Pulmonary surfactant protein D binds MD-2 through the carbohydrate recognition domain

Xiaomeng Nie, Chiaki Nishitani, Masami Yamazoe, Shigeru Ariki, Motoko Takahashi, Takeyuki Shimizu, Hiroaki Mitsuzawa, Kaku Sawada, Kelly Smith, Erika Crouch, Hisato Nagae, Hiroki Takahashi, Yoshio Kuroki

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24 Scopus citations

Abstract

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 Ca2+-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 Ca2+. 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 Asn26 → Ala and Asn114 → 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.

Original languageEnglish
Pages (from-to)12878-12885
Number of pages8
JournalBiochemistry
Volume47
Issue number48
DOIs
StatePublished - Dec 2 2008

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    Nie, X., Nishitani, C., Yamazoe, M., Ariki, S., Takahashi, M., Shimizu, T., Mitsuzawa, H., Sawada, K., Smith, K., Crouch, E., Nagae, H., Takahashi, H., & Kuroki, Y. (2008). Pulmonary surfactant protein D binds MD-2 through the carbohydrate recognition domain. Biochemistry, 47(48), 12878-12885. https://doi.org/10.1021/bi8010175