Monomeric Recombinant MD-2 Binds Toll-like Receptor 4 Tightlyand Confers Lipopolysaccharide Responsiveness*Received for publication, March 15, 2002, and in revised form, April 19, 2002Published, JBC Papers in Press, April 25, 2002, DOI 10.1074/jbc.M202554200Fabio Re‡§ and Jack L. Strominger‡¶From the ‡Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts 02115and the¶Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138In order to mediate cellular response to lipopolysac-charide (LPS), Toll-like receptor (TLR) 4 must interactwith MD-2, a secreted protein. In this study, a biochem-ical assay was developed to demonstrate that recombi-nant MD-2 can interact with the extracellular portion ofTLR4 in solution. The ability of MD-2 to multimerize wasconfirmed, and MD-1 was also shown to possess thisability. Through site-directed mutagenesis, more thantwo intermolecular disulfide bonds were found to stabi-lize the MD-2 multimer. MD-2’s abilities to confer LPSresponsiveness and to bind TLR4 were strongly associ-ated functions. Remarkably, although the majority ofrecombinant MD-2 exists in multimeric form, mono-meric MD-2 was found to preferentially bind TLR4 andto confer LPS responsiveness more efficiently thanMD-2 multimers.Innate immunity relies on the ability of cells of the immunesystem to recognize molecular motifs that distinguish classes ofmicroorganisms through germ line-encoded pattern recognitionreceptors (1, 2). The lipopolysaccharide (LPS)1found in theouter membrane of Gram-negative bacteria is among the bestcharacterized pathogen-associated molecular patterns. An ex-tremely low concentration of LPS can be sensed by immuno-competent cells, such as macrophages and dendritic cells, lead-ing to the activation of intracellular signaling pathways andthe production of cytokines and other molecules that are re-quired to fight the invading microorganism and activate theadaptive immune system.At least four cellular proteins are known to participate inLPS recognition and cellular activation (3–5). CD14 is a glyco-sylphosphatidylinositol-linked cell surface protein that servesas the major LPS receptor but is itself devoid of signalingcapacity. CD14 is aided in LPS recognition by LPS-bindingprotein, a serum protein that acts as a lipid transferase. Thesignaling subunit of the LPS receptor is TLR4, a member of theTLR family. TLR4 is a type I transmembrane protein thatcontains in the cytoplasmic region a Toll-interleukin-1 receptordomain that links the receptor to the signaling machineryshared by interleukin-1 and interleukin-18. Lastly, MD-2, asecreted protein that is retained on the cell surface throughinteraction with TLR4, is also indispensable for TLR4 signalingin response to LPS. MD-2 shares sequence homology withMD-1, a protein that binds RP105, a TLR family memberpreferentially expressed on B cells (6). Although it has beenshown that CD14, TLR4, and MD-2 all lie in close proximity tothe LPS molecule (7), the relative contribution of each receptorsubunit to the recognition event and signal transduction is notentirely understood. The role of MD-2 is particularly puzzling.Both biochemical (8) and genetic (9) evidence demonstrated theabsolute requirement of this protein for TLR4 function. How-ever, MD-2 lacks structural features that would suggest itsdirect involvement in the transduction of the signal. Rather,MD-2 appears to be involved in agonist recognition; the abilityof Taxol to stimulate mouse but not human TLR4 is dependenton the N-terminal region of mouse MD-2 (10); moreover, MD-2directly binds LPS with an affinity similar to that of CD14 (11).The role of MD-2 is rendered even more confusing by the factthat most TLRs, with the exception of TLR4 and RP105, do notseem to rely on MD-2 or MD-2-like proteins for their function.Recently, MD-2 was reported to form disulfide-linked multim-ers (12), adding a further level of complexity.In this study, the interaction between MD-2 and solubleTLR4 and their relationship to LPS responsiveness have beenexamined in some detail. Remarkably, although the majority ofMD-2 exists in multimeric form, monomeric MD-2 was shownto preferentially bind TLR4 and to confer LPS responsivenessmore efficiently than MD-2 multimers.EXPERIMENTAL PROCEDURESCells and Transfection—HeLa cells were grown in Dulbecco’s modi-fied Eagle’s medium supplemented with 10% fetal calf serum. HeLacells (7 ⫻ 106) were transfected by electroporation (250 V, 950 micro-farads) with 30␮g of plasmid mixture in Dulbecco PBS/1.25% Me2SO.Cells were replated in a 10-cm plate in fetal calf serum-containingmedium to allow recovery and cell adhesion. After 12 h, plates wererinsed in PBS, and 6 ml of serum-free medium 293 SFM (Invitrogen)were added. Media were collected 24 – 48 h later. The HeLa-TLR4 cellline was described previously (13).Luciferase Assay—HeLa-TLR2, HeLa-TLR4, or HeLa-TLR4/MD2cells were transiently transfected in 6-well plates using Superfect rea-gent (Qiagen, Valencia, CA) with 0.5␮g of ELAM-luciferase and 1␮gofpcDNA-CD14 (kindly provided by Dr. D. Golenbock, University of Mas-sachusetts) and 0.1␮g of CMV-␤-galactosidase. Luciferase assay wasperformed using Promega (Madison, WI) reagents according to themanufacturer’s recommendations. Efficiency of transfection was nor-malized by measuring␤-galactosidase in cell lysates.Expression Vectors—Expression vectors for FLAG-TLR4, Myc-TLR4,and FLAG-TLR2 were described previously (13). Soluble forms of TLR4and TLR2 were expressed using these vectors after the following mod-ifications. FLAG-sTLR4-His and Myc-sTLR4-His were generated byadding a 6-histidine tag after amino acid 634 of TLR4. The EcoRV-BamHI fragment of pCMV-FLAG-TLR4 or pCMV-Myc-TLR4 was re-* This work was supported in part by National Institutes of HealthGrants 5R35-CA47554 and N01-AI45198 (to J. L. S.). The costs of pub-lication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.§ Supported by a National Research Service Award postdoctoraltraining grant in AIDS Research (Dana-Farber Cancer Institute/Na-tional Institutes of Health). To whom correspondence should be ad-dressed: Dept. of Cancer Immunology and AIDS, Dana-Farber CancerInstitute, 44 Binney St., Dana-1414, Boston, MA 02115. Tel.: