The functional and structural properties of MD-2 required for lipopolysaccharide binding are absent in MD-1.
MD-1 and MD-2 are secretory glycoproteins that exist on the cell surface in complexes with transmembrane proteins. MD-1 is anchored by radioprotective 105 (RP105), and MD-2 is associated with TLR4. In vivo studies revealed that MD-1 and MD-2 have roles in responses to LPS. Although the direct binding function of MD-2 to LPS has been observed, the physiological function of MD-1 remains unknown. In this study, we compared the LPS-binding functions of MD-1 and MD-2. LPS binding to cell surface complexes was detected for cells transfected with TLR4/MD-2. In contrast, binding was not observed for RP105/MD-1-transfected cells. When rMD-2 protein was expressed in Escherichia coli, it was purified in complexes containing LPS. In contrast, preparations of MD-1 did not contain LPS. When rMD-2 protein was prepared in a mutant strain lacking the lpxM gene, LPS binding disappeared. Therefore, the secondary myristoyl chain attached to the (R)-3-hydroxymyristoyl chain added by LpxM is required for LPS recognition by MD-2, under these conditions. An amphipathic cluster composed of basic and hydrophobic residues in MD-2 has been suggested to be the LPS-binding site. We specifically focused on two Phe residues (119 and 121), which can associate with fatty acids. A mutation at Phe(191) or Phe(121) strongly reduced binding activity, and a double mutation at these residues prevented any binding from occurring. The Phe residues are present in MD-2 and absent in MD-1. Therefore, the LPS recognition mechanism by RP105/MD-1 is distinct from that of TLR4/MD-2.
Tsuneyoshi
Naoko
N
Department of Immunology, Saga Medical School, 5-1-1 Nabeshima, Saga 849-8501, Japan.
Fukudome
Kenji
K
Kohara
Jun
J
Tomimasu
Rika
R
Gauchat
Jean-Francois
JF
Nakatake
Hiroshi
H
Kimoto
Masao
M
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D000954:Antigens, Surface / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000502:physiology; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D008070:Lipopolysaccharides / Q000378:metabolism*; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010649:Phenylalanine / Q000235:genetics; D011956:Receptors, Cell Surface / Q000378:metabolism; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014162:Transfection
D015703:Antigens, CD; D000954:Antigens, Surface; C092722:CD180 protein, human; D002352:Carrier Proteins; C115213:LY86 protein, human; C495377:LY96 protein, human; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D010649:Phenylalanine