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1.
Glycobiology ; 29(7): 576-587, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-30913288

RESUMO

Fucosylation of the N-glycan core via the α1-6 linkage (core fucosylation) is detected in specific types of cancers and related diseases, and thereby serves for a relevant biomarker. The lectin from a mushroom Pholiota squarrosa (PhoSL) shows a clear specificity to core fucosylation, without recognizing those with other types of fucosylation, such as the H type via the α1-2 linkage or the Lewis type via the α1-3 or α1-4 linkage. Here we determined the crystal structure of the PhoSL trimer in complex with a disaccharide fucose(α1-6)N-acetylglucosamine (GlcNAc). In the three sugar-binding pockets of PhoSL, extensive hydrophobic and hydrogen-bonding contacts were formed with the fucose moiety. In contrast, the GlcNAc moiety showed only a few hydrophobic and hydrogen-bonding contacts. To elucidate the mechanism for the specificity, we performed molecular dynamics simulations on this disaccharide and a trisaccharide fucose(α1-6)[GlcNAc(ß1-4)]GlcNAc in complex with PhoSL. It was observed that the GlcNAc corresponding to the outer one of the N-glycan core entered the sugar-binding pocket with the N-acetyl group placed stably at the bottom, forming extensive hydrophobic and hydrogen-bonding interactions. In addition, these glycans adopted unstressed favorable conformations when bound to PhoSL. In contrast, H- and Lewis-types of fucosylated trisaccharides adopting favorable conformations caused inevitable steric hindrance with the steep edge of the binding pocket, when docked with PhoSL. Therefore, the specificity to core fucosylation of PhoSL was achieved by a combination of these preferential and exclusive mechanisms.


Assuntos
Fucose/metabolismo , Pholiota/metabolismo , Polissacarídeos/metabolismo , Configuração de Carboidratos , Fucose/química , Simulação de Dinâmica Molecular , Polissacarídeos/química
2.
Sci Rep ; 8(1): 7740, 2018 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-29773815

RESUMO

The core α1-6 fucosylation-specific lectin from a mushroom Pholiota squarrosa (PhoSL) is a potential tool for precise diagnosis of cancers. This lectin consists of only 40 amino acids and can be chemically synthesized. We showed here that a synthesized PhoSL peptide formed a trimer by gel filtration and chemical cross-linking assays, and determined a structure of the PhoSL trimer by NMR. The structure possesses a ß-prism motif with a three-fold rotational symmetry, where three antiparallel ß-sheets are tightly connected by swapping of ß-strands. A triad of Trp residues comprises the structural core, forming NH-π electrostatic interactions among the indole rings. NMR analysis with an excess amount of fucose revealed the structural basis for the molecular recognition. Namely, fucose deeply enters a pocket formed at a junction of ß-sheet edges, with the methyl group placed at the bottom. It forms a number of hydrophobic and hydrogen-bonding interactions with PhoSL residues. In spite of partial similarities to the structures of other functionally related lectins, the arrangement of the antiparallel ß-sheets in the PhoSL trimer is novel as a structural scaffold, and thus defines a novel type of lectin structure.


Assuntos
Fucose/metabolismo , Lectina de Ligação a Manose/química , Lectina de Ligação a Manose/metabolismo , Fragmentos de Peptídeos/química , Pholiota/metabolismo , Multimerização Proteica , Sequência de Aminoácidos , Sítios de Ligação , Fucose/química , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Manose/química , Manose/metabolismo , Modelos Moleculares , Fragmentos de Peptídeos/metabolismo , Conformação Proteica
3.
Glycoconj J ; 34(4): 537-544, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28455724

RESUMO

An increase in Lewis- and core-type fucosylation of haptoglobin has been reported in patients with pancreatic cancer (PC), suggesting that fucosylated haptoglobin is a candidate PC biomarker. Previously, we developed a Pholiota squarrosa lectin antibody enzyme-linked immunosorbent assay (PhoSL-ELISA) system for the detection of core-fucosylated haptoglobin. However, with this methodology, positive results were only obtained for some patients with PC, demonstrating the need for a more sensitive detection system. In the current study, we developed an improved PhoSL-ELISA system with higher sensitivity to detect core-fucosylated haptoglobin using high-concentration urea as a denaturing agent with lectin to facilitate detection. We then reevaluated the performance of PhoSL reactive-core-fucosylated haptoglobin (PhoSL-HP) as a PC biomarker using the improved PhoSL-ELISA system. PhoSL-HP levels in the sera of patients with PC were significantly higher than those in healthy volunteers, with an area under the curve (AUC) value of 0.753. Furthermore, the AUC value of CA19-9 improved from 0.793 to 0.907 when combined with PhoSL-HP. Additionally, several CA19-9-negative cases among the patients with PC were diagnosed as positive for PhoSL-HP. In conclusion, PhoSL-HP detection using our improved ELISA system might allow PhoSL-HP to serve as a potential biomarker for PC and thus might be useful to complement the detection of CA19-9 in PC diagnosis.


Assuntos
Biomarcadores Tumorais/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Haptoglobinas/metabolismo , Lectinas/metabolismo , Neoplasias Pancreáticas/metabolismo , Pholiota/metabolismo , Adulto , Idoso , Antígenos Glicosídicos Associados a Tumores/metabolismo , Biomarcadores Tumorais/sangue , Biotinilação , Feminino , Células Hep G2 , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/sangue , Desnaturação Proteica
4.
J Basic Microbiol ; 56(9): 1036-45, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27106661

RESUMO

The α-glucosidase gene from Pholiota microspora, designated PnGcs, was amplified and characterized. The open reading frame region of PnGcs, from ATG to the stop codon, is 2937 bp and encodes a protein of 979 amino acids with a signal peptide of 20 amino acids at the N-terminus. The predicted amino acid sequence of PnGcs indicated that it is a glycoside hydrolase family 31 protein. Quantitative reverse transcription PCR was used to investigate PnGcs expression in mycelia cultured in minimal medium containing various carbon sources, as well as in tissue during different stages of development of fruiting bodies. When P. microspora was grown in minimal medium supplemented with different carbon sources, PnGcs expression was highest when induced by maltose. During cultivation on sawdust medium, PnGcs expression increased dramatically at the fruiting body formation stage compared with the mycelial growth stage, which implied that PnGcs is closely associated with fruiting body development.


Assuntos
Regulação Fúngica da Expressão Gênica , Maltose/metabolismo , Micélio/metabolismo , Pholiota/metabolismo , alfa-Glucosidases/biossíntese , Sequência de Aminoácidos , DNA Fúngico/genética , Genoma Fúngico/genética , Pholiota/citologia , Pholiota/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , alfa-Glucosidases/genética
5.
Chem Commun (Camb) ; 52(10): 2169-72, 2016 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-26698630

RESUMO

A purposefully-designed anthraquinone-Pholiota squarrosa lectin (PhoSL) hybrid effectively degraded α-fetoprotein-L3 (AFP-L3) associated with liver cancer. Degradation was achieved under light irradiation in the absence of any additives and under neutral pH conditions. Moreover, the hybrid effectively exhibited selective photo-cytotoxicity against HuH-7 hepatocarcinoma cells upon photo-irradiation.


Assuntos
Antraquinonas/química , Carcinoma Hepatocelular/química , Proteínas Fetais/química , Lectinas/química , Neoplasias Hepáticas/química , Neoplasias Hepáticas/metabolismo , Pholiota/química , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proteínas Fetais/metabolismo , Humanos , Lectinas/metabolismo , Dados de Sequência Molecular , Pholiota/metabolismo , Fotólise , Ligação Proteica
6.
J Biol Chem ; 287(41): 33973-82, 2012 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-22872641

RESUMO

Fucα1-6 oligosaccharide has a variety of biological functions and serves as a biomarker for hepatocellular carcinoma because of the elevated presence of fucosylated α-fetoprotein (AFP) in this type of cancer. In this study we purified a novel Fucα1-6-specific lectin from the mushroom Pholiota squarrosa by ion-exchange chromatography and affinity chromatography on thyroglobulin-agarose. The purified lectin was designated as PhoSL (P. squarrosa lectin). SDS-PAGE, MALDI-TOF mass spectrometry, and N-terminal amino acid sequencing indicate that PhoSL has a molecular mass of 4.5 kDa and consists of 40 amino acids (NH(2)-APVPVTKLVCDGDTYKCTAYLDFGDGRWVAQWDTNVFHTG-OH). Isoelectric focusing of the lectin showed bands near pI 4.0. The lectin activity was stable between pH 2.0 and 11.0 and at temperatures ranging from 0 to 100 °C for incubation times of 30 min. When PhoSL was investigated with frontal affinity chromatography using 132 pyridylaminated oligosaccharides, it was found that the lectin binds only to core α1-6-fucosylated N-glycans and not to other types of fucosylated oligosaccharides, such as α1-2-, α1-3-, and α1-4-fucosylated glycans. Furthermore, PhoSL bound to α1-6-fucosylated AFP but not to non-fucosylated AFP. In addition, PhoSL was able to demonstrate the differential expression of α1-6 fucosylation between primary and metastatic colon cancer tissues. Thus, PhoSL will be a promising tool for analyzing the biological functions of α1-6 fucosylation and evaluating Fucα1-6 oligosaccharides as cancer biomarkers.


Assuntos
Fucose/química , Proteínas Fúngicas/química , Lectinas/química , Oligossacarídeos/química , Pholiota/química , Sequência de Aminoácidos , Antígenos de Neoplasias/metabolismo , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Fucose/genética , Fucose/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Humanos , Lectinas/genética , Lectinas/isolamento & purificação , Lectinas/metabolismo , Neoplasias Hepáticas/metabolismo , Dados de Sequência Molecular , Oligossacarídeos/genética , Oligossacarídeos/metabolismo , Pholiota/genética , Pholiota/metabolismo , Ligação Proteica , Estabilidade Proteica
7.
Int J Biol Macromol ; 50(1): 164-70, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-22044749

RESUMO

Some physico-chemical characterizations of Pholiota nameko polysaccharides (PNPS-1) were studied, including sulfate content, UV/visible and infrared spectra, also the variation of cytokine communication network in serum to clarify the pharmacological effects of PNPS-1 by determination of 39 cytokines in serum of healthy volunteers. The result proved that PNPS-1 possessed significant anti-inflammatory activity. Further, we use Microsoft Visio 2007 software to map out the cell-cell communication network diagram. The analysis to the diagram suggested that PNPS-1 could take effect on the innate and adaptive immunity and hematopoiesis of volunteers.


Assuntos
Citocinas/biossíntese , Pholiota/metabolismo , Polissacarídeos/química , Adulto , Comunicação Celular , Físico-Química/métodos , Citocinas/metabolismo , Feminino , Humanos , Inflamação , Masculino , Modelos Estatísticos , Ligação Proteica , Software , Espectrofotometria Ultravioleta/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Fatores de Tempo
8.
J Biosci Bioeng ; 111(1): 50-4, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20801714

RESUMO

Response surface methodology (RSM) was used to optimize the extraction parameters for Pholiota adiposa SX-02 intracellular polysaccharide (IPS) produced during submerged culture. The optimum conditions of IPS extraction were predicted to be, ultrasonic power at 564.93 W, precipitation time 30.34 h and pH 8.28, and IPS yield was estimated at 19.75%. The actual value of IPS under these conditions was 20.51%. The in vitro antioxidant results showed that the inhibition effects of IPS at a dosage of 250 mg/l on superoxide anion, hydroxyl and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical were 74.66 ± 5.31%, 69.20 ± 5.13%, and 75.20 ± 6.73%, respectively, which were 27.51 ± 2.23%, 16.58 ± 1.33%, and 9.46 ± 0.72% higher than that of butylated hydroxytoluene (BHT), respectively. The reducing power of IPS was 0.32 ± 0.02 (absorbance at 700 nm), 39.13 ± 3.47% higher than that of BHT. The results provide a reference for large-scale extraction of IPS by P. adiposa SX-02 in industrial fermentation and the IPS can be used as a potential antioxidant which enhances adaptive immune responses.


Assuntos
Depuradores de Radicais Livres/isolamento & purificação , Pholiota/metabolismo , Polissacarídeos/isolamento & purificação , Compostos de Bifenilo/metabolismo , Meios de Cultura , Depuradores de Radicais Livres/farmacologia , Concentração de Íons de Hidrogênio , Radical Hidroxila/metabolismo , Picratos/metabolismo , Polissacarídeos/farmacologia
9.
J Microbiol Biotechnol ; 20(4): 835-43, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20467262

RESUMO

The objectives of this study were to investigate the effect of fermentation medium on the hydroxyl radical scavenging activity of exo-polysaccharides from Inonotus obliquus by response surface methodology. A two-level fractional factorial design was used to evaluate the effect of different components of medium. Corn flour, peptone, and KH2PO4 were important factors significantly affecting hydroxyl radical scavenging activity. These selected variables were subsequently optimized using path of steepest ascent (descent), a central composite design, and response surface analysis. The optimal medium composition was (% w/v): corn flour 5.30, peptone 0.32, KH2PO4 0.26, MgSO4 0.02, and CaCl2 0.01. Under the optimal condition, the hydroxyl radical scavenging rate (49.4%) was much higher than that using either basal fermentation medium (10.2%) and single variable optimization of fermentation medium (35.5%). The main monosaccharides components of the RSM optimized polysaccharides are rhamnose, arabinose, xylose, mannose, glucose and galactose with molar proportion at 1.45%, 3.63%, 2.17%, 15.94%, 50.00%, and 26.81%.


Assuntos
Depuradores de Radicais Livres/metabolismo , Radical Hidroxila/metabolismo , Microbiologia Industrial/métodos , Pholiota/metabolismo , Polissacarídeos/metabolismo , Fermentação , Modelos Estatísticos
10.
Sheng Wu Gong Cheng Xue Bao ; 20(3): 414-22, 2004 May.
Artigo em Chinês | MEDLINE | ID: mdl-15971616

RESUMO

Our previous work has indicated that mycelium growth and exopolysaccharide accumulation in submerged fermentation by Pholiota squarrosa (Pers. ex Fr.) Quel. AS 5.245 are strongly affected by many internal and external factors, including medium constituents and fermentation conditions. In this study, we use an effective two-phase statistical approach to enhance exopolysaccharide production. In the first phase, Plackett-Burman design was undertaken to evaluate the effects of the twenty factors, i.e., glucose, fructose, maltose, yeast extract, tryptone, K2HPO4, KH2PO4, (NH4)2SO4, NaNO3, FeSO4, MgSO4, MnCl2, ZnCl2, FeCl3, CuSO4.5H2O, vitamin B1, initial pH, the temperature, the medium volume and the duration, to the fermentation. By regression analysis, yeast extract, tryptone, fructose, MgSO4, MnCl2, initial pH and temperature were found to be important for exopolysaccharide production, while glucose, maltose, NaNO3, ZnCl2, vitamin B1, the duration and the volume are important to the mycelium biomass. In the second phase of the optimization process, a response surface methodology (RSM) was used to optimize the above critical internal factors, and to find out the optimal concentration levels and the relationships between these factors. Based on the results of the first phase, a five-level six-factor (yeast extract, fructose, MgSO4, maltose, ZnCl2 and initial pH) central composite rotatable design (CCRD) was employed. By solving the quadratic regression model equation using appropriate statistic methods, the optimal concentrations for obtaining 876.32 microg exopolysaccharide per milliliter of fermentation liquor were calculated as: 6.0g/L yeast extract, 11.5g/L fructose, 0.5g/L MgSO4, 9.6g/L maltose, 38.6mg/L ZnCl2 and with the initial pH 5.3. The experimental data under various conditions have validated the theoretical values.


Assuntos
Meios de Cultura , Fermentação , Pholiota/metabolismo , Polissacarídeos/biossíntese , Frutose/metabolismo , Concentração de Íons de Hidrogênio , Maltose/metabolismo , Pholiota/crescimento & desenvolvimento , Polissacarídeos/análise , Temperatura
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