Secreted MD-2 is a Large Polymeric Protein That Efficiently Confers Lipopolysaccharide Sensitivity to Toll-like Receptor 4

• Published in: Proceedings of the National Academy of Sciences - PNAS 2001-10, Vol.98 (21), p.12156-12161
• Main Authors: Visintin, Alberto, Mazzoni, Alessandra, Spitzer, Jessica A., Segal, David M.
• Format: Article
• Language: English
• Subjects: Antigens, Surface - biosynthesis; Antigens, Surface - genetics; Antigens, Surface - immunology; Biological Sciences; Cell Line; Cell Line, Transformed; Cell lines; Cells; Cultured cells; Dendritic Cells - drug effects; Dendritic Cells - immunology; Dendritic Cells - metabolism; Dimers; Disulfides; Drosophila Proteins; Endoplasmic Reticulum - metabolism; Gels; HEK293 cells; Humans; Immunology; lipopolysaccharides; Lipopolysaccharides - immunology; Lipopolysaccharides - pharmacology; Lymphocyte Antigen 96; MD-2 protein; Membrane Glycoproteins - genetics; Membrane Glycoproteins - immunology; Molecular weight; Molecules; Oligomers; Oligopeptides; Polymers; Proteins; Receptors, Cell Surface - genetics; Receptors, Cell Surface - immunology; TLR4 protein; Toll-Like Receptor 4; Toll-Like Receptors
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.211445098

Toll-like receptor 4 (TLR4), the principal signaling receptor for lipopolysaccharide (LPS) in mammals, requires the binding of MD-2 to its extracellular domain for maximal responsiveness. MD-2 contains a leader sequence but lacks a transmembrane domain, and we asked whether it is secreted into the medium as an active protein. As a source of secreted MD-2 (sMD-2), we used culture supernatants from cells stably transduced with epitope-tagged human MD-2. We show that sMD-2 exists as a heterogeneous collection of large disulfide-linked oligomers formed from stable dimeric subunits and that concentrations of sMD-2 as low as 50 pM enhance the responsiveness of TLR4 reporter cells to LPS. An MD-2-like activity is also released by monocyte-derived dendritic cells from normal donors. When coexpressed, TLR4 indiscriminately associates in the endoplasmic reticulum/cis Golgi with differentsized oligomers of MD-2, and excess MD-2 is secreted into the medium. We conclude that normal and transfected cells secrete a soluble form of MD-2 that binds with high affinity to TLR4 and that could play a role in regulating responses to LPS and other pathogen-derived substances in vivo.