Affinage

Showing LY96 — MD-2 is a alias.

LY96

Lymphocyte antigen 96 · UniProt Q9Y6Y9

Length
160 aa
Mass
18.5 kDa
Annotated
2026-06-10
100 papers in source corpus 45 papers cited in narrative 44 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 8/8 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

MD-2 (LY96) is the essential LPS-binding co-receptor of the innate immune endotoxin sensor, a secreted β-sandwich glycoprotein that constitutively associates with the extracellular domain of TLR4 and renders TLR4-expressing cells responsive to LPS (PMID:10359581). MD-2 is required for TLR4 to exit the Golgi and reach the plasma membrane, and genetic loss of MD-2 abolishes LPS responses and protects mice from endotoxic shock (PMID:12055629, PMID:26076332). MD-2 binds LPS directly with nanomolar affinity, independently of LBP and CD14, and is the principal endotoxin-binding subunit of the receptor (PMID:11500507), its crystal structure revealing a deep hydrophobic cavity that buries the acyl chains of lipid A while presenting the phosphorylated glucosamine head groups at the cavity rim (PMID:17569869). High-sensitivity detection proceeds by ordered, sequential transfer of endotoxin from LBP to CD14 to MD-2, producing a monomeric endotoxin–MD-2 complex that activates TLR4 at picomolar concentrations (PMID:15010525). In the assembled TLR4–MD-2–LPS structure, five of six LPS acyl chains are enclosed in the MD-2 pocket while the exposed sixth chain and LPS phosphates bridge a second TLR4–MD-2 unit, building the active m-shaped 2:2:2 signaling complex (PMID:19252480, PMID:19321453). Functionally distinct MD-2 surfaces separate TLR4 binding (the Cys95–Cys105 disulfide loop) from LPS binding and receptor clustering (hydrophobic pocket residues and the F126/G129 region), and species-specific differences in MD-2 pocket geometry and surface charge dictate whether partial lipid A structures such as lipid IVa act as agonists or antagonists (PMID:14607928, PMID:16670331, PMID:22532668, PMID:20592019). Beyond LPS, the MD-2 pocket accommodates diverse exogenous and endogenous ligands—paclitaxel, sulfatides, Gb4, heme, SAA3, sCD83—and small-molecule inhibitors targeting the pocket or covalently modifying Cys133 block TLR4 activation, establishing MD-2 as a tractable anti-inflammatory drug target (PMID:18650420, PMID:34290146, PMID:37487070, PMID:23858030). Soluble MD-2 additionally functions as an acute-phase opsonin that coats gram-negative bacteria and promotes their TLR4-dependent phagocytosis and killing (PMID:18056837, PMID:18203953).

Mechanistic history

Synthesis pass · year-by-year structured walk · 12 steps
  1. 1999 High

    Established that TLR4 alone is insufficient for LPS sensing and that a physically associated partner, MD-2, is required—defining the existence of a co-receptor.

    Evidence Co-immunoprecipitation and MD-2 transfection conferring LPS responsiveness via NF-κB reporter

    PMID:10359581

    Open questions at the time
    • Did not resolve whether MD-2 binds LPS directly or only stabilizes TLR4
    • No structural detail of the interaction
  2. 2001 High

    Showed MD-2 is itself a genuine LPS-binding protein, localizing endotoxin recognition to MD-2 rather than TLR4.

    Evidence Recombinant MD-2 binding assays with Kd determination and LBP competition

    PMID:11274165 PMID:11500507

    Open questions at the time
    • Structural basis of the binding pocket not yet known
    • Stoichiometry within the activated receptor undefined
  3. 2001 Medium

    Defined MD-2 quaternary state and its trafficking origin, showing monomeric MD-2 is the functional TLR4-binding species assembled in the ER/Golgi.

    Evidence SDS-PAGE oligomer characterization, TLR4 binding assays, and subcellular fractionation

    PMID:11593030

    Open questions at the time
    • Functional significance of disulfide-linked oligomers unresolved
    • Single-lab biochemical characterization
  4. 2002 High

    Demonstrated genetically that MD-2 is essential in vivo for LPS responses and for delivering TLR4 to the plasma membrane.

    Evidence MD-2 knockout mice with LPS challenge plus fractionation/microscopy of fibroblasts showing Golgi retention of TLR4

    PMID:12055629

    Open questions at the time
    • Molecular chaperone mechanism of TLR4 export not defined
    • Whether secreted vs membrane MD-2 drives trafficking unclear
  5. 2003 High

    Mapped MD-2 into two separable functional surfaces—one for TLR4 binding (Cys95-Cys105 disulfide region) and one for LPS responsiveness—and established that MD-2 must dock TLR4 before LPS-driven clustering.

    Evidence Site-directed mutagenesis with co-IP, LPS binding, confocal clustering and NF-κB reporter assays

    PMID:12642668 PMID:12960171 PMID:14607928

    Open questions at the time
    • Conformational changes accompanying activation not visualized
    • Order of LPS loading vs TLR4 dimerization only inferred
  6. 2004 High

    Reconstituted the ordered LBP→CD14→MD-2 transfer pathway, explaining how the system achieves picomolar LPS sensitivity.

    Evidence Purified endotoxin–MD-2 complex, TLR4-dependent activation assay, and inhibition by excess soluble MD-2

    PMID:15010525

    Open questions at the time
    • Kinetics of each transfer step not fully quantified
    • Structural intermediates of transfer not captured
  7. 2007 High

    Provided the structural foundation, revealing the MD-2 hydrophobic cavity that fully encloses lipid A acyl chains.

    Evidence X-ray crystallography of apo MD-2 and the lipid IVa complex

    PMID:17569869

    Open questions at the time
    • Did not show how MD-2-bound LPS engages a second TLR4
    • Active receptor multimer not yet resolved
  8. 2009 High

    Resolved the active receptor architecture, showing LPS bridges two TLR4-MD-2 units into an m-shaped 2:2:2 complex via an exposed acyl chain and phosphate contacts.

    Evidence Crystal structure of the TLR4-MD-2-LPS complex plus mutagenesis of the hydrophobic dimerization interface (MD-2 V82/M85/L87, TLR4 F440/F463)

    PMID:19252480 PMID:19321453

    Open questions at the time
    • Dynamics of dimerization in membranes not addressed
    • Link between clustering and downstream adaptor recruitment not structurally defined
  9. 2012 High

    Explained species-specific agonism/antagonism structurally, showing lipid IVa adopts opposite poses in mouse versus human MD-2.

    Evidence Crystal structures of mouse TLR4/MD-2 with LPS and with lipid IVa, complemented by combinatorial charge-reversal mutagenesis

    PMID:16407172 PMID:18606678 PMID:20592019 PMID:22532668

    Open questions at the time
    • Full conformational switching mechanism in MD-2 not directly observed
    • Generalization to other partial lipid A structures incomplete
  10. 2016 High

    Established that the MD-2 pocket can be engaged by non-LPS chemotypes to drive canonical TLR4 signaling, validating it as a versatile and druggable ligand-binding module.

    Evidence Crystal structure of Neoseptin-3 bound to mouse MD-2 with MyD88/TRIF-KO and CD14-independence validation; pocket binding of paclitaxel, curcumin, opioids, sulfatides and inhibitors

    PMID:17609337 PMID:18650420 PMID:19679181 PMID:26831104 PMID:34290146

    Open questions at the time
    • Endogenous physiological ligands beyond LPS incompletely catalogued
    • In vivo relevance of some pocket ligands uncertain
  11. 2020 Medium

    Extended MD-2 ligand repertoire to endogenous danger and acute-phase molecules (heme, SAA3, sCD83, Gb4), positioning MD-2 as a hub for sterile and endogenous TLR4 activation.

    Evidence Direct binding/pull-down, SPR, co-IP and mutagenesis with NF-κB readouts and KO mouse models

    PMID:23471986 PMID:23858030 PMID:28193829 PMID:32695117

    Open questions at the time
    • Single-lab binding evidence for several ligands
    • Physiological concentrations and competition with LPS not resolved
  12. 2023 High

    Demonstrated therapeutic targeting of MD-2, with covalent Cys133 modification blocking TLR4 dimerization and reducing inflammation in disease models.

    Evidence Disulfiram covalent modification of Cys133, dimerization/reporter assays, macrophage cytokine assays and an MPTP Parkinson's mouse model

    PMID:26076332 PMID:37487070

    Open questions at the time
    • Selectivity of covalent inhibitors for MD-2 in vivo not fully established
    • Translation to human disease unproven

Open questions

Synthesis pass · forward-looking unresolved questions
  • How MD-2 conformational switching couples ligand binding within the pocket to productive TLR4 dimerization and adaptor recruitment, and how the diverse endogenous ligands are physiologically prioritized, remain incompletely defined.
  • No time-resolved structural view of pocket-to-dimerization signal transmission
  • Competition hierarchy among endogenous MD-2 ligands unresolved
  • Mechanism by which secreted MD-2 chaperones TLR4 export not molecularly defined

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0008289 lipid binding 3 GO:0001618 virus receptor activity 2 GO:0038024 cargo receptor activity 2 GO:0060089 molecular transducer activity 2
Localization
GO:0005794 Golgi apparatus 3 GO:0005886 plasma membrane 3 GO:0005576 extracellular region 2 GO:0005783 endoplasmic reticulum 1
Pathway
R-HSA-162582 Signal Transduction 3 R-HSA-168256 Immune System 3
Partners
CD14CD83LBPPTX3SAA3TLR2TLR4
Complex memberships
LBP-CD14-MD-2 endotoxin transfer systemTLR4-MD-2 LPS receptor complexTLR4-MD-2-LPS 2:2:2 signaling complex

Evidence

Reading pass · 44 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1999 MD-2 (LY96) is physically associated with the extracellular domain of TLR4 on the cell surface and is requisite for LPS signaling by TLR4; transfection of MD-2 confers LPS responsiveness on TLR4-expressing cells that otherwise lack it. Co-immunoprecipitation, transfection-based cell activation assay (NF-κB reporter) The Journal of experimental medicine High 10359581
2002 MD-2 knockout mice do not respond to LPS and survive endotoxic shock; in MD-2-deficient fibroblasts, TLR4 fails to reach the plasma membrane and is retained in the Golgi apparatus, demonstrating that MD-2 is essential for correct intracellular trafficking and plasma membrane localization of TLR4. MD-2 knockout mouse (in vivo LPS challenge, Salmonella infection); subcellular fractionation and microscopy of embryonic fibroblasts Nature immunology High 12055629
2001 MD-2 directly binds LPS (apparent KD ~65 nM) independently of LBP and CD14; LBP competes with MD-2 for LPS binding, establishing MD-2 as a genuine LPS-binding protein. Recombinant protein production, five independent binding assays (including equilibrium binding with Kd determination); competition assay with LBP The Journal of biological chemistry High 11500507
2001 LPS is cross-linked directly to both TLR4 and MD-2 (and CD14) when co-expressed with CD14, demonstrating LPS is in close proximity to each member of the tripartite receptor complex; CD14 is required for LPS transfer to TLR4 and MD-2. Radioiodinated photoactivatable LPS cross-linking assay in transiently transfected HEK293 cells The Journal of biological chemistry High 11274165
2007 Crystal structure of human MD-2 (2.0 Å) reveals a deep hydrophobic cavity formed by two beta-sheets; in complex with tetra-acylated lipid IVa, all four acyl chains are fully enclosed in this cavity with the phosphorylated glucosamine moieties at the cavity entrance, establishing MD-2 as the principal endotoxin-binding subunit. X-ray crystallography at 2.0 Å (apo MD-2) and 2.2 Å (lipid IVa complex) Science (New York, N.Y.) High 17569869
2009 Crystal structure of the TLR4-MD-2-LPS complex at ~3 Å resolution reveals an m-shaped 2:2:2 receptor multimer; five of six LPS lipid chains are buried in MD-2's hydrophobic pocket while the sixth is exposed and contacts conserved TLR4 phenylalanines; LPS directly bridges two TLR4-MD-2 units; the MD-2 F126 loop undergoes localized conformational change to support TLR4 dimerization; phosphate groups of LPS form ionic interactions with positively charged residues in TLR4 and MD-2 to stabilize the active complex. X-ray crystallography of TLR4-MD-2-LPS complex Nature High 19252480
2004 Purified monomeric endotoxin-MD-2 complex (generated by sequential transfer from LBP→sCD14→MD-2) activates TLR4-expressing cells at picomolar endotoxin concentrations; excess free MD-2 inhibits delivery of the endotoxin-MD-2 complex to TLR4 cells, establishing ordered sequential transfer as the mechanism for high-sensitivity LPS detection. Purification of recombinant endotoxin-MD-2 complex; TLR4-dependent cell activation assay; inhibition studies with excess soluble MD-2 Proceedings of the National Academy of Sciences of the United States of America High 15010525
2003 Cell surface LPS-TLR4-MD-2 complexes form with a Kd ~3 nM; CD14 greatly enhances LPS loading onto TLR4-MD-2 but is not retained in the final complex; detergent disrupts LPS-CD14 but not LPS-TLR4-MD-2 interaction. Co-immunoprecipitation of LPS-TLR4-MD-2 complexes from cell surface; competition binding with lipid A antagonist E5531; Kd estimation The Journal of experimental medicine High 14517279
2001 MD-2 exists predominantly as large disulfide-linked oligomers in solution; monomeric MD-2 preferentially binds TLR4 and confers LPS responsiveness more efficiently than multimeric forms; MD-2 associates with TLR4 in the ER/cis-Golgi before being secreted. Secreted MD-2 characterization by SDS-PAGE, TLR4 binding assays; LPS responsiveness reporter assay; subcellular fractionation Proceedings of the National Academy of Sciences of the United States of America Medium 11593030
2001 N-linked glycosylation at Asn26 and Asn114 of MD-2 is not required for cell surface expression, but the double glycosylation mutant of MD-2 fails to support LPS-induced NF-κB activation, IL-8 secretion, or JNK activation, demonstrating that N-linked carbohydrates of MD-2 are essential for functional LPS receptor integrity. Site-directed mutagenesis of N-glycosylation sites; UV-crosslinking LPS binding assay; NF-κB/IL-8/JNK activation assays in transfected HeLa cells The Journal of biological chemistry High 11706042
2003 Lysines 128 and 132 of MD-2 are required for LPS binding; MD-2 must be surface-bound to TLR4 before LPS binding and TLR4 receptor cluster formation can occur; CD14 enhances LPS binding to MD-2 but is not essential for cellular activation. Site-directed mutagenesis; LPS binding assay; confocal microscopy of TLR4 clustering; NF-κB reporter assay The Journal of biological chemistry High 12960171
2003 Separate domains of human MD-2 mediate TLR4 binding and LPS responsiveness; Cys95 and Cys105 (forming an intramolecular disulfide) and surrounding residues (R90, K91, D100, Y102) are required for TLR4 binding; a distinct basic/aromatic region is responsible for conferring LPS responsiveness. Site-directed mutagenesis; co-immunoprecipitation; NF-κB reporter assay; dominant-negative inhibition in primary endothelial cells Journal of immunology (Baltimore, Md. : 1950) High 14607928
2003 The intramolecular disulfide bond between Cys95 and Cys105 of MD-2 is essential for LPS responsiveness; substitution of either Cys95 or Cys105 alone abolishes activity, whereas simultaneous substitution of both partially restores it; most Cys residues can participate in intermolecular oligomer formation. 17 single and multiple Cys-to-Ser substitution mutants analyzed by SDS-PAGE and NF-κB reporter assay; structural analysis of disulfide connectivity Proceedings of the National Academy of Sciences of the United States of America High 12642668
2001 Human MD-2 determines species-specific LPS recognition: hMD-2 confers responsiveness to lipid A but not to lipid IVa when associated with mouse TLR4, whereas mouse MD-2 confers responsiveness to both, demonstrating that MD-2 directly controls the fine ligand specificity of TLR4. Chimeric receptor (mTLR4/hMD-2) transfection and NF-κB activation assay; pharmacological analysis with lipid IVa as agonist/antagonist probe International immunology High 11717200
2000 Co-expression of mouse TLR4 and mouse MD-2 is required for Taxol-induced NF-κB signaling; human TLR4/hMD-2 does not confer Taxol responsiveness; an LPS antagonist also blocks Taxol-induced signaling via TLR4/MD-2, indicating TLR4/MD-2 is the shared signaling complex for both Taxol and LPS in mice and that species specificity resides in MD-2. Ba/F3 transfectants expressing species-specific TLR4/MD-2 combinations; NF-κB reporter assay; pharmacological blockade The Journal of biological chemistry High 10644670 11581576
2008 Paclitaxel binds directly to recombinant human MD-2 protein (dose-dependent, blocked by anti-MD-2 antibody); murine MD-2, not human MD-2, is required for TLR4 activation by paclitaxel; species-specific differences in pocket size, surface charge, and position of binding within the MD-2 pocket — particularly involving Phe126 as a dimerization bridge — explain species specificity. ELISA-based binding assay with recombinant hMD-2; HEK293 transfections with human/murine TLR4/MD-2 chimeras; computational docking to crystal structures The Journal of biological chemistry Medium 18650420
2002 TLR4-CD14-MD-2 complexes rapidly and constitutively recycle between the plasma membrane and the Golgi apparatus; LPS follows these trafficking pathways in CD14-positive cells; MyD88 translocates to the cell surface upon LPS exposure; Golgi-localized TLR4 is not required for LPS signaling (brefeldin A disrupts Golgi TLR4 but preserves signaling). Fluorescent TLR4 live-cell confocal imaging; brefeldin A treatment; MyD88 translocation assay; cross-linking-induced signaling The Journal of biological chemistry High 12324469
2006 MD-2 residues Phe126 and Gly129 are required for LPS-induced TLR4 receptor clustering but not for LPS binding; Gly59 is a novel critical residue for LPS binding; receptor clustering and LPS-receptor dissociation depend on TLR4 signaling and require endosomal acidification. MD-2 alanine-scanning mutagenesis; LPS binding assay; TLR4 clustering assay by microscopy; pharmacological blockade of endosomal acidification Journal of immunology (Baltimore, Md. : 1950) High 16670331
2009 Hydrophobic residues Val82, Met85, and Leu87 in the MD-2 hairpin loop (β5–β6) are essential for transfer of endotoxin from CD14 to monomeric MD-2 and for TLR4 activation; conserved hydrophobic residues Phe440 and Phe463 in TLR4 LRR16–17 are also essential for activation; these residues form a hydrophobic interface between the exposed acyl chain of LPS-bound MD-2 and the neighboring TLR4. Site-directed mutagenesis of MD-2 and TLR4; endotoxin transfer assay from CD14 to MD-2; NF-κB reporter assay in transfected cells The Journal of biological chemistry High 19321453
2012 Crystal structures of mouse TLR4/MD-2/LPS (2.5 Å) and TLR4/MD-2/lipid IVa (2.7 Å) complexes show that lipid IVa in mouse MD-2 occupies nearly the same space as LPS and induces an agonistic 2:2:2 complex similar to LPS; human MD-2 binds lipid IVa in a completely different, antagonistic manner, providing structural basis for species-specific agonism/antagonism. X-ray crystallography of mouse TLR4/MD-2/LPS and TLR4/MD-2/lipid IVa complexes Proceedings of the National Academy of Sciences of the United States of America High 22532668
2016 Neoseptin-3, a peptidomimetic with no structural similarity to LPS, activates mouse TLR4/MD-2 by binding as an asymmetrical dimer within the MD-2 hydrophobic pocket; crystal structure at 2.57 Å shows an active 2:2 receptor complex similar to lipid A; activation is CD14-independent and triggers canonical MyD88- and TRIF-dependent signaling. X-ray crystallography at 2.57 Å; NF-κB/IRF3 reporter assays; MyD88/TRIF knockout validation; CD14-independence experiments Proceedings of the National Academy of Sciences of the United States of America High 26831104
2021 Short-chain sulfatides directly bind the MD-2 hydrophobic pocket and activate mouse TLR4-MD-2 (MyD88- and TRIF-dependent) independently of CD14; crystal structure of mouse TLR4-MD-2/C16-sulfatide at 2.6 Å shows three sulfatide molecules in the MD-2 pocket inducing an active 2:2 complex; sulfatides antagonize human TLR4-MD-2, with agonism/antagonism dependent on the sulfate group and inversely related to FA chain length. Crystal structure; macrophage activation assays; MyD88/TRIF-knockout validation; structure-activity relationship with sulfatide variants Proceedings of the National Academy of Sciences of the United States of America High 34290146
2007 Curcumin binds MD-2 at submicromolar affinity in its hydrophobic pocket (fluorescence blue-shift assay); the binding site overlaps with LPS; curcumin inhibits both MyD88-dependent and -independent TLR4 signaling; C133F mutant MD-2 retains curcumin binding, and curcumin does not form a covalent bond to the free thiol of MD-2. Fluorescence spectroscopy; competition binding with LPS; NF-κB reporter assay; C133F mutagenesis Journal of leukocyte biology Medium 17609337
2023 Disulfiram (DSF) covalently modifies Cys133 of MD-2, blocking LPS-induced TLR4 dimerization, cell surface expression, and downstream NF-κB/IRF3 signaling; this mechanism suppresses inflammatory cytokine/interferon production by macrophages in vitro and reduces neuroinflammation and dopaminergic neuron loss in an MPTP mouse model of Parkinson's disease. Covalent modification identification; TLR4 dimerization assay; NF-κB/IRF3 reporter; macrophage cytokine assay; MPTP mouse model in vivo Proceedings of the National Academy of Sciences of the United States of America High 37487070
2004 Structural model of MD-2 (based on NPC2/Der p2 homology) predicted a beta-sandwich hydrophobic pocket; two basic residue clusters (Lys89-Arg90-Lys91 and Lys125-Lys132) are required for LPS signal transduction upon co-expression with TLR4 or as soluble protein added to TLR4-expressing cells; a peptide spanning the Cys95-Cys105 loop inhibited LPS-induced TNF-α and IL-8 production. Homology modeling; CD spectroscopy (confirming beta-sheet content); site-directed mutagenesis; NF-κB reporter assay; TNF-α/IL-8 inhibition with synthetic peptide The Journal of biological chemistry Medium 15111623
2001 MD-2 physically associates with TLR2 (in addition to TLR4), enabling TLR2 to respond to LPS and lipid A structures that are otherwise non-activating; the MD-2-TLR2 interaction is weaker than the MD-2-TLR4 interaction; MD-2 enhances expression of both TLR2 and TLR4. Co-immunoprecipitation; NF-κB reporter assays in transfected cells; flow cytometry for surface expression Journal of immunology (Baltimore, Md. : 1950) Medium 11160242
2004 Structural regions 57-79 and 108-135 of MD-2 determine the agonist-antagonist activity of lipid IVa; single amino acid substitutions at Thr57, Val61, and Glu122 in mouse MD-2 impair lipid IVa agonism while preserving LPS (E. coli lipid A) activation. Human/mouse chimeric MD-2 expression; NF-κB reporter assay in HEK293 cells; point mutagenesis The Journal of biological chemistry Medium 16407172
2010 MD-2 residues Tyr42, Arg69, Asp122, and Leu125 determine lipid IVa species specificity; E122K mutation in mouse MD-2 substantially reduces lipid IVa response; combining MD-2 and TLR4 charge-reversal mutations can completely convert murine receptor response to a human-like pattern (and vice versa), demonstrating that MD-2 surface charges at two distinct interfaces (pocket entrance and MD-2/TLR4 contact surface) govern species-specific activation. Site-directed mutagenesis; stable TLR4-expressing cell lines; purified monomeric MD-2; MD-2-deficient bone marrow macrophages; NF-κB reporter assay The Journal of biological chemistry High 20592019
2009 Morphine and structurally diverse opioids activate TLR4 signaling non-stereoselectively; in silico docking indicates opioids bind preferentially to the LPS-binding pocket of MD-2 rather than TLR4; naloxone blocks this signaling non-stereoselectively; TLR4 knockout mice show a threefold leftward shift in morphine analgesia dose-response. In vitro TLR4 reporter assay; pharmacological blockade (naloxone, classical TLR4 antagonist); TLR4 knockout mouse; in silico docking to MD-2 pocket Brain, behavior, and immunity Medium 19679181
2013 Serum amyloid A3 (SAA3) synthetic peptide (aa 20-86) directly binds MD-2 (not TLR4) with KD ~2.2 μM as measured by surface plasmon resonance; FLAG-tagged SAA3 co-precipitates with protein A-tagged MD-2 in baculovirus co-infection experiments; SAA3-MD-2 interaction activates MyD88-dependent TLR4 signaling (p38, NF-κB, Rho GTPase) but not TRIF-dependent IFN-β. Surface plasmon resonance; co-immunoprecipitation in baculovirus system; NF-κB/p38 activation assays; TLR4/MyD88 KO validation Journal of immunology (Baltimore, Md. : 1950) High 23858030
2017 Soluble CD83 (sCD83) binds MD-2 as a high-affinity partner; sCD83 binding to MD-2 on monocytes rapidly degrades IRAK-1 and induces anti-inflammatory mediators (IDO, IL-10, PGE2 via COX-2), leading to T cell unresponsiveness. Pull-down/co-IP identifying MD-2 as sCD83 binding partner; IRAK-1 degradation assay; IDO/IL-10/PGE2 measurement; T cell proliferation assay Journal of immunology (Baltimore, Md. : 1950) Medium 28193829
2014 PTX3 directly binds MD-2 in vitro; in MD-2-knockout mice, PTX3 fails to confer immune protection against Aspergillus fumigatus; MD-2-competent PMN adoptive transfer restores protection; PTX3-opsonized conidia activate TLR4/MD-2/TRIF-dependent signaling converging on IL-10. In vitro binding assay (PTX3-MD-2); MD-2 knockout mouse model; adoptive transfer of MD-2-competent PMN; cytokine profiling Journal of immunology (Baltimore, Md. : 1950) Medium 25049357
2013 Globotetraosylceramide (Gb4) binds to TLR4-MD-2 (co-precipitated with recombinant MD-2; confirmed by native PAGE and docking); Gb4 competes with LPS for TLR4-MD-2 binding; A4galt-deficient mice lacking Gb4 show increased LPS sensitivity; exogenous Gb4 protects mice from LPS-induced mortality. Co-precipitation of Gb4 with recombinant MD-2; native PAGE; A4galt-knockout mouse; in vivo LPS challenge; gene expression assay Proceedings of the National Academy of Sciences of the United States of America Medium 23471986
2020 Heme binds MD-2 and activates TLR4 signaling; MD-2 is required for heme-mediated NF-κB activation (absent without MD-2); heme binding site involves residues W23 and Y34 (mutagenesis reduces heme pull-down and NF-κB response); site Y36A increases heme-induced NF-κB signaling without affecting LPS response. Heme-agarose/biotin-heme pull-down of recombinant MD-2; UV/visible spectroscopy; site-directed mutagenesis; NF-κB luciferase reporter in HEK293; in silico docking Frontiers in immunology Medium 32695117
2002 Monomeric recombinant MD-2 can interact with the soluble extracellular domain of TLR4 in solution; MD-2's ability to confer LPS responsiveness and to bind TLR4 are strongly associated; more than two intermolecular disulfide bonds stabilize MD-2 multimers. Biochemical binding assay (TLR4 ectodomain-MD-2 interaction in solution); site-directed mutagenesis of Cys residues; NF-κB reporter assay The Journal of biological chemistry Medium 11976338
2011 Intracellular TLR4/MD-2 (present in the absence of cell-surface TLR4) can sense phagocytosed gram-negative bacteria and activate MyD88-dependent chemokine production (CCL2, CCL5) and co-stimulatory molecule upregulation (CD40, CD86) independently of TRIF/TICAM-1; intracellular TLR4/MD-2 requires PRAT4A-independent compartment; TRIF-dependent type I IFN production depends on surface TLR4. PRAT4A knockout macrophages (abolishing surface TLR4); intracellular LPS sensing assay; cytokine/chemokine measurement; co-stimulatory molecule flow cytometry International immunology Medium 21712422
2002 MD-2 expression in intestinal epithelial cells (IECs) is regulated epigenetically; IFN-γ positively regulates the MD-2 promoter through JAK-STAT signaling (blocked by STAT inhibitor SOCS3); IFN-γ and TNF-α sensitize IECs to LPS-dependent IL-8 secretion by upregulating MD-2. RT-PCR; Western blot; MD-2 promoter reporter assay; SOCS3 overexpression; IL-8 secretion assay The Journal of biological chemistry Medium 11923281
2009 Epigenetic silencing (CpG methylation and histone deacetylation) of the MD-2 promoter underlies low MD-2 expression and LPS unresponsiveness in intestinal epithelial cells; inhibition of methylation (5-azacytidine) or histone deacetylation (trichostatin A) increases MD-2 mRNA expression; LPS responsiveness is polarized to the basolateral membrane of IECs. Bisulfite sequencing of MD-2 promoter; 5-azacytidine and trichostatin A treatment; MD-2 mRNA quantification; NF-κB reporter assay Innate immunity Medium 19710105
2007 Soluble MD-2 is a type II acute-phase protein: mRNA and protein levels rise in mouse liver during acute-phase response; IL-6 upregulates sMD-2 secretion from hepatocytes; sMD-2 binds gram-negative (but not gram-positive) bacteria and functions as an opsonin, enhancing phagocytosis by neutrophils and serving as a cofactor for TLR4-expressing cell activation by gram-negative bacteria. Mouse acute-phase response model; IL-6 stimulation of hepatocytes; bacterial binding assay; phagocytosis assay; TLR4-dependent cell activation assay Blood Medium 18056837
2008 Soluble MD-2 binds to the surface of live gram-negative bacteria; MD-2-coated bacteria show enhanced cellular activation, bacterial internalization, and intracellular killing, all in a TLR4-signaling-dependent manner (absent in Lpsd macrophages with signaling-deficient TLR4), confirming sMD-2 as an opsonin that bridges bacteria to TLR4. MD-2 binding to live bacteria (binding assay); phagocytosis/killing assays in WT vs. Lpsd macrophages; TLR4-dependence controls Blood Medium 18203953
2015 The chalcone derivative L6H21 inserts into the hydrophobic pocket of MD-2, forming hydrogen bonds with Arg90 and Tyr102; it suppresses LPS-induced TLR4/MD-2 complex formation and downstream MAPK/NF-κB signaling; MD-2 knockout mice are universally protected from LPS-induced septic shock, validating MD-2 as the essential therapeutic target. SPR binding assay; ELISA; fluorescence measurement; flow cytometry; computer docking; Western blot/EMSA; MD-2 knockout mouse model British journal of pharmacology Medium 26076332
2021 Zebrafish possess an MD-2 ortholog encoded by the ly96 gene; zebrafish Md-2 and Tlr4ba form a functional complex that activates NF-κB signaling in response to LPS; ly96 loss-of-function in larval zebrafish perturbs LPS-induced cytokine production, establishing an ancestral Tlr4/Md-2 LPS-sensing complex in teleosts. Bioinformatic identification; single-cell RNA sequencing; functional NF-κB reporter assay with zebrafish Md-2/Tlr4ba co-expression; ly96 loss-of-function mutation analysis Journal of immunology (Baltimore, Md. : 1950) Medium 33472906
2008 MD-2 residues 57-66 and 82-89 (horse vs. human) and a single residue in the glycan-free flank of TLR4 solenoid determine whether lipid IVa acts as agonist or antagonist; replacing horse MD-2 residues 57-66 and 82-89 with human equivalents confers constitutive activity, suggesting conformational switching in MD-2 is important for ligand-induced activation. Horse/human chimeric MD-2 and TLR4 constructs; NF-κB reporter assay; surface charge analysis Journal of immunology (Baltimore, Md. : 1950) Medium 18606678
2004 A single coding mutation in human MD-2 (Thr35Ala, A→G at position 103) results in reduced LPS-induced signaling in reporter gene assays, demonstrating that Thr35 contributes to MD-2 function. SSCP mutation screening; Lightcycler/FRET genotyping; NF-κB reporter gene assay; in vitro LPS stimulation (TNF-α measurement) Genes and immunity Low 15057266

Source papers

Stage 0 corpus · 100 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2009 The structural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex. Nature 1889 19252480
1999 MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. The Journal of experimental medicine 1580 10359581
2002 Essential role of MD-2 in LPS responsiveness and TLR4 distribution. Nature immunology 793 12055629
2001 Lipopolysaccharide is in close proximity to each of the proteins in its membrane receptor complex. transfer from CD14 to TLR4 and MD-2. The Journal of biological chemistry 483 11274165
2009 Evidence that opioids may have toll-like receptor 4 and MD-2 effects. Brain, behavior, and immunity 435 19679181
2007 Crystal structures of human MD-2 and its complex with antiendotoxic lipid IVa. Science (New York, N.Y.) 373 17569869
2000 Cutting edge: cell surface expression and lipopolysaccharide signaling via the toll-like receptor 4-MD-2 complex on mouse peritoneal macrophages. Journal of immunology (Baltimore, Md. : 1950) 358 10725698
2002 Lipopolysaccharide rapidly traffics to and from the Golgi apparatus with the toll-like receptor 4-MD-2-CD14 complex in a process that is distinct from the initiation of signal transduction. The Journal of biological chemistry 352 12324469
2003 Lipopolysaccharide interaction with cell surface Toll-like receptor 4-MD-2: higher affinity than that with MD-2 or CD14. The Journal of experimental medicine 319 14517279
2002 TLR4 and MD-2 expression is regulated by immune-mediated signals in human intestinal epithelial cells. The Journal of biological chemistry 294 11923281
2012 Structural basis of species-specific endotoxin sensing by innate immune receptor TLR4/MD-2. Proceedings of the National Academy of Sciences of the United States of America 283 22532668
2004 Isolation of an endotoxin-MD-2 complex that produces Toll-like receptor 4-dependent cell activation at picomolar concentrations. Proceedings of the National Academy of Sciences of the United States of America 282 15010525
2000 Mouse toll-like receptor 4.MD-2 complex mediates lipopolysaccharide-mimetic signal transduction by Taxol. The Journal of biological chemistry 282 10644670
2004 Innate recognition of lipopolysaccharide by Toll-like receptor 4-MD-2. Trends in microbiology 236 15051069
2001 MD-2 binds to bacterial lipopolysaccharide. The Journal of biological chemistry 231 11500507
2013 Recognition of lipid A variants by the TLR4-MD-2 receptor complex. Frontiers in cellular and infection microbiology 217 23408095
2001 MD-2 and TLR4 N-linked glycosylations are important for a functional lipopolysaccharide receptor. The Journal of biological chemistry 205 11706042
2001 Human MD-2 confers on mouse Toll-like receptor 4 species-specific lipopolysaccharide recognition. International immunology 196 11717200
2006 Analysis of TLR4 polymorphic variants: new insights into TLR4/MD-2/CD14 stoichiometry, structure, and signaling. Journal of immunology (Baltimore, Md. : 1950) 187 16785528
2009 Lactate boosts TLR4 signaling and NF-kappaB pathway-mediated gene transcription in macrophages via monocarboxylate transporters and MD-2 up-regulation. Journal of immunology (Baltimore, Md. : 1950) 180 19201903
2001 Secreted MD-2 is a large polymeric protein that efficiently confers lipopolysaccharide sensitivity to Toll-like receptor 4. Proceedings of the National Academy of Sciences of the United States of America 177 11593030
2003 Lysines 128 and 132 enable lipopolysaccharide binding to MD-2, leading to Toll-like receptor-4 aggregation and signal transduction. The Journal of biological chemistry 174 12960171
2001 MD-2 enables Toll-like receptor 2 (TLR2)-mediated responses to lipopolysaccharide and enhances TLR2-mediated responses to Gram-positive and Gram-negative bacteria and their cell wall components. Journal of immunology (Baltimore, Md. : 1950) 168 11160242
2010 Evidence that intrathecal morphine-3-glucuronide may cause pain enhancement via toll-like receptor 4/MD-2 and interleukin-1beta. Neuroscience 146 19833175
2006 Regulatory roles for MD-2 and TLR4 in ligand-induced receptor clustering. Journal of immunology (Baltimore, Md. : 1950) 137 16670331
2004 Essential role of MD-2 in TLR4-dependent signaling during Helicobacter pylori-associated gastritis. Journal of immunology (Baltimore, Md. : 1950) 137 15240737
2016 TLR4/MD-2 activation by a synthetic agonist with no similarity to LPS. Proceedings of the National Academy of Sciences of the United States of America 133 26831104
2007 MD-2 as the target of curcumin in the inhibition of response to LPS. Journal of leukocyte biology 127 17609337
2008 Unique properties of the chicken TLR4/MD-2 complex: selective lipopolysaccharide activation of the MyD88-dependent pathway. Journal of immunology (Baltimore, Md. : 1950) 125 18768894
2006 Surfactant protein A directly interacts with TLR4 and MD-2 and regulates inflammatory cellular response. Importance of supratrimeric oligomerization. The Journal of biological chemistry 117 16754682
2000 Regulatory roles for CD14 and phosphatidylinositol in the signaling via toll-like receptor 4-MD-2. Biochemical and biophysical research communications 115 10652232
2008 Elucidation of the MD-2/TLR4 interface required for signaling by lipid IVa. Journal of immunology (Baltimore, Md. : 1950) 112 18606678
2014 Polymorphisms in the inflammatory pathway genes TLR2, TLR4, TLR9, LY96, NFKBIA, NFKB1, TNFA, TNFRSF1A, IL6R, IL10, IL23R, PTPN22, and PPARG are associated with susceptibility of inflammatory bowel disease in a Danish cohort. PloS one 105 24971461
2004 Structural model of MD-2 and functional role of its basic amino acid clusters involved in cellular lipopolysaccharide recognition. The Journal of biological chemistry 104 15111623
2007 TLR4/MD-2 monoclonal antibody therapy affords protection in experimental models of septic shock. Journal of immunology (Baltimore, Md. : 1950) 95 17947685
2003 Innate recognition of lipopolysaccharide by CD14 and toll-like receptor 4-MD-2: unique roles for MD-2. International immunopharmacology 94 12538042
2009 Essential roles of hydrophobic residues in both MD-2 and toll-like receptor 4 in activation by endotoxin. The Journal of biological chemistry 93 19321453
2012 Glycyrrhizin and isoliquiritigenin suppress the LPS sensor toll-like receptor 4/MD-2 complex signaling in a different manner. Journal of leukocyte biology 91 22422925
2003 Separate functional domains of human MD-2 mediate Toll-like receptor 4-binding and lipopolysaccharide responsiveness. Journal of immunology (Baltimore, Md. : 1950) 88 14607928
2002 Monomeric recombinant MD-2 binds toll-like receptor 4 tightly and confers lipopolysaccharide responsiveness. The Journal of biological chemistry 86 11976338
2009 MD-2-mediated ionic interactions between lipid A and TLR4 are essential for receptor activation. The Journal of biological chemistry 85 20018893
2004 MD-2: the Toll 'gatekeeper' in endotoxin signalling. Trends in biochemical sciences 83 15276183
2020 Immunoinformatics approach to understand molecular interaction between multi-epitopic regions of SARS-CoV-2 spike-protein with TLR4/MD-2 complex. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 82 33039603
2015 MD-2 as the target of a novel small molecule, L6H21, in the attenuation of LPS-induced inflammatory response and sepsis. British journal of pharmacology 82 26076332
2004 Endotoxin recognition molecules, Toll-like receptor 4-MD-2. Seminars in immunology 79 14751758
2014 Role of berberine in anti-bacterial as a high-affinity LPS antagonist binding to TLR4/MD-2 receptor. BMC complementary and alternative medicine 76 24602493
2008 Paclitaxel binding to human and murine MD-2. The Journal of biological chemistry 74 18650420
2000 Role of MD-2 in TLR2- and TLR4-mediated recognition of Gram-negative and Gram-positive bacteria and activation of chemokine genes. Journal of endotoxin research 74 11521063
2004 Soluble MD-2 activity in plasma from patients with severe sepsis and septic shock. Blood 72 15328161
2015 Eritoran inhibits S100A8-mediated TLR4/MD-2 activation and tumor growth by changing the immune microenvironment. Oncogene 67 26165843
2013 Serum amyloid A3 binds MD-2 to activate p38 and NF-κB pathways in a MyD88-dependent manner. Journal of immunology (Baltimore, Md. : 1950) 66 23858030
2007 Kinetics of binding of LPS to recombinant CD14, TLR4, and MD-2 proteins. Molecules and cells 66 17846506
2009 Regulation of Toll-like receptor 4-associated MD-2 in intestinal epithelial cells: a comprehensive analysis. Innate immunity 65 19710105
2007 Protein-energy malnutrition decreases the expression of TLR-4/MD-2 and CD14 receptors in peritoneal macrophages and reduces the synthesis of TNF-alpha in response to lipopolysaccharide (LPS) in mice. Cytokine 65 17950615
2012 Humanized TLR4/MD-2 mice reveal LPS recognition differentially impacts susceptibility to Yersinia pestis and Salmonella enterica. PLoS pathogens 62 23071439
2003 The role of disulfide bonds in the assembly and function of MD-2. Proceedings of the National Academy of Sciences of the United States of America 60 12642668
2005 Structural regions of MD-2 that determine the agonist-antagonist activity of lipid IVa. The Journal of biological chemistry 59 16407172
2006 MD-2. Immunobiology 58 16920483
2023 Disulfiram blocks inflammatory TLR4 signaling by targeting MD-2. Proceedings of the National Academy of Sciences of the United States of America 57 37487070
2017 Soluble CD83 Inhibits T Cell Activation by Binding to the TLR4/MD-2 Complex on CD14+ Monocytes. Journal of immunology (Baltimore, Md. : 1950) 57 28193829
2011 MD-2 as the target of nonlipid chalcone in the inhibition of endotoxin LPS-induced TLR4 activity. The Journal of infectious diseases 56 21402551
2003 Overexpression of CD14, TLR4, and MD-2 in HEK 293T cells does not prevent induction of in vitro endotoxin tolerance. Journal of endotoxin research 56 12691621
2003 Role of TLR4/MD-2 and RP105/MD-1 in innate recognition of lipopolysaccharide. Scandinavian journal of infectious diseases 56 14620136
2002 Identification of LPS-binding peptide fragment of MD-2, a toll-receptor accessory protein. Biochemical and biophysical research communications 56 11944896
2000 Innate recognition of lipopolysaccharide by Toll-like receptor 4/MD-2 and RP105/MD-1. Journal of endotoxin research 54 11521060
2017 MD-2 regulates LPS-induced NLRP3 inflammasome activation and IL-1beta secretion by a MyD88/NF-κB-dependent pathway in alveolar macrophages cell line. Molecular immunology 52 28654770
2021 Sulfatides are endogenous ligands for the TLR4-MD-2 complex. Proceedings of the National Academy of Sciences of the United States of America 51 34290146
2019 LPS-induced CXCR7 expression promotes gastric Cancer proliferation and migration via the TLR4/MD-2 pathway. Diagnostic pathology 51 30636642
2013 TLR4-MD-2 complex is negatively regulated by an endogenous ligand, globotetraosylceramide. Proceedings of the National Academy of Sciences of the United States of America 49 23471986
2005 Inhibition of TLR-4/MD-2 signaling by RP105/MD-1. Journal of endotoxin research 49 16303092
2014 PTX3 binds MD-2 and promotes TRIF-dependent immune protection in aspergillosis. Journal of immunology (Baltimore, Md. : 1950) 48 25049357
2014 The molecular mechanism of species-specific recognition of lipopolysaccharides by the MD-2/TLR4 receptor complex. Molecular immunology 47 25037631
2013 Modulation of CD14 and TLR4·MD-2 activities by a synthetic lipid A mimetic. Chembiochem : a European journal of chemical biology 47 24339336
2010 Neisseria meningitidis capsular polysaccharides induce inflammatory responses via TLR2 and TLR4-MD-2. Journal of leukocyte biology 47 21191086
2001 Involvement of TLR4/MD-2 complex in species-specific lipopolysaccharide-mimetic signal transduction by Taxol. Journal of endotoxin research 46 11581576
2004 Analysis of chicken TLR4, CD28, MIF, MD-2, and LITAF genes in a Salmonella enteritidis resource population. Poultry science 44 15109052
2007 Soluble MD-2 is an acute-phase protein and an opsonin for Gram-negative bacteria. Blood 41 18056837
2015 Plasminogen activator inhibitor-1 regulates LPS-induced TLR4/MD-2 pathway activation and inflammation in alveolar macrophages. Inflammation 40 25342286
2016 Atractylenolide I modulates ovarian cancer cell-mediated immunosuppression by blocking MD-2/TLR4 complex-mediated MyD88/NF-κB signaling in vitro. Journal of translational medicine 39 27118139
2010 MD-2 residues tyrosine 42, arginine 69, aspartic acid 122, and leucine 125 provide species specificity for lipid IVA. The Journal of biological chemistry 39 20592019
2008 Lack of MD-2 expression in human corneal epithelial cells is an underlying mechanism of lipopolysaccharide (LPS) unresponsiveness. Immunology and cell biology 38 18936773
2011 Intracellular TLR4/MD-2 in macrophages senses Gram-negative bacteria and induces a unique set of LPS-dependent genes. International immunology 37 21712422
2008 MD-2-dependent pulmonary immune responses to inhaled lipooligosaccharides: effect of acylation state. American journal of respiratory cell and molecular biology 37 18203970
2020 Identification of a Heme Activation Site on the MD-2/TLR4 Complex. Frontiers in immunology 35 32695117
2008 Phagocytosis and intracellular killing of MD-2 opsonized gram-negative bacteria depend on TLR4 signaling. Blood 35 18203953
2002 Toll-like receptor 4-MD-2 complex mediates the signal transduction induced by flavolipin, an amino acid-containing lipid unique to Flavobacterium meningosepticum. Journal of immunology (Baltimore, Md. : 1950) 35 11884465
2007 MD-2 controls bacterial lipopolysaccharide hyporesponsiveness in human intestinal epithelial cells. Life sciences 34 18215718
2012 Inhibition of LPS binding to MD-2 co-receptor for suppressing TLR4-mediated expression of inflammatory cytokine by 1-dehydro-10-gingerdione from dietary ginger. Biochemical and biophysical research communications 33 22387540
2011 Electrochemical endotoxin sensors based on TLR4/MD-2 complexes immobilized on gold electrodes. Biosensors & bioelectronics 33 21816600
2021 Identification and Characterization of Zebrafish Tlr4 Coreceptor Md-2. Journal of immunology (Baltimore, Md. : 1950) 32 33472906
2005 The functional and structural properties of MD-2 required for lipopolysaccharide binding are absent in MD-1. Journal of immunology (Baltimore, Md. : 1950) 32 15611257
2017 Loss of BMI-1 dampens migration and EMT of colorectal cancer in inflammatory microenvironment through TLR4/MD-2/MyD88-mediated NF-κB signaling. Journal of cellular biochemistry 31 28815730
2006 MD-2 expression is not required for cell surface targeting of Toll-like receptor 4 (TLR4). Journal of leukocyte biology 31 16946018
2015 MD-2 determinants of nickel and cobalt-mediated activation of human TLR4. PloS one 30 25803856
2007 Human MD-2 discrimination of meningococcal lipid A structures and activation of TLR4. Glycobiology 30 17545685
2005 MD-2 and Der p 2 - a tale of two cousins or distant relatives? Journal of endotoxin research 30 15949148
2004 A coding mutation within the first exon of the human MD-2 gene results in decreased lipopolysaccharide-induced signaling. Genes and immunity 30 15057266
2016 Flavonoids from Radix Tetrastigmae inhibit TLR4/MD-2 mediated JNK and NF-κB pathway with anti-inflammatory properties. Cytokine 29 27235587
2011 Partially glycosylated dendrimers block MD-2 and prevent TLR4-MD-2-LPS complex mediated cytokine responses. PLoS computational biology 29 21738462
2004 Endotoxin recognition molecules MD-2 and toll-like receptor 4 as potential targets for therapeutic intervention of endotoxin shock. Current drug targets. Inflammation and allergy 28 15379597

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