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Comparison of lipopolysaccharide-binding functions of CD14 and MD-2
Jun Koraha1, Naoko Tsuneyoshi, Masao Kimoto
1Department of Immunology, Saga Medical School, 5-1-1 Nabeshima, Saga 849-8501, Japan.
Abstract:
Prior to being recognized by the cell surface Toll-like receptor 4/MD-2 complex, lipopolysaccharide (LPS) in the bacterial outer membrane has to be processed by LPS-binding protein and CD14. CD14 forms a complex with monomeric LPS extracted by LPS-binding protein and transfers LPS to the cell surface signaling complex. In a previous study, we prepared a functional recombinant MD-2 using a bacterial expression system. We expressed the recombinant protein in Escherichia coli as a fusion protein with thioredoxin and demonstrated specific binding to LPS. In this study, we prepared recombinant CD14 fusion proteins using the same approach. Specific binding of LPS was demonstrated with a recombinant protein containing 151 amino-terminal residues. The region contained a hydrophilic region and the first three leucine-rich repeats (LRRs). The LRRs appeared to contribute to the binding because removal of the region resulted in a reduction in the binding function. LPS binding to the recombinant MD-2 was resistant to detergents. On the other hand, the binding to CD14 was prevented in the presence of low concentrations of detergents. In the case of human MD-2, the secondary myristoyl chain of LPS added by LpxM was required for the binding. A nonpathogenic penta-acyl LPS mutant lacking the myristoyl chain did not bind to MD-2 but did so normally to CD14. The broader LPS-binding spectrum of CD14 may allow recognition of multiple pathogens, and the lower affinity for LPS binding of CD14 allows transmission of captured materials to MD-2.
Insights
Researchers developed recombinant CD14 fusion proteins to study lipopolysaccharide (LPS) binding. CD14
Area of Science:
- Immunology
- Microbiology
- Biochemistry
Background:
- Lipopolysaccharide (LPS) recognition by Toll-like receptor 4/MD-2 complex involves LPS-binding protein and CD14.
- CD14 facilitates LPS transfer from LPS-binding protein to the cell surface signaling complex.
- Previous work established functional recombinant MD-2 with specific LPS binding.
Purpose of the Study:
- To prepare and characterize recombinant CD14 fusion proteins for LPS binding studies.
- To investigate the structural regions of CD14 involved in LPS binding.
- To compare LPS binding characteristics of CD14 and MD-2.
Main Methods:
- Preparation of recombinant CD14 fusion proteins using a bacterial expression system (Escherichia coli).
- Assessment of LPS binding to recombinant CD14 and MD-2 proteins.
- Evaluation of the effect of detergents and LPS acyl chain modifications on binding.
Main Results:
- A recombinant CD14 protein containing the N-terminal 151 residues, including the first three leucine-rich repeats (LRRs), specifically bound LPS.
- Removal of the LRR region reduced CD14's LPS binding function.
- CD14 LPS binding was inhibited by detergents, unlike MD-2 binding.
- Human MD-2 required the secondary myristoyl chain of LPS for binding, while CD14 bound LPS mutants lacking this chain.
- CD14 demonstrated a broader LPS-binding spectrum and lower affinity compared to MD-2.
Conclusions:
- The N-terminal LRRs of CD14 are crucial for LPS binding.
- CD14 and MD-2 exhibit distinct LPS binding properties, influenced by detergents and LPS structure.
- CD14's broad specificity and lower affinity facilitate pathogen recognition and transfer to MD-2.
