MD-2 is necessary for the toll-like receptor 4 protein to undergo glycosylation essential for its translocation to the cell surface.
MD-2 has been reported to be required for the translocation of the Toll-like receptor 4 (TLR4) to the cell surface. However, the mechanism by which MD-2 promotes TLR4 translocation is unknown. We identified the presence of two forms of TLR4 with different molecular masses (approximately 110 and 130 kDa) when TLR4 was expressed together with MD-2. Expressing TLR4 alone produced only the 110-kDa form. Using a membrane-impermeable biotinylation reagent, we found that only the 130-kDa form of TLR4 was expressed on the cell surface. When a cellular extract prepared from cells expressing TLR4 and MD-2 was treated with N-glycosidase, the two forms of TLR4 converged into a single band whose size was smaller than the 110-kDa form of TLR4. Mutation of TLR4 at Asn(526) or Asn(575) resulted in the disappearance of the 130-kDa form and prevented TLR4 from being expressed on the cell surface without affecting the ability of TLR4 to associate with MD-2. These results indicate that TLR4 is able to undergo multiple glycosylations without MD-2 but that the specific glycosylation essential for cell surface expression requires the presence of MD-2.
Ohnishi
Takahiro
T
Division of Microbiology, National Institute of Health Sciences, Setagaya, Tokyo 158-8501, Japan.
Muroi
Masashi
M
Tanamoto
Ken-Ichi
K
D000954:Antigens, Surface / Q000502:physiology*; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D021381:Protein Transport; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014162:Transfection
D000954:Antigens, Surface; C495377:LY96 protein, human; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D009699:N-Glycosyl Hydrolases