A conserved motif within the vitamin K-dependent carboxylase gene is widely distributed across animal phyla.

The vitamin K-dependent gamma-glutamyl carboxylase catalyzes the posttranslational conversion of glutamic acid to gamma-carboxyglutamic acid, an amino acid critical to the function of the vitamin K-dependent blood coagulation proteins. Given the functional similarity of mammalian vitamin K-dependent carboxylases and the vitamin K-dependent carboxylase from Conus textile, a marine invertebrate, we hypothesized that structurally conserved regions would identify sequences critical to this common functionality. Furthermore, we examined the diversity of animal species that maintain vitamin K-dependent carboxylation to generate gamma-carboxyglutamic acid. We have cloned carboxylase homologs in full-length or partial form from the beluga whale (Delphinapterus leucas), toadfish (Opsanus tau), chicken (Gallus gallus), hagfish (Myxine glutinosa), horseshoe crab (Limulus polyphemus), and cone snail (Conus textile) to compare these structures to the known bovine, human, rat, and mouse cDNA sequences. Comparison of the predicted amino acid sequences identified a nearly perfectly conserved 38-amino acid residue region in all of these putative carboxylases. In addition, this amino acid motif is also present in the Drosophila genome and identified a Drosophila homolog of the gamma-carboxylase. Assay of hagfish liver demonstrated vitamin K-dependent carboxylase activity in this hemichordate. These results demonstrate the broad distribution of the vitamin K-dependent carboxylase gene, including a highly conserved motif that is likely critical for enzyme function. The vitamin K-dependent biosynthesis of gamma-carboxyglutamic acid appears to be a highly conserved function in the animal kingdom.

Structure determination of two conotoxins from Conus textile by a combination of matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization mass spectrometry and biochemical methods.

Two highly modified conotoxins from the mollusc Conus textile, epsilon-TxIX and Gla(1)-TxVI, were characterized by matrix-assisted laser desorption/ionization and electrospray mass spectrometry and also by electrospray ionization tandem and triple mass spectrometry in combination with enzymatic cleavage and chemical modification reactions. The mass spectrometric studies allowed the confirmation of the sequence determined by Edman degradation and assignment of unidentified amino acid residues, among which bromotryptophan residues and an O-glycosylated threonine residue were observed. Methyl esterification was found necessary for the site-specific assignment of the Gla residues in the peptides.

Hydrophobic amino acids define the carboxylation recognition site in the precursor of the gamma-carboxyglutamic-acid-containing conotoxin epsilon-TxIX from the marine cone snail Conus textile.

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.

A conotoxin from Conus textile with unusual posttranslational modifications reduces presynaptic Ca2+ influx.

Cone snails are gastropod mollusks of the genus Conus that live in tropical marine habitats. They are predators that paralyze their prey by injection of venom containing a plethora of small, conformationally constrained peptides (conotoxins). We report the identification, characterization, and structure of a gamma-carboxyglutamic acid-containing peptide, conotoxin epsilon-TxIX, isolated from the venom of the molluscivorous cone snail, Conus textile. The disulfide bonding pattern of the four cysteine residues, an unparalleled degree of posttranslational processing including bromination, hydroxylation, and glycosylation define a family of conotoxins that may target presynaptic Ca2+ channels or act on G protein-coupled presynaptic receptors via another mechanism. This conotoxin selectively reduces neurotransmitter release at an Aplysia cholinergic synapse by reducing the presynaptic influx of Ca2+ in a slow and reversible fashion. The three-dimensional structure, determined by two-dimensional 1H NMR spectroscopy, identifies an electronegative patch created by the side chains of two gamma-carboxyglutamic acid residues that extend outward from a cavernous cleft. The glycosylated threonine and hydroxylated proline enclose a localized hydrophobic region centered on the brominated tryptophan residue within the constrained intercysteine region.

Pharmacological characterization of I1 and I2 imidazoline receptors in human striatum.

[3H]RX821002, [3H]clonidine and [3H]idazoxan have previously been shown to selectively label alpha 2-adrenergic receptors, I1 and I2 imidazoline receptors in the human central nervous system, respectively. Idazoxan shows relatively high affinity for all three receptors. We investigated the possible selectivity of several compounds towards one of those receptors in human striatum. Addition of an alkoxy group at the 2-position of the benzodioxan moiety of idazoxan (ethoxy-idazoxan, methoxy-idazoxan) increases the alpha 2-selectivity in human brain. Efaroxan is also alpha 2-selective. On the contrary, BU224, BU239, cirazoline and RX801077 display imidazoline receptor selectivity. Our results indicate that for all molecules tested, idazoxan and 'flat' analogs possess I1/I2 receptor selectivity. A 'bulky' substituent at the 2-position of the benzodioxan ring gives rise to alpha 2-adrenergic receptor selectivity. Until now, we found no more than 3-fold difference in IC50 between both imidazoline receptors. Both receptors also display similar stereoselectivity, suggesting that they might be 'interconnected' in the human striatum.

Neuropeptide Y receptors from calf brain: effect of crude Conus venom preparations on [3H]NPY binding.

NPY receptors are identified in calf frontal cortex and hippocampus membrane preparations by binding of N-[propionyl-3H] neuropeptide Y. Saturation and competition binding data with PYY, NPY-(18-36) and NPY itself fit with a single class of sites: for the radioligand KD = 1.4 +/- 0.5 nM, Bmax = 434 +/- 180 fmol/mg protein in frontal cortex, KD = 0.7 +/- 0.2 nM, Bmax = 267 +/- 50 fmol/mg protein in hippocampus. Competition curves of the Y1-subtype selective agonist [Leu31, Pro34]NPY are biphasic in both membrane preparations: high affinity sites (i.e. Y1-subtype) amount to 80% in frontal cortex and 23% in hippocampus. The remaining sites are of the Y2-subtype. Out of 23 Conus venom preparations, 17 inhibit the binding of [3H]NPY in both membrane preparations, but only two of them (from Conus aulicus and C. pennaceus) do so with high potency (IC50 < 5 micrograms protein/ml). Only one venom preparation (from C. mercator) had weak discriminatory properties (IC50Y2/IC50Y1 = 6). Venom from C. anemone increased the [3H]NPY binding 5-fold and with an IC50 of 15-18 micrograms protein/ml. This binding occurred to the venom itself and was unrelated to the NPY receptors since it was equally potent when displaced by [Leu31, Pro34]NPY, NPY-(18-36), PYY and NPY.

High-affinity binding of [3H]neuropeptide Y to a polypeptide from the venom of Conus anemone.

Venom preparation from Conus anemone contains a component that binds radiolabeled neuropeptide Y ([3H]neuropeptide Y) with high affinity (KD = 2.9 nM +/- 0.2 nM, Bmax = 15.2 +/- 0.5 pmol/mg protein). Binding of [3H]neuropeptide Y to the venom component is displaced with nanomolar affinity of unlabeled human and porcine neuropeptide Y, porcine [Leu31-Pro34]neuropeptide Y, peptide YY, avian and bovine pancreatic polypeptide, and the (18-36) and (25-36) C-terminal fragments from neuropeptide Y. No displacement is found with the (1-24) N-terminal neuropeptide Y fragment, human secretin, porcine dynorphin A and Boc-DAKLI (bolton Hunter coupled dynorphin A analog kappa ligand) nor with the non-peptide neuropeptide Y receptor antagonist BIBP3266. Gel filtration chromatography and denaturing (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) show that the [3H]neuropeptide Y-binding component is very likely a single-chain polypeptide with a molecular mass of 18.5 kDa.

Identification and characterization of imidazoline-binding sites from calf striatum.

"Non-adrenoceptor'-binding sites for [3H]clonidine (I1-sites) and [3H]idazoxan (I2-sites) are identified in calf striatum membranes. The pharmacological profile of both subtypes was investigated by competition binding with the imidazolines idazoxan, cirazoline, Bu 224 (2-(4,5-dihydroimidaz-2-yl)-quinoline) and Bu 239 (2-(4,5-dihydroimidaz-2-yl)-quinoxaline); the guanidino derivatives clonidine, moxonidine, guanabenz, amiloride and agmatine; the oxazoline rilmenidine and the imidazole histamine. The competition experiments indicate that both populations of imidazoline-binding sites in calf striatum consist of a high- (H) and a low-affinity (L) compartment. The monoamine oxidase (MAO) inhibitors pargyline (non-selective) and deprenyl (MAO-B-selective) have micromolar affinity for the I1-sites and much lower affinity for the I2-sites. The venom of the marine snail Conus geographus is the most potent of the 13 tested Conus venom preparations. None of the tested venoms is able to discriminate between both sites.

Selective masking of M1-receptors in calf retina membranes by the venom of the marine snail Conus tessulatus.

The non discriminatory antagonist [3H]QNB labels M1- and M2-muscarinic receptors in calf retina membranes. Crude venom from the marine gastropod Conus tessulatus produces a partial decrease in [3H]QNB binding. The total number of sites (560 +/- 13 fmol/mg protein in control experiments) decreases to 370 +/- 10 fmol/mg protein whereas the affinity of the radioligand is unaffected (KD = 0.42 +/- 0.01 nM and 0.46 +/- 0.02 nM, respectively). This process is venom concentration-dependent, quasi-irreversible, and calcium-dependent. Proteolytic activity can not be detected. The partial effect of the venom is related to preferential masking of the M1-receptors. Competition curves of the M1-selective antagonist pirenzepine are shallow in control experiments: 45% of the receptors are of the M1-type (Ki = 45 +/- 6 nM) while the remaining are of the M2-type (Ki = 1.0 +/- 0.2 microM). In venom-treated membranes, only a low affinity site (M2-receptors, Ki = 1.5 +/- 0.4 microM) is detected by pirenzepine competition binding. Saturation binding experiments reveal that the venom causes a substantial decrease in the number of high affinity sites for [3H]pirenzepine without affecting its KD (23 +/- 4 nM and 20 +/- 6 nM in control- and venom-treated membranes respectively). The venom produces a leftward shift of the carbachol/[3H]QNB competition binding curve, but the ability of 0.1 mM GTP to confer a rightward shift of the competition curve is not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

[3H]rauwolscine behaves as an agonist for the 5-HT1A receptors in human frontal cortex membranes.

The alpha 2 adrenergic antagonist [3H]rauwolscine binds with comparable nanomolar affinity to alpha 2 adrenoceptors and the nonadrenergic 5-HT1A receptors sites in human frontal cortex membranes. Addition of 0.5 mM GTP into the incubation medium produces a significant decrease in the amount of [3H]rauwolscine binding sites (Bmax = 230 +/- 16 and 115 +/- 11 fmol/mg protein in the absence and presence of GTP, respectively). The affinity for [3H]rauwolscine remains unchanged (i.e. KD = 40 +/- 0.9 nM and 4.1 +/- 1 nM). This effect of GTP can be attributed to decreased binding of the radioligand to the 5-HT1A receptors. GTP decreases binding of [3H]rauwolscine to nearly the same level as the one corresponding to the alpha 2 adrenoceptors in membranes from both the human frontal cortex and hippocampus. The venom of the marine cone snail, Conus tessulatus, preferentially inhibits [3H]rauwolscine binding to 5-HT1A receptors as compared with the alpha 2 adrenoceptors. Following complete masking of the 5-HT1A receptors by this venom. GTP no longer affects the saturation binding characteristics of [3H]rauwolscine for the remaining alpha 2 adrenoceptors. Nucleotides decrease the binding of [3H]rauwolscine to the 5-HT1A receptors with an order of potencies (i.e. GTP gamma S greater than GPP(NH)P much greater than GDP greater than GTP much greater than ATP) that is typical for nucleotide-mediated receptor-G protein dissociation. This suggests that [3H]rauwolscine is a 5-HT1A receptor agonist and this conclusion is compatible with earlier functional studies, indicating that rauwolscine (as well as yohimbine) has agonistic properties at the level of 5-HT autoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Conus venom interaction with ?(2)-adrenergic receptors in calf retina membranes.

?(2)-Adrenergic receptors were identified in calf retina membranes by the specific binding of the radiolabelled antagonist [(3)H]RX 781094. Crude venoms from various Conus species did not interact with the radioligand but were able to inhibit radioligand binding to the ?(2)-receptors with the following order of potency: C. planorbis (IC(50) = 2.1 ?g protein/ml) ? C. tessulatus (IC(50) = 2.7) >C. eburneus (IC(50) = 19) >C. textile (IC(50) = 54) >C. geographus (IC(50) = 130). Venom from 17 other species was less or not active at all. Venom competition binding curves were steep and not affected by GTP. In contrast, the ( ? )-epinephrine competition binding curve was shallow and underwent a rightward shift and steepening in the presence of GTP. The venom-?(2)-receptor interaction was completely inhibited by the calcium chelating reagent EGTA. These data suggest that the venom of certain Conus species contains peptide toxins which are capable of shielding the binding site of ?(2)-receptors in an antagonistic manner.