Lipopolysaccharide is in close proximity to each of the proteins in its membrane receptor complex. transfer from CD14 to TLR4 and MD-2.
The structural features of some proteins of the innate immune system involved in mediating responses to microbial pathogens are highly conserved throughout evolution. Examples include members of the Drosophila Toll (dToll) and the mammalian Toll-like receptor (TLR) protein families. Activation of Drosophila Toll is believed to occur via an endogenous peptide rather than through direct binding of microbial products to the Toll protein. In mammals there is a growing consensus that lipopolysaccharide (LPS) initiates its biological activities through a heteromeric receptor complex containing CD14, TLR4, and at least one other protein, MD-2. LPS binds directly to CD14 but whether LPS then binds to TLR4 and/or MD-2 is not known. We have used transient transfection to express human TLRs, MD-2, or CD14 alone or in different combinations in HEK 293 cells. Interactions between LPS and these proteins were studied using a chemically modified, radioiodinated LPS containing a covalently linked, UV light-activated cross-linking group ((125)I-ASD-Re595 LPS). Here we show that LPS is cross-linked specifically to TLR4 and MD-2 only when co-expressed with CD14. These data support the contention that LPS is in close proximity to the three known proteins of its membrane receptor complex. Thus, LPS binds directly to each of the members of the tripartite LPS receptor complex.
Scripps Research Institute, Department of Immunology, La Jolla, California 92037, USA.
J
J
da Silva Correia
K
K
Soldau
U
U
Christen
P S
PS
Tobias
R J
RJ
Ulevitch
D000818:Animals; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D003432:Cross-Linking Reagents; D003847:Deoxyglucose / Q000494:pharmacology; D004330:Drosophila; D029721:Drosophila Proteins; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000737:chemistry / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism*; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019810:Sodium Azide / Q000494:pharmacology; D012969:Sodium Fluoride / Q000494:pharmacology; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014162:Transfection; D014466:Ultraviolet Rays
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D003432:Cross-Linking Reagents; D029721:Drosophila Proteins; C495377:LY96 protein, human; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D012969:Sodium Fluoride; D019810:Sodium Azide; D003847:Deoxyglucose