Preparation and characterization of truncated human lipopolysaccharide-binding protein in Escherichia coli.
Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria, and is the causative agent of endotoxin shock. LPS induces signal transduction in immune cells when it is recognized by the cell surface complex of toll-like receptor 4 (TLR4) and MD-2. The complex recognizes the lipid A structure in LPS, which is buried in the membrane of the outer envelope. To present the Lipid A structure to the TLR4/MD-2, processing of LPS by LPS-binding protein (LBP) and CD14 is required. In previous studies, we expressed recombinant proteins of human MD-2 and CD14 as fusion proteins with thioredoxin in Escherichia coli, and demonstrated their specific binding abilities to LPS. In this study, we prepared a recombinant fusion protein containing 212 amino terminal residues of human LBP (HLB212) by using the same expression system. The recombinant protein expressed in E. coli was purified as a complex form with host LPS. The binding was not affected by high concentrations of salt, but was prevented by low concentrations of various detergents. Both rough-type LPS lacking the O antigen and smooth-type LPS with the antigen bound to HLBP212. Therefore, oligosaccharide repeats appeared to be unnecessary for the binding. A nonpathogenic penta-acylated LPS also bound to HLBP212, but the binding was weaker than that of the wild type. The hydrophobic interaction between the LBP and acyl chains of lipid A appears to be important for the binding. The recombinant proteins of LPS-binding molecules would be useful for analyzing the defense mechanism against infections.
Kohara
Jun
J
Department of Immunology, Saga Medical School, 5-1-1 Nabeshima, Saga 849-8501, Japan.
Tsuneyoshi
Naoko
N
Gauchat
Jean-François
JF
Kimoto
Masao
M
Fukudome
Kenji
K
D000209:Acute-Phase Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008050:Lipid A / Q000737:chemistry / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D051218:Lymphocyte Antigen 96 / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D019081:O Antigens / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000378:metabolism; D000099231:Lipopolysaccharide-Binding Protein
D000209:Acute-Phase Proteins; D002352:Carrier Proteins; D008050:Lipid A; D018950:Lipopolysaccharide Receptors; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins; D019081:O Antigens; D011994:Recombinant Proteins; D051197:Toll-Like Receptor 4; D000099231:Lipopolysaccharide-Binding Protein; C495377:LY96 protein, human; C495345:TLR4 protein, human