RESUMEN
Linaclotide is a 14-amino acid residue peptide approved by the FDA for the treatment of irritable bowel syndrome with constipation (IBS-C), which activates guanylate cyclase C to accelerate intestinal transit. Here we show a new method for the synthesis of linaclotide through the completely selective formation of three disulfide bonds in satisfactory overall yields via mild oxidation reactions of the solid phase and liquid phase, using 4-methoxytrityl (Mmt), diphenylmethyl (Dpm) and 2-nitrobenzyl (O-NBn) protecting groups of cysteine as substrate, respectively.
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Estreñimiento , Síndrome del Colon Irritable , Humanos , Cisteína , Síndrome del Colon Irritable/tratamiento farmacológico , Péptidos , Resultado del TratamientoRESUMEN
BACKGROUND & OBJECTIVE: Contryphan-Bt is a D-tryptophan-containing disulfide-constrained decapeptide recently isolated from the venom of Conus betulinus. The molecular targets of contryphans are controversial, and the identification of its interacting proteins may be of great importance. METHODS: His-tag pull-down assays were performed to investigate intracellular binding proteins of contryphan-Bt from rat brain lysate. Bt-Acp-[His]6, a contryphan-Bt derivative containing hexahistidine tag, was synthesized and used as the bait. As a control, Acp-[His]6 was used to exclude nonspecific bindings. RESULTS: Glutamine synthetase was identified as a potential contryphan-Bt binding protein by pull-- down assays and subsequent LC-MS/MS. The binding of contryphan-Bt to glutamine synthetase was confirmed and determined using microscale thermophoresis, with a Kd of 74.02 ± 2.8 µM. The binding did not affect glutamine synthetase activity, suggesting that the interaction site was distinct from the catalytic center. CONCLUSION: Glutamine synthetase was identified as a novel contryphan-Bt binding protein. This is the first report in which the conopeptide binds to an intracellular protein.
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Glutamato-Amoníaco Ligasa , Venenos de Moluscos , Secuencia de Aminoácidos , Animales , Proteínas Portadoras , Cromatografía Liquida , Glutamina , Venenos de Moluscos/química , Péptidos Cíclicos , Ratas , Espectrometría de Masas en TándemRESUMEN
Although there are various Conus species with publicly available transcriptome and proteome data, no genome assembly has been reported yet. Here, using Chinese tubular cone snail (C. betulinus) as a representative, we sequenced and assembled the first Conus genome with original identification of 133 genome-widely distributed conopeptide genes. After integration of our genomics, transcriptomics, and peptidomics data in the same species, we established a primary genetic central dogma of diverse conopeptides, assuming a rough number ratio of ~1:1:1:10s for the total genes: transcripts: proteins: post-translationally modified peptides. This ratio may be special for this worm-hunting Conus species, due to the high diversity of various Conus genomes and the big number ranges of conopeptide genes, transcripts, and peptides in previous reports of diverse Conus species. Only a fraction (45.9%) of the identified conotopeptide genes from our achieved genome assembly are transcribed with transcriptomic evidence, and few genes individually correspond to multiple transcripts possibly due to intraspecies or mutation-based variances. Variable peptide processing at the proteomic level, generating a big diversity of venom conopeptides with alternative cleavage sites, post-translational modifications, and N-/C-terminal truncations, may explain how the 133 genes and ~123 transcripts can generate thousands of conopeptides in the venom of individual C. betulinus. We also predicted many conopeptides with high stereostructural similarities to the putative analgesic ω-MVIIA, addiction therapy AuIB and insecticide ImI, suggesting that our current genome assembly for C. betulinus is a valuable genetic resource for high-throughput prediction and development of potential pharmaceuticals.
RESUMEN
Jellyfish grow rapidly and have a strong regenerative ability, indicating that they may express high levels of growth factors. Therefore, the aim of this research was to isolate the growth-promoting components from the jellyfish Cyanea capillata (C. capillata) and to further explore the underlying mechanisms. In this study, we first isolated and identified a novel polypeptide from C. capillata tentacles using size-exclusion chromatography followed by reverse-phase HPLC. This peptide, consisting of 58 amino acids (MW 5782.9â¯Da), belonged to the granulin (GRN) family of growth factors; thus, we named it Cyanea capillata granulin-1 (CcGRN-1). Second, using CCK-8 assay and flow cytometry, we verified that CcGRN-1 at the 0.5⯵g/ml concentration could promote cell proliferation and increase the expression of cell-cycle proteins (CyclinB1 and CyclinD1). Third, signaling pathways studies showed that CcGRN-1 could activate the PI3K/Akt- and ERK1/2 MAPK-signaling pathways but not the JNK MAPK- or NF-κB-signaling pathways. Subsequently, we further confirmed that the CcGRN-1-induced cell proliferation and migration were associated only with the ERK1/2 MAPK-signaling pathway. Considering all of these factors, CcGRN-1, as the first jellyfish-derived GRN homologue, possesses growth-promoting properties and may be a candidate for novel therapeutics to promote human wound healing in unfavorable conditions.
Asunto(s)
Proteínas de Peces/aislamiento & purificación , Proteínas de Peces/farmacología , Granulinas/aislamiento & purificación , Granulinas/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Escifozoos , Secuencia de Aminoácidos , Animales , Ciclo Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Proteínas de Peces/química , Granulinas/química , Humanos , Péptidos/química , Péptidos/aislamiento & purificación , Péptidos/farmacología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Cicatrización de Heridas/efectos de los fármacosRESUMEN
The venom of each Conus species consists of a diverse array of neurophysiologically active peptides, which are mostly unique to the examined species. In this study, we performed high-throughput transcriptome sequencing to extract and analyze putative conotoxin transcripts from the venom ducts of 3 vermivorous cone snails (C. caracteristicus, C. generalis, and C. quercinus), which are resident in offshore waters of the South China Sea. In total, 118, 61, and 48 putative conotoxins (across 22 superfamilies) were identified from the 3 Conus species, respectively; most of them are novel, and some possess new cysteine patterns. Interestingly, a series of 45 unassigned conotoxins presented with a new framework of C-C-C-C-C-C, and their mature regions were sufficiently distinct from any other known conotoxins, most likely representing a new superfamily. O- and M-superfamily conotoxins were the most abundant in transcript number and transcription level, suggesting their critical roles in the venom functions of these vermivorous cone snails. In addition, we identified numerous functional proteins with potential involvement in the biosynthesis, modification, and delivery process of conotoxins, which may shed light on the fundamental mechanisms for the generation of these important conotoxins within the venom duct of cone snails.
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Conotoxinas/genética , Caracol Conus/genética , Animales , China , Conotoxinas/metabolismo , Caracol Conus/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Filogenia , Análisis de Secuencia de ARN , TranscriptomaRESUMEN
A new member of the contryphans family was isolated from the venom of Conus betilinus, a vermivorous species distributed in the South China Sea. Its sequence, ZSGCO(D-W)KPWC-NH2 (Z, pyroglutamic acid), was established by a combination of de novo MS/MS sequencing and venom-duct transcriptome sequencing. The occurrence of D-Trp6 was confirmed by chemical synthesis and HPLC behavior comparison. Like known contryphans, contryphan-Bt produces the "stiff-tail" syndrome in mice and contains one disulfide bond, a hydroxyproline, a D-tryptophan, and an amidated C-terminus. However, contryphan-Bt differs from previously identified contryphans by a pyroglutamic acid at the N terminus. CD spectrum reveals that contryphan-Bt possess ß-turn in solution.
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Caracol Conus/química , Péptidos Cíclicos/química , Péptidos Cíclicos/aislamiento & purificación , Animales , Ratones , Venenos de Moluscos/química , Péptidos Cíclicos/toxicidad , Estructura Secundaria de Proteína , Ácido Pirrolidona Carboxílico/química , TranscriptomaRESUMEN
µ-Conotoxin GIIIA, a peptide toxin isolated from Conus geographus, preferentially blocks the skeletal muscle sodium channel NaV1.4. GIIIA folds compactly to a pyramidal structure stabilized by three disulfide bonds. To assess the contributions of individual disulfide bonds of GIIIA to the blockade of NaV1.4, seven disulfide-deficient analogues were prepared and characterized, each with one, two, or three pairs of disulfide-bonded Cys residues replaced with Ala. The inhibitory potency of the analogues against NaV1.4 was assayed by whole cell patch-clamp on rNaV1.4, heterologously expressed in HEK293 cells. The corresponding IC50 values were 0.069 ± 0.005 µM for GIIIA, 2.1 ± 0.3 µM for GIIIA-1, 3.3 ± 0.2 µM for GIIIA-2, and 15.8 ± 0.8 µM for GIIIA-3 (-1, -2 and -3 represent the removal of disulfide bridges Cys3-Cys15, Cys4-Cys20 and Cys10-Cys21, respectively). Other analogues were not active enough for IC50 measurement. Our results indicate that all three disulfide bonds of GIIIA are required to produce effective inhibition of NaV1.4, and the removal of any one significantly lowers its sodium channel binding affinity. Cys10-Cys21 is the most important for the NaV1.4 potency.
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Conotoxinas/farmacología , Caracol Conus/metabolismo , Disulfuros/metabolismo , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/metabolismo , Animales , Línea Celular , Células HEK293 , Humanos , Músculo Esquelético/efectos de los fármacos , Unión ProteicaRESUMEN
BACKGROUND: The venom of predatory marine cone snails mainly contains a diverse array of unique bioactive peptides commonly referred to as conopeptides or conotoxins. These peptides have proven to be valuable pharmacological probes and potential drugs because of their high specificity and affinity to important ion channels, receptors and transporters of the nervous system. Most previous studies have focused specifically on the conopeptides from piscivorous and molluscivorous cone snails, but little attention has been devoted to the dominant vermivorous species. RESULTS: The vermivorous Chinese tubular cone snail, Conus betulinus, is the dominant Conus species inhabiting the South China Sea. The transcriptomes of venom ducts and venom bulbs from a variety of specimens of this species were sequenced using both next-generation sequencing and traditional Sanger sequencing technologies, resulting in the identification of a total of 215 distinct conopeptides. Among these, 183 were novel conopeptides, including nine new superfamilies. It appeared that most of the identified conopeptides were synthesized in the venom duct, while a handful of conopeptides were identified only in the venom bulb and at very low levels. CONCLUSIONS: We identified 215 unique putative conopeptide transcripts from the combination of five transcriptomes and one EST sequencing dataset. Variation in conopeptides from different specimens of C. betulinus was observed, which suggested the presence of intraspecific variability in toxin production at the genetic level. These novel conopeptides provide a potentially fertile resource for the development of new pharmaceuticals, and a pathway for the discovery of new conotoxins.
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Conotoxinas/genética , Caracol Conus/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Transcriptoma , Secuencia de Aminoácidos , Animales , China , Caracol Conus/clasificación , Perfilación de la Expresión Génica/métodos , Variación Genética , Datos de Secuencia Molecular , Océanos y Mares , Péptidos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad de la EspecieRESUMEN
Dengue virus (DENV) infection is a serious public health threat worldwide that demands effective treatment. In the search for potent virus protease inhibitors, several cone snail venoms were screened against serotype 2 DENV NS2B-NS3 protease, and one conotoxin, MrIA, was identified to have inhibitory activity. The inhibitory activity was attributed to a disulfide bond-mediated loop, from which rational optimization was made to improve the potency and stability. An eight-residue cyclic peptide inhibitor was finally obtained with high potency (inhibitory constant 2.2 µM), stability, and cell permeability. This inhibitor can thus serve as a good lead for DENV drug development. In addition, this work highlights the critical effect of peptide cyclization on the potency of oligopeptide inhibitors against DENV protease, which may advance the design of peptide inhibitors for homologous virus proteases.
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Antivirales/química , Conotoxinas/química , Virus del Dengue/enzimología , Oligopéptidos/química , Péptidos Cíclicos/química , Serina Endopeptidasas , Inhibidores de Serina Proteinasa/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Antivirales/síntesis química , Antivirales/farmacología , Línea Celular , Permeabilidad de la Membrana Celular , Chlorocebus aethiops , Conotoxinas/farmacología , Cricetinae , Ciclización , Modelos Moleculares , Oligopéptidos/síntesis química , Oligopéptidos/farmacología , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/farmacología , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/síntesis química , Inhibidores de Serina Proteinasa/farmacología , Relación Estructura-Actividad , Proteínas no Estructurales Virales/metabolismoRESUMEN
O-superfamily conotoxins include several families that have diverse pharmacological activity on Na+, K+ or Ca2+ channels. These superfamily toxins have been mainly found in fish-hunting and mollusk-hunting Conus species. Here, we reported two novel O-superfamily conotoxins, vx6a and vx6b, purified from a worm-hunting cone snail, Conus vexillum. Though their cysteine framework and signal peptides share high similarity with those of other members of O-superfamily, the mature vx6a and vx6b both have a low sequence homology with others. To test the biological function of vx6a, the toxin was chemically synthesized and then tested on the locust dorsal unpaired median (DUM) neuron system which containing various ion channels. Although no any activity on ion channels was found on the DUM neuron system, vx6a could clearly elicit a series of symptoms in mouse via intracranial injection, such as quivering, climbing, scratching, barrel rolling and paralysis of limbs at different dose.
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Conotoxinas/toxicidad , Canales Iónicos/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación de Organismos , Conotoxinas/síntesis química , Conotoxinas/aislamiento & purificación , Caracol Conus , Relación Dosis-Respuesta a Droga , Ratones , Datos de Secuencia Molecular , FilogeniaRESUMEN
Cone snails are tropical marine mollusks that envenomate prey with a complex mixture of neuropharmacologically active compounds for the purpose of feeding and defence, each evolved to act in a highly specific manner on different parts of the nervous system. Here, we report the peptide purification, molecular cloning, chemical synthesis, and functional characterization of a structurally unique toxin isolated from the venom of Conus vexillum. The novel peptide, designated Vx2, was composed of 21 amino acid residues cross-linked by 3 disulfide bonds (WIDPSHYCCCGGGCTDDCVNC). Intriguingly, its mature peptide sequence shows low level of similarity with other identified conotoxins, and its unique motif (-CCCGGGC-) was not reported in other Conus peptides. However, its signal peptide sequence shares high similarity with those of the M-superfamily conotoxins. Hence, Vx2 could be classified into a new family of the M-superfamily.
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Conotoxinas/química , Conotoxinas/clasificación , Caracol Conus/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Conotoxinas/genética , Conotoxinas/aislamiento & purificación , Caracol Conus/genética , ADN Complementario/genética , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
AIM: To study the antitumor peptide components in the stems and leaves of mistletoe (Viscum coloratum (Kom.) Nakai), the primary structure of the novel peptide was elucidated. METHODS: Cation exchange, gel filtration and HPLC were employed for isolation and purification. Matrix Assisted Laser Desorption Ionization-Time of Flight-Mass Spectrometry was used to determine the mass. The complete amino acid sequence of the novel peptide was obtained by Edman degradation combined with enzyme digestion. The antitumor activity of the peptide in vitro was studied with MTT method. RESULTS: The primary stucture of the peptide named as viscotoxin B2 is KSCCKNTTGRNIYNTCRFAGGSRERCAKLSGCKIISASTCPSDYPK. The IC50 value of viscotoxin B2 on the Rat Osteoblast-like Sarcoma 17/2.8 cells in vitro is 1.6 mg x L(-1). CONCLUSION: Viscotoxin B2 in V. coloratum, which has high similarity with viscotoxins from V. album, showed antitumor activity.
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Antineoplásicos Fitogénicos/aislamiento & purificación , Péptidos/química , Proteínas de Plantas/química , Proteínas de Plantas/farmacología , Plantas Medicinales/química , Viscum/química , Secuencia de Aminoácidos , Animales , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Neoplasias Óseas/patología , Concentración 50 Inhibidora , Datos de Secuencia Molecular , Peso Molecular , Osteosarcoma/patología , Péptidos/aislamiento & purificación , Péptidos/farmacología , Hojas de la Planta/química , Proteínas de Plantas/aislamiento & purificación , Tallos de la Planta/química , Ratas , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
A novel conotoxin, kappa-conotoxin (kappa-BtX), has been purified and characterized from the venom of a worm-hunting cone snail, Conus betulinus. The toxin, with four disulfide bonds, shares no sequence homology with any other conotoxins. Based on a partial amino acid sequence, its cDNA was cloned and sequenced. The deduced sequence consists of a 26-residue putative signal peptide, a 31-residue mature toxin, and a 13-residue extra peptide at the C terminus. The extra peptide is cleaved off by proteinase post-processing. All three Glu residues are gamma-carboxylated, one of the two Pro residues is hydroxylated at position 27, and its C-terminal residue is Pro-amidated. The monoisotopic mass of the toxin is 3569.0 Da. Electrophysiological experiments show that: 1) among voltage-gated channels, kappa-BtX is a specific modulator of K(+) channels; 2) among the K channels, kappa-BtX specifically up-modulates the Ca(2+)- and voltage-sensitive BK channels (252 +/- 47%); 3) its EC(50) is 0.7 nm with a single binding site (Hill = 0.88); 4) the time constant of wash-out is 8.3 s; and 5) kappa-BtX has no effect on single channel conductance, but increases the open probability of BK channels. It is concluded that kappa-BtX is a novel specific biotoxin against BK channels.