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Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14
E Hailman1, H S Lichenstein, M M Wurfel
1Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York 10021.
Insights
CD14 protein directly binds bacterial lipopolysaccharide (LPS) without needing LPS-binding protein (LBP). This binding activates immune cells, showing CD14 acts as a direct LPS receptor.
Area of Science:
- Immunology
- Molecular Biology
Background:
- CD14 is a protein involved in cellular responses to bacterial lipopolysaccharide (LPS).
- Both cell-surface and soluble CD14 participate in LPS-mediated signaling.
- The precise interaction mechanism between CD14, LPS, and LPS-binding protein (LBP) remained unclear.
Purpose of the Study:
- To investigate the direct binding of CD14 to LPS.
- To determine the role of LPS-binding protein (LBP) in the CD14-LPS interaction.
- To assess the functional consequences of CD14-LPS binding independent of LBP.
Main Methods:
- Nondenaturing polyacrylamide gel electrophoresis (native PAGE) to analyze protein-ligand interactions.
- Use of recombinant soluble CD14 (rsCD14) and recombinant LBP (rLBP).
- Functional assays measuring integrin function on polymorphonuclear leukocytes (PMN) and E-selectin expression on endothelial cells.
Main Results:
- Recombinant soluble CD14 (rsCD14) directly binds LPS in the absence of LBP.
- The CD14-LPS complex is stable and forms at a low stoichiometry.
- LBP accelerates, but is not essential for, LPS binding to CD14 and subsequent cell activation.
Conclusions:
- CD14 functions as a direct receptor for LPS, both in soluble and cell-surface forms.
- LPS-binding protein (LBP) likely acts as a catalyst to facilitate LPS binding to CD14.
- CD14-mediated cellular responses to LPS can occur independently of LBP.
Abstract:
CD14 is a 55-kD protein found as a glycosylphosphatidylinositol (GPI)-anchored protein on the surface of monocytes, macrophages, and polymorphonuclear leukocytes, and as a soluble protein in the blood. Both forms of CD14 participate in the serum-dependent responses of cells to bacterial lipopolysaccharide (LPS). While CD14 has been described as a receptor for complexes of LPS with LPS-binding protein (LBP), there has been no direct evidence showing whether a ternary complex of LPS, LBP, and CD14 is formed, or whether CD14 binds LPS directly. Using nondenaturing polyacrylamide gel electrophoresis (native PAGE), we show that recombinant soluble CD14 (rsCD14) binds LPS in the absence of LBP or other proteins. Binding of LPS to CD14 is stable and of low stoichiometry (one or two molecules of LPS per rsCD14). Recombinant LBP (rLBP) does not form detectable ternary complexes with rsCD14 and LPS, but it does accelerate the binding of LPS to rsCD14. rLBP facilitates the interaction of LPS with rsCD14 at substoichiometric concentrations, suggesting that LBP functions catalytically, as a lipid transfer protein. Complexes of LPS and rsCD14 formed in the absence of LBP or other serum proteins strongly stimulate integrin function on PMN and expression of E-selectin on endothelial cells, demonstrating that LBP is not necessary for CD14-dependent stimulation of cells. These results suggest that CD14 acts as a soluble and cell surface receptor for LPS, and that LBP may function primarily to accelerate the binding of LPS to CD14.