Molecular characterization of a novel fungal immunomodulatory protein from Ganoderma cf. resinaceum: cloning, heterologous expression and bioactivity

Fungal immunomodulatory proteins from Ganoderma species are promising macromolecular candidates for biotherapeutics and functional food development. However, the protein repertoire of Ganoderma resinaceum remains largely unexplored, and the known Ganoderma members of this family cluster phylogenetically around the LZ-8 lineage, leaving its diversity insufficiently charted. This study aimed to identify a novel fungal immunomodulatory protein from G. cf. resinaceum and to characterize its heterologous production and functional properties. A novel gene, FIP-gre2 , was cloned from the genomic DNA of G. cf. resinaceum by thermal asymmetric interlaced polymerase chain reaction genome walking, together with its upstream and downstream flanking regions. The gene encodes a 111-amino-acid polypeptide that adopts the canonical Fve-type fold yet is phylogenetically distinct from the LZ-8 cluster. The recombinant protein was secreted by Pichia pastoris , purified, and verified by LC-MS/MS peptide mapping (53.2% sequence coverage, including peptides spanning two of the three residues distinguishing FIP-gre2 from its closest homolog FIP-gmi). In RAW 264.7 macrophages, FIP-gre2 acted as a stimulus-dependent immunopotentiator: at non-cytotoxic concentrations (0.5–2 µg/mL) it did not affect the basal phagocytic activity of resting cells, but under lipopolysaccharide-induced inflammatory conditions it enhanced phagocytosis in a dose-dependent manner and attenuated LPS-induced redox imbalance by reducing malondialdehyde accumulation and recovering superoxide dismutase activity. At higher concentrations, FIP-gre2 inhibited the proliferation of T-47D and MCF-7 human breast cancer cells in a dose- and time-dependent manner (half-maximal inhibitory concentrations of 3.66 µg/mL at 24 h and 8.81 µg/mL at 48 h, respectively) and suppressed their long-term clonogenic survival at sub-inhibitory concentrations. FIP-gre2 is a phylogenetically novel Ganoderma immunomodulatory protein with dose-stratified dual functionality, being immunomodulatory at low doses without basal immune activation and antiproliferative at higher doses. This profile mitigates the concern of indiscriminate immune stimulation associated with this protein family and positions FIP-gre2 as a candidate food-derived bioactive ingredient for functional food and nutraceutical development, warranting further mechanistic and in vivo evaluation.