N-terminal of FIP-fve is essential for its immune activity

FIP-fve, an immunomodulatory protein isolated from Flammulina velutipes, has been demonstrated to have activities of immunomodulatory, anti-cancer, anti-allergic and suppression of inflammation. Based on a secondary structure analysis, we known that two FIP-fve monomers form homodimer through the hydrophobic interaction between N-terminal α-helics. Previous studies suggested that FIP-fve can mediate T cells activation through the linkage between T cell receptors (TCR) on T cells and MHC molecules on antigen present cells (APCs) without processing procedure of APCs. This process is similar to the mechanism of superantigen-mediated immune response. The aim of this study was to demonstrate the necessity of dimer structure for FIP-fve immunomodulatory activity and to prove the directly binding of FIP-fve with MHC and TCR. To study the relationship between the dimer structure and immunomodulatory activity of FIP-fve, we constructed the full length 1-114 FIP-fve cDNA and truncated 14-114 FIP-fve cDNA on pET-32a(+) vector and expressed the His-fusion protein His-FIP-fve 1-114 and His-FIP-fve 14-114. After digested by enterokinase to remove the His-tag residue, rFIP-fve 1-114 and rFIP-fve 14-114 were purified with FPLC system. The ability of rFIP-fve 14-114 to stimulate IFN-γ production on murine splenocytes was lower than rFIP-fve 1-114. Incubating murine splenocytes with rFIP-fve 14-114 could not elevate the gene expression of IFN-γ and IL-2. According to the above results, we suggested that the dimerization might be critical for FIP-fve immunomodulatory activity. On the other hand, FIP-fve and RAW 264.7 cell lysate were co-immunoprecipitated to determine superatigen-like characterization of FIP-fve. Results indicated that FIP-fve could interact with MHC class II molecule directly. Interestingly, we also found that FIP-fve was pulled down by both anti-MHC class I antibody and IgG2a which was isotype of anti-MHC class I antibody. However, no co-immunoprecipitated evidence showed that FIP-fve bound with TCR in this study. In conclusion, we proved that FIP-fve directly bind with MHC class II molecule.