MD-2 and TLR4 N-linked glycosylations are important for a functional lipopolysaccharide receptor.
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.
da Silva Correia
Jean
J
Scripps Research Institute, Department of Immunology, La Jolla, California 92037, USA.
Ulevitch
Richard J
RJ
D000954:Antigens, Surface / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D017931:DNA Primers; D029721:Drosophila Proteins; D006031:Glycosylation; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D008070:Lipopolysaccharides / Q000378:metabolism; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D011233:Precipitin Tests; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors
D000954:Antigens, Surface; D017931:DNA Primers; D029721:Drosophila Proteins; C495377:LY96 protein, human; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors