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Molecular cloning and function characterization of a new macrophage-activating protein from Tremella fuciformis
Chih-Liang Hung1, An-Ju Chang, Xhao-Kai Kuo
1Department of Horticulture and Landscape Architecture and ‡Center for Biotechnology, National Taiwan University , No. 1, Sec. 4, Roosevelt Road, Taipei 10673, Taiwan, R.O.C.
Abstract:
Silver ear mushroom ( Tremella fuciformis ) is an edible fungus with health benefits. In this study, we purified a new T. fuciformis protein (TFP) and demonstrated its ability to activate primary murine macrophages. The isolation procedure involved ammonium sulfate fractionation and ion exchange chromatography. TFP naturally formed a 24 kDa homodimeric protein and did not contain glycan residues. The TFP gene was cloned using the rapid amplification of cDNA ends method, and the cDNA sequence of TFP was composed of 408 nucleotides with a 336 nucleotide open reading frame encoding a 112 amino acid protein. TFP was capable of stimulating TNF-α, IL-1β, IL-1ra, and IL-12 production in addition to CD86/MHC class II expression, mRNA expression of M1-type chemokines, and nuclear NF-κB accumulation in murine peritoneal macrophage cells. Furthermore, TFP failed to stimulate TLR4-neutralized and TLR4-knockout macrophages, suggesting that TLR4 is a required receptor for TFP signaling on macrophages. Taken together, these results indicate that TFP may be an important bioactive compound from T. fuciformis that induces M1-polarized activation through a TLR4-dependent NF-κB signaling pathway.
Insights
A novel protein from Silver ear mushroom (Tremella fuciformis), termed TFP, activates immune cells called macrophages. This protein stimulates an M1 immune response via a pathway involving Toll-like receptor 4 (TLR4).
Area of Science:
- Immunology
- Mycology
- Biochemistry
Background:
- Silver ear mushroom (Tremella fuciformis) is an edible fungus with known health benefits.
- Understanding the bioactive compounds within T. fuciformis can reveal new therapeutic agents.
- Macrophages play a crucial role in the innate and adaptive immune system.
Purpose of the Study:
- To purify and characterize a novel protein from T. fuciformis.
- To investigate the immunomodulatory effects of this protein on murine macrophages.
- To elucidate the signaling pathway involved in TFP-mediated macrophage activation.
Main Methods:
- Protein purification using ammonium sulfate fractionation and ion exchange chromatography.
- Gene cloning via rapid amplification of cDNA ends (RACE).
- Macrophage activation assays measuring cytokine production, surface marker expression, and NF-κB activation.
- Utilized TLR4-neutralized and TLR4-knockout macrophages to assess receptor involvement.
Main Results:
- A 24 kDa homodimeric protein, TFP, was purified from T. fuciformis.
- TFP treatment stimulated the production of pro-inflammatory cytokines (TNF-α, IL-1β, IL-12) and M1-associated markers.
- TFP-induced macrophage activation was dependent on Toll-like receptor 4 (TLR4) and involved NF-κB signaling.
Conclusions:
- TFP is a bioactive protein from T. fuciformis with potent immunomodulatory properties.
- TFP induces M1-polarized activation of macrophages.
- The TFP-mediated immune response is dependent on TLR4 and the NF-κB signaling pathway.
