Sialyl Residues Modulate LPS-Mediated Signaling through the Toll-Like Receptor 4 Complex

dc.contributor.authorFeng, Chiguang
dc.contributor.authorStamatos, Nicholas M.
dc.contributor.authorDragan, Anatoliy
dc.contributor.authorMedvedev, Andrei
dc.contributor.authorWhitford, Melissa
dc.contributor.authorZhang, Lei
dc.contributor.authorSong, Chang
dc.contributor.authorRallabhandi, Prasad
dc.contributor.authorCole, Leah
dc.contributor.authorNhu, Quan M.
dc.contributor.authorVogel, Stefanie N.
dc.contributor.authorGeddes, Chris
dc.contributor.authorCross, Alan S.
dc.date.accessioned2024-10-01T18:05:32Z
dc.date.available2024-10-01T18:05:32Z
dc.date.issued2012-04-09
dc.description.abstractWe previously reported that neuraminidase (NA) pretreatment of human PBMCs markedly increased their cytokine response to lipopolysaccharide (LPS). To study the mechanisms by which this occurs, we transfected HEK293T cells with plasmids encoding TLR4, CD14, and MD2 (three components of the LPS receptor complex), as well as a NFₖB luciferase reporting system. Both TLR4 and MD2 encoded by the plasmids are α-2,6 sialylated. HEK293T cells transfected with TLR4/MD2/CD14 responded robustly to the addition of LPS; however, omission of the MD2 plasmid abrogated this response. Addition of culture supernatants from MD2 (sMD2)-transfected HEK293T cells, but not recombinant, non-glycosylated MD2 reconstituted this response. NA treatment of sMD2 enhanced the LPS response as did NA treatment of the TLR4/CD14-transfected cell supplemented with untreated sMD2, but optimal LPS-initiated responses were observed with NA-treated TLR4/CD14-transfected cells supplemented with NA-treated sMD2. We hypothesized that removal of negatively charged sialyl residues from glycans on the TLR4 complex would hasten the dimerization of TLR4 monomers required for signaling. Co-transfection of HEK293T cells with separate plasmids encoding either YFP- or FLAG-tagged TLR4, followed by treatment with NA and stimulation with LPS, led to an earlier and more robust time-dependent dimerization of TLR4 monomers on co-immunoprecipitation, compared to untreated cells. These findings were confirmed by fluorescence resonance energy transfer (FRET) analysis. Overexpression of human Neu1 increased LPS-initiated TLR4-mediated NFₖB activation and a NA inhibitor suppressed its activation. We conclude that (1) sialyl residues on TLR4 modulate LPS responsiveness, perhaps by facilitating clustering of the homodimers, and that (2) sialic acid, and perhaps other glycosyl species, regulate MD2 activity required for LPS-mediated signaling. We speculate that endogenous sialidase activity mobilized during cell activation may play a role in this regulation.
dc.description.sponsorshipThese studies were supported by National Institutes of Health (NIH) grant HL086933-01A1 to ASC. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
dc.description.urihttps://journals.plos.org/plosone/article?id=10.1371/journal.pone.0032359
dc.format.extent12 pages
dc.genrejournal articles
dc.identifierdoi:10.13016/m2luis-jpmm
dc.identifier.citationFeng, Chiguang, Nicholas M. Stamatos, Anatoliy I. Dragan, Andrei Medvedev, Melissa Whitford, Lei Zhang, Chang Song, et al. “Sialyl Residues Modulate LPS-Mediated Signaling through the Toll-Like Receptor 4 Complex.” PLOS ONE 7, no. 4 (April 9, 2012): e32359. https://doi.org/10.1371/journal.pone.0032359.
dc.identifier.urihttps://doi.org/10.1371/journal.pone.0032359
dc.identifier.urihttp://hdl.handle.net/11603/36586
dc.language.isoen_US
dc.publisherPLOS
dc.relation.isAvailableAtThe University of Maryland, Baltimore County (UMBC)
dc.relation.ispartofUMBC Institute of Fluorescence (IoF)
dc.relation.ispartofUMBC Faculty Collection
dc.relation.ispartofUMBC Chemistry & Biochemistry Department
dc.rightsAttribution 4.0 International
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.subjectImmune receptor signaling
dc.subjectLuciferase
dc.subjectFluorescence resonance energy transfer
dc.subjectDimerization
dc.subjectImmunoprecipitation
dc.subjectGlycosylation
dc.subjectToll-like receptors
dc.subjectTransfection
dc.titleSialyl Residues Modulate LPS-Mediated Signaling through the Toll-Like Receptor 4 Complex
dc.typeText
dcterms.creatorhttps://orcid.org/0000-0002-1679-8307
dcterms.creatorhttps://orcid.org/0000-0002-9110-6374

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