Peptides 41 (2013) 8–16 Contents lists available at SciVerse ScienceDirect Peptides j ournal homepage: www.elsevier.com/locate/peptides Unraveling the peptidome of the South African cone snails Conus pictus and Conus natalis a a b c a Steve Peigneur , Annelies Van Der Haegen , Carolina Möller , Etienne Waelkens , Elia Diego-García , b d a,∗ Frank Marí , Ryno Naudé , Jan Tytgat a Laboratory of Toxicology, University of Leuven (K.U. Leuven), O&N 2, PO Box 922, Herestraat 49, 3000 Leuven, Belgium b Department of Chemistry and Biochemistry, Florida Atlantic University (FAU), 777 Glades Road, Boca Raton, FL 33431, USA c Laboratory of Protein Phosphorylation and Proteomics, University of Leuven (K.U. Leuven), O&N 1, PO Box 901, Herestraat 49, 3000 Leuven, Belgium d Department of Biochemistry and Microbiology, Nelson Mandela Metropolitan University, PO Box 77000, Port Elizabeth 6031, South Africa a r t i c l e i n f o a b s t r a c t Article history: Venoms from cone snails (genus Conus) can be seen as an untapped cocktail of biologically active com- Received 31 May 2012 pounds, being increasingly recognized as an emerging source of peptide-based therapeutics. Cone snails Received in revised form 2 July 2012 are considered to be specialized predators that have evolved the most sophisticated peptide chemistry Accepted 2 July 2012 and neuropharmacology system for their own biological purposes by producing venoms which contains Available online 7 July 2012 a structural and functional diversity of neurotoxins. These neurotoxins or conotoxins are often small cysteine-rich peptides which have shown to be highly selective ligands for a wide range of ion chan- Keywords: nels and receptors. Local habitat conditions have constituted barriers preventing the spreading of Conus Conus pictus species occurring along the coast of South Africa. Due to their scarceness, these species remain, there- Conus natalis fore, extremely poorly studied. In this work, the venoms of two South African cone snails, Conus pictus, Cone snail Conotoxin a vermivorous snail and Conus natalis, a molluscivorous snail, have been characterized in depth. In total, A-, M-, O-superfamily 26 novel peptides were identified. Comparing the venoms of both snails, interesting differences were observed regarding venom composition and molecular characteristics of these components. © 2012 Published by Elsevier Inc. 1. Introduction precursor that is proteolytically cleaved at specific sites to yield the mature toxin. Although hypermutation of peptide The marine gastropods known as cone snails (Conus) are one sequences is the main cause for conopeptide diversity, post- of the largest single genus of living marine invertebrates. All cone translational modifications provide an overlying level of diversity snails are venomous predators and possess a very complex venom [28]. apparatus. They use their venom for capturing prey, but also to Conopeptides can be classified into two major groups: the defend themselves. In addition, it is probably used in other biotic disulfide-poor and the disulfide-rich conopeptides, the latter interactions (e.g. interactions with competitors). Based on their also called conotoxins. These are classified into superfamilies, prey, cone snails can be divided into three groups: vermivorous based on a conserved signal sequence found in the precur- (worm-hunting), piscivorous (fish-hunting), and molluscivorous sor peptide and in their cysteine framework, and subsequently (mollusc-hunting) cone snails [26]. into pharmacological families based on the targets they interact More than 700 different cone snail species are known today with. At present, 22 cysteine frameworks have been identified and each species produces an unprecedented molecular diversity [16]. of up to 1000 distinct pharmacologically active components. In this work, two cone snails endemic to South Africa were stud- The venom of each different Conus species has a different set of ied. Conus pictus is a vermivorous cone snail, while Conus natalis peptides, resulting in an array of more than 150,000 peptides is a molluscivorous cone snail. Their habitats are restricted to the in total. Each conopeptide is encoded by a single messenger coastal regions of South Africa, in rather shallow water. A mass- ribonucleic acid (mRNA) and translated as a prepropeptide fingerprint study of the venom of C. pictus was already published [18] and recently, we reported the first characterized peptide from C. pictus, pc16a [32]. In this work, we identified 11 novel conopep- ∗ tides in C. pictus venom and 15 novel peptides in the venom of C. Corresponding author. Tel.: +32 16 32 34 03; fax: +32 16 32 34 05. natalis, totally or partially characterized by amino acid sequence E-mail addresses: [email protected], [email protected] (J. Tytgat). determination. The characterization of novel peptides from both 0196-9781/$ – see front matter © 2012 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.peptides.2012.07.002 S. Peigneur et al. / Peptides 41 (2013) 8–16 9 Table 1 venoms expands the knowledge about the South-African cone Mass spectrometric analysis of purified Conus pictus and Conus natalis venom. snails, whose venoms are unexplored to date. Masses are shown as average masses. ESI-MS [M] (Da) of C. ESI-MS [M] (Da) of C. natalis venom pictus venom 2. Materials and methods 1062.3 3232.5 1161.5 2546.5 3808.8 1135.6 3312.1 1258.3 2603.1 3980.3 2.1. Materials 1151.8 3325.6 1389.8 2618.4 3985.0 1249.5 3328.8 1428.0 2695.7 4017.6 Specimens were collected from the coastal region of Port 1258.3 3333.8 1631.9 2705.8 4033.6 Elizabeth, South-Africa. Venom ducts and bulbs were dissected, 1259.2 3343.4 1642.5 2723.6 4099.1 ◦ lyophilized and stored at −20 C. 1288.2 3376.0 1664.4 2836.1 4127.5 1303.5 3391.5 1680.8 2879.8 4212.9 1324.8 3873.5 1698.6 2890.7 4429.7 1341.0 3901.3 1705.2 3003.2 4627.5 2.2. Extraction of crude venom 1403.5 3953.3 1717.5 3018.8 4627.6 1460.5 3960.8 1721.0 3071.5 4638.6 Venom bulbs and ducts of 6 (C. pictus) and 5 specimens 1509.3 4042.6 1731.8 3087.9 4671.1 (C. natalis) were macerated and extracted with 30% acetonitrile 1566.7 8565.1 1741.8 3168.8 4683.4 1621.3 1742.7 3276.5 4737.7 (ACN) containing 0.1% trifluoroacetic acid (TFA) and centrifuged at ◦ 1640.8 1758.4 3305.2 4749.0 16,100 × g at 4 C for 30 min. The supernatant was lyophilized and 1661.8 1798.8 3329.5 4782.7 − ◦ stored at 20 C. 1678.0 1818.8 3431.1 4935.3 1855.0 1825.1 3451.0 4963.5 1932.4 1834.3 3470.7 5039.4 1965.7 1904.4 3546.4 5052.2 2.3. Purification 1986.0 1954.8 3553.9 5084.9 2134.3 1998.0 3572.9 5097.2 Lyophilized crude venom extract was dissolved in 30% ACN, 2149.8 2014.6 3585.6 7911.2 containing 0.1% TFA. Size exclusion chromatography was per- 2186.6 2137.9 3590.1 7955.6 TM formed on a Superdex Peptide 10/300 GL column (GE Healthcare 2259.6 2139.5 3675.7 8008.8 2295.2 2408.7 3701.7 10,460.9 Bio-Sciences AB, Sweden) at room temperature at a flow rate of 2599.6 2453.2 3720.4 11,071.4 0.5 ml/min using an isocratic elution with 30% ACN, containing 2618.5 2467.3 3742.3 11,086.4 0.1% TFA. A second purification was performed on a reversed-phase 2922.7 2493.6 3765.1 11,819.2 × Vydac C18 column (Grace, USA, 218MS54, 4.6 mm 250 mm, 5 ␮m Total masses = 44 Total masses = 90 particle size) at room temperature at a flow rate of 1 ml/min using a linear gradient from 0 to 70% solution B in 280 min. Solutions were as follows: (A) 0.1% (v/v) TFA in water; (B) 0.085% (v/v) TFA 3. Results in ACN. Absorbance was monitored at 214 nm for all purifications and peaks were collected using a PrepFC collector (Gilson, USA). 3.1. Chromatographic separation and mass fingerprinting of C. pictus and C. natalis venom 2.4. Mass spectrometry Crude venoms of C. pictus and C. natalis were fractionated into five fractions by SE-HPLC using a Superdex Peptide column Molecular mass analysis was performed on a LCQ Deca XP elec- (Fig. 1A and Fig. 2A, respectively). Each fraction was lyophilized trospray ionization – quadrupole ion trap – mass spectrometer and further purified by RP-HPLC using a Vydac C18 column (Thermo Finnigann, USA), using a positive-ionization mode. The (Fig. 1B–D and Fig. 2B and C). Only the chromatograms of the frac- sample was dissolved in 50% aqueous ACN containing 0.1% acetic tions in which several peptides were characterized are shown. acid. A flow rate of 7.5 ␮l/min, a spray voltage of 4 kV, a capillary ◦ temperature of 142 C, a capillary voltage of 38.5 V and a tube lens 3.2. Mass spectrometric analysis of purified C. pictus and C. offset of 152 V were employed. A full scan from 400 to 2000 m/z was TM natalis venom performed and the data were deconvoluted using the ProMass Deconvolution software giving the monoisotopic mass of the pep- tide. Due to limited native material, we were not able to determine the molecular masses of all compounds present in the venom of C. pictus and C. natalis. In total, we detected 44 components in the purified fractions of the venom of C.