RESUMEN
A multi-specific fungal galectin from the mushroom Agrocybe cylindracea (ACG) binds a broad range of ß-galactosides, as well as their derivative GalNAcα1-3Gal. Site-directed mutagenesis of the hydrophilic residues His, Asn, Arg, and Glu, involved in carbohydrate recognition, abolished the binding affinity of the derived mutants to ß-galactosides, whereas only N46A caused increased affinity to GalNAcα1-3Gal-containing oligosaccharides and loss of ß-galactoside-binding activity. Detailed structural analysis revealed that Pro45, the preceding residue of Asn46 of the wild-type ACG, takes the cis imide conformation to tether Asn46 onto a loop region to make new hydrogen bonds with ß-galactosides and to compensate for the lack of evolutionarily conserved Asn. In contrast, in the N46A mutant, Pro45 takes the more stable trans conformation, resulting in "switched" specificity to αGalNAc. Such an altered recognition system in the binding specificity of galectins can be observed in other lectin molecules not only in nature but will also be observed in those engineered in the future.
Asunto(s)
Agrocybe , Agrocybe/genética , Agrocybe/metabolismo , Galectinas/metabolismoRESUMEN
This study aimed to investigate the potential anti-aging mechanisms of Agrocybe cylindracea crude polysaccharides (APS), when used synergistically with Lactobacillus rhamnosus GG (APS + LGG) in a D-galactose-induced aging mouse model. In the Morris water maze test, APS + LGG showed a significantly higher memory and learning capacity compared to untreated, APS only treated and LGG treated mice. This was thought to be mediated by increased levels of brain-derived neurotrophic factor, which decreased escape latency. In addition to this, in the aging mouse model, APS + LGG co-treatment markedly alleviated liver oxidation and metabolism by enhancing the antioxidant activity of enzymes; this decreased the lipid metabolism and peroxidation levels. Furthermore, high throughput sequencing analysis revealed that an APS + LGG supplemented feed increased the relative abundance of positive bacteria in the gut microbiota such as Alloprevotella and Parvibacter. Importantly, Alloprevotella and Parvibacter showed a negative relationship with low density lipoprotein-cholesterol in the Spearman correlation analysis. These results illustrate that APS, in combination with LGG, postponed aging related oxidative stress when used as a prebiotic. The proposed mechanism for this is the reduction in liver oxidation and lipid metabolism, as well as the regulation of gut microbiota.
Asunto(s)
Envejecimiento/efectos de los fármacos , Agrocybe/metabolismo , Antioxidantes/farmacología , Lacticaseibacillus rhamnosus/metabolismo , Estrés Oxidativo/efectos de los fármacos , Polisacáridos/farmacología , Prebióticos/administración & dosificación , Animales , Antioxidantes/metabolismo , Modelos Animales de Enfermedad , Masculino , Ratones , Polisacáridos/metabolismoRESUMEN
The edible mushroom Agrocybe aegerita produces a ribotoxin-like protein known as Ageritin. In this work, the gene encoding Ageritin was characterized by sequence analysis. It contains several typical features of fungal genes such as three short introns (60, 55 and 69 bp) located at the 5' region of the coding sequence and typical splice junctions. This sequence codes for a precursor of 156 amino acids (~17-kDa) containing an additional N-terminal peptide of 21 amino acid residues, absent in the purified toxin (135 amino acid residues; ~15-kDa). The presence of 17-kDa and 15-kDa forms was investigated by Western blot in specific parts of fruiting body and in mycelia of A. aegerita. Data show that the 15-kDa Ageritin is the only form retrieved in the fruiting body and the principal form in mycelium. The immunolocalization by confocal laser scanning microscopy and transmission electron microscopy proves that Ageritin has vacuolar localization in hyphae. Coupling these data with a bioinformatics approach, we suggest that the N-terminal peptide of Ageritin (not found in the purified toxin) is a new signal peptide in fungi involved in intracellular routing from endoplasmic reticulum to vacuole, necessary for self-defense of A. aegerita ribosomes from Ageritin toxicity.
Asunto(s)
Agrocybe/genética , Citotoxinas/genética , Cuerpos Fructíferos de los Hongos/metabolismo , Proteínas Fúngicas/genética , Micelio/metabolismo , Ribonucleasas/genética , Agrocybe/metabolismo , Agrocybe/ultraestructura , Secuencia de Aminoácidos , Biología Computacional , Citotoxinas/biosíntesis , Citotoxinas/aislamiento & purificación , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Exones , Cuerpos Fructíferos de los Hongos/ultraestructura , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/aislamiento & purificación , Expresión Génica , Intrones , Micelio/ultraestructura , Sistemas de Lectura Abierta , Señales de Clasificación de Proteína/genética , Transporte de Proteínas , Ribonucleasas/biosíntesis , Ribonucleasas/aislamiento & purificación , Ribosomas/genética , Ribosomas/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Vacuolas/metabolismo , Vacuolas/ultraestructuraRESUMEN
Agrocybe cylindracea, an edible mushroom, is widely cultivated for its abundance of nutrients and flavor, and many of its metabolites are reported to have beneficial roles, such as medicinal effects on tumors and chronical illnesses. However, the lack of genomic information has hindered further molecular studies on this fungus. Here, we present a genome assembly of A. cylindracea together with comparative genomics and pathway analyses of Agaricales species. The draft, generated from both next-generation sequencing (NGS) and single-molecule real-time (SMRT) sequencing platforms to overcome high genetic heterozygosity, is composed of a 56.5 Mb sequence and 15,384 predicted genes. This mushroom possesses a complex reproductive system, including tetrapolar heterothallic and secondary homothallic mechanisms, and harbors several hydrolases and peptidases for gradual and effective degradation of various carbon sources. Our pathway analysis reveals complex processes involved in the biosynthesis of polysaccharides and other active substances, including B vitamins, unsaturated fatty acids, and N-acetylglucosamine. RNA-seq data show that A. cylindracea stipes tend to synthesize carbohydrate for carbon sequestration and energy storage, whereas pilei are more active in carbon utilization and unsaturated fatty acid biosynthesis. These results reflect diverse functions of the two anatomical structures of the fruiting body. Our comprehensive genomic and transcriptomic data, as well as preliminary comparative analyses, provide insights into the molecular details of the medicinal effects in terms of active compounds and nutrient components.
Asunto(s)
Agrocybe/genética , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Genómica/métodos , Redes y Vías Metabólicas , Transcriptoma , Agrocybe/clasificación , Agrocybe/metabolismo , Biología Computacional , Secuenciación de Nucleótidos de Alto Rendimiento , Secuenciación Completa del GenomaRESUMEN
Terpenoids constitute a structurally diverse group of natural products with wide applications in the pharmaceutical, nutritional, flavor and fragrance industries. Fungi are known to produce a large variety of terpenoids, yet fungal terpene synthases remain largely unexploited. Here, we report the sesquiterpene network and gene clusters of the black poplar mushroom Agrocybe aegerita. Among 11 putative sesquiterpene synthases (STSs) identified in its genome, nine are functional, including two novel synthases producing viridiflorol and viridiflorene. On this basis, an additional 1133 STS homologues from higher fungi have been curated and used for a sequence similarity network to probe isofunctional STS groups. With the focus on two STS groups, one producing viridiflorene/viridiflorol and one Δ6-protoilludene, the isofunctionality was probed and verified. Three new Δ6-protoilludene synthases and two new viridflorene/viridiflorol synthases from five different fungi were correctly predicted. The study herein serves as a fundamental predictive framework for the discovery of fungal STSs and biosynthesis of novel terpenoids. Furthermore, it becomes clear that fungal STS function differs between the phyla Ascomycota and Basidiomycota with the latter phylum being more dominant in the overall number and variability. This study aims to encourage the scientific community to further work on fungal STS and the products, biological functions, and potential applications of this vast source of natural products.
Asunto(s)
Agrocybe/enzimología , Transferasas Alquil y Aril/metabolismo , Productos Biológicos/química , Sesquiterpenos/química , Agrocybe/genética , Agrocybe/metabolismo , Transferasas Alquil y Aril/genética , Secuencia de Aminoácidos , Secuencia de Bases , Basidiomycota/enzimología , Basidiomycota/genética , Basidiomycota/metabolismo , Productos Biológicos/metabolismo , Vías Biosintéticas , Clonación Molecular , Escherichia coli/genética , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Estructura Molecular , Familia de Multigenes , Homología de Secuencia de Ácido Nucleico , Sesquiterpenos/metabolismoRESUMEN
Fungi produce various defense proteins against antagonists, including ribotoxins. These toxins cleave a single phosphodiester bond within the universally conserved sarcin-ricin loop of ribosomes and inhibit protein biosynthesis. Here, we report on the structure and function of ageritin, a previously reported ribotoxin from the edible mushroom Agrocybe aegerita The amino acid sequence of ageritin was derived from cDNA isolated from the dikaryon A. aegerita AAE-3 and lacks, according to in silico prediction, a signal peptide for classical secretion, predicting a cytoplasmic localization of the protein. The calculated molecular weight of the protein is slightly higher than the one reported for native ageritin. The A. aegerita ageritin-encoding gene, AaeAGT1, is highly induced during fruiting, and toxicity assays with AaeAGT1 heterologously expressed in Escherichia coli showed a strong toxicity against Aedes aegypti larvae yet not against nematodes. The activity of recombinant A. aegerita ageritin toward rabbit ribosomes was confirmed in vitro Mutagenesis studies revealed a correlation between in vivo and in vitro activities, indicating that entomotoxicity is mediated by ribonucleolytic cleavage. The strong larvicidal activity of ageritin makes this protein a promising candidate for novel biopesticide development.IMPORTANCE Our results suggest a pronounced organismal specificity of a protein toxin with a very conserved intracellular molecular target. The molecular details of the toxin-target interaction will provide important insight into the mechanism of action of protein toxins and the ribosome. This insight might be exploited to develop novel bioinsecticides.
Asunto(s)
Agaricales/metabolismo , Agrocybe/metabolismo , Micotoxinas/metabolismo , Micotoxinas/toxicidad , Ribonucleasas/metabolismo , Ribonucleasas/toxicidad , Agaricales/genética , Agrocybe/genética , Secuencia de Aminoácidos , Animales , Culicidae/efectos de los fármacos , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Larva/efectos de los fármacos , Mutagénesis , Mutación , Micotoxinas/química , Micotoxinas/genética , Proteínas Recombinantes , Ribonucleasas/química , Ribonucleasas/genética , Ribosomas/efectos de los fármacos , Células Sf9/efectos de los fármacosRESUMEN
Oxylipins are metabolites with a variety of biological functions. However, the biosynthetic pathway is widely unknown. It is considered that the first step is the oxygenation of polyunsaturated fatty acids like linoleic acid. Therefore, a lipoxygenase (LOX) from the edible basidiomycete Agrocybe aegerita was investigated. The AaeLOX4 was heterologously expressed in E. coli and purified via affinity chromatography and gel filtration. Biochemical properties and kinetic parameters of the purified AaeLOX4 were determined with linoleic acid and linolenic acid as substrates. The obtained Km, vmax and kcat values for linoleic acid were 295.5 µM, 16.5 µM · min-1 · mg-1 and 103.9 s-1, respectively. For linolenic acid Km, vmax and kcat values of 634.2 µM, 19.5 µM · min-1 · mg-1 and 18.3 s-1 were calculated. Maximum activities were observed at pH 7.5 and 25 °C. The main product of linoleic acid conversion was identified with normal-phase HPLC. This analysis revealed an explicit production of 13-hydroperoxy-9,11-octadecadienoic acid (13-HPOD). The experimental regio specificity is underpinned by the amino acid residues W384, F450, R594 and V635 considered relevant for regio specificity in LOX. In conclusion, HPLC-analysis and alignments revealed that AaeLOX4 is a 13-LOX.
Asunto(s)
Agrocybe/enzimología , Proteínas Fúngicas/metabolismo , Lipooxigenasas/metabolismo , Agrocybe/metabolismo , Secuencias de Aminoácidos , Dominio Catalítico , Proteínas Fúngicas/química , Cinética , Ácido Linoleico/metabolismo , Lipooxigenasas/química , Especificidad por SustratoRESUMEN
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
8-Hydroxy-2,4,6-octatriynamide, a natural polyacetylene with inhibitory activities against rice pathogens, was isolated from the liquid fermentation broth of strain Agrocybe sp. YB2005 during screening for new natural chemical agents to control rice pathogens. 8-hydroxy-2,4,6-octatriynamide was purified by consecutive chromatography over a Cl8 reversed phase silica gel, sephadex LH-20 and silica gel. The chemical structure of 8-hydroxy-2,4,6-octatriynamide was elucidated through spectroscopic analyses, including 1D- and 2D-NMR, ESI mass spectrometry and X-ray single crystal diffraction. Bioassays showed that 8-hydroxy-2,4,6-octatriynamide could significantly inhibit growth of Xanthomonas oryzae with an MIC of 53.1 µM in a 96-well plate and the growth of Rhizoctonia solani at 1.02 mM in a 24-well plate. When rice leaves were inoculated with Magnaporthe grisea and cultured in artificial nutrition liquid containing 0.34 mM 8-hydroxy-2,4,6-octatriynamide, no rice blast was observed. The present study implied that 8-hydroxy-2,4,6-octatriynamide could be a candidate agent against rice pathogens.
Asunto(s)
Agrocybe/metabolismo , Antiinfecciosos/farmacología , Medios de Cultivo/química , Poliinos/aislamiento & purificación , Poliinos/farmacología , Agrocybe/crecimiento & desarrollo , Antiinfecciosos/aislamiento & purificación , Cromatografía Liquida , Magnaporthe/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Oryza/microbiología , Enfermedades de las Plantas/microbiología , Rhizoctonia/efectos de los fármacos , Análisis Espectral , Xanthomonas/efectos de los fármacosRESUMEN
There is an increasing interest in enzymes that catalyze the hydroxylation of naphthalene under mild conditions and with minimal requirements. To address this challenge, an extracellular fungal aromatic peroxygenase with mono(per)oxygenase activity was engineered to convert naphthalene selectively into 1-naphthol. Mutant libraries constructed by random mutagenesis and DNA recombination were screened for peroxygenase activity on naphthalene together with quenching of the undesired peroxidative activity on 1-naphthol (one-electron oxidation). The resulting double mutant (G241D-R257K) obtained from this process was characterized biochemically and computationally. The conformational changes produced by directed evolution improved the substrate's catalytic position. Powered exclusively by catalytic concentrations of H2 O2 , this soluble and stable biocatalyst has a total turnover number of 50 000, with high regioselectivity (97 %) and reduced peroxidative activity.
Asunto(s)
Agrocybe/enzimología , Evolución Molecular Dirigida , Oxigenasas de Función Mixta/metabolismo , Naftalenos/metabolismo , Naftoles/metabolismo , Ingeniería de Proteínas , Agrocybe/genética , Agrocybe/metabolismo , Oxigenasas de Función Mixta/genética , Modelos Moleculares , Mutación PuntualRESUMEN
O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a reversible post-translational modification that plays essential roles in many cellular pathways. Research in this field, however, is hampered by the lack of suitable probes to identify, accumulate, and purify the O-GlcNAcylated proteins. We have previously reported the identification of a lectin from the mushroom Agrocybe aegerita, i.e., Agrocybe aegerita lectin 2, or AAL2, that could bind terminal N-acetylglucosamine with higher affinities and specificity than other currently used probes. In this paper, we report the crystal structures of AAL2 and its complexes with GlcNAc and GlcNAcß1-3Galß1-4GlcNAc and reveal the structural basis of GlcNAc recognition by AAL2 and residues essential for the binding of terminal N-acetylglucosamine. Study on AAL2 may enable us to design a protein probe that can be used to identify and purify O-GlcNAcylated proteins more efficiently.
Asunto(s)
Acetilglucosamina/química , Agrocybe/metabolismo , Lectinas/química , Acetilglucosamina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Glicosilación , Lectinas/metabolismo , Metales/química , Metales/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Polisacáridos/química , Polisacáridos/metabolismo , Unión Proteica , Conformación Proteica , Alineación de Secuencia , Relación Estructura-Actividad , Especificidad por Sustrato , Resonancia por Plasmón de SuperficieRESUMEN
The large number of spores produced by edible mushrooms cause many problems, including causing lung disease, depleting natural genetic diversity, and reduced quality of fruiting bodies. Obtaining spore-deficient strains and understanding the underlying molecular mechanisms of such strains are important for breeding work. In this study, we crossed monokaryotic strains isolated from the edible fungi Agrocybe salicacola to obtain three spore-deficient strains with losses of the sterigmata on the surface of the lamella. A mating test revealed that recessive alleles distributed in some strains might control sterigmata development during the mitotic or meiotic phases. Transcriptome analysis revealed that the majority of the genes involved in DNA mismatch repair, base excision repair, and homologous recombination exhibited down-regulated expression patterns in the mutant fruiting bodies. Five genetic fragments, which were highly similar to the GTP-cyclohydrolase encoding gene, the DNA repair gene rad 8, and cell wall integrity and stress response component-encoding genes, were all expressed exclusively in the wild-type strains; these findings provide important information for the study of the spore development of edible fungi.
Asunto(s)
Agrocybe/genética , Proteínas Fúngicas/genética , Esporas Fúngicas/crecimiento & desarrollo , Transcriptoma , Agrocybe/crecimiento & desarrollo , Agrocybe/metabolismo , Proteínas Fúngicas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Esporas Fúngicas/genética , Esporas Fúngicas/metabolismoRESUMEN
Galectins are ß-galactoside binding proteins which have the ability to serve as potent antitumor, cancer biomarker, and induce tumor cell apoptosis. Agrocybe cylindracea galectin (ACG) is a fungal galectin which specifically recognizes α(2,3)-linked sialyllactose at the cell surface that plays extensive roles in the biological recognition processes. To investigate the change in glycan-binding specificity upon mutations, single point and double point site-directed in silico mutations are performed at the binding pocket of ACG. Molecular dynamics (MD) simulation studies are carried out for the wild-type (ACG) and single point (ACG1) and double point (ACG2) mutated ACGs to investigate the dynamics of substituted mutants and their interactions with the receptor sialyllactose. Plausible binding modes are proposed for galectin-sialylglycan complexes based on the analysis of hydrogen bonding interactions, total pair-wise interaction energy between the interacting binding site residues and sialyllactose and binding free energy of the complexes using molecular mechanics-Poisson-Boltzmann surface area. Our result shows that high contribution to the binding in different modes is due to the direct and water-mediated hydrogen bonds. The binding specificity of double point mutant Y59R/N140Q of ACG2 is found to be high, and it has 26 direct and water-mediated hydrogen bonds with a relatively low-binding free energy of -47.52 ± 5.2 kcal/mol. We also observe that the substituted mutant Arg59 is crucial for glycan-binding and for the preference of α(2,3)-linked sialyllactose at the binding pocket of ACG2 galectin. When compared with the wild-type and single point mutant, the double point mutant exhibits enhanced affinity towards α(2,3)-linked sialyllactose, which can be effectively used as a model for biological cell marker in cancer therapeutics.
Asunto(s)
Agrocybe/metabolismo , Galectinas/metabolismo , Simulación de Dinámica Molecular , Polisacáridos/metabolismo , Galectinas/química , Galectinas/genética , Enlace de Hidrógeno , Lactosa/análogos & derivados , Lactosa/química , Lactosa/metabolismo , Mutación , Polisacáridos/química , Unión Proteica , Ácidos Siálicos/química , Ácidos Siálicos/metabolismoRESUMEN
The aim of the study was to evaluate the possibility of supplementation with inorganic forms of selenium (Na2SeO4 and Na2SeO3) in concentrations of 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.5 mM of three medicinal mushroom species: Agrocybe aegerita, Hericium erinaceus and Ganoderma lucidum. Tested mushroom species grew in Se additions of 0-0.6 mM (A. aegerita and H. erinaceus), while growth of G. lucidum bodies was observed for 0-0.8 mM. For the latter mushroom species, the total Se content was the highest. Content of Seorg was diverse; for control bodies it was the highest for G. lucidum (only organic forms were present), lower for A. aegerita (84% organic forms) and the lowest for H. erinaceus (56% organic forms). Accumulation of Se(IV) was generally significantly higher than Se(VI) for all tested mushroom species. There was no significant decrease of A. aegerita or G. lucidum biomass with the exception of G. lucidum bodies growing under 0.8 mM of Se species addition (15.51 ± 6.53 g). Biomass of H. erinaceus bodies was the highest under 0.2 (197.04 ± 8.73 g), control (191.80 ± 6.06 g) and 0.1 mM (185.04 ± 8.73 g) of both inorganic salts. The addition to the medium of Se salts brought about macroscopic changes in the fruiting bodies of the examined mushrooms. Concentrations exceeding 0.4 mM caused diminution of carpophores or even their total absence. In addition, colour changes of fruiting bodies were also recorded. At Se concentrations of 0.4 and 0.6 mM, A. aegerita fruiting bodies were distinctly lighter and those of H. erinaceus changed colour from purely white to white-pink.
Asunto(s)
Agrocybe/efectos de los fármacos , Basidiomycota/efectos de los fármacos , Suplementos Dietéticos , Plantas Medicinales/efectos de los fármacos , Reishi/efectos de los fármacos , Compuestos de Selenio/farmacología , Agrocybe/crecimiento & desarrollo , Agrocybe/metabolismo , Basidiomycota/crecimiento & desarrollo , Basidiomycota/metabolismo , Biomasa , Alimentos Formulados , Plantas Medicinales/crecimiento & desarrollo , Plantas Medicinales/metabolismo , Reishi/crecimiento & desarrollo , Reishi/metabolismo , Ácido Selénico/farmacología , Ácido Selenioso/farmacología , Selenio/farmacocinética , Selenito de Sodio/farmacologíaRESUMEN
BACKGROUND: Agrocybe aegerita Lectin (AAL) has been identified to have high affinity for sulfated and α2-3- linked sialic acid glycoconjugates, especially the sulfated and sialyl TF (Thomsen-Friedenreich) disaccharide. This study was conducted to investigate the clinicopathological and prognostic value of AAL in identifying aberrant glycosylation in colorectal cancer (CRC). MATERIALS AND METHODS: Glycoconjugate expression in 59 CRC tissues were detected using AAL-histochemistry. Clinicopathological associates of expression were analyzed with chi- square test or Fisher's exact test. Relationships between expression and the various clinicopathological parameters was estimated using Kaplan-Meier analysis and Cox regression models. RESULTS: AAL specific glycoconjugate expression was significantly higher in tumor than corresponding normal tissues (66.1% and 46.1%, respectively, p=0.037), correlating with depth of invasion (p=0.015) and TNM stage (p=0.024). Patients with lower expression levels had a significantly higher survival rate than those with higher expression (p=0.046 by log rank test and p=0.047 by Breslow test for overall survival; p=0.054 by log rank test and P=0.038 by Breslow test for progress free survival). A marginally significant association was found between AAL specific glycoconjugate expression and overall survival by univariate Cox regression analysis (p=0.059). CONCLUSIONS: Lower AAL specific glycoconjugate expression is a significant favorable prognostic factor for overall and progress free survival in CRC. This is the first report about the employment of AAL for histochemical analysis of cancer tissues. The binding characteristics of AAL means it has potential to become a powerful tool for the glycan investigation and clinical application.
Asunto(s)
Agrocybe/metabolismo , Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Neoplasias Colorrectales/mortalidad , Lectinas/metabolismo , Neoplasias Colorrectales/patología , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Femenino , Glicosilación , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Pronóstico , Modelos de Riesgos Proporcionales , Tasa de SupervivenciaRESUMEN
Agrocybe praecox is a litter-decomposing Basidiomycota species of the order Agaricales, and is frequently found in forests and open woodlands. A. praecox grows in leaf-litter and the upper soil and is able to colonize bark mulch and wood chips. It produces extracellular manganese peroxidase (MnP) activities and mineralizes synthetic lignin. In this study, the A. praecox MnP1 isozyme was purified, cloned and enzymatically characterized. The enzyme catalysed the oxidation of Mn(2+) to Mn(3+), which is the specific reaction for manganese-dependent class II heme-peroxidases, in the presence of malonate as chelator with an activity maximum at pH 4.5; detectable activity was observed even at pH 7.0. The coding sequence of the mnp1 gene demonstrates a short-type of MnP protein with a slightly modified Mn(2+) binding site. Thus, A. praecox MnP1 may represent a novel group of atypical short-MnP enzymes. In lignocellulose-containing cultures composed of cereal bran or forest litter, transcription of mnp1 gene was followed by quantitative real-time RT-PCR. On spruce needle litter, mnp1 expression was more abundant than on leaf litter after three weeks cultivation. However, the expression was constitutive in wheat and rye bran cultures. Our data show that the atypical MnP of A. praecox is able to catalyse Mn(2+) oxidation, which suggests its involvement in lignocellulose decay by this litter-decomposer.
Asunto(s)
Agrocybe/enzimología , Peroxidasas/genética , Peroxidasas/metabolismo , Agrocybe/genética , Agrocybe/metabolismo , Clonación Molecular , ADN de Hongos/química , ADN de Hongos/genética , Fibras de la Dieta/metabolismo , Fibras de la Dieta/microbiología , Estabilidad de Enzimas , Expresión Génica , Perfilación de la Expresión Génica , Concentración de Iones de Hidrógeno , Manganeso/metabolismo , Datos de Secuencia Molecular , Oxidación-Reducción , Peroxidasas/química , Peroxidasas/aislamiento & purificación , Hojas de la Planta/metabolismo , Hojas de la Planta/microbiología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Agrocybe aegerita, the black poplar mushroom, has been highly valued as a functional food for its medicinal and nutritional benefits. Several bioactive extracts from A. aegerita have been found to exhibit antitumor and antioxidant activities. However, limited genetic resources for A. aegerita have hindered exploration of this species. METHODOLOGY/PRINCIPAL FINDINGS: To facilitate the research on A. aegerita, we established a deep survey of the transcriptome and proteome of this mushroom. We applied high-throughput sequencing technology (Illumina) to sequence A. aegerita transcriptomes from mycelium and fruiting body. The raw clean reads were de novo assembled into a total of 36,134 expressed sequences tags (ESTs) with an average length of 663 bp. These ESTs were annotated and classified according to Gene Ontology (GO), Clusters of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways. Gene expression profile analysis showed that 18,474 ESTs were differentially expressed, with 10,131 up-regulated in mycelium and 8,343 up-regulated in fruiting body. Putative genes involved in polysaccharide and steroid biosynthesis were identified from A. aegerita transcriptome, and these genes were differentially expressed at the two stages of A. aegerita. Based on one-dimensional gel electrophoresis (1-DGE) coupled with electrospray ionization liquid chromatography tandem MS (LC-ESI-MS/MS), we identified a total of 309 non-redundant proteins. And many metabolic enzymes involved in glycolysis were identified in the protein database. CONCLUSIONS/SIGNIFICANCE: This is the first study on transcriptome and proteome analyses of A. aegerita. The data in this study serve as a resource of A. aegerita transcripts and proteins, and offer clues to the applications of this mushroom in nutrition, pharmacy and industry.
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Agrocybe/genética , Agrocybe/metabolismo , Proteoma/análisis , Transcriptoma/genética , Electroforesis en Gel de Poliacrilamida , Metabolismo Energético/genética , Etiquetas de Secuencia Expresada , Cuerpos Fructíferos de los Hongos/genética , Cuerpos Fructíferos de los Hongos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Micelio/genética , Micelio/metabolismo , Polisacáridos/biosíntesis , Proteómica/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Ionización de Electrospray , Esteroides/biosíntesis , Espectrometría de Masas en Tándem , Vocabulario ControladoRESUMEN
A novel lectin was isolated from the mushroom Agrocybe aegerita (designated AAL-2) by affinity chromatography with GlcNAc (N-acetylglucosamine)-coupled Sepharose 6B after ammonium sulfate precipitation. The AAL-2 coding sequence (1224 bp) was identified by performing a homologous search of the five tryptic peptides identified by MS against the translated transcriptome of A. aegerita. The molecular mass of AAL-2 was calculated to be 43.175 kDa from MS, which was consistent with the data calculated from the amino acid sequence. To analyse the carbohydrate-binding properties of AAL-2, a glycan array composed of 465 glycan candidates was employed, and the result showed that AAL-2 bound with high selectivity to terminal non-reducing GlcNAc residues, and further analysis revealed that AAL-2 bound to terminal non-reducing GlcNAc residues with higher affinity than previously well-known GlcNAc-binding lectins such as WGA (wheatgerm agglutinin) and GSL-II (Griffonia simplicifolia lectin-II). ITC (isothermal titration calorimetry) showed further that GlcNAc bound to AAL-2 in a sequential manner with moderate affinity. In the present study, we also evaluated the anti-tumour activity of AAL-2. The results showed that AAL-2 could bind to the surface of hepatoma cells, leading to induced cell apoptosis in vitro. Furthermore, AAL-2 exerted an anti-hepatoma effect via inhibition of tumour growth and prolongation of survival time of tumour-bearing mice in vivo.
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Acetilglucosamina/metabolismo , Agrocybe/metabolismo , Proteínas Fúngicas/metabolismo , Lectinas/metabolismo , Secuencia de Aminoácidos , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proteínas Fúngicas/química , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/uso terapéutico , Humanos , Lectinas/química , Lectinas/aislamiento & purificación , Lectinas/uso terapéutico , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Datos de Secuencia Molecular , Unión Proteica , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Sialic acid represents a critical sugar component located at the outermost position of glycoconjugates, playing important roles in extensive biological processes. To date, however, there have been only few probes which show affinity to α(2,3)-linked sialic acid-containing glycoconjugates. Agrocybe cylindracea galectin is known to have a relatively high affinity towards Neu5Acα(2,3)Galß(1,4)Glc (3'-sialyl lactose), but it significantly recognizes various ß-galactosides, such as Galß(1,4)GlcNAcß (LacNAc) and Galß(1,3)GalNAcα (T-antigen). To eliminate this background specificity, we focused an acidic amino acid residue (Glu86), which interacts with the glucose unit of 3'-sialyl lactose and substituted it with all other amino acids. Carbohydrate-binding specificity of the derived 14 mutants was analysed by surface plasmon resonance, and it was found that E86D mutant (Glu86 substituted with Asp) substantially lost the binding ability to LacNAc and T-antigen, while it retained the high affinity for 3'-sialyl lactose. Further, frontal affinity chromatography analysis using 132 pyridylaminated oligosaccharides confirmed that the E86D mutant had a strong preference for α(2,3)-disialo biantennary N-linked glycan. However, it showed the large decrease in the affinity for any of the asialo complex-type N-glycans and the glycolipid-type glycans. Thus, the developed mutant E86D will be of practical use in various fields relevant to cell biology and glycotechnology.
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Agrocybe/metabolismo , Galectinas/metabolismo , Mutagénesis/fisiología , Ácido N-Acetilneuramínico/metabolismo , Polisacáridos/metabolismo , Ingeniería de Proteínas/métodos , Agrocybe/genética , Galectinas/química , Galectinas/genética , Enlace de Hidrógeno , Mutagénesis/genética , Ácido N-Acetilneuramínico/química , Unión ProteicaRESUMEN
Mycelia of basidiomycetes differentiating into fruiting body is a controlled developmental process, however the underlying molecular mechanism remains unknown. In previous work, a novel fungal Agrocybe aegerita galectin (AAL) was isolated from A. aegerita in our laboratory. AAL was shown to promote mycelial differentiation in A. aegerita and Auricularia polytricha, indicating that AAL might function as a conserved fruiting initiator during basidiomycete mycelia development. In the current work, we investigate the role of AAL in mycelia differentiation and fruiting body formation. First, the expression and localization of AAL in mycelia, primordium and fruiting body were assessed by Western blotting and immunohistochemistry. AAL was found to be ubiquitously expressed in the primordium and fruiting body but not in the mycelia. AAL facilitated mycelia congregation and promoted fruiting body production when AAL was applied on mycelia. At the same time, when AAL was spread on potato dextrose agar (PDA) medium prior to mycelia inoculation, mycelia exhibited slowed growth rates, resulting in mycelia cords formation and inhibition of fruiting body formation. The 5' regulatory sequence of aal was cloned by 'genome walking'. Here, we show that aal lack introns in the coding region and the upstream 740 bp sequence was characterized by the existence of core promoter elements, which included: two CCAAT boxes (-535/-280), a GC box (-145), a TATA box (-30) and a fungal leader intron within the 5' UTR. The identification of regulatory expression elements may provide an explanation to the stage-specific and high-level expression of aal during fruiting development.