Synthesis And Function Study Of Mutants Of α-conotoxin LvIA

Marine snails of Conus are genus of predatory marine gastropod.Around 700 species of Conus are mainly distributed throughout tropical and subtropical waters.Conus species can use venom to capture prey and defend against predators.The venom of each Cone snail may have 2000 distinct,biologically active small peptides,which named conotoxins or conopeptides.Conotoxins are also distinctive by virtue of their small size,rich Cysteine,variable sequences and specific targets and so on.CTxs have been divided into several superfamilies according to signal sequences,such as A-,O-,M-,P-,I-,T etc.The superfamily may be subdivided into several families with distinct pharmacological activities,for example the A-superfamily(α-,αA-,κA-and ρ-Conotoxins),M-superfamily(μ-and Ψ-Conotoxins),O-superfamily(ω-,μO,δ-,and κ-Conotoxins),T-superfamily(τ-and χ-Conotoxins)and so on.Individual CTxs are selectively targeted to specific voltage-gated and ligand-gated ion channels.The α-CTxs are important class of CTxs,they can specifically target different subtypes of nicotinic acetylcholine receptors(nAChRs).Due to their extraordinary potential and pharmacological properties,CTxs are increasingly used as tools in neuroscience and as drugs or drug leads in clinical trials.lvla is a noval α-CTxs precursor gene in our lab,which was cloned form C.lividus genomic DNA native to Hainan.LvIA mature peptide sequence was showed:GCCSHPACNVDHPEIC#(#denotes an amidated C-terminus).Preliminary studies in our laboratory found LvIA have highest affinity for α3β2 nAChRs.They have ability to discriminate between similar neuronal nAChRs subtypes,for example,α3β2 andα6/α3β2β3 nAChRs;α3β2 and α3β4 nAChRs.LvIA is a α4/7-CTxs and has some similar amino acids with previously reported α4/7-CTxs.The mature peptide of α-CTx LvIA and mutants were obtained by chemical synthesis and two-step oxidation.The pharmacological targets of LvIA mutants were screened.Xenopus laevis oocytes are optimal system for the expression of receptor and channel protein.When the nAChRs subunit cRNA were prepared and injected into Xenopus oocytes,the nAChRs could be expressed on oocytes membrane.By this method,we constructed 20 kinds of human source and rat source nAChRs model expressed in oocytes,such as α3β2,α3β4,α7,α9α10 etc.The pharmacological activity of α-CTx LvIA and mutants on different nAChRs heterologously expressed in Xenopus oocytes were analyzed.We identified critical amino acid residues in α-CTx LvIA by alanine scanning.When the His5 or Pro6 position was substituted with Ala respectively,LvIA lost its activity at all nAChRs subtypes completely.When the His12 was substituted with Ala,the potency of LvIA mutant had decreased greatly.When the Asrn9 or Asp11 was substituted with Ala respectively,the activity of LvIA mutants at rα3β2 nAChRs had increased greatly.LvIA(N9A)and LvIA(D11A)gave IC50 values of 2.14 nM and 11.35 nM respectively at rα3β2 nAChRs.The ability of LvIA(N9A)to discriminate between rα3β2 and rα3β4 nAChRs had improved.The generation LvIA mutants at position 9 and 11 were designed.When the Asn9 was substituted with Arg,the activity of LvIA mutants at rα3β2 nAChRs had increased with an IC50 value was 2.12 nM.In the mean time,the target of LvIA(N9R)increased to ra7 nAChRs with an IC50 98.6 nM.We also find the SHPA as a conserved motif is important for α4/7-CTxs pharmacological activity including for LvIA.Modified amino acids in LvIA sequence were studied.When the Pro6 was substituted with Hyp(O),LvIA lost its activity at all nAChRs subtypes.When the Pro13 was substituted with Hyp,the activity of LvIA(P13O)at rα3β2 nAChRs had decreased slightly with an IC50 value was 51.92 nM,but there was no obvious change at rα3β4 nAChRs.C-terminal amidation not only had an impact on the activity of LvIA,but played a pivotal role in the folding tendency of LvIA.When the modification of C-terminal amidation was eliminated,LvIA inhibited the rα3β2 and rα3β4 nAChRs with IC50 of 298.8 nM and 1.65μM respectively,a 10-fold potency than native LvIA.Tandem repeats of lvla gene fragment were constructed and ligated with prokaryotic expression vector.The LvIA was expressed successfully in E.coli.Ivla mature peptide gene was situated downstream of the ketosteroid isomerase(ksi)gene and upstream of a his6 gene.A KSI-(rLvIA)n-His6 fusion protein was overproduced in E.coli,which was purified by Ni chelate chromatography.95%purity recombinant LvIA(rLvIA)were obtained by CNBr cleavage and purification,40 mg rLvIA could be obtained from 1 L media eventually.Mice hot plate test and electrophysiology results showed that rLvIA had good analgesic activity.This study not only provide an effective way to obtain a-CTx LvIA but also provide useful method to obtain other small CTxs,which have great significance for CTxs development and application in future.In summary,we combined molecular biology,peptide chemistry and patch clamp electrophysiology technique to determine the pharmacological target,key amino acids and expression method of a4/7-CTx LvIA thoroughly.These research results will have important significance for the LvIA application in neuroscience and development in new drugs.