title: Sialyl residues modulate LPS-mediated signaling through the Toll-like receptor 4 complex.
abstract: We 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 NFκB 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 NFκB 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.
authors.affiliation: Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Chiguang
authors.initials: C
authors.lastname: Feng
authors.forename: Nicholas M
authors.initials: NM
authors.lastname: Stamatos
authors.forename: Anatoliy I
authors.initials: AI
authors.lastname: Dragan
authors.forename: Andrei
authors.initials: A
authors.lastname: Medvedev
authors.forename: Melissa
authors.initials: M
authors.lastname: Whitford
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Chang
authors.initials: C
authors.lastname: Song
authors.forename: Prasad
authors.initials: P
authors.lastname: Rallabhandi
authors.forename: Leah
authors.initials: L
authors.lastname: Cole
authors.forename: Quan M
authors.initials: QM
authors.lastname: Nhu
authors.forename: Stefanie N
authors.initials: SN
authors.lastname: Vogel
authors.forename: Chris D
authors.initials: CD
authors.lastname: Geddes
authors.forename: Alan S
authors.initials: AS
authors.lastname: Cross
mesh_terms: D015153:Blotting, Western; D002450:Cell Communication; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005786:Gene Expression Regulation; D006031:Glycosylation; D006801:Humans; D047468:Immunoprecipitation; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000235:genetics / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D051218:Lymphocyte Antigen 96 / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D009439:Neuraminidase / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D012794:Sialic Acids / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*
chemical_list: D016207:Cytokines; C495377:LY96 protein, human; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D016328:NF-kappa B; D012333:RNA, Messenger; D012794:Sialic Acids; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; C486385:NEU1 protein, human; C465436:Neu3 protein, human; D009439:Neuraminidase