[ 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
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
Cyclic peptides are an attractive class of bioactive molecules whose proven utility in the research lab and clinic has fueled the development of new candidates with diverse chemical structures and functions.However,th...Cyclic peptides are an attractive class of bioactive molecules whose proven utility in the research lab and clinic has fueled the development of new candidates with diverse chemical structures and functions.However,these candidates have been largely limited to naturally evolved products or their derivatives.Herein,we describe the de novo discovery of bicyclic peptides using a phage-encoded library of peptides possessing the characteristic framework of naturally occurringα-conotoxins(α-Ctxs).Selection againstα7 nicotinic acetylcholine receptor yielded a bicyclic peptide possessing an uncanonical disulfide connectivity rarely found in naturally occurringα-Ctxs.The chemically synthesized analogue of the hit demonstrated a more potent inhibitory effect on the receptor(IC_(50)=8.6 nmol/L)compared to the majority of known naturalα-Ctxs.Cryo-EM structural studies revealed that the binding mode of this selected peptide to the receptor differed from that of naturalα-Ctxs,based on the orientation of the peptide in the binding pocket and its contacts with the receptor.Our work highlights the utility of a natural toxin-based directed evolution strategy to identify conformationally constrained peptide binders of target proteins,and is expected to accelerate the discovery of well-behaved peptides for use as biological probes and therapeutics.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.
基金supported by the Science and Technological Fund of Anhui Province for Outstanding Youth(2208085J21)the National Natural Science Foundation of China(22227810,21825703,22137005,and 92253302)the Beijing Life Science Academy Scientific Research Program(2023000CA0070)。
文摘Cyclic peptides are an attractive class of bioactive molecules whose proven utility in the research lab and clinic has fueled the development of new candidates with diverse chemical structures and functions.However,these candidates have been largely limited to naturally evolved products or their derivatives.Herein,we describe the de novo discovery of bicyclic peptides using a phage-encoded library of peptides possessing the characteristic framework of naturally occurringα-conotoxins(α-Ctxs).Selection againstα7 nicotinic acetylcholine receptor yielded a bicyclic peptide possessing an uncanonical disulfide connectivity rarely found in naturally occurringα-Ctxs.The chemically synthesized analogue of the hit demonstrated a more potent inhibitory effect on the receptor(IC_(50)=8.6 nmol/L)compared to the majority of known naturalα-Ctxs.Cryo-EM structural studies revealed that the binding mode of this selected peptide to the receptor differed from that of naturalα-Ctxs,based on the orientation of the peptide in the binding pocket and its contacts with the receptor.Our work highlights the utility of a natural toxin-based directed evolution strategy to identify conformationally constrained peptide binders of target proteins,and is expected to accelerate the discovery of well-behaved peptides for use as biological probes and therapeutics.
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.
