Masao Kimoto

Essential role of MD-2 in LPS responsiveness and TLR4 distribution

Toll-like receptor 4 (TLR4) mediates lipopolysaccharide (LPS) signaling in a variety of cell types. MD-2 is associated with the extracellular domain of TLR4 and augments TLR4-dependent LPS responses in vitro. We show here that MD-2−/− mice do not respond to LPS, do survive endotoxic shock but are susceptible to Salmonella typhimurium infection. We found that in MD-2−/− embryonic fibroblasts, TLR4 was not able to reach the plasma membrane and predominantly resided in the Golgi apparatus, whereas TLR4 was distributed at the leading edge surface of cells in wild-type embryonic fibroblasts. Thus, MD-2 is essential for correct intracellular distribution and LPS-recognition of TLR4.

Role of TLR4/MD-2 and RP105/MD-1 in Innate Recognition of Lipopolysaccharide

TLR4 and RP105 are unique members of the Toll-like receptor (TLR) family molecules. They are associated with small molecules called MD-2 and MD-1, respectively, to form heterodimers (TLR4/MD-2 and RP105/MD-1) and function as recognition/signaling molecules of lipopolysaccharide (LPS), a membrane component of Gram-negative bacteria. Analysis of transfectant cell lines and gene-targeted mice revealed that both MD-2 and MD-1 are involved in the recognition of LPS as well as in the regulation of intracellular distribution and the surface expression of TLR4 and RP105, respectively. Since RP105 or MD-1-deficient mice show a reduced but not complete lack of LPS responsiveness, there may be functional associations between TLR4/MD-2 and RP105/MD-1. In addition, there was an increased frequency of RP105-negative B-lymphocytes in the peripheral blood in several rheumatic diseases, such as systemic lupus erythematosus, suggesting the involvement of RP105 in the pathophysiology of autoimmunity. Further analysis of the structure and function of TLR4/MD-2 and RP105/MD-1 will provide a better understanding of the pathophysiology, and a chance to develop evidence-based treatments for septic shock syndrome and autoimmunity.

The Functional and Structural Properties of MD-2 Required for Lipopolysaccharide Binding Are Absent in MD-1

MD-1 and MD-2 are secretory glycoproteins that exist on the cell surface in complexes with transmembrane proteins. MD-1 is anchored by radioprotective 105 (RP105), and MD-2 is associated with TLR4. In vivo studies revealed that MD-1 and MD-2 have roles in responses to LPS. Although the direct binding function of MD-2 to LPS has been observed, the physiological function of MD-1 remains unknown. In this study, we compared the LPS-binding functions of MD-1 and MD-2. LPS binding to cell surface complexes was detected for cells transfected with TLR4/MD-2. In contrast, binding was not observed for RP105/MD-1-transfected cells. When rMD-2 protein was expressed in Escherichia coli, it was purified in complexes containing LPS. In contrast, preparations of MD-1 did not contain LPS. When rMD-2 protein was prepared in a mutant strain lacking the lpxM gene, LPS binding disappeared. Therefore, the secondary myristoyl chain attached to the (R)-3-hydroxymyristoyl chain added by LpxM is required for LPS recognition by MD-2, under these conditions. An amphipathic cluster composed of basic and hydrophobic residues in MD-2 has been suggested to be the LPS-binding site. We specifically focused on two Phe residues (119 and 121), which can associate with fatty acids. A mutation at Phe191 or Phe121 strongly reduced binding activity, and a double mutation at these residues prevented any binding from occurring. The Phe residues are present in MD-2 and absent in MD-1. Therefore, the LPS recognition mechanism by RP105/MD-1 is distinct from that of TLR4/MD-2.

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

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.

Expression of CD180, a toll-like receptor homologue, is up-regulated in children with Kawasaki disease

Kawasaki disease (KD) is an acute febrile illness in childhood characterized by the formation of aneurysms in coronary arteries. It is believed that KD is caused by infectious agents because of its epidemic waves and high incidence of familial occurrence. Because an increase in the levels and dysfunction of B cells in peripheral blood was reported in KD, we investigated the expression of cluster of differentiation 180 (CD180), a toll-like receptor homologue, in the B cells of children with KD, and in those with bacterial or viral infections. The percentages of CD180 positive B cells were significantly higher in children with KD or viral infections than in those with bacterial infections or in healthy controls. When the expression levels of CD180 were compared by using the mean fluorescent intensity ratio of patients to healthy controls, the level of CD180 expression was also significantly up-regulated in children with KD or viral infections. To clarify the effect of viral infection on the expression of CD180, B cells were stimulated with poly inosinic-cytidyric acid [poly(IC)], a synthetic double-stranded RNA. Poly(IC) clearly enhanced CD180 expression in B cells in vitro, both at the protein and messenger RNA levels. These results suggest that similar mechanisms may be involved in the up-regulation of B cell CD180 expression in patients with either KD or viral infections.