fills the lumen of the proboscis and is forced through the radula tooth into the prey.5 Once injected, the venom causes paralysis and the prey is engulfed through the largely expanded mouth.7 How sufficient pressure is generated to force the venom from the gland into the radula tooth is unknown. The distal end the venom gland of cone snails dilates into an oval structure called the venom bulb (Figure 1B). Due to its muscular nature the bulb has been suggested to function in venom transport,8 presumably as a peristaltic pump. In araneomorph spiders, venom injection is driven by contraction of the bulbous ampulla, a muscular dilation of the venom duct close to the spider’s mouth.9 Due to the columnar-shaped nature of its epithelial layer, the ampulla of spiders has been suggested to participate in venom secretion as well as transport.10 Whether the venom bulb of cone snails also engages in venom biosynthesis is unknown. To date, studies addressing the role of the venom bulb relied upon morphological examinations.8,11 With the advances in proteomics, organ-specific proteome profiling has become a 10.1021/pr100431x

 2010 American Chemical Society

Venom Delivery in Marine Cone Snails

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Figure 1. Venom apparatus of cone snails. (A) Cartoon depicting a cone snail hunting a marine worm. (B) Schematic of the cone snail venom apparatus. The venom is synthesized in the tubular venom gland and translocated into the proboscis prior to injection. The venom bulb is located at the distal end of the venom gland. Harpoon-like radular teeth are synthesized in the radular sac. The radular teeth are moved into the proboscis and propelled into the prey by a high speed hydraulic mechanism. Venom is then injected into the prey through the hollow radular tooth. (C) Cross section through the venom bulb of Conus novaehollandiae stained with Mallory’s trichrome stain to clearly differentiate muscle (red) from collagen (blue). Scale bar: 50 µm. The complementary schematic representation of the venom bulb depicts the orientation of the collagen sheet and fibres (white). (D) Longitudinal section through the venom bulb. Scale bar: 50 µm (E) Magnification of the cross-section through the bulb. The radial (dashed line), circular (light blue area) and longitudinal (white circles) muscle fibres are depicted. (F) Comparison of the epithelial layers of the venom bulb (Fi and Fii) and venom gland (Fiii and Fiv). Venom granules that fill the lumen and the secretory cells of the venom gland (purple and yellow ovoid shapes in Fiii and Fiv) are absent from the bulb. The circular (light blue area) and radial (dashed line) muscle fibres of the bulb are depicted.

widely used technique for the characterization and differentiation of a variety of animal and plant tissue types.12-15 Identification of the most abundant proteins expressed by a certain tissue type can be used to assess its predominant biological function. Although the venom gland proteome of cone snails has not been fully profiled, a number of studies have demonstrated high abundances of proteins involved in conotoxin folding and modification.16,17 If the major role of the venom bulb was venom biosynthesis, the proteome would be similar to that of the venom gland and proteomic analyses would identify proteins characteristic for conotoxin biosynthesis. Alternatively, a predominant role of this organ as a muscular pump would be reflected by high abundances of proteins typical for muscle movement such as myosin and actin (for review, see ref 18). Given the immense speed at which injection of venom into the prey takes place it is tempting to hypothesize that envenomation involves explosive burst contractions of the bulb. A well characterized system of burst contraction in invertebrates is the rapid swimming response of the giant scallop Placopecten magellanicus19,20 and the squid Loligo pealei.21 Scallops and squids swim using jet propulsion. In scallops, rapid clapping

or closing of the valve is driven by burst contraction of the adductor muscle.22 These muscles generally show high abundances of glycolytic enzymes such as phosphofructokinase (PFK) and glyceraldehyde 3-phosphate dehydrogenase (G3PD).23 Another key enzyme of muscles undergoing burst contraction is arginine kinase (AK), a member of the phosphagen kinases. Arginine kinase catalyzes the reversible transfer from phospho-L-arginine to APD to form ATP and is important for buffering ATP levels during burst muscle contraction in invertebrates.20,24 To shed light on the role of the venom bulb, we analyzed the bulb proteomes of the Australian cone species Conus novaehollandiae and Conus victoriae by two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry. Protein profiling clearly demonstrated a role of this organ in motor activity and further suggested its ability to rapidly contract, as evident by high abundances of proteins characteristic for burst contractions such as PFK, G3PD and AK. Further characterization of AK, the second most abundant soluble protein after myosin, revealed high mRNA expression levels in the bulb when compared to the venom gland and foot muscle. High AK concentrations have also been reported in Journal of Proteome Research • Vol. 9, No. 11, 2010 5611

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the adductor muscle of scallops, the tail muscles of several crustacean species25,26 and the mantle muscle of squids.27 Proteomic and molecular analysis was strongly supported by histological examination of the bulb. The presence of a variety of muscle fibres of different orientations concomitant with collagen fibres very similar to those found in the squid mantle underpinned the ability of the bulb to perform repeated explosive bursts. On the basis of these findings, we suggest that venom translocation into the radula tooth and subsequent injection into the prey is at least partially driven by burst contraction of the venom bulb. Arginine kinase buffers ATP levels during this energy demanding event. To our knowledge, this is the first study to comprehensively assess venom translocation from the gland to the injection site in predatory marine cone snails.

Material and Methods Specimen Collection, Tissue Preparation and Histology. Live specimens of Conus novaehollandiae (Adams 1853) and Conus victoriae (Reeve 1843) were collected from Broome, Western Australia. For protein and RNA extractions, venom bulbs and ducts were dissected, immediately snap-frozen in liquid nitrogen and stored at -80 °C until further processing. For histological preparations, venom bulbs were dissected from C. novaehollandiae (n ) 3), placed into 4% paraformaldehyde/ phosphate buffered saline (PBS) for 4 h at 4 °C and processed for routine histology. Bulbs were sectioned (7 µm) and stained with H&E or Mallory’s trichrome stain28 following routine histological procedures. Protein Extraction and 2-Dimensional Gel Electrophoresis (2DE). Frozen venom bulbs were pooled (a total of 2 samples pooled from 3 snails each), ground under liquid nitrogen, resuspended in 1 mL of cell lysis buffer [10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 1 mM NaF, 20 mM Na4P2O7, 1% TritonX, 10% glycerol, 0.1% SDS, 1 mM PMSF, and 1× Complete protease inhibitor cocktail (Roche, Kew, Victoria, Australia), pH 7.6] and incubated for 30 min on ice. Cell lysates were centrifuged and proteins quantified (BCA assay kit, Thermo Fisher Scientific, Sydney, NSW, Australia). Proteins were precipitated from the supernatants (2-D Clean-up Kit, GE Healthcare, Rydalmere, NSW, Australia) and reconstituted in rehydration buffer (8 M urea, 2% CHAPS, 0.002% bromophenol blue, 20 mM DTT, 0.5% immobilized pH gradient (IPG) buffer pH 3-11 (GE Healthcare)) at a final concentration of 1 mg/ mL in preparation for isoelectric focusing (IEF). Two-hundred micrograms of protein was applied onto a nonlinear, pH 3-11 IPG strip (Immobiline, GE Healthcare), and rehydrated overnight. IEF was performed on the Ettan IPGphor II IEF System (GE Healthcare). Running conditions were 500 V for 1 h at 0.5 kVh, 1000 V for 1 h at 0.8 kVh, 6000 V for 2 h at 7.0 kVh and 6000 V for 40 min at 0.7-3.7 kVh. Following IEF, strips were reduced in equilibration buffer (75 mM Tris-HCl, 6 M urea, 30% glycerol, 2% SDS, 0.002% bromophenol blue) containing 65 mM DTT for 15 min followed by alkylation for 15 min in the presence of 80 mM iodoacetamide. Second dimension gel electrophoreses were performed on 8-16% Tris-HCl SDS-PAGE gels (Criterion, Bio-Rad, Gladesville, NSW, Australia) for 50 min at 200 V. Gels were stained with Coomassie brilliant blue G-250 (Bio-Rad). In-gel digestion and protein identification was performed as previously described.17 Briefly, 2DE gel spots were excised, washed in 50% acetonitrile/TEAB (triethylammonium bicarbonate), reduced with 20 mM DTT followed by alkylation in 100 mM iodoacetamide. In-gel digestion was performed 5612

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using sequencing grade trypsin (Sigma-Aldrich, Sydney, NSW, Australia) at a final concentration of 10 µg/mL in 25 mM TEAB. Peptides extracted after overnight digestion were separated on a C18 reversed-phase column (ProteCol nano column, particle size 300 Å and 3 µm, dimensions: 75 µm × 100 mm, SGE Analytical Sciences, Victoria, Australia) and analyzed using a Hybrid Quadrupole-TOF LC-MS/MS mass spectrometer (QSTAR Elite, AB SCIEX, Foster City, CA). Solvent A contained 0.1% formic acid and solvent B consisted of 95% ACN/0.1% formic acid. Separation was performed with a solvent B gradient of 5-60% over 40 min. Acquired data were analyzed using Analyst QS software (version 2.0, AB SCIEX). MS/MS data were used to search the UniProt nonredundant protein database using Mascot (version 2.2, www.matrixscience.com, Matrix Science, Boston, MA) with the following settings: trypsin, 1 missed cleavage, carbamidomethyl as a fixed and oxidation of methionine as a variable modification, 1.2 Da peptide tolerance, 0.8 Da MS/MS tolerance, error tolerant search included. One Dimensional Gel Electrophoresis of Venom Gland and Bulb Proteins. Proteins were extracted from venom glands and bulbs of C. novaehollandiae and C. victoriae, quantified, purified and resuspended in rehydration buffer as described above. Five micrograms of protein were separated on a 4-12% Bis-Tris SDS-PAGE gel (NuPage, Invitrogen, Mount Waverley, Victoria, Australia) for 50 min at 200 V. Gels were stained with Coomassie brilliant blue G-250 (Bio-Rad). In-gel digestion and protein identification was performed as described above. cDNA Isolation and Identification of Conus Arginine Kinase and BiP. Rapid amplification of cDNA ends (RACE) is a PCR-based technique that can be used to obtain full cDNA transcripts of a protein of interest. This technique was utilized to sequence the complete cDNA transcripts of Conus AK and immunoglobulin binding protein (BiP). Briefly, two frozen venom bulbs and glands from C. novaehollandiae were pooled and ground under liquid nitrogen. Total RNA was extracted and reverse transcribed as previously described.17 For rapid amplification of cDNA ends (RACE), 5′ and 3′ universal primeradapted cDNA was prepared from 1000 ng of total RNA using the SMART RACE cDNA Amplification Kit (Clontech Laboratories, Mountain View, California, USA). Primary PCR reactions were performed in volumes of 50 µL containing 2.5 µL of 1:10 diluted cDNA, 0.5 µL of TITANIUM Taq DNA Polymerase (Clontech), 1X Advantage 2 PCR buffer (Clontech), 200 µM of each deoxynucleotide triphosphate (dNTPs), 5 µL of 10× universal primer A mix (Clontech) and 0.2 µM of a gene-specific oligonucleotide (Suppl. Table 1). If the PCR reaction did not produce a visible PCR amplicon, a nested PCR using 5 µL of the primary PCR reaction was performed. All PCR amplicons were analyzed by gel electrophoresis, cloned into pGEM-T plasmid vectors (Promega, Annandale, NSW, Australia) and subsequently sequenced. Nucleotide sequencing was performed using the ABI Prism BigDye Terminator (version 3.1) Cycle Sequencing Kit (ABI) with the SP6 promoter oligonucleotide (Promega). Samples were analyzed on an ABI 3730xl DNA analyzer (ABI). Sequences analyzed in this study were deposited in GenBank (National Center for Biotechnology Information, U.S. National Library of Medicine, Bethesda, MD). All nucleotide data were translated into the predicted amino acid sequences and comparative alignment of the protein sequences were performed using MAFFT E-INS-i sequence alignment by means of local pairwise alignment information.29 Reverse Transcription PCR (RT-PCR). Total RNA was prepared as described above. cDNA was reverse transcribed from


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Figure 2. Two dimensional gel images of proteins extracted from the venom bulb of (A) Conus novaehollandiae and (B) Conus victoriae showing high conservation of proteins between the two species. 200 µg of total protein was loaded onto nonlinear pH 3-11 IEF strips and separated on 8-16% Tris-HCl SDS-PAGE gels. Gel spots were excised, reduced and alkylated, trypsin digested and analyzed by LC-MS/MS. Mass spectrometry results were searched against the UniProt protein database using Mascot software. Proteins with g2 peptide matches and Mascot scores >60 are depicted. Circles represent proteins for which tryptic digestion provided numerous peptide sequences that could not be identified due to insufficient homology to protein sequences available in public databases. (C) Proteins identified for C. novaehollandiae were grouped according to their gene ontologies (Swiss-Prot ExPASy database). ACT, actin; AK, arginine kinase; AST, aspartate aminotransferase; BACT, beta actin; CALP, calponin; ENO, enolase; G3PD; glyceraldehyde 3-phosphate dehydrogenase; MYO, myosin; TPI, triosephosphate isomerase; TPN, troponin; TPM, tropomyosin; TUB, tubulin; PFK, phosphofructokinase; PMYO, paramyosin.

720 ng of DNase-treated total RNA extracted from venom ducts, bulbs and muscle (total of 2 samples per tissue, pooled from 3 snails each), using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche). Primary RT-PCR reactions were performed in volumes of 30 µL containing 2 µL of cDNA (60 ng), 0.3 µL of TITANIUM Taq DNA Polymerase (Clontech), 1X Advantage 2 PCR buffer (Clontech), 200 µM of each dNTP, 0.2 µM of forward and reverse oligonucleotides (Supplemental Table 1, Supporting Information). To rule out false amplification of genomic DNA, a negative control was performed using the reaction from which the enzyme reverse transcriptase was excluded. To ensure linearity of the reaction and rule out PCR inhibition, RT-PCR on all oligonucleotides and tissues was performed with different concentrations of template cDNA (0.6, 6, 60, and 600 ng) for 25, 30, and 35 amplification cycles (data not shown). All PCR amplicons were analyzed by gel electrophoresis and subsequently sequenced as described above. In situ Hybridization of the Venom Bulb Using an Arginine Kinase Specific Probe. Locked nucleic acid (LNA) containing oligonucleotide probes that hybridize to arginine kinase from C. novaehollandiae were designed following established guidelines (Hugenholtz et al., 2001), synthesized and 5′ end-labeled with digoxygenin (DIG) (Thermo Electron, Hamburg, Germany). Unstained tissue sections (7 µm) were placed onto coated glass slides (Polysine, Menzel-Gla¨ser, Braunschweig, Germany) and dried overnight at 37 °C. Unless specified, all washes were performed at room temperature. Sections were dewaxed, rehydrated and sequentially washed twice for 5 min each with diethyl pyrocarbonate (Sigma)-treated PBS (DEPC-PBS), 10 min with DEPC-PBS containing 100 mM glycine, 15 min with DEPC-PBS containing 0.3% Triton X-100

(Sigma) and twice for 5 min each with DEPC-PBS. Sections were then permeabilised with 5 µg/mL of RNase-free proteinase K (Amresco, Solon, OH) at 37 °C for 30 min, postfixed for 5 min in DEPC-PBS containing 4% paraformaldehyde (ProSciTech, Townsville, Queensland, Australia) at 4 °C then washed in DEPC-PBS twice for 5 min each. Sections were acetylated in 0.1 M triethanolamine (Sigma) buffer, pH 8.0, containing 0.25% (v/v) acetic anhydride (Fluka, Castle Hill, New South Wales, Australia) for 10 min on a rocking platform. Tissue was overlaid with 80 µL prehybridization buffer [2 × saline sodium citrate (SSC), 1 × Denhardt’s solution, 10% dextran sulfate (Sigma), 50 mM phosphate buffer, pH 7.0, 50 mM dithiothreitol (Sigma), 500 µg/mL of denatured and sheared cod DNA and 47% deionized formamide (dF) (Sigma). A coverslip was added and slides were incubated in a humid chamber at 37 °C for 2 h. Coverslips were removed by immersing sections in 2 × SSC for 5 min. Sections were then overlaid with 80 µL hybridization buffer with a probe (prehybridization buffer and 4 ng/µL of sense or antisense probe) or without a probe (no probe controls). Slides were incubated in a humid chamber at 50 °C for 17 h. Coverslips were again removed in 2 × SSC then the slides were sequentially washed on a shaking platform at 37 °C in 2 × SSC twice for 15 min each, 1 × SSC twice for 15 min each and 0.25 × SSC twice for 15 min each. DIG-labeled probe detection was performed using a 5-bromo- 4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) immunological method. Slides were washed on a shaking platform in trisbuffered saline (TBS) (100 mM Tris-HCl, pH 7.5 and 150 mM NaCl) for 20 min. Sections were then covered with blocking solution (TBS containing 0.1% Triton X-100 and 2% normal sheep serum) and incubated for 30 min. The blocking solution Journal of Proteome Research • Vol. 9, No. 11, 2010 5613

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Table 1. Proteins Identified in This Study spot

no of peptides

function

Conus novaehollandiae Crassostrea gigas ACQ72912 Crassostrea gigas ACQ72912 Liolophura japonica BAA22871 Liolophura japonica BAA22871 Actinobacillus pleuropneumoniae ABY69221 Taenia asiatica ABN14959 Ciona intestinalis XP_002123605 Lysiphlebus testaceipes AAY63977

16 22 7 3 2 2 2 3

cytoskeleton cytoskeleton kinase activity kinase activity metabolism motor activity motor activity glycolysis

51 45 16 10 3 11 7 9

1188 930 602 226 100 90 92 230

Pinctada fucata Mytilus galloprovincialis Aplysia californica Mytilus galloprovincialis Salmo salar Neptunea polycostata Patinopecten yessoensis Xenopus tropicalis Aplysia californica Conus marmoreus Cellana grata

AAZ79490 CAB64662 CAA88366 BAA36517 ACM09835 BAH10150 O15987 AA135707 Q16956 ABF48564 BAB41096

3 6 2 2 2 16 3 9 5 12 25

motor activity motor activity motor activity motor activity glycolysis motor activity motor activity structure protein folding protein folding kinase activity

21 3 34 1 12 42 10 20 6 23 17

78 373 203 97 190 904 234 560 118 449 241

Conus victoriae Crassostrea gigas Liolophura japonica Liolophura japonica Tetraodon nigroviridis Schistosoma mansoni Strongylocentrotus purpuratus Branchiostoma floridae Syrrhopodon albovaginatus

BAB84579 BAA22871 BAA22871 CAF98918 AAB47536 XP_001196848 XP_002614035 AAN16044

17 4 4 2 2 2 7 3

cytoskeleton kinase activity kinase activity metabolism motor activity motor activity metabolism glycolysis

67 16 15 5 6 6 14 14

1931 526 434 80 64 77 279 153

Schistosoma japonicum Mytilus galloprovincialis Aplysia californica Strongylocentrotus purpuratus Strongylocentrotus purpuratus Mytilus galloprovincialis Neptunea polycostata Chlamys nipponensis akazara Saccostrea kegaki Crassostrea gigas Conus marmoreus Haliotis madaka

AAP06506 CAB64662 CAA88366 AAT06231 AAT06231 BAA36517 BAH10150 BAA23775 BAG55008 BAD15288 ABF48564 BAA05100

3 7 3 2 2 3 14 2 20 3 4 27

motor activity motor activity motor activity glycolysis glycolysis motor activity motor activity motor activity structure protein folding protein folding kinase activity

25 3 44 9 9 3 40 7 45 2 5 11

106 344 213 85 73 124 800 174 1482 80 120 313

protein ID

ACT ACT2 AK1 AK2 AST CALP1 CALP2 G3PD

Actin Actin Arginine kinase Arginine kinase Aspartate aminotransferase Calponin Calponin Glyceraldehyde 3-phosphate dehydrogenase MYO elc Myosin essential light chain MYO hc Myosin heavy chain MYO rlc Myosin regulatory light chain PMYO Paramyosin TPI Triosephosphate isomerase TPM Tropomyosin TPN Troponin TUB Beta-tubulin 1 78 kDa glucose-regulated protein 2 Protein disulfide isomerase 3 Arginine kinase ACT AK1 AK2 ASP CALP1 CALP2 ENO G3PD

Actin Arginine kinase Arginine kinase Aspartate aminotransferase Calponin homologue Calponin Enolase Glyceraldehyde 3-phosphate dehydrogenase MYO elc Myosin light chain 1 MYO hc Myosin heavy chain MYO rlc Myosin regulatory light chain PFK Phosphofructokinase PFK2 Phosphofructokinase PMYO Paramyosin TPM Tropomyosin TPN Troponin TUB Beta-tubulin 1 78 kDa glucose-regulated protein 2 Protein disulfide isomerase 3 Arginine kinase

species

acc no

coverage score

a MS/MS data were searched against the UniProtKB/Swiss-Prot non-redundant protein database using Mascot (version 2.2) with the following settings: trypsin, 1 missed cleavage, carbamidomethyl as a fixed and oxidation of methionine as a variable modification, 1.2 Da peptide tolerance, 0.8 Da MS/MS tolerance, error tolerant search included. Proteins with a minimum of two peptide matches were accepted (individual peptide scores > 60, p < 0.05).

was decanted and slides were covered with TBS containing 0.1% Triton X-100, 1% sheep serum and a 1:500 dilution of sheep anti-DIG-alkaline phosphatase (Roche) for 2 h in a humid chamber. Slides were washed in TBS for 20 min on a shaking platform, then incubated for 10 min with TBS-MgCl2 (100 mM Tris- HCl, 100 mM NaCl and 50 mM MgCl2, pH 9.5). The TBSMgCl2 was decanted and sections were overlaid with a premixed BCIP/NBT solution (Sigma) and incubated for up to 3 h. The reaction was monitored under a light microscope and stopped by briefly washing slides in TE buffer (10 mM TrisHCl, pH 8.1 and 1 mM EDTA). Sections were counterstained for 5 min in 0.1% nuclear fast red (Sigma), dehydrated and mounted (Vecta- Mount, Vector Laboratories, Burlingame, CA).

Results and Discussion Cone snail venoms have received much attention over the last few decades mainly due to the therapeutic potential of their 5614


main constituents, the conotoxins. While venom biosynthesis is known to occur in the venom gland (for review, see ref 30) and, to a much lesser extent, in the salivary gland31 the role of the venom bulb in venom biosynthesis, transport and delivery remained elusive. To comprehensively assess the function of the venom bulb a methodology combining proteome profiling, molecular biology and morphological examination was applied. Proteome Profiling. The transcriptome of Conus has not been sequenced. Identification of proteins therefore required an error-tolerance based approach as previously described.32 Current approaches to sequence the genome and transcriptome of cone snails will greatly advance proteomic based protein identifications currently restricted by the lack of database information available. Using the error-tolerant approach we were able to identify 47% (16 proteins) and 50% (17 proteins) of the 34 gel spots excised for C. novaehollandiae and C. victoriae respectively (Figure 2, Table 1). Proteome profiling


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Figure 3. Comparative protein alignment of arginine kinase (AK) identified in the venom duct of Conus novaehollandiae (GenBank accession number: GU936709) with other molluscan AKs obtained from the public NCBI protein database. Alignment was performed using MAFFT EINS-I by means of local pairwise alignment information.43 Dashes denote gaps. Amino acid conservations are denoted by an asterisk (*), whereas colons (:) and full stops (.) represent a high and low degree of similarity respectively.

revealed high conservation of proteins between the two species (Figure 2 and Table 1) suggesting an evolutionary conserved function of this organ. The majority of proteins identified had high similarities to those found in other molluscan species such as the Pacific oyster Crassostrea giga and the chiton Liolophura japonica. Identified proteins were categorized into 3 functional groups: (i) cytoskeletal proteins, and proteins involved in (ii) motor activity or (iii) energy metabolism (Figure 2C). High abundances of proteins functioning in cell structure and motor activity such as myosin, paramyosin and calponin unambiguously demonstrated a predominant role of the bulb in muscular movement (Figure 2A and B). Interestingly, we identified calponin, a marker of smooth muscle in invertebrates and troponin, a protein characteristic for striated muscle.18 Upon histological examination the bulb appeared to consist of smooth muscle only (Figure 1C and D). In invertebrates differentiation between smooth and striated muscle based on morphology alone is known to bear difficulties,18 particularly in molluscs.33 The identification of troponin in the bulb of Conus therefore suggested the presence of elements of striated muscle as well as smooth muscle. Arginine kinase (AK) was identified as the second most abundant protein after myosin (Figure 2A and 2B). While AK is known to maintain ATP levels in a variety of different invertebrate tissues,34-36 the amount of AK observed in the venom bulbs of both species was surprising. High levels of AK have been reported for muscles undergoing burst contraction such as the adductor muscle of the giant scallop,19,20 the tail muscle of several crustacean species25,26 and the mantle of squids.27 Burst contraction requires high levels of ATP generated via AK-mediated phosphorylation of ADP. Thus, high abundance of AK in the venom bulb of Conus suggests that this organ experiences episodes of burst muscle contractions. Among the proteins involved in

energy metabolism were high levels of key proteins of glycolysis such as phosphofructokinase (PFK) and glyceraldehyde 3-phosphate dehydrogenase (G3PD). Significant glycolytic potentials have previously been reported for rapidly contracting muscles in molluscs such as in the adductor muscle of scallops.23 To further interrogate the bulb’s potential to perform burst contractions, histology was performed. Morphological Examination. Concurrent with previous reports on the structure of the venom bulb in Conus anemone37 and in members of the closely related family of Turridae8 histological examination of the bulb of C. novaehollandiae demonstrated the muscular nature of this organ. The bulb was previously suggested to push the venom through the gland by peristaltic movement, however the presence of distinct muscle layers separated by a tunic-like collagen sheet and the unique orientation of collagen fibres in the outer musculature infer a more sophisticated function of this organ (Figure 1C-E). The bulb of C. novaehollandiae consists of three layers of muscle, of which the outer two are separated by a collagenous sheet (Figure 1 C and D). The innermost layer consists of circular fibers followed by a prominent layer of radial muscle interspersed with longitudinal fibres. The outermost muscle is similar in composition but further comprises radial collagen fibres originating from the collagenous sheet and spiralling outward in a clock-wise direction. Interestingly, the organization of muscle and collagen fibers in the venom bulb is similar to that observed in the mantle of squids, animals that are capable of explosive bursts of their mantle muscle.38 In squids, the inner and outer surfaces of the muscular mantle are lined with layers of collagen called the tunics.38,39 Tunics are stronger than the muscles they replace and prevent the muscle from stretching longitudinally when the radial muscles contract to fill the mantle cavity with water. During constriction of the Journal of Proteome Research • Vol. 9, No. 11, 2010 5615

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Figure 4. (A) In situ hybridization using on the venom duct, bulb and foot tissue of Conus novaehollandiae using an arginine kinase (AK) specific oligonucleotide probe (antisense). Tissue structures were visualized with H&E stain and control hybridizations were performed using a sense probe. Intense staining is present in the cell layer located between the two muscle layers of the bulb. Staining is also present in the foot muscle tissue and some coloration is apparent in the epithelial layer of the duct. Scale bars: venom duct 30 µm (i, iv, vii, x), venom bulb 100 µm (ii, v, vii, ix), foot muscle 100 µm (iii, vi, ix, xii). (B) Reverse transcription (RT) PCR on venom gland, bulb and foot tissue using ferritin as a housekeeping gene (n ) 2). RT-PCR confirmed high expression of AK in the bulb tissue of C. novaehollandiae. nRT, RT reaction from which the enzyme reverse transcriptase was omitted; NTC, no template control.

mantle lumen dispersed radial collagen fibres are compressed. These fibres are thinner than the tunics and able to store elastic energy that is rapidly released to help expand the mantle during refilling. Detailed analysis of the squid collagen fibres revealed that they are dispersed throughout the muscle and found in multiple orientations.39 Although the distribution of collagen fibres in the venom bulb of cone snails is less complex, the mechanism of expansion and constriction is likely to be similar. The thick collagen sheet provides an anchoring site for the radial muscle fibres (Figure 1E) and ensures that the bulb does not expand longitudinally during contraction of the inner radial muscles. We hypothesize that when these muscles contract the wall of the bulb becomes thinner and venom can fill the lumen. Subsequently, relaxation of these muscles concomitant with contraction of the inner circular and the outermost radial muscles leads to a constriction of the bulb. The venom is expelled rapidly while radial collagen fibres in the outermost layer store the elastic energy to ensure rapid dilation of the bulb for the next cycle of expansion and constriction. Based on proteomic and morphological interrogations we propose that the venom bulb of cone snails is capable of repeated burst contractions rapidly moving the venom through the gland toward its injection site. Arginine kinase provides ATP during this energy demanding event. High abundances of this enzyme in the bulb were subsequently confirmed by RT-PCR and in situ hybridization (ISH). 5616


Investigation of Arginine Kinase (AK). Using information inferred from proteomic analysis we sequenced the full length contiguous transcript for Conus AK by RACE-PCR (GenBank accession number GU936709). The open reading frame was 1056 bases in length encoding a protein with a predicted molecular mass of 39 379 Da. Protein sequence alignment revealed high similarities to AKs from other molluscs (Figure 3) suggesting high conservation of this protein within the molluscan phylum. Sequence information was used to perform differential mRNA expression studies on AK in the venom bulb, venom gland and foot muscle of C. novaehollandiae. Reversetranscription PCR revealed highest mRNA expression levels in the venom bulb followed by the foot muscle and the venom gland (Figure 4B). This finding was further confirmed by in situ hybridization studies using a Conus AK specific probe (Figure 4A, vii-xii). AK expression was observed throughout the bulb but appeared to be highest in the outer muscle layer close to the collagen sheet and fibres suggesting that these areas have high ATP demands (Figure 4A, viii and xi). In the foot muscle AK expression was mainly located in the muscle layer adjacent to the foot’s outer epithelium. Some AK was also evident in the venom gland, particularly in the basal squamous epithelial cells where it may provide ATP for metabolic processes (Figure 4A, vii and x). To test the specificity of the hybridization reaction the antisense probe was ommitted and/or replaced by a sense probe as previously described.40,41 No staining was


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observed in no-probe controls (data not shown) nor in sense probe reactions (Figure 4A, iv-vi). Comparative Analysis of the Venom Bulb and Gland Tissue. To investigate whether the bulb also plays a role in venom biosynthesis comparative analysis between this tissue and the venom gland was performed by morphological examination, one-dimensional gel electrophoresis and RT-PCR. In araneomorph spiders the epithelial cells of the bulbous ampulla are columnar shaped, thus very similar to the cells of the venom secreting poison gland.10 In C. novaehollandiae however, the bulb’s epithelial layer constitutes simple squamous epithelial cells unlikely to be specialized in secretory processes (Figure 1Fi and ii). Contrary, the epithelial cells of the venom gland are of columnar nature and filled with granules that presumably contain toxins and/or toxin precursors (Figure 1Fiii and iv). One dimensional gel electrophoresis of venom gland and bulb proteins revealed significant differences in the proteomes between these two tissue types (Figure 5A). Proteins highly abundant in the venom gland such as protein disulfide isomerase (PDI16,17) and immunoglobulin binding protein (BiP) could not be detected in the bulb tissue by in gel tryptic digest and LC-MS/MS analysis. This finding suggested very low abundances of proteins important for toxin biosynthesis. To further inform on the differential expression of these proteins, RT-PCR was performed on the venom gland, bulb and foot muscle of C. novaehollandiae (Figure 5B). While the cDNA sequence of Conus PDI was publicly available (GenBank accession number DQ486867,42) Conus BiP had to be sequenced. The full length contiguous transcript of Conus BiP has an open reading frame of 1989 bases encoding a protein with a predicted molecular mass of 70,964 Da, an N-terminal signal sequence (residue 1-23) and the characteristic ER retention signal KDEL (GenBank accession number GU936709). RT-PCR on Conus BiP and PDI confirmed low expression levels of these proteins in the bulb and foot muscle when compared to the venom gland (Figure 5B). Similar findings were reported for Conus peptidyl-prolyl isomerase (PPI), another important protein for conotoxin biosynthesis.17 While the bulb may engage in low level conotoxin biosynthesis, it is unlikely to be a major role of this organ. Mass spectrometric analyses of the bulb’s peptidome will shed light on the presence of toxins in this tissue. Mechanisms of Venom Injection. In most venomous animals translocation of venom from the synthesizing gland to the site of injection is driven by muscle contraction events. In viperid, elapid and atractaspid snakes venom expulsion is a result of contracting skeletal muscles surrounding the venom gland.43 While the skeletal muscles are not directly attached to the venom gland, muscle contraction causes sufficient pressure to force the venom through the duct toward the fangs.44,45 In most spiders, the ducts of the paired cylindrical venom glands lead to the apex of the chelicera, fanglike appendages near the mouth.10 Contraction of the bulbous ampulla, a dilation of the duct proximal to the chelicera, is believed to push the venom into the chelicera for injection.9,46 Similar to the ampulla of spiders, the venom bulb of cone snails represents a dilation of the venom gland, however unlike in spiders, it is located at the gland’s distal end. Based on our findings we suggest that in addition to acting as a peristaltic pump as previously proposed,37 the venom bulb of cone snails also participates in venom injection events. Recordings on the feeding strategies of C. catus5 and juveniles of C. pennaceus6 revealed that prior to tooth ejection the lumen of the proboscis

Figure 5. One dimensional gel images of proteins extracted from the venom gland and bulb of Conus novaehollandiae (C. nov.) and Conus victoriae (C. vic.). Five micrograms of reduced protein was loaded per lane and separated on a 4-12% Bis-Tris SDSPAGE gel. Gel spots labeled 1-3 were excised, reduced and alkylated, trypsin digested and analyzed by LC-MS/MS. Mass spectrometry results were searched against the UniProtKB/SwissProt protein database using Mascot software. Gel slices were subsequently identified as 1, Immunoglobulin binding protein (BiP); 2, Protein disulfide isomerase (PDI); and 3, Arginine kinase (AK) (see Table 1 for details). Ladder, Unstained protein ladder (Invitrogen). (B) Reverse transcription (RT) PCR on PDI and BiP in the venom gland, bulb and foot tissue using ferritin as a housekeeping gene (n ) 2). RT-PCR confirmed that BiP and PDI are highly expressed in the venom gland but only present in low abundances in the bulb and foot muscle. nRT, RT reaction from which the enzyme reverse transcriptase was omitted; NTC, no template control.

is pressurized via a build-up of liquid of unknown origin. During this pressurization the venom density in the proboscis rapidly increases and continues to do so while the radular tooth is propelled into the prey. Shortly after ejection the venom flow decreases and comes to a halt.6 We hypothesize that repeated burst contractions of the venom bulb concomitant with relaxation of the proboscis leads to a sudden ballistic discharge of the radula tooth as previously documented.5,6 The tooth is shot into the prey and filled with venom by ongoing repeated burst contractions of the bulb. It is tempting to further hypothesize that these contractions also play a role in the early pressurization event. However, it was previously suggested that the liquid responsible for pressurizing the proboscis is of Journal of Proteome Research • Vol. 9, No. 11, 2010 5617

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nonvenom origin, thus the bulb might not be involved in the initial proboscis pressurization. Further studies investigating contraction and relaxation of the bulb and the proboscis during venom injection are required to determine whether this mechanism is plausible. This study is the first to combine detailed proteomic analysis of different regions of the venom apparatus of Conus species and in combination with histological and molecular biological tools has shed light on the mechanism of venom injection. Due to the lack of genomic sequences and failure of most commercial antibodies to react to Conus proteins; proteomics used in combination with these other techniques provided the ideal tool to dissect this important aspect of Conus biology. We therefore confirm what has been hypothesized from light microscopy of the Conus venom apparatus that the venom bulb is primarily a muscular organ that has the capacity to undergo rapid contractile bursts to load venom into the venom delivery apparatus upon injection.

Supporting Information Available: Supplementary Table 1. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Kohn, A. J. Tempo and mode of evolution in Conidae. Malacologia 1990, 32 (1), 55–67. (2) Olivera, B. M. Conus Venom Peptides, Receptor and Ion Channel Targets, and Drug Design: 50 Million Years of Neuropharmacology. Mol. Biol. Cell 1997, 8, 2101–2109. (3) Marshall, J.; Kelley, W. P.; Rubakhin, S. S.; Bingham, J.-P.; Sweedler, J. V.; Gilly, W. F. Anatomical Correlates of Venom Production in Conus californicus. Biol. Bull. 2002, 203, 27–41. (4) Freeman, S. E.; Turner, R. J.; Silva, S. R. The venom and venom apparatus of the marine gastropod Conus striatus Linne. Toxicon 1974, 12, 587–592. (5) Schulz, J. R.; Norton, A. G.; Gilly, W. F. The projectile tooth of a fish-hunting cone snail: Conus catus injects venom into fish prey using a high-speed ballistic mechanism. Biol. Bull. 2004, 207, 77– 79. (6) Salisbury, S. M.; Martin, G. G.; Kier, W. M.; Schulz, J. R. Venom kinematics during prey capture in Conus: the biomechanics of a rapid injection system. J. Exp. Biol. 2010, 213 (5), 673–682. (7) Olivera, B. M.; Rivier, J.; Clark, C.; Ramilo, C. A.; Corpuz, G. P.; Abogadie, F. C.; Mena, E. E.; Wooward, S. R.; Hillyard, D. R.; Cruz, L. J. Diversity of Conus neuropeptides. Science 1990, 249, 257– 263. (8) Medinskaya, A. I. Structure of the venom gland - muscular bulb complex in the family Turridae (Gastropoda, Conoidea). Ruthenica 2002, 12 (2), 125–133. (9) Boeve´, J. L.; Kuhn-Nentwig, L.; Keller, S.; Nentwig, W. Quantity and quality of venom released by a spider (Cupiennius salei, Ctenidae). Toxicon 1995, 33 (10), 1347–1357. (10) Millot, J. Les glandes venimeuses des arantides. Ann. Sci. Naturelles 1931, 10, 113–147. (11) Le Gall, F.; Favreau, P.; Richard, G.; Letourneux, Y.; Molgo´, J. The strategy used by some piscivorous cone snails to capture their prey: the effects of their venoms on vertebrates and on isolated neuromuscular preparations. Toxicon 1999, 37 (7), 985–998. (12) Pu, L.; Brady, S. Systems Biology Update: Cell Type-Specific Transcriptional Regulatory Networks. Plant Physiol. 2010, 152 (2), 411. (13) Brechenmacher, L.; Lee, J.; Sachdev, S.; Song, Z.; Nguyen, T. H.; Joshi, T.; Oehrle, N.; Libault, M.; Mooney, B.; Xu, D. Establishment of a protein reference map for soybean root hair cells. Plant Physiol. 2009, 149, 670–682. (14) Su ¨ ss, C.; Solimena, M. Proteomic profiling of beta-cells using a classical approach - two-dimensional gel electrophoresis. Exp. Clin. Endocrinol. Diabetes 2008, 116, 13–20. (15) Jahn, O.; Tenzer, S.; Hauke, W. B. Myelin Proteomics: Molecular Anatomy of an Insulating Sheath. Mol. Neurobiol. 2009, 40 (1), 55–72. (16) Gowd, K. H.; Krishnan, K. S.; Balaram, P. I. Identification of Conus amadis disulfide isomerase: minimum sequence length of peptide fragments necessary for protein annotation. Mol. Biosyst. 2007, 3, 554–566.

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Journal of Proteome Research • Vol. 9, No. 11, 2010 5619

Proteomic Interrogation of Venom Delivery in Marine Cone Snails

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