Molecular Cloning, Expression and Macrophage Activation of an Immunoregulatory Protein from Cordyceps militaris.

Abstract

Protein components of C. militaris have been reported to possess various biological activities. In our previous research, a Cordyceps militaris-derived immunoregulatory protein (CMIP) was naturally isolated and showed the activity of inhibiting the metastasis of breast cancer cells. This study aimed to obtain recombinant CMIP (rCMIP) using recombinant expression and elucidate its ability to activate macrophages. Recombinant CMIP showed one band at approximately 15 kDa or 30 kDa, or two bands at 15 kDa and 30 kDa, under different denaturation conditions of electrophoresis. The cell binding assay showed that rCMIP selectively binds to the surface of macrophages. After adhesion, it did not induce the apoptosis of RAW 264.7 cells, but promoted their proliferation. Moreover, rCMIP significantly induced the expression of M1 macrophage polarization-related molecules. The mean fluorescence intensity (MFI) of CD 86 was enhanced by 2.1-fold and 3.2-fold under 0.64 μM and 1.6 μM of rCMIP treatment, respectively. Cytokines typically expressed in M1 macrophages, such as TNF-α, iNOS, IL-6, CCL 4, CCL 5 and CXCL 10, were also considerably induced by rCMIP, while the expression of cytokines in typical M2 macrophages, like Arg-1, CCL17 and CCL22, were not changed or slightly decreased. Under rCMIP treatment, the release of NO was also appreciably induced. In the present study, we reported cloning, expression and functional characterization of rCMIP, which was naturally isolated from the fruiting body of C. militaris in our previous study. The data imply that rCMIP possesses immunomodulatory activity in macrophages.

• Authors: Qing Yang, Binmei Jia, Xiaomei Liu, Jialing Fang, Luyang Zhao, Lin Xu, Min Fang, Zhiyong Gong, Hui Sun
• Journal: Molecules (Basel, Switzerland) (Molecules) Vol. 26 Issue 23 (Nov 24 2021) ISSN: 1420-3049 [Electronic] Switzerland
• PMID: 34885688 (Publication Type: Journal Article)

Chemical References

• Cytokines
• Immunologic Factors
• NF-kappa B
• Recombinant Proteins
• Tumor Necrosis Factor-alpha
• Nitric Oxide

Topics

• Animals
• Apoptosis (drug effects)
• Cloning, Molecular
• Cordyceps (chemistry, genetics)
• Cytokines (genetics)
• Gene Expression Regulation (drug effects, immunology)
• Humans
• Immunologic Factors (genetics, immunology, isolation & purification, pharmacology)
• Macrophage Activation (drug effects, immunology)
• Macrophages (drug effects, immunology)
• Mice
• NF-kappa B (genetics, immunology)
• Nitric Oxide (genetics, immunology)
• RAW 264.7 Cells
• Recombinant Proteins (genetics, immunology, isolation & purification, pharmacology)
• Tumor Necrosis Factor-alpha (genetics, immunology)