Surfactant protein A directly interacts with TLR4 and MD-2 and regulates inflammatory cellular response. Importance of supratrimeric oligomerization.
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-kappaB 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-kappaB activation and tumor necrosis factor-alpha 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.
Yamada
Chieko
C
Department of Biochemistry, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan; Core Research for Engineering, Science, and Technology, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan; Department of Otolaryngology, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan.
Sano
Hitomi
H
Department of Biochemistry, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan; Core Research for Engineering, Science, and Technology, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan. Electronic address: sanohito@sapmed.ac.jp.
Shimizu
Takeyuki
T
Department of Biochemistry, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan; Core Research for Engineering, Science, and Technology, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan.
Mitsuzawa
Hiroaki
H
Department of Biochemistry, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan; Core Research for Engineering, Science, and Technology, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan.
Nishitani
Chiaki
C
Department of Biochemistry, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan; Core Research for Engineering, Science, and Technology, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan.
Himi
Tetsuo
T
Department of Otolaryngology, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan.
Kuroki
Yoshio
Y
Department of Biochemistry, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan; Core Research for Engineering, Science, and Technology, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan.
D001665:Binding Sites; D002118:Calcium; D002241:Carbohydrates; D002460:Cell Line; D019281:Dimerization; D006801:Humans; D007249:Inflammation / Q000378:metabolism / Q000473:pathology*; D008070:Lipopolysaccharides / Q000378:metabolism / Q000494:pharmacology; D051218:Lymphocyte Antigen 96 / Q000378:metabolism*; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D010446:Peptide Fragments; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D037662:Pulmonary Surfactant-Associated Protein A / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D051197:Toll-Like Receptor 4 / Q000378:metabolism*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D002241:Carbohydrates; C495377:LY96 protein, human; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D016328:NF-kappa B; D010446:Peptide Fragments; D037662:Pulmonary Surfactant-Associated Protein A; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; D002118:Calcium