RESUMO
The full-length cDNAs of two A-superfamily conotoxins, kappaA-SIVA and alpha-SII, were respectively cloned and sequenced from Conus striatus using 3' RACE and 5' RACE. The cDNA of kappaA-SIVA encodes a precursor of 68 residues, including a signal peptide of 21 residues, a pro-peptide of 17 residues, and a mature peptide of 30 residues with an additional residue Gly which is prerequisite for the amidation of the preceding C-terminal Cys. The cDNA-deduced sequence of alpha-SII is composed of a signal peptide of 21 residues, a pro-peptide of 29 residues, a mature peptide of 19 residues and three additional residues Arg-Thr-Ile at the C-terminus. This tripeptide might be cleaved off by proteolytic processing. Although these two conotoxins belong to different families and target voltage-gated potassium channel and nicotinic acetylcholine receptor, respectively, they share the same signal sequence, and both are processed at the common signal site -X-Arg- immediately before the mature peptide sequences. The length of 3' untranslational region of alpha-conotoxin SII was extraordinarily large about 10 times longer than that of kappaA-SIVA with 770 and 75 bp, respectively. The elucidated cDNAs of these two toxins will facilitate a better understanding of the process of their post-translational modifications.
Assuntos
DNA Complementar/genética , Moluscos/genética , Venenos de Moluscos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Evolução Molecular , Dados de Sequência Molecular , Venenos de Moluscos/classificaçãoRESUMO
To identify the amino acid sequence of the precursor of the Gla-containing peptide, epsilon-TxIX, from the venom of the marine snail Conus textile, the cDNA encoding this peptide was cloned from a C. textile venom duct library. The cDNA of the precursor form of epsilon-TxIX encodes a 67 amino acid precursor peptide, including an N-terminal prepro-region, the mature peptide, and four residues posttranslationally cleaved from the C-terminus. To determine the role of the propeptide in gamma-carboxylation, peptides were designed and synthesized based on the propeptide sequence of the Gla-containing conotoxin epsilon-TxIX and used in assays with the vitamin K-dependent gamma-glutamyl carboxylase from C. textile venom ducts. The mature acarboxy peptide epsilon-TxIX was a high K(M) substrate for the gamma-carboxylase. Synthetic peptides based on the precursor epsilon-TxIX were low K(M) substrates (5 microM) if the peptides included at least 12 residues of propeptide sequence, from -12 to -1. Leucine-19, leucine-16, asparagine-13, leucine-12, leucine-8 and leucine-4 contribute to the interaction of the pro-conotoxin with carboxylase since their replacement by aspartic acid increased the K(M) of the substrate peptide. Although the Conus propeptide and the propeptides of the mammalian vitamin K-dependent proteins show no obvious sequence homology, synthetic peptides based upon the structure of pro-epsilon-TxIX were intermediate K(M) substrates for the bovine carboxylase. The propeptide of epsilon-TxIX contains significant alpha-helix, as estimated by measurement of the circular dichroism spectra, but the region of the propeptide that plays the dominant role in directing carboxylation does not contain evidence of helical structure. These results indicate that the gamma-carboxylation recognition site is defined by hydrophobic residues in the propeptide of this conotoxin precursor.
Assuntos
Conotoxinas/química , Venenos de Moluscos/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Bovinos , Dicroísmo Circular , Clonagem Molecular , Conotoxinas/genética , Conotoxinas/metabolismo , Primers do DNA/genética , DNA Complementar/genética , Dados de Sequência Molecular , Venenos de Moluscos/genética , Venenos de Moluscos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Precursores de Proteínas/química , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Caramujos/genéticaRESUMO
The venom of predatory marine snails is a rich source of natural products that act on specific receptors and ion channels within the mammalian nervous system. A 41-amino acid peptide, final sigma-conotoxin GVIIIA, was purified on the basis of its ability to inactivate the 5-HT3 receptor, an excitatory serotonin-gated ion channel. final sigma-Conotoxin contains a brominated tryptophan residue, which may be important for peptide activity because the endogenous ligand for the 5-HT3 receptor is a hydroxylated derivative of tryptophan. final sigma-Conotoxin inactivates the 5-HT3 receptor through competitive antagonism and is a highly selective inhibitor of this receptor. Serotonin receptors can now be included among the molecular targets of natural polypeptide neurotoxins.