| Summary: | 碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 91 === Flammulina velutipes is an edible mushroom, called as golden-needle mushroom. A fungal immunomodulatory protein (FIP), FIP-fve with a molecular mass of 13 kDa, was isolated and purified from Flammulina velutipes. The amino acid sequence and secondary structure of FIP-fve are similar to those of variable region of immuno-globulin heavy chain. The secondary structure of FIP-fve is rich in b-structure, and contains sevenb-strands, two a-helices, and one b-turn.
FIP-fve was able to agglutinate hman red blood cells. The immunomodulatory activity of FIP-fve was demonstrated by its stimulatory activity toward T cells. In these studies, we investigated the molecular mechanism of immunomodulatory activity of FIP-fve by examining the profiles of tyrosine-phosphorylated proteins of hPBMCs treated with FIP-fve. Two-dimensional gel electrophoresis and Western blotting analysis were employed to study the change of the profile of tyrosine-phosphorylated proteins of hPBMC after treated with FIP-fve. Proteins such as Lnk etc were found to be probably phosphorylated at tyrosine residues. It suggests that FIP-fve exerts its activity by inducing signal transduction in hPBMC.
The localization of FIP-fve in Jurkat T or U937 (monocyte) cells was studied by using confocal microscopy analysis. FIP-fve was able to bind on the cell membrane of Jurkat T cells, and no translocation of FIP-fve into cytosol was observed 30 min after the treatment. However, FIP-fve bound on the cell membrane of U937 cells after treatment for 5 min, and it was translocated into the cytosol 30 min after the treatment. It suggests that FIP-fve can get into U937 cells and exerts its activity, while for Jurkat T cells, no translocation of FIP-fve into the cytosol occurs.
Furthermore, the protein(s) of hPBMC interacted with FIP-fve was studied by GST pull-down assay followed by LC MS/MS analysis. We found that FIP-fve interacted probably with integrin b-3. According to previous studies and the result that FIP-fve probably interacts with integrin β-3, we inferred that FIP-fve probably acts on CD4+ T cells.
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