FIP-VVO, A NEW FUNGAL IMMUNOMODULATORY PROTEIN ISOLATED FROM VOLVARIELLA-VOLVACEA

A new fungal immunomodulatory protein (Fip) has been purified from the edible mushroom, Volvariella volvacea, and designated Fip-vvo. Analys is of the purified protein by SDS/PAGE followed by Coomassie Blue stai ning demonstrated that Fip-vvo is a single polypeptide with an apparen t molecular mass of 15 kDa. Periodic acid/Schiff staining showed that this single polypeptide lacks carbohydrates. Using an in vitro bioassa y measuring blast-formation stimulatory activity, Fip-vvo was shown to stimulate the maximum proliferation of human peripheral blood lymphoc ytes at a concentration of 5 mu g/ml. Fip-vvo was capable of agglutina ting rat red blood cells. Neither haemagglutination nor mitogenic acti vities were inhibited by mono- or dimeric sugars. In vivo, repeat admi nistration of Fip-vvo greatly reduced the production of BSA-induced Ar thus reaction in mice, whereas little effect was observed on the preve ntion of systemic anaphylaxis reactions. The selectively enhanced tran scriptional expression of interleukin (IL)-2, IL-4, interferon-gamma, tumour necrosis factor-a, lymphotoxin and IL-2 receptor by Fip-vvo was also demonstrated by reverse transcriptase-PCR. This finding suggests that Fip-vvo exerts its immunomodulatory effects via cytokine regulat ion. In addition, the complete amino acid sequence of Fip-vvo was obta ined by direct protein sequencing. This protein consists of 112 amino acid residues with a blocked N-terminal end and has a calculated molec ular mass of 12667 Da not including the N-terminal blocking group. By gel filtration analysis, Fip-vvo exhibited a molecular mass of 26 kDa for the native molecules in PBS. This result indicates that native Fip -vvo is most likely a non-covalently associated homodimeric molecule.