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Characterization, Recombinant Production, and Bioactivity of a Novel Immunomodulatory Protein from Hypsizygus
Shuhui Yu1,2, Ying Wang2, Yingying Wu2
1School of Chemistry and Life Sciences, Changchun University of Technology, Changchun 130012, China.
Abstract:
A novel fungal immunomodulatory protein (FIP), identified as FIP-hma, was discovered in the genome of an edible mushroom Hypsizygus marmoreus. Bioinformatics analysis suggested FIP-hma contained the cerato-platanin (CP) conserved domain and was categorized into Cerato-type FIP. In phylogenetic analysis, FIP-hma was clustered into a new branch of the FIP family, displaying large system divergence from most of the other FIPs. The higher gene expression of FIP-hma was observed during the vegetative growth stages than that during the reproductive growth stages. In addition, the cDNA sequence of FIP-hma was cloned and successfully expressed in Escherichia coli (E. coli) BL21(DE3). The recombinant protein of FIP-hma (rFIP-hma) was neatly purified and isolated by Ni-NTA and SUMO-Protease. The iNOS, IL-6, IL-1β, and TNF-α levels of RAW 264.7 macrophages were upregulated by rFIP-hma, indicating its activation of an immune response by regulating central cytokines. No cytotoxic effects were observed in an MTT test. The findings of this work discovered a novel immunoregulatory protein from H. marmoreus, provided a systematic bioinformatic profile, suggested an effective approach for its heterologous recombinant production, and reported its potent immunoregulatory activity in macrophages. This study sheds light on the physiological function research of FIPs and their further industrial utilization.
Insights
A new fungal immunomodulatory protein (FIP), FIP-hma, from Hypsizygus marmoreus, activates immune responses in macrophages by upregulating cytokines. This discovery offers potential for industrial applications of fungal proteins.
Area of Science:
- Mycology
- Immunology
- Biochemistry
Background:
- Fungal immunomodulatory proteins (FIPs) are crucial for immune system regulation.
- The edible mushroom Hypsizygus marmoreus is a potential source of novel bioactive compounds.
- Understanding FIP diversity and function is essential for their therapeutic and industrial applications.
Purpose of the Study:
- To discover and characterize a novel FIP from Hypsizygus marmoreus.
- To investigate the phylogenetic position and gene expression of the novel FIP.
- To explore the immunomodulatory activity and recombinant production of the novel FIP.
Main Methods:
- Bioinformatics analysis for conserved domains and phylogenetic relationships.
- Gene expression analysis during different growth stages.
- Heterologous expression of FIP-hma in Escherichia coli and protein purification.
- In vitro assessment of FIP-hma's effect on macrophage cytokine production (iNOS, IL-6, IL-1β, TNF-α) and cytotoxicity (MTT assay).
Main Results:
- A novel FIP, FIP-hma, belonging to the Cerato-type FIP family, was identified in Hypsizygus marmoreus.
- FIP-hma exhibited distinct phylogenetic placement and higher gene expression during vegetative growth.
- Recombinant FIP-hma (rFIP-hma) significantly upregulated pro-inflammatory cytokines in macrophages without exhibiting cytotoxicity.
Conclusions:
- FIP-hma is a novel immunomodulatory protein with potent immune-activating properties in macrophages.
- The study provides a comprehensive profile of FIP-hma, including its bioinformatics, expression, and functional activity.
- This research supports the potential industrial utilization of FIP-hma and contributes to the understanding of FIPs' physiological roles.
