A novel immunomodulatory protein from Poria cocos induces Toll-like receptor 4-dependent activation within mouse peritoneal macrophages.
Poria cocos is an important Oriental medical fungus with multiple functionalities, yet its bioactive substances and the mechanisms involved have not been fully characterized. A novel immunomodulatory protein (P. cocos immunomodulatory protein; PCP) was purified from the dried sclerotium of P. cocos (Schw.) Wolf using DE-52 cellulose and gel filtration chromatography. Chromatography and electrophoresis results indicated that the native PCP (35.6 kDa) is a disulfide-linked heterodimeric glycoprotein consisting of 14.3 and 21.3 kDa subunits with N- and O-glycosylation. PCP was capable of stimulating RAW 264.7 macrophages in vitro through the induction of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) as well as the regulation of nuclear factor-kappa B (NF-kappaB)-related gene expression. In primary mouse macrophages, PCP directly activated peritoneal cavity macrophages to induce Toll-like receptor 4 (TLR4)-mediated myeloid differentiation factor 88 (MyD88)-dependent signaling. This study demonstrated the cell surface interactions of PCP with TLR4 and the capacity of PCP for TLR4 tyrosine phosphorylation. Results obtained with peritoneal macrophages from TLR4-deficient C57BL/10ScN mice revealed that PCP-induced activation and PCP cell surface binding were significantly attenuated. Moreover, enzymatic deglycosylation decreased PCP-mediated responses, indicating that the glycosylated portion of PCP was a key factor in PCP signaling through TLR4 in peritoneal macrophages. These findings suggest that PCP is a new potential immune stimulator within P. cocos and that TLR4 is primarily responsible for PCP signaling in murine macrophages.
Chang
Hui-Hsin
HH
Department of Horticulture, National TaiwanUniversity, Taipei 10673, Taiwan, Republic of China.
Yeh
Chen-Hao
CH
Sheu
Fuu
F
D000818:Animals; D002460:Cell Line; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects; D006031:Glycosylation; D007155:Immunologic Factors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D008262:Macrophage Activation / Q000187:drug effects* / Q000235:genetics; D008264:Macrophages / Q000187:drug effects / Q000276:immunology; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D016328:NF-kappa B / Q000502:physiology; D055441:Poria / Q000737:chemistry*; D015398:Signal Transduction / Q000187:drug effects; D051197:Toll-Like Receptor 4 / Q000172:deficiency / Q000502:physiology*
D005656:Fungal Proteins; D007155:Immunologic Factors; D016328:NF-kappa B; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4