Pulmonary surfactant protein D binds MD-2 through the carbohydrate recognition domain.
Pulmonary surfactant protein D binds MD-2 through the carbohydrate recognition domain.
复制标题
DOI：
10.1021/bi8010175
复制
发表时间：
2008-12
期刊：
影响因子：
2.9
通讯作者：
X. Nie;C. Nishitani;Masami Yamazoe;S. Ariki;Motoko Takahashi;Takeyuki Shimizu;H. Mitsuzawa;Kaku Sawada;Kelly A. Smith;E. Crouch;H. Nagae;Hiroki Takahashi;Y. Kuroki
中科院分区：
文献类型：
--
作者：
X. Nie;C. Nishitani;Masami Yamazoe;S. Ariki;Motoko Takahashi;Takeyuki Shimizu;H. Mitsuzawa;Kaku Sawada;Kelly A. Smith;E. Crouch;H. Nagae;Hiroki Takahashi;Y. Kuroki
肺表面活性物质蛋白D（pulmonary surfactant protein D，SP-D）是胶原凝集素家族的一员，在肺的天然免疫中起重要作用。我们以前已经表明，表面活性蛋白A（SP-A），同源聚集蛋白，与MD-2相互作用，改变脂多糖信号。在这项研究中，我们检查和特点的结合SP-D MD-2使用可溶性形式的重组MD-2（sMD-2）。SP-D以浓度和Ca（2+）依赖性方式与包被在微量滴定威尔斯孔上的sMD-2结合。过量的甘露糖破坏了SP-D与sMD-2的结合。在溶液中，SP-D与sMD-2在Ca（2+）存在下共沉淀。通过BIAcore分析证实SP-D与sMD-2的直接结合。抗SP-D单克隆抗体识别SP-D的糖识别结构域（CRD），可显著抑制SP-D与sMD-2的结合，表明CRD参与了SP-D与sMD-2的结合。配体印迹分析显示SP-D与N-糖肽酶F处理的sMD-2结合。此外，生物素化的SP-D使具有Asn（26）-> Ala和Asn（114）-> Ala取代的突变体sMD-2下降，其缺乏N-糖基化的共有序列。此外，sMD-2突变体共沉淀SP-D。这些结果表明SP-D通过CRD与MD-2直接相互作用。
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.