Specialisation of the Venom Gland Proteome in Predatory Cone

ARTICLE pubs.acs.org/jpr

Specialisation of the Venom Gland Proteome in Predatory Cone Snails Reveals Functional Diversification of the Conotoxin Biosynthetic Pathway Helena Safavi-Hemami,†,‡ William A. Siero,§ Dhana G. Gorasia,†,‡ Neil D. Young,|| David MacMillan,§ Nicholas A. Williamson,‡ and Anthony W. Purcell*,†,‡ †

Department of Biochemistry and Molecular Biology, ‡The Bio21 Molecular Science and Biotechnology Institute, Department of Biology, and Department of Veterinary Science, University of Melbourne, Victoria, Australia )

§

bS Supporting Information ABSTRACT: Conotoxins, venom peptides from marine cone snails, diversify rapidly as speciation occurs. It has been suggested that each species can synthesize between 1000 and 1900 different toxins with little to no interspecies overlap. Conotoxins exhibit an unprecedented degree of post-translational modifications, the most common one being the formation of disulfide bonds. Despite the great diversity of structurally complex peptides, little is known about the glandular proteins responsible for their biosynthesis and maturation. Here, proteomic interrogations on the Conus venom gland led to the identification of novel glandular proteins of potential importance for toxin synthesis and secretion. A total of 161 and 157 proteins and protein isoforms were identified in the venom glands of Conus novaehollandiae and Conus victoriae, respectively. Interspecies differences in the venom gland proteomes were apparent. A large proportion of the proteins identified function in protein/ peptide translation, folding, and protection events. Most intriguingly, however, we demonstrate the presence of a multitude of isoforms of protein disulfide isomerase (PDI), the enzyme catalyzing the formation and isomerization of the native disulfide bond. Investigating whether different PDI isoforms interact with distinct toxin families will greatly advance our knowledge on the generation of cone snail toxins and disulfide-rich peptides in general. KEYWORDS: cone snails, conotoxins, venom gland, diversification, protein disulfide isomerase, protein/peptide folding

’ INTRODUCTION Venoms from marine cone snails have received much attention over the last few decades due to their extraordinary complexity and diversity. Although there is evidence for the presence of larger proteins in the venom of cone snails,1,2 by far the best studied and most abundant venom components are small, mostly disulfide-rich peptides called conotoxins or conopeptides. Each of the ∼700 Conus species synthesizes its own characteristic repertoire of ∼10001900 toxin peptides and post-translational peptide variants, and it has been estimated that the toxin library of cone snails comprises as many as 500 000 different bioactive compounds.3,4 Various target receptors and ion channels have been identified for these peptides including different subtypes of the ligand-gated nicotinic acetylcholine receptor,5 voltage-gated sodium channels6 and the adrenergic receptor.7 Due to their remarkable target specificity, conotoxins are indispensable tools in ion channel research and as therapeutic agents.8 Such is the recent interest in conotoxins that the growing number of identified novel peptides with new or improved biological activity seems incessant. It is commonly believed that the peptide diversity in Conus is driven by an accelerated evolution of a few gene superfamilies.9,10 Each conotoxin gene encodes a prepropeptide consisting of an r 2011 American Chemical Society

N-terminal signal sequence, an intermediate spacer region and a single copy of the toxin at the C-terminus. While the signal and spacer regions are highly conserved within the same toxin superfamily the amino acid residues of the mature toxin region are hypervariable between a conserved pattern of cysteine residues.9 The exact mechanism for this coordinated diversification of the mature toxin alongside with conservation of the signal region is unknown. Gene duplication and focal hypermutation events have been suggested in the past10,11 with recent evidence pointing toward strong positive selection as the major driving force for Conus venom diversification.12,13 Whether the accelerated evolution of cone snail peptides also applies to the proteins important for toxin biosynthesis has not previously been addressed. Venom biosynthesis, modification, and transport occur in the convoluted venom gland of the cone snail. Following biosynthesis in the secretory cells, the venom is packaged into granules and secreted into the lumen of the gland. Little is known about the glandular proteins responsible for toxin maturation, processing, and packaging. The enzyme protein disulfide isomerase (PDI) was identified as one of the most abundant glandular protein in Conus1416 and shown to catalyze the oxidation and Received: December 30, 2010 Published: June 27, 2011 3904

dx.doi.org/10.1021/pr1012976 | J. Proteome Res. 2011, 10, 3904–3919

Journal of Proteome Research isomerization of the native disulfide bonds in vitro.17 Other venom gland specific proteins involved in the in vitro folding, modification, and processing of conotoxins include peptidyl-prolyl cistrans isomerase (PPI),18 the cysteine-rich protease Tex3119 and γ-carboxylase, the enzyme that catalyzes the carboxylation of glutamate residues.20 Molecular approaches such as generation of cDNA libraries have focused on the peptide component and since the genome/transcriptome of Conus is yet to be comprehensively sequenced information on the proteins expressed in the epithelial cells of the venom gland is sparse. To further the knowledge on conotoxin biosynthesis, maturation and secretion and to elucidate interspecies variations, we interrogated the gross morphology, the peptidome, and the proteome of the venom gland of two Australian cone snails, Conus novaehollandiae and Conus victoriae. We confirm interspecies diversification of the venom peptidome by liquid chromatography and mass spectrometry and demonstrate convergent strategies for venom packaging and secretion. Proteomic profiling led to the identification of novel Conus proteins and protein isoforms and revealed differences in the protein profiles between the two species. We further confirm high abundances of PDI and identify a multitude of different PDI isoforms in both species. On the basis of our findings, we propose that the diversification of conotoxins may also apply to proteins that are crucial for toxin biosynthesis.

’ MATERIAL AND METHODS Specimen Collection, Tissue Preparation and Histology

Live specimens of C. novaehollandiae and C. victoriae were collected from Broome, Western Australia. For protein extractions, venom glands were dissected, immediately snap-frozen in liquid nitrogen and stored at 80 °C until further processing. Crude venom was manually squeezed from dissected glands of C. victoriae, immediately snap-frozen in liquid nitrogen and stored at 80 °C. In addition, venom glands were dissected, placed into 4% paraformaldehyde/phosphate buffered saline (PBS) for 4 h at 4 °C and processed for routine histology. Glands were sectioned (7 μm) and stained with Mallory’s trichrome stain21 following routine histological procedures. Venom Extraction and Analysis

Venom components were squeezed from the dissected venom glands of C. victoriae (n = 3) and C. novaehollandiae (n = 3), pooled and extracted in water with sonication for 20 min. Insoluble material was pelleted by centrifugation and the supernatants were lyophilized. Pellets were resuspended in 20% acetonitrile (ACN)/water and venom components were extracted by sonication as described above. Samples were centrifuged, supernatants lyophilized and pellets resuspended in 40% ACN/water. Lyophilized supernatants were pooled and reconstituted in 5% ACN/0.1% trifluoroacetic acid (TFA)/water prior to liquid chromatography/mass spectrometry (LCMS) analysis. Protein concentrations were determined using Bradford reagent (Sigma-Aldrich) with bovine serum albumin (Sigma-Aldrich) as standard. Forty μg of extracted venom was separated on a C18 column (3 μm particle size, dimensions: 15 cm x 2.61 mm, ProteCol-G C18 HQ303 column, SGE Analytical Science) using the Ettan LC system (GE Healthcare) with a linear gradient from 10 to 100% buffer B (80% ACN/0.1% TFA/water, buffer A: 0.1% TFA/water) over 80 min. Reverse phase fractions were

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lyophilized and reconstituted in 10 μL of 50% ACN/0.1% TFA/ water. One microliter of each fraction was analyzed by MALDITOF MS (Microflex MALDI-TOF, Bruker Daltonics) using alpha-cyano-4-hydroxycinnamic acid (CHCA, Bruker Daltonics) as matrix. Detection was performed in positive reflector mode with a range of 5005000 m/z using flexControl software (version 3.0, Bruker Daltonics). Peak lists were generated in flexAnalysis software (version 3.0, Bruker Daltonics) using the Snap peak detection algorithm with default settings. Peak lists were corrected for (di)sodium (+22 Da, +44 Da) and potassium (+38 Da) adducts and matrix ions. Mass matches were determined in Excel (version 12.2.6, Microsoft) and defined as identical if within (0.5 Da. Graphs were constructed in Prism (version 5.01, GraphPad Software). Protein Extraction and Two-Dimensional Gel Electrophoresis (2DGE)

Protein extraction and 2DGE were performed as previously described.16 Briefly, frozen squeezed venom and venom glands were ground under liquid nitrogen and resuspended in lysis buffer (10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 1 mM NaF, 20 mM Na4P2O7, 1% Triton-X, 10% glycerol, 0.1% SDS, 1 mM PMSF, and 1 Complete protease inhibitor cocktail (Roche Applied Sciences), pH 7.6). Proteins were precipitated from supernatants (2D Clean-up Kit, GE Healthcare) 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 47 (GE Healthcare). Protein concentrations were determined using Bradford reagent with bovine serum albumin as standard. Three-hundred micrograms of protein was applied onto nonlinear, pH 47 IPG strips (Immobiline, GE Healthcare), and rehydrated overnight. Isoelectric focusing (IEF) was performed on the Ettan IPGphor II IEF System (GE Healthcare) with the following running conditions 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 0.73.7 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 electrophoresis was performed on 816% TrisHCl SDS-PAGE gels (Criterion, Bio-Rad) for 50 min at 200 V. Gels were stained with Coomassie brilliant blue G-250 (Bio-Rad). In-Gel Digestion and Protein Identification

In-gel digestion was performed as previously described.16 Briefly, 2DGE gel spots were excised and washed in 50% ACN/ triethylammonium bicarbonate (TEAB). In-gel digestion was performed using sequencing grade trypsin (Sigma-Aldrich) at a final concentration of 10 μg/mL in 25 mM TEAB. Peptides extracted after overnight digestions 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) and analyzed using a Hybrid Quadrupole-TOF LCMS/MS mass spectrometer (QSTAR Elite, AB SCIEX). Solvent A contained 0.1% formic acid/water and solvent B consisted of 95% ACN/0.1% formic acid/water. Separation was performed with a solvent B gradient of 560% over 40 min. Acquired data were analyzed using Analyst QS software (version 2.0, AB SCIEX). MS/MS data were used to search the Swiss-Prot protein database using Mascot (version 2.2, www. matrixscience.com, Matrix Science) with the following settings: trypsin, 1 missed cleavage, carbamidomethyl as a fixed and 3905

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Journal of Proteome Research

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Figure 1. Analysis and variation of the venom peptidome between Conus novaehollandiae and Conus victoriae. Reversed-phase chromatograms of venom extracted from (A) C. novaehollandiae and (B) C. victoriae overlaid with the peptide profiles obtained by MALDI-TOF analysis (m/z on the right y-axis). (C) Bar graph showing mass distribution of peptides. Overlap of (D) reversed-phase chromatograms and (E) peptide profiles illustrating interspecies differences in the venom peptidome. A total of 365 and 258 peptides with unique masses (mass tolerance: (0.5 Da) were identified in C. victoriae (gray) and C. novaehollandiae (orange) respectively with 90 mass matches between the two species (F).

oxidation of methionine as a variable modification, 1.2 Da peptide tolerance, 0.8 Da MS/MS tolerance, error tolerant search included.22 Additionally, an in-house database was collated which contained all molluscan proteins submitted to Swiss-Prot (n = 47 252) and a second nonredundant database was generated by translating the recently published Conus bullatus venom gland transcriptome sequences into their longest putative open reading frames.23 Data were searched against these databases using Protein Pilot software (version 3.0, AB SCIEX) with the following selections: iodoacetamide, trypsin gel-based identification, biological modifications, thorough ID. Based on their homology to Swiss-Prot entries gene ontology (GO) annotations were collected and proteins were linked to their biological pathways using KEGG.24 Immunoblotting

Proteins were transferred from 2DGE gels onto nitrocellulose membranes, blocked with 5% skim milk in PBS/0.05% Tween-20 (PBS-T) followed by immunoblotting (primary antibodies: rabbit polyclonal PDI antibody raised against recombinant rat liver PDI (NP_037130), courtesy of Prof M. Hubbard; secondary

antibody: sheep anti-rabbit HRP-conjugated, Abcam). Incubations were carried out at 25 °C for 1 h. Antibody stocks were diluted 1:2000 in 2.5% skim milk/PBS-T. Detection was performed with ECL reagent (Western Lightning Plus ECL, PerkinElmer).

’ RESULTS AND DISCUSSION Interspecies differences in the venom repertoire have been reported for many cone snail species,3,10 however the mechanism for this accelerated diversification remains unclear. The proteins responsible for the biosynthesis, modification and secretion of such a great diversity of compounds have been largely unexplored. To identify glandular Conus proteins, and further investigate if interspecies differences in the venom peptidome are also reflected in the venom gland proteome, proteomic interrogations were performed on the venom glands of two different species of Conus, C. novaehollandiae and C. victoriae. Interspecies Differences in the Venom Peptidome

Prior to investigations of the venom gland proteome, differences in the venom repertoire between the C. novaehollandiae 3906



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Figure 2. Light micrographs and schematic representations showing the venom apparatus of (A, B) Conus novaehollandiae and (C) Conus victoriae. The radula sac, venom bulb, esophagus, salivary gland and numerous sections through the venom gland are marked. (A1, B1, C1) Magnifications of the venom gland with its four distinct zones: (i) an outermost connective layer, (ii) a layer of squamous epithelial cells, (iii) the secretory columnar epithelium, and (iv) the inner lumen filled with granular material. Both species possess ovoid, yellow-stained granules that in case of C. victoriae are densely packed into larger agglomerates (A2, B2, C2). Sections were cut at 7 μm and stained with Mallory’s trichrome stain. Scale bars are 500 μm for B and C, 100 μm for B1 and C1, and 10 μm for B2 and C2.

and C. victoriae were investigated by liquid chromatography and MALDI-TOF mass spectrometry. Reversed-phase fractionation revealed a complex mixture of compounds in both species (Figure 1A and B). The majority of peptides eluted within 20 60% ACN and were between 1000 and 2000 Da in mass as determined by mass spectrometric analysis (Figure 1C). Overlaps of reversed-phase and mass spectrometric profiles from the two species revealed distinct differences in the venom composition

(Figure 1D and E). A total of 650 peptides were identified in the venom of C. victoriae, of which 455 had unique mass values within the same sample. The venom of C. novaehollandiae was less complex with 515 peptides identified, of which 348 were unique. Mass matches could either represent isobaric peptides with the same molecular weight but different amino acid compositions, identical peptides eluting in adjacent reversed-phase fractions, peptides with identical sequences but different disulfide 3907



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Figure 3. Two-dimensional gel electrophoresis (2DGE) of proteins extracted from the venom glands of (A) Conus novaehollandiae, (B) Conus victoriae and (C) the squeezed venom of C. victoriae. Gel spots were excised from 2DGE gels, digested with trypsin and identified by LCMS/MS. Mass spectrometry data were searched against the Swiss-Prot protein database using Mascot (Matrix Science) software and against the Conus bullatus transcriptome72 and an in-house mollusc database using Protein Pilot (AB SCIEX). Database searching led to the identification of 161 and 157 proteins in C. novaehollandiae and C. victoriae respectively (see Table 1, Supplemental Figure 1 and Supplemental File 1 for details, Supporting Information). Proteins of interest are a CRAMP1-like protein (A, black arrow, spot 243), a kallikrein-like protein (A, white arrow, spot 255), two isoforms of a Tex31 homologue (B and C, white arrows, spot 363 and 366), potential isoforms of BiP (A, B, gray arrows, spots 258, 260, 285, 286, 287 and 288) and PPI (A, B, striped arrows, spots 197 and 371). A number of unidentified proteins were enriched in the squeezed venom of C. victoriae (C, black arrows). Proteins identified from (D) C. novaehollandiae and (E) C. victoriae were grouped into 7 categories according to their gene ontology (GO) annotation (Uniprot/ TrEMBL). (F) Bar chart showing comparison of protein identifications between the two species. (G) Schematic depicting the ER protein processing pathway generated using KEGG software.24 Proteins identified in the venom gland of C. victoriae and C. novaehollandiae are boxed in gray and those identified in C. victoriae only are highlighted black. A number of these proteins were present in multiple isoforms, that is, BiP, PDI, PDIA3 (ERP57), PDI A4, PDIA6 and ERP44. Small circles represent other compounds, for example, ions and small molecules.

connectivities or peptides with different molecular masses not resolved by the MALDI-TOF instrument. Between-species comparison

revealed 90 peptide matches (mass tolerance: (0.5 Da; Figure 1F). MS analysis on a higher accuracy instrument in addition to 3908



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Table 1. Selected Proteins Identified in This Studya spot

protein description

accession no

no of peptides

protein score

coverage (%)

database

Conus novaehollandiae 196

ATP-dependent molecular chaperone HSC82

HSC82_YEAST

2

135

3

Swiss-Prot

196

Heat shock protein 90

HSP90_BRUPA

8

203

7.9

Swiss-Prot

197

Calmodulin-A

CALMA_HALRO

7

202

197

Peptidyl-prolyl cistrans isomerase B

PPIB_HUMAN

2

42

9.3

Swiss-Prot

199

Calreticulin

CALR_CRIGR

7

161

14.6

Swiss-Prot

200

30S ribosomal protein S20

RS20_SYNPW

2

39

9.8

Swiss-Prot

201

50S ribosomal protein L15

RL15_ALISL

3

30

5.6

Swiss-Prot

201 202

Valyl-tRNA synthetase Protein disulfide isomerase

SYV_LEGPL Q1HGL1_CONMR

2 4

50

1.8

Swiss-Prot Conus

202


RL9_POLNA

3

30

10.7

Swiss-Prot

203

Protein disulfide isomerase

Q1HGL1_CONMR

5

16.6

Mollusca

204


Q1HGL1_CONMR

7

25.2

Mollusca

205

Hemolysin

HLYA_SERMA

2

39

2.8

Swiss-Prot

205

Probable inactive ser/thr-protein kinase

BUB1_DICDI

2

64

0.6

Swiss-Prot

205

Transcription factor ACEII

ACE2_TRIRE

2

29

11.4

Swiss-Prot

207 207

Electron transfer ubiquinone oxidoreductase Spectrin alpha chain

ETFD_SCHPO SPTCA_DROME

2 17 (14)

41 218

3.8 3.3

209

ATP phosphoribosyltransferase regulatory sub.

HISZ_AZOSB

209

Spectrin alpha chain

SPTA2_HUMAN

210

Desmin

DESM_CHICK

212

Radixin

RADI_BOVIN

213

Ezrin

EZRI_BOVIN

215

V-type proton ATPase catalytic sub. A

VATA_ANOGA

215 217

Prolyl 4-hydroxylase Pyridoxine/pyridoxamine 50 -phosphate oxidase

Q95P11_BRUMA PDXH_NITWN

5 2

33

218

Glycyl-tRNA synthetase

SYG_METM5

2

38

5.4

Swiss-Prot

219


RL2_METI4

2

42

2.1

Swiss-Prot

219

60 kDa chaperonin

CH60_CHRSD

2

39

7.7

Swiss-Prot

219

Protein disulfide isomerase A6

B0WJ11_CULQU

5

220

GDI-1 GDP dissociation inhibitor

B7PIZ1_IXOSC

7

220

Probable E3 ubiquitin-protein ligase MGRN1

MGRN1_MOUSE

2

33

1.5

Swiss-Prot

221 222

Rab GDP dissociation inhibitor alpha Omega-Crystallin

GDIA_BOVIN CROM_OCTDO

4 10

106 89

5.4 6.9

Swiss-Prot Conus

222

Sortin nexin-6

Q6P845_XENTR

222

Aldehyde dehydrogenase

ALDH_ENCBU

4 (2)

101

2.8

Swiss-Prot (Conus)

222

Probable U3 nucleolar RNA-associated protein

UTP11_LEIMA

2

55

4.7

Swiss-Prot

223

Protein disulfide-isomerase A3

A5LHW1_HAELO

3

223

Signal recognition particle protein

SRP54_RICPR

2

39

5.8

224


A5LHW1_HAELO

2

224 225

Trigger factor Protein disulfide-isomerase A3

TIG_LACPL A5LHW1_HAELO

2 4

225

Coiled-coil domain-containing protein 157

CC157_BOVIN

2

57

3.1

Swiss-Prot

226


PDIA3_CERAE

3

78

5.3

Swiss-Prot

226

Prohormone convertase 1

D0EP83_HALDV

2

228

Alpha-enolase

ENOA_PYTRG

4

67

3.5

Swiss-Prot

228

Enolase

ENO_LOLPE

4

65

6.5

Swiss-Prot

228

Glutamate dehydrogenase 1, mitochondrial

DHE3_HUMAN

4

158

6.1

Swiss-Prot

229 229

Alpha-enolase ATP-dependent hsl protease ATP-binding

ENOA_XENLA HSLU_NATTJ

3 (5) 2

94 45

6.2 4

Swiss-Prot (Conus) Swiss-Prot

229

Elongation factor 1-alpha

EF1A_APIME

7 (3)

228

12.8

Swiss-Prot (Conus)

230

ATP-dependent RNA helicase FAL1

FAL1_CRYNE

3

107

9.8

Swiss-Prot

230

Eukaryotic initiation factor 4A-III-A

I4A3A_XENLA

5

93

7.5

Swiss-Prot

49

Swiss-Prot Swiss-Prot (Conus)

2

36

2.1

Swiss-Prot

17

407

4.8

Swiss-Prot

2

113

1.9

9 (12)

307

15.1

Swiss-Prot (Conus)

12 (11)

333

10.7

Swiss-Prot (Conus)

5 (7)

301

10.1

Swiss-Prot (Conus)

10.3

Conus Swiss-Prot

Swiss-Prot

Conus Conus

Conus

9

3909

Swiss-Prot

Conus Swiss-Prot Conus 51

49.8

Swiss-Prot Conus

Conus



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Table 1. Continued spot

protein description

accession no

no of peptides

GELS2_LUMTE SEPT2_DROME

2 (9) 2

protein score

coverage (%)

database

55 35

5.5 1.4


230 230

Gelsolin-like protein 2 Septin-2

230

Proliferation-associated protein

E2ASD7_9HYME

231

ATP-dependent helicase/deoxyribonuclease

ADDB_STRPB

2

34

2.1

Swiss-Prot

231

Cytochrome P450 6B1

CP6B1_PAPPO

2

62

1.4

Swiss-Prot

231

Thioredoxin peroxidase

B1N694_HALDI

233

14-3-3 protein zeta/delta

1433Z_BOVIN

17 (5)

259

20.4

Swiss-Prot (Conus)

233

Peroxiredoxin-1

PRDX1_HUMAN

5 (3)

116

14.1

Swiss-Prot (Conus)

234 236

14-3-3 protein homologue 14-3-3 protein zeta/delta

1433_TRIHA 1433Z_BOVIN

17 4

145 191

9.2 13.5

Swiss-Prot Swiss-Prot

236

Multifunctional chaperone, putative

B7Q5I4_IXOSC

18

236

Tropomyosin

TPM_HELAS

2

140

6.7

237

ATP phosphoribosyltransferase regulatory sub.

HISZ_RHISN

2

32

8.2

Swiss-Prot

238

50S ribosomal protein L5P

RL5_METAC

2

29

13.3

Swiss-Prot

240

60S acidic ribosomal protein P0

RLA0_DANRE

6 (5)

165

11.3

Swiss-Prot (Conus)

240

Eukaryotic translation initiation factor 3 sub. I

EIF3I_BOVIN

3 (2)

84

11.4

241 242

Crystallin J1A Glycerol-3-phosphate dehydrogenase

B5XGK6_SALSA GPDA_GLUOX

243

Guanine nucleotide-binding protein sub. beta

GBB_LOLFO

243

Protein cramped-like

CRML_MOUSE

244

Coatomer sub. epsilon

COPE_BOVIN

246

Leucyl-tRNA synthetase

SYL_RHOPA

246


Q1HGL1_CONMR

246

Gelsolin

Q703I5_SUBFI

4

247 247

50S ribosomal protein L11 Leucyl-tRNA synthetase

RL11_PROM4 SYL_RHOPA

2 2

247


Q1HGL1_CONMR

247

Protein disulfide-isomerase

PDIA1_CHICK

20

84

2.7

Swiss-Prot

247

SWI/SNF-related actin-dependent regulator

SMAL1_DANRE

2

51

2.1

Swiss-Prot

248


RL11_PROM4

2

52

12.1

Swiss-Prot

248

Arginyl-tRNA synthetase

SYR_LEUCK

2

48

3.4

Swiss-Prot

248

Collagen alpha-1(XIV) chain

COEA1_MOUSE

3

41

25.3

Swiss-Prot

248 248

Protein disulfide isomerase Ubiquitin-like modifier-activating enzyme 5

Q1HGL1_CONMR UBA5_CHICK

16 6 (9)

172

15.8 6.3

Mollusca Swiss-Prot (Conus)

250

Chaperone surA

SURA_YERPA

2

32

4.4

Swiss-Prot

250

Formin-like protein 16

FH16_ORYSJ

2

39

3.2

Swiss-Prot

250

Probable E3 ubiquitin-protein ligase HERC2

HERC2_DROME

2

45

0.5

Swiss-Prot

250


Q1HGL1_CONMR

16 (11)

250

Tubulin beta chain

TBB_PARLI

15 (14)

842

34.9

Swiss-Prot (Conus)

251

Tubulin beta chain

TBB_PSEAM

15

821

35.1

Swiss-Prot

252 253

Uncharacterized protein C4B3.18 Actin

YJ2I_SCHPO ACT_LUMRU

2 35 (21)

44 912

5.4 50.3


253

Type III intermediate filament

IF3T_TORCA

2

75

3.7

Swiss-Prot

254

Protein recA

RECA_DESPS

2

49

7.6

Swiss-Prot

255

DNA polymerase IV

DPO4_PHOLL

2

37

5.1

Swiss-Prot

255

Kallikrein 1-related peptidase b24

K1B24_MOUSE

2

41

3.8

Swiss-Prot

255

Proteasome component C7-alpha

PSA6_YEAST

2

65

7.9

258

78 kDa glucose-regulated protein

GRP78_APLCA

19

258 259

Prohibitin-2 Protein disulfide-isomerase

PHB2_CHICK PDIA1_BOVIN

2 4 (5)

45 51

8 1.4


260


GRP78_APLCA

30 (28)

687

25.3

Swiss-Prot (Conus)

261


Q1HGL1_CONMR

16.6

Mollusca (Conus)

262

Desmin

DESM_CHICK

262

Intermediate filament protein

Q45RT7_BIOGL

Conus

14

Conus

4

3 2

44

11 (8)

222

2

48

3.4 32 1.6

Swiss-Prot

Swiss-Prot (Conus) Conus Swiss-Prot Swiss-Prot (Conus) Swiss-Prot

2 (12)

60

3.9

Swiss-Prot (Conus)

2

68

1.9

Swiss-Prot

20 (19)

52.4 50 69

10.6 1.9


51.6

Mollusca (Conus)

15.8

Mollusca (Conus)

Swiss-Prot Conus

3 (5) 17

Mollusca (Conus) Conus

20 (15)

2

3910

Conus

136

1.9

Swiss-Prot Conus



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protein description

accession no

no of peptides

protein score

coverage (%)

database

2 22 (19)

84 1004

5.1 53.4


72

3.7


262 262

Retrograde protein of 51 kDa Tubulin alpha-1A chain

RGP51_LYMST TBA1A_CRIGR

262


Q1HGL1_CONMR

6

263

Glial fibrillary acidic protein

GFAP_HUMAN

2

263

Retrograde protein of 51 kDa

RGP51_LYMST

2

68

5.1

263

Tubulin alpha chain

TBA_BOMMO

32 (19)

1020

57.1

264

Tubulin alpha chain

TBA_BOMMO

18 (15)

729

41.6

265

Prolyl 4-hydroxylase

B0XDL6_CULQU

7

267 267

Heat shock cognate protein 70 Protein disulfide-isomerase A4

C1KC83_HALDV PDIA4_BOVIN

20 (18) 3

853

22.1

267

Serine/threonine-protein phosphatase 2A

2AAA_HUMAN

4

210

8.3

268


Q45RT7_BIOGL

11

268

Desmin

DESM_CHICK

268

Heat shock-related 70 kDa protein 2

HSP72_HUMAN

268

Phosphoribosylformylglycinamidine synthase 2

268

Protein QN1 homologue

269 270

Retrograde protein of 51 kDa Aldehyde dehydrogenase, mitochondrial

270 270

Conus

Swiss-Prot (Conus) Swiss-Prot (Conus) Conus Swiss-Prot (Conus) Conus Swiss-Prot Conus

2

116

20

624

1.9

PURL_THEFY

2

61

3.1

Swiss-Prot

QN1_MOUSE

2

92

0.7

Swiss-Prot

RGP51_LYMST ALDH2_HORSE

2 12

80 60

5.1 6.8


DNA-directed RNA polymerase sub. alpha

RPOA_NICSY

12

48

2.1

Crystallin

CROM_OCTDO

271

Tropomyosin

B7XC64_9CAEN

9

271

Aldehyde dehydrogenase

ALDH_ENCBU

12

102

2.8

272

Tropomyosin-2

TPM2_BIOGL

25 (18)

855

42.3

273

FKBP-type peptidyl-prolyl isomerase

D3TSE2_GLOMM

273 275

Alanyl-tRNA synthetase Glucose-regulated protein 94

SYA_SULDN A5LGG7_CRAGI

38

3.7

277

Thioredoxin domain-containing protein 16

TXD16_HUMAN

277

Paramyosin

MYSP_MYTGA

495

14.8

278

Valosin-containing protein

Q5ZMU9_CHICK

278

Transitional endoplasmic reticulum ATPase

TERA_DANRE

13

279

Transmembrane emp24 domain-containing prot.

TMEDA_BOVIN

14

23

Conus Swiss-Prot Swiss-Prot (Conus) Conus

3

Swiss-Prot Conus Conus

6 18

Swiss-Prot Conus

6

2 11


Swiss-Prot Conus

5 669

16.5

Swiss-Prot Conus

Conus victoriae 282 283

Endoplasmin, Glucose-regulated protein 94 ATP-dependent molecular chaperone HSC82

ENPL_RAT HSC82_YEAST

12 (22) 2

446 119

12.1 3.7


283

Elongation factor 2

EF2_NEUCR

283

Endoplasmin, Glucose-regulated protein 94

ENPL_BOVIN

283

Heat shock-like 85 kDa protein

283

Thioredoxin domain-containing protein 16

284

Glucosidase 2 sub. beta

GLU2B_MOUSE

286


GRP78_APLCA

23 (18)

767

31.6

Swiss-Prot (Conus)

286 287

78 kDa glucose-regulated protein 78 kDa glucose-regulated protein

GRP78_APLCA GRP78_APLCA

24 (22) 22 (17)

767 820

31.6 33.4

Swiss-Prot (Conus) Swiss-Prot (Conus)

288


GRP78_APLCA

23 (20)

806

29.5

Swiss-Prot (Conus)

288

Thioredoxin-1

THIO_ECOLI

3

221

47.7

Swiss-Prot

288


Q1HGL1_CONMR

289

Calreticulin

CALR_MOUSE

6 (18)

184

11.1

290

Nesprin-1

SYNE1_MOUSE

2

55

0.2

290

Tropomyosin

TPM_HELAS

19 (13)

836

38.7

Swiss-Prot (Conus)

291 292

Tropomyosin 14-3-3 protein sigma

TPM_HELAS 1433S_HUMAN

18 (15) 2

262 94

28.5 7.7


292

Multifunctional chaperone, putative

B7Q5I4_IXOSC

18

292

DNA-directed RNA polymerase I sub. RPA2

RPA2_YEAST

3

47

1.2

Swiss-Prot

292

Tropomyosin

TPM_HELAS

9

319

14.8

Swiss-Prot

293

14-3-3 protein zeta/delta

1433Z_BOVIN

16

326

18

Swiss-Prot

4

79

2.5

Swiss-Prot

4 (6)

163

5.1

Swiss-Prot (Conus)

HSP85_TRYCR

3

83

3.4

TXD16_MOUSE

12

Conus

8

3911

Swiss-Prot Conus

Conus

5


Conus



ARTICLE


protein description

accession no

no of peptides 12 10

protein score

coverage (%)

183

38.8

database Conus Swiss-Prot

293 294

Multifunctional chaperone, putative 14-3-3 protein epsilon

B7Q5I4_IXOSC 1433E_BOVIN

294

Charged multivesicular body protein 4b

CHM4B_CHICK

3 (8)

53

16.3

294

Probable DNA polymerase yorL

YORL_BACSU

2

89

1.1

294

Protein BMH1

BMH1_YEAST

16

252

294

Serine/threonine-protein kinase 38-like

ST38L_HUMAN

2

90

295

Endoplasmic reticulum resident protein 44

ERP44_MOUSE

9

Conus

297

CG17271 isoform B

Q0KI39_DROME

8

Conus

300 301

Endoplasmic reticulum resident protein 44 Endoplasmic reticulum resident protein 44

ERP44_MOUSE ERP44_MOUSE

12 11

Conus Conus

305

G protein beta sub.

Q5GIS3_PINFU

4

Conus

308

Similar to nucleosome remodeling factor

CG4634_PA

6

Conus

311

Alternative splicing factor

B7QKF8_IXOSC

3

Conus

314


D0EP83_HALDV

4

315


Q1HGL1_CONMR

3 (3)

6.6

316


Q1HGL1_CONMR

3 (7)

6.6

316 318

Peptidase D Actin, cytoskeletal 1A

B1PMA7_CHICK ACTA_STRPU

5 9

367

318

Janus kinase and microtubule-interacting prot. 1

JKIP1_BOVIN

2

47

3

Swiss-Prot

318

Probable protein disulfide-isomerase A4

PDIA4_CAEEL

6

44

3.1

Swiss-Prot

318


Q1HGL1_CONMR

5

319

Carbamoyl-phosphate synthase large chain

CARB_MACCJ

2

49

1.7

Swiss-Prot

319


PDIA4_CAEEL

4

44

3.1

Swiss-Prot

319

Tubulin alpha chain

TBA_BOMMO

5

320 320

Protein disulfide isomerase Tubulin alpha chain, testis-specific

Q1HGL1_CONMR TBAT_ONCMY

321

Desmin

DESM_CHICK

321


Q45RT7_BIOGL

321

Pre-mRNA-processing RNA helicase PRP5

PRP5_CANGA

3

62

4.5

Swiss-Prot

321

Protein QN1 homologue

QN1_MOUSE

2

91

0.7

Swiss-Prot

322

Tubulin alpha-1A chain

TBA1A_CRIGR

3 (10)

132

9.8

Swiss-Prot (Conus)

323

Flap structure-specific endonuclease

FEN_PYRFU

2

46

5.3

Swiss-Prot

323 324

Pre-mRNA-splicing factor CLF1 Translation initiation factor IF-2

CLF1_ASHGO IF2_TOLAT

2 2

41 43

1 2.5


325

60 kDa heat shock protein homologue 2

CH60C_DROME

25 (21)

334

15.5

326


PDIA4_CAEEL

5

44

3.1

326


Q1HGL1_CONMR

13.6

Mollusca (Conus)

326

Tubulin alpha chain, testis-specific

TBAT_ONCMY

6

267

12.4

Swiss-Prot

327

Desmoglein-3

DSG3_HUMAN

2

63

2.5

Swiss-Prot

327


PDIA4_CAEEL

6

50

3.1

327 328

Tubulin alpha-3C/D chain Protein disulfide-isomerase

TBA3C_HUMAN PDIA1_BOVIN

11 (12) 5 (7)

603 54

26.4 1.6

329


Q1HGL1_CONMR

329

Niemann-Pick type C1 containing protein

B7PPF5_IXOSC

330

ATP phosphoribosyltransferase regulatory

HISZ_BORA1

2

46

5.2

Swiss-Prot

330

Histone-lysine N-methyltransferase

DOT1L_HUMAN

2

48

1.2

Swiss-Prot

330 331

Protein disulfide-isomerase Protein disulfide isomerase

PDIA1_BOVIN Q1HGL1_CONMR

2 2

56

1.4 4.6

Swiss-Prot Mollusca

332 332

14-3-3 protein zeta/delta 30S ribosomal protein S2P

1433Z_BOVIN RS2_METM6

2 (13) 2

63 49

6.5 10.4

332

Tubulin beta chain (Fragment)

TBB_HALDI

3 (8)

146

11.1

Swiss-Prot (Conus)

333

Glyoxalase domain-containing protein 4

GLOD4_MOUSE

6

80

14.1

Swiss-Prot

333

Ubiquitin C-terminal hydrolase, putative

B7QGG8_IXOSC

5

334

Glutamyl-tRNA synthetase 1

SYE1_LACBA

2

40

3.8

18 1.5


Conus

21.3

Mollusca (Conus) Mollusca (Conus) Conus Swiss-Prot

Conus

215

10.4

Swiss-Prot

3 (9) 5

221

19.2 12

Mollusca (Conus) Swiss-Prot

3

161

3.9

Swiss-Prot Conus

14

5 (5)

5 (5)

16.8


Swiss-Prot Swiss-Prot (Conus) Swiss-Prot (Conus) Mollusca (Conus) Conus

6

3912



Conus Swiss-Prot



ARTICLE


protein description

accession no

no of peptides

protein score 37 51

coverage (%)

database

2.7 1.4


335 335

GTP-binding protein era homologue Protein disulfide-isomerase

ERA_EHRCJ PDIA1_BOVIN

2 2 (3)

335

Cathepsin D

C6ZP36_9BIVA

2

336

40S ribosomal protein SA

RSSA_PINFU

3

336


Q1HGL1_CONMR

2

337

Glycosylation-dependent cell adhesion molecule

GLCM1_BOVIN

4

337

DnaJ homologue subfamily B member 11

DJB11_RAT

3

337

Twinfilin

Q6NWD9_DANRE

7

338 338

Calreticulin Glycosylation-dependent cell adhesion molecule

CALR_ONCVO GLCM1_BOVIN

2 4

338

Probable proline iminopeptidase

PIP_PLEBO

339

ATP synthase sub. alpha, mitochondrial

ATPA_DROME

339


Q1HGL1_CONMR

340

Thioredoxin-1

THIO_ECOLI

3

342

Actin, cytoplasmic 1

ACTB_BOSMU

3

342

Elongation factor 1-alpha 2

EF1A2_DROME

7 (4)

293

14.5

342 342

Eukaryotic translation initiation factor 3 sub. A Protein disulfide-isomerase A6

EIF3A_ASHGO E2AZ08_9HYME

2 6

36

1.8

Swiss-Prot Conus

343


PDIA1_BOVIN

26 (13)

57

2.9

Swiss-Prot (Conus)

343

Ribosome production factor 2 homologue

RPF2_ORYSJ

2

45

4.8

344


Q1HGL1_CONMR

344

Trigger factor

TIG_ACIBL

345


Q1HGL1_CONMR

346

Nesprin-1

SYNE1_MOUSE

3

346 346

Gelsolin Protein disulfide-isomerase

Q703I5_SUBFI PDIA1_BOVIN

6 4

347 347 347

Cytochrome P450 1A1 mRNA 30 -end-processing protein RNA14

CP1A1_FELCA RNA14_CANAL

2 2

Prolyl-tRNA synthetase

SYP_SHELP

2

347

Ribosomal RNA small sub. methyltransferase G

RSMG_MARMS

2

347

Prolyl 4-hydroxylase

B0XDL6_CULQU

9

Conus

347


PDIA4_BOVIN

3

Conus

350 350

GDI-1 GDP dissociation inhibitor Protein disulfide-isomerase A6

B7PIZ1_IXOSC E2AZ08_9HYME

5 7

Conus Conus

353

FKBP-type peptidyl-prolyl isomerase

D3TSE2_GLOMM

14

355

Glutamine-rich protein 2

QRIC2_HUMAN

2

59

1

355

Serine/threonine-protein phosphatase 2A

2AAA_HUMAN

9

388

16.6

Swiss-Prot

356

Juvenile hormone epoxide hydrolase 2

HYEP2_CTEFE

2

43

6

Swiss-Prot

356

Protein rhsC

RHSC_ECOLI

3

40

4.2

Swiss-Prot

356

Mediator of RNA polymerase II transc. sub. 15

MED15_DICDI

2

54

2.4

356 358

Prolyl 4-hydroxylase Prolyl 4-hydroxylase

B0XDL6_CULQU B0XDL6_CULQU

7 3

357

Titin

TITIN_DROME

2

361


D0EP83_HALDV

6

Conus 114

12 6.4

117

29.4

Swiss-Prot Mollusca Swiss-Prot Conus Conus

38 117

4.1 29.4

Swiss-Prot Swiss-Prot Swiss-Prot

3

73

4

21 (14)

606

21.2

Swiss-Prot (Conus)

41.4

Mollusca (Conus)

205

33.9

Swiss-Prot

151

8.3

Swiss-Prot

10 (7)

31 (24) 2

59.8 37

31 (23)

2.5 59.8

54

Swiss-Prot (Conus)

Swiss-Prot Mollusca (Conus) Swiss-Prot Mollusca (Conus)

0.3

Swiss-Prot

56

3.7

Conus Swiss-Prot

45 57

4.1 1.9


43

4

Swiss-Prot

40

12.5

Swiss-Prot

Conus Swiss-Prot

Swiss-Prot Conus Conus

39

0.1

Swiss-Prot Conus

363

Substrate-specific endoprotease Tex31

TX31_CONTE

4

87

366

Substrate-specific endoprotease Tex31

TX31_CONTE

11

115

13 50.3


371

Peptidyl-prolyl cistrans isomerase

PPIA_BLAGE

4

165

18

Swiss-Prot

a

Gel spots were excised from 2DGE gels, digested with trypsin and identified by mass spectrometry. Data were searched against the Swiss-Prot protein database using Mascot (Matrix Science) software and against the Conus bullatus transcriptome72 and an in-house mollusc database using Protein Pilot (AB SCIEX). All peptides identified using Protein Pilot had confidence scores of >95%. Coverage is not provided for searches against the Conus database as contigs do not span the entire protein length. Total protein scores are provided for Mascot searches. See Supplemental Figure 1 for annotated 2DGE gels and Supplemental File 1 for complete list of proteins identified in this study, Supporting Information.

MS/MS sequencing is likely to reduce the number of inter- and intraspecies mass matches. A recent study on the venom

compositions of Conus textile, Conus imperialis and Conus marmoreus identified a total of 2428, 845, and 1147 peptides, 3913


Journal of Proteome Research respectively.3 Venoms from three individuals were analyzed for each species by LCESIMS on a QqTOF instrument.3 Remarkable inter- and intraspecies differences were observed with little to no overlap between the different species examined.3 Analysis of venoms extracted from additional individuals of C. novaehollandiae and C. victoriae is therefore likely to result in further peptide identifications. Consistent with previous reports,3,25,26 venom profiling confirmed the extraordinary complexity of cone snail venom and corroborated striking interspecies variations. Interspecies Differences in Venom Packaging and Secretion

To investigate potential differences in the gross morphology of the venom apparatus, histological examinations were performed. Light microscopy showed multiple cross sections through the convoluted venom gland in the same plane (Figure 2). The venom bulb, a muscular dilation located at the distal end of the gland, is also apparent in both species with its connection point to the venom gland visible in the micrograph for C. novaehollandiae (Figure 2B). Other identified structures include the radula sac, where the harpoon-like radula teeth are synthesized and stored, the salivary gland and the esophagus. While the overall organization of the venom apparatus appeared to be identical between the two species, further interrogation of the venom gland revealed distinct differences in venom packaging. The venom glands of both species consist of four zones: (i) an outermost connective layer; (ii) a single-cell lining of squamous epithelial cells; (iii) the secretory columnar epithelium; and, (iv) the inner lumen filled with granules that presumably contain venom27,28 (Figure 2A1, B1 and C1). Some cellular material appears to be present in the lumen suggesting that venom granules are released by holocrine secretion as previously suggested,28,29 that is, secretion occurs via rupture of the cell membrane. Both species possess ovoid granules of the same size in the glandular lumen that stain yellow with Mallory’s trichrome stain (Figure 2B2 and C2). These granules appear to have a similar gross morphological structure as those described previously28,30 and most likely contain conotoxins and/or toxin precursors. Interestingly, the yellow ovoid shaped granules present in C. victoriae are densely packed into larger assemblages (Figure 2C2) not previously described in Conus. Packaging appears to occur in the epithelial cells, as large granules can be seen within these cells (Figure 2C1). Serial sections through the entire venom gland of specimens of C. novaehollandiae (n = 2) provided no evidence of packaging of yellow granules into these large granular agglomerations. Interspecies variations in granule gross morphology have been observed previously. The venom gland of Conus californicus harbors two types of ultrastructurally distinct granules28 that differ from the “immature” and very large, “mature” granules observed in C. magus.27 Neither species appeared to secrete large granule agglomerations similar to those observed in C. victoriae (Figure 2C1). Interspecies variations in venom packaging could be associated with the diversification of venom peptides. Purification of granules from different species and subsequent analysis of their content could be informative in this context. Specialisation of the Venom Gland Proteome

Two-dimensional gel electrophoresis coupled with LCMS/ MS analyses lead to the identification of 161 and 157 proteins and protein isoforms in C. novaehollandiae and C. victoriae respectively (Figure 3, Table 1, Supplemental Figure 1, Supplemental File 1, Supporting Information). This constituted around 60% of gel spots excised (∼250 spots each). Searching against the recently published Conus bullatus database23 provided

ARTICLE

additional hits for proteins with very little sequence homology to proteins present in the Swiss-Prot database (see Table 1). It is anticipated that more comprehensive future analyses of the Conus venom gland genome/transcriptome will result in improved proteomics-based protein identifications. Proteins identified in the current study were grouped into seven major gene ontology and molecular/biological function categories (Figure 3D, E and F; Uniprot/TrEMBL31). Among the many structural proteins identified in C. novaehollandiae was collagen (Table 1, Supplemental Figure 1, Supporting Information). Histological examination of the venom gland showed bright blue staining of the outer layer in both species when stained with Mallory’s trichrome stain, typical of collagen.21 Collagen-like fibrils interspersed with muscle-like cells were previously observed in the outer layer of the venom gland of C. californicus by electron microscopy.28 In addition to collagen, proteins characteristic of muscular movement such as actin, tubulin, myosin and tropomyosin, were also identified in the venom glands of C. victoriae and C. novaehollandiae (Table 1, Supplemental Figure 1, Supporting Information). Rapid translocation and injection of venom were previously suggested to be driven by burst contractions of the venom bulb.16 Concerted contractions of the collagen embedded muscle cells of the venom gland may provide a rigid structure that prevents the dilation of the tubular venom gland during these burst contractions. Indeed, specialized collagen fibres were observed within the bulb, similar to those surrounding the venom gland, and suggested to ensure structural integrity and elasticity during bulb muscle contraction.16 The combination of proteomics and histology therefore proved to be a powerful tool for the identification of cellular and subcellular structures of the Conus venom apparatus. The columnar epithelial cells of the venom glands of both species were completely filled with secretory vesicles (Figure 2B1 and C1). Proteomics analyses further illuminated the secretory nature of these cells by identifying an extensive network of proteins important for vesicular trafficking. Proteins of interest included coatomers, retrograde transport proteins, desmin and a large number of actin-binding proteins such as formin, gelsolin, spectrin and ezrin/radixin/moesin proteins (ERM) (Table 1, Supplemental Figure 1, Supporting Information). Formin is important for the polymerization of actin32 while gelsolin mediates actin filament assembly and disassembly.33 ERM proteins cross-link assembled actin filaments with the plasma membrane.34 Spectrin binds short actin filaments and forms a cytoskeletal network that contributes to the maintenance of Golgi structure and protein trafficking in the early secretory pathway.35 Spectrin has further been localized to chromaffin granules and small synaptic vesicles and was suggested to play a role in vesicle secretion events.36,37 Together, the secretory cells of the venom gland comprise a complex network of actin and actin-binding proteins that likely play an important role for the trafficking and sorting of the secretory granules. A notably large proportion of the proteins present were involved in gene transcription and translation processes, including different subunits of DNA polymerase, various tRNAsynthetases and the mRNA splicing factor CLF1 (Table 1, Supplemental Figure 1, Supporting Information). This is not surprising considering the high protein and peptide turnover rates in the venom gland. Interestingly, a protein with sequence similarities to CRAMP1, an antimicrobial protein found in mice, was identified in the venom gland of C. novaehollandiae (Figure 3A, black arrow). 3914



ARTICLE

Figure 4. Identification of multiple isoforms of protein disulfide isomerase (PDI). Gel spots identified as PDIA1 in (A) Conus novaehollandiae and (D) Conus victoriae by in-gel tryptic digests and LCMS/MS analysis are shown in white. (B, E) 2DGE gels were immunoblotted using a polyclonal rat PDIA1 antibody. (C, F) PDI isoforms only identified by immunoblotting (black spots) or tryptic-digest (white spots) and those identified by both techniques (striped spots).

CRAMP-1 is a potent antibiotic agent against Gram-negative bacteria.38 Antimicrobial peptides and proteins are widely distributed in the animal and plant kingdom39,40 but have not yet been identified in cone snails. Conolysin-Mt is a hemolytic peptide discovered in the venom of Conus mustelinus.41 ConolysinMT disrupted eukaryotic membranes with high potency but had low antimicrobial activity.41 Future studies are required to characterize the antimicrobial activity of the C. novaehollandiae CRAMP-like protein identified herein, to confirm whether it truly represents the first class of antimicrobial proteins identified in Conus. Proteomic analyses further identified a number of novel proteases not previously described in Conus. The venom gland of C. novaehollandiae harbored high abundances of a kallikreinlike protein (Figure 3A, white arrow). Kallikreins are a group of glandular proteins that are related to trypsin and other serine proteases. The major mammalian kallikrein which is found in the pancreas, kidney and salivary gland cleaves the precursor kininogen to release bradykinin, a vasoactive peptide important for blood flow.42 Kallikreins have been found in the venoms of a variety of animals including insects43,44 and reptiles45,46 where they exert various functions, such as the liberation of kinins and

fibrinogens.45 To our knowledge, this is the first study to show the presence of a kallikrein-like protein in cone snails. Given its high abundance in the venom gland of C. novaehollandiae, this enzyme is likely to play an important role in the proteolytic cleavage of conotoxins or, if injected, of proteins in the prey. Similarly, a novel Cathepsin D-like protease was identified in the venom gland of C. victoriae (Table 1, Supplemental Figure 1, Supporting Information). Cathepsin D is an aspartyl protease that cleaves a number of different substrates including fibronectin.47 Fibronectin processing enzymes have been found in other animal venoms48,49 and could be injected by cone snails to elicit degradation of the extracellular matrix in the prey. Further analysis however, could not confirm enrichment of this protein in the venom fluid of C. victoriae (Supplemental Figure 1B, spot no 335, Supporting Information) suggesting that this enzyme may be involved in intracellular processes instead. Additionally, the venom gland of C. victoriae contained two proline peptidases (Table 1, Supplemental Figure 1B, spot no 316 and 338, Supporting Information), enzymes that hydrolyze proline containing peptide bonds and play a role in a number of crucial cellular processes such as collagen biosynthesis.50 As cleavage of conotoxins generally occurs after basic and/or dibasic 3915



ARTICLE

Figure 5. Analysis of peptide matches identified as PDIA1 by in-gel tryptic digests and LCMS/MS. Peptides obtained for (A) Conus victoriae and (B) Conus novaehollandiae were aligned with Conus marmoreus PDI (GenBank accession no: ABF48564). The font size represents the number of peptide matches obtained in each species. Amino acids with no matches are shown in the smallest font while those with nine matching peptides per species are depicted in the largest font. Sequence analysis revealed highly conserved regions for the different PDIA1 isoforms in both species (e.g., FVQDFLDGK and MDATANEIEEVK for C. victoriae and DQESTGALAFK for C. novaehollandiae).

residues these peptidases are unlikely to be involved in conotoxin maturation events. Interestingly, a novel prohormone convertase 1-like enzyme was identified in both species (Supplemental Figure 1A and B, spot no 314 and 226, Supporting Information). Prohormone convertase 1 is an important enzyme of insulin maturation and belongs to the family of subtilisin-like enzymes that cleave after basic residues.51,52 Proteolytic cleavage of conotoxin precursors by a subtilisin-like protease has previously been suggested.53 The prohormone convertase 1-like enzyme identified here is therefore likely to play a role in conotoxin maturation. Analysis of the squeezed venom isolated from C. victoriae revealed enrichment of a number of proteins (Figure 3C, black arrows) when compared to the whole venom gland protein extract (Figure 3B). These proteins are likely to be secreted venom components. Based on the small size of the venom gland of C. novaehollandiae, the quantities of squeezed venom obtained for this species did not allow for 2DGE analysis. Due to low sequence similarities with proteins available in public protein database only one of the C. victoriae venom proteins was identified. This protein shared high sequence similarity with Tex31, a protease sequenced from the venom gland of C. textile (Figure 3C, white arrow).19 The C. victoriae protein appears to be present in at least two isoforms. These isoforms are better resolved on whole gland 2DGE gels, probably due to lower abundances in these preparations (Figure 3B, white arrows, Table 1 and Supplemental Figure 1, Supporting Information). Tex31 was originally purified from the venom gland of C. textile and subsequently sequenced, expressed and functionally characterized.19 Recombinant Tex31 possessed in vitro proteolytic activity and substrate specificity for conotoxin-like peptide substrates,19 and may thus be important for the liberation of mature conotoxins.19 While its presence in the venom (Figure 3C and ref 19) suggests that proteolytic cleavage of mature toxins occurs post-secretion, controversy still exists on the function of Tex31. Recently, a homologue of Tex31 was identified in the venom gland of C. marmoreus, showing very low proteolytic activity in vitro.54 Interestingly, Tex31 belongs to the family of cysteine-rich secretory proteins (CRISP) that have been found in the injected venoms of various animals where they play a role as calcium and/or potassium channel blockers.45 Tex31 may

function as a neurotoxin rather than a protease.54 Further characterisations are needed to clarify the molecular function of Tex31 and its homologues. Interestingly, we were not able to detect Tex31 in the venom gland of C. novaehollandiae suggesting comparatively lower expression levels of this enzyme. Alternatively, given the high abundance of the kallikrein-like protease found in this species, different species may utilize their own characteristic set of proteases, a hypothesis that warrants further investigation. A large proportion of proteins identified in the current study were those involved in protein processing events in the ER. Biological pathway analysis was performed to illustrate the major molecular function of these proteins within the ER.24 Among the proteins identified were those involved in protein/peptide synthesis, folding, misfolding and degradation such as the ERresident heat shock proteins BiP (GRP78) and glucose binding protein 94 (GRP94), calreticulin, elongation factors, PPI and multiple isoforms of PDI (Figure 1G, Table 1, Supplemental Figure 1, Supporting Information). PPIs are ubiquitous enzymes found in vertebrates, invertebrates, plants and bacteria and are present in almost all cellular compartments (for review, see ref 55). Many functions have been described for these diverse proteins including their role as chaperones and folding catalysts.5658 PPIs catalyze the cis trans isomerization of peptidyl-prolyl bonds, an otherwise slow process that can impede protein folding. In fact, multiple isoforms of this enzyme were recently sequenced from the venom gland of C. novaehollandiae and shown to facilitate the oxidative folding of conotoxin μ-GIIIA, a conotoxin containing three disulfide bonds and three hydroxylated proline residues.18 This finding alongside with the presence of PPIs in the venom glands of C. novaehollandiae and C. victoriae (Figure 3A and B, striped arrows) suggests that these enzymes play a role in the oxidative folding of conotoxins in vivo. BiP is an ER-resident member of the heat shock protein 70 family (HSP70) found in almost all organisms and often comprising multiple members.59 The HSP70 family chaperones have two major functional domains, an N-terminal ATPase domain and C-terminal protein-binding domain.60,61 In the ER, BiP facilitates protein folding by preventing the aggregation of proteins through binding to unfolded or partially folded proteins.62 3916


Journal of Proteome Research A number of gel spots were identified as BiP in both species indicating the presence of multiple isoforms of this enzyme (Figure 3A and B, gray arrows). Conus BiP may therefore play a role in facilitating folding and/or preventing misfolding of conotoxins in the ER of the venom gland cells. Presence of Multiple PDI Isoforms

The presence of such a great number of PDI isoforms identified by 2DGE was surprising (Table 1, Supplemental Figure 1, Supporting Information) and therefore further investigated. PDI was first characterized as a chaperone of reduction, oxidation and isomerization of disulfide bonds.63 Since then, various other functions have been described for this ubiquitous protein. PDI also catalyzes folding of proteins without disulfides and is a subunit of prolyl 4-hydroxylase (P4H), the enzyme catalyzing hydroxylation of proline residues.64 PDI is a member of the thioredoxin superfamily and contains four thioredoxin-like domains (a, b, a0 , b0 ), two of which have the catalytic CGHC motifs.65 To date, 19 members of the PDI family have been identified in humans that differ significantly in their primary amino acid sequence, domain organization, catalytic activity and molecular mass.66,67 The first identified, and by far the best-studied PDI family member is PDIA1, or simply PDI.66 Several studies have shown high abundances of PDIA1 in the venom gland of various cone snail species,15,17 including two isoforms in the venom gland of C. textile.14 Other PDI family members such as PDIA4 or ERp44 have not been described in Conus. Here, a multitude of Conus PDIA1 isoforms was discovered by 2DGE in-gel tryptic digestion (Figure 4A and D) and 2DGE immunoblotting (Figure 4B and E). Mass spectrometric analyses identified 7 isoforms of PDIA1 in C. novaehollandiae and 10 in C. victoriae (Figure 4A and D). Unique peptide matches from gel spots identified as PDIA1 were aligned with the C. marmoreus enzyme to visualize conserved regions (Figure 5). Sequence analyses revealed a number of conserved peptides within and between the two cone snail species (Figure 5). Immunoblotting using a polyclonal anti-rat PDIA1-specific antibody further confirmed the presence of multiple isoforms in the venom glands of both species and led to the identification of additional protein spots likely due to the higher sensitivity of this technique (Figure 4B and D). A number of gel spots identified as PDI by immunoblotting could not be visually detected on the Coomassie-stained gels and were therefore not selected for in-gel tryptic digestion. On the other hand, not all spots identified as PDI by mass spectrometry were detected by immunoblotting. This could be explained by a lack of sequence homology between the rat and the cone snail protein (57% sequence identity, BLASTp, NCBI). A highly conservative approach accepting only gel spots identified by mass spectrometry and immunoblotting demonstrated the presence of at least 5 isoforms of PDIA1 in each species with species-specific expression profiles (Figure 4C and F). In-gel tryptic digestion further identified P4H, the enzyme associated with PDIA1 in the hydroxylation of prolines, and a number of other PDI family members, that is, PDIA4, PDIA6 and several isoforms of PDIA3 in C. novaehollandiae and multiple isoforms of ERP44, PDIA4 and PDIA6 in C. victoriae (Table 1). These PDIs did not stain immunopositive using the anti-PDIA1 antibody confirming the specificity of this antibody. Several studies have addressed substrate specificities of the different PDI family members. Examples include PDIp, a pancreas specific PDI that recognizes tyrosine and tryptophan

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residues68 or ERP57 that exclusively interacts with glycosylated proteins.69 In case of ERP57 substrate specificity is mediated indirectly through its interaction with the glycoprotein-binding protein calnexin.70 Similarly, substrate specificity of PDIA6 was suggested to be at least partially determined by its binding to BiP.71 Investigating whether different Conus PDI family members as well as different isoforms of the same PDI type exhibit direct and/or indirect substrate specificities for certain conotoxin families or toxin-associated chaperones will greatly advance our knowledge on conotoxins synthesis and the generation of disulfide-rich peptides in general. Comparisons of the venom gland proteomes demonstrated obvious variations in the overall expression profiles between the two species (Figure 3A and B). Recent proteomic analysis of the venom bulb showed high conservation of proteins between C. novaehollandiae and C. victoriae indicating a conserved function of this organ.16 In contrast, interspecies differences in the venom gland proteome suggest that glandular proteins experience a higher rate of adaptation and/or acclimatization. This may not be a reflection of the genetic diversity due to the potential differences in the timing and the level of protein expression and post-translational modification of proteins in the venom gland. While it is tempting to hypothesize that the accelerated diversification of conopeptides also applies to the proteins important for conopeptides biosynthesis such as PDI, genome sequencing alongside with proteomic interrogations are needed to investigate the potential accelerated evolution of venom gland proteins.

’ CONCLUSIONS This study comprehensively assessed the proteins expressed in the venom gland of cone snails and, in combination with histological assessment and peptidomic analysis, shed light on interspecies differences in venom composition, biosynthesis, packaging and secretion. Proteomic analyses led to the identification of a number of interesting proteins not previously described in Conus, such as the kallikrein-like and CRAMP1-like proteins found in C. novaehollandiae. We further identified two isoforms of a homologue of the Tex31 protease in C. victoriae and a number of other proteases that may play a role in conotoxin processing. Finally, we identify a multitude of PDI isoforms in the venom glands of both species potentially indicating an accelerated evolution of glandular proteins in cone snails. Future characterization of the different PDI isoforms will elucidate potential conotoxin-PDI (co)evolution events and advance our knowledge on conotoxin biosynthesis and the generation of biodiversity in these animals. ’ ASSOCIATED CONTENT

bS

Supporting Information Supplemental Figure 1 and Supplemental File 1 include annotated 2DGE images and full list of proteins identified in this study. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Anthony W. Purcell, E-mail: [email protected]. Phone +613 8344 2288. Fax +613 9348 142. 3917



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Specialisation of the Venom Gland Proteome in Predatory Cone

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