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Analysis of lipopolysaccharide binding by CD14
T N Kirkland1, F Finley, D Leturcq
1Department of Pathology and Medicine, University of California, San Diego.
The Journal of Biological Chemistry
|November 25, 1993
Summary
Bacterial lipopolysaccharide (LPS) binds to CD14 on immune cells, requiring LPS-binding protein (LBP). This study quantifies LPS-CD14 interactions, revealing a non-1:1 binding ratio suggesting complex molecular mechanisms.
Area of Science:
- Immunology and Molecular Biology
- Cell Surface Receptor Interactions
- Host-Pathogen Interactions
Background:
- The cell surface protein CD14 plays a crucial role in innate immunity by binding bacterial lipopolysaccharide (LPS).
- LPS binding to CD14, facilitated by LPS-binding protein (LBP), is essential for activating mammalian myeloid cells.
- Understanding the quantitative aspects of this interaction is key to deciphering LPS-induced cellular responses.
Purpose of the Study:
- To quantitatively analyze the binding kinetics and stoichiometry of 3H-labeled LPS to human CD14.
- To investigate the role of CD14 expression and LBP in LPS binding.
- To explore the influence of temperature and cellular energy status on LPS-CD14 complex formation.
Main Methods:
- Quantitative binding assays using 3H-labeled LPS on Chinese hamster ovary (CHO) cells expressing human CD14 and THP-1 macrophage cells.
- Inhibition studies using anti-CD14 monoclonal antibodies.
- Assessment of LPS release after phosphatidylinositol-specific phospholipase C (PI-PLC) treatment to determine binding association.
- Determination of apparent dissociation constants (Kd) and molar ratios of LPS bound per surface CD14 at different temperatures (10°C and 37°C).
Main Results:
- LPS binding to CD14 required CD14 expression and the presence of LBP, and was inhibited by specific anti-CD14 antibodies.
- Binding occurred rapidly (within 10 min) and was largely temperature-independent between 4-37°C.
- PI-PLC treatment released 75-90% of bound LPS, indicating association with glycosylphosphatidylinositol-anchored CD14.
- Apparent dissociation constants for LPS binding to CD14 were in the range of 2.7-4.9 x 10^-8 M at 10°C and 2.7 x 10^-8 M at 37°C.
- The molar ratio of LPS bound per surface CD14 was approximately 20:1 at saturating concentrations at 10°C and 8:1 at 37°C, significantly deviating from a 1:1 interaction.
Conclusions:
- CD14, in conjunction with LBP, mediates LPS binding to myeloid cells with high affinity.
- The observed supra-stoichiometric binding of LPS to CD14 suggests complex molecular interactions, potentially involving CD14's leucine-rich motifs.
- These findings provide quantitative insights into the initial steps of LPS recognition, crucial for understanding innate immune responses.