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Identification of CD14 residues involved in specific lipopolysaccharide recognition
R A Shapiro1, M D Cunningham, K Ratcliffe
1Inflammation Department, Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121, USA.
Abstract:
CD14 is a key molecule responsible for the innate host inflammatory response to microbial infection. It is able to bind a wide variety of microbial ligands and facilitate the activation of both myeloid and nonmyeloid cells. However, its specific contribution to the innate recognition of bacteria is not known. Presently there is no information on the contribution of individual CD14 residues to Escherichia coli lipopolysaccharide (LPS) binding or on the molecular basis of the interaction between CD14 and LPS from other bacteria. LPS obtained from Porphyromonas gingivalis, a bacterium associated with chronic inflammatory disease, binds CD14 and activates myeloid cells but does not facilitate the activation of nonmyeloid cells. The transfer and binding of these two LPS species to soluble CD14 recombinant globulin proteins with single point mutations was examined. Functional activity of the mutant proteins was monitored by E-selectin expression on human umbilical cord endothelial cells. The analysis identified a charge reversal mutation in a single residue, E47, that demonstrated selective binding to E. coli LPS but not to P. gingivalis LPS. E-selectin activation assays indicated that proteins with mutations at position E47 maintained their structural integrity. Other mutations, including a charge reversal mutation of residue E58, did not significantly reduce the binding of either LPS ligand or the ability of the molecule to facilitate E-selectin activation. These data demonstrate that CD14 can selectively recognize different LPS ligands.
Insights
CD14 binds microbial lipopolysaccharides (LPS) to trigger inflammation. Researchers identified a specific CD14 mutation (E47) that selectively binds E. coli LPS, revealing CD14
Area of Science:
- Immunology
- Microbiology
- Molecular Biology
Background:
- CD14 is crucial for innate immune responses to microbial infections, binding various ligands.
- Its precise role in bacterial recognition and specific interactions with lipopolysaccharide (LPS) remain unclear.
- Different bacterial LPS molecules, like those from E. coli and P. gingivalis, exhibit distinct binding and activation patterns with CD14.
Purpose of the Study:
- To investigate the molecular basis of CD14's interaction with different bacterial LPS ligands.
- To identify specific CD14 residues responsible for selective LPS binding and downstream cellular activation.
- To understand how mutations in CD14 affect its ability to bind and respond to various LPS types.
Main Methods:
- Site-directed mutagenesis was used to create single-point mutations in soluble CD14 recombinant proteins.
- The binding affinity of wild-type and mutant CD14 to LPS from E. coli and P. gingivalis was assessed.
- Functional activity was measured by monitoring E-selectin expression on human umbilical cord endothelial cells.
Main Results:
- A charge reversal mutation at residue E47 of CD14 resulted in selective binding to E. coli LPS, but not P. gingivalis LPS.
- Mutations at E47 did not compromise the structural integrity of the CD14 protein.
- Other mutations, including E58, did not significantly alter LPS binding or E-selectin activation.
Conclusions:
- CD14 possesses the ability to selectively recognize and bind different LPS ligands.
- The E47 residue plays a critical role in the differential recognition of LPS from various bacterial species.
- These findings provide molecular insights into CD14's specificity in innate immune recognition.