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 involv ed in mediating responses to microbial pathogens are highly conserved throu ghout evolution. Examples include members of the Drosophila Toll (dToll) an d the mammalian Toll-like receptor (TLR) protein families. Activation of Dr osophila Toll is believed to occur via an endogenous peptide rather than th rough direct binding of microbial products to the Toll protein. In mammals there is a growing consensus that lipopolysaccharide (LPS) initiates its bi ological 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 tran sient transfection to express human TLRs, MD-2, or CD14 alone or in differe nt combinations in HEK 293 cells, Interactions between LPS and these protei ns were studied using a chemically modified, radioiodinated LPS containing a covalently linked, UV light-activated crosslinking group (I-125-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 LP S is in close proximity to the three known proteins of its membrane recepto r complex. Thus, LPS binds directly to each of the members of the tripartit e LPS receptor complex.