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At least 19 recordsLinked to original sources

PubMed2006

Discovery and characterization of the short kappaA-conotoxins: a novel subfamily of excitatory conotoxins.

We have characterized the defining members of a novel subfamily of excitatory conotoxins, the short kappaA-conotoxins (kappaA(S)-conotoxins). kappaA-conotoxins PIVE and PIVF (kappaA-PIVE and kappaA-PIVF) were purified from Conus purpurascens venom. Both peptides elicited excitatory activity upon injection into fish. kappaA-PIVE was synthesized for further characterization. The excitatory effects of kappaA-PIVE in vivo were dose dependent, causing hyperactivity at low doses and rapid immobilization at high doses, symptomatic of a type of excitotoxic shock. Consistent with these observations, kappaA-PIVE caused repetitive action potentials in frog motor axons in vitro. Similar results have been reported for other structurally distinct conotoxin families; such peptides appear to be required by most fish-hunting cone snails for the rapid immobilization of prey. Unexpected structure-function relationships were revealed between these peptides and two families of homologous conotoxins: the alphaA-conotoxins (muscle nAChR antagonists) and kappaA-conotoxins (excitotoxins), which all share a common arrangement of cysteine residues (CC-C-C-C-C). Biochemically, the kappaA(S)-conotoxins more closely resemble the alphaA(S)-conotoxins than the other kappaA-conotoxin subfamily, the long kappaA-conotoxins (kappaA(L)-conotoxins); however, kappaA(S)- and alphaA(S)-conotoxins produce different physiological effects. In contrast, the kappaA(S)-and kappaA(L)-conotoxins that diverge in several biochemical characteristics are clearly more similar in their physiological effects.

Amino Acid Sequence↗

PubMed2004

AlphaA-Conotoxin OIVA defines a new alphaA-conotoxin subfamily of nicotinic acetylcholine receptor inhibitors.

The venoms of cone snails are rich in multiply disulfide-crosslinked peptides, the conotoxins. Conotoxins are grouped into families on the basis of shared cysteine patterns and homologous molecular targets. For example, both the kappaA- and alphaA-conotoxin families share the same Class IV Cys pattern (-CC-C-C-C-C-), but differ in their molecular targets. The kappaA-conotoxins are excitatory toxins that purportedly block potassium channels, while the alphaA-conotoxins are paralytic conotoxins that inhibit nicotinic acetylcholine receptors (nAChRs). In this work, we describe the isolation and characterization of a novel Conus peptide from venom milked from Hawaiian specimens of Conus obscurus. This peptide shares the Class IV Cys pattern but differs from both previously characterized alphaA- and kappaA-conotoxins in the spacing of amino acids between Cys resides. However, the peptide is similar to previously characterized alphaA-conotoxins in its paralytic effects on fish and its antagonist activity on the neuromuscular nAChR. Unexpectedly, the peptide differs in its disulfide bonding from alphaA-conotoxin PIVA. We have named this unique peptide alphaA-conotoxin OIVA, and we consider it the defining member of a subfamily of alphaA-conotoxins that we designate the alphaA(1-3)-conotoxins to identify them by their unique disulfide bonding framework. These results indicate that the alphaA-conotoxin family is both more structurally diverse and broadly distributed than previously believed.

Amino Acid Sequence↗

PubMed1992

Immunoreactivity, epitope mapping and protection studies with anti-conotoxin GI sera and various conotoxins.

Goat or rabbit anti-conotoxin GI sera recognized native or dithiothreitol-reduced alpha-conotoxins GI, MI and SI in an enzyme-linked immunosorbent assay (ELISA). Native mu-conotoxin GIIIA or omega-conotoxin GVIA did not react with either anti-conotoxin GI serum in the assay. The goat anti-conotoxin GI serum neutralized 2.5 LD50S of alpha-conotoxins GI or MI in mouse lethal assays, while the rabbit antiserum had little protective capabilities. Epitope mapping of synthesized conotoxin peptide fragments revealed that both anti-conotoxin GI sera recognized linear sequences from five different alpha-conotoxins: GI, GIA, GII, MI and SI. The CCNPAC sequence was optimally recognized by both antisera.

Amino Acid Sequence↗

PubMed2005

Alpha S-conotoxin RVIIIA: a structurally unique conotoxin that broadly targets nicotinic acetylcholine receptors.

We report the purification and characterization of a new conotoxin from the venom of Conus radiatus. The peptide, alphaS-conotoxin RVIIIA (alphaS-RVIIIA), is biochemically unique with respect to its amino acid sequence, post-translational modification, and molecular targets. In comparison to other nicotinic antagonists from Conus venoms, alphaS-RVIIIA exhibits an unusually broad targeting specificity for nicotinic acetylcholine receptor (nAChR) subtypes, as assayed by electrophysiology. The toxin is paralytic to mice and fish, consistent with its nearly irreversible block of the neuromuscular nAChR. Similar to other antagonists of certain neuronal nAChRs, the toxin also elicits seizures in mice upon intracranial injection. The only previously characterized conotoxin from the S superfamily, sigma-conotoxin GVIIIA, is a specific competitive antagonist of the 5-HT3 receptor; thus, alphaS-RVIIIA defines a novel family of nicotinic antagonists within the S superfamily. All previously characterized competitive conotoxin nAChR antagonists have been members of the A superfamily of conotoxins. Our working hypothesis is that the particular group of fish-hunting Conus species that includes Conus radiatus uses the alphaS-conotoxin family to target the muscle nAChR and paralyze prey.

Amino Acid Sequence↗

PubMed1997

NMR structure determination of a novel conotoxin, [Pro 7,13] alpha A-conotoxin PIVA.

A high-resolution solution conformation of a novel conotoxin, [Pro 7,13] alpha A-conotoxin PIVA, GCCGSYPNAACHPCSCKDROSYCGQ-NH2, has been determined by two-dimensional 1H NMR methods and distance geometry calculations. The total of 324 NOE-derived interproton distance restraints including 33 long-range NOE restraints as well as 11 phi and 7 chi 1 torsion angle restraints was used for computation of structures. Back-calculation from the experimental NOE spectrum has provided 49 new NOE restraints and yielded the final R-factors of Ra = 0.641 and Rb = 0.157. The final RMSD values are 0.90 and 1.16 A for the backbone and the heavy atoms, respectively. The C-terminal half of the molecule involving the residues 12-24 is extremely well-defined with a backbone RMSD value of 0.56 A, whereas the N-terminal 3-11 disulfide loop is relatively flexible, possessing a backbone RMSD value of 1.09 A. The [Pro 7,13] alpha A-conotoxin PIVA does not contain any significant secondary structure although the 21S-24G nearly completes one turn of a 3(10) helix. The overall protein fold is largely maintained by the three disulfide bridges of 2-16, 3-11, and 14-23. The presence of the three disulfide bridges imposes geometric constraints that force the molecule to form six continuous bends involving the following residues: 3C-5S, 7P-10A, 12H-14C, 15S-17K, 17K-19R, and 21S-25Q. The overall shape of the [Pro 7,13] alpha A-conotoxin PIVA can be described as an "iron". Residues 15S-19R form a loop that protrudes out of the "bottom plate" formed by the rest of the protein and constitute the handle of the iron. The N-terminal tip of the molecule is relatively immobile due to attractive electrostatic interactions between the gamma-hydroxyl group of 20 Hyp and the phenolic hydroxyl group of 22Y. The flexible 3-11 disulfide loop consists mostly of hydrophobic residues, while the best-defined 14-23 disulfide loop contains the highly charged hydrophilic 15S-19R "handle" domain exposed to the exterior of the protein. Binding to nicotinic acetylcholine receptor can be mediated through two different types of interactions: one involving the aromatic hydrophobic residues such as 6Y and 12H and the other involving the positively charged hydrophilic side chain of the 19R. The side chain of the 19R in the [Pro 7, 13] alpha A-conotoxin PIVA and that of the 9R of the alpha-conotoxin G1, and also the side chains of the 12H and 6Y in the former and those of 10H and 11Y in the latter can be aligned to point to the same direction when the corresponding backbone atoms are superimposed to an RMSD value of 2.5 A.

Amino Acid Sequence↗

PubMed2000

lambda-conotoxins, a new family of conotoxins with unique disulfide pattern and protein folding. Isolation and characterization from the venom of Conus marmoreus.

Conotoxins are multiple disulfide-bonded peptides isolated from marine cone snail venom. These toxins have been classified into several families based on their disulfide pattern and biological properties. Here, we report a new family of Conus peptides, which have a novel cysteine motif. Three peptides of this family (CMrVIA, CMrVIB, and CMrX) have been purified from Conus marmoreus venom, and their structures have been determined. Their amino acid sequences are VCCGYK-LCHOC (CMrVIA), NGVCCGYKLCHOC (CMrVIB), and GICCGVSFCYOC (CMrX), where O represents 4-trans-hydroxyproline. Two of these peptides (CMrVIA and CMrX) have been chemically synthesized. Using a selective protection and deprotection strategy during disulfide bond formation, peptides with both feasible cysteine-pairing combinations were generated. The disulfide pattern (C(1)-C(4), C(2)-C(3)) in native toxins was identified by their co-elution with the synthetic disulfide-isomeric peptides on reverse-phase high pressure liquid chromatography. Although cysteine residues were found in comparable positions with those of alpha-conotoxins, these toxins exhibited a distinctly different disulfide bonding pattern; we have named this new family "lambda -conotoxins." CMrVIA and CMrX induced different biological effects when injected intra-cerebroventricularly in mice; CMrVIA induces seizures, whereas CMrX induces flaccid paralysis. The synthetic peptide with lambda-conotoxin folding is about 1150-fold more potent in inducing seizures than the mispaired isomer with alpha-conotoxin folding. Thus it appears that the unique disulfide pattern, and hence the "ribbon" conformation, in lambda-conotoxins is important for their biological activity.

Amino Acid Sequence↗

PubMed2006

A novel conotoxin inhibitor of Kv1.6 channel and nAChR subtypes defines a new superfamily of conotoxins.

Using assay-directed fractionation of the venom from the vermivorous cone snail Conus planorbis, we isolated a new conotoxin, designated pl14a, with potent activity at both nicotinic acetylcholine receptors and a voltage-gated potassium channel subtype. pl14a contains 25 amino acid residues with an amidated C-terminus, an elongated N-terminal tail (six residues), and two disulfide bonds (1-3, 2-4 connectivity) in a novel framework distinct from other conotoxins. The peptide was chemically synthesized, and its three-dimensional structure was demonstrated to be well-defined, with an alpha-helix and two 3(10)-helices present. Analysis of a cDNA clone encoding the prepropeptide precursor of pl14a revealed a novel signal sequence, indicating that pl14a belongs to a new gene superfamily, the J-conotoxin superfamily. Five additional peptides in the J-superfamily were identified. Intracranial injection of pl14a in mice elicited excitatory symptoms that included shaking, rapid circling, barrel rolling, and seizures. Using the oocyte heterologous expression system, pl14a was shown to inhibit both a K+ channel subtype (Kv1.6, IC50 = 1.59 microM) and neuronal (IC50 = 8.7 microM for alpha3beta4) and neuromuscular (IC50 = 0.54 microM for alpha1beta1 epsilondelta) subtypes of the nicotinic acetylcholine receptor (nAChR). Similarities in sequence and structure are apparent between the middle loop of pl14a and the second loop of a number of alpha-conotoxins. This is the first conotoxin shown to affect the activity of both voltage-gated and ligand-gated ion channels.

Amino Acid Sequence↗

PubMed1997

Crystal structure at 1.1 A resolution of alpha-conotoxin PnIB: comparison with alpha-conotoxins PnIA and GI.

Conotoxins are small, cysteine-rich peptides isolated from the venom of Conus spp. of predatory marine snails, which selectively target specific receptors and ion channels critical to the functioning of the neuromuscular system. alpha-Conotoxins PnIA and PnIB are both 16-residue peptides (differing in sequence at only two positions) isolated from the molluscivorous snail Conus pennaceus. In contrast to the muscle-selective alpha-conotoxin GI from Conus geographus, PnIA and PnIB block the neuronal nicotinic acetylcholine receptor (nAChR). Here, we describe the crystal structure of PnIB, solved at a resolution of 1.1 A and phased using the Shake-and-Bake direct methods program. PnIB crystals are orthorhombic and belong to the space group P212121 with the following unit cell dimensions: a = 14.6 A, b = 26.1 A, and c = 29.2 A. The final refined structure of alpha-conotoxin PnIB includes all 16 residues plus 23 solvent molecules and has an overall R-factor of 14.7% (R-free of 15.9%). The crystal structures of the alpha-conotoxins PnIB and PnIA are solved from different crystal forms, with different solvent contents. Comparison of the structures reveals them to be very similar, showing that the unique backbone and disulfide architecture is not strongly influenced by crystal lattice constraints or solvent interactions. This finding supports the notion that this structural scaffold is a rigid support for the presentation of important functional groups. The structures of PnIB and PnIA differ in their shape and surface charge distribution from that of GI.

Amino Acid Sequence↗

PubMed1999

NMR solution structure of alpha-conotoxin ImI and comparison to other conotoxins specific for neuronal nicotinic acetylcholine receptors.

Alpha-Conotoxins, peptides produced by predatory species of Conus marine snails, are potent antagonists of nicotinic acetylcholine receptors (nAChRs), ligand-gated ion channels involved in synaptic transmission. We determined the NMR solution structure of the smallest known alpha-conotoxin, ImI, a 12 amino acid peptide that binds specifically to neuronal alpha7-containing nAChRs in mammals. Calculation of the structure was based on a total of 80 upper distance constraints and 31 dihedral angle constraints resulting in 20 representative conformers with an average pairwise rmsd of 0.44 A from the mean structure for the backbone atoms N, Calpha, and C' of residues 2-11. The structure of ImI is characterized by two compact loops, defined by two disulfide bridges, which form distinct subdomains separated by a deep cleft. Two short 310-helical regions in the first loop are followed by a C-terminal beta-turn in the second. The two disulfide bridges and Ala 9 form a rigid hydrophobic core, orienting the other amino acid side chains toward the surface. Comparison of the three-dimensional structure of ImI to those of the larger, 16 amino acid alpha-conotoxins PnIA, PnIB, MII, and EpI-also specific for neuronal nAChRs-reveals remarkable similarity in local backbone conformations and relative solvent-accessible surface areas. The core scaffold is conserved in all five conotoxins, whereas the residues in solvent-exposed positions are highly variable. The second helical region, and the specific amino acids that the helix exposes to solvent, may be particularly important for binding and selectivity. This comparative analysis provides a three-dimensional structural basis for interpretation of mutagenesis data and structure-activity relationships for ImI as well other neuronal alpha-conotoxins.

Amino Acid Sequence↗

PubMed1999

NMR solution conformation of an antitoxic analogue of alpha-conotoxin GI: identification of a common nicotinic acetylcholine receptor alpha 1-subunit binding surface for small ligands and alpha-conotoxins.

The three-dimensional solution conformation of an 11-residue antitoxic analogue of alpha-conotoxin GI, des-Glu1-[Cys3Ala]-des-Cys13-conotoxin GI (CANPACGRHYS-NH(2), designated "GI-15" henceforth), has been determined using two-dimensional (1)H NMR spectroscopy. The disulfide loop region (1C-6C) and the C-terminal tail (8R-11S) are connected by a flexible hinge formed near 7G, and the pairwise backbone rmsds for the former and the latter are 0.58 and 0.65 A, respectively. Superpositioning GI-15 with the structure of alpha-conotoxin GI shows that the two share an essentially identical fold in the common first disulfide loop region (1C-6C). However, the absence of the second disulfide loop in GI-15 results in segmental motion of the C-terminal half, causing the key receptor subtype selectivity residue 8R (Arg9 in alpha-conotoxin GI) to lose its native spatial orientation. The combined features of structural equivalence in the disulfide loop and a mobile C-terminal tail appear to be responsible for the activity of GI-15 as a competitive antagonist against native toxin. Electrostatic surface potential comparisons of the first disulfide region of GI-15 with other alpha-conotoxins or receptor-bound states of acetylcholine and d-tubocurarine show a common protruding surface that may serve as the minimal binding determinant for the neuromuscular acetylcholine receptor alpha 1-subunit. On the basis of the original "Conus toxin macrosite model" [Olivera, B. M., Rivier, J., Scott, J. K., Hillyard, D. R., and Cruz, L. J. (1991) J. Biol. Chem. 266, 1923-1936], we propose a revised binding model which incorporates these results.

Amino Acid Sequence↗

PubMed1995

A new conotoxin affecting sodium current inactivation interacts with the delta-conotoxin receptor site.

We describe a new peptide conotoxin affecting sodium current inactivation, that competes on binding with delta-conotoxin TxVIA (delta TxVIA). The amino acid sequence of the new toxin, designated conotoxin NgVIA (NgVIA), is SKCFSOGTFCGIKOGLCCSVRCFSLFCISFE (where O is trans-4-hydroxyproline). The primary structure of NgVIA has an identical cysteine framework and similar hydrophobicity as delta TxVIA but differs in its net charge. NgVIA competes with delta TxVIA on binding to rat brain synaptosomes and molluscan central nervous system and strongly inhibits sodium current inactivation in snail neurons, as does delta TxVIA. In contrast to delta TxVIA, NgVIA is a potent paralytic toxin in vertebrate systems, its binding appears to be voltage-dependent, and it synergically increases veratridine-induced sodium influx to rat brain synaptosomes. delta TxVIA acts as a partial antagonist to NgVIA in rat brain in vivo. NgVIA appears to act via a receptor site distinct from that of delta TxVIA but similar to that of Conus striatus toxin. This new toxin provides a lead for structure-function relationship studies in the delta-conotoxins and will enable analysis of the functional significance of this complex of receptor sites in gating mechanisms of sodium channels.

Amino Acid Sequence↗

PubMed1999

Differential blockade of rat alpha3beta4 and alpha7 neuronal nicotinic receptors by omega-conotoxin MVIIC, omega-conotoxin GVIA and diltiazem.

Rat alpha3beta4 or alpha7 neuronal nicotinic acetylcholine receptors (AChRs) were expressed in Xenopus laevis oocytes, and the effects of various toxins and non-toxin Ca2+ channel blockers studied. Nicotinic AChR currents were elicited by 1 s pulses of dimethylphenylpiperazinium (DMPP, 100 microM) applied at regular intervals. The N/P/Q-type Ca2+ channel blocker omega-conotoxin MVIIC inhibited alpha3beta4 currents with an IC50 of 1.3 microM; the blockade was non-competitive and reversible. The alpha7 currents were unaffected. At 1 microM, omega-conotoxin GVIA (N-type Ca2+ channel blocker) inhibited by 24 and 20% alpha3beta4 and alpha7 currents, respectively. At 1 microM, omega-agatoxin IVA (a P/Q-type Ca2+ channel blocker) did not affect alpha7 currents and inhibited alpha3beta4 currents by only 15%. L-type Ca2+ channel blockers furnidipine, verapamil and, particularly, diltiazem exhibited a preferential blocking activity on alpha3beta4 nicotinic AChRs. The mechanism of alpha3beta4 currents blockade by omega-conotoxins and diltiazem differed in the following aspects: (i) the onset and reversal of the blockade was faster for toxins; (ii) the blockade by the peptides was voltage-dependent, while that exerted by diltiazem was not; (iii) diltiazem promoted the inactivation of the current while omega-toxins did not. These data show that, at concentrations currently employed as Ca2+ channel blockers, some of these compounds also inhibit certain subtypes of nicotinic AChR currents. Our data calls for caution when interpreting many of the results obtained in neurons and other cell types, where nicotinic receptor and Ca2+ channels coexist.

Animals↗

PubMed2002

Generation of polyclonal antibody against mu-conotoxin GIIIA using an immunogen of [Cys(5)]mu-conotoxin GIIIA site-specifically conjugated with bovine serum albumin.

mu-Conotoxin GIIIA, one of the strong peptide toxins in the cone shell, preferentially blocks the skeletal muscle-type sodium channels in vertebrates. The toxicity of mu-conotoxin GIIIA is nearly equal to that of tetrodotoxin. The generation of an antibody for the native toxins is analytically useful, but practically difficult due to its high toxicity to animals. In this study, we generated the polyclonal antibody for mu-conotoxin GIIIA using a specific conjugation method in which the immunogen was detoxified while retaining the active-site structure for the sodium channels. ELISA analysis showed that the generated antibody recognized the native toxin folded with three disulfide bridges, but not the linear one. Furthermore, the physiologically active mutants of GIIIA were recognized while the inactive mutants were not, suggesting that the newly generated antibody can selectively recognize the physiologically active toxins. These methods for generating an antibody against peptide toxins will be applicable to other peptide toxins.

Amino Acid Sequence↗

PubMed1996

Conotoxin-sensitive and conotoxin-resistant Ca2+ currents in fish retinal ganglion cells.

Using whole-cell patch-clamp methods, we tested whether omega-toxins from Conus block voltage-gated Ca2+ currents in teleost central neurons. The fractions omega-CTx-GVIA and omega-CTx-MVIIC, together with omega-toxins from Agelenopsis, the dihydropyridine BAY-K-8644, and voltage steps, produced effects indicating three types of Ca2+ current in dissociated goldfish retinal ganglion cells. One was activated by depolarization of most cells beyond -65 mV, primed at -95 mV but not at -45 mV, reduced by Ni2+, and unchanged by conotoxins, agatoxins, or BAY-K-8644. The second type constituted more than three-quarters of the total Ca2+ current in all cells, and at test potentials more positive than -30 mV, was reduced consistently by omega-CTx-GVIA, omega-CTx-MVIIC, and omega-Aga-IA, but not omega-Aga-IVA. The third Ca2+ current type was augmented by BAY-K-8644 at test potentials as negative as -45 mV, even in the presence of omega-CTx-GVIA. Replacement of extracellular Ca2+ by Ba2+ augmented current amplitude and slowed current decay. Conditioning depolarizations reduced Ca2+ current amplitude less than did omega-CTx-GVIA, and slowed current decay to imperceptible rates. These results provide the first description of conotoxin-sensitive, voltage-gated Ca2+ current recorded from teleost central neurons. Although most of the high-threshold Ca2+ current in these cells is blocked by omega-CTx-GVIA, it is also Ni(2+)-sensitive, and relatively resistant to omega-Aga-IIIA. The voltage sensitivities of low-and high-threshold Ca2+ current may suit current recruitment in situ after light-evoked hyperpolarizations end, and after light-evoked depolarizations begin, respectively.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed2006

Screening for post-translational modifications in conotoxins using liquid chromatography/mass spectrometry: an important component of conotoxin discovery.

Mass spectrometry has emerged as an important technique for conotoxin analysis due to its capacity for selective, sensitive, information-rich analyses. Using liquid chromatography/mass spectrometry, Conus venom can be fractionated and the peptides surveyed for specific post-translational modifications, indicating those toxin components likely to have an important biological function. With Conus striatus and Conus victoriae venom as models, bromination, carboxylation and glycosylation modifications are identified through characteristics such as isotopic distribution and labile losses observed during mass spectrometric analysis. This modification screening approach enables the identification of a C. victoriae bromo-carboxy-conotoxin, designated vc5c, as a candidate for detailed mass spectrometric analysis. Using a cDNA sequence coupled with liquid chromatography/mass spectrometry and nanoelectrospray ionization-ion trap-mass spectrometry, the sequence of vc5c is determined to be ICCYPNXWCCD, where W is 6-bromotryptophan, X is gamma-carboxy glutamate and C is disulfide-linked cysteine. This represents the ninth T-superfamily (-CC-CC- scaffold) toxin that has been isolated from venom and characterized.

Amino Acid Sequence↗

PubMed2002

A new level of conotoxin diversity, a non-native disulfide bond connectivity in alpha-conotoxin AuIB reduces structural definition but increases biological activity.

alpha-Conotoxin AuIB and a disulfide bond variant of AuIB have been synthesized to determine the role of disulfide bond connectivity on structure and activity. Both of these peptides contain the 15 amino acid sequence GCCSYPPCFATNPDC, with the globular (native) isomer having the disulfide connectivity Cys(2-8 and 3-15) and the ribbon isomer having the disulfide connectivity Cys(2-15 and 3-8). The solution structures of the peptides were determined by NMR spectroscopy, and their ability to block the nicotinic acetylcholine receptors on dissociated neurons of the rat parasympathetic ganglia was examined. The ribbon disulfide isomer, although having a less well defined structure, is surprisingly found to have approximately 10 times greater potency than the native peptide. To our knowledge this is the first demonstration of a non-native disulfide bond isomer of a conotoxin exhibiting greater biological activity than the native isomer.

Amino Acid Sequence↗

PubMed2005

Characterization of D-amino-acid-containing excitatory conotoxins and redefinition of the I-conotoxin superfamily.

Post-translational isomerization of l-amino acids to d-amino acids is a subtle modification, not detectable by standard techniques such as Edman sequencing or MS. Accurate predictions require more sequences of modified polypeptides. A 46-amino-acid-long conotoxin, r11a, belonging to the I-superfamily was previously shown to have a d-Phe residue at position 44. In this report, we characterize two related peptides, r11b and r11c, with d-Phe and d-Leu, respectively, at the homologous position. Electrophysiological tests show that all three peptides induce repetitive activity in frog motor nerve, and epimerization of the single amino acid at the third position from the C-terminus attenuates the potency of r11a and r11b, but not that of r11c. Furthermore, r11c (but neither r11a nor r11b) also acts on skeletal muscle. We identified more cDNA clones encoding conopeptide precursors with Cys patterns similar to r11a/b/c. Although the predicted mature toxins have the same cysteine patterns, they belong to two different gene superfamilies. A potential correlation between the identity of the gene superfamily to which the I-conotoxin belongs and the presence or absence of a d-amino acid in the primary sequence is discussed. The great diversity of I-conopeptide sequences provides a rare opportunity for defining parameters that may be important for this most stealthy of all post-translational modifications. Our results indicate that neither the chemical nature of the side chain nor the precise vicinal sequence around the modified residue seem to be critical, but there may be favored loci for isomerization to a d-amino acid.

Amino Acid Sequence↗

PubMed1991

omega-Conotoxin GVIA receptors of Discopyge electric organ. Characterization of omega-conotoxin binding to the nicotinic acetylcholine receptor.

A peptide toxin from a Conus marine snail, omega-conotoxin GVIA (omega-CgTx) has been used extensively as a probe for certain types of neuronal calcium channels. It is often assumed that omega-CgTx interacts with Ca2+ channels exclusively. We have tested this assumption in a study of omega-CgTx-binding sites in the electric organ of Discopyge ommata. Synaptosomal membranes from this tissue contain low affinity omega-CgTx receptor sites (Kd = 0.6 microM) in great abundance (280 pmol/mg of protein), as first reported by Ahmad and Miljanich (Ahmad, S. N., and Miljanich, G.P. (1988) Brain Res. 453, 247-256). However, we find that a large majority of these omega-CgTx-binding sites co-purify with the nicotinic acetylcholine receptor (nAChR) and can be immunoprecipitated by monoclonal antibodies generated against the nAChR of Torpedo. Cross-linking experiments with radiolabeled omega-CgTx show pronounced specific labeling of the alpha-subunit of the nAChR but not other subunits. Specific omega-CgTx binding to the nAChR is reduced by millimolar Ca2+ but not by alpha- or kappa-bungarotoxin, alpha-conotoxin, or carbamylcholine. Cross-linking experiments also reveal omega-CgTx-binding proteins of 170 and 60 kDa. The characteristics of the 170-kDa protein make it a likely candidate for the alpha 1-subunit of an N-type Ca2+ channel.

Affinity Labels↗