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Clinical and Diagnostic Laboratory Immunology, November 2005, p. 1292-1297, Vol. 12, No. 11
1071-412X/05/$08.00+0     doi:10.1128/CDLI.12.11.1292-1297.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Comparison of Lipopolysaccharide-Binding Functions of CD14 and MD-2

Jun Koraha,^1, Naoko Tsuneyoshi,^1, Masao Kimoto,¹ Jean-Francois Gauchat,² Hiroshi Nakatake,³ and Kenji Fukudome^1*

Department of Immunology, Saga Medical School, 5-1-1 Nabeshima, Saga 849-8501, Japan,¹ Department of Pharmacology, University of Montreal, C.P. 6128, Succursale Centre-ville, Montreal, Quebec H3C 3J7, Canada,² First Research Department, The Chemo-Sero-Therapeutic Research Institute, Kikuchi, Kumamoto 869-1298, Japan³

Received 1 July 2005/ Returned for modification 11 August 2005/ Accepted 31 August 2005

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.

J.K. and N.T. contributed equally to this paper.