The First γ-Carboxyglutamic Acid-containing Contryphan
2004; Elsevier BV; Volume: 279; Issue: 31 Linguagem: Inglês
10.1074/jbc.m313825200
ISSN1083-351X
AutoresKarin Hansson, Xiaosong Ma, Lena Eliasson, Eva Czerwiec, Bruce Furie, Barbara C. Furie, Patrik Rorsman, Johan Stenflo,
Tópico(s)Ion channel regulation and function
ResumoContryphans constitute a group of conopeptides that are known to contain an unusual density of post-translational modifications including tryptophan bromination, amidation of the C-terminal residue, leucine, and tryptophan isomerization, and proline hydroxylation. Here we report the identification and characterization of a new member of this family, glacontryphan-M from the venom of Conus marmoreus. This is the first known example of a contryphan peptide carrying glutamyl residues that have been post-translationally carboxylated to γ-carboxyglutamyl (Gla) residues. The amino acid sequence of glacontryphan-M was determined using automated Edman degradation and electrospray ionization mass spectrometry. The amino acid sequence of the peptide is: Asn-Gla-Ser-Gla-Cys-Pro-d-Trp-His-Pro-Trp-Cys. As with most other contryphans, glacontryphan-M is amidated at the C terminus and maintains the five-residue intercysteine loop. The occurrence of a d-tryptophan residue was confirmed by chemical synthesis and HPLC elution profiles. Using fluorescence spectroscopy we demonstrated that the Gla-containing peptide binds calcium with a KD of 0.63 mm. Cloning of the full-length cDNA encoding glacontryphan-M revealed that the primary translation product carries an N-terminal signal/propeptide sequence that is homologous to earlier reported contryphan signal/propeptide sequences up to 10 amino acids preceding the toxin region. Electrophysiological experiments, carried out on mouse pancreatic B-cells, showed that glacontryphan-M blocks L-type voltage-gated calcium ion channel activity in a calcium-dependent manner. Glacontryphan-M is the first contryphan reported to modulate the activity of L-type calcium ion channels. Contryphans constitute a group of conopeptides that are known to contain an unusual density of post-translational modifications including tryptophan bromination, amidation of the C-terminal residue, leucine, and tryptophan isomerization, and proline hydroxylation. Here we report the identification and characterization of a new member of this family, glacontryphan-M from the venom of Conus marmoreus. This is the first known example of a contryphan peptide carrying glutamyl residues that have been post-translationally carboxylated to γ-carboxyglutamyl (Gla) residues. The amino acid sequence of glacontryphan-M was determined using automated Edman degradation and electrospray ionization mass spectrometry. The amino acid sequence of the peptide is: Asn-Gla-Ser-Gla-Cys-Pro-d-Trp-His-Pro-Trp-Cys. As with most other contryphans, glacontryphan-M is amidated at the C terminus and maintains the five-residue intercysteine loop. The occurrence of a d-tryptophan residue was confirmed by chemical synthesis and HPLC elution profiles. Using fluorescence spectroscopy we demonstrated that the Gla-containing peptide binds calcium with a KD of 0.63 mm. Cloning of the full-length cDNA encoding glacontryphan-M revealed that the primary translation product carries an N-terminal signal/propeptide sequence that is homologous to earlier reported contryphan signal/propeptide sequences up to 10 amino acids preceding the toxin region. Electrophysiological experiments, carried out on mouse pancreatic B-cells, showed that glacontryphan-M blocks L-type voltage-gated calcium ion channel activity in a calcium-dependent manner. Glacontryphan-M is the first contryphan reported to modulate the activity of L-type calcium ion channels. The vitamin K-dependent γ-glutamyl carboxylase catalyzes the post-translational conversion of glutamyl residues to γ-carboxyglutamyl (Gla) 1The abbreviations used are: Gla, γ-carboxyglutamic acid; HPLC, high performance liquid chromatography; NanoESI-MS, nanoelectrospray ionization mass spectrometry. 1The abbreviations used are: Gla, γ-carboxyglutamic acid; HPLC, high performance liquid chromatography; NanoESI-MS, nanoelectrospray ionization mass spectrometry. residues in precursor proteins that contain the appropriate γ-carboxylation recognition site within the propeptide of the precursor (1Stenflo J. Suttie J.W. Annu. Rev. Biochem. 1977; 46: 157-172Google Scholar, 2Suttie J.W. Annu. Rev. Biochem. 1985; 54: 459-477Google Scholar, 3Furie B. Bouchard B.A. Furie B.A. Blood. 1999; 93: 1798-1808Google Scholar, 4Stenflo J. Crit. Rev. Eukaryot. Gene. Expr. 1999; 9: 59-88Google Scholar). Among the classes of proteins that contain Gla, the vitamin K-dependent blood coagulation proteins have been most thoroughly studied. Upon addition of calcium they undergo a conformational transition that is a prerequisite for their interaction with biological membranes, and hence crucially important for their biological activity. Subsequent to its initial discovery, Gla was shown in 1984 to be present in venom peptides of highly specialized invertebrate systems, marine snails of the genus Conus (5McIntosh J.M. Olivera B.M. Cruz L.J. Gray W.R. J. Biol. Chem. 1984; 259: 14343-14346Google Scholar, 6Olivera B.M. Rivier J. Clark C. Ramilo C.A. Corpuz G.P. Abogadie F.C. Mena E.E. Woodward S.R. Hillyard D.R. Cruz L.J. Science. 1990; 20: 257-263Google Scholar). It was found that a neuroactive Conus peptide, conantokin-G (17 amino acid residues) contained five residues of Gla. These discoveries indicated that the role of this post-translational modification in blood coagulation represents only a subset of Gla function in animal phyla. The cone snail venoms contain a diverse array of paralyzing peptides (conotoxins) that are injected into prey after a cone snail harpoons its victim. The peptides specifically bind to a variety of receptors and ion channels in the neuromuscular system and interfere with their function. The Gla content varies from species to species but is especially high in the venoms of Conus textile and Conus marmoreus (7Hauschka P.V. Mullen E.A. Hintsch G. Jazwinski S. Suttie J.W. Current Advances in Vitamin K Research. Elsevier, New York1988: 237-243Google Scholar). Cloning and expression of the Conus (8Begley G.S. Furie B.C. Czerwiec E. Taylor K.L. Furie G.L. Bronstein L. Stenflo J. Furie B. J. Biol. Chem. 2000; 275Google Scholar, 9Bandyopadhyay P.K. Garrett J.E. Shetty R.P. Keate T. Walker C.S. Olivera B.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1264-1269Google Scholar, 10Czerwiec E. Begley G.S. Bronstein M. Stenflo J. Taylor K. Furie B.C. Furie B. Eur. J. Biochem. 2002; 269: 6162-6172Google Scholar) and Drosophila (11Tao L. Yang C-T. Jin D. Stafford W. J. Biol. Chem. 2000; 275: 18291-18296Google Scholar, 12Walker C.S. Shetty R.P. Clark K. Kazuko S.G. Letsou A. Olivera B.M. Bandyopadhyay P.K. J. Biol. Chem. 2001; 276: 7769-7774Google Scholar) γ-glutamyl carboxylases has revealed a marked conservation of this gene in the animal kingdom. Experiments with crude preparations of Conus carboxylase have shown that this enzymatic reaction requires vitamin K (7Hauschka P.V. Mullen E.A. Hintsch G. Jazwinski S. Suttie J.W. Current Advances in Vitamin K Research. Elsevier, New York1988: 237-243Google Scholar, 13Stanley T.B. Stafford D.W. Olivera B.M. Bandyopadhyay P.K. FEBS Lett. 1997; 407: 85-88Google Scholar). Like its mammalian counterpart the Conus carboxylase requires a recognition site that resides on the propeptide of the precursor form of the toxin (14Bandyopadhyay P.K. Colledge C.J. Walker C.S. Zhou L. Hillyard D.R. Olivera B.M. J. Biol. Chem. 1998; 273: 5447-5450Google Scholar, 15Bush K. Stenflo J. Roth D.A. Czerwiec E. Harrist A. Begley G.S. Furie B.C. Furie B. Biochemistry. 1999; 38: 14660-14666Google Scholar). Although the γ-glutamyl carboxylases are highly conserved, the Conus and mammalian carboxylase binding sites do not bear any obvious sequence resemblance. To date several Gla-containing conotoxins have been isolated (16Haack J.A. Rivier J. Parks T.N. Mena E.E. Cruz L.J. Olivera B.M. J. Biol. Chem. 1990; 265: 6025-6029Google Scholar, 17Fainzilber M. Gordon D. Hasson A. Spira M.E. Zlotkin E. Eur. J. Biochem. 1991; 202: 589-595Google Scholar, 18Nakamura T. Yu Z. Fainzilber M. Burlingame A.L. Protein Sci. 1996; 5: 524-530Google Scholar, 19Fainzilber M. Nakamura T. Lodder J.C. Zlotkin E. Kits K.S. Burlingame A.L. Biochemistry. 1998; 37: 1470-1477Google Scholar, 20Rigby A.C. Lucas-Meunier E. Kalume D.E. Czerwiec E. Hambe B. Dahlqvist I. Fossier P. Baux G. Roepstorff P. Baleja J.D. Furie B.C. Furie B. Stenflo J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5758-5763Google Scholar, 21Kalume D.E. Stenflo J. Czerwiec E. Hambe B. Furie B.C. Furie B. Roepstorff P. J. Mass Spectr. 2000; 35: 145-156Google Scholar). The metal binding properties and the three-dimensional structure of some of these conotoxins suggest a specific structural role for Gla (22Prorok M. Warder S.E. Blandl T. Castellino F.J. Biochemistry. 1996; 35: 16528-16534Google Scholar, 23Rigby A.C. Baleja J.D. Furie B.C. Furie B. Biochemistry. 1997; 36: 6906-6914Google Scholar, 24Rigby A. Baleja L. Li L. Pedersen L.G. Furie B.C. Furie B. Biochemistry. 1997; 36: 15677-15684Google Scholar, 25Skjaerbaek N. Nielsen K.J. Lewis R.J. Alewood P. Craik D.J. J. Biol. Chem. 1997; 272: 2291-2299Google Scholar, 26Chen Z. Blandl T. Prorok M. Warder S.E. Li L. Zhu Y. Pedersen L.G. Ni F. Castellino F.J. J. Biol. Chem. 1998; 26: 16248-16258Google Scholar, 27Prorok M. Castellino F.J. J. Biol. Chem. 1998; 273: 19573-19578Google Scholar, 28Blandl T. Warder S.E. Prorok M. Castellino F.J. FEBS Lett. 2000; 470: 139-146Google Scholar). With the exception of conantokin-G the function of Gla in the conotoxins is, however, still unknown (5McIntosh J.M. Olivera B.M. Cruz L.J. Gray W.R. J. Biol. Chem. 1984; 259: 14343-14346Google Scholar, 29Chandler P. Pennington M. Maccecchini M.L. Nashed N.T. Skolnick P. J. Biol. Chem. 1993; 268: 17173-17178Google Scholar, 30Zhou L.M. Szendrei G.I. Fossom L.H. Maccecchini M.L. Skolnick P. Otvos Jr., L. Neurochemistry. 1996; 66: 620-628Google Scholar). The contryphan family of conopeptides isolated from piscivorous (31Jimenez E.C. Craig A.G. Watkins M. Hillyard D.R. Gray W.R. Gulyas J. Rivier J.E. Cruz L.J. Olivera B.M. Biochemistry. 1997; 36: 989-994Google Scholar, 32Jacobsen R. Jimenez E.C. Grilley M. Watkins M. Hillyard D. Cruz L.J. Olivera B.M. J. Pept. Res. 1998; 51: 173-179Google Scholar), molluscivorous (33Jimenez E.C. Watkins M. Juszczak L.J. Cruz L.J. Olivera B.M. Toxicon. 2001; 39: 803-808Google Scholar), and vermivourous (34Massilia G.R. Schininá M.E. Ascenzi P. Polticelli F. Biochem. Biophys. Res. Commun. 2001; 288: 908-913Google Scholar) cone snails are distinct for their unusual density of post-translational modifications. These include bromination of tryptophan, proline hydroxylation, C-terminal amidation, and leucine and tryptophan L to D isomerization. Contryphans cause the so-called "stiff-tail syndrome" when injected intracranially in mice (35Jimenez E.C. Olivera B.M. Gray W.R. Cruz L.J. J. Biol. Chem. 1996; 271: 28002-28005Google Scholar) and body tremor and secretion of mucous substances when injected into fish (36Jacobsen R. Jimenez E.C. De la Cruz R.G.C. Gray W.R. Cruz L.J. Olivera B.M. J. Pept. Res. 1999; 54: 93-99Google Scholar). Recently, the first functional target for a contryphan was reported (37Massilia G.R. Eliseo T. Grolleau F. Lapied B. Barbier J. Bournaud R. Molgó J. Cicero D.O. Paci M. Schininá M.E. Ascenzi P. Poltizelli F. Biochem. Biophys. Res. Commun. 2003; 303: 238-246Google Scholar) as Contryphan-Vn from the wormhunting C. ventricosus was demonstrated to affect both voltage-gated and calcium-dependent K+ channels. Here we describe the identification, purification, and characterization of a novel contryphan peptide, glacontryphan-M, extracted from the venom of the molluscivorous C. marmoreus. Glacontryphan-M is the first example of a contryphan peptide containing Gla residues endowing it with calcium binding properties. Cloning of the cDNA of glacontryphan-M showed a high density of arginyl and lysyl residues in the propetide region compared with the propeptide regions of the earlier described contryphans. This might indicate that these basic residues are part of the γ-carboxylation recognition site. Patch-clamp recordings carried out on insulin-secreting B-cells from the islets of Langerhans demonstrate that glacontryphan-M, in a calcium-dependent manner, specifically antagonizes L-type Ca2+ channel activity. Like in other neuro- and endocrine cells (38Burgoyne R.D. Morgan A. Biochem. J. 1993; 293: 305-316Google Scholar) exocytosis from pancreatic B-cells is determined by calcium influx through voltage-dependent Ca2+ channels (39Barg S. Ma X. Eliasson L. Galvanovskis J. Göpel S.O. Obermuller S. Platzer J. Renström E. Trus M. Atlas D. Striessnig J. Rorsman P. Biophys. J. 2001; 81: 3308-3323Google Scholar). This is the first Gla-containing conopeptide reported to modulate the activity of this type of calcium ion channels. Conotoxin Purification—Frozen cone snails (C. marmoreus) were obtained from Vietnam. Lyophilized venom extract (1000 mg, from five cone snails) was dissolved in 0.2 m ammonium acetate buffer (pH 7.5) (17Fainzilber M. Gordon D. Hasson A. Spira M.E. Zlotkin E. Eur. J. Biochem. 1991; 202: 589-595Google Scholar, 20Rigby A.C. Lucas-Meunier E. Kalume D.E. Czerwiec E. Hambe B. Dahlqvist I. Fossier P. Baux G. Roepstorff P. Baleja J.D. Furie B.C. Furie B. Stenflo J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5758-5763Google Scholar) and chromatographed on a Sephadex G-50 superfine column (2.5 × 92 cm) equilibrated with 0.2 m ammonium acetate buffer (pH 7.5) and eluted with a flow rate of 10.3 ml/h (Fig. 1A). The material in the major Gla-containing peak was separated on a reversed-phase HPLC column (HyChrom C18, 5 μm; 10 × 250 mm) in 0.1% (v/v) trifluoroacetic acid and eluted with a linear acetonitrile gradient at a flow rate of 2 ml/min (Fig. 1B). A total of 30 nmol of Glacontryphan-M was obtained from a second reversed-phase HPLC purification step (Vydac C18 column, 5 μm; 4.6 × 250 mm) in 0.1% trifluoroacetic acid using a linear acetonitrile gradient at a flow rate of 0.5 ml/min (Fig. 1C). Amino Acid Analysis and Sequencing—The amino acid composition of glacontryphan-M was determined after acid hydrolysis, except for Gla, which was measured after alkaline hydrolysis (40Stenflo J. Fernlund P. Egan W. Roepstorff P. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 2730-2733Google Scholar, 41Fernlund P. Stenflo J. J. Biol. Chem. 1975; 250: 6125-6133Google Scholar). For peptide sequencing a PerkinElmer Life Sciences (Foster City, CA) ABI Procise 494 sequencer was used according to the protocol from the manufacturer. To identify the Gla residues in the sequence, the sample was methyl-esterfied with methanolic HCl prior to sequencing (42Cairns J.R. Williamson M.K. Price P.A. Anal. Biochem. 1991; 199: 93-97Google Scholar). Reduction and Alkylation of Disulfide Bonds—For reduction and alkylation of cysteine residues with iodoacetic acid (BDH Chemicals Ltd, Poole, England), the peptides were dissolved in 250 μl of 6 m guanidine hydrochloride in 1 m Tris-HCl, 10 mm EDTA (pH 8.6). Dithiothreitol (ICN Biomedicals Inc.) was added to 20 mm followed by incubation at 37 °C for 2 h, after which iodoacetic acid was added to a final concentration of 50 mm. After incubation at room temperature for 30 min, β-mercaptoethanol (BDH Chemicals Ltd) was added to a final concentration of 1% to quench the reaction. The sample was then dialyzed against 3.5 m acetic acid, and the peptide was purified by reversed-phase HPLC. Peptide Synthesis—Peptides covering the mature toxin region of glacontryphan-M were synthesized either with a dor l-tryptophan at position 7. The peptides were synthesized with DPfd l- and d-Fmoc amino acids (Perseptive Biosystems, Framingham, MA) on a Milligen 9050 Plus peptide synthesizer (PerkinElmer Life Sciences). The peptides were deprotected and cleaved from the resin by treatment with 95% anhydrous trifluoroacetic acid containing relevant scavengers. Each peptide was then applied to a C8 preparative reversed-phase HPLC column and eluted with an acetonitrile gradient in 0.1% (v/v) trifluoroacetic acid. For each of the synthesized peptides the major peak corresponding to the peptide fraction was added dropwise to an equal volume of 1 mm iodine in 20% acetonitrile in 0.1% (v/v) trifluoroacetic acid. After 15 min the reactions were quenched with a few drops of 0.1 m ascorbic acid. Each of the oxidized peptides was applied to a reversed-phase C8 preparative column and eluted with an acetonitrile gradient in 0.1% (v/v) trifluoroacetic acid. Major peaks were analyzed with nanoelectrospray ionization mass spectrometry (NanoESI-MS). The synthesized d-tryptophan-containing peptide was further purified on a reversed-phase C18 column using an acetonitrile gradient supplied with 0.1% (v/v) trifluoroacetic acid, and the synthesis was confirmed by NanoESI-MS analysis and amino acid sequencing. For the synthesized l-tryptophan-containing peptide, NanoESI-MS analysis revealed that only dimers had formed during the folding step. A peptide, glucontryphan-M, covering the mature toxin region of glacontryphan-M but having the Gla residues at positions 2 and 4 replaced with glutamic acid residues was synthesized, folded, and purified according to the above procedure. The synthesis was confirmed by NanoESI-MS analysis and amino acid sequencing. Mass Spectrometry—NanoESI mass spectra were acquired on an API QSTAR Pulsar-i quadrapole time-of-flight mass spectrometer (Applied Biosystems/MDS Sciex, Toronto, Canada) equipped with a NanoSpray ion source (MDS Proteomics, Odense, Denmark). The samples were sprayed from a silver-coated glass EconoTip nanospray capillary supplied by New Objective (Woburn, MA). Spectra were obtained in either positive or negative ion mode. A potential of 800–1100 V was applied to the nanoflow tip in the ion source using a curtain gas (N2) flow rate of 1.3 liter/min. The acquisition and the deconvolution of data were performed on an AnalystQS Windows PC data system. Bioanalyst version 1.0 software (Applied Biosystems/MDS Sciex) was used to analyze the mass spectra. Samples were dried and dissolved (∼1–5 pmol/μl) in 50% (v/v) methanol supplied with 1% (v/v) formic acid for NanoESI-MS and MS/MS analysis in positive ion mode and in 50% acetonitrile for NanoESI-MS analysis in negative ion mode. Parent ions (identified in a time-of-flight MS survey scan) for MS/MS analysis were selected in Q1, and product ions were generated in Q2 using N2 as the collision gas and collision energies of 14–35 eV. Identification and Sequencing of a cDNA Clone Encoding Glacontryphan-M—On the basis of the amino acid sequence of the isolated contryphan peptide from C. marmoreus and the well conserved signal peptide of the previously identified contryphans, oligonucleotide primers were designed for PCR (amplification of the corresponding cDNA from a Lambda ZAP II Custom cDNA library (Stratagene). Two oligonucleotide primers with degenerate nucleotide sequences were synthesized. p95–1, 5′-ATG GGN AAR YTN ACN ATH YTN G-3′; p95–2, 5′-CCR CAC CAN GGR TGC CAN GGR CAY TC-3′(where n = A, T, C or G, Y = T or C, H = A, C or T, r = A or G) represent sequences complementary to the sequences at the N terminus signal peptide and the C terminus toxin region of the peptide, respectively. The amplification parameters for the PCR reactions were 94 °C for 5 min; 35 cycles of 94 °C for 1 min, 46 °C for 1 min and 72 °C for 1 min; and a 10-min extension at 72 °C. The PCR product was TA-cloned into the pGEM-T vector (Promega), and the resulting plasmid DNA sequenced using M13 forward, 5′-GTT TTC CCA GTC ACG AC-3′ and reversed 5′-CAG GAA ACA GCT ATG AC-3′, primers. Gene-specific oligonucleotide primers, p95–3, 5′-TCT CGC TCT ACC TGG GTT ATC GTC-3′and p95–4, 5′-AGC GAG AAG AAA GCG CAT GAA AG-3′ (Fig. 6) were used in combination with the vector-specific M13 forward and reverse primers for PCR amplification of the 5′- and the 3′-ends of the cDNA encoding glacontryphan-M, respectively. The PCR products were cloned in vector pGEM-T and sequenced using M13 reverse and forward primers. The entire cDNA sequence of glacontryphan-M was assembled from the overlapping sequences and is predicted to contain the amino acid sequence of the mature contryphan toxin. The obtained cDNA sequence was confirmed by cloning (in pGEM-T) and sequencing (using M13 reverse and forward primers) of the PCR products obtained with oligonucleotide primer p95–6, 5′-AAC CTT TAT CAT GG-3′, in combination with primer p95–7, 5′-CAA TCG TGG ATT CCG ATC-3′, or primer p95–8, 5′-GGA CAT TCA GAC TCA TTT AG-3′ (Fig. 6). Calcium Binding Measurements—Tryptophan fluorescence was measured at 20 °C in a Spex Fluoromax-3 spectrofluorometer (Jobin Yvon-Spex, Instruments s.a., Inc.) equipped with a 150 watt Xenon lamp. The peptide concentrations were 2 μm, and the solvent was metal-free 50 mm Tris-HCl, pH 7.5 containing 0.1 m NaCl. The samples were subjected to exciting light for a minimum of time to avoid photodecomposition. Fluorescence emission spectra were recorded between 295 and 425 nm with an excitation wavelength of 280 nm and excitation and emission bandwidths of 2 and 8 nm, respectively. The Ca2+ dependence of the intrinsic fluorescence was measured by addition of 0.3–1 μl portions of 0.11 or 0.77 mm stock solutions of CaCl2 to 1 ml of sample and by averaging 30 signals readings of 0.25 s each. The excitation wavelength was 280 nm and the emission wavelength 355.5 nm (corresponding to the maximum fluorescence emission intensity of the peptides). The excitation and emission bandwidths were 2 and 8 nm, respectively. The data were expressed as 1 – F/F0 where F and F0 are the fluorescence intensities in the presence and absence of Ca2+ ions, respectively. The dissociation constant was determined by non-linear regression to fit the data to the equilibrium binding equation (43Nordenman B. Danielsson A. Björk I. Eur. J. Biochem. 1978; 90: 1-6Google Scholar). Electrophysiology—Isolated pancreatic islets from NMRI mice were dispersed into single cells using a Ca2+-free solution as described elsewhere (44Eliasson L. Ma X. Renström E. Barg S. Berggren P.O. Galvanovskis J. Gromada J. Jing X. Lundquist I. Salehi A. Sewing S. Rorsman P. J. Gen. Physiol. 2003; 121: 181-197Google Scholar). The insulin-secreting B-cells were identified by their absence of Na+ current at physiological membrane-potentials (45Barg S. Galvanovskis J. Göpel S.O. Rorsman P. Eliasson L. Diabetes. 2000; 49: 1500-1510Google Scholar). Patchclamp electrodes were made from borosilicate glass capillaries coated with sylgard and fire-polished. The pipette resistance ranged between 3 and 6 MΩ when filled with pipette solution as specified below. Whole cell currents were recorded using an EPC-9 patch clamp amplifier and the software Pulse ver 8.31 (Heka Elektronik, Germany). All experiments were conducted using the perforated patch whole cell configuration in which the cytoplasm retains intact. The standard extracellular solution consisted of 118 mm NaCl, 20 mm tetraethyl-ammonium chloride (TEA-Cl; to block voltage-gated K+ currents), 5.6 mm KCl, 2.6 mm CaCl2, 1.2 mm MgCl2, 5 mm glucose, and 5 mm HEPES (pH 7.4 using NaOH). In the Ca2+-free extracellular solution CaCl2 was substituted with 11.4 mm sucrose (to retain similar osmolarity) and 2 mm EGTA (to bind Ca2+). Glacontryphan-M, glucontryphan-M, and isradipine were added to the extracellular solution as indicated in the figures. The pipette solution contained 76 mm Cs2SO4, 10 mm NaCl, 10 mm KCl, 1 mm MgCl2, 5 mm HEPES (pH 7.35 using CsOH). Electrical contact was established by addition of the pore-forming compound amphotericin B to the pipette solution (final concentration 0.24 mg/ml; Ref. 46Rae J. Cooper K. Gates P. Watsky M. J. Neurosci. Methods. 1991; 37: 15-26Google Scholar). Data are presented as mean values ± S.E., and statistical significance was evaluated using the Student′s t test. Purification and Characterization of Glacontryphan-M— Crude C. marmoreus venom was extracted and chromatographed on a Sephadex G-50 superfine column (Fig. 1A). Every third collected fraction was analyzed for Gla content after alkaline hydrolysis. From the major Gla-containing peak, glacontryphan-M was purified by a two-step reversed phase HPLC procedure (Fig. 1, B and C). The primary sequence for the native form of the peptide was determined by automated Edman degradation to be Asn-Xxx-Ser-Xxx-Xxx-Pro-Trp-His-Pro-Trp-Xxx. Sequencing demonstrated partial deamidation (8–11%) of the N-terminal Asn residue. The presence of cysteines at positions 5 and 11 was deduced by sequencing the carboxymethylated peptide. At positions 2 and 4, γ-carboxyglutamic acid residues were identified in a peptide that had been methyl-esterfied prior to sequencing. The amino acid composition of the peptide analyzed after acid or alkaline hydrolysis was consistent with the following sequence: Asn-Gla-Ser-Gla-CysPro-Trp-His-Pro-Trp-Cys. The sequence shows high similarity to the amino acid sequences earlier described for peptides of the contryphan family of conotoxins (Table I). To further investigate the primary sequence of glacontryphan-M the peptide was analyzed by NanoESI-MS.Table IComparison of mature contryphan sequencesPeptideSequenceaO, 4-trans-hydroxyproline; W, d-tryptophan; L, d-leucine; X, bromotryptophan; γ, gamma-carboxyglutamic acid.SpeciesRef.Des(Gly1)contryphan-RCOWEPWC-NH2C. radiatus35Jimenez E.C. Olivera B.M. Gray W.R. Cruz L.J. J. Biol. Chem. 1996; 271: 28002-28005Google ScholarContryphan-RGCOWEPWC-NH2C. radiatus35Jimenez E.C. Olivera B.M. Gray W.R. Cruz L.J. J. Biol. Chem. 1996; 271: 28002-28005Google ScholarBromocontyphan-RGCOWEPXC-NH2C. radiatus31Jimenez E.C. Craig A.G. Watkins M. Hillyard D.R. Gray W.R. Gulyas J. Rivier J.E. Cruz L.J. Olivera B.M. Biochemistry. 1997; 36: 989-994Google ScholarContryphan-SmGCOWQPWC-NH2C. stercusmuscarum32Jacobsen R. Jimenez E.C. Grilley M. Watkins M. Hillyard D. Cruz L.J. Olivera B.M. J. Pept. Res. 1998; 51: 173-179Google ScholarContryphan-PGCOWDPWC-NH2C. purpurascens32Jacobsen R. Jimenez E.C. Grilley M. Watkins M. Hillyard D. Cruz L.J. Olivera B.M. J. Pept. Res. 1998; 51: 173-179Google ScholarContryphan-R/TxGCOWEPWC-NH2C. textile33Jimenez E.C. Watkins M. Juszczak L.J. Cruz L.J. Olivera B.M. Toxicon. 2001; 39: 803-808Google ScholarContryphan-TxGCOWQPYC-NH2C. textile33Jimenez E.C. Watkins M. Juszczak L.J. Cruz L.J. Olivera B.M. Toxicon. 2001; 39: 803-808Google ScholarContryphan-VnGDCPWKPWC-NH2C. ventricosus34Massilia G.R. Schininá M.E. Ascenzi P. Polticelli F. Biochem. Biophys. Res. Commun. 2001; 288: 908-913Google ScholarLeu-contryphan-PGCVLLPWC-OHC. purpurascens36Jacobsen R. Jimenez E.C. De la Cruz R.G.C. Gray W.R. Cruz L.J. Olivera B.M. J. Pept. Res. 1999; 54: 93-99Google ScholarLeu-contryphan-TxCVLYPWC-NH2C. textile33Jimenez E.C. Watkins M. Juszczak L.J. Cruz L.J. Olivera B.M. Toxicon. 2001; 39: 803-808Google ScholarGlacontryphan-MNγSγCPWHPWC-NH2C. marmoreusThis worka O, 4-trans-hydroxyproline; W, d-tryptophan; L, d-leucine; X, bromotryptophan; γ, gamma-carboxyglutamic acid. Open table in a new tab Analysis of Glacontryphan-M by NanoESI-MS—The native form of glacontryphan-M was analyzed by NanoESI-MS in negative and positive ion modes. The mass spectrum acquired in negative ion mode is shown in Fig. 2A (m/z 1400–1480) and B (m/z 680–750). The signal observed at m/z 1470.47 Da (M-1H, Fig. 2A) corresponds to the molecular ion of the peptide and the major ion peak at m/z 734.74 Da (Fig. 2B), most likely contain the doubly (M-2H) charged ion of glacontryphan-M. The unusual isotope distribution with the second peak being more intense than the first (Fig. 2, A and B) is in all likelihood caused by the deamidation of the N-terminal Asn residue. The presence of two Gla residues in the sequence is notable in the mass spectrum via decarboxylation of the peptide resulting in a loss of 44 Da for each Gla residue (Fig. 2B). This loss of CO2 is observed at m/z 712.74 Da and 690.75 Da for the doubly charged ion. The monoisotopic molecular mass of native glacontryphan-M was determined from the negative ion mode NanoESI-MS experiments to be 1471.47 Da. These data fit well with the theoretically calculated monoisotopic molecular mass (1471.48 Da) of the peptide, assuming disulfide bonding of the cysteine residues and, like most conopeptides, amidation of the C-terminal carboxyl group (Fig. 2C). The observed molecular mass of native glacontryphan-M was further confirmed by NanoESI-MS experiments performed in the positive ion mode (not shown). Reduction and alkylation of cysteines in glacontryphan-M with iodoacetic acid resulted in a mass increase of 118.13 Da (theoretical 118.01 Da) as compared with the native compound, consistent with two cysteine residues in the sequence (the monoisotopic molecular mass was determined to be 1589.60 Da; Fig. 3A). The S-carboxymethylated peptide was analyzed by MS/MS spectrometry in the positive ion mode. The fragment ion spectrum obtained for the selected trapped ion at m/z 795.80 Da (Fig. 3B) revealed b and y ions in agreement with the proposed sequence. The mass of the y1 ion further confirms that glacontryphan-M contains an amidated C terminus. The isotope pattern of the b ions was consistent with partial deamidation of the N-terminal Asn residue (Fig. 3B). The y ions gave no indication of deamidation of the C-terminal amide. Glacontryphan-M Contains a d-Tryptophan—The sequence similarity of the C. marmoreus contryphan with the previously identified contryphans suggested the presence of a d-tryptophan residue at position 7 (Table I). Therefore, a synthetic peptide covering the sequence of glacontryphan-M with a d-tryptophan at position 7 and having the C-terminal carboxyl group amidated was prepared and folded. Analysis of the synthetic material by automated Edman degradation and NanoESI-MS confirmed the sequence of the peptide. Moreo
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