Project description:Lignosus rhinocerotis (Cooke) Ryvarden (Polyporales, Basidiomycota), also known as the tiger milk mushroom, has received much interest in recent years owing to its wide-range ethnobotanical uses and the recent success in its domestication. The sclerotium is the part with medicinal value. Using two-dimensional gel electrophoresis coupled with mass spectrometry analysis, a total of 16 non-redundant, major proteins were identified with high confidence level in L. rhinocerotis sclerotium based on its genome as custom mapping database. Some of these proteins, such as the putative lectins, immunomodulatory proteins, superoxide dismutase, and aegerolysin may have pharmaceutical potential; while others are involved in nutrient mobilization and the protective antioxidant mechanism in the sclerotium. The findings from this study provide a molecular basis for future research on potential pharmacologically active proteins of L. rhinocerotis.
Project description:Lignosus rhinocerotis (Tiger milk mushroom) is an important folk medicine for indigenous peoples in Southeast Asia. We previously reported its de novo assembled 34.3?Mb genome encoding a repertoire of proteins including a putative bioactive fungal immunomodulatory protein. Here we report the cDNA of this new member (FIP-Lrh) with a homology range of 54-64% to FIPs from other mushroom species, the closest is with FIP-glu (LZ-8) (64%) from Ganoderma lucidum. The FIP-Lrh of 112 amino acids (12.59?kDa) has a relatively hydrophobic N-terminal. Its predicted 3-dimensional model has identical folding patterns to FIP-fve and contains a partially conserved and more positively charged carbohydrates binding pocket. Docking predictions of FIP-Lrh on 14 glycans commonly found on cellular surfaces showed the best binding energy of -3.98?kcal/mol to N-acetylgalactosamine and N-acetylglucosamine. Overexpression of a 14.9?kDa soluble 6xHisFIP-Lrh was achieved in pET-28a(+)/BL21 and the purified recombinant protein was sequence verified by LC-MS/MS (QTOF) analysis. The ability to haemagglutinate both mouse and human blood at concentration ?0.34??M, further demonstrated its lectin nature. In addition, the cytotoxic effect of 6xHisFIP-Lrh on MCF-7, HeLa and A549 cancer cell lines was detected at IC50 of 0.34??M, 0.58??M and 0.60??M, respectively.
Project description:BACKGROUND:Genome mining facilitated by heterologous systems is an emerging approach to access the chemical diversity encoded in basidiomycete genomes. In this study, three sesquiterpene synthase genes, GME3634, GME3638, and GME9210, which were highly expressed in the sclerotium of the medicinal mushroom Lignosus rhinocerotis, were cloned and heterologously expressed in a yeast system. RESULTS:Metabolite profile analysis of the yeast culture extracts by GC-MS showed the production of several sesquiterpene alcohols (C15H26O), including cadinols and germacrene D-4-ol as major products. Other detected sesquiterpenes include selina-6-en-4-ol, ?-elemene, ?-cubebene, and cedrene. Two purified major compounds namely (+)-torreyol and ?-cadinol synthesised by GME3638 and GME3634 respectively, are stereoisomers and their chemical structures were confirmed by 1H and 13C NMR. Phylogenetic analysis revealed that GME3638 and GME3634 are a pair of orthologues, and are grouped together with terpene synthases that synthesise cadinenes and related sesquiterpenes. (+)-Torreyol and ?-cadinol were tested against a panel of human cancer cell lines and the latter was found to exhibit selective potent cytotoxicity in breast adenocarcinoma cells (MCF7) with IC50 value of 3.5 ± 0.58 ?g/ml while ?-cadinol is less active (IC50 = 18.0 ± 3.27 ?g/ml). CONCLUSIONS:This demonstrates that yeast-based genome mining, guided by transcriptomics, is a promising approach for uncovering bioactive compounds from medicinal mushrooms.
Project description:Lignosus rhinocerotis has a long history of use by the indigenous community within East Asia to treat a range of health conditions including asthma and chronic cough. To date, there is limited scientific evidence to support its therapeutic effects in relieving these airways conditions. In this study, we examined the effects of the different molecular weight fractions [high-molecular-weight (HMW), medium-molecular-weight (MMW), and low-molecular-weight (LMW)] obtained from the cold water sclerotial extract (CWE) of L. rhinocerotis on airways patency using airway segments isolated from Sprague Dawley rat in an organ bath set-up. It is demonstrated that the HMW and MMW fractions exhibited higher efficacy in relaxing the pre-contracted airways when compared to the CWE and LMW fraction. In addition, the HMW fraction markedly supressed carbachol-, 5-hydroxytrptamine-, and calcium-induced airway contractions. CWE demonstrated a lower efficacy than the HMW fraction but it also significantly attenuated carbachol- and calcium-induced airway contractions. Results showed that the bronchorelaxation effect of CWE and fractions is mediated via blockade of extracellular Ca2+ influx. The composition analysis revealed the following parts of carbohydrate and proteins, respectively: HMW fraction: 71 and 4%; MMW fraction: 35 and 1%; and LMW fraction: 22 and 0.3%. Our results strongly suggest that the polysaccharide-protein complex or proteins found in the HMW and MMW fractions is likely to contribute to the bronchorelaxation effect of CWE.