Novel Ganoderma protein shows dual immune and anticancer activity in cell studies

Researchers have identified and characterized a previously unknown fungal immunomodulatory protein from the mushroom Ganoderma cf. resinaceum, a species whose protein repertoire in this family had remained largely unexplored. The new gene, named FIP-gre2, was cloned from the fungus’s genomic DNA using thermal asymmetric interlaced polymerase chain reaction genome walking, an approach that also captured the upstream and downstream flanking regions of the gene. The work, published in BMC Biotechnology, positions FIP-gre2 as a phylogenetically distinct member of a protein family that has attracted attention for biotherapeutic and functional food applications.

Sequencing revealed that FIP-gre2 encodes a polypeptide of 111 amino acids that adopts the canonical Fve-type fold characteristic of fungal immunomodulatory proteins. Importantly, phylogenetic analysis showed that the protein sits apart from the LZ-8 cluster, the lineage around which known Ganoderma members of this family have tended to group. This distinction matters because the diversity of Ganoderma immunomodulatory proteins has been insufficiently charted, and most characterized members have clustered tightly around a single lineage.

To produce the protein in quantity, the team expressed it in the yeast Pichia pastoris, which secreted the recombinant protein into the culture medium. After purification, the identity of the product was confirmed by LC-MS/MS peptide mapping, which achieved 53.2 percent sequence coverage. Notably, the mapped peptides spanned two of the three residues that distinguish FIP-gre2 from its closest known homolog, FIP-gmi, providing direct molecular evidence that the expressed protein matched the cloned gene rather than a contaminating homolog.

Functional testing in RAW 264.7 macrophage cells revealed a strikingly dose-dependent behavior. At concentrations of 0.5 to 2 micrograms per milliliter, which were non-cytotoxic, FIP-gre2 did not alter the basal phagocytic activity of resting macrophages. This absence of effect on unstimulated cells is significant because indiscriminate immune activation has been a concern for this protein family. However, when the cells were stimulated with lipopolysaccharide to induce inflammatory conditions, FIP-gre2 enhanced phagocytosis in a dose-dependent manner, acting as a stimulus-dependent immunopotentiator rather than a blanket activator of the immune response.

The protein also modulated oxidative stress in the activated macrophages. Under lipopolysaccharide-induced inflammatory conditions, FIP-gre2 attenuated the resulting redox imbalance by reducing the accumulation of malondialdehyde, a marker of lipid peroxidation, and by recovering superoxide dismutase activity, an antioxidant enzyme that is typically depleted during inflammatory stress. Together, these findings suggest that the protein can support immune cell function while simultaneously buffering the oxidative damage that accompanies inflammatory activation, at least in this cell model.

At higher concentrations, FIP-gre2 displayed a second, distinct activity: it inhibited the proliferation of two human breast cancer cell lines, T-47D and MCF-7, in both a dose- and time-dependent manner. The half-maximal inhibitory concentrations were 3.66 micrograms per milliliter at 24 hours and 8.81 micrograms per milliliter at 48 hours. Beyond blocking proliferation, the protein suppressed the long-term clonogenic survival of these cells even at sub-inhibitory concentrations, meaning that doses below those needed to halt proliferation still reduced the ability of individual cancer cells to form colonies over time.

The authors summarize this behavior as dose-stratified dual functionality: immunomodulatory at low doses without basal immune activation, and antiproliferative at higher doses. According to the study’s conclusions, this profile mitigates the concern of indiscriminate immune stimulation associated with the fungal immunomodulatory protein family and positions FIP-gre2 as a candidate food-derived bioactive ingredient for functional food and nutraceutical development.

Several caveats apply to these findings. All functional results come from cell culture experiments, specifically mouse-derived macrophage cells and two human breast cancer cell lines, and the study’s authors explicitly note that further mechanistic and in vivo evaluation is warranted. The published version is an early-release, peer-reviewed accepted manuscript that remains subject to further editorial processing before the final version of record appears. Effects observed in RAW 264.7 macrophages and cancer cell lines cannot be assumed to translate directly to immune function or cancer outcomes in humans, and no animal or clinical data are reported in this study.

Nevertheless, the molecular characterization is thorough for a discovery-stage study. The combination of genome walking to capture the full gene with flanking regions, heterologous secretion in Pichia pastoris, and peptide-level confirmation by mass spectrometry provides a reproducible pipeline for producing and verifying the protein. The phylogenetic novelty of FIP-gre2, distinct from the LZ-8 cluster, adds a new branch to the known diversity of Ganoderma immunomodulatory proteins and suggests that further exploration of undercharacterized Ganoderma species may yield additional members with distinct functional profiles.

For researchers in the functional food and biotherapeutics fields, the study offers a concrete example of a protein whose immune-stimulating activity is conditional on inflammatory context rather than universal, a property the authors argue reduces safety concerns around indiscriminate immune stimulation. Whether that property holds in living organisms, and whether the antiproliferative effects observed in breast cancer cell lines extend to clinically relevant settings, remain open questions that the authors flag for future work.

Subject of Research: Cloning, heterologous expression and cell-based bioactivity characterization of the novel fungal immunomodulatory protein FIP-gre2 from Ganoderma cf. resinaceum

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

Article References: Shen, Y., Zuo, Z., Zhou, Z., Zhang, Z., Zhang, Q., Jing, S., Wang, Y., Zhu, J., & Li, Q. (2026). Molecular characterization of a novel fungal immunomodulatory protein from Ganoderma cf. resinaceum: cloning, heterologous expression and bioactivity. BMC Biotechnology. https://doi.org/10.1186/s12896-026-01224-z

Image Credits: AI Generated

DOI: 10.1186/s12896-026-01224-z

Keywords: FIP-gre2, Ganoderma cf. resinaceum, fungal immunomodulatory proteins, Pichia pastoris, LC-MS/MS, macrophages, phagocytosis, oxidative stress, breast cancer cell lines, clonogenic survival, functional food, nutraceuticals