Mutational Analysis of Membrane and Soluble Forms of Human MD-2
2006; Elsevier BV; Volume: 281; Issue: 17 Linguagem: Inglês
10.1074/jbc.m511627200
ISSN1083-351X
AutoresSuganya Viriyakosol, Peter S. Tobias, Theo N. Kirkland,
Tópico(s)Pneumonia and Respiratory Infections
ResumoToll-like receptor 4 and MD-2 form a receptor for lipopolysaccharide (LPS), a major constituent of Gram-negative bacteria. MD-2 is a 20-25-kDa extracellular glycoprotein that binds to Tolllike receptor 4 (TLR4) and LPS and is a critical part of the LPS receptor. Here we have shown that the level of MD-2 expression regulates TLR4 activation by LPS. Using site-directed mutagenesis, we have found that glycosylation has no effect on MD-2 function as a membrane receptor for LPS. We used alanine-scanning mutagenesis to identify regions of human MD-2 that are important for TLR4 and LPS binding. We found that mutation in the N-terminal 46 amino acids of MD-2 did not substantially diminish LPS activation of Chinese hamster ovary (CHO) cells co-transfected with TLR4 and mutant MD-2. The residues 46-50 were important for LPS activation but not LPS binding. The residues 79-83, 121-124, and 125-129 are identified as important in LPS activation but not surface expression of membrane MD-2. The function of soluble MD-2 is somewhat more sensitive to mutation than membrane MD-2. Our results suggest that the 46-50 and 127-131 regions of soluble MD-2 bind to TLR4. The region 79-120 is not involved in LPS binding but affects monomerization of soluble MD-2 as well as TLR4 binding. We define the LPS binding region of monomeric soluble MD-2 as a cluster of basic residues 125-131. Studies on both membrane and soluble MD-2 suggest that domains of MD-2 for TLR4 and LPS binding are separate as well as overlapping. By mapping these regions on a three-dimensional model, we show the likely binding regions of MD-2 to TLR4 and LPS. Toll-like receptor 4 and MD-2 form a receptor for lipopolysaccharide (LPS), a major constituent of Gram-negative bacteria. MD-2 is a 20-25-kDa extracellular glycoprotein that binds to Tolllike receptor 4 (TLR4) and LPS and is a critical part of the LPS receptor. Here we have shown that the level of MD-2 expression regulates TLR4 activation by LPS. Using site-directed mutagenesis, we have found that glycosylation has no effect on MD-2 function as a membrane receptor for LPS. We used alanine-scanning mutagenesis to identify regions of human MD-2 that are important for TLR4 and LPS binding. We found that mutation in the N-terminal 46 amino acids of MD-2 did not substantially diminish LPS activation of Chinese hamster ovary (CHO) cells co-transfected with TLR4 and mutant MD-2. The residues 46-50 were important for LPS activation but not LPS binding. The residues 79-83, 121-124, and 125-129 are identified as important in LPS activation but not surface expression of membrane MD-2. The function of soluble MD-2 is somewhat more sensitive to mutation than membrane MD-2. Our results suggest that the 46-50 and 127-131 regions of soluble MD-2 bind to TLR4. The region 79-120 is not involved in LPS binding but affects monomerization of soluble MD-2 as well as TLR4 binding. We define the LPS binding region of monomeric soluble MD-2 as a cluster of basic residues 125-131. Studies on both membrane and soluble MD-2 suggest that domains of MD-2 for TLR4 and LPS binding are separate as well as overlapping. By mapping these regions on a three-dimensional model, we show the likely binding regions of MD-2 to TLR4 and LPS. Innate immunity is the first line of defense against pathogens. A key component of the mammalian innate immune system is a family of Toll-like receptors (TLRs) 3The abbreviations used are: TLR, Toll-like receptor; LPS, lipopolysaccharide; LBP, LPS-binding protein; mAb, monoclonal antibody; CHO, Chinese hamster ovary; ELISA, enzyme-linked immunosorbent assay: FACS, fluorescence-activating cell sorter; HA, hemagglutinin; IL, interleukin; MCN, mean channel number; WT, wild type; MES, 4-morpholineethanesulfonic acid. 3The abbreviations used are: TLR, Toll-like receptor; LPS, lipopolysaccharide; LBP, LPS-binding protein; mAb, monoclonal antibody; CHO, Chinese hamster ovary; ELISA, enzyme-linked immunosorbent assay: FACS, fluorescence-activating cell sorter; HA, hemagglutinin; IL, interleukin; MCN, mean channel number; WT, wild type; MES, 4-morpholineethanesulfonic acid. (1Medzhitov R. Preston-Hurlburt P. Janeway Jr., C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4378) Google Scholar, 2Aderem A. Ulevitch R.J. Nature. 2000; 406: 782-787Crossref PubMed Scopus (2601) Google Scholar). Lipopolysaccharide (LPS), a major component of Gram-negative bacteria, activates a variety of cells to produce inflammatory cytokines that can lead to septic shock in humans. The innate immune mechanism that recognizes LPS involves a transfer of LPS to a pattern recognition molecule CD14 (3Wright S.D. Ramos R.A. Tobias P.S. Ulevitch R.J. Mathison J.C. Science. 1990; 249: 1431-1433Crossref PubMed Scopus (3375) Google Scholar) by lipopolysaccharide-binding protein (LBP) (4Tobias P.S. Ulevitch R.J. Immunobiology. 1993; 187: 227-232Crossref PubMed Scopus (150) Google Scholar). CD14 has no transmembrane domain and is not capable of signaling. TLR4 is a type 1 transmembrane protein that has extracellular leucine-rich repeats and an intracellular signaling domain that is responsible for LPS signaling (5Poltorak A. He X. Smirnova I. Liu M.Y. Van Huffel C. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6379) Google Scholar, 6Qureshi S.T. Lariviere L. Leveque G. Clermont S. Moore K.J. Gros P. Malo D. J. Exp. Med. 1999; 189: 615-625Crossref PubMed Scopus (1343) Google Scholar). TLR4 forms a complex with MD-2, a 22-25-kDa glycoprotein, on the cell surface (7Shimazu R. Akashi S. Ogata H. Nagai Y. Fukudome K. Miyake K. Kimoto M. J. Exp. Med. 1999; 189: 1777-1782Crossref PubMed Scopus (1730) Google Scholar). A cascade of events leading to maximal cellular activation is likely to involve transferring of LPS by LBP to CD14 and then to TLR4·MD-2. Although CD14 and LBP enhance cellular activation, activation of TLR4 by LPS absolutely requires MD-2 (8Akashi S. Shimazu R. Ogata H. Nagai Y. Takeda K. Kimoto M. Miyake K. J. Immunol. 2000; 164: 3471-3475Crossref PubMed Scopus (422) Google Scholar).MD-2 can be found on the cell surface in association with TLR4 or as a secreted protein. It shares a sequence homology to MD-1, a protein that binds to another TLR family member, RP105 (9Miyake K. Shimazu R. Kondo J. Niki T. Akashi S. Ogata H. Yamashita Y. Miura Y. Kimoto M. J. Immunol. 1998; 161: 1348-1353PubMed Google Scholar), which constitutes an LPS signaling complex on B-cells (10Miyake K. Ogata H. Nagai Y. Akashi S. Kimoto M. J. Endotoxin Res. 2000; 6: 389-391Crossref PubMed Google Scholar). MD-2 contributes to the ligand recognition of TLR4. It binds LPS with high affinity (11Viriyakosol S. Tobias P.S. Kitchens R.L. Kirkland T.N. J. Biol. Chem. 2001; 276: 38044-38051Abstract Full Text Full Text PDF PubMed Google Scholar) and is responsible for the different responses of human and mouse to lipid IVA and taxol (12Kawasaki K. Akashi S. Shimazu R. Yoshida T. Miyake K. Nishijima M. J. Biol. Chem. 2000; 275: 2251-2254Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). Interaction of the cell surface TLR4·MD-2 complex with LPS induces clustering of TLR4 and may be the mechanism that triggers cellular activation (13Visintin A. Latz E. Monks B.G. Espevik T. Golenbock D.T. J. Biol. Chem. 2003; 278: 48313-48320Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar).Although proper glycosylation and trafficking of TLR4 to the cell surface requires intracellular association with MD-2 (14Nagai Y. Akashi S. Nagafuku M. Ogata M. Iwakura Y. Akira S. Kitamura T. Kosugi A. Kimoto M. Miyake K. Nat. Immunol. 2002; 3: 667-672Crossref PubMed Scopus (831) Google Scholar, 15Ohnishi T. Muroi M. Tanamoto K. Clin. Diagn. Lab. Immunol. 2003; 10: 405-410Crossref PubMed Scopus (76) Google Scholar), functional TLR4 can be presented on the cell surface without MD-2 in both transfected cells and human airway epithelial cells (11Viriyakosol S. Tobias P.S. Kitchens R.L. Kirkland T.N. J. Biol. Chem. 2001; 276: 38044-38051Abstract Full Text Full Text PDF PubMed Google Scholar, 16Jia H.P. Kline J.N. Penisten A. Apicella M.A. Gioannini T.L. Weiss J. McCray Jr., P.B. Am. J. Physiol. 2004; 287: L428-L437Crossref PubMed Scopus (140) Google Scholar). These cells can respond to LPS in the presence of soluble MD-2. Whereas soluble MD-2 is essential for LPS-induced activation of cells expressing only TLR4, high levels of soluble MD-2 can inhibit cellular responses (11Viriyakosol S. Tobias P.S. Kitchens R.L. Kirkland T.N. J. Biol. Chem. 2001; 276: 38044-38051Abstract Full Text Full Text PDF PubMed Google Scholar). Soluble MD-2 exists as a heterogeneous collection of monomer and oligomers through inter- and intrachain disulfide bonds (17Mullen G.E. Kennedy M.N. Visintin A. Mazzoni A. Leifer C.A. Davies D.R. Segal D.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3919-3924Crossref PubMed Scopus (64) Google Scholar), and one group has presented data that argues that monomeric MD-2 preferentially binds to TLR4 and functions as a co-receptor with TLR4 (18Re F. Strominger J.L. J. Biol. Chem. 2002; 277: 23427-23432Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar).Human MD-2 contains 160 amino acid residues, including the N-terminal 17 amino acid signal sequence, with 7 cysteine residues and 2 N-glycosylation sites. Regions of functional importance on human and mouse MD-2 have been identified using peptide fragments (19Mancek M. Pristovsek P. Jerala R. Biochem. Biophys. Res. Commun. 2002; 292: 880-885Crossref PubMed Scopus (66) Google Scholar), mutation analysis (13Visintin A. Latz E. Monks B.G. Espevik T. Golenbock D.T. J. Biol. Chem. 2003; 278: 48313-48320Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 20Kawasaki K. Nogawa H. Nishijima M. J. Immunol. 2003; 170: 413-420Crossref PubMed Scopus (72) Google Scholar, 21Schromm A.B. Lien E. Henneke P. Chow J.C. Yoshimura A. Heine H. Latz E. Monks B.G. Schwartz D.A. Miyake K. Golenbock D.T. J. Exp. Med. 2001; 194: 79-88Crossref PubMed Scopus (237) Google Scholar, 22Re F. Strominger J.L. J. Immunol. 2003; 171: 5272-5276Crossref PubMed Scopus (97) Google Scholar, 23da Silva Correia J. Ulevitch R.J. J. Biol. Chem. 2002; 277: 1845-1854Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 24Ohnishi T. Muroi M. Tanamoto K. J. Immunol. 2001; 167: 3354-3359Crossref PubMed Scopus (76) Google Scholar), and structural modeling (25Gruber A. Mancek M. Wagner H. Kirschning C.J. Jerala R. J. Biol. Chem. 2004; 279: 28475-28482Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). In this study, we have investigated the structure/function relationships of membrane and soluble MD-2 by alanine-scanning mutagenesis.MATERIALS AND METHODSReagents—Salmonella minnesota Re 595 LPS (Re LPS) was prepared as previously described (26Viriyakosol S. Kirkland T.N. J. Biol. Chem. 1995; 270: 361-368Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Recombinant soluble CD14 with C-terminal His tags were prepared as described previously (27Viriyakosol S. Mathison J.C. Tobias P.S. Kirkland T.N. J. Biol. Chem. 2000; 275: 3144-3149Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Anti-His tag and anti-HA tag were from Qiagen and Roche Diagnostics, respectively. Control mouse IgG1 and rabbit IgG were obtained from Caltag. All protein biotinylations were done using the EZ-Link Sulfo-NHS-LC biotinylation kit (Pierce). All reagents were tested for LPS contamination with Limulus Amoebocyte Lysate (BioWhittaker). All reagents had <0.02 pg of endotoxin/μg of protein. When necessary, LPS was removed from the reagents using END-X (Associates of Cape Cod, Inc.). Mutagenesis of MD-2—The human MD-2 gene with the gp64 signal peptide sequence as described in Ref. 11Viriyakosol S. Tobias P.S. Kitchens R.L. Kirkland T.N. J. Biol. Chem. 2001; 276: 38044-38051Abstract Full Text Full Text PDF PubMed Google Scholar was subcloned into the EcoRI and AgeI site of the plasmid pcDNA4/V5His (Invitrogen) for expression of secreted C-terminal His-tagged protein in mammalian cells. The MD-2 amino acids were changed using the QuikChange site-directed mutagenesis kit (Stratagene). The mutant MD-2 constructs were transfected into a CHO cell line stably transfected with TLR4 containing an N-terminal HA tag and a CD25 reporter plasmid as described previously (11Viriyakosol S. Tobias P.S. Kitchens R.L. Kirkland T.N. J. Biol. Chem. 2001; 276: 38044-38051Abstract Full Text Full Text PDF PubMed Google Scholar). Stably transfected cell lines were generated by selection with Zeocin followed by immunomagnetic sorting using anti-His tag mAb (Qiagen).Cell Culture and Transfection—Cell lines were maintained in the laboratory as previously described (26Viriyakosol S. Kirkland T.N. J. Biol. Chem. 1995; 270: 361-368Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). EL1, a CHO cell line stably transfected with inducible membrane CD25 under the transcriptional control of a human E-selectin promoter containing NFκB binding sites, was a gift from Dr. D. Golenbock (28Delude R.L. Yoshimura A. Ingalls R.R. Golenbock D.T. J. Immunol. 1998; 161: 3001-3009PubMed Google Scholar). Plasmid DNA was prepared using an EndoFree kit (Qiagen). Stably transfected cell lines were generated using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. The cells expressing TLR proteins were sorted by immunomagnetic beads (Dynal) using anti-HA mAb (Roche Diagnostics). The stably transfected lines were generated by selection with G418 (Invitrogen). Transfected cells were assayed for surface expression of the HA-epitope by FACS analysis using anti-HA mAb followed by a F(ab′)2 fragment of goat anti-mouse Ig fluorescein isothiocyanate (Caltag).Analysis of NFκB Activity by Flow Cytometry—CHO cells carrying NFκB reporter plasmids to express surface CD25 were plated in a 24-well plate 1 day prior to activation. The cells were stimulated overnight, harvested, and stained with phycoerythrin-CD25 mAb (BD Biosciences) and analyzed by FACS as previously described (11Viriyakosol S. Tobias P.S. Kitchens R.L. Kirkland T.N. J. Biol. Chem. 2001; 276: 38044-38051Abstract Full Text Full Text PDF PubMed Google Scholar).Expression of Soluble MD-2 and MD-1—Wild-type (WT) and mutant MD-2 (11Viriyakosol S. Tobias P.S. Kitchens R.L. Kirkland T.N. J. Biol. Chem. 2001; 276: 38044-38051Abstract Full Text Full Text PDF PubMed Google Scholar) and MD-1 genes (Invivogen) were subcloned into pBlueBac4.5/V5-His (Invitrogen) or pBac11 (Novagen) and recombinant virus generated by the manufacturers' protocols. Recombinant virus stock was verified to contain the correct mutation by sequencing PCR-amplified inserts. Expressed protein in the insect cell supernatant was purified by nickel-nitrilotriacetic acid affinity chromatography as described previously (11Viriyakosol S. Tobias P.S. Kitchens R.L. Kirkland T.N. J. Biol. Chem. 2001; 276: 38044-38051Abstract Full Text Full Text PDF PubMed Google Scholar). The purity of all proteins was determined by Coomassie Blue staining of protein electrophoresed on a Nu-polyacrylamide gel (Invitrogen). The protein concentrations were determined by BCA assay (Pierce), ELISA using biotinylated anti-His tag, and Western blotting of the protein with anti-His tag. The control His tag protein was produced using control recombinant virus supplied by Novagen.LPS Binding Assays—The assay for MD-2 binding using immobilized LPS was done in a similar manner to the method described previously (11Viriyakosol S. Tobias P.S. Kitchens R.L. Kirkland T.N. J. Biol. Chem. 2001; 276: 38044-38051Abstract Full Text Full Text PDF PubMed Google Scholar).Activation of U373 Cells—The cells were cultured in a 96-well plate and activated with various reagents in minimal essential medium with Earle's salts and glutamine supplemented with 10 mg/ml human serum albumin. The supernatant was harvested after 16 h of activation and assayed for IL-6 by ELISA as described previously (11Viriyakosol S. Tobias P.S. Kitchens R.L. Kirkland T.N. J. Biol. Chem. 2001; 276: 38044-38051Abstract Full Text Full Text PDF PubMed Google Scholar).Analysis of Soluble MD-2 Binding to TLR4—TLR4-transfected CHO cells with or without the CD25 reporter gene were incubated with various amounts of soluble MD-2 in RPMI 1640 medium with 10% fetal calf serum for 15 min or 16 h at 20 °C. After washing off the excess protein with medium, the cells were stained with mAb anti-His (Qiagen) to detect MD-2 or anti-HA to detect TLR4, followed by rabbit anti-mouse-Ig-fluorescein isothiocyanate and analyzed by FACS. To quantitate the amount of MD-2 and TLR4 on the cell surface, the mean channel number (MCN) of fluorescence intensity was compared with the standard curve of Simply Cellular Microbeads (Bangs Laboratories) stained with the corresponding antibodies.Antibody Sandwich ELISA for the Detection of Soluble MD-2—A 96-well microtiter plate (Immulon, Dynex) was coated with 1 μg/ml of three different monoclonal antibodies against soluble MD-2 developed in our laboratory and diluted in carbonate buffer, pH 9.6, overnight at 4 °C. The plate was washed once with phosphate-buffered saline and 0.01% Tween (PBST) and blocked with 1% casein in phosphate-buffered saline for 1 h at room temperature. The culture supernatant was added to each well and the plate incubated for 1.5 h at 37 °C. The wells were washed four times in PBST, and 2.5 μg/ml biotinylated polyclonal rabbit anti-soluble MD-2 was added to each well. After incubation for 1 h at 37 °C, the wells were washed five times, and 80 ng/ml Streptavidinhorseradish peroxidase conjugate (Zymed Laboratories Inc.) was added for 45 min at 37 °C. The wells were washed five times, and the substrate (1% tetramethylbenzidine) was added. After 15 min, the reaction was stopped by adding 1.2 m H2SO4 to each well, and the absorbance was measured at 450 nm. The concentration of soluble MD-2 in the culture supernates was derived from a standard curve using purified wild-type-soluble MD-2 expressed by baculovirus.Immunoprecipitation and Western Blotting—CHO cells stably transfected with TLR4 were incubated with 1 μg of soluble MD-2 for 15 min at 25 °C. The cells were washed three times in Dulbecco's modified Eagle's medium/F-12 medium (Invitrogen) with 10% fetal calf serum and lysed in 50 mm Hepes, 0.1% Nonidet P-40, 1 mm EDTA, and HALT proteinase inhibitor mixture (Pierce). The cell lysate was incubated with anti-HA-agarose (Profound HA, Pierce) overnight at 4 °C and the precipitated protein eluted according to the manufacturer's protocol. The eluted protein was electrophoresed on a reduced 4-12% Nu-polyacrylamide gel (Invitrogen) and transferred to a polyvinylidene difluoride membrane. The soluble MD-2 and TLR4 were detected using the biotinylated anti-His or anti-HA antibody, respectively. Streptavidin-horseradish peroxidase (Zymed Laboratories Inc.) and the enhanced chemiluminescence system (ECL, Amersham Biosciences) were used to detect the bound antibodies.RESULTSMD-2 Influences LPS Activation—To investigate the role of MD-2 LPS receptor function, we varied the amount of soluble MD-2 and examined the response of TLR4-transfected EL1 cells (a CHO cell line stably transfected with a NFκB/CD25 reporter plasmid) (28Delude R.L. Yoshimura A. Ingalls R.R. Golenbock D.T. J. Immunol. 1998; 161: 3001-3009PubMed Google Scholar) to LPS. The amount of soluble MD-2 that bound to TLR4-transfected cells well as the TLR4 expression level were assayed by FACS analysis using antibody to His and HA tags, respectively. The cells were also activated with 100 ng/ml of LPS, and NFκB activation was assayed by FACS analysis of surface CD25 expression. These data plotted versus the concentration of soluble MD-2 are shown in Fig. 1. As the soluble MD-2 concentration increased, more MD-2 could be detected on the cell surface, whereas the TLR4 receptor number remained constant. At a concentration of 0.5 nm soluble MD-2, the calculated ratio of surface MD-2 per TLR4 was 0.5, yet a small amount of LPS-induced NFκB activation occurred. The level of activation increased with more MD-2 on the cell surface and reached a maximum when the calculated ratio of MD-2 per TLR4 was ∼5. This suggests that optimal receptor function requires multiple molecules of MD-2 bound to each TLR4.N-linked Glycosylation of Membrane MD-2 Has Little Effect on Cellular Activation—There have been several reports in the literature that the glycosylation state of MD-2 was important for receptor function (23da Silva Correia J. Ulevitch R.J. J. Biol. Chem. 2002; 277: 1845-1854Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 24Ohnishi T. Muroi M. Tanamoto K. J. Immunol. 2001; 167: 3354-3359Crossref PubMed Scopus (76) Google Scholar). To test this hypothesis, we mutated the two N-glycosylation sites by replacing them with glutamines. The mutated MD-2 gene pcDNA/V5His was transfected into the TLR4/CHO reporter cell line, NQ) expressed unglycosylated MD-2 protein, as shown by Western blotting of the cellular extract with antibody to His tag (Fig. 2A). The TLR4·MD-2 NQ expressed the MD-2 protein as a value 17 Kd protein, whereas the wild type expressed glycoforms of ∼20-27 Kd. Because the level of MD-2 expression on the cell surface influences LPS activation, it was important that we compare the function of this mutant to the wild type expressed at a similar level. To achieve equal expression of mutant and WT MD-2, we sorted the TLR4·MD-2 NQ cell line several times using immunomagnetic beads to select cells expressing high levels of MD-2 NQ. Fig. 2B shows FACS analysis of the surface expression of TLR4 and MD-2 on the TLR4·MD-2 NQ and WT cell lines. The TLR4·MD-2 NQ and WT cell lines were activated with LPS, and NFκB activation was assayed by CD25 surface expression. Fig. 3A shows that 100 ng/ml LPS activated the non-glycosylated mutant cells to express surface CD25 at the same level as the WT cells. We compared the level of CD25 expression after both cell lines were activated with concentrations of LPS stimulation from 1 ng/ml to 1 μg/ml and found similar levels of LPS activation, as shown in Fig. 3B. These data show that glycosylation is not important for the LPS receptor function of membrane MD-2.FIGURE 2Expression of the unglycosylated mutant of MD-2. A, CHO (EL1) cells were stably transfected with TLR4 (lane 1), TLR4 and MD-2 wild type (TLR4·MD-2 WT) (lane 2), and TLR4 and MD-2 unglycosylated mutant (TLR4·MD-2 NQ) (lane 3). The cells were lysed and cell extracts immunoprecipitated with mAb against MD-2, 5H10 (developed in our laboratory). Immunoprecipitates were Western blotted and probed with anti-His tag antibody. B, surface expression of TLR4 and MD-2 on the cell lines was analyzed by FACS using anti-HA tag and anti-His tag staining for TLR4 and MD-2, respectively. MW, molecular weight.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3The unglycosylated mutant of MD-2 functions normally. A, CHO (EL1) cells stably transfected with TLR4 (dotted line), TLR4·MD-2 wild type (solid line), and TLR4 and the unglycosylated mutant TLR4·MD-2 NQ (bolded line) were stimulated with 100 ng/ml LPS and NFκB activation assayed by FACS analysis of CD25 surface expression using anti-CD25-phycoerythrin. B, CHO (EL1) cells stably transfected with TLR4 (▪), TLR4·MD2 WT (▴), and TLR4·MD2 NQ (○) were activated with various doses of LPS, and NFκB activation was assayed by FACS analysis of CD25 surface expression. MCN of fluorescence intensity was normalized by MCN of CD25 expression of cells activated with 100 ng/ml IL-1β. Data are presented as means ± S.D. of triplicate samples.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Critical Regions of Human Membrane MD-2 for TLR4 Binding and Cellular Activation by LPS—We analyzed regions of functional importance of human MD-2 by site-directed mutagenesis. The protein sequence of human MD-2 was analyzed for Kyte-Dolittle hydrophilicity with McVector Software. Mutagenesis was designed by replacing blocks of 4-5 amino acids with alanine, as shown in Fig. 4. The expression constructs with C-terminal His tags were stably transfected into TLR4/EL1 cells. We analyzed cell surface TLR4 and MD-2 by FACS using anti-HA and anti-His tags, respectively. TLR4 surface expression levels were relatively constant (data not shown), whereas MD-2 levels varied widely. (Fig. 5). Alanine replacement mutagenesis in the N-terminal 46 amino acids of MD-2 produced mutants that expressed as well or better than the wild type. Mutation in the rest of the molecule yielded protein that was expressed poorly on the cell surface, except for three regions: amino acids 79-83, 106-110, and 121-129. This suggests that most of the MD-2 sequence after amino acid 61 is important for cell surface expression. Fig. 5, left column, shows that all mutants were synthesized and secreted as soluble protein into the medium. The levels of secreted MD-2 varied among the mutants and were not correlated with the cell surface expression levels. These data rule out the possibility that MD-2 was not found on the cell surface, because the engineered mutants were not synthesized.FIGURE 4Alanine replacement mutagenesis of human MD-2. The amino acid sequence of the predicted mature MD-2 protein, starting at amino acid 17, is shown in gray. The 7 cysteine residues are shown in black. The two N-glycosylation sites are underlined. A set of 4-5 alanines replaces the wild-type residues in the positions as shown. The blocks of alanine shown in underlined letters replace the hydrophilic regions of MD2, whereas the ones shown in italics replace the hydrophobic regions. The alanines in lowercase letters replace the regions in between.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5Analysis of membrane MD-2 mutants. Mutant MD-2 in pcDNA V5-His were created by alanine replacement mutagenesis at the amino acid numbers and sequences, as indicated, and transfected into TLR4/EL1 cells. Cell lines expressing maximal amounts of membrane MD-2 expression were selected by immunomagnetic bead sorting with anti-His antibody. Expression of MD-2 on the cell surface was analyzed by FACS using anti-His. MCN of fluorescent intensity of the staining was expressed as fold difference compared with the TLR4·MD-2 wild type. Stably transfected EL1 cells with TLR4 and MD-2 mutants were activated with 100 ng/ml LPS, and NFκB activation was assayed by FACS analysis of surface CD25 staining. MCN of LPS-induced CD25 expression was normalized against IL-1β-induced CD25 expression. The data are presented as the mean ± S.D. from at least three experiments. Left column, expression level of soluble MD-2 in the supernatant of these cell lines was assayed by ELISA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The stably transfected cells expressing TLR4 and MD-2 mutants were activated with 100 ng/ml LPS and NFκB activation assayed by CD25 expression. Each mutant was assayed in triplicate at least twice. Fig. 5 shows the LPS activation levels of different mutants as compared with the surface expression level. All mutants in the N-terminal 61 amino acids, which expressed on the cell surface, were responsive to LPS. Among this group, mutant 19-23 is more responsive to LPS than one would predict from the surface expression level. Mutants in the region 42-61 were expressed at a higher level than WT but responded to LPS less than half as well as WT. The three mutants at the C-terminal region (amino acids 79-83, 121-125, and 125-129) were expressed well but were not responsive to LPS. Mutants in the regions 66-79, 87-102, 116-122, and 127-150 were not expressed well on the cell surface.Conformation of Soluble MD-2 Mutants—Selected MD-2 mutants with different levels of expression on the cell surface, mutants that expressed well but were poorly activated by LPS, as well as the unglycosylated mutant were chosen for expression as soluble protein. The mutant genes were subcloned into baculoviral plasmids and soluble proteins expressed in insect cells. All recombinant viruses used were purified and DNA sequenced to confirm the correct mutation. The protein was purified by nickel-nitrilotriacetic acid affinity chromatography. The purified protein was analyzed on non-reducing PAGE. Fig. 6 shows isoforms of different soluble MD-2 mutants on non-reduced PAGE. Soluble mutants 38-42, 46-50, and 125-129 are most similar to the WT MD-2 in the amount of monomer. These mutants also expressed well on the cell surface. Mutants 75-79, 79-83, 106-110, and 116-120 did not express well on the cell surface. The soluble forms of these mutants contained very little monomer. These data suggest that monomer formation may correlate positively with membrane MD-2 expression.FIGURE 6Effect of alanine substitution and N-glycosylation mutation on soluble MD-2 oligomerization. Soluble proteins from selected mutations were expressed and purified from insect cells. MD-2 wild-type (WT), alanine replacement mutant, as indicated, and the unglycosylated mutant (NQ) proteins wer
Referência(s)