Surfactant Protein A Directly Interacts with TLR4 and MD-2 and Regulates Inflammatory Cellular Response: IMPORTANCE OF SUPRATRIMERIC OLIGOMERIZATION

• Published in: The Journal of biological chemistry Vol. 281; no. 31; pp. 21771 - 21780
• Main Authors: Yamada, Chieko, Sano, Hitomi, Shimizu, Takeyuki, Mitsuzawa, Hiroaki, Nishitani, Chiaki, Himi, Tetsuo, Kuroki, Yoshio
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 04.08.2006
• Subjects: Binding Sites; Calcium; Carbohydrates; Cell Line; Dimerization; Humans; Inflammation - metabolism; Inflammation - pathology; Lipopolysaccharides - metabolism; Lipopolysaccharides - pharmacology; Lymphocyte Antigen 96 - metabolism; Macrophages - cytology; Macrophages - metabolism; NF-kappa B - metabolism; Peptide Fragments; Protein Binding; Protein Structure, Tertiary; Pulmonary Surfactant-Associated Protein A - chemistry; Pulmonary Surfactant-Associated Protein A - metabolism; Pulmonary Surfactant-Associated Protein A - physiology; Toll-Like Receptor 4 - metabolism; Transfection; Tumor Necrosis Factor-alpha - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M513041200

The purpose of the current study was to examine the binding of pulmonary surfactant protein A (SP-A) to TLR4 and MD-2, which are critical signaling receptors for lipopolysaccharides (LPSs). The direct binding of SP-A to the recombinant soluble form of extracellular TLR4 domain (sTLR4) and MD-2 was detected using solid-phase binding, immunoprecipitation, and BIAcore. SP-A bound to sTLR4 and MD-2 in a Ca²⁺-dependent manner, and an anti-SP-A monoclonal antibody whose epitope lies in the region Thr¹⁸⁴-Gly¹⁹⁴ blocked the SP-A binding to sTLR4 and MD-2, indicating the involvement of the carbohydrate recognition domain (CRD) in the binding. SP-A avidly bound to the deglycosylated forms of sTLR4 and MD-2, suggesting a protein/protein interaction. In addition, SP-A attenuated cell surface binding of smooth LPS and smooth LPS-induced NF-κB activation in TLR4/MD-2-expressing cells. To know the role of oligomerization in the interaction of SP-A with TLR4 and MD-2, the collagenase-resistant fragment (CRF), which consisted of CRD plus neck domain of SP-A, was isolated. CRF assembled as a trimer, whereas SP-A assembled as a higher order oligomer. Although CRD was suggested to be involved in the binding, CRF exhibited approximately 600- and 155-fold higher KD for the binding to TLR4 and MD-2, respectively, when compared with SP-A. Consistently significantly higher molar concentrations of CRF were required to inhibit smooth LPS-induced NF-κB activation and tumor necrosis factor-α secretion. These results demonstrate for the first time the direct interaction between SP-A and TLR4/MD-2 and suggest the importance of supratrimeric oligomerization in the immunomodulatory function of SP-A.