Separate functional domains of human MD-2 mediate Toll-like receptor 4-binding and lipopolysaccharide responsiveness.
Cellular responses to LPS are mediated by a cell surface receptor complex consisting of Toll-like receptor 4 (TLR4), MD-2, and CD14. MD-2 is a secreted protein that interacts with the extracellular portion of TLR4. Site-directed mutagenesis was used to identify the regions of human MD-2 involved in its ability to bind TLR4 and confer LPS responsiveness. A separate region of MD-2 was found to mediate each function. MD-2 binding to TLR4 was dependent on Cys(95) and Cys(105), which might form an intramolecular disulfide bond. Hydrophilic and charged residues surrounding this area, such as R90, K91, D100, and Y102, also contributed to the formation of the TLR4-MD-2 complex. A different region of MD-2 was found to be responsible for conferring LPS responsiveness. This region is not involved in TLR4 binding and is rich in basic and aromatic residues, several of which cooperate for LPS responsiveness and might represent a LPS binding site. Disruption of the endogenous MD-2-TLR4 complex by expression of mutant MD-2 inhibited LPS responses in primary human endothelial cells. Thus, our data indicate that MD-2 interaction with TLR4 is necessary but not sufficient for cellular response to LPS. Either of the two functional domains of MD-2 can be disrupted to impair LPS responses and therefore represent attractive targets for therapeutic interventions.
Re
Fabio
F
Department of Cancer Immunology and AIDS, Dana Farber Cancer Institute, Boston, MA 02115, USA. fabio_re@dfci.harvard.edu
Strominger
Jack L
JL
D000954:Antigens, Surface / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D006367:HeLa Cells; D006801:Humans; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D011956:Receptors, Cell Surface / Q000378:metabolism*; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014162:Transfection
D000954:Antigens, Surface; C495377:LY96 protein, human; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors