Novel natriuretic peptides from the venom of the inland taipan (Oxyuranus microlepidotus): isolation, chemical and biological characterisation

Three natriuretic-like peptides (TNP-a, TNP-b, and TNP-c) were isolated from the venom of Oxyuranus microlepidotus (inland taipan) and were also present in the venoms of Oxyuranus scutellatus canni (New Guinea taipan) and Oxyuranus scutellatus scutellatus (coastal taipan). They were isolated by HPLC, characterised by mass spectrometry and Edman analysis, and consist of 35-39 amino acid residues. These molecules differ from ANP/BNP through replacement of invariant residues within the 17-membered ring structure and by inclusion of proline residues in the C-terminal tail. TNP-c was equipotent to ANP in specific GC-A assays or aortic ring assays whereas TNP-a and TNP-b were either inactive (GC-A over-expressing cells and endothelium-denuded aortic rings) or weakly active (endothelium-intact aortic rings). TNP-a and TNP-b were also unable to competitively inhibit the binding of TNP-c in endothelium-denuded aortae (GC-A) or endothelium-intact aortae (NPR-C). Thus, these naturally occurring isoforms provide a new platform for further investigation of structure-function relationships of natriuretic peptides.     

Subtype-selective noncompetitive or competitive inhibition of human alpha1-adrenergic receptors by rho-TIA

The 19-amino acid conopeptide (rho-TIA) was shown previously to antagonize noncompetitively alpha(1B)-adrenergic receptors (ARs). Because this is the first peptide ligand for these receptors, we compared its interactions with the three recombinant human alpha(1)-AR subtypes (alpha(1A), alpha(1B), and alpha(1D)). Radioligand binding assays showed that rho-TIA was 10-fold selective for human alpha(1B)-over alpha(1A)- and alpha(1D)-ARs. As observed with hamster alpha(1B)-ARs, rho-TIA decreased the number of binding sites (B(max)) for human alpha(1B)-ARs without changing affinity (K(D)), and this inhibition was unaffected by the length of incubation but was reversed by washing. However, rho-TIA had opposite effects at human alpha(1A)-ARs and alpha(1D)-ARs, decreasing K(D) without changing B(max), suggesting it acts competitively at these subtypes. rho-TIA reduced maximal NE-stimulated [(3)H]inositol phosphate formation in HEK293 cells expressing human alpha(1B)-ARs but competitively inhibited responses in cells expressing alpha(1A)- or alpha(1D)-ARs. Truncation mutants showed that the amino-terminal domains of alpha(1B)- or alpha(1D)-ARs are not involved in interaction with rho-TIA. Alanine-scanning mutagenesis of rho-TIA showed F18A had an increased selectivity for alpha(1B)-ARs, and F18N also increased subtype selectivity. I8A had a slightly reduced potency at alpha(1B)-ARs and was found to be a competitive, rather than noncompetitive, inhibitor in both radioligand and functional assays. Thus rho-TIA noncompetitively inhibits alpha(1B)-ARs but competitively inhibits the other two subtypes, and this selectivity can be increased by mutation. These differential interactions do not involve the receptor amino termini and are not because of the charged nature of the peptide, and isoleucine 8 is critical for its noncompetitive inhibition at alpha(1B)-ARs.     

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.     

Isolation and characterisation of conomap-Vt, a D-amino acid containing excitatory peptide from the venom of a vermivorous cone snail

Cone snail venom is a rich source of bioactives, in particular small disulfide rich peptides that disrupt synaptic transmission. Here, we report the discovery of conomap-Vt (Conp-Vt), an unusual linear tetradecapeptide isolated from Conus vitulinus venom. The sequence displays no homology to known conopeptides, but displays significant homology to peptides of the MATP (myoactive tetradecapeptide) family, which are important endogenous neuromodulators in molluscs, annelids and insects. Conp-Vt showed potent excitatory activity in several snail isolated tissue preparations. Similar to ACh, repeated doses of Conp-Vt were tachyphylactic. Since nicotinic and muscarinic antagonists failed to block its effect and Conp-Vt desensitised tissue remained responsive to ACh, it appears that Conp-Vt contractions were non-cholinergic in origin. Finally, biochemical studies revealed that Conp-Vt is the first member of the MATP family with a d-amino acid. Interestingly, the isomerization of L-Phe to D-Phe enhanced biological activity, suggesting that this post-translational modified conopeptide may have evolved for prey capture.     

Identification of a novel class of nicotinic receptor antagonists: dimeric conotoxins VxXIIA, VxXIIB, and VxXIIC from Conus vexillum

The venoms of predatory marine snails (Conus spp.) contain diverse mixtures of peptide toxins with high potency and selectivity for a variety of voltage-gated and ligand-gated ion channels. Here we describe the chemical and functional characterization of three novel conotoxins, alphaD-VxXIIA, alphaD-VxXIIB, and alphaD-VxXIIC, purified from the venom of Conus vexillum. Each toxin was observed as an approximately 11-kDa protein by LC/MS, size exclusion chromatography, and SDS-PAGE. After reduction, the peptide sequences were determined by Edman degradation chemistry and tandem MS. Combining the sequence data together with LC/MS and NMR data revealed that in solution these toxins are pseudo-homodimers of paired 47-50-residue peptides. The toxin subunits exhibited a novel arrangement of 10 conserved cystine residues, and additional post-translational modifications contributed heterogeneity to the proteins. Binding assays and two-electrode voltage clamp analyses showed that alphaD-VxXIIA, alphaD-VxXIIB, and alphaD-VxXIIC are potent inhibitors of nicotinic acetylcholine receptors (nAChRs) with selectivity for alpha7 and beta2 containing neuronal nAChR subtypes. These dimeric conotoxins represent a fifth and highly divergent structural class of conotoxins targeting nAChRs.     

Isolation,structure, and activity of GID,a novel alpha 4/7-conotoxin with an extended N-terminal sequence

Using assay-directed fractionation of Conus geographus crude venom, we isolated alpha-conotoxin GID, which acts selectively at neuronal nicotinic acetylcholine receptors (nAChRs). Unlike other neuronally selective alpha-conotoxins, alpha-GID has a four amino acid N-terminal tail, gamma-carboxyglutamate (Gla), and hydroxyproline (O) residues, and lacks an amidated C terminus. GID inhibits alpha 7 and alpha 3 beta 2 nAChRs with IC(50) values of 5 and 3 nm, respectively and is at least 1000-fold less potent at the alpha 1 beta 1 gamma delta, alpha 3 beta 4, and alpha 4 beta 4 combinations. GID also potently inhibits the alpha 4 beta 2 subtype (IC(50) of 150 nm). Deletion of the N-terminal sequence (GID Delta 1-4) significantly decreased activity at the alpha 4 beta 2 nAChR but hardly affected potency at alpha 3 beta 2 and alpha 7 nAChRs, despite enhancing the off-rates at these receptors. In contrast, Arg(12) contributed to alpha 4 beta 2 and alpha 7 activity but not to alpha 3 beta 2 activity. The three-dimensional structure of GID is well defined over residues 4-19 with a similar motif to other alpha-conotoxins. However, despite its influence on activity, the tail appears to be disordered in solution. Comparison of GID with other alpha 4/7-conotoxins which possess an NN(P/O) motif in loop II, revealed a correlation between increasing length of the aliphatic side-chain in position 10 (equivalent to 13 in GID) and greater alpha 7 versus alpha 3 beta 2 selectivity.     

Mast cell and monocyte recruitment by S100A12 and its hinge domain

S100A12 is expressed at sites of acute, chronic, and allergic inflammation. S100 proteins have regions of high sequence homology, but the "hinge" region between the conserved calcium binding domains is structurally and functionally divergent. Because the murine S100A8 hinge domain (mS100A8(42-55)) is a monocyte chemoattractant whereas the human sequence (hS100A8(43-56)) is inactive, we postulated that common hydrophobic amino acids within the S100A12 hinge sequence may be functional. The hinge domain, S100A12(38-53), was chemotactic for human monocytes and murine mast cells in vitro. S100A12(38-53) provoked an acute inflammatory response similar to that elicited by S100A12 in vivo and caused edema and leukocyte and mast cell recruitment. Circular dichroism studies showed that S100A12(38-53) had increased helical structure in hydrophobic environments. Mutations in S100A12(38-53) produced using an alanine scan confirmed that specific hydrophobic residues (I44A, I47A, and I53A) on the same face of the helix were critical for monocyte chemotaxis in vitro and generation of edema in vivo. In a hydrophobic environment such as the cell membrane, these critical residues would likely align on one face of an alpha-helix to facilitate receptor interaction. Interaction is unlikely to occur via the receptor for advanced glycation end products but, rather, via a G-protein-coupled mechanism.