Engineering And Identification Of?-conotoxins From Cone Snails Native To Hainan

The cone snails (genus Conus) are venomous marine molluscs that use small, structured peptide toxins (conotoxins) for prey capture, defense, and competitor deterrence. Each of the700Conus can produce excess of1000different conopeptides, with little overlap between species. Because the majority of conopeptides discriminate between closely related subtypes of ion channels, they are widely used as pharmacological agents in ion channel research, and several have direct diagnostic and therapeutic potential.a-Conotoxins are a specific class of short, disulfide-constrained peptides with a conserved Cys-framework, CCXnCXmC. Xn and Xm represent the number of amino acids and are used to subclassify peptides such as?4/3, a4/4, a4/6and a4/7, which specifically target various nAChR isoforms.Our lab identified a a-4/6-conotoxin (CTx) TxID from Conus textile. The new toxin consists of15amino acid residues with two disulfide bonds. TxID was synthesized using solid phase methods, and the synthetic peptide was functionally tested on nAChRs heterologously expressed in Xenopus laevis oocytes. TxID blocked rat a3(34nAChRs with a12.5nM IC50, which places it among the most potent ?3?4nAChR antagonists. TxID also blocked the closely related a6/a3?4with a94nM IC50but showed little activity on other nAChR subtypes. a-CTx TxID is a novel tool with which to probe the structure and function of a3?4nAChRs.We performed alanine scanning mutagenesis of TxID, homology modeling, and molecular docking to identify the structural determinants of the TxID and-a3?4nAChR interaction. Five alanine-substituted TxID analogues,[H5A]TxID,[P6A]TxID,[V7A]TxID [M11A]TxID and [P13A]TxID, did not inhibit the a3?4nAChR. Because methionine is easy to suffer from oxidation, we also redesigned synthesis of seven mutants of TxID, which methionine was replaced by other amino acids. Electrophysiological recordings indicated that [M11L]TxID and [S9A/M11L]TxID mutants increased IC50-values significantly. At the same time, we reengineered the Ile-14which located in superficial of structure. All analogues displayed inhibitory activity toward a3?4nAChRs that was comparable to TxID. Significantly, TxID[I10D] demonstrated moderately improved activity toward a3?4nAChRs when compared with TxID. As molecular docking studies also suggested that His-5, Val-7and Met-11might be a key residue engendering a3?4nAChRs activity. Homology modeling and molecular docking also suggested that amno acids on Loop-B, Loop-C of a3nAChR subunit and Ile-111, Gln-119and Leu-121on the ?4nAChR subunit played a key role in activity. These findings provide insights into the inhibition and mechanism of antagonist properties of the a-CTx TxID on nAChRs and might help improve the design of analgesic conopeptides.Here, we used reversed-phase high performance liquid chromatography (RP-HPLC) and electrospray ionization-mass spectrometry (ESI-MS) to explore the venom peptide diversity of Conus textile, a species of cone snail native to Hainan, China. One fraction ofC. textile crude venom potently blocked a3?2nAChRs. Subsequent purification, synthesis, and tandem mass spectrometric analysis demonstrated that the most active compound in this fraction was identical to a-CTx TxIA, an antagonist of a3?2nAChRs.In order to know the effect of disulfide-pairing and C-Terminal Amidation on a-CTx TxIA and TxID, three disulfide isoforms and no amidation mutants of these conotxin were synthesized. Then their activities were investigated systematically for the first time. As we observed, disulfide isomerisation was particularly important for a-CTx TxID potency, and non-native Disulfide Bond Connectivity in Tx?D lost its biological activity. Like TxIA, globular TxIA potently inhibited a3?2nAChRs with an IC50of5.4nM, while ribbon TxIA had an IC50of430nM. In contrast, beads isomer had little activity towards a3?2nAChRs. The amide modification had less influence on TxID inhibited a3?4nAChRs, but activity of TxIA with no amidation was down about10times. This study gives further insight into the influence of disulfide linkage and C-terminal amidation on conotoxin pharmacology.RP-HPLC studies on TxID were performed in a solution containing glutathione and in human serum at physiological pH. The result showed TxID produced disulfide bond scrambling, and the degradation of the conotoxin TxID was obvious after12hours. From this result, we plan to redesign TxID in depth to increase stability in physiological environment.