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- Title
Radioiodination of an endotoxin·MD-2 complex generates a novel sensitive, high-affinity ligand for TLR4.
- Authors
Teghanemt, Athmane; Weiss, Jerrold P; Gioannini, Theresa L
- Abstract
A purified complex of metabolically labeled [3H]lipooligosaccharide (LOS) and recombinant human myeloid differentiation factor 2 (MD-2), [3H]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 [125I] to sMD-2 with 20-fold higher specific radioactivity and TLR4-activating properties comparable to metabolically-labeled LOS·MD-2. LOS·MD-2[125I] and [3H]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[125I] binding to TLR4 indicating the pM affinity binding of LOS·MD-2[125I] is agonist-independent. LOS·MD-2[125I] 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[125I] 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.
- Subjects
RADIOIODINATION; ENDOTOXINS; LIGANDS (Biochemistry); LIPOOLIGOSACCHARIDES; CELL membranes; TYROSINE; QUANTITATIVE research
- Publication
Innate Immunity, 2013, Vol 19, Issue 5, p545
- ISSN
1753-4259
- Publication type
Article
- DOI
10.1177/1753425913475688