[ Objective] The disulfide-rich conotoxin MrV1B was produced by simple and fast genetic engineering method, to find new efficient ways for the synthesis of natural active conotoxins. [Method] Primers of conotoxin gene...[ Objective] The disulfide-rich conotoxin MrV1B was produced by simple and fast genetic engineering method, to find new efficient ways for the synthesis of natural active conotoxins. [Method] Primers of conotoxin gene MrVIB were synthesized to construct expression vectors pET22b（ ＋ ）/His-Xa-MrVIB and pET32a/Trx-EK-MrV1B, which were transformed into BL21 （DE3）pLysS and expressed under induction by IPTG. Recombinant proteins were purified by affinity chromatography using Ni-NTA agarose column, and the expression of the recombinant proteins was analyzed by Tricine-SDS-PAGE electrophoresis. [ Result] The recombinant conotoxins His-Xa-MrVIB and Trx-EK-MrVIB were effectively expressed in E. coli, and purified by one-step affinity chromatography, and the purity of the recombinant conotoxins was greater than 90%. [ Conclusion] The conotoxin MrVIB was effectively secreted and expressed by genetic engineering method, which could solve the problems in chemical synthesis of conotoxins including low yield, high cost and difficult purification.展开更多
Objective:To determine the new M-superfamily conotoxins from molluscivorous snail Conus bandanus in Vietnam.Methods:Conus bandanus venom was fractionated and purified on HPLC system with an analytical reversed-phase C...Objective:To determine the new M-superfamily conotoxins from molluscivorous snail Conus bandanus in Vietnam.Methods:Conus bandanus venom was fractionated and purified on HPLC system with an analytical reversed-phase C18 column in order to screen small conotoxins.The primary structure of peptide was analyzed by matrix-assisted laser desorption/ionization time of flight tandem mass spectrometry using collision-induced dissociation and confirmed by Edman’s degradation method.Results:Five new conotoxins were biochemically characterized from the crude venom of the mollusk-hunting cone snail Conus bandanus,which were collected at Ke Ga reef of the Nha Trang Bay(Vietnam).Each conotoxin had 15 or 16 amino acid residues and shared the same characteristic cysteine framework V as–CC–C–C–CC–.They were termed as Bn3 b,Bn3 c,Bn3 d,Bn3 e and Bn3 f following the conotoxins nomenclature.Conclusions:The conotoxins Bn3 b,Bn3 e,and Bn3 f are categorized in the mini-M conotoxins of the M1 branch,while conotoxins Bn3 c and Bn3 d are categorized in the mini-M conotoxins of the M2 branch.The homological analysis reveals that these conotoxins could serve as promising probe compounds for voltage-gated sodium channels.展开更多
Nicotinic acetylcholine receptors(nAChRs) are widely distributed ligand gated ion channels throughout the peripheral and central nervous systems of mammals.There are 16 different n AChR subunits,α1-α7,α9,α10 and ...Nicotinic acetylcholine receptors(nAChRs) are widely distributed ligand gated ion channels throughout the peripheral and central nervous systems of mammals.There are 16 different n AChR subunits,α1-α7,α9,α10 and β1-β4,as well as γ,δ,and ε,which assemble into pentamers to form different nAChR subtypes with distinct pharmacological properties in mammals.Among them α6β2*(*designates other possible subunit),α3β4 and α4β2 nAChR subtypes are potential therapeutic targets for the treatment of addiction.However,various n AChR subtypes are very difficult to pharmacologically distinguish from each other.The α6* n AChRs are expressed by dopaminergic neurons in the central nervous system,which modulate the release of dopamine and are believed to be important in mediating tobacco,morphine,cocaine and ethanol addiction.The α3β4 nAChRs present in the medial habenula with important role in influencing nicotine addiction.Blockage of α3β4 nAChRs in the medial habenula decreased the dose of nicotine that rodents would self-administer.Thus,new antagonists of α6β2* or α3β4 nA ChR subtypes are of considerable interest,which would give strategies to selectively modulate α6β2* or α3β4 nA ChR function.We characterized an α-conotoxin(α-CTx)TxIB with 16 amino acids and an α-CTx TxID with 15 amino acids from Conus textile.The sequence of TxIB is GCCSDPPCRNKHPDLCamide.The sequence of TxID is GCCSHPVCSAMSPIC with C-terminal amidation too.Both peptides with a Ⅰ-Ⅲ and Ⅱ-Ⅳ disulfide con-nectivity were chemically synthesized.The residues between Cys-Ⅱ and Cys-Ⅲ and Cys-Ⅲand Cys-Ⅳ of α-CTx are commonly referred to as loops 1 and 2,respectively.The number of residues in each of these loops is used to further classify the α-CTx.So TxIB is classified as a 4/7α-CTx,whereas the α-CTx TxIB has a 4/6 spacing.Both peptides were tested on rat nAChRs heterologously expressed in Xenopus laevis oocytes.The α-CTx TxIB blocked α6/α3β2β3 nAChR with an IC50 of 28 nmol·L^(-1),which showed little or no block of all the other tested subtypes at concentrations up to 10 μmol·L^(-1).TxIB blocking α6/α3β2β3 nAChR is rapidly reversed after toxin washout.The ability ofα-CTx TxIB to discriminate between α6/α3β2β3 and the other nAChR receptors is unique.There are no small molecules have this selectivity profile.Previously described α-CTx that potently blockα6/α3β2β3 nA ChR s also block either α6/α3β4 nAChRs,α3β2 nAChRs and(or) other nAChRs subtypes.TxID was the very potent α3β4 nAChR antagonists blocking rat α3β4 n AChRs with an IC-50 of 12.5 nmol·L1.However,TxID also blocked the closely related α6/α3β4 with an IC50 of 94 nmol·L^(-1).In fact,the expression profile ofα3β4 nAChRs and α6/α3β4 nAChRs overlap in a variety of tissues.So TxI D can′t differentiate α3β4 nA ChR from α6/α3β4 nA ChR effectively.To distinguish between these two close subtypes,positional-scanning mutagenesis of TxID was performed to identify critical residues that confer potency for α3β4 nAChRs,and hope to obtain more selective mutant to discriminate between these two close subtypes.The effects of 15 analogues and TxID were tested on both α3β4 and α6/α3β4 nAChRs.An analogue,ie [S9 A]TxID had46-fold greater potency for α3β4 versus α6/α3β4 nAChRs,which showed significantly improved selectivity for α3β4 versus α6/α3β4 nAChRs.Both TxI D and [S9 A]TxI D had little activity on other nA ChR subtypes.The three-dimensional solution structures of TxIB,TxID and [S9 A]TxID were determined using NMR spectroscopy.α-CTx TxI B,TxID and [S9 A]TxID represent uniquely selective ligand for probing the structure and function of α6β2*and α3β4 nA ChR s respectively.It is known about20% people have used drugs recreationally resulting in a substance use disorder finally.Therefore,structural insights derived from these ligands may facilitate the development of novel therapeutics for addiction involving α6β2* and α3β4 nA ChR s.展开更多
Conus loroisii is a marine vermivorous snail found profusely in the southern seas of India.They harbor several toxic peptide components commonly called as‘conotoxins’.In this study,we have identified and sequenced f...Conus loroisii is a marine vermivorous snail found profusely in the southern seas of India.They harbor several toxic peptide components commonly called as‘conotoxins’.In this study,we have identified and sequenced five conotoxins using proteome based tandem mass spectrometry analysis through Data analysis 4.1 software.Among them,we found Lo959 as contryphan which is previously described.All other conotoxins Lo1702,Lo1410,Lo1385 and Lo1686 belong to M-Superfamily conotoxins and novel to C.loroisii.Lo1410 is completely novel to conotoxin research with 3 disulfides and the amino acid sequence is derived as CCSTNCAVCIPCCP.All the identified M-Superfamily conotoxins are sub categorised to mini M2 superfamily conotoxins.Lo1702 and Lo1686 possess C-terminal amidation which is the key feature in conotoxins.Moreover,we have screened the natural venom for the occurrence of toxicity in the zebrafish model and brine shrimp.展开更多
Conotoxins are short peptide-toxins with specific targets and large diversity. They are usetul in analgesia, neuroprotection, detection of some kinds of deseases, and receptor and ion channel study. in order to explor...Conotoxins are short peptide-toxins with specific targets and large diversity. They are usetul in analgesia, neuroprotection, detection of some kinds of deseases, and receptor and ion channel study. in order to explore the conotoxin resourses of Chinese oceans, rapid amplification of 3’ cDNA ends (RACE) method was utilized to systemically analyze the O-superfamily conotoxin content of Conus striatus inhabited near Chinese Hainan Island. Six new O-superfamily conopeptides were identified, one of which is highly homologous to MVIlA, an N-type calcium channel antagonist.展开更多
Conotoxins are marine peptide toxins from marine cone snails.Theα-conotoxin RegIIA can selectively act on human(h)α3β4 nicotinic acetylcholine receptor(nAChR),and is an important lead for drug development.The high-...Conotoxins are marine peptide toxins from marine cone snails.Theα-conotoxin RegIIA can selectively act on human(h)α3β4 nicotinic acetylcholine receptor(nAChR),and is an important lead for drug development.The high-resolution cryo-electron microscopy structure of theα3β4 nAChR demonstrates several carbohydrates are located near the orthosteric binding sites,which may affectα-conotoxin binding.Oligosaccharide chains can modify the physical and chemical properties of proteins by changing the conformation,hydrophobicity,quality and size of the protein.The purpose of this study is to explore the effect of oligosaccharide chains on the binding modes and activities of RegIIA and its derivatives at hα3β4 nAChRs.Through computational simulations,we designed and synthesized RegIIA mutants at position 14 to explore the importance of residue H14 to the activity of the peptide.Molecular dynamics simulations suggest that the oligosaccharide chains affect the binding of RegIIA at the hα3β4 nAChR through direct interactions with H14 and by affecting the C-loop conformation of the binding sites.Electrophysiology studies on H14 analogues suggest that in addition to forming direct interactions with the carbohydrates,the residue might play an important role in maintaining the conformation of the peptide.Overall,this study further clarifies the structure–activity relationship ofα-conotoxin RegIIA at the hα3β4 nAChR and,also provides important experimental and theoretical basis for the development of new peptide drugs.展开更多
Cone snails (Conus) elaborate a series of conotoxin (CTX) peptides in their venoms to paralyze their prey. Among these toxins, w-CTX抯 specifically target to presynaptic voltage-gated calcium channel subsets, causing ...Cone snails (Conus) elaborate a series of conotoxin (CTX) peptides in their venoms to paralyze their prey. Among these toxins, w-CTX抯 specifically target to presynaptic voltage-gated calcium channel subsets, causing inhibition of neurotransmitter release. w-CTX SO3 was isolated from the venom of Conus striatus, which is the only available fish-hunting snail near the coast of the South China Sea. The three-dimensional solution structure of w-CTX SO3, a peptide which is the only w-conotoxin reported to show high homology with another w-CTX (MVIIA from C. Magus), has been determined by 1H NMR techniques. The molecular structure of w-CTX SO3 is stabilized by three disulfide bridges and a short triple-stranded antiparallel b-sheet with four turns. A comprehensive comparison suggested that the backbone conformation of w-CTXs was quite conserved, while the length of b-sheet and the type of some turns might have minor differences.展开更多
基金Supported by Natural Science Foundation of China(81560611)Natural Science Foundation of Hainan Province(No.317170)
文摘[ Objective] The disulfide-rich conotoxin MrV1B was produced by simple and fast genetic engineering method, to find new efficient ways for the synthesis of natural active conotoxins. [Method] Primers of conotoxin gene MrVIB were synthesized to construct expression vectors pET22b（ ＋ ）/His-Xa-MrVIB and pET32a/Trx-EK-MrV1B, which were transformed into BL21 （DE3）pLysS and expressed under induction by IPTG. Recombinant proteins were purified by affinity chromatography using Ni-NTA agarose column, and the expression of the recombinant proteins was analyzed by Tricine-SDS-PAGE electrophoresis. [ Result] The recombinant conotoxins His-Xa-MrVIB and Trx-EK-MrVIB were effectively expressed in E. coli, and purified by one-step affinity chromatography, and the purity of the recombinant conotoxins was greater than 90%. [ Conclusion] The conotoxin MrVIB was effectively secreted and expressed by genetic engineering method, which could solve the problems in chemical synthesis of conotoxins including low yield, high cost and difficult purification.
基金funded by Vietnam National Foundation for Science and Technology Development(NAFOSTED)under grant number 106-NN.02-2015.14
文摘Objective:To determine the new M-superfamily conotoxins from molluscivorous snail Conus bandanus in Vietnam.Methods:Conus bandanus venom was fractionated and purified on HPLC system with an analytical reversed-phase C18 column in order to screen small conotoxins.The primary structure of peptide was analyzed by matrix-assisted laser desorption/ionization time of flight tandem mass spectrometry using collision-induced dissociation and confirmed by Edman’s degradation method.Results:Five new conotoxins were biochemically characterized from the crude venom of the mollusk-hunting cone snail Conus bandanus,which were collected at Ke Ga reef of the Nha Trang Bay(Vietnam).Each conotoxin had 15 or 16 amino acid residues and shared the same characteristic cysteine framework V as–CC–C–C–CC–.They were termed as Bn3 b,Bn3 c,Bn3 d,Bn3 e and Bn3 f following the conotoxins nomenclature.Conclusions:The conotoxins Bn3 b,Bn3 e,and Bn3 f are categorized in the mini-M conotoxins of the M1 branch,while conotoxins Bn3 c and Bn3 d are categorized in the mini-M conotoxins of the M2 branch.The homological analysis reveals that these conotoxins could serve as promising probe compounds for voltage-gated sodium channels.
文摘Nicotinic acetylcholine receptors(nAChRs) are widely distributed ligand gated ion channels throughout the peripheral and central nervous systems of mammals.There are 16 different n AChR subunits,α1-α7,α9,α10 and β1-β4,as well as γ,δ,and ε,which assemble into pentamers to form different nAChR subtypes with distinct pharmacological properties in mammals.Among them α6β2*(*designates other possible subunit),α3β4 and α4β2 nAChR subtypes are potential therapeutic targets for the treatment of addiction.However,various n AChR subtypes are very difficult to pharmacologically distinguish from each other.The α6* n AChRs are expressed by dopaminergic neurons in the central nervous system,which modulate the release of dopamine and are believed to be important in mediating tobacco,morphine,cocaine and ethanol addiction.The α3β4 nAChRs present in the medial habenula with important role in influencing nicotine addiction.Blockage of α3β4 nAChRs in the medial habenula decreased the dose of nicotine that rodents would self-administer.Thus,new antagonists of α6β2* or α3β4 nA ChR subtypes are of considerable interest,which would give strategies to selectively modulate α6β2* or α3β4 nA ChR function.We characterized an α-conotoxin(α-CTx)TxIB with 16 amino acids and an α-CTx TxID with 15 amino acids from Conus textile.The sequence of TxIB is GCCSDPPCRNKHPDLCamide.The sequence of TxID is GCCSHPVCSAMSPIC with C-terminal amidation too.Both peptides with a Ⅰ-Ⅲ and Ⅱ-Ⅳ disulfide con-nectivity were chemically synthesized.The residues between Cys-Ⅱ and Cys-Ⅲ and Cys-Ⅲand Cys-Ⅳ of α-CTx are commonly referred to as loops 1 and 2,respectively.The number of residues in each of these loops is used to further classify the α-CTx.So TxIB is classified as a 4/7α-CTx,whereas the α-CTx TxIB has a 4/6 spacing.Both peptides were tested on rat nAChRs heterologously expressed in Xenopus laevis oocytes.The α-CTx TxIB blocked α6/α3β2β3 nAChR with an IC50 of 28 nmol·L^(-1),which showed little or no block of all the other tested subtypes at concentrations up to 10 μmol·L^(-1).TxIB blocking α6/α3β2β3 nAChR is rapidly reversed after toxin washout.The ability ofα-CTx TxIB to discriminate between α6/α3β2β3 and the other nAChR receptors is unique.There are no small molecules have this selectivity profile.Previously described α-CTx that potently blockα6/α3β2β3 nA ChR s also block either α6/α3β4 nAChRs,α3β2 nAChRs and(or) other nAChRs subtypes.TxID was the very potent α3β4 nAChR antagonists blocking rat α3β4 n AChRs with an IC-50 of 12.5 nmol·L1.However,TxID also blocked the closely related α6/α3β4 with an IC50 of 94 nmol·L^(-1).In fact,the expression profile ofα3β4 nAChRs and α6/α3β4 nAChRs overlap in a variety of tissues.So TxI D can′t differentiate α3β4 nA ChR from α6/α3β4 nA ChR effectively.To distinguish between these two close subtypes,positional-scanning mutagenesis of TxID was performed to identify critical residues that confer potency for α3β4 nAChRs,and hope to obtain more selective mutant to discriminate between these two close subtypes.The effects of 15 analogues and TxID were tested on both α3β4 and α6/α3β4 nAChRs.An analogue,ie [S9 A]TxID had46-fold greater potency for α3β4 versus α6/α3β4 nAChRs,which showed significantly improved selectivity for α3β4 versus α6/α3β4 nAChRs.Both TxI D and [S9 A]TxI D had little activity on other nA ChR subtypes.The three-dimensional solution structures of TxIB,TxID and [S9 A]TxID were determined using NMR spectroscopy.α-CTx TxI B,TxID and [S9 A]TxID represent uniquely selective ligand for probing the structure and function of α6β2*and α3β4 nA ChR s respectively.It is known about20% people have used drugs recreationally resulting in a substance use disorder finally.Therefore,structural insights derived from these ligands may facilitate the development of novel therapeutics for addiction involving α6β2* and α3β4 nA ChR s.
文摘Conus loroisii is a marine vermivorous snail found profusely in the southern seas of India.They harbor several toxic peptide components commonly called as‘conotoxins’.In this study,we have identified and sequenced five conotoxins using proteome based tandem mass spectrometry analysis through Data analysis 4.1 software.Among them,we found Lo959 as contryphan which is previously described.All other conotoxins Lo1702,Lo1410,Lo1385 and Lo1686 belong to M-Superfamily conotoxins and novel to C.loroisii.Lo1410 is completely novel to conotoxin research with 3 disulfides and the amino acid sequence is derived as CCSTNCAVCIPCCP.All the identified M-Superfamily conotoxins are sub categorised to mini M2 superfamily conotoxins.Lo1702 and Lo1686 possess C-terminal amidation which is the key feature in conotoxins.Moreover,we have screened the natural venom for the occurrence of toxicity in the zebrafish model and brine shrimp.
文摘Conotoxins are short peptide-toxins with specific targets and large diversity. They are usetul in analgesia, neuroprotection, detection of some kinds of deseases, and receptor and ion channel study. in order to explore the conotoxin resourses of Chinese oceans, rapid amplification of 3’ cDNA ends (RACE) method was utilized to systemically analyze the O-superfamily conotoxin content of Conus striatus inhabited near Chinese Hainan Island. Six new O-superfamily conopeptides were identified, one of which is highly homologous to MVIlA, an N-type calcium channel antagonist.
基金supported by the National Key Research and Development Program(2019YFC0312601)the grant from the Fundamental Research Funds for the Central Universities(201762011 and 201941012)+1 种基金National Natural Science Foundation of China(NSFC)(No.81502977 and 41830535)an Australian Research Council(ARC)Discovery Project Grant(DP150103990 awarded to Prof D.J.Adams)。
文摘Conotoxins are marine peptide toxins from marine cone snails.Theα-conotoxin RegIIA can selectively act on human(h)α3β4 nicotinic acetylcholine receptor(nAChR),and is an important lead for drug development.The high-resolution cryo-electron microscopy structure of theα3β4 nAChR demonstrates several carbohydrates are located near the orthosteric binding sites,which may affectα-conotoxin binding.Oligosaccharide chains can modify the physical and chemical properties of proteins by changing the conformation,hydrophobicity,quality and size of the protein.The purpose of this study is to explore the effect of oligosaccharide chains on the binding modes and activities of RegIIA and its derivatives at hα3β4 nAChRs.Through computational simulations,we designed and synthesized RegIIA mutants at position 14 to explore the importance of residue H14 to the activity of the peptide.Molecular dynamics simulations suggest that the oligosaccharide chains affect the binding of RegIIA at the hα3β4 nAChR through direct interactions with H14 and by affecting the C-loop conformation of the binding sites.Electrophysiology studies on H14 analogues suggest that in addition to forming direct interactions with the carbohydrates,the residue might play an important role in maintaining the conformation of the peptide.Overall,this study further clarifies the structure–activity relationship ofα-conotoxin RegIIA at the hα3β4 nAChR and,also provides important experimental and theoretical basis for the development of new peptide drugs.
基金This work was sup-ported by the“985"Project THSJZ of Tsinghua University.
文摘Cone snails (Conus) elaborate a series of conotoxin (CTX) peptides in their venoms to paralyze their prey. Among these toxins, w-CTX抯 specifically target to presynaptic voltage-gated calcium channel subsets, causing inhibition of neurotransmitter release. w-CTX SO3 was isolated from the venom of Conus striatus, which is the only available fish-hunting snail near the coast of the South China Sea. The three-dimensional solution structure of w-CTX SO3, a peptide which is the only w-conotoxin reported to show high homology with another w-CTX (MVIIA from C. Magus), has been determined by 1H NMR techniques. The molecular structure of w-CTX SO3 is stabilized by three disulfide bridges and a short triple-stranded antiparallel b-sheet with four turns. A comprehensive comparison suggested that the backbone conformation of w-CTXs was quite conserved, while the length of b-sheet and the type of some turns might have minor differences.
