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A new fungal immunomodulatory protein (FIP-fie) has been isolated and purified from the edible golden needle mushroom (Flammulina velutipes). The apparent molecular mass of FIP-fie determined by SDS/PAGE agrees well with the value of 12704 Da calculated from its amino acid composition and sequence. The complete amino acid sequence of FIP-fie was elucidated by protein sequencing techniques. FIP-fie consists of 114 amino acid residues with an acetylated amino end, and lacks methionine, halfcystine and histidine residues. FIP-fie was able to hemagglutinate human red blood cells. The immunomodulatory activity of FIP-fie was demonstrated by its stimulatory activity toward human peripheral blood lymphocytes, and its suppression of systemic anaphylaxis reactions and local swelling of mouse footpads. FIP-fie was found to enhance the transcriptional expression of interleukin-2 and interferon-y. Keywords. Flarnmulina velutipesGolden needle mushrooms (Flammulina velutipes) are a popular edible mushroom in Asia. We previously reported the isolation of a cardiotoxin, flammutoxin, from this mushroom [l]. Many polysaccharide biological response modifiers with antitumor activities, such as lentinan [2, 31 Schizophyllan [4] and OK-432 [5], have been isolated from fungi or bacteria. An anticarcinogenic and immunomodulatory protein has also been isolated from the edible grass mushroom, Volvariella volvacea [6]. Another fungal immunomodulatory protein (FIP), Ling Zhi-8, was isolated from the mycelia of Ganoderma. lucidum [7]. In this study, we describe the isolation and characterization of FIP-fie from the edible golden needle mushroom, E velutipes. The immunomodulatory and hemagglutinating activities of this FIP were studied, and the mechanism of immunornodulatory activity was examined. The complete amino acid sequence was determined by protein sequencing techniques. MATERIALS AND METHODSIsolation of FIP-fie. The fruit bodies of E velutipes (400 g) were homogenized with 1 1 ice-cold 5% acetic acid in the presence of 0.05 M 2-mercaptoethanol [6], and soluble proteins in the supernatant were precipitated by addition of ammonium sul-Correspondence to J. Y. Lin, Abbreviations. FIP-fie, fungal immunomodulatory protein isolated from Flammulina velutipes; IL-2, interleukin-2 ; IFN-y, interferon-y ; RT, reverse transcriptase.

Autophagy is a self-digestive process that degrades the cytoplasmic constituents. Immunomodulatory protein, one major bioactive component of Ganoderma, has antitumor activity. In this study, recombinant fungal immunomodulatory protein, GMI, was cloned from Ganoderma microsporum and purified. We demonstrated that GMI induces lung cancer cell death by activating autophagy, but does not induce apoptotic cell death. On western blot, GMI increased LC3 conversion and decreased p53 expression in a time- and concentration-dependent manner. Cytoplasmic calcium chelator BAPTA-AM was used to prove that GMI promotes autophagy via a calcium-mediated signaling pathway. 3-methyladenine (3-MA), an autophagy inhibitor, enhanced the cytotoxicity of GMI on cell viability assay. Using VZV-G pseudotyped lentivirus-shRNA system for autophagy-related genes silencing, the capabilities of GMI to reduce cell viability and colony formation were abolished in autophagy-defective cells. Furthermore, GMI did not stimulate apoptosis after blocking of autophagy by 3-MA or shRNA knockdown system. In xenograft studies, oral administration of GMI inhibited the tumor growth and induced autophagy significantly in nude mice that had received a subcutaneous injection of A549 cells. This is the first study to reveal the novel function of GMI in activating autophagy. GMI may be a potential chemopreventive agent against non-small cell lung cancer.

LGG had an anti-inflammatory effect on OVA-induced airway inflammation and might be an additional or supplementary therapy for allergic airway diseases.