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Md-2 and tlr4 n-linked glycosylations are important for a functional lipopolysaccharide receptor

Academic Article
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Overview

authors

  • da Silva Correia, J.
  • Ulevitch, Richard

publication date

  • 2002

journal

  • Journal of Biological Chemistry  Journal

abstract

  • The lipopolysaccharide (LPS) receptor is a multi-protein complex that consists of at least three proteins, CD14, TLR4, and MD-2. Because each of these proteins is glycosylated, we have examined the functional role of N-linked carbohydrates of both MD-2 and TLR4. We demonstrate that MD-2 contains 2 N-glycosylated sites at positions Asn(26) and Asn(114), whereas the amino-terminal ectodomain of human TLR4 contains 9 N-linked glycosylation sites. Site-directed mutagenesis studies showed that cell surface expression of MD-2 did not depend on the presence of either N-linked site, whereas in contrast, TLR4 mutants carrying substitutions in Asn(526) or Asn(575) failed to be transported to the cell surface. Using a UV-activated derivative of Re595 LPS (ASD-Re595 LPS) in cross-linking assays, we demonstrated a critical role of MD-2 and TLR4 carbohydrates in LPS cross-linking to the LPS receptor. The ability of the various glycosylation mutants to support cell activation was also evaluated in transiently transfected HeLa cells. The double mutant of MD-2 failed to support LPS-induced activation of an interleukin-8 (IL-8) promoter-driven luciferase reporter to induce IL-8 secretion or to activate amino-terminal c-Jun kinase (JNK). Similar results were observed with TLR4 mutants lacking three or more N-linked glycosylation sites. Surprisingly, the reduction in activation resulting from expression of the Asn mutants of MD-2 and TLR4 can be partially reversed by co-expression with CD14. This suggests that the functional integrity of the LPS receptor depends both on the surface expression of at least three proteins, CD14, MD-2, and TLR4, and that N-linked sites of both MD-2 and TLR4 are essential in maintaining the functional integrity of this receptor.

subject areas

  • Antigens, CD14
  • Antigens, Surface
  • Base Sequence
  • Blotting, Western
  • Cell Line
  • DNA Primers
  • Drosophila Proteins
  • Glycosylation
  • Humans
  • Lipopolysaccharides
  • Lymphocyte Antigen 96
  • Membrane Glycoproteins
  • Mutagenesis, Site-Directed
  • Precipitin Tests
  • Receptors, Cell Surface
  • Signal Transduction
  • Toll-Like Receptor 4
  • Toll-Like Receptors
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Identity

International Standard Serial Number (ISSN)

  • 0021-9258

Digital Object Identifier (DOI)

  • 10.1074/jbc.M109910200

PubMed ID

  • 11706042
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Additional Document Info

start page

  • 1845

end page

  • 1854

volume

  • 277

issue

  • 3

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