Abstract
Ricin toxin binding subunit B (RTB) is a galactose-binding lectin protein derived from the beans of the castor oil plant (Ricinus communis). Our previous studies have reported a direct immunomodulatory effect of recombinant RTB, which stimulates RAW264.7 cells to produce cytokines including TNF-α. However, the role of RTB in innate immune response and its specific mechanism have not been reported in detail. In this work, the results showed that RTB treatment of macrophages significantly increased TLR4 protein levels. RTB also activated TLR4 downstream events, including MyD88, IRAK, and TRAF6, resulting in macrophage activation and TNF-α production. This process is reflected in the increase of IκB phosphorylation. TLR4 knockdown macrophages treated with RTB exhibited greatly reduced IκB phosphorylation and TNF-α secretion. Moreover, treatment with MyD88 inhibitor also suppressed TNF-α production. The docking of RT and TLR4 was simulated by computer, and the contact residues were concentrated on RTB. Our results suggest that recombinant RTB can activate mouse macrophages to secrete TNF-α through activation of NF-κB via the TLR4 signaling pathways.
Highlights
Ricin is a plant toxin extracted from castor bean (Ricinus communis), which belongs to type II ribosome-inactivating proteins
To study whether Tumor necrosis factor (TNF)-a production by RAW264.7 cells induced by Ricin toxin binding subunit B (RTB) is related to the TLR4 signaling pathway, we first determined the expression of TLR4
To determine whether RTB activates the TLR4 signaling pathway, we examined whether downstream signaling molecules myeloid differentiation primary response protein 88 (MyD88), IL1R–associated kinases (IRAKs), and TNF receptor associated factor 6 (TRAF6) were stimulated by RTB
Summary
Ricin is a plant toxin extracted from castor bean (Ricinus communis), which belongs to type II ribosome-inactivating proteins. The ricin holotoxin consists of a toxic moiety A chain (RTA) and a lectin moiety B chain (RTB) linked via a disulfide bond. The A-chain has N-glycosidase enzymatic activity and causes protein synthesis arrest in mammalian cells. The Bchain is a galactose-binding lectin protein that binds to galactosyl moieties on the eukaryotic cell membrane (Stirpe and Barbieri, 1986; Lord et al, 2003). Our previous studies reported that recombinant RTB is an immunomodulatory stimulus that stimulates the RAW264.7 mouse macrophage cell line to produce inducible NOS (iNOS), tumor necrosis factor–a (TNF-a) and. Ricin Toxin Binding Subunit B interleukin 6 (IL-6) via signaling pathways that may involve protein tyrosine kinase, NF-kB, and JAK-STAT activation (Liu et al, 2013; Xu et al, 2013). The role of RTB in innate immune response and its specific mechanism have not been understood very well
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