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1
المصدر: Journal of Proteomics. 164:73-84
مصطلحات موضوعية: 0301 basic medicine, Biophysics, Hyaluronoglucosaminidase, Mollusk Venoms, Venom, Biochemistry, Conus purpurascens, Cone snail, 03 medical and health sciences, Protein Domains, Hyaluronidase, Conus, medicine, Animals, Amino Acid Sequence, Asparagine, Conotoxin, chemistry.chemical_classification, 030102 biochemistry & molecular biology, biology, Conus Snail, biology.organism_classification, Molecular biology, 030104 developmental biology, Enzyme, chemistry, medicine.drug
الوصف: Hyaluronidases are ubiquitous enzymes commonly found in venom and their main function is to degrade hyaluran, which is the major glycosaminoglycan of the extracellular matrix in animal tissues. Here we describe the purification and characterization of a 60 kDa hyaluronidase found in the injected venom from Conus purpurascens , Conohyal-P1. Using a combined strategy based on transcriptomic and proteomic analysis, we determined the Conohyal-P1 sequence. Conohyal-P1 has conserved consensus catalytic and positioning domain residues characteristic of hyaluronidases and a C-terminus EGF-like domain. Additionally, the enzyme is expressed as a mixture of glycosylated isoforms at five asparagine sites. The activity of the native Conohyal-P1 was assess MS-based methods and confirmed by classical turbidimetric methods. The MS-based assay is particularly sensitive and provides the first detailed analysis of a venom hyaluronidase activity monitored with this method. The discovery of new hyaluronidases and the development of techniques to evaluate their performance can advance several therapeutic procedures, as these enzymes are widely used for enhanced drug delivery applications. Biological significance Cone snail venom is a remarkable source of therapeutically important molecules, as is the case of conotoxins, which have undergone extensive clinical trials for several applications. In addition to the conotoxins, a large array of proteins have been reported in the venom of several species of cone snails, including enzymes that were found in dissected and injected Conus venom. Here we describe the isolation and characterization of the hyaluronidase Conohyal-P1 from the injected venom of C. purpurascens . We employed a combined transcriptomic and proteomic analysis to obtain the full sequence of this hyaluronidase. The activity of Conohyal-P1 was assessed by a mass spectrometry-based method, which provide the first detailed venom hyaluronidase activity analysis monitored by mass spectrometry allowing the visualization of the substrate degradation by the enzyme.
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::6ca0ec0b4e086858ee029100bef58260Test
https://doi.org/10.1016/j.jprot.2017.05.002Test -
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المساهمون: Ministerio de Ciencia e Innovación (España)
المصدر: Repositório Científico de Acesso Aberto de Portugal
Repositório Científico de Acesso Aberto de Portugal (RCAAP)
instacron:RCAAP
Digital.CSIC. Repositorio Institucional del CSIC
instnameمصطلحات موضوعية: 0301 basic medicine, Species complex, Messinian salinity crisis, Conus, Snails, Zoology, Lautoconus, Messinian Salinity Crisis, Neogastropoda, Evolutionary history, 03 medical and health sciences, Radula, Marine gastropod, Phylogenetics, Genetics, Animals, Mixed models, Conidae, Endemism, Cape-Verde conus, Molecular Biology, Phylogeny, Ecology, Evolution, Behavior and Systematics, Caenogastropoda, Base Sequence, Phylogenetic tree, biology, Gastropod genus conus, Ecology, Plio-Pleistocene transition, Genetic Variation, Species diversity, biology.organism_classification, Senegal, Africa, Western, 030104 developmental biology, Mitochondrial genomes, Spain, Genome, Mitochondrial, Late miocene, Convergence, North-Atlantic, Rocky shores
الوصف: Cone snails attain in Senegal one of their highest peaks of species diversity throughout the continental coast of Western Africa. A total of 15 endemic species have been described, all placed in the genus Lautoconus. While there is ample data regarding the morphology of the shell and the radular tooth of these species, virtually nothing is known regarding the genetic diversity and phylogenetic relationships of one of the most endangered groups of cones. In this work, we determined the complete or near-complete (only lacking the control region) mitochondrial (mt) genomes of 17 specimens representing 11 endemic species (Lautoconus belairensis, Lautoconus bruguieresi, Lautoconus cacao, Lautoconus cloveri, Lautoconus cf. echinophilus, Lautoconus guinaicus, Lautoconus hybridus, Lautoconus senegalensis, Lautoconus mercator, Lautoconus taslei, and Lautoconus unifasciatus). We also sequenced the complete mt genome of Lautoconus guanche from the Canary Islands, which has been related to the cones endemic to Senegal. All mt genomes share the same gene arrangement, which conforms to the consensus reported for Conidae, Neogastropoda and Caenogastropoda. Phylogenetic analyses using probabilistic methods recovered three major lineages, whose divergence coincided in time with sea level and ocean current changes as well as temperature fluctuations during the Messinian salinity crisis and the Plio-Pleistocene transition. Furthermore, the three lineages corresponded to distinct types of radular tooth (robust, small, and elongated), suggesting that dietary specialization could be an additional evolutionary driver in the diversification of the cones endemic to Senegal. The reconstructed phylogeny showed several cases of phenotypic convergence (cryptic species) and questions the validity of some species (ecotypes or phenotypic plasticity), both results having important taxonomic and conservation consequences.
This work was supported by the Spanish Ministry of Science and Innovation, Spain (CGL2013-45211-C2-2-P to RZ; BES-2014-069575 to SA).الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::fe45dd29ad3afb0c533ec4c771c697c7Test
https://doi.org/10.1016/j.ympev.2017.04.020Test -
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المؤلفون: Julita S. Imperial, Baldomero M. Olivera, Taleen Dilanyan, Manuel B. Aguilar, Pradip K. Bandyopadhyay, Samuel S. Espino, Russell W. Teichert
المصدر: Toxicon. 113:11-17
مصطلحات موضوعية: 0301 basic medicine, Glycine, Mollusk Venoms, Venom, Peptide, Toxicology, Bioinformatics, Article, Mice, 03 medical and health sciences, 0302 clinical medicine, Species Specificity, Dorsal root ganglion, Ganglia, Spinal, medicine, Animals, Amino Acid Sequence, Conotoxin, chemistry.chemical_classification, biology, Biological activity, biology.organism_classification, Molecular biology, Conus virgo, 030104 developmental biology, medicine.anatomical_structure, chemistry, Mollusca, Conotoxins, Sequence Alignment, 030217 neurology & neurosurgery, Conus terebra
الوصف: Cone snails in the Virgiconus clade prey on marine worms. Here, we identify six related conotoxins in the O1-superfamily from three species in this clade, Conus virgo, Conus terebra and Conus kintoki. One of these peptides, vi6a, was directly purified from the venom of C. virgo by following its activity using calcium imaging of dissociated mouse dorsal root ganglion (DRG) neurons. The purified peptide was biochemically characterized, synthesized and tested for activity in mice. Hyperactivity was observed upon both intraperitoneal and intracranial injection of the peptide. The effect of the synthetic peptide on DRG neurons was identical to that of the native peptide. Using the vi6a sequence, five other homologs were identified. These peptides define a glycine-rich subgroup of the O1-superfamily from the Virgiconus clade, with biological activity in mice.
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::071d0988637e4b02d38e4b6e41b42fc6Test
https://doi.org/10.1016/j.toxicon.2016.02.001Test -
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المؤلفون: Ying Wu, Ling Jiang, Qiuyun Dai, Jiuping Ding, Zhuguo Liu, Tianpeng Du, Sheng Wang, Shuo Wang
المصدر: Biochemical and Biophysical Research Communications. 454:151-156
مصطلحات موضوعية: Models, Molecular, BK channel, Conus textile, Protein Conformation, Xenopus, Biophysics, Receptors, Nicotinic, complex mixtures, Biochemistry, Animals, Humans, Amino Acid Sequence, Conotoxin, Nuclear Magnetic Resonance, Biomolecular, Molecular Biology, Cells, Cultured, Neurons, Binding Sites, biology, Chemistry, Sodium channel, Calcium channel, HEK 293 cells, Conus Snail, Cell Biology, biology.organism_classification, Recombinant Proteins, Nicotinic acetylcholine receptor, HEK293 Cells, nervous system, Oocytes, biology.protein, Female, Pharmacophore, Conotoxins
الوصف: T-superfamily conotoxins have a typical cysteine pattern of "CC-CC", and are known to mainly target calcium or sodium ion channels. Recently, we screened the targets of a series of T-superfamily conotoxins and found that a new T-superfamily conotoxin TxVC (KPCCSIHDNSCCGL-NH2) from the venom of Conus textile. It selectively targeted the neuronal nicotinic acetylcholine receptor (nAChR) subtypes alpha 4 beta 2 and alpha 3 beta 2, with IC50 values of 343.4 and 1047.2 nM, respectively, but did not exhibit obvious pharmacological effects on voltage-gated potassium, sodium or calcium channel in DRG cells, the BK channels expressed in HEK293 cells, or the Kv channels in L beta T2 cells. The changes in the inhibitory activities of its Ala mutants, the NMR structure, and molecular simulation results based on other conotoxins targeting nAChR alpha 4 beta 2, all demonstrated that the residues Ile(6) and Leu(14) were the main hydrophobic pharmacophores. To our best knowledge, this is the first T-superfamily conotoxin that inhibits neuronal nAChRs and possesses high binding affinity to alpha 4 beta 2. This finding will expand the knowledge of the targets of T-superfamily conotoxins and the motif information could help the design of new nAChR inhibitors. (C) 2014 Elsevier Inc. All rights reserved.
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::547e06cc1ceaa0f155377d06f4bbef61Test
https://doi.org/10.1016/j.bbrc.2014.10.055Test -
5
المؤلفون: Aiping Lu, Shaoqiong Xu, Longjin Yang, Chunguang Wang
المصدر: Molecular & Cellular Proteomics. 13:105-118
مصطلحات موضوعية: Signal peptide, Proteome, Neurotoxins, Computational biology, Protein Sorting Signals, Conus flavidus, Tandem mass spectrometry, complex mixtures, Biochemistry, Mass Spectrometry, Analytical Chemistry, Complementary DNA, Conus, RNA Precursors, Animals, Cysteine, RNA, Messenger, Conotoxin, Molecular Biology, biology, Venoms, Research, Conus Snail, biology.organism_classification, Molecular biology, Conotoxins, Protein Processing, Post-Translational
الوصف: Conotoxins are peptide neurotoxins produced by predatory cone snails. They are mostly cysteine-rich short peptides with remarkable structural diversity. The conserved signal peptide sequences of their mRNA-encoded precursors have enabled the grouping of known conotoxins into a limited number of superfamilies. However, the conotoxins within each superfamily often present variable sequences, cysteine frameworks, and post-translational modifications. To understand better how conotoxins are diversified, we performed a venomic study with C. flavidus, an uninvestigated vermivorous Conus species, by combining transcriptomic and proteomic analyses. In order to obtain the full-length conotoxin sequences, protease digestion was not performed with the venom extraction prior to spectra acquisition via tandem mass spectrometry (MS/MS). Because conotoxins are produced from mRNA-encoded precursors by means of proteolytic cleavage, nonspecific digestion of precursors was applied during the database search. Special attention was also paid in interpreting the MS/MS spectra. All together, these analyses identified 69 nonredundant cDNA sequences and 31 conotoxin components with confident MS/MS spectra. A new Q-superfamily was also identified. More importantly, this study revealed that conotoxin-encoding transcripts are diversified by hypermutation, fragment insertion/deletion, and mutation-induced premature termination, and that a single mRNA species can produce multiple toxin products through alternative post-translational modifications and alternative cleavages of the translated precursor. These multiple diversification strategies at different levels may explain, at least in part, the diversity of conotoxins, and provide the basis for further investigation.
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::4238a2e95b39b96f1d6b62a7191b5d07Test
https://doi.org/10.1074/mcp.m113.028647Test -
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المؤلفون: Balasubramanian Thangavel, Giji Sadhasivam, Ramya Rajasekaran, Abirami Pachaiyappan, Arumugam Muthuvel
المصدر: Asian Pacific Journal of Tropical Disease, Vol 4, Iss Sup 1, Pp 102-110 (2014)
مصطلحات موضوعية: Microbiology (medical), lcsh:Arctic medicine. Tropical medicine, lcsh:RC955-962, Hemolytic assay, Hyaluronidase, lcsh:Medicine, Venom, Phospholipase, Conus, medicine, MTT assay, chemistry.chemical_classification, biology, Conus betulinus, Fibrinogenolytic, lcsh:R, Proteolytic, biology.organism_classification, Molecular biology, Infectious Diseases, Enzyme, chemistry, Biochemistry, Chemical fingerprinting, medicine.drug
الوصف: Objective To study the biochemical and biomedical properties of predatory gastropod Conus betulinus venom. Methods Molecular weight of the crude venom extract was determined by using SDS-PAGE. Toxicity studies were carried out using haemolytic and brine shrimp lethality assays. Fibrin plate assay and substrate SDS-PAGE were used to determine the effect of sample on fibrin(ogen)olytic and proteolytic activity. The FTIR characterisation and chemical fingerprinting of amino acid were done with HPTLC. Enzymatic activities like phospholipase and hyaluronidase were measured spectrophotometrically and calculated in units. Anticancer screening was carried out by MTT assay. Results Studies on this deadly venom revealed six different molecular weight proteins of medical significance ranging between 20.0 kDa and 97.4 kDa. The protein content was estimated as 0.9 mg/mL. Haemolytic activity in erythrocytes was recorded and LC50 (Artemia) at 31.5 μg/mL. Further the venom showed considerable enzymatic properties like gelatinolytic, caesinolytic, fibrinolytic and fibrinogenolytic activities. The hyaluronidase and phospholipase activities were recorded at meagre range. The venom exhibited significant activity against HeLa cell lines. Moreover the evolution of venom is the crucial nature of Conus peptides in their challenging ecosystem. Periodical study on these peptides will unveil more peptides of biomedical use. Conclusions Although quite a lot of works have dealt with paralytic effects of Conus venom, it still remains as an unexplored cocktail with promising molecules for drug development.
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::f6881805cbb2900dbdbcf6add45e3fc0Test
https://doi.org/10.1016/s2222-1808Test(14)60423-0 -
7
المصدر: Journal of Biological Chemistry. 290:18156-18172
مصطلحات موضوعية: Conantokins, Protein subunit, Molecular Sequence Data, Allosteric regulation, Mollusk Venoms, Peptide, Molecular Dynamics Simulation, Receptors, N-Methyl-D-Aspartate, Biochemistry, Protein Structure, Secondary, Divalent, Mice, Neurobiology, mental disorders, Animals, Magnesium, Amino Acid Sequence, Molecular Biology, Cells, Cultured, Ion channel, Neurons, chemistry.chemical_classification, Chemistry, musculoskeletal, neural, and ocular physiology, Conus Snail, Cell Biology, Amino acid, Hydroxyproline, nervous system, Biophysics, NMDA receptor, biological phenomena, cell phenomena, and immunity, Conotoxins, Peptides, psychological phenomena and processes
الوصف: Conantokins are ~20-amino acid peptides present in predatory marine snail venoms that function as allosteric antagonists of ion channels of the N-methyl-d-aspartate receptor (NMDAR). These peptides possess a high percentage of post-/co-translationally modified amino acids, particularly γ-carboxyglutamate (Gla). Appropriately spaced Gla residues allow binding of functional divalent cations, which induces end-to-end α-helices in many conantokins. A smaller number of these peptides additionally contain 4-hydroxyproline (Hyp). Hyp should prevent adoption of the metal ion-induced full α-helix, with unknown functional consequences. To address this disparity, as well as the role of Hyp in conantokins, we have solved the high resolution three-dimensional solution structure of a Gla/Hyp-containing 18-residue conantokin, conRl-B, by high field NMR spectroscopy. We show that Hyp(10) disrupts only a small region of the α-helix of the Mn(2+)·peptide complex, which displays cation-induced α-helices on each terminus of the peptide. The function of conRl-B was examined by measuring its inhibition of NMDA/Gly-mediated current through NMDAR ion channels in mouse cortical neurons. The conRl-B displays high inhibitory selectivity for subclasses of NMDARs that contain the functionally important GluN2B subunit. Replacement of Hyp(10) with N(8)Q results in a Mg(2+)-complexed end-to-end α-helix, accompanied by attenuation of NMDAR inhibitory activity. However, replacement of Hyp(10) with Pro(10) allowed the resulting peptide to retain its inhibitory property but diminished its GluN2B specificity. Thus, these modified amino acids, in specific peptide backbones, play critical roles in their subunit-selective inhibition of NMDAR ion channels, a finding that can be employed to design NMDAR antagonists that function at ion channels of distinct NMDAR subclasses.
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::482437aceccd43eef47fd12dcd020168Test
https://doi.org/10.1074/jbc.m115.650341Test -
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المؤلفون: Sébastien Dutertre, Quentin Kaas, Alun Jones, Paul F. Alewood, Ai-Hua Jin, Richard J. Lewis
المساهمون: Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM), Institut for Molecular Bioscience, University of Queensland [Brisbane]
المصدر: Molecular and Cellular Proteomics
Molecular and Cellular Proteomics, American Society for Biochemistry and Molecular Biology, 2013, 12 (2), pp.312-329. ⟨10.1074/mcp.M112.021469⟩مصطلحات موضوعية: Proteomics, Spectrometry, Mass, Electrospray Ionization, DNA, Complementary, Sequence analysis, Molecular Sequence Data, Venom, Computational biology, Bioinformatics, Biochemistry, Analytical Chemistry, Cone snail, 03 medical and health sciences, Tandem Mass Spectrometry, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Animals, Amino Acid Sequence, Conotoxin, Protein Precursors, Molecular Biology, Peptide sequence, Conus marmoreus, Chromatography, High Pressure Liquid, 030304 developmental biology, 0303 health sciences, biology, Research, 030302 biochemistry & molecular biology, Conus Snail, Sequence Analysis, DNA, biology.organism_classification, Molecular Weight, Gene Expression Regulation, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Conotoxins, Peptides, Transcriptome, Protein Processing, Post-Translational, Sequence Alignment
الوصف: International audience; Cone snails produce highly complex venom comprising mostly small biologically active peptides known as conotoxins or conopeptides. Early estimates that suggested 50-200 venom peptides are produced per species have been recently increased at least 10-fold using advanced mass spectrometry. To uncover the mechanism(s) responsible for generating this impressive diversity, we used an integrated approach combining second-generation transcriptome sequencing with high sensitivity proteomics. From the venom gland transcriptome of Conus marmoreus, a total of 105 conopeptide precursor sequences from 13 gene superfamilies were identified. Over 60% of these precursors belonged to the three gene superfamilies O1, T, and M, consistent with their high levels of expression, which suggests these conotoxins play an important role in prey capture and/or defense. Seven gene superfamilies not previously identified in C. marmoreus, including five novel superfamilies, were also discovered. To confirm the expression of toxins identified at the transcript level, the injected venom of C. marmoreus was comprehensively analyzed by mass spectrometry, revealing 2710 and 3172 peptides using MALDI and ESI-MS, respectively, and 6254 peptides using an ESI-MS TripleTOF 5600 instrument. All conopeptides derived from transcriptomic sequences could be matched to masses obtained on the TripleTOF within 100 ppm accuracy, with 66 (63%) providing MS/MS coverage that unambiguously confirmed these matches. Comprehensive integration of transcriptomic and proteomic data revealed for the first time that the vast majority of the conopeptide diversity arises from a more limited set of genes through a process of variable peptide processing, which generates conopeptides with alternative cleavage sites, heterogeneous post-translational modifications, and highly variable N- and C-terminal truncations. Variable peptide processing is expected to contribute to the evolution of venoms, and explains how a limited set of ∼ 100 gene transcripts can generate thousands of conopeptides in a single species of cone snail.
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::6eb0a139dd5eb520ba2a634e1bf0f2e8Test
https://doi.org/10.1074/mcp.m112.021469Test -
9
المؤلفون: J. Michael McIntosh, Sulan Luo, Xiaopeng Zhu, Yong Wu, Dongting Zhangsun, David J. Craik, Yuanyan Hu, Muharrem Akcan, Sean Christensen, Melissa McIntyre
المصدر: Journal of Biological Chemistry. 288:894-902
مصطلحات موضوعية: Conus textile, genetic structures, Protein subunit, Molecular Conformation, Receptors, Nicotinic, Biology, complex mixtures, Polymerase Chain Reaction, Biochemistry, parasitic diseases, Conus, Animals, Neurotoxin, Conotoxin, Cloning, Molecular, Nuclear Magnetic Resonance, Biomolecular, Molecular Biology, Chromatography, High Pressure Liquid, DNA Primers, Acetylcholine receptor, Base Sequence, Peptide chemical synthesis, Conus Snail, Cell Biology, biology.organism_classification, Nicotinic agonist, nervous system, Protein Structure and Folding, sense organs, Conotoxins, Oxidation-Reduction
الوصف: α6β2 Nicotinic acetylcholine receptors (nAChRs) expressed by dopaminergic neurons in the CNS are potential therapeutic targets for the treatment of several neuropsychiatric diseases, including nicotine addiction and Parkinson disease. However, recent studies indicate that the α6 subunit can also associate with the β4 subunit to form α6β4 nAChRs that are difficult to pharmacologically distinguish from α6β2, α3β4, and α3β2 subtypes. The current study characterized a novel 16-amino acid α-conotoxin (α-CTx) TxIB from Conus textile whose sequence is GCCSDPPCRNKHPDLC-amide as deduced from gene cloning. The peptide and an analog with an additional C-terminal glycine were chemically synthesized and tested on rat nAChRs heterologously expressed in Xenopus laevis oocytes. α-CTx TxIB blocked α6/α3β2β3 nAChR with an IC(50) of 28 nm. In contrast, the peptide showed little or no block of other tested subtypes at concentrations up to 10 μm. The three-dimensional solution structure of α-CTx TxIB was determined using NMR spectroscopy. α-CTx TxIB represents a uniquely selective ligand for probing the structure and function of α6β2 nAChRs.
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::8c7b9124019ad6166102db5f8a4ecaeeTest
https://doi.org/10.1074/jbc.m112.427898Test -
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المؤلفون: Sulan Luo, Yuanyan Hu, Yong Wu, Quentin Kaas, J. Michael McIntosh, David J. Craik, Dongting Zhangsun, Xiaopeng Zhu
المصدر: Journal of Biological Chemistry. 290:9855-9862
مصطلحات موضوعية: Models, Molecular, Patch-Clamp Techniques, Protein subunit, Molecular Sequence Data, Plasma protein binding, Receptors, Nicotinic, Biology, Binding, Competitive, Biochemistry, Membrane Potentials, Xenopus laevis, Neurobiology, mental disorders, Animals, Amino Acid Sequence, Conotoxin, Amino Acids, Binding site, Receptor, Molecular Biology, Acetylcholine receptor, Binding Sites, Dose-Response Relationship, Drug, Sequence Homology, Amino Acid, fungi, Conus Snail, food and beverages, Cell Biology, Protein Structure, Tertiary, Rats, Protein Subunits, Nicotinic acetylcholine receptor, Nicotinic agonist, nervous system, Mutation, Oocytes, Female, sense organs, Conotoxins, Peptides, Protein Binding
الوصف: α-Conotoxin LvIA (α-CTx LvIA) is a small peptide from the venom of the carnivorous marine gastropod Conus lividus and is the most selective inhibitor of α3β2 nicotinic acetylcholine receptors (nAChRs) known to date. It can distinguish the α3β2 nAChR subtype from the α6β2* (* indicates the other subunit) and α3β4 nAChR subtypes. In this study, we performed mutational studies to assess the influence of residues of the β2 subunit versus those of the β4 subunit on the binding of α-CTx LvIA. Although two β2 mutations, α3β2[F119Q] and α3β2[T59K], strongly enhanced the affinity of LvIA, the β2 mutation α3β2[V111I] substantially reduced the binding of LvIA. Increased activity of LvIA was also observed when the β2-T59L mutant was combined with the α3 subunit. There were no significant difference in inhibition of α3β2[T59I], α3β2[Q34A], and α3β2[K79A] nAChRs when compared with wild-type α3β2 nAChR. α-CTx LvIA displayed slower off-rate kinetics at α3β2[F119Q] and α3β2[T59K] than at the wild-type receptor, with the latter mutant having the most pronounced effect. Taken together, these data provide evidence that the β2 subunit contributes to α-CTx LvIA binding and selectivity. The results demonstrate that Val(111) is critical and facilitates LvIA binding; this position has not previously been identified as important to binding of other 4/7 framework α-conotoxins. Thr(59) and Phe(119) of the β2 subunit appear to interfere with LvIA binding, and their replacement by the corresponding residues of the β4 subunit leads to increased affinity.
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::ddb77c1eea3d7231203107c1b515b49fTest
https://doi.org/10.1074/jbc.m114.632646Test
