Identification and Characterization of New Framework 14 Conotoxins from Two Vermivorous Conus Species,as Novel Antagonists of Nicotinic Acetylcholine Receptors

【作者】 彭灿； 叶铭宇； 汪燕芳； 邵晓霞； 袁多多； Edward Hawrot； 戚正武； 王春光；

【Author】 Can Peng,Mingyu Ye,Yanfang Wang,Xiaoxia Shao,Duoduo Yuan,Jing Liu,Edward Hawrot,Chengwu Chi,Chunguang Wang 1 Institute of Protein Research,College of Life Sciences and Technology,Tongji University, Shanghai 200092,China 2 Institute of Biochemistry and Cell Biology,Shanghai Institute of Biological Sciences,Chinese Academy of Sciences,Graduate School of the Chinese Academy of Sciences Shanghai 200031, China 3 Department of Molecular Pharmacology,Physiology and Biotechnology,Brown Medical School, Providence,Rhode Island 02912,USA

【机构】 Institute of Protein Research,College of Life Sciences and Technology,Tongji University； Department of Molecular Pharmacology,Physiology and Biotechnology,Brown Medical School Providence； Institute of Biochemistry and Cell Biology,Shanghai Institute of Biological Sciences,Chinese Academy of Sciences,Graduate School of the Chinese Academy of Sciences；

【摘要】 <正>As part of studies of the venom components present in Conus pulicarius and Conus tessulatus, two vermivorous Conus species collected from the South China Sea,we identified two new conotoxins,designated pu14a and ts14a,by cDNA cloning and peptide purification,respectively. The predicted mature toxin region of pu14a contains 19 amino acid（aa） residues with two disulfide bonds（1-3,2-4 connectivity） in a pattern of C(X₁₀)C（X₁）C（X₃）C（framework 14）. However,the 69-amino acid pu14a precursor surprisingly exhibits an identical signal sequence to corresponding regions of previously characterizedα-conotoxins,revealing a new level of conotoxin diversity and a plausible evolution mechanism of Conus prepropeptide recombination. The peptide pu14a has very unique primary structure and intercysteine spacing pattern with its first loop rich in proline residues.This peptide was chemically synthesized to further characterize its function.The synthetic pu14a induces a sleeping phenotype in mice,and it is also toxic to freshwater goldfish upon intramuscular injection.Using the Xenopus oocyte heterologous expression system,pu14a（1μM） was shown to inhibit both rat neuronalα3β2-containing and mouse neuromuscularα1β1γδsubtypes of nicotinic acetylcholine receptors（nAChRs）.It blocked the ACh-induced currents（51%anc 55%） in recombinantα3β2 andα6α3β2 nAChRs,respectively, and the toxin dissociated rapidly from the receptor.A block of approximately 82%of the ACh-evoked current was obtained inα1β1γδ,with a similar dissociation rate.The 20-aa polypeptide chain of ts14a with a 1-3/2-4 cysteine pairing shows high sequence similarity with pu14a,suggesting that it may also inhibit nAChRs.Thus,pu14a is a defining member of a novel family of framework 14 conotoxins in the A gene superfamily.更多还原

【Abstract】 As part of studies of the venom components present in Conus pulicarius and Conus tessulatus, two vermivorous Conus species collected from the South China Sea,we identified two new conotoxins,designated pu14a and ts14a,by cDNA cloning and peptide purification,respectively. The predicted mature toxin region of pu14a contains 19 amino acid（aa） residues with two disulfide bonds（1-3,2-4 connectivity） in a pattern of C(X₁₀)C（X₁）C（X₃）C（framework 14）. However,the 69-amino acid pu14a precursor surprisingly exhibits an identical signal sequence to corresponding regions of previously characterizedα-conotoxins,revealing a new level of conotoxin diversity and a plausible evolution mechanism of Conus prepropeptide recombination. The peptide pu14a has very unique primary structure and intercysteine spacing pattern with its first loop rich in proline residues.This peptide was chemically synthesized to further characterize its function.The synthetic pu14a induces a sleeping phenotype in mice,and it is also toxic to freshwater goldfish upon intramuscular injection.Using the Xenopus oocyte heterologous expression system,pu14a（1μM） was shown to inhibit both rat neuronalα3β2-containing and mouse neuromuscularα1β1γδsubtypes of nicotinic acetylcholine receptors（nAChRs）.It blocked the ACh-induced currents（51%anc 55%） in recombinantα3β2 andα6α3β2 nAChRs,respectively, and the toxin dissociated rapidly from the receptor.A block of approximately 82%of the ACh-evoked current was obtained inα1β1γδ,with a similar dissociation rate.The 20-aa polypeptide chain of ts14a with a 1-3/2-4 cysteine pairing shows high sequence similarity with pu14a,suggesting that it may also inhibit nAChRs.Thus,pu14a is a defining member of a novel family of framework 14 conotoxins in the A gene superfamily.更多还原