Abstract
Ganoderma leucocontextum, a newly identified species from the Tibetan Plateau, has been mainly studied for its polysaccharides and triterpenoids, with no prior reports on fungal immunomodulatory proteins (FIPs). This study explores the biological activity of FIP-gle2, cloned from G. leucocontextum and expressed in Pichia pastoris. The effects and mechanisms of recombinant FIP-gle2 (rFIP-gle2) on cell activity and melanin synthesis in mouse melanoma B16-F10 cells were investigated in vitro. The results showed that the FIP-gle2 gene, with an open reading frame (ORF) of 333 bp, encodes a 111-amino acid polypeptide with a molecular weight of 12.60 kDa and an isoelectric point of 4.48. We achieved a yield of 184.18 mg/L of rFIP-gle2. In vitro functional experiments showed that rFIP-gle2 significantly inhibited the proliferation of B16-F10 melanoma cells and induced apoptosis in a dose-dependent manner, particularly at concentrations above 1 μg/mL. At 3 μg/mL, rFIP-gle2 effectively inhibited tyrosinase activity and reduced melanin content, downregulating microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), and tyrosinase-related proteins (TRP-1 and TRP-2). Furthermore, RNA-seq analysis indicated that differentially expressed genes in treated cells were enriched in the mitogen-activated protein kinase (MAPK) signaling pathway, with Western blotting confirming enhanced phosphorylation of JNK, ERK, and p38 proteins. Thus, P. pastoris is an effective host for rFIP-gle2 production, which shows potential for applications in pharmaceuticals, cosmeceuticals, and food fields.