RESUMEN
Agrocybe cylindracea dietary fiber (ADF) contains 95% water-insoluble dietary fiber, resulting in poor application performance. To address this issue, ADF was modified by four methods (cellulase, sodium hydroxide, high-temperature, and Lactobacillus fermentation) in this paper. By comparing the physicochemical properties, microstructures, monosaccharide compositions, and functional characteristics (antioxidant and α-glucosidase inhibitory activities in vitro) of all modified ADF samples, the optimal modification method was selected. Results showed that sodium hydroxide treatment was deemed the most effective modification method for ADF, as alkali-treated ADF (ADF-A) revealed a higher oil-holding capacity (2.02 g/g), swelling capacity (8.38 mL/g), cholesterol adsorption (6.79 mg/g), and α-glucosidase inhibitory activity (more than 70% at 0.4-0.6 mg/mL) than the other modified samples. The looser microstructure in ADF-A might be attributed to molecular rearrangement and spatial structure disruption, which resulted in smaller molecular sizes and decreased viscosity, hence improving ADF's physicochemical and functional qualities. All these findings indicate the greater application potential of modified ADF products in food and weight-loss industries, providing a comprehensive reference for the industrial application of ADF.
Asunto(s)
Agrocybe , Celulasa , Fibras de la Dieta , Fermentación , Lactobacillus , Hidróxido de Sodio , Fibras de la Dieta/análisis , Lactobacillus/enzimología , Hidróxido de Sodio/química , Hidróxido de Sodio/farmacología , Celulasa/metabolismo , Celulasa/química , Agrocybe/química , Calor , Antioxidantes/farmacología , Antioxidantes/química , Inhibidores de Glicósido Hidrolasas/farmacología , Inhibidores de Glicósido Hidrolasas/químicaRESUMEN
Agrocybe aegerita, one of the edible mushroom varieties, is popular among consumers for its umami taste. Umami peptides, including EV, EG, EY, ENG, ECG, DEL, DDL, PEG, PEEL, DGPL, and EDCS are the main umami compounds in A. aegerita. In this study, when the concentration of these 11 umami peptides was 5 mg/mL, the corresponding relative umami intensity (measured by MSG concentration) ranged from 4.457 to 5.240 mg/mL, with DDL being the highest. All umami peptides exhibited better umami taste under neutral and weakly acidic conditions (pH 6-7). EY and ENG, with a higher umami intensity at 70 °C, were more suitable for a wide application in thermally processed foods. Additionally, the relationship between the structure and strength of umami peptides was explored using a three-dimensional quantitative structure-activity relationship model with an R2 of 0.987. Overall, umami peptides in A. aegerita possess strong potential for application in food processing.
Asunto(s)
Agrocybe , Péptidos , Relación Estructura-Actividad Cuantitativa , Gusto , Péptidos/química , Agrocybe/química , Humanos , Aromatizantes/químicaRESUMEN
This study explored the physicochemical properties and structural characteristics of Agrocybe cylindracea polysaccharides at four developmental stages, as well as their dynamic evolution during maturation. Results showed that the polysaccharides from A. cylindracea water extract exhibited similar structural characteristics across all four maturity stages, despite a significant reduction in yields. Four water-soluble heteroglycans, including one high molecular weight (ACPM-Et50-I) and three low molecular weight (ACPM-Et50-II, ACPM-Et60, ACPM-Et80), were isolated from A. cylindracea at each maturity stage. ACPM-Et50-I was identified as branched heterogalactans, while ACPM-Et60 and ACPM-Et80 were branched heteroglucans. However, ACPM-Et50-II was characterized as a branched glucuronofucogalactoglucan at the tide-turning stage but a glucuronofucoglucogalactan at the pileus expansion stage due to the increase of its α-(1 â 6)-D-Galp. In general, although the structural skeletons of most A. cylindracea heteroglycans were similar during maturation as shown by their highly consistent glycosyl linkages, there were still differences in the distribution of some heteroglucans. This work has for the first time reported a glucuronofucogalactoglucan in A. cylindracea and its dynamic evolution during maturation, which may facilitate the potential application of A. cylindracea in food and biomedicine industries.
Asunto(s)
Agrocybe , Agua , Agua/química , Agrocybe/química , Glucanos/química , Polisacáridos/química , Peso MolecularRESUMEN
Polysaccharides derived from Agrocybe cylindracea have been demonstrated to exhibit various bioactivities. However, studies on their structural characteristics during the growth process are limited. This study aimed to compare the physicochemical properties and structural characteristics of alkali-extracted polysaccharides from A. cylindracea fruiting bodies (JACP) across four growth stages. Results showed that the extraction yields and protein levels of JACP declined along with the growth of A. cylindracea, while the contents of neutral sugar and glucose increased significantly. However, JACP exhibited structural characteristics similar to those across the four stages. Four polysaccharide subfractions were isolated from each growth stage, including JACP-Et30, JACP-Et50, JACP-Et60, and JACP-Et70. JACP-Et30 from the four stages and JACP-Et50 from the initial three stages were identified as heteroglucans with ß-1,3-d-Glcp and ß-1,6-d-Glcp residues as main chains, respectively. However, other subfractions were considered as ß-1,6-d-glucans containing minor glucuronic acid. These subfractions were predominantly replaced by Glcp residues at the O-3 and O-6 positions. Overall, while JACP exhibited variable physicochemical properties, its structural characteristics remained stable during the growth process, offering new insights into its potential applications in the food and medicinal industries.
Asunto(s)
Agrocybe , Cuerpos Fructíferos de los Hongos , Polisacáridos , Agrocybe/química , Agrocybe/crecimiento & desarrollo , Cuerpos Fructíferos de los Hongos/química , Cuerpos Fructíferos de los Hongos/crecimiento & desarrollo , Polisacáridos/química , Polisacáridos/aislamiento & purificación , Álcalis/químicaRESUMEN
Polysaccharides are the important bioactive macromolecules in Agrocybe cylindracea, but their changes are as yet elusive during developmental process. This study investigated the dynamic changes of polysaccharides from A. cylindracea fruiting body water extract at four developmental stages and its structure characteristics. Results revealed that the polysaccharides from A. cylindracea water extract significantly increased at the pileus expansion stage and the increased fraction could be α-glucan. The further purification and identification indicated that this α-glucan was a glycogen. It had typical morphology of ß particles with a molecular weight of 1375 kDa. Its backbone comprised α-D-(1 â 4)-Glcp and α-D-(1 â 4,6)-Glcp residues at a ratio of 5:1, terminated by α-D-Glcp residue. Rheological behavior suggested that it was a Newtonian fluid at the concentration of 1 %. In addition, despite both the glycogen and natural starch were composed of D-glucose, they exhibited the entirely distinct Maltese cross characteristic and unique crystalline structure. This study is the first to demonstrate the presence of abundant glycogen in the pileus expansion stage of A. cylindracea, which provides new insights on the change patterns of fungal polysaccharides.
Asunto(s)
Agrocybe , Glucanos , Agua , Glucanos/química , Agua/química , Agrocybe/química , Peso Molecular , Glucógeno/metabolismo , Glucógeno/química , Cuerpos Fructíferos de los Hongos/química , ReologíaRESUMEN
Ageritin is the prototype of a new ribotoxin-like protein family, which has been recently identified also in basidiomycetes. The protein exhibits specific RNase activity through the cleavage of a single phosphodiester bond located at sarcin/ricin loop of the large rRNA, thus inhibiting protein biosynthesis at early stages. Conversely to other ribotoxins, its activity requires the presence of divalent cations. In the present study, we report the activity of Ageritin on both prokaryotic and eukaryotic cells showing that the protein has a prominent effect on cancer cells viability and no effects on eukaryotic and bacterial cells. In order to rationalize these findings, the ability of the protein to interact with various liposomes mimicking normal, cancer and bacterial cell membranes was explored. The collected results indicate that Ageritin can interact with DPPC/DPPS/Chol vesicles, used as a model of cancer cell membranes, and with DPPC/DPPG vesicles, used as a model of bacterial cell membranes, suggesting a selective interaction with anionic lipids. However, a different perturbation of the two model membranes, mediated by cholesterol redistribution, was observed and this might be at the basis of Ageritin selective toxicity towards cancer cells.
Asunto(s)
Membrana Celular/metabolismo , Micotoxinas/farmacología , Neoplasias/metabolismo , Ribonucleasas/farmacología , Agrocybe/química , Animales , Antineoplásicos/farmacología , Bacterias/efectos de los fármacos , Basidiomycota/química , Calorimetría/métodos , Línea Celular , Membrana Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colesterol/metabolismo , Liposomas/metabolismo , Ratones , Micotoxinas/toxicidad , Neoplasias/tratamiento farmacológico , Biosíntesis de Proteínas/efectos de los fármacos , ARN Ribosómico/metabolismo , Ribonucleasas/metabolismo , Ribonucleasas/toxicidad , Ribosomas/metabolismoRESUMEN
Agrocybe cylindracea is a common source of active polysaccharides, but their fine structures are not clearly elucidated. In the present study, four fractions were purified from the alkaline extract of A. cylindracea (JACP), and their chemical components and structures were compared by HPAEC-PAD, methylation combined with GC-MS, and 1D/2D NMR analysis. Results showed the purified fractions' physicochemical properties, including monosaccharide compositions, molecular weights, viscosities and surface morphology considerably varied. JACP-30 was identified as a fucoglucogalactan with a α-(1 â 6)-galactopyranosyl as main chain. JACP-50p and JACP-80r were characterized as ß-(1 â 6)-glucans with side chains composed of terminal and 3-substituted ß-glucopyranosyl residues attached at O-3 for every three residues. Similarly, the backbone of JACP-80 was ß-(1 â 6)-linked glucopyranosyl and ß-(1 â 3,6)-linked glucopyranosyl residues at a ratio of 4:1. This work provides more information to the understanding of polysaccharides from A. cylindracea, further guiding its biological researches and developing the application in food and biomedicine industries.
Asunto(s)
Agrocybe/química , Polisacáridos Fúngicos/química , Extracción Líquido-Líquido/métodos , Monosacáridos/química , beta-Glucanos/química , Secuencia de Carbohidratos , Polisacáridos Fúngicos/clasificación , Polisacáridos Fúngicos/aislamiento & purificación , Humanos , Metilación , Peso Molecular , Monosacáridos/clasificación , Monosacáridos/aislamiento & purificación , Hidróxido de Sodio/química , beta-Glucanos/clasificación , beta-Glucanos/aislamiento & purificaciónRESUMEN
This study aimed at determining hepatoprotective and antioxidants effects of Lactarius deliciosus and Agrocybe cylindracea against CCl4-induced oxidative stress (OS) in rats. Herein, 36 rats were divided into 6 groups: (I) Control, (II) CCl4, (III) CCl4 + L. deliciosus, (IV) L. deliciosus, (V) CCl4 + A. cylindracea, and (VI) A. cylindracea. According to the 7-week study results, aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutathione S-transferase (GST) (brain), superoxide dismutase (SOD) (kidney), malondialdehyde (MDA) (kidney), MDA (spleen), MDA (erythrocyte), GST (erythrocyte), and GST (liver) in group III (at 10%, 26%, 34%, 57%, 7%, 25%, 42%, 27%, and 35%, respectively) were significantly lower than in group II. Moreover, the reduced glutathione (GSH) (spleen) and catalase (spleen) in group III (at 20% and 38%, respectively) were significantly higher than in group II. AST, ALT, triglyceride, glutathione reductase (GR) (brain), GR (kidney), SOD (spleen), and SOD (liver) in group V (at 194%, 147%, 38%, 36%, 66%, 8%, and 7%, respectively) were significantly higher than in group II. However, MDA (kidney), MDA (erythrocyte), (GSH) (erythrocyte), MDA (liver), GSH (liver), and GST (liver) in group V (at 48%, 35%, 34%, 15%, 30%, and 41%, respectively) were significantly lower than in group II. Hence, while L. deliciosus may exhibit tissue-protective against CCl4-induced OS in rats, A. cylindracea does not.
Asunto(s)
Agrocybe , Antioxidantes/farmacología , Basidiomycota , Estrés Oxidativo/efectos de los fármacos , Sustancias Protectoras/farmacología , Agrocybe/química , Animales , Basidiomycota/química , Tetracloruro de Carbono , Suplementos Dietéticos , Femenino , Ratas , Ratas Wistar , TurquíaRESUMEN
The aim of the present study is to characterize the structure of a novel natural polysaccharide from Agrocybe aegirita (AAPS) and evaluate its anti-aging activity. The MALLS and GC-MS analysis indicated that the AAPS with molecular weights of 1.81â¯×â¯104 Da was mainly composed by rhamnose, fucose, mannose, and glucose in a molar ratio of 2.90:10.25:3.70:38.27. The FT-IR and NMR analysis showed that the backbone of AAPS was α-L-Rhap-(1â6)-ß-D-Glcp-(1â2)-α-L-Fucp-(1â6)-α-D-Glcp-(1â5)-α-L-Araf-(1â4)-ß-D-GlcpA-(1â5)-α-L-Araf-(1â6)-α-D-Manp-(1â6)-α-D-Manp-(1â2)-α-L-Fucp-(1â6)-ß-D-Glap-(1â2)-α-L-Rhap-(1â6)-ß-D-Galp-(1â, which linked with two side chains α-L-Fucp-(1â6)-ß-D-Glcp-(1â6)-ß-D-Manp-(1â and α-D-Xylp-(1â2)-α-L-Fucp-(1â5) -α-D-Araf-(1â6)-ß-D-Galp-(1â at OH2 at H-4-arabinose and the terminal Galp residues, respectively. The MRC-5 cells induced by H2O2 were used to explore the anti-ageing effect and its underlying mechanism of AAPS. It showed a potent anti-ageing activity, representing by the increased cell viability and ß-Gal viability, prevented G1-phase cell-cycle arrest, and decreased mitochondrial membrane potential. The polysaccharides extracted from A. aegirita might be applied in functional food as anti-ageing ingredient.
Asunto(s)
Envejecimiento/efectos de los fármacos , Agrocybe/química , Antioxidantes/farmacología , Polisacáridos/farmacología , Antioxidantes/química , Antioxidantes/aislamiento & purificación , Secuencia de Carbohidratos , Línea Celular , Fenómenos Químicos , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Polisacáridos/química , Polisacáridos/aislamiento & purificaciónRESUMEN
A bienzymatic cascade for selective sulfoxidation is presented. The evolved recombinant peroxygenase from Agrocybe aegeritra catalyses the enantioselective sulfoxidation of thioanisole whereas the choline oxidase from Arthrobacter nicotianae provides the H2 O2 necessary via reductive activation of ambient oxygen. The reactions are performed in choline chloride-based deep eutectic solvents serving as co-solvent and stoichiometric reductant at the same time. Very promising product concentrations (up to 15â mM enantiopure sulfoxide) and catalyst performances (turnover numbers of 150,000 and 2100 for the peroxygenase and oxidase, respectively) have been achieved.
Asunto(s)
Agrocybe/enzimología , Oxidorreductasas de Alcohol/metabolismo , Productos Biológicos/química , Micrococcaceae/enzimología , Oxigenasas de Función Mixta/metabolismo , Safrol/análogos & derivados , Sulfuros/química , Agrocybe/química , Biocatálisis , Colina/química , Hidrógeno/química , Peróxido de Hidrógeno/química , Micrococcaceae/química , Oxidación-Reducción , Oxígeno/química , Procesos Fotoquímicos , Safrol/química , Solventes/química , EstereoisomerismoRESUMEN
Ageritin, a specific ribonuclease, damaging the largest rRNA in the highly conserved α-sarcin/ricin stem-loop (SRL) has been well characterized from edible mushroom Agrocybe aegerita. Given its peculiar characteristic, Ageritin is the prototype of a new ribotoxins family expressed in basidiomycetes. In this framework, we report the characterization of Met-Ageritin, an isoform of Ageritin with an additional N-terminal methionyl residue. This difference affects the enzymatic features of this toxin despite is able to release α-fragment when acting on yeast, rabbit or Trichoderma asperellum ribosomes. Met-Ageritin inhibits protein synthesis in vitro with an IC50â¯=â¯2.8â¯nM that is 21-fold lower than that of Ageritin, while not show endonuclease activity on DNA. Subsequently, we explored the antifungal activity of both isoforms against T. asperellum, pathogen for A. aegerita and Saccharomyces cerevisiae, used as eukaryotic model microorganism. The presence of an additional N-terminal methionyl residue in Met-Ageritin abolishes antifungal activity towards T. asperellum, while neither of two isoforms is able to inhibit the growth of S. cerevisiae. Overall, these data highlight the importance of the N-terminal region of this toxin that likely alters the conformational state of this enzyme considering the presence in this region of metal binding sites necessary for explicate enzymatic activity.
Asunto(s)
Agrocybe/química , Antifúngicos/farmacología , Ribonucleasas/farmacología , Antifúngicos/química , Antifúngicos/aislamiento & purificación , Cuerpos Fructíferos de los Hongos/química , Conformación Proteica , Ribonucleasas/química , Ribonucleasas/aislamiento & purificaciónRESUMEN
Ribotoxins make up a group of extracellular rRNA endoribonucleases produced by ascomycetes that display cytotoxicity toward animal cells, having been proposed as insecticidal agents. Recently, the ribotoxin Ageritin has been isolated from the basidiomycetes Agrocybe aegerita (poplar mushroom), suggesting that ribotoxins are widely distributed among fungi. To gain insights into the protective properties of Ageritin against pathogens and its putative biotechnological applications, we have tested several biological activities of Ageritin, comparing them with those of the well-known ribotoxin α-sarcin, and we found that Ageritin displayed, in addition to the already reported activities, (i) antibacterial activity against Micrococcus lysodeikticus, (ii) activity against the tobacco mosaic virus RNA, (iii) endonuclease activity against a supercoiled plasmid, (iv) nuclease activity against genomic DNA, (v) cytotoxicity to COLO 320, HeLa, and Raji cells by promoting apoptosis, and (vi) antifungal activity against the green mold Penicillium digitatum. Therefore, Ageritin and α-sarcin can induce resistance not only to insects but also to viruses, bacteria, and fungi. The multiple biological activities of Ageritin could be exploited to improve resistance to different pathogens by engineering transgenic plants. Furthermore, the induction of cell death by different mechanisms turns these ribotoxins into useful tools for cancer therapy.
Asunto(s)
Agrocybe/química , Proliferación Celular/efectos de los fármacos , Citotoxinas/farmacología , Ribonucleasas/farmacología , Antiinfecciosos/farmacología , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Pruebas de Sensibilidad Microbiana , Micrococcus/efectos de los fármacos , Ribonucleasas/aislamiento & purificación , Virus del Mosaico del Tabaco/efectos de los fármacosRESUMEN
A pot experiment was conducted to investigate the detoxification mechanism of Agrocybe aegerita (A. aegerita). The physiological responses, subcellular distribution and chemical forms of cadmium (Cd) in A. aegerita grown in Cd stress were analyzed. The results showed that the biomass was decreased under Cd stress, while the production of malonaldehyde, thiols, and low-molecular-weight organic acids (LWMOAs) as well as the antioxidant enzymes in A. aegerita was increased compared with control group. The HPLC results showed that nine LWMOAs were found in A. aegerita with critic acid as the dominant and they played important role in the detoxification and accumulation of Cd in A. aegerita. More Cd was accumulated in pileus than in stipe. Differential centrifugation technique showed that the majority of Cd was compartmentalized in the soluble fraction (53-75%) and bound to the cell wall (19-42%). The proportion of Cd in the cell wall increased with the increase of the accumulation of Cd in the fruiting body, but in the soluble fraction showed an opposite trend. Furthermore, most of the Cd in A. aegerita was mainly in the forms of NaCl- (29-49%) and ethanol-extractable Cd (20-40%). The ethanol- and water-extractable Cd in stipe (60-66%) was higher than in pileus (43-49%). Thus intracellular detoxification mechanisms of Cd in A. aegerita is related to subcellular partitioning and chemical forms of Cd and well-coordinated physiological responses.
Asunto(s)
Agrocybe/metabolismo , Cadmio/metabolismo , Agrocybe/química , Antioxidantes/metabolismo , Biomasa , Cadmio/análisis , Pared Celular/química , Ácido Cítrico/metabolismo , Frutas/química , Inactivación Metabólica , Malondialdehído/metabolismo , Compuestos de Sulfhidrilo/metabolismoRESUMEN
There is an increasing interest in the application of peroxygenases in biocatalysis, because of their ability to catalyse the oxyfunctionalisation reaction in a stereoselective fashion and with high catalytic efficiencies, while using hydrogen peroxide or organic peroxides as oxidant. However, enzymes belonging to this class exhibit a very low stability in the presence of peroxides. With the aim of bypassing this fast and irreversible inactivation, we study the use of a gradual supply of hydrogen peroxide to maintain its concentration at stoichiometric levels. In this contribution, we report a multienzymatic cascade for in situ generation of hydrogen peroxide. In the first step, in the presence of NAD+ cofactor, formate dehydrogenase from Candida boidinii (FDH) catalysed the oxidation of formate yielding CO2. Reduced NADH was reoxidised by the reduction of the flavin mononucleotide cofactor bound to an old yellow enzyme homologue from Bacillus subtilis (YqjM), which subsequently reacts with molecular oxygen yielding hydrogen peroxide. Finally, this system was coupled to the hydroxylation of ethylbenzene reaction catalysed by an evolved peroxygenase from Agrocybe aegerita (rAaeUPO). Additionally, we studied the influence of different reaction parameters on the performance of the cascade with the aim of improving the turnover of the hydroxylation reaction.
Asunto(s)
Proteínas Bacterianas/química , FMN Reductasa/química , Formiato Deshidrogenasas/química , Proteínas Fúngicas/química , Peróxido de Hidrógeno/síntesis química , Oxigenasas de Función Mixta/química , Agrocybe/química , Agrocybe/enzimología , Bacillus subtilis/química , Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , Derivados del Benceno/química , Derivados del Benceno/metabolismo , Biocatálisis , Candida/química , Candida/enzimología , Dióxido de Carbono/química , Dióxido de Carbono/metabolismo , Coenzimas/química , Coenzimas/metabolismo , FMN Reductasa/metabolismo , Mononucleótido de Flavina/química , Mononucleótido de Flavina/metabolismo , Formiato Deshidrogenasas/metabolismo , Formiatos/química , Formiatos/metabolismo , Proteínas Fúngicas/metabolismo , Peróxido de Hidrógeno/metabolismo , Hidroxilación , Cinética , Oxigenasas de Función Mixta/metabolismo , NAD/química , NAD/metabolismo , Oxidación-Reducción , Oxígeno/química , Oxígeno/metabolismo , EstereoisomerismoRESUMEN
The inhibition of arginase from Leishmania spp. is considered a promising approach to the leishmaniasis treatment. In this study, the potential of a fucogalactan isolated from the medicinal mushroom Agrocybe aegerita was evaluated against arginase (ARG) from Leishmania amazonensis. The polysaccharide was obtained via aqueous extraction, and purified by freeze thawing and precipitation with Fehling solution. Its chemical structure was established by monosaccharide composition, methylation analysis, partial acid hydrolysis, and NMR spectroscopy. The data indicated that it is a fucogalactan (FG-Aa; Mw = 13.8 kDa), having a (1â6)-linked α-D-Galp main-chain partially substituted in O-2 by non-reducing end-units of α-L-Fucp. FG-Aa showed significant inhibitory activity on ARG with IC50potency of 5.82 ± 0.57 µM. The mechanism of ARG inhibition by the heterogalactan was the competitive type, with Kiof 1.54 ± 0.15 µM. This is the first report of an inhibitory activity of arginase from L. amazonensis by biopolymers, which encourages us to investigate further polysaccharides as a new class of ARG inhibitors.
Asunto(s)
Agrocybe/química , Arginasa/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Polisacáridos Fúngicos/química , Galactanos/química , Leishmania/enzimología , Proteínas Protozoarias/antagonistas & inhibidores , Arginasa/química , Proteínas Protozoarias/químicaRESUMEN
Ageritin has been recently described as the first ribotoxin-like from Basidiomycota division (mushroom Agrocybe aegerita) with known antitumor activity (BBA 2017, 1861: 1113-1121). By investigating structural, catalytic and binding properties, we demonstrate that Ageritin is a unique ribotoxin-like protein. Indeed, typical of the ribotoxin family, it shows the specific ribonucleolytic activity against the ribosomal Sarcin-Ricin Loop in a rabbit reticulocytes assay. However, it displays several elements of novelty, as this activity is strongly metal-dependent and completely suppressed in the presence of EDTA, different from other representative members of the ribotoxin family. Consistently, we prove that Ageritin is able to bind magnesium ions with low micromolar affinity. We also show that Ageritin is significantly more stable than other ribotoxins in thermal and chemical denaturation experiments. These peculiar features make Ageritin the prototype of a new ribotoxin-like family present in basidiomycetes. Finally, given its high stability, this enzyme is a promising candidate as a new tool in immunoconjugates and nanoconstructs.
Asunto(s)
Agrocybe/química , Antineoplásicos/química , Antineoplásicos/farmacología , Magnesio/metabolismo , Ribonucleasas/química , Ribonucleasas/farmacología , Ribosomas/efectos de los fármacos , Toxinas Biológicas/química , Toxinas Biológicas/farmacología , Animales , Calorimetría , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Unión Proteica , Estabilidad Proteica , Estructura Secundaria de Proteína , Conejos , Ribosomas/metabolismo , Espectrofotometría UltravioletaRESUMEN
O-linked N-acetylglucosamine (O-GlcNAcylation) is an important post-translational modification on serine or threonine of proteins, mainly observed in nucleus or cytoplasm. O-GlcNAcylation regulates many cell processes, including transcription, cell cycle, neural development and nascent polypeptide chains stabilization. However, the facile identification of O-GlcNAc is a major bottleneck in O-GlcNAcylation research. Herein, we report that a lectin, Agrocybe aegerita GlcNAc-specific lectin (AANL), also reported as AAL2, can be used as a powerful probe for O-GlcNAc identification. Glycan array analyses and surface plasmon resonance (SPR) assays show that AANL binds to GlcNAc with a dissociation constant (KD) of 94.6 µM, which is consistent with the result tested through isothiocyanate (ITC) assay reported before (Jiang S, Chen Y, Wang M, Yin Y, Pan Y, Gu B, Yu G, Li Y, Wong BH, Liang Y, et al. 2012. A novel lectin from Agrocybe aegerita shows high binding selectivity for terminal N-acetylglucosamine. Biochem J. 443:369-378.). Confocal imaging shows that AANL co-localizes extensively with NUP62, a heavily O-GlcNAcylated and abundant nuclear pore glycoprotein. Furthermore, O-GlcNAc-modified peptides could be effectively enriched in the late flow-through peak from simple samples by using affinity columns Sepharose 4B-AANL or POROS-AANL. Therefore, using AANL affinity column, we identified 28 high-confidence O-linked HexNAc-modified peptides mapped on 17 proteins involving diverse cellular progresses, including transcription, hydrolysis progress, urea cycle, alcohol metabolism and cell cycle. And most importantly, major proteins and sites were not annotated in the dbOGAP database. These results suggest that the AANL lectin is a new useful tool for enrichment and identification of O-GlcNAcylated proteins and peptides.
Asunto(s)
Acetilglucosamina/metabolismo , Proteínas Fúngicas/química , Glicómica/métodos , Lectinas/química , Procesamiento Proteico-Postraduccional , Acetilglucosamina/análisis , Agrocybe/química , Proteínas Fúngicas/metabolismo , Glicosilación , Células HeLa , Humanos , Lectinas/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/metabolismo , Unión ProteicaRESUMEN
Two extractable polysaccharides of Ac-MPS and Al-MPS were extracted from the mycelium of Agrocybe aegerita (Brig.) Sing and the in vitro antioxidant activities and in vivo anti-aging activities in a d-gal-induced aging mice were investigated in the present study. The in vitro antioxidant analysis showed that Ac-MPS showed superior scavenging effects on hydroxyl of 68.62±2.59% and DPPH of 85.19±3.26% at the concentration of 4500µg/mL. Biochemical indices and antioxidant analysis in vivo showed that Ac-MPS had better effects in raising hepatic activities of SOD, CAT, GPx and T-AOC, inhibiting hepatic contents of LPO and MDA, as well as improving serum lipid levels of LDL-C, HDL-C, LDL-C/HDL-C, TG and TC. Furthermore, HYP measurements suggested that skin collagen in aging mice could be maintained. This study suggested that both Ac-MPS and Al-MPS could be used as potential natural agents or dietary supplements in attenuating aging and its complications.
Asunto(s)
Envejecimiento/efectos de los fármacos , Agrocybe/química , Antioxidantes/farmacología , Polisacáridos/farmacología , Ácidos/química , Envejecimiento/patología , Álcalis/química , Antioxidantes/química , Antioxidantes/aislamiento & purificación , Humanos , Micelio/química , Polisacáridos/química , Polisacáridos/aislamiento & purificaciónRESUMEN
CONTEXT: Accumulated evidence has indicated that recombinant Agrocybe aegerita lectin (AAL) possesses immunoadjuvant activity to enhance antigen-specific immune responses. However, the mechanism of how AAL regulates immune response remains poorly defined. AIM: This study is aimed to reveal the mechanism of AAL's immunoadjuvant activity. METHODS: In this study, AAL alone or combined with inactivated avian influenza virus H9N2 was immunized to mice and the transcriptome profile of immunized mice was analyzed. RESULTS: In line with previous studies, our results showed that H9N2-specific IgG level was significantly increased in AAL-treated mice, suggesting the immunoadjuvant activity of AAL. More importantly, transcriptome data revealed that genes participating in the primary adherence, lymphocyte activation, secondary adherence and transmembrane migration of leukocyte migration, were up-regulated by AAL. CONCLUSION: These findings suggest that AAL exerts immunoadjuvant effects by promoting chemotaxis and phagotrophy activity of neutrophil leucocyte and macrophage to improve innate immunity and antigen presentation.
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Adyuvantes Inmunológicos/farmacología , Agrocybe/química , Presentación de Antígeno/efectos de los fármacos , Proteínas Fúngicas/farmacología , Inmunidad Innata/efectos de los fármacos , Subtipo H9N2 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/farmacología , Lectinas/farmacología , Adyuvantes Inmunológicos/química , Agrocybe/genética , Agrocybe/inmunología , Animales , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/inmunología , Vacunas contra la Influenza/química , Vacunas contra la Influenza/inmunología , Lectinas/química , Lectinas/genética , Lectinas/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacologíaRESUMEN
The in vitro antioxidant and in vivo anti-aging activities in D-galactose-induced aging mice of exopolysaccharides (EPS) and its fractions (EPS-1, EPS-2 and EPS-3) by Agrocybe cylindraceas were investigated presently. The scavenging effects of EPS-2 on hydroxyl (HO) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals were 97.1±4.25% and 90.23±3.25%, while the reducing power reached 1.18±0.08 at 120mg/L, which were higher than EPS, EPS-1 and EPS-3 at 1000mg/L. The EPS showed potential anti-aging activities via reducing the contents of malonaldehyde (MDA) and total cholesterol (TC), as well as increasing the activities of superoxide dismutase (SOD) activities, glutathione peroxidase (GSH-Px) activities, and total antioxidant capacity (T-AOC). Additionally, the monosaccharide analysis showed EPS contained rhamnose and glucose with the molar ratio of 2.8:1. These results demonstrated that EPS could be used as functional foods and natural drugs on preventing and mitigating the aging process.