Secreted MD-2 is a large polymeric protein that efficiently confers lipopolysaccharide sensitivity to Toll-like receptor 4

Abstract

Toll-like receptor 4 (TLR4), the principal signaling receptor for lipopolysaccharide (LPS) in mammals, requires the binding of MD-2 to its extracellular domain for maximal responsiveness. MD-2 contains a leader sequence but lacks a transmembrane domain, and we asked whether it is secreted into the medium as an active protein. As a source of secreted MD-2 (sMD-2), we used culture supernatants from cells stably transduced with epitope-tagged human MD-2. We show that sMD-2 exists as a heterogeneous collection of large disulfide-linked oligomers formed from stable dimeric subunits and that concentrations of sMD-2 as low as 50 pM enhance the responsiveness of TLR4 reporter cells to LPS. An MD-2-like activity is also released by monocyte-derived dendritic cells from normal donors. When coexpressed, TLR4 indiscriminately associates in the endoplasmic reticulum͞cis Golgi with differentsized oligomers of MD-2, and excess MD-2 is secreted into the medium. We conclude that normal and transfected cells secrete a soluble form of MD-2 that binds with high affinity to TLR4 and that could play a role in regulating responses to LPS and other pathogen-derived substances in vivo.M ammalian cells respond to lipopolysaccharide (LPS) (1), a major component of the outer membrane of Gramnegative bacterial cell walls, by activating Toll-like receptor 4 (TLR4) (1-5). TLR4 was first described as a member of a family of type I integral membrane proteins that contain numerous leucine-rich motifs on their extracellular portions and an intracellular signaling domain homologous to that of the IL-1 receptor (6, 7). In response to LPS, TLR4 initiates a cascade of serine͞threonine kinases that eventually leads to the transcription of genes involved in inflammation (8, 9). To respond efficiently to LPS, TLR4 requires an accessory protein, MD-2 (10). MD-2 is a 20-30-kDa glycoprotein that was originally discovered by sequence homology with MD-1, a protein found associated with a B cell homologue of TLR4, RP105 (11,12). MD-1 binds to the extracellular domain of RP105 and enhances its surface expression. Similarly, MD-2 binds to the extracellular domains of both TLRs 2 and 4 and causes their surface expression levels to increase (10, 13). Photoaffinity labeling studies have recently shown that LPS binds directly to the TLR4͞MD-2 complex and that both molecules are in close proximity to the bound LPS (14).MD-2, which contains a leader sequence but lacks a transmembrane domain, is secreted into the medium by transfected kidney epithelial cells (15). However, the structure and function of secreted MD-2 (sMD-2) have not been defined, nor is it known whether MD-2 is released into the medium by normal cells. To examine the properties of sMD-2, we have stably transfected HEK293 cells with an epitope-tagged version of MD-2 and have examined the secreted protein in culture supernatants. Based on previous findings from this laboratory that immature dendritic cells (iDC) express elevated levels of MD-2 message and decreased levels of TLRs 2 and 4 relative to mon...

Identification of Mouse MD-2 Residues Important for Forming the Cell Surface TLR4-MD-2 Complex Recognized by Anti-TLR4-MD-2 Antibodies, and for Conferring LPS and Taxol Responsiveness on Mouse TLR4 by Alanine-Scanning Mutagenesis

“…Human MD-2 contains seven cysteine residues, which are conserved between mouse and man, and these residues are important for the formation of large disulfide-linked oligomers (26,31). Our results showed that Cys residues, such as Cys 37 , Cys 95 , Cys 105 , and Cys 148 of mouse MD-2 are important for conferring LPS and Taxol responsiveness on mouse TLR4 and for forming the cell surface TLR4-MD-2 complex recognized by MTS510.…”

“…The Ab was raised against mouse TLR4, was reported to react with TLR4 but not with MD-2, and was suggested to recognize the specific conformation of mouse TLR4 that is associated with mouse MD-2 (11). Therefore, 293/mTLR4/luc cells that express a mutant mouse MD2 but do not react with MTS510 might be defective in the TLR4-MD-2 complex formation on the cell surface because of a defect in the physical association of the mutant MD-2 with TLR4, and/or a defect in the translocation of the TLR4-MD-2 complex, which has been shown to be formed in the endoplasmic reticulum (26) to the cell surface. Alternatively, such cells might have lost the conformational epitope recognized by the Ab.…”

“…Alanine is usually chosen for replacement because it eliminates the side chain beyond the ␤ carbon yet does not alter the main-chain conformation or have an extreme electrostatic or steric effect (21,25). Because MD-2 is a secretory protein (26), and the site between Thr 16 and Glu 17 of mouse MD-2 was predicted to be a possible cleavage site of the leader sequence with the PSORT II program (http://psort.ims.u-tokyo.ac.jp/), we analyzed the amino acid residues from Glu 17 to C-terminal Asn 160 . The expression constructs were introduced into 293/mTLR4/luc cells, and cell surface TLR4-MD-2 complex formation was measured by flow cytometry using MTS510 (Fig.…”

The Ectodomain of the Toll-like Receptor 4 Prevents Constitutive Receptor Activation

“…Thus, as MD-2-and CD14-based TLR4 chimeras are constitutively active, they must be present in the cell in the form of dimeric or oligomeric clusters. As MD-2 is known to form dimers and higher-order multimers (32,33), and CD14 was in fact crystallized as a dimer (26), a dimeric or oligomeric state of the chimeric receptors could lead to the oligomerization of the ectodomains of these constructs, bringing the TIR domains close together. This is supported by the previous report on the addition of the CD4 dimerization domain to the transmembrane and cytoplasmic domains of TLR4, which also resulted in constitutive activity of CD4-TLR4 chimeras (17).…”

Heat shock proteins and innate immunity

“…The TLR4 response in turn is significantly augmented by the molecule MD-2. This is produced by the responding cell and associates with TLR4, augmenting responses even when present at picomolar concentrations [23]. These two amplification mechanisms, LPS binding protein and MD-2, suggest that there might be scope for hsp to act in similar ways rather than as direct ligands of the signalling molecule, in addition to the postulated role of 'chaperoning' bacterial components to TLRs.…”