Purification, cloning, and functional characterization of a novel immunomodulatory protein from Antrodia camphorata (bitter mushroom) that exhibits TLR2-dependent NF-κB activation and M1 polarization within murine macrophages.
A new immunomodulatory protein, designated ACA, was purified from the mycelium extract of Antrodia camphorata , a well-known folk medicine bitter mushroom in Taiwan, and N-terminally sequenced. By taking advantage of its N-terminal amino acid sequence, the full-length ACA gene was cloned using rapid amplification of cDNA ends (RACE) approach. This gene encodes a 136 amino acid protein that is homologous to the phytotoxic proteins from fungi. On the basis of the data of N-terminal sequencing and N-glycosidase F treatment, the native ACA was confirmed to be a glycoprotein. The similarity in activation of TLR4-deficient macrophages by both the native ACA and recombinant ACA (rACA) suggested that the glycosyl group(s) of the native ACA was insignificant in macrophage activation. Moreover, the failure of rACA to induce TLR2-deficient macrophages and to activate the RAW 264.7 macrophages transfected with the dominate-negative MyD88 (dnMyD88) indicated that the ACA-mediated macrophage activation was TLR2/MyD88 dependent. Microarray assay of the ACA-activated NFκB-related gene expression showed that rACA demonstrated a LPS-mimetic proinflammatory response toward RAW 264.7 macrophages. Furthermore, rACA enhanced phagocytosis activity and CD86 (B7-2) expression as well as induced TNF-α and IL-1β production within murine peritoneal macrophages. A time-dependent induction of mRNA expression of cytokines TNF-α, IL-1β, IL-6, and IL-12 as well as chemokines CCL3, CCL4, CCL5, and CCL10, but not IL-10, CCL17, CCL22, and CCL24, was observed after the ACA treatment of the macrophages. These results proposed that ACA exhibited M1 polarization and differentiation in macrophages. Thus, ACA is an important immunomodulatory protein of A. camphorata.
Sheu
Fuu
F
Research Center of Food and Biomolecules, Chinese Culture University, Taiwan, Republic of China. fsheu@ntu.edu.tw
Chien
Po-Jung
PJ
Hsieh
Kuang-Yang
KY
Chin
Kah-Lock
KL
Huang
Wan-Ting
WT
Tsao
Chiao-Yin
CY
Chen
Yin-Fang
YF
Cheng
Hui-Chung
HC
Chang
Hui-Hsin
HH
D000595:Amino Acid Sequence; D000818:Animals; D055491:Antrodia / Q000737:chemistry*; D001483:Base Sequence; D002460:Cell Line; D018925:Chemokines / Q000235:genetics; D003001:Cloning, Molecular; D016207:Cytokines / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects; D007155:Immunologic Factors / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D008262:Macrophage Activation / Q000187:drug effects; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D025282:Mycelium / Q000737:chemistry; D016328:NF-kappa B / Q000187:drug effects* / Q000502:physiology; D051193:Toll-Like Receptors / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D014162:Transfection
C558313:ACA (immunomodulatory protein), Antrodia camphorata; D018925:Chemokines; D016207:Cytokines; D005656:Fungal Proteins; D007155:Immunologic Factors; D016328:NF-kappa B; C539092:Tlr12 protein, mouse; D051193:Toll-Like Receptors