Abstract
Regions of mouse CD14 required for Toll-like receptor 2 (TLR2)- and TLR4-mediated activation of NF-kappaB were studied in transiently transfected 293 cells. Wild-type CD14 enhanced lipopolysaccharide (LPS)-induced NF-kappaB-dependent reporter activity in cells expressing TLR4/MD-2, and deletion of amino acid regions 35-44, 144-153, 235-243, and 270-275 impaired the TLR4-mediated activation. Unlike human CD14, mouse CD14 truncated at amino acid 151 lost the activity. Deletion of amino acids 35-44 or 235-243 also abrogated TLR2-mediated activation of NF-kappaB, whereas mutants lacking 144-153 and 270-275 retained the activity. Deletion and alanine substitution experiments revealed that amino acids 151-153 and 273-275 were required for the TLR4-mediated activation. Both deletion mutants lacking amino acids 35-44 and 235-243 and alanine substitution mutants in regions 151-153 and 273-275 were expressed on the cell surface and retained the ability to associate with TLR4. A cross-linking study with photoreactive LPS showed that the labeling intensities to CD14 mutants/TLR4/MD-2 were paralleled by the ability of CD14 mutants to increase TLR4-mediated activation. These results indicate that different regions of mouse CD14 are required for TLR4- and TLR2-mediated activation of NF-kappaB and suggest that amino acids 35-44, 151-153, 235-243, and 273-275 of mouse CD14 play an important role in LPS binding and its transfer to TLR4/MD-2.
Highlights
Bacterial lipopolysaccharide (LPS)1 is a constituent of the outer membrane of the cell wall of Gram-negative bacteria and plays a major role in septic shock in humans [1, 2]
These results indicate that different regions of mouse CD14 are required for Toll-like receptor 4 (TLR4)- and Toll-like receptor 2 (TLR2)-mediated activation of NF-B and suggest that amino acids 35– 44, 151–153, 235– 243, and 273–275 of mouse CD14 play an important role in LPS binding and its transfer to TLR4/MD-2
Effects of CD14 Deletion Mutants on TLR4- and TLR2-mediated Activation of NF-B—To explore the structure-activity relationship of mouse CD14, we first examined the effect of wild-type CD14 on TLR4- and TLR2-mediated activation of NF-B
Summary
Bacterial lipopolysaccharide (LPS) is a constituent of the outer membrane of the cell wall of Gram-negative bacteria and plays a major role in septic shock in humans [1, 2]. Toll-like receptor 4 (TLR4) has been identified as another molecule that transmits LPS signaling into intracellular components [7, 8]. TLR2 was initially recognized as a signaling molecule for LPS [15, 16], analyses of TLR2-deficient mice [14, 17] and modified phenol extraction studies [18, 19] showed that an endotoxic substance(s) other than LPS is responsible for TLR2-mediated signaling. Since no structural studies of mouse CD14, especially the regions of the mouse CD14 molecule required for TLR4/MD-2- or TLR2-mediated activation of NF-B, had ever been performed, in the present study we investigated these regions by transiently transfecting 293 cells as a first step in clarifying the speciesspecific actions of Salmonella lipid A
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