Purified monomeric ligand.MD-2 complexes reveal molecular and structural requirements for activation and antagonism of TLR4 by Gram-negative bacterial endotoxins

Gioannini, Theresa L.; Teghanemt, Athmane; Zhang, Desheng; Esparza, Gregory A; Yu, Liping; Weiss, Jerrold

doi:10.1007/s12026-014-8543-y

Cited by 25 publications

(27 citation statements)

References 44 publications

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“…15 These properties of the radiolabeled LOS have greatly facilitated measurements of the physical size of complexes containing LOS and the stoichiometry and affinity of interactions with specific endotoxin binding proteins. 1,13,14,20,21 …”

Section: Resultsmentioning

confidence: 99%

High-affinity caspase-4 binding to LPS presented as high molecular mass aggregates or in outer membrane vesicles

et al. 2017

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Caspases of the non-canonical inflammasome (caspases-4/5/11) directly bind endotoxin (LOS/LPS) and can be activated in the absence of any co-factors. Models of LPS-induced caspase activation have postulated that 1:1 binding of endotoxin monomers to caspase trigger caspase oligomerization and activation, analogous to that established for endotoxin-induced activation of MD-2/TLR4. However, using metabolically radiolabeled LOS and LPS, we now show high affinity and selective binding of caspase-4 to high molecular-mass aggregates of purified endotoxin and to endotoxin-rich outer membrane vesicles without formation of 1:1 endotoxin:caspase complexes. Our findings thus demonstrate markedly different endotoxin recognition properties of caspase-4 from that of MD-2/TLR4 and strongly suggest that activation of caspase-4 (and presumably -5 and -11) are mediated by interactions with activating endotoxin-rich membrane interfaces rather than by endotoxin monomers.

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Section: Resultsmentioning

confidence: 99%

High-affinity caspase-4 binding to LPS presented as high molecular mass aggregates or in outer membrane vesicles

et al. 2017

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“…LPS can activate human cells through binding to hMD-2 that is already associated with TLR4 ecd or through binding of the hMD-2/LPS complex to the ectodomain of TLR4 (22, 24, 31). Murine MD-2 differs from its human orthologue as it does not form a detectable mMD-2/LPS complex (17, 20).…”

Section: Resultsmentioning

confidence: 99%

Molecular Basis of the Functional Differences between Soluble Human Versus Murine MD-2: Role of Val135 in Transfer of Lipopolysaccharide from CD14 to MD-2

Vašl

Oblak

Peternelj

et al. 2016

The Journal of Immunology

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Myeloid differentiation factor (MD-2) is an extracellular protein, associated with the ectodomain of Toll-like receptor 4 (TLR4), that plays a critical role in the recognition of bacterial lipopolysaccharide (LPS). Despite high overall structural and functional similarity, human (h) and murine (m) MD-2 exhibit several species-related differences. hMD-2 is capable of binding LPS in the absence of TLR4, whereas mMD-2 supports LPS responsiveness only when mMD-2 and mTLR4 are coexpressed in the same cell. Previously, charged residues at the edge of LPS binding pocket have been attributed to this difference. In this study, site-directed mutagenesis was used to explore the hydrophobic residues within MD-2 binding pocket, as the source of functional differences between hMD-2 and mMD-2. While decreased hydrophobicity of residues 61 and 63 in hMD-2 binding pocket retained the characteristics of wild type hMD-2, a relatively minor change of valine to alanine at position 135 completely abolished the binding of LPS to the hMD-2 mutant. The mutant, however, retained the LPS binding in complex with TLR4 and also cell activation, resulting in a murine-like phenotype. These results were supported by the molecular dynamics simulation. We propose that the residue at position 135 of MD-2 governs the dynamics of the binding pocket and its ability to accommodate lipid A, which is allosterically affected by bound TLR4.

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“…The mechanisms underlying immunomodulation by endotoxin are not entirely clear, but they are thought to include regulatory T lymphocytes and both proinflammatory and antiinflammatory cytokines and their upstream and downstream signaling pathways (26). The binding of LPS-binding protein to CD14 associated with lymphocyte antigen 96 (also referred to as LY96 or MD2) and TLR4 triggers a cascade that includes nuclear factor-kB and activation of type 1 T helper cell and regulatory T lymphocyte pathways (27,28). Exposures lead to chronic, low-level activation of innate immunity, which regulates immune responses away from type 2 inflammation pathways (29).…”

Section: Discussionmentioning

confidence: 99%

Endotoxin Exposure: Predictors and Prevalence of Associated Asthma Outcomes in the United States

Thorne

Mendy

Metwali

et al. 2015

Am J Respir Crit Care Med

108

122

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Rationale: Inhaled endotoxin induces airway inflammation and is an established risk factor for asthma. The -2006 National Health and Nutrition Examination Survey included measures of endotoxin and allergens in homes as well as specific IgE to inhalant allergens.Objectives: To understand the relationships between endotoxin exposure, asthma outcomes, and sensitization status for 15 aeroallergens in a nationally representative sample.Methods: Participants were administered questionnaires in their homes. Reservoir dust was vacuum sampled to generate composite bedding and bedroom floor samples. We analyzed 7,450 National Health and Nutrition Examination Survey dust and quality assurance samples for their endotoxin content using extreme quality assurance measures. Data for 6,963 subjects were available, making this the largest study of endotoxin exposure to date. Log-transformed endotoxin concentrations were analyzed using logistic models and forward stepwise linear regression. Analyses were weighted to provide national prevalence estimates and unbiased variances.Measurements and Main Results: Endotoxin exposure was significantly associated with wheeze in the past 12 months, wheeze during exercise, doctor and/or emergency room visits for wheeze, and use of prescription medications for wheeze. Models adjusted for age, sex, race and/or ethnicity, and poverty-to-income ratio and stratified by allergy status showed that these relationships were not dependent upon sensitization status but were worsened among those living in poverty. Significant predictors of higher endotoxin exposures were lower family income; Hispanic ethnicity; participant age; dog(s), cat(s), cockroaches, and/or smoker(s) in the home; and carpeted floors.Conclusions: In this U.S. nationwide representative sample, higher endotoxin exposure was significantly associated with measures of wheeze, with no observed protective effect regardless of sensitization status.

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Purified monomeric ligand.MD-2 complexes reveal molecular and structural requirements for activation and antagonism of TLR4 by Gram-negative bacterial endotoxins

Cited by 25 publications

References 44 publications

High-affinity caspase-4 binding to LPS presented as high molecular mass aggregates or in outer membrane vesicles

High-affinity caspase-4 binding to LPS presented as high molecular mass aggregates or in outer membrane vesicles

Molecular Basis of the Functional Differences between Soluble Human Versus Murine MD-2: Role of Val135 in Transfer of Lipopolysaccharide from CD14 to MD-2

Endotoxin Exposure: Predictors and Prevalence of Associated Asthma Outcomes in the United States

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