Chemical Punch Packed in Venoms Makes Centipedes Excellent Predators
2012; Elsevier BV; Volume: 11; Issue: 9 Linguagem: Inglês
10.1074/mcp.m112.018853
ISSN1535-9484
AutoresShilong Yang, Zhonghua Liu, Yao Xiao, Yuan Li, Mingqiang Rong, Songping Liang, Zhiye Zhang, Haining Yu, Glenn F. King, Ren Lai,
Tópico(s)Nicotinic Acetylcholine Receptors Study
ResumoCentipedes are excellent predatory arthropods that inject venom to kill or immobilize their prey. Although centipedes have long been known to be venomous, their venoms remain largely unexplored. The chemical components responsible for centipede predation and the functional mechanisms are unknown. Twenty-six neurotoxin-like peptides belonging to ten groups were identified from the centipede venoms, Scolopendra subspinipes mutilans L. Koch by peptidomics combined with transcriptome analysis, revealing the diversity of neurotoxins. These neurotoxins each contain two to four intramolecular disulfide bridges, and in most cases the disulfide framework is different from that found in neurotoxins from the venoms of spiders, scorpions, marine cone snails, sea anemones, and snakes (5S animals). Several neurotoxins contain potential insecticidal abilities, and they are found to act on voltage-gated sodium, potassium, and calcium channels, respectively. Although these neurotoxins are functionally similar to the disulfide-rich neurotoxins found in the venoms of 5S animals in that they modulate the activity of voltage-gated ion channels, in almost all cases the primary structures of the centipede venom peptides are unique. This represents an interesting case of convergent evolution in which different venomous animals have evolved different molecular strategies for targeting the same ion channels in prey and predators. Moreover, the high level of biochemical diversity revealed in this study suggests that centipede venoms might be attractive subjects for prospecting and screening for peptide candidates with potential pharmaceutical or agrochemical applications. Centipedes are excellent predatory arthropods that inject venom to kill or immobilize their prey. Although centipedes have long been known to be venomous, their venoms remain largely unexplored. The chemical components responsible for centipede predation and the functional mechanisms are unknown. Twenty-six neurotoxin-like peptides belonging to ten groups were identified from the centipede venoms, Scolopendra subspinipes mutilans L. Koch by peptidomics combined with transcriptome analysis, revealing the diversity of neurotoxins. These neurotoxins each contain two to four intramolecular disulfide bridges, and in most cases the disulfide framework is different from that found in neurotoxins from the venoms of spiders, scorpions, marine cone snails, sea anemones, and snakes (5S animals). Several neurotoxins contain potential insecticidal abilities, and they are found to act on voltage-gated sodium, potassium, and calcium channels, respectively. Although these neurotoxins are functionally similar to the disulfide-rich neurotoxins found in the venoms of 5S animals in that they modulate the activity of voltage-gated ion channels, in almost all cases the primary structures of the centipede venom peptides are unique. This represents an interesting case of convergent evolution in which different venomous animals have evolved different molecular strategies for targeting the same ion channels in prey and predators. Moreover, the high level of biochemical diversity revealed in this study suggests that centipede venoms might be attractive subjects for prospecting and screening for peptide candidates with potential pharmaceutical or agrochemical applications. Venomous animals have developed sophisticated chemical strategies to capture prey and defend themselves from predators (1Fry B.G. Roelants K. Champagne D.E. Scheib H. Tyndall J.D. King G.F. Nevalainen T.J. Norman J.A. Lewis R.J. Norton R.S. Renjifo C. de la Vega R.C. The toxicogenomic multiverse: Convergent recruitment of proteins into animal venoms.Annu. Rev. Genomics Hum. Genet. 2009; 10: 483-511Crossref PubMed Scopus (562) Google Scholar, 2Fry B.G. Vidal N. Norman J.A. Vonk F.J. Scheib H. Ramjan S.F. Kuruppu S. Fung K. Hedges S.B. Richardson M.K. Hodgson W.C. Ignjatovic V. Summerhayes R. Kochva E. Early evolution of the venom system in lizards and snakes.Nature. 2006; 439: 584-588Crossref PubMed Scopus (441) Google Scholar). Most of them secrete venoms containing substances with a wide variety of pharmacological activities to acutely interfere with the physiology of the prey or predator. The most thoroughly studied venomous animals are 5S animals (cone snails, scorpions, sea anemones, snakes, and spiders) (3Norton R.S. Structures of sea anemone toxins.Toxicon. 2009; 54: 1075-1088Crossref PubMed Scopus (60) Google Scholar, 4Terlau H. Olivera B.M. Conus venoms: A rich source of novel ion channel-targeted peptides.Physiol. Rev. 2004; 84: 41-68Crossref PubMed Scopus (804) Google Scholar, 5Koh C.Y. Kini R.M. From snake venom to therapeutics: Cardiovascular examples.Toxicon. 2011; 59: 497-506Crossref PubMed Scopus (157) Google Scholar, 6Calvete J.J. Juárez P. Sanz L. Snake venomics. Strategy and applications.J. Mass Spectrom. 2007; 42: 1405-1414Crossref PubMed Scopus (283) Google Scholar, 7Escoubas P. Diochot S. Corzo G. Structure and pharmacology of spider venom neurotoxins.Biochimie. 2000; 82: 893-907Crossref PubMed Scopus (209) Google Scholar, 8Rash L.D. Hodgson W.C. Pharmacology and biochemistry of spider venoms.Toxicon. 2002; 40: 225-254Crossref PubMed Scopus (284) Google Scholar, 9Vassilevski A.A. Kozlov S.A. Grishin E.V. Molecular diversity of spider venom.Biochemistry. 2009; 74: 1505-1534PubMed Google Scholar, 10Rodríguez de la Vega R.C. Possani L.D. Current views on scorpion toxins specific for K+-channels.Toxicon. 2004; 43: 865-875Crossref PubMed Scopus (306) Google Scholar, 11Rodríguez de la Vega R.C. Possani L.D. Overview of scorpion toxins specific for Na+ channels and related peptides: Biodiversity, structure-function relationships and evolution.Toxicon. 2005; 46: 831-844Crossref PubMed Scopus (303) Google Scholar, 12Fuller M.D. Thompson C.H. Zhang Z.R. Freeman C.S. Schay E. Szakács G. Bakos E. Sarkadi B. McMaster D. French R.J. Pohl J. Kubanek J. McCarty N.A. State-dependent inhibition of cystic fibrosis transmembrane conductance regulator chloride channels by a novel peptide toxin.J. Biol. Chem. 2007; 282: 37545-37555Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), frogs (13You D. Hong J. Rong M. Yu H. Liang S. Ma Y. Yang H. Wu J. Lin D. Lai R. The first gene-encoded amphibian neurotoxin.J. Biol. Chem. 2009; 284: 22079-22086Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), and insects including ants, bees, and wasps (14dos Santos L.D. Santos K.S. Pinto J.R. Dias N.B. de Souza B.M. dos Santos M.F. Perales J. Domont G.B. Castro F.M. Kalil J.E. Palma M.S. Profiling the proteome of the venom from the social wasp Polybia paulista: A clue to understand the envenoming mechanism.J. Proteome Res. 2010; 9: 3867-3877Crossref PubMed Scopus (61) Google Scholar). However, there are many other venomous animals including anguimorphs, birds, corals, fishes, jellyfishes, mammals, ticks, and also centipedes. Centipedes have long been known to be venomous, but their venoms have been little studied (15Rates B. Bemquerer M.P. Richardson M. Borges M.H. Morales R.A. De Lima M.E. Pimenta A.M. Venomic analyses of Scolopendra viridicornis nigraScolopendra angulata (Centipede, Scolopendromorpha): Shedding light on venoms from a neglected group.Toxicon. 2007; 49: 810-826Crossref PubMed Scopus (52) Google Scholar, 16González-Morales L. Diego-García E. Segovia L. Gutiérrez Mdel C. Possani L.D. Venom from the centipede Scolopendra viridis Say: Purification, gene cloning and phylogenetic analysis of a phospholipase A2.Toxicon. 2009; 54: 8-15Crossref PubMed Scopus (31) Google Scholar, 17Undheim E.A. King G.F. On the venom system of centipedes (Chilopoda), a neglected group of venomous animals.Toxicon. 2011; 57: 512-524Crossref PubMed Scopus (88) Google Scholar). One of the most obvious similarities shared by most of 5S venoms is that they contain many disulfide-rich peptide neurotoxins that act on ion channels or receptors (18Sollod B.L. Wilson D. Zhaxybayeva O. Gogarten J.P. Drinkwater R. King G.F. Were arachnids the first to use combinatorial peptide libraries?.Peptides. 2005; 26: 131-139Crossref PubMed Scopus (172) Google Scholar). Spider venoms contain an extreme diversity of small peptide neurotoxins (20–50 amino acids with three to five disulfide bridges) that are targeted to neuronal receptors and ion channels (Ca2+, Na+, K+, and Cl− channels) (7Escoubas P. Diochot S. Corzo G. Structure and pharmacology of spider venom neurotoxins.Biochimie. 2000; 82: 893-907Crossref PubMed Scopus (209) Google Scholar, 8Rash L.D. Hodgson W.C. Pharmacology and biochemistry of spider venoms.Toxicon. 2002; 40: 225-254Crossref PubMed Scopus (284) Google Scholar, 9Vassilevski A.A. Kozlov S.A. Grishin E.V. Molecular diversity of spider venom.Biochemistry. 2009; 74: 1505-1534PubMed Google Scholar). Scorpion venoms also contain many peptide neurotoxins, including short chain peptides (22–47 amino acids) acting on K+ channels (10Rodríguez de la Vega R.C. Possani L.D. Current views on scorpion toxins specific for K+-channels.Toxicon. 2004; 43: 865-875Crossref PubMed Scopus (306) Google Scholar) and long chain peptides (58–76 amino acids) acting on Na+ channels (11Rodríguez de la Vega R.C. Possani L.D. Overview of scorpion toxins specific for Na+ channels and related peptides: Biodiversity, structure-function relationships and evolution.Toxicon. 2005; 46: 831-844Crossref PubMed Scopus (303) Google Scholar). Some of them can interact with Ca2+ or Cl− channels (12Fuller M.D. Thompson C.H. Zhang Z.R. Freeman C.S. Schay E. Szakács G. Bakos E. Sarkadi B. McMaster D. French R.J. Pohl J. Kubanek J. McCarty N.A. State-dependent inhibition of cystic fibrosis transmembrane conductance regulator chloride channels by a novel peptide toxin.J. Biol. Chem. 2007; 282: 37545-37555Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Conus venoms are rich chemical cocktails with diverse range of peptides. Most disulfide-rich conotoxins from Conus venoms are 12–30 residues in length, and they mostly act on ligand-gated or voltage-gated ion channels (4Terlau H. Olivera B.M. Conus venoms: A rich source of novel ion channel-targeted peptides.Physiol. Rev. 2004; 84: 41-68Crossref PubMed Scopus (804) Google Scholar). Several superfamilies have been defined according to differences in their signal sequence and disulfide framework (4Terlau H. Olivera B.M. Conus venoms: A rich source of novel ion channel-targeted peptides.Physiol. Rev. 2004; 84: 41-68Crossref PubMed Scopus (804) Google Scholar). The venoms of Elapidae snakes are a rich source of three-fingered neurotoxins, 60–70-residue polypeptides that act on particular subtypes of voltage-gated ion channels in the brain and at neuromuscular junctions (19Kini R.M. Doley R. Structure, function and evolution of three-finger toxins: Mini proteins with multiple targets.Toxicon. 2010; 56: 855-867Crossref PubMed Scopus (261) Google Scholar). At least two classes of neurotoxins have been identified from sea anemones that act on sodium or potassium channels (20Suput D. In vivo effects of cnidarian toxins and venoms.Toxicon. 2009; 54: 1190-1200Crossref PubMed Scopus (74) Google Scholar). Despite their abundance and often painful encounters with humans, little is known about the composition of centipede venom (15Rates B. Bemquerer M.P. Richardson M. Borges M.H. Morales R.A. De Lima M.E. Pimenta A.M. Venomic analyses of Scolopendra viridicornis nigraScolopendra angulata (Centipede, Scolopendromorpha): Shedding light on venoms from a neglected group.Toxicon. 2007; 49: 810-826Crossref PubMed Scopus (52) Google Scholar, 16González-Morales L. Diego-García E. Segovia L. Gutiérrez Mdel C. Possani L.D. Venom from the centipede Scolopendra viridis Say: Purification, gene cloning and phylogenetic analysis of a phospholipase A2.Toxicon. 2009; 54: 8-15Crossref PubMed Scopus (31) Google Scholar, 17Undheim E.A. King G.F. On the venom system of centipedes (Chilopoda), a neglected group of venomous animals.Toxicon. 2011; 57: 512-524Crossref PubMed Scopus (88) Google Scholar). Centipede envenomations are capable of inflicting severe symptoms in humans, including myocardial ischemia and infarction, hemoglobinuria and hematuria, hemorrhage, and rhabdomyolysis, itching, fever and chills, general rash, eosinophilic cellulitis, and anaphylaxis. The long list of symptoms and complications induced by centipede envenomations suggests that centipede venoms contain a variety of different components with diverse functions (15Rates B. Bemquerer M.P. Richardson M. Borges M.H. Morales R.A. De Lima M.E. Pimenta A.M. Venomic analyses of Scolopendra viridicornis nigraScolopendra angulata (Centipede, Scolopendromorpha): Shedding light on venoms from a neglected group.Toxicon. 2007; 49: 810-826Crossref PubMed Scopus (52) Google Scholar, 16González-Morales L. Diego-García E. Segovia L. Gutiérrez Mdel C. Possani L.D. Venom from the centipede Scolopendra viridis Say: Purification, gene cloning and phylogenetic analysis of a phospholipase A2.Toxicon. 2009; 54: 8-15Crossref PubMed Scopus (31) Google Scholar, 17Undheim E.A. King G.F. On the venom system of centipedes (Chilopoda), a neglected group of venomous animals.Toxicon. 2011; 57: 512-524Crossref PubMed Scopus (88) Google Scholar). Centipedes are excellent predators. Their prey includes both vertebrates and invertebrates, including bats, rats, amphibians, reptiles, and insects. Centipede venom causes instant rigid paralysis in envenomated houseflies, cockroaches, and crickets, implying that there are neurotoxins in centipede venoms that provide an efficient means of rapidly paralyzing prey (15Rates B. Bemquerer M.P. Richardson M. Borges M.H. Morales R.A. De Lima M.E. Pimenta A.M. Venomic analyses of Scolopendra viridicornis nigraScolopendra angulata (Centipede, Scolopendromorpha): Shedding light on venoms from a neglected group.Toxicon. 2007; 49: 810-826Crossref PubMed Scopus (52) Google Scholar, 16González-Morales L. Diego-García E. Segovia L. Gutiérrez Mdel C. Possani L.D. Venom from the centipede Scolopendra viridis Say: Purification, gene cloning and phylogenetic analysis of a phospholipase A2.Toxicon. 2009; 54: 8-15Crossref PubMed Scopus (31) Google Scholar, 17Undheim E.A. King G.F. On the venom system of centipedes (Chilopoda), a neglected group of venomous animals.Toxicon. 2011; 57: 512-524Crossref PubMed Scopus (88) Google Scholar). However, until now, no convincing information has been available about the molecular nature of these neurotoxins. Based on the observed symptoms induced by centipede envenomations and the fast acting manner in which prey is subdued, we hypothesized that centipede venoms contain neurotoxins acting on ion channels and/or receptors that induce rapid paralysis. In the current work, we attempt to provide the first molecular evidence for the presence of neurotoxins in centipede venoms and to investigate the effect of these neurotoxins on insects and vertebrate ion channels. Assay-guided fractionation was used to purify 10 peptide neurotoxins from the venom of the centipede Scolopendra subspinipes mutilans. Complete amino acid sequences were determined for each of these toxins using a combination of Edman degradation and RACE analysis of a venom gland cDNA library. The cDNA sequences for the complete toxin transcripts indicated that each of these toxins is produced as a larger precursor that is post-translationally processed to yield the mature neurotoxin. The mature toxins ranged in size from 31 to 83 residues, and contained two to four disulfide bonds, which is similar to the mass range observed for neurotoxic peptides in arachnid venoms (21Escoubas P. Sollod B. King G.F. Venom landscapes: Mining the complexity of spider venoms via a combined cDNA and mass spectrometric approach.Toxicon. 2006; 47: 650-663Crossref PubMed Scopus (164) Google Scholar). Adult S. subspinipes mutilans L. Koch (both sexes, n = 3000) were purchased from Jiangsu Province of China. Venom was collected manually by stimulating the venom glands in the first pair forceps of centipedes using a 3 V alternating current as in our previous report (22Peng K. Kong Y. Zhai L. Wu X. Jia P. Liu J. Yu H. Two novel antimicrobial peptides from centipede venoms.Toxicon. 2010; 55: 274-279Crossref PubMed Scopus (62) Google Scholar). The venoms were stored at −20 °C until further use. Each milking occurred 1 week after the previous milking. One milliliter of venom was diluted with 3 ml of 0.1 m phosphate buffer, pH 6.0 (PBS) and mixed with 20 μl of proteinase inhibitor mixture (Sigma; P8340-5). The diluted venom (4 ml, total absorbance at 280 nm is 200) was applied to a Sephadex G-50 (Superfine; Amersham Biosciences; 2.6 × 100 cm) gel filtration column equilibrated with 0.1 m PBS. Elution was performed with the same buffer, collecting fractions of 3.0 ml. The absorbance of the eluate was monitored at 280 nm. The effects of eluted fractions on ion channel currents were assayed using patch clamp electrophysiology as described below. The fractions containing interesting activity were pooled (30 ml), lyophilized, resuspended in 2 ml of 0.1 m PBS, and purified further using C18 reverse phase (RP) 1The abbreviations used are:RPreverse phaseDRGdorsal root ganglionTTX-Stetrodotoxin-sensitiveTTX-Rtetrodotoxin-resistant. HPLC (Gemini C18 column, 5-μm particle size, 110 Å pore size, 250 × 4.6 mm). reverse phase dorsal root ganglion tetrodotoxin-sensitive tetrodotoxin-resistant. Lyophilized HPLC fractions were dissolved in 0.1% (v/v) trifluoroacetic acid/water, and 0.5 μl was spotted onto a MALDI-TOF plate with 0.5 μl of α-cyano-4-hydroxycinnamic acid matrix (10 mg/ml in 60% acetonitrile). The spots were analyzed by an UltraFlex I mass spectrometer (Bruker Daltonics) in a positive ion mode. Partial or complete amino acid sequences of purified neurotoxins were determined by Edman degradation using a pulsed liquid phase Procise® Sequencer, model 491 (Applied Biosystems). cDNA was prepared as we described previously (23Li J. Xu X. Xu C. Zhou W. Zhang K. Yu H. Zhang Y. Zheng Y. Rees H.H. Lai R. Yang D. Wu J. Anti-infection peptidomics of amphibian skin.Mol. Cell. Proteomics. 2007; 6: 882-894Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Total RNA was extracted from the venom glands of 20 centipedes using TRIzol (Invitrogen), and this was used to prepare cDNA using a SMARTTM PCR cDNA synthesis kit (Clontech). The first strand was synthesized by using the 3′ SMART CDS Primer II A (5′-AAGCAGTGGTATCAACGCAGAGTACT(30)N−1N-3′, where N = A, C, G, or T and N−1 = A, G, or C) and SMART II A oligonucleotide, (5′-AAGCAGTGGTATCAACGCAGAGTACGCGGG-3′). The 5′ PCR primer II A (5′-AAGCAGTGGTATCAACGCAGAGT-3′) provided by the kit was used to synthesize the second strand using Advantage polymerase (Clontech). RACE was used to clone transcripts encoding venom neurotoxins from the venom gland cDNA library (24Frohman M.A. Rapid amplification of complementary DNA ends for generation of full-length complementary DNAs: Thermal RACE.Methods Enzymol. 1993; 218: 340-356Crossref PubMed Scopus (465) Google Scholar). Sense direction primers were designed according to the amino acid sequences determined by Edman degradation (see supplemental Table S1 for a list of primers). These primers were used in conjunction with an antisense SMARTTM II A primer II in PCRs to screen for transcripts encoding neurotoxins. PCR was performed using Advantage polymerase (Clontech) using the following conditions: 2 min at 94 °C, followed by 30 cycles of 10 s at 92 °C, 30 s at 50 °C, 40 s at 72 °C. Finally, the PCR products were cloned into pGEM®-T Easy vector (Promega, Madison, WI). DNA sequencing was performed on an ABI PRISM 377 DNA sequencer (Applied Biosystems). Rat DRG neurons were acutely dissociated and maintained in a short term primary culture according to procedures adapted from Xiao et al. (25Xiao Y. Tang J. Yang Y. Wang M. Hu W. Xie J. Zeng X. Liang S. Jingzhaotoxin-III, a novel spider toxin inhibiting activation of voltage-gated sodium channel in rat cardiac myocytes.J. Biol. Chem. 2004; 279: 26220-26226Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Ca2+, Na+, and K+ currents were recorded from cells using the whole cell patch clamp technique using an Axon Multiclamp 700B amplifier (Molecular Devices). The P/4 protocol was used to subtract linear capacitive and leakage currents. The experiment data were acquired and analyzed by using the program Clampfit10.0 (Molecular Devices) and Sigmaplot (Sigma). All of the experimental protocols using animals were approved by the animal care and use committee at Kunming Institute of Zoology, Chinese Academy of Sciences (2011-162). Purified neurotoxins were dissolved in insect saline (concentrations in deionized water: 140 mm NaCl, 5 mm KCl, 4 mm NaHCO3, 1 mm MgCl2, 0.75 mm CaCl2, 5 mm HEPES) and injected into American cockroaches (Periplaneta americana; mass 700–900 mg), houseflies (Musca domestica larvae and adults; mass 35–45 mg and 15–25 mg, respectively), and mealworms (Tenebrio molitor larvae; mass 190–210 mg). The median lethal dose (LD50) was determined according to the method described by Tedford et al. (26Tedford H.W. Fletcher J.I. King G.F. Functional significance of the beta hairpin in the insecticidal neurotoxin omega-atracotoxin-Hv1a.J. Biol. Chem. 2001; 276: 26568-26576Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Fractionation of venom from the scolopendromorph centipede S. subspinipes mutilans using Sephadex G-50 gel filtration yielded seven protein fractions (Fig. 1A). Fraction V activated voltage-gated Ca2+ (CaV) channels in rat DRGs, whereas fraction VI inhibited K+ currents and tetrodotoxin-sensitive (TTX-S) voltage-gated Na+ (NaV) currents in rat DRGs (data not shown). Fraction V from the gel filtration separation was subjected to C18 RP-HPLC chromatography as illustrated in Fig. 1B. The peak eluting at 39 min ('5p-1g' in Fig. 1B) activated CaV currents in rat DRGs. This peak (5p-1g) was fractionated further by C18 RP-HPLC using a shallower acetonitrile gradient (Fig. 1C). The peak eluting at 25.5 min in the chromatogram shown in Fig. 1C (peak 5p-1g-4) activated CaV currents in rat DRGs (as described below), and MALDI-TOF-MS analysis (supplemental Fig. S1E) revealed it was a homogenous peptide with a mass of 8810.4 Da. Fraction VI from the gel filtration separation was subjected to C18 RP-HPLC chromatography as illustrated in Fig. 1D, resulting in the elution of more than 20 peaks. Peaks 6p-1g, 6p-3g, and 6p-6g were found to inhibit K+ currents in DRG neurons, peak 6p-4g was found to inhibit TTX-S NaV channel currents, and peak 6p-5g was found to activate CaV channel currents. All of these fractions were purified further using C18 RP-HPLC (Fig. 1, E–I). Peaks 6p-1g-2 (6050.2 Da), 6p-3g-1 (3465.8 Da), 6p-4g-1 (3762.5 Da), 6p-5g-2 (6015.2 Da), and 6p-6g-1 (7989.07 Da) were shown to be homogeneous by MALDI-TOF-MS analysis (supplemental Fig. S1). As described below, 6p-1g-2, 6p-3g-1, and 6p-6g-1 are neurotoxins that act on K+ channels. 6p-4g-1 inhibits TTX-S NaV channels, and 6p-5g-2 activates CaV channels. The other four peptides indicated as PN1, PN4, PN5, and PN9 are also purified, but their functions are unknown. Their partial amino acid sequences were determined by Edman degradation as illustrated in Fig. 8. 6p-4g-1 (named μ-scoloptoxin-Ssm1a; μ-SLPTX-Ssm1a; Fig. 2A) was found to inhibit TTX-S NaV channel currents in rat DRGs as illustrated in Fig. 2. NaV currents were elicited by a depolarization to −10 mV from a holding potential of −80 mV. As seen in Fig. 2, 10 nm μ-SLPTX-Ssm1a inhibited TTX-S NaV current amplitude by 60 ± 5%, whereas the TTX-S NaV current amplitude was depressed by almost 100% in the presence of 10 μm μ-SLPTX-Ssm1a (Fig. 2B). Because both TTX-S and TTX-resistant (TTX-R) NaV channels are expressed in small DRG neurons, we added 200 nm TTX to the extracellular solution to dissect out TTX-R NaV currents. The application of 10 μm toxin had virtually no effect on TTX-R NaV currents (Fig. 2C). We conclude based on the concentration-response curve shown in Fig. 2D that μ-SLPTX-Ssm1a specifically inhibits TTX-S NaV channels with an IC50 of ∼9 nm. The complete amino acid sequence of μ-SLPTX-Ssm1a determined by Edman degradation was ADNKFENSLRREIACGQCRDKVKCDPYFYHCG (Fig. 2A), yielding a predicted mass of 3763.2 Da for the fully oxidized form of the peptide. This corresponds closely to the mass of 3762.5 Da determined for the native toxin using MALDI-TOF-MS analysis (supplemental Fig. S1A). This indicates that the four cysteine residues in μ-SLPTX-Ssm1a form two disulfide bonds. A BLAST search of the UniProt database revealed that μ-SLPTX-Ssm1a is a novel peptide. Three peptide neurotoxins (6p-1g-2, κ-SLPTX-Ssm1a; 6p-3g-1, κ-SLPTX-Ssm2a; and 6p-6g-1, κ-SLPTX-Ssm3a) were purified and found to inhibit K+ channel currents in DRG neurons. The sequence of κ-SLPTX-Ssm1a (51 residues) was determined to be TDDESSNKCAKTKRRENVCRVCGNRSGNDEYYSECCESDYRYHRCLDLLRN by Edman degradation, mass spectrometry, and cDNA cloning (Fig. 3A and GenBankTM accession number JN646114). Four homologues of κ-SLPTX-Ssm1a (κ-SLPTX-Ssm1b-1e) were identified by cDNA cloning (GenBankTM accession number JQ757061-4). The complete toxin transcript cloned from the venom gland cDNA library revealed that the toxin is transcribed as a larger precursor containing a 23-residue prepro-region that is cleaved off during post-translational processing. There are six half-cysteines in the sequence. The molecular mass of κ-SLPTX-Ssm1a predicted from the sequence, assuming that the six Cys residues form three disulfide bridges, is 6050.6 Da, which represents a difference of only +0.4 from the mass of 6050.2 Da observed by MS analysis (supplemental Fig. S1B). We therefore conclude that the six Cys residues in κ-scoloptoxin-Ssm1a form three intramolecular disulfide bridges. A BLAST search of the UniProt database revealed no sequences similar to κ-SLPTX-Ssm1a. As seen in Fig. 3B, 8 nm κ-SLPTX-Ssm1a inhibited K+ current amplitude in DRG neurons by 25 ± 5%, but almost 100% of the K+ current amplitude was depressed when the toxin concentration was increased to 1 μm. Based on the concentration response shown in Fig. 3C, we conclude that κ-SLPTX-Ssm1a inhibits K+ currents in DRG neurons with an IC50 of ∼44.2 nm. The sequence of κ-SLPTX-Ssm2a (31 residues) was determined to be AQNHYCKEHNCPPGKHCPKVPIVCVYGPCCF by Edman degradation, mass spectrometry, and cDNA cloning (Fig. 4A). The complete toxin transcript (GenBankTM accession number JN646115) cloned from the venom gland cDNA library revealed that the toxin is transcribed as a larger precursor containing a 19-residue signal peptide (MLVFYALLFVTVFSNTVMG) and a 24-residue propeptide (ATIDKPIPKPILREAIEEIEVNKR) that are removed during post-translational processing. The C terminus of the propeptide contains a classical dibasic processing site (KR), and like most spider venom propeptides, it is highly acidic (18Sollod B.L. Wilson D. Zhaxybayeva O. Gogarten J.P. Drinkwater R. King G.F. Were arachnids the first to use combinatorial peptide libraries?.Peptides. 2005; 26: 131-139Crossref PubMed Scopus (172) Google Scholar, 27Wang X.H. Connor M. Wilson D. Wilson H.I. Nicholson G.M. Smith R. Shaw D. Mackay J.P. Alewood P.F. Christie M.J. King G.F. Discovery and structure of a potent and highly specific blocker of insect calcium channels.J. Biol. Chem. 2001; 276: 40306-40312Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Five homologues of κ-SLPTX-Ssm2a (predicted κ-SLPTX-Ssm2b-2f) were identified by cDNA cloning (JQ757065-9). The molecular mass of κ-SLPTX-Ssm2a calculated from the sequence, assuming that the six Cys residues form three intramolecular disulfide bridges, is 3467.1 Da, which represents a difference of +1.3 Da from the mass (3465.8 Da) determined by MS analysis (supplemental Fig. S1C); this suggests that these Cys residues in this toxin form three intramolecular disulfide bridges. A BLAST search of the UniProt database revealed no sequences similar to κ-SLPTX-Ssm2a. As seen in Fig. 4B, 200 nm κ-SLPTX-Ssm2a inhibited K+ current amplitude in DRG neurons by 45 ± 5%, and the current was inhibited by almost 80% when the toxin concentration was increased to 1 μm. Based on the concentration response shown in Fig. 4C, we conclude that κ-SLPTX-Ssm2a inhibits K+ currents in DRG neurons with an IC50 of ∼570 nm. The amino acid sequence of κ-SLPTX-Ssm3a (68 residues) was determined to be EVIAIDGLEICSNDQLHVTIYSIFSPLFDKPKLNYYFNCSCPGSIYISDVYPKYFNDIAHIEYRCKLT (Fig. 5A). The complete toxin transcript (GenBankTM accession number JN646116) cloned from the venom gland cDNA library revealed that the toxin is transcribed as a larger precursor containing a 16-residue signal peptide (MSWMFYSFIVFTLAIK) that is removed during post-translational processing. MS analysis of the native peptide yielded a mass of 7989.07 Da (supplemental Fig. S1D), which matches well with the predicted mass based on the sequence (7989.05 Da), assuming that the four Cys residues from two intramolecular disulfide bridges. 200 nm κ-SLPTX-Ssm3a inhibited K+ current amplitude in DRG neurons by only 25 ± 5% and even at concentrations up to 5 μm κ-SLPTX-Ssm3a did not inhibit all K+ currents. However, the toxin shows an obvious difference in its ability to inhibit peak currents and slowly activated delayed rectifier K+ currents (Fig. 5B). Two peptide neurotoxins (5p-1g-4, ω-SLPTX-Ssm1a; and 6p-5g-2, ω-SLPTX-Ssm2a) were purified and found to activate or inhibit CaV channel currents. The amino acid sequence of ω-SLPTX-Ssm1a (83 residues) was determined to be EECPSLSGGSSNYCSKKETFTSNGNLEQTRRYCNSVAAPSTACTDLKTGGKLCEYSCNTDGCNSVAGMEPTRAVYFIAILMLA (Fig. 5C). The complete toxin transcript (GenBankTM accession number JN646117) cloned from the venom gland cDNA library revealed that the t
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