Serum Amyloid A3 Binds MD-2 To Activate p38 and NF-κB Pathways in a MyD88-Dependent Manner

Abstract

Serum amyloid A (SAA) 3 is a major component of the acute phase of inflammation. We previously reported that SAA3 served as an endogenous peptide ligand for TLR4 to facilitate lung metastasis. Because these experiments were performed with SAA3 recombinant proteins purified from Escherichia coli or mammalian cells, we could not rule out the possibility of LPS contamination. In this study, we used SAA3 synthetic peptides to eliminate the presence of LPS in SAA3. We found that the SAA3 synthetic peptide (aa 20–86) (20–86) stimulated cell migration and activated p38 in a manner dependent on TLR4, MD-2, and MyD88. SAA3 (20–86) also activated NF-κB and Rho small GTPase. Using surface plasmon resonance analysis, the binding constant KD values between SAA3 (20–86) or SAA3 (43–57) and TLR4/MD-2 protein highly purified by the baculovirus system were 2.2 and 30 μM, respectively. FLAG-tagged SAA3 tightly bound to protein A–tagged MD-2, but not to TLR4 in baculovirus coinfection experiments. Although SAA3 (20–86) caused a low, but appreciable level of endocytosis in TLR4, it induced the upregulation of both IL-6 and TNF-α, but not IFN-β1. An i.v. injection of SAA3 (43–57) induced the lung recruitment of CD11b+Gr-1+ cells at an estimated serum concentration around its KD value toward TLR4/MD-2. Taken together, these results suggest that SAA3 directly binds MD-2 and activates the MyD88-dependent TLR4/MD-2 pathway.

Eritoran inhibits S100A8-mediated TLR4/MD-2 activation and tumor growth by changing the immune microenvironment

“…We previously confirmed that murine SAA3 is an endogenous ligand of TLR4/MD-2 using SAA3 (20-86) peptides. 28 In case of SAA3, MD-2 is crucial for SAA3 binding to TLR4/MD-2. 28 As shown in Figure 2b, the results suggested that S100A8 also bound to MD-2, but the molecular docking simulation indicated that S100A8 is likely to bind to TLR4 directly, and that the involvement of MD-2 seems to be lesser extent than the case of SAA3 (Figure 3, and Table 1).…”

“…28 In case of SAA3, MD-2 is crucial for SAA3 binding to TLR4/MD-2. 28 As shown in Figure 2b, the results suggested that S100A8 also bound to MD-2, but the molecular docking simulation indicated that S100A8 is likely to bind to TLR4 directly, and that the involvement of MD-2 seems to be lesser extent than the case of SAA3 (Figure 3, and Table 1). Thus, both SAA3 and S100A8 functions as endogenous TLR4 ligand, but the binding style of S100A8 might be different from that of SAA3.…”

“…43 We more recently reported that SAA3 activates TLR4/MD-2 pathway in a MyD88-dependent, but TRIF-independent manner by using an SAA3 peptide. 28 In contrast, in case of S100A8, RAGE and TLR4 are identified as S100A8/A9 receptors. However, the mechanism(s) which signaling mediated by S100A8 mainly has a role in inflammatory response is still unclear.…”

“…In addition to PAMPs, TLRs also recognize damageassociated molecular patterns. Heat-shock protein (Hsp) 60, 24 Hsp70, 25 high-mobility group box 1, 26 S100A8/S100A9, 27 serum amyloid A3 (SAA3), 28,29 Fetuin-A, 30 defensin β, 31 fibrinogen 32 and fibronectin, 33 as well as polysaccharides such as hyaluronan, 34 heparan sulfate, 35 biglycan 36 and decorin 37 appear to be endogenous ligands of TLR4. The ligand-induced dimerization of TLRs triggers the recruitment of adaptor proteins to intracellular TIR (Toll/interleukin-1 receptor) domains to initiate signaling.…”

Human Coagulation Factor X-Adenovirus Type 5 Complexes Poorly Stimulate an Innate Immune Response in Human Mononuclear Phagocytes

“…TLR4 forms a complex with MD-2 for efficient binding on the cell surface (43) and with CD14 for the internalization of TLR4 (35). MD-2 acts as a coreceptor for recognition of both exogenous ligands (43,44) and endogenous ligands (45,46). In addition, TLR4 can bind bacterial and viral PAMPs and, under inflammatory conditions, also endogenous damage-associated molecular pattern molecules (9).…”