title: Radioiodination of an endotoxin·MD-2 complex generates a novel sensitive, high-affinity ligand for TLR4.
abstract: A purified complex of metabolically labeled [(3)H]lipooligosaccharide (LOS) and recombinant human myeloid differentiation factor 2 (MD-2), [(3)H]LOS·MD-2, has been used to demonstrate pM affinity binding interactions with soluble TLR4 ectodomain (TLR4ecd). For measurement of the binding parameters of membrane-bound TLR4, we took advantage of the stability of endotoxin·MD-2 and tyrosine(s) present on the surface of MD-2 to radioiodinate LOS·MD-2. Radioiodinated LOS·MD-2 generated a reagent with an estimated 1:1 molar ratio of [(125)I] to sMD-2 with 20-fold higher specific radioactivity and TLR4-activating properties comparable to metabolically-labeled LOS·MD-2. LOS·MD-2[(125)I] and [(3)H]LOS·MD-2 have similar affinities for soluble (FLAG) TLR4ecd and for membrane-bound TLR4 in HEK293T/TLR4 cells. In a similar dose-dependent manner, sMD-2 and LOS·MD-2 inhibit LOS·MD-2[(125)I] binding to TLR4 indicating the pM affinity binding of LOS·MD-2[(125)I] is agonist-independent. LOS·MD-2[(125)I] allowed measurement of low levels of cell-surface human or murine TLR4 expressed by stable cell lines (2000-3000 sites/cell) and quantitatively measures low levels of 'MD-2-free' TLR4 (est. 250 molecules/cell) in cells co-expressing TLR4 and MD-2. Occupation of 50-100 TLR4/cell by LOS·MD-2 is sufficient to trigger measurable TLR4-dependent cell activation. LOS·MD-2[(125)I] provides a powerful reagent to measure quantitatively functional human and murine cell-surface TLR4, including in cells where surface TLR4 is potentially functionally significant but not detectable by other methods.
authors: 1Inflammation Program, Department of Internal Medicine, Roy A. and Lucille J. Carver College of Medicine, University of Iowa, Iowa City, IA, USA.
authors: Athmane
authors: A
authors: Teghanemt
authors: Jerrold P
authors: JP
authors: Weiss
authors: Theresa L
authors: TL
authors: Gioannini
mesh_terms: D000818:Animals; D005240:Feasibility Studies; D057809:HEK293 Cells; D006801:Humans; D007113:Immunity, Innate; D007457:Iodine Radioisotopes / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000378:metabolism*; D051218:Lymphocyte Antigen 96 / Q000378:metabolism*; D051379:Mice; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011869:Radioligand Assay; D012680:Sensitivity and Specificity; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D019076:Transgenes / Q000235:genetics
chemical_list: D007457:Iodine Radioisotopes; C495377:LY96 protein, human; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D046912:Multiprotein Complexes; D051197:Toll-Like Receptor 4
keywords: Endotoxin; LPS; MD-2; TLR4