PMID- 23439691
OWN - NLM
STAT- MEDLINE
DCOM- 20140422
LR  - 20211021
IS  - 1753-4267 (Electronic)
IS  - 1753-4259 (Print)
IS  - 1753-4259 (Linking)
VI  - 19
IP  - 5
DP  - 2013 Oct
TI  - Radioiodination of an endotoxin.MD-2 complex generates a novel sensitive, 
      high-affinity ligand for TLR4.
PG  - 545-60
LID - 10.1177/1753425913475688 [doi]
AB  - 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.
FAU - Teghanemt, Athmane
AU  - Teghanemt A
AD  - 1Inflammation Program, Department of Internal Medicine, Roy A. and Lucille J. 
      Carver College of Medicine, University of Iowa, Iowa City, IA, USA.
FAU - Weiss, Jerrold P
AU  - Weiss JP
FAU - Gioannini, Theresa L
AU  - Gioannini TL
LA  - eng
GR  - P30 ES005605/ES/NIEHS NIH HHS/United States
GR  - AI088372/AI/NIAID NIH HHS/United States
GR  - R01 AI088372/AI/NIAID NIH HHS/United States
GR  - R01 AI059372/AI/NIAID NIH HHS/United States
GR  - I01 BX000949/BX/BLRD VA/United States
GR  - AI059372/AI/NIAID NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, U.S. Gov't, Non-P.H.S.
DEP - 20130225
PL  - United States
TA  - Innate Immun
JT  - Innate immunity
JID - 101469670
RN  - 0 (Iodine Radioisotopes)
RN  - 0 (LY96 protein, human)
RN  - 0 (Lipopolysaccharides)
RN  - 0 (Lymphocyte Antigen 96)
RN  - 0 (Multiprotein Complexes)
RN  - 0 (Toll-Like Receptor 4)
SB  - IM
MH  - Animals
MH  - Feasibility Studies
MH  - HEK293 Cells
MH  - Humans
MH  - Immunity, Innate
MH  - Iodine Radioisotopes/*metabolism
MH  - Lipopolysaccharides/*metabolism
MH  - Lymphocyte Antigen 96/*metabolism
MH  - Mice
MH  - Multiprotein Complexes/chemistry/*metabolism
MH  - Protein Binding
MH  - Radioligand Assay
MH  - Sensitivity and Specificity
MH  - Toll-Like Receptor 4/genetics/*metabolism
MH  - Transgenes/genetics
PMC - PMC3779530
MID - NIHMS480603
OTO - NOTNLM
OT  - Endotoxin
OT  - LPS
OT  - MD-2
OT  - TLR4
COIS- The authors have no conflicts of interest to report.
EDAT- 2013/02/27 06:00
MHDA- 2014/04/23 06:00
PMCR- 2014/10/01
CRDT- 2013/02/27 06:00
PHST- 2013/02/27 06:00 [entrez]
PHST- 2013/02/27 06:00 [pubmed]
PHST- 2014/04/23 06:00 [medline]
PHST- 2014/10/01 00:00 [pmc-release]
AID - 1753425913475688 [pii]
AID - 10.1177/1753425913475688 [doi]
PST - ppublish
SO  - Innate Immun. 2013 Oct;19(5):545-60. doi: 10.1177/1753425913475688. Epub 2013 Feb 
      25.