Glycan array analysis of Pholiota squarrosa lectin and other fucose-oriented lectins.

The α(1,6)fucose residue attached to the N-glycoprotein core is suspected to play an essential role in the progression of several types of cancer. Lectins remain the first choice for probing glycan modifications, although they may lack specificity. Thus, efforts have been made to identify new lectins with a narrower core fucose (CF) detection profile. Here, we present a comparison of the classical Aleuria aurantia lectin (AAL), Lens culinaris agglutinin (LCA) and Aspergillus oryzae lectin (AOL) with the newer Pholiota squarrosa lectin (PhoSL), which has been described as being specific for core fucosylated N-glycans. To this end, we studied the binding profiles of the four lectins using mammalian glycan arrays from the Consortium of Functional Glycomics. To validate their glycan specificity, we probed AOL, LCA and PhoSL in western-blot assays using protein extracts from eight common colorectal cancer (CRC) lines and colorectal biopsies from a small cohort of patients with CRC. The results showed that (i) LCA and PhoSL were the most specific lectins for detecting the presence of CF in a concentration-dependent manner; (ii) PhoSL exhibited the highest N-glycan sequence restriction, with preferential binding to core fucosylated paucimannosidic-type N-glycans, (iii) the recognition ability of PhoSL was highly influenced by the presence of terminal N-acetyl-lactosamine; (iv) LCA bound to paucimannosidic, bi-antennary and tri-antennary core fucosylated N-glycans and (v) AOL and AAL exhibited broader specificity towards fucosylation. Together, our results support the choice of LCA as the most appropriate lectin for CF detection, as validated in protein extracts from CRC cell lines and tissue specimens from patients with CRC.

Serum core-type fucosylated prostate-specific antigen index for the detection of high-risk prostate cancer.

It is known that core-type fucosylation is higher in prostate cancer cells than in other cancer cell types and is associated with high-risk prostate cancer. Here, we developed an automated microcapillary electrophoresis-based immunoassay system for measuring serum core-type fucosylated prostate-specific antigen (PSA) and evaluated whether the serum fucosylated PSA index (FPI) can detect high-risk prostate cancer. Core-type fucosylated-free PSA was measured by our automated microcapillary electrophoresis-based immunoassay system with Pholiota squarrosa lectin. The FPI was calculated from total PSA and the percentage of fucosylated-free PSA. The optimum model to predict Gleason grade (GG) ≥2 was constructed by multivariate logistic regression analysis. Discrimination was assessed by determining the area under the receiver operator characteristic curve (AUC). The study included 252 men who underwent prostate needle biopsy due to elevated serum PSA levels (4-20 ng/mL), including 138 with GG ≥2. A higher FPI was significantly associated with GG (P < .0001). Multivariate logistic regression analysis showed that age, prostate volume and FPI were significant predictors of GG ≥2. The AUC of FPI and the model were 0.729 (95% confidence interval [CI]: 0.668-0.790) and 0.837 (95% CI: 0.788-0.886), respectively, compared to 0.629 (95% CI: 0.561-0.698) for PSA. Decision curve analysis showed the superior benefit of FPI and the model when compared to PSA. In a cohort with serum PSA levels <20 ng/mL, FPI could differentiate high-risk prostate cancer from biopsy-negative or low-risk prostate cancer. Therefore, FPI could be a useful adjunct in prostate biopsy counseling for men with abnormal PSA levels.

Structural basis for specific recognition of core fucosylation in N-glycans by Pholiota squarrosa lectin (PhoSL).

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.

Reevaluation of Pholiota squarrosa lectin-reactive haptoglobin as a pancreatic cancer biomarker using an improved ELISA system.

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.

Expression of α-glucosidase during morphological differentiation in the basidiomycetous fungus Pholiota microspora.

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.

A novel core fucose-specific lectin from the mushroom Pholiota squarrosa.

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.

Chemical Components Analyzed by UPLC-QTOF-MS and In Vitro Antitumor Effects of the Chestnut Mushroom Pholiota adiposa (Agaricomycetes).

We investigated the chemical constituents and anti-tumor activity of cultivated Pholiota adiposa in vitro using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry. HepG-2, A549, HeLa, and MCF-7 cells, which are 4 kinds of human cancer cell lines, were cultured in vitro, treated with different concentrations of the ethanol extract of Ph. adiposa (EPA), and cytotoxicity was determined using the cell counting kit-8 assay. Flow cytometry was used to analyze the apoptosis of HepG-2 cells via annexin V-fluorescein isothiocyanate/propidium iodide double staining. Expression levels of apoptosis-associated proteins were determined via Western blotting analysis. Thirty-five components were consistent with those recorded in the chemical composition database, with sterols, fatty acids, and polysaccharide compounds accounting for a relatively high proportion. EPA showed the strongest cytotoxicity against HepG-2 cells, increasing the apoptosis rate up to 23.71 ± 1.59% at a concentration of 50 µg/mL. Ph. adiposa has various functional chemical constituents and potential anti-tumor applications. We found that the functional constituents exerted anti-tumor activity by inducing apoptosis. Furthermore, the expression levels of BCL-2-associated X were increased, whereas those of BCL-2 were decreased in cells after treatment with EPA. These results suggest that EPA induces HepG-2 cell apoptosis via a caspase-mediated pathway.

Anti-diabetic effects of fungal Ergosta-4, 6, 8(14), 22-tetraen-3-one from Pholiota adiposa.

OBJECTIVE: Pholiota adiposa is a valuable edible and medicinal fungus. In this research, Ergosta-4, 6, 8(14), 22-tetraen-3-one (ETO) was obtained from Pholiota adiposa which is the first time to study the anti-diabetic and related mechanism. METHODS: Ergosta-4, 6, 8 (14), 22-tetraen-3-one (ETO) was defined by IR and NMR. Relevant biochemical indicators were detected by ELISA assay, hematoxylin-eosin staining (H & E), fasting glucose levels (FBG), oral glucose tolerance test (OGTT), tissue homogenate biochemical measurements, immunohistochemical staining and western blot. RESULTS: In this research, the ETO treatment groups exhibited a significant reduction in fasting blood glucose (FBG) levels, the High dose group (HD) was about 10 mmol/L lower than the diabetic control group (DC), and increase in body weight, the HD group weighed about 5 g more than the DC group on average. Also, the levels of triacylglycerol (TG), total cholesterol (TC), and low-density lipoprotein-cholesterol (LDL-C) were found to be decreased, while the levels of high-density superoxide dismutase (SOD), lipoproteincholesterol (HDL-C), catalase (CAT), and glutathione peroxidase (GSH-Px) were increased in the ETO treatment groups. The pancreatic and liver sections of diabetic control group (DC) exhibited several histopathological changes, but the ETO treatment groups exhibited improvements. ETO treatment led to the significant restoration of islet morphology and function. Moreover, the results of the western blot analysis indicate that ETO could be used for the treatment of diabetes, since it modifies part of the IRS1 / PI3K / AKT signaling pathway.

Study on the physicochemical properties and immunomodulatory anti-tumor effect of the Pholiota adiposa polysaccharide.

In this study, the extraction, purification, physical and chemical properties, and biological activity of the Pholiota adiposa (PAP) polysaccharide were investigated. One fraction (PAP-1a) of Pholiota adiposa polysaccharides was isolated using DEAE Sepharose™ Fast Flow and Sephacryl™ S-300 High-Resolution columns. The HPLGPC results revealed that the molecular weight of PAP-1a was 16.453 kDa. PAP-1a was composed of mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose and their molar % was 33.41, 0.53, 1.33, 0.07, 0.27, 5.28, 38.31, 0.83, 18.04 and 2.23, respectively. PAP-1a could activate macrophages to secrete NO and cytokines such as TNF-a, IL-6, and IL-12p70. When hepatocellular carcinoma cells (HCCs) and macrophages were co-cultured, it was observed that PAP-1a inhibited the growth of Hep-G2, Hep-3B, and Huh7 via immunoregulation. It triggered cell apoptosis by blocking the cell cycle in the G0/G1 stage. Furthermore, PAP-1a had no direct cytotoxicity against the hepatocyte cell line L02 and macrophages RAW264.7.

Optimization of hydroxyl radical scavenging activity of exo-polysaccharides from Inonotus obliquus in submerged fermentation using response surface methodology.

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%.

One new sterpurane sesquiterpene from cultures of the basidiomycete Pholiota nameko.

One new sterpurane sesquiterpene (1), named (3R,6S,7S,8R,10S)-3,7,14-trihydroxy-1-sterpurene was isolated from cultures of the basidiomycete Pholiota nameko. The structure of new compound was elucidated by extensive spectroscopic. Additionally, a single crystal X-ray diffraction not only confirmed the structure, but also determined the absolute configuration of the new compound. The compound was evaluated for cytotoxicity against five human cancer cell lines, but no significant cytotoxicity were found (IC50 values > 40 µM).

A novel antitumor protein from the mushroom Pholiota nameko induces apoptosis of human breast adenocarcinoma MCF-7 cells in vivo and modulates cytokine secretion in mice bearing MCF-7 xenografts.

Breast cancer is the most common malignant tumor in women and one of the three most common cancers worldwide. It is a life-threatening disease among women and the leading cause of death among women. New drugs or new drug translations and laboratory clinical studies are ongoing. A new antitumor protein (PNAP) purified from edible fungus Pholiota nameko has potential for treating breast cancer. We have previously found that PNAP exhibits anti-proliferative and apoptosis-inducing activities in a human breast cancer cell line (MCF-7). In this study, we constructed a BALB/c mouse model of MCF-7 tumor xenografts. In vivo experiments show that PNAP can effectively inhibit the malignant proliferation of MCF-7 solid tumors. This is because PNAP can successfully activate the death receptor pathway and mitochondrial apoptosis pathway of MCF-7 tumor cells in vivo, and induce tumor cells to wither. It is estimated that PNAP may also have an immunoregulatory ability to indirectly inhibit malignant proliferation of tumors. We also found that PNAP may also have the immunomodulatory ability to indirectly inhibit the malignant proliferation of tumors, which can shift the balance of Th1/Th2 to Th1 and eventually inhibit the growth of tumors. The study reveals a new therapeutic approach for breast cancer patient.

The trimeric solution structure and fucose-binding mechanism of the core fucosylation-specific lectin PhoSL.

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.

Target-selective photo-degradation of AFP-L3 and selective photo-cytotoxicity against HuH-7 hepatocarcinoma cells using an anthraquinone-PhoSL hybrid.

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.

Effects of the polysaccharide from Pholiota nameko on human cytokine network in serum.

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.

Extraction and in vitro antioxidant activity of intracellular polysaccharide by Pholiota adiposa SX-02.

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.

[Media optimization for exopolysaccharide by Pholiota squarrosa (Pers. ex Fr.) Quel. AS 5.245 on submerged fermentation].

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.