Secreted MD-2 is a large polymeric protein that efficiently confers lipopolysaccharide sensitivity to Toll-like receptor 4.
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 different-sized 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.
Visintin
A
A
Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-1360, USA.
Mazzoni
A
A
Spitzer
J A
JA
Segal
D M
DM
D000954:Antigens, Surface / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D002460:Cell Line; D002461:Cell Line, Transformed; D003713:Dendritic Cells / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D004220:Disulfides; D029721:Drosophila Proteins; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D008070:Lipopolysaccharides / Q000276:immunology* / Q000494:pharmacology; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology*; D009842:Oligopeptides; D011956:Receptors, Cell Surface / Q000235:genetics / Q000276:immunology*; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors
D000954:Antigens, Surface; D004220:Disulfides; D029721:Drosophila Proteins; C495377:LY96 protein, human; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins; D009842:Oligopeptides; D011956:Receptors, Cell Surface; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors