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Martin Hofrichter - One of the best experts on this subject based on the ideXlab platform.

Hui Sun - One of the best experts on this subject based on the ideXlab platform.

  • aanl Agrocybe aegerita lectin 2 is a new facile tool to probe for o glcnacylation
    Glycobiology, 2018
    Co-Authors: Wei Liu, Shuai Jiang, Guanghui Han, Yalin Yin, Qing Yang, Yajun Yang, Gerald W Hart, Hui Sun
    Abstract:

    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.

  • A novel lectin from Agrocybe aegerita shows high binding selectivity for
    2016
    Co-Authors: Barry Hon, Yi Liang, Cheung Wong, Hui Sun
    Abstract:

    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. Key words: Agrocybe aegerita, anti-hepatoma activity, fungal lectin, glycan-binding property, N-acetylglucosamine

  • structural basis of specific recognition of non reducing terminal n acetylglucosamine by an Agrocybe aegerita lectin
    PLOS ONE, 2015
    Co-Authors: Xiaoming Ren, Hui Sun, Shuai Jiang, Xianqing Lan, Dacheng Wang
    Abstract:

    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 GlcNAcβ1-3Galβ1-4GlcNAc 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.

  • transcriptome and proteome exploration to provide a resource for the study of Agrocybe aegerita
    PLOS ONE, 2013
    Co-Authors: Man Wang, Barry Hon Cheung Wong, Yijie Chen, Shuai Jiang, Yi Liang, Yalin Yin, Jie Huang, Yongfu Pan, Hui Sun
    Abstract:

    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.

  • crystallization and preliminary crystallographic studies of the recombinant antitumour lectin from the edible mushroom Agrocybe aegerita
    Biochimica et Biophysica Acta, 2005
    Co-Authors: Na Yang, Hui Sun, Xin Tong, Ye Xiang, Ying Zhang, Dacheng Wang
    Abstract:

    The antitumour lectin from Agrocybe aegerita, named AAL, shows strong inhibition effects on human and mouse tumour cells via apoptosis induction activity. Recombinant AAL (rAAL) has been expressed and purified. Both rAAL and rAAL-lactose complex have been crystallized and their X-ray diffraction data were collected to resolutions of 1.9 A and 1.6 A, respectively. Both crystals belong to space group P2(1) with unit cell parameters a = 53.20 A, b = 66.01 A, c = 57.86 A, beta = 109.38 and a = 53.38 A, b = 66.29 A, c = 58.02 A, beta = 109.03, respectively.

Rene Ullrich - One of the best experts on this subject based on the ideXlab platform.

  • Enzymatic Preparation of 2,5-Furandicarboxylic Acid (FDCA)—A Substitute of Terephthalic Acid—By the Joined Action of Three Fungal Enzymes
    Microorganisms, 2018
    Co-Authors: Rene Ullrich, Alexander Karich, Sebastian Bruno Kleeberg, Martin Hofrichter
    Abstract:

    Enzymatic oxidation of 5-hydroxymethylfurfural (HMF) and its oxidized derivatives was studied using three fungal enzymes: wild-type aryl alcohol oxidase (AAO) from three fungal species, wild-type peroxygenase from Agrocybe aegerita (AaeUPO), and recombinant galactose oxidase (GAO). The effect of pH on different reaction steps was evaluated and apparent kinetic data (Michaelis-Menten constants, turnover numbers, specific constants) were calculated for different enzyme-substrate ratios and enzyme combinations. Finally, the target product, 2,5-furandicarboxylic acid (FDCA), was prepared in a multi-enzyme cascade reaction combining three fungal oxidoreductases at micro-scale. Furthermore, an oxidase-like reaction is proposed for heme-containing peroxidases, such as UPO, horseradish peroxidase, or catalase, causing the conversion of 5-formyl-2-furancarboxylic acid into FDCA in the absence of exogenous hydrogen peroxide.

  • enzymatic preparation of 2 5 furandicarboxylic acid fdca a substitute of terephthalic acid by the joined action of three fungal enzymes
    Microorganisms, 2018
    Co-Authors: Alexander Karich, Rene Ullrich, Sebastian Bruno Kleeberg, Martin Hofrichter
    Abstract:

    Enzymatic oxidation of 5-hydroxymethylfurfural (HMF) and its oxidized derivatives was studied using three fungal enzymes: wild-type aryl alcohol oxidase (AAO) from three fungal species, wild-type peroxygenase from Agrocybe aegerita (AaeUPO), and recombinant galactose oxidase (GAO). The effect of pH on different reaction steps was evaluated and apparent kinetic data (Michaelis-Menten constants, turnover numbers, specific constants) were calculated for different enzyme-substrate ratios and enzyme combinations. Finally, the target product, 2,5-furandicarboxylic acid (FDCA), was prepared in a multi-enzyme cascade reaction combining three fungal oxidoreductases at micro-scale. Furthermore, an oxidase-like reaction is proposed for heme-containing peroxidases, such as UPO, horseradish peroxidase, or catalase, causing the conversion of 5-formyl-2-furancarboxylic acid into FDCA in the absence of exogenous hydrogen peroxide.

  • directed evolution of unspecific peroxygenase from Agrocybe aegerita
    Applied and Environmental Microbiology, 2014
    Co-Authors: Patricia Molinaespeja, Rene Ullrich, Martin Hofrichter, Eva Garciaruiz, David Gonzalezperez, Miguel Alcalde
    Abstract:

    Unspecific peroxygenase (UPO) represents a new type of heme-thiolate enzyme with self-sufficient mono(per)oxygenase activity and many potential applications in organic synthesis. With a view to taking advantage of these properties, we subjected the Agrocybe aegerita UPO1-encoding gene to directed evolution in Saccharomyces cerevisiae. To promote functional expression, several different signal peptides were fused to the mature protein, and the resulting products were tested. Over 9,000 clones were screened using an ad hoc dual-colorimetric assay that assessed both peroxidative and oxygen transfer activities. After 5 generations of directed evolution combined with hybrid approaches, 9 mutations were introduced that resulted in a 3,250-fold total activity improvement with no alteration in protein stability. A breakdown between secretion and catalytic activity was performed by replacing the native signal peptide of the original parental type with that of the evolved mutant; the evolved leader increased functional expression 27-fold, whereas an 18-fold improvement in the kcat/Km value for oxygen transfer activity was obtained. The evolved UPO1 was active and highly stable in the presence of organic cosolvents. Mutations in the hydrophobic core of the signal peptide contributed to enhance functional expression up to 8 mg/liter, while catalytic efficiencies for peroxidative and oxygen transfer reactions were increased by several mutations in the vicinity of the heme access channel. Overall, the directed-evolution platform described is a valuable point of departure for the development of customized UPOs with improved features and for the study of structure-function relationships.

  • Benzene oxygenation and oxidation by the peroxygenase of Agrocybe aegerita
    AMB Express, 2013
    Co-Authors: Alexander Karich, Rene Ullrich, Martin Kluge, Martin Hofrichter
    Abstract:

    Aromatic peroxygenase (APO) is an extracellular enzyme produced by the agaric basidiomycete Agrocybe aegerita that catalyzes diverse peroxide-dependent oxyfunctionalization reactions. Here we describe the oxygenation of the unactivated aromatic ring of benzene with hydrogen peroxide as co-substrate. The optimum pH of the reaction was around 7 and it proceeded via an initial epoxide intermediate that re-aromatized in aqueous solution to form phenol. Identity of the epoxide intermediate as benzene oxide was proved by a freshly prepared authentic standard using GC-MS and LC-MS analyses. Second and third [per]oxygenation was also observed and resulted in the formation of further hydroxylation and following [per]oxidation products: hydroquinone and p -benzoquinone, catechol and o -benzoquinone as well as 1,2,4-trihydroxybenzene and hydroxy- p -benzoquinone, respectively. Using H_2 ^18O_2 as co-substrate and ascorbic acid as radical scavenger, inhibiting the formation of peroxidation products (e.g., p -benzoquinone), the origin of the oxygen atom incorporated into benzene or phenol was proved to be the peroxide. Apparent enzyme kinetic constants (k_cat, K_m) for the peroxygenation of benzene were estimated to be around 8 s^-1 and 3.6 mM. These results raise the possibility that peroxygenases may be useful for enzymatic syntheses of hydroxylated benzene derivatives under mild conditions.

  • stereoselective benzylic hydroxylation of alkylbenzenes and epoxidation of styrene derivatives catalyzed by the peroxygenase of Agrocybe aegerita
    Green Chemistry, 2012
    Co-Authors: Martin Kluge, Rene Ullrich, Katrin Scheibner, Martin Hofrichter
    Abstract:

    Here we report on the stereoselective benzylic hydroxylation and C1–C2 epoxidation of alkylbenzenes and styrene derivatives, respectively, by a heme-thiolate peroxygenase (EC 1.11.2.1) from the fungus Agrocybe aegerita. Benzylic hydroxylation led exclusively to the (R)-1-phenylalkanols. For (R)-1-phenylethanol, (R)-1-phenylpropanol and (R)-1-tetralol, the ee reached >99%. For longer chain lengths, the enantiomeric excesses (ee) and total turnover numbers (TTN) decreased while the number of by-products, e.g. 1-phenylketones, increased. Epoxidation of straight chain and cyclic styrene derivatives gave a heterogeneous picture and resulted in moderate to excellent ee values and TTN: e.g., in the case of (1R,2S)-cis-β-methylstyrene oxide formation, an ee >99% and a TTN of 110 000 was achieved. Hydroxylation and epoxidation were true peroxygenations, which was demonstrated by the incorporation of 18O from H218O2 into the products. The use of fed-batch devices and varying feeding strategies for the substrate and co-substrate turned out to be a suitable approach to optimize peroxygenase catalysis.

Shu-yao Tsai - One of the best experts on this subject based on the ideXlab platform.

  • non volatile taste components of agaricus blazei Agrocybe cylindracea and boletus edulis
    Food Chemistry, 2008
    Co-Authors: Shu-yao Tsai, Huili Tsai
    Abstract:

    Abstract Three species of dried mushrooms are commercially available in Taiwan, namely Agaricus blazei (Brazilian mushroom), Agrocybe cylindracea (black popular mushroom) and Boletus edulis (king bolete), and their non-volatile taste components were studied. All mushrooms were high in contents of carbohydrate, crude fiber and protein but low in contents of crude ash and fat. Arabitol, myo-inositol, mannitol and trehalose were detected in these three mushrooms, whereas glucose was not found in B. edulis . Contents of total soluble sugars and polyols ranged from 150.33 to 225.08 mg/g. Total free amino acid contents were low in these three mushrooms and ranged from 8.97 to 14.91 mg/g. The contents of MSG-like components ranged from 1.24 to 4.40 mg/g were in the descending order of the A. blazei , A. cylindracea and B. edulis . Total 5′-nucleotides contents of A. blazei and A. cylindracea were higher than that of B. edulis whereas flavor 5′-nucleotides content of A. blazei was higher than those of A. cylindracea and B. edulis . Equivalent umami concentrations values in three mushrooms ranged from 10.46 to 135.90 g per 100 g. Overall, these three mushrooms possessed highly umami taste.

  • antioxidant properties of agaricus blazei Agrocybe cylindracea and boletus edulis
    Lwt - Food Science and Technology, 2007
    Co-Authors: Shu-yao Tsai, Huili Tsai
    Abstract:

    Abstract Three mushrooms are currently available in Taiwan, including Agaricus blazei, Agrocybe cylindracea, and Boletus edulis. Their ethanolic and hot water extracts were prepared and antioxidant properties studied. Ethanolic extracts from three mushrooms were more effective than hot water extracts in antioxidant activity using the conjugated diene method and scavenging ability on 1,1-diphenyl-2-picrylhydrazyl radicals whereas hot water extracts were more effective in reducing power, scavenging ability on hydroxyl radials and chelating ability on ferrous ions as evidenced by their lower EC50 values. Overall, for both extracts, B. edulis was more effective among antioxidant properties assayed. Naturally occurring antioxidant components including total tocopherols (3.18–6.18 mg/g) and total phenols (5.67–5.81 mg/g) were found in the extracts and their contents were associated (r=0.636–0.907) with EC50 value of antioxidant properties. Based on the results obtained, both extracts from these three mushrooms were effective in antioxidant properties.

  • Antioxidant properties of methanolic extracts from Agrocybe cylindracea
    Lwt - Food Science and Technology, 2006
    Co-Authors: Shih-jeng Huang, Shu-yao Tsai, Jeng-leun Mau
    Abstract:

    Abstract Agrocybe cylindracea (DC: Fr.) Mre. (Bolbitiaceae) was available in the form of fruit bodies, mycelia and fermentation filtrate. From these three forms, methanolic extracts were prepared and their antioxidant properties studied. The methanolic extract from fruit bodies showed high inhibitory ability on lipid oxidation (90.0–97.3%) at 5–20 mg/ml and had an EC 50 value of 0.94 mg extract/ml. EC 50 values in reducing power were 2.36, 6.89 and 5.00 mg extract/ml for fruit bodies, mycelia and filtrate, respectively. The scavenging ability of the methanolic extract from fruit bodies on 1,1-diphenyl-2-picrylhydrazyl radicals was 89.0% at 1 mg/ml whereas those from mycelia and filtrate was 91.4% and 94.9% at 10 mg/ml, respectively. The chelating abilities of methanolic extracts from fruit bodies, mycelia and filtrate on ferrous ions were 90.6%, 84.6% and 96.3% at 5 mg/ml, respectively. Total phenols were the major detected naturally occurring antioxidant components found in methanolic extracts from A. cylindracea and in the range of 15.55–23.47 mg/g. Based on EC 50 values, A. cylindracea was good in antioxidant properties, except for the scavenging ability on hydroxyl radicals.

  • Antioxidant properties of hot water extracts from Agrocybe cylindracea
    Food Chemistry, 2006
    Co-Authors: Shu-yao Tsai, Shih-jeng Huang, Jeng-leun Mau
    Abstract:

    Abstract Agrocybe cylindracea (DC: Fr.) Mre. was available in the form of fruit bodies, mycelia and fermentation filtrate. From these three forms, hot-water extracts were prepared and their antioxidant properties were studied. Antioxidant activities of hot-water extracts from fruit bodies, mycelia and filtrate were 63.6%, 81.6% and 56.8% at 20 mg ml−1, respectively. EC50 values in reducing power were 2.72, 3.97 and 3.09 mg ml−1 whereas those in scavenging abilities of 1,1-diphenyl-2-picrylhydrazyl radicals were 0.62, 1.66 and 0.82 mg ml−1 for fruit bodies, mycelia and filtrate, respectively. At 20 mg ml−1, the scavenging abilities of hydroxyl radicals were 80.1%, 57.0% and 54.3% for fruit bodies, mycelia and filtrate, respectively. With regard to EC50 values in chelating abilities on ferrous ions, the hot-water extract from filtrate was better than that from mycelia. Total phenols were the major naturally occurring antioxidant components found in hot-water extracts and in the range of 23.74–30.16 mg g−1. From EC50 values obtained, it can be concluded that hot-water extracts from three forms of A. cylindracea were good in antioxidant properties.

Dacheng Wang - One of the best experts on this subject based on the ideXlab platform.

  • structural basis of specific recognition of non reducing terminal n acetylglucosamine by an Agrocybe aegerita lectin
    PLOS ONE, 2015
    Co-Authors: Xiaoming Ren, Hui Sun, Shuai Jiang, Xianqing Lan, Dacheng Wang
    Abstract:

    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 GlcNAcβ1-3Galβ1-4GlcNAc 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.

  • importance of nuclear localization for the apoptosis induced activity of a fungal galectin aal Agrocybe aegerita lectin
    Biochemical and Biophysical Research Communications, 2009
    Co-Authors: Yi Liang, Shuai Jiang, Xin Tong, Lei Feng, Kun Wang, Jia Cheng Lin, Zi Jian Tang, Hong Hong Liu, Lin Guo, Dacheng Wang
    Abstract:

    Agrocybe aegerita lectin (AAL) was identified previously in our group as a novel galectin from medicinal fungi Agrocybe aegerita, and has been shown to effectively induce cancer cell cycle arrest and apoptosis in vitro and tumor regression in vivo. Here, AAL was observed to translocate into the HeLa cell nucleus and induce cell apoptosis when it was predominantly in the nucleus. The N-terminus and C-terminus of AAL were required for nuclear localization. Site mutated proteins were generated based on AAL structure. Dimer interface mutant I25G, carbohydrate recognition domain (CRD) mutant R63H, and loop region mutant L33A could not enter the nucleus and lost the ability to induce apoptosis. CRD mutant H59Q and loop region mutant I144G maintained nuclear localization activity, and H59Q retained residual bioability but I144G had no activity, indicating that nuclear localization is important but not sufficient for AAL to become apoptotically active. Our findings provide a novel antitumor mechanism of fungal galectin.

  • crystallization and preliminary crystallographic studies of the recombinant antitumour lectin from the edible mushroom Agrocybe aegerita
    Biochimica et Biophysica Acta, 2005
    Co-Authors: Na Yang, Hui Sun, Xin Tong, Ye Xiang, Ying Zhang, Dacheng Wang
    Abstract:

    The antitumour lectin from Agrocybe aegerita, named AAL, shows strong inhibition effects on human and mouse tumour cells via apoptosis induction activity. Recombinant AAL (rAAL) has been expressed and purified. Both rAAL and rAAL-lactose complex have been crystallized and their X-ray diffraction data were collected to resolutions of 1.9 A and 1.6 A, respectively. Both crystals belong to space group P2(1) with unit cell parameters a = 53.20 A, b = 66.01 A, c = 57.86 A, beta = 109.38 and a = 53.38 A, b = 66.29 A, c = 58.02 A, beta = 109.03, respectively.

  • molecular character of the recombinant antitumor lectin from the edible mushroom Agrocybe aegerita
    Journal of Biochemistry, 2005
    Co-Authors: Na Yang, Hui Sun, Xin Tong, Yi Liang, Ye Xiang, Ying Zhang, Dacheng Wang
    Abstract:

    The lectin from Agrocybe aegerita (AAL) has been found to possess potent tumor-suppressing function and tumor cell apoptosis-inducing activity. In this paper, we report the full sequence, the active expression of the gene encoding AAL at a high level and bioassay of the binding property with lactose, apoptosis-inducing activity and DNase activity of recombinant AAL (rAAL). The results reveal that AAL is a member of the galectin family and the dimeric form is the active unit for the functional performance. The rAAL showed comparable tumor cell apoptosis-inducing activity with the wild AAL but no DNase activity at all. The molecular characters revealed by this study are significant for the in-depth investigation of the functional mechanism of this interesting protein.