Nie Xiaomeng, Nishitani Chiaki, Yamazoe Masami, Ariki Shigeru, Takahashi Motoko, Shimizu Takeyuki, Mitsuzawa Hiroaki, Sawada Kaku, Smith Kelly, Crouch Erika, Nagae Hisato, Takahashi Hiroki, Kuroki Yoshio
Department of Biochemistry and Third Department of Internal Medicine, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan.
Biochemistry. 2008 Dec 2;47(48):12878-85. doi: 10.1021/bi8010175.
Pulmonary surfactant protein D (SP-D) is a member of the collectin family and plays crucial roles in the innate immunity of the lung. We have previously shown that surfactant protein A (SP-A), a homologous collectin, interacts with MD-2 and alters lipopolysaccharide signaling. In this study, we examined and characterized the binding of SP-D to MD-2 using a soluble form of recombinant MD-2 (sMD-2). SP-D bound in a concentration- and Ca(2+)-dependent manner to sMD-2 coated onto microtiter wells. Excess mannose abolished the binding of SP-D to sMD-2. In solution, SP-D cosedimented with sMD-2 in the presence of Ca(2+). The direct binding of SP-D to sMD-2 was confirmed by BIAcore analysis. Anti-SP-D monoclonal antibody that recognizes the carbohydrate recognition domain (CRD) of SP-D significantly inhibited the binding of SP-D to sMD-2, indicating the involvement of the CRD for the binding to sMD-2. Ligand blot analysis revealed that SP-D bound to N-glycopeptidase F-treated sMD-2. In addition, the biotinylated SP-D pulled down the mutant sMD-2 with Asn(26) --> Ala and Asn(114) --> Ala substitutions, which lacks the consensus for N-glycosylation. Furthermore, the sMD-2 mutant cosedimented SP-D. These results demonstrate that SP-D directly interacts with MD-2 through the CRD.
肺表面活性蛋白D（SP-D）是凝集素家族的成员，在肺的固有免疫中发挥关键作用。我们之前已经表明，表面活性蛋白A（SP-A），一种同源凝集素，与MD-2相互作用并改变脂多糖信号传导。在本研究中，我们使用重组MD-2的可溶性形式（sMD-2）检测并表征了SP-D与MD-2的结合。SP-D以浓度和Ca(2+)依赖性方式与包被在微量滴定孔上的sMD-2结合。过量的甘露糖消除了SP-D与sMD-2的结合。在溶液中，在Ca(2+)存在下，SP-D与sMD-2共沉降。通过BIAcore分析证实了SP-D与sMD-2的直接结合。识别SP-D碳水化合物识别域（CRD）的抗SP-D单克隆抗体显著抑制了SP-D与sMD-2的结合，表明CRD参与了与sMD-2的结合。配体印迹分析显示SP-D与N-糖苷酶F处理的sMD-2结合。此外，生物素化的SP-D下拉了具有Asn(26)→Ala和Asn(114)→Ala替换的突变体sMD-2，该突变体缺乏N-糖基化的共有序列。此外，sMD-2突变体使SP-D共沉降。这些结果表明SP-D通过CRD直接与MD-2相互作用。
