Pulmonary surfactant protein D binds MD-2 through the carbohydrate recognition domain.
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.
Nie
Xiaomeng
X
Department of Biochemistry and Third Department of Internal Medicine, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan.
Nishitani
Chiaki
C
Yamazoe
Masami
M
Ariki
Shigeru
S
Takahashi
Motoko
M
Shimizu
Takeyuki
T
Mitsuzawa
Hiroaki
H
Sawada
Kaku
K
Smith
Kelly
K
Crouch
Erika
E
Nagae
Hisato
H
Takahashi
Hiroki
H
Kuroki
Yoshio
Y
D050260:Carbohydrate Metabolism; D004586:Electrophoresis; D006801:Humans; D051218:Lymphocyte Antigen 96 / Q000737:chemistry / Q000378:metabolism*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D037663:Pulmonary Surfactant-Associated Protein D / Q000737:chemistry* / Q000378:metabolism*; D012995:Solubility
D051218:Lymphocyte Antigen 96; D037663:Pulmonary Surfactant-Associated Protein D; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase