N-linked glycosylations at Asn(26) and Asn(114) of human MD-2 are required for toll-like receptor 4-mediated activation of NF-kappaB by lipopolysaccharide.
MD-2 is physically associated with Toll-like receptor 4 (TLR4) and is required for TLR4-mediated LPS signaling. Western blotting analysis revealed the presence of three forms of human (h)MD-2 with different electrophoretic mobilities. After N-glycosidase treatment of the cellular extract prepared from cells expressing hMD-2, only a single form with the fastest mobility was detected. Mutation of either one of two potential glycosylation sites (Asn(26) and Asn(114)) of MD-2 resulted in the disappearance of the slowest mobility form, and only the fastest form was detected in hMD-2 carrying mutations at both Asn(26) and Asn(114). Although these mutants were expressed on the cell surface and maintained its ability to associate with human TLR4, these mutations or tunicamycin treatment substantially impaired the ability of MD-2 to complement TLR4-mediated activation of NF-kappaB by LPS. LPS binding to cells expressing CD14, TLR4, and MD-2 was unaffected by these mutations. These observations demonstrate that hMD-2 undergoes N-linked glycosylation at Asn(26) and Asn(114), and that these glycosylations are crucial for TLR4-mediated signal transduction of LPS.
Ohnishi
T
T
Division of Microbiology, National Institute of Health Sciences, Tokyo, Japan.
Muroi
M
M
Tanamoto
K
K
D000209:Acute-Phase Proteins; D000954:Antigens, Surface / Q000737:chemistry* / Q000502:physiology; D001216:Asparagine / Q000737:chemistry*; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D029721:Drosophila Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D006031:Glycosylation; D006801:Humans; D007668:Kidney; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011956:Receptors, Cell Surface / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000502:physiology; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014162:Transfection; D000099231:Lipopolysaccharide-Binding Protein
D000209:Acute-Phase Proteins; D000954:Antigens, Surface; D001216:Asparagine; D002352:Carrier Proteins; D029721:Drosophila Proteins; D008070:Lipopolysaccharides; D008156:Luciferases; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D000099231:Lipopolysaccharide-Binding Protein; C495377:LY96 protein, human; C495345:TLR4 protein, human