Identification of a Heme Activation Site on the MD-2/TLR4 Complex.

Frontiers in Immunology
John D BelcherGregory M Vercellotti

Abstract

Myeloid differentiation factor-2 (MD-2) binds lipopolysaccharide (LPS) and initiates toll-like receptor-4 (TLR4) pro-inflammatory signaling. Heme also activates TLR4 signaling, but it is unknown if heme interacts with MD-2. Therefore, we examined MD-2 for a potential heme activation site. Heme-agarose and biotin-heme/streptavidin-agarose pulled down recombinant MD-2, which was inhibited by excess free heme. UV/visible spectroscopy confirmed MD-2-heme binding. To determine whether MD-2 was required for heme-mediated TLR4 signaling, HEK293 cells were transfected with MD-2, TLR4, CD14, and an NF-κB luciferase reporter, and then stimulated with heme or LPS. Heme or LPS treatment elicited robust reporter activity. Absence of MD-2, TLR4 or CD14 plasmid abolished NF-κB reporter responses to heme or LPS. In silico analysis identified two potential heme docking sites on MD-2 near conserved amino acids W23/S33/Y34 and Y36/C37/I44. Heme-induced NF-κB activity was reduced by 39 and 78% in HEK293 cells transfected with MD-2 mutants W23A and Y34A, respectively, compared to WT-MD-2. NF-κB activation by LPS was not affected by the same mutants. Biotinyl-heme/streptavidin-agarose pulled down 68% less W23A and 80% less W23A/S33A/Y34A mutant MD-2...Continue Reading

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Citations

Sep 29, 2020·Frontiers in Immunology·Sabina JanciauskieneStephan Immenschuh
Dec 15, 2020·The FEBS Journal·Olivia MayMarie Frimat
Feb 6, 2021·Frontiers in Immunology·Joan D BeckmanGregory M Vercellotti
Jun 3, 2021·International Journal of Molecular Sciences·Stefan W Ryter
Sep 15, 2021·JCI Insight·Ghee Rye LeeLeo E Otterbein

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Methods Mentioned

BETA
electrophoresis
column chromatography
Pull-Down
glycosylations
glycosylation
circular dichroism
isothermal titration calorimetry
surface plasmon resonance

Software Mentioned

SigmaStat
HemeBind
QuickChange

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