A coding mutation within the first exon of the human MD-2 ... - Nature

Genes and Immunity (2004) 5, 283–288 & 2004 Nature Publishing Group All rights reserved 1466-4879/04 $30.00 www.nature.com/gene

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A coding mutation within the first exon of the human MD-2 gene results in decreased lipopolysaccharide-induced signaling L Hamann1, O Kumpf2, M Mu¨ller1, A Visintin3, J Eckert1, PM Schlag2 and RR Schumann1 Institute for Microbiology and Hygiene, Charite´ Medical Center, Humboldt-University, Berlin, Germany; 2Robert-Ro¨ssle-Clinic, Department of Surgery and Surgical Oncology, Charite´ Medical Center, Humboldt-University, Berlin, Germany; 3Division of Infectious Diseases, Medical School, University of Massachusetts, Worcester, USA 1

MD-2 is an accessory protein of the Toll-like receptor (TLR)-4, necessary for assembling a receptor complex to sense low quantities of lipopolysaccharide in order to subsequently trigger innate immune responses. MD-2 and TLR-4 are expressed on a variety of immunocompetent cells. Mutations within the TLR-4 gene have been shown to attenuate immune responses against lipopolysaccharide in mice. In humans, a TLR-4 polymorphism has been associated with a higher risk for developing severe Gram-negative sepsis and with a lower risk for atherosclerosis. Since MD-2 is an essential part of the lipopolysaccharide receptor complex, we screened 20 patients that underwent surgical cancer therapy for novel MD-2 mutations by a single-strand conformation polymorphism technique. In one patient we found an A-G substitution at position 103, resulting in an amino-acid exchange from Thr 35 to Ala. Reporter gene assays revealed that this mutation resulted in a reduced lipopolysaccharide-induced signaling. The patient displayed an uneventful postoperative course, with the exception of slightly decreased TNF-a levels after in vitro stimulation with LPS as compared to wt patients. Genotyping of a further 41 patients by a newly developed Lightcycler/FRET method failed to detect any additional polymorphism carriers, indicating that this is a rare mutation. Genes and Immunity (2004) 5, 283–288. doi:10.1038/sj.gene.6364068 Published online 1 April 2004 Keywords: MD-2; TLR-4; inflammation; LPS; polymorphism

Introduction Innate immunity is the first line of a host’s defense against invading microbial pathogens and triggers the adaptive immune system.1,2 It involves the release of cytokines, chemokines and activation of macrophages and monocytes.3 Bacteria and bacterial cell wall components are recognized by lipopolysaccharide (LPS)-binding protein,4 CD14,5 Toll-like receptors (TLRs) and MD2.6 TLR-2 serves as the receptor for cell wall components originating from Gram-positive bacteria, mycobacteria and spirochetes, that is, peptidoglycan, lipoteichoic acid or lipoproteins.7–9 Gram-negative bacteria and their major outer membrane constituent LPS are recognized by TLR-4 as shown by hyporesponsive C3H/HeJ mice carrying a mutation in the TLR-4 gene, and by TLR-4 knockout mice.10,11 However, TLR-4 needs the association of MD-2 with its extracellular domain in order to Correspondence: Dr L Hamann, Institute for Microbiology and Hygiene, Charite´ Medical Center, Humboldt University, Dorotheenstrasse 96, 10117 Berlin, Germany. E-mail: [email protected] All authors disclose any financial interest that are relevant to the research or constitute a conflict of interest. Received 11 December 2003; revised 09 February 2004; accepted 09 February 2004; published online 1 April 2004

function as a high-affinity receptor for bacterial LPS.12 MD-2 knockout mice failed to respond to LPS and survived an otherwise lethal endotoxic shock.13 Furthermore, co-expression of MD-2 and TLR-4 seems to be crucial for membrane localization of TLR-4: in MD-2/ cells, TLR-4 resides predominantly in the Golgiapparatus and is not efficiently transported to the cell surface.13 MD-2 is also present as monomeric and multimeric soluble molecule (sMD-2) that confers LPS responsiveness to MD-2-negative, TLR-4-expressing cells.14 Several polymorphisms within the TLR-4 gene have been described,15 with at least one of them, Asp299Gly, resulting in loss of function.16 Furthermore, this polymorphism has been associated with an increased risk for sepsis caused by Gram-negative bacteria,17 and a lower risk for atherosclerosis potentially due to decreased inflammatory signaling.18 Several other polymorphisms are also associated with altered responses of innate immunity influencing a variety of diseases.19 Since MD-2 is essential for the induction of innate immune responses by Gram-negative bacteria or LPS, we screened 20 patients with a high risk of septic complications due to major tumor surgery for novel mutations of the MD-2 gene by single-strand conformation polymorphism (SSCP) analysis. We found one A to G exchange at

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position 103 (A of the start codon was set as 1), resulting in an exchange of Thr 35 to Ala. To extend this analysis, we established a rapid Lightcycler/FRET method for genotyping this mutation. Genotyping of a further 41 patients by the Lightcycler assay failed to detect additional polymorphism carriers, indicating that the Thr35Ala mutation of the MD-2 gene is a rare mutation. Employing a reporter-gene assay, a reduced LPS-induced signaling by the mutant MD-2 protein was found. Furthermore, the patient carrying the mutation showed a decreased TNF-a secretion after in vitro whole-blood stimulation with LPS as compared to control patients.

Table 1 Conserved amino-acid region around position 37 Species Human Mouse Hamster Rabbit Mutation

Amino-acid sequence SYTYCD SYSYCD SYSYCD SYTYCD SYAYCD

The residue exchanged by the new mutation is shown in bold.

Results A mutation in the human MD-2 gene causes a threonine to alanine substitution in the first exon A study group consisting of 61 patients that underwent surgical cancer therapy were investigated regarding genetic risk factors for postoperative mortality and developing infectious complications. We screened these patients for novel MD-2 polymorphisms. PCR fragments covering all five exons, respectively, of the MD-2 gene were amplified from 20 patients and subjected to SSCP analysis. We found one SSCP in exon 1 (Figure 1, sample 7). To confirm this SSCP we cloned the PCR fragments from samples 7 and 6 as control and sequenced eight independent clones, respectively. All clones derived from sample 6 revealed wild-type sequence, whereas only four clones derived from sample 7 did so. The remaining clones showed an A-G transition at position 103, indicating heterozygosity at this position. This sequence change causes an amino-acid substitution at position 35 from threonine to alanine. Residue 35 lies within a highly conserved region containing a cysteine (Cys 37) residue possibly involved in building a disulfide bridge (Table 1). MD-2 Thr35Ala is a rare mutation To analyze whether the Thr35Ala mutation is a common single nucleotide polymorphism requiring a frequency above 1%, we developed a method for rapid genotyping

Figure 1 MD-2 genotype assessment by PCR/DNA single-strand conformation polymorphism analysis. SSCP analysis of PCR fragments encompassing exon 1 of four different patients is shown. One additional band in patient 7 is marked by an arrow and indicates a mutation. Genes and Immunity

Figure 2 Genotyping for MD-2 Thr35Ala polymorphism. Representative melting curves (d(F2/F1)/dT) of three wild-type samples and the sample heterozygous for the Thr35Ala mutation analyzed (dashed line) previously by SSCP. Wild-type samples give rise to a single melting peak at 57.21C, whereas the sample heterozygous for the polymorphism exhibits an additional melting peak at 63.81C.

of this mutation employing fluorescence resonance energy transfer and subsequent melting curve analysis using the LightCyclert (Roche Diagnostics). Wild-type samples result in a single melting peak at 57.21C, whereas samples heterozygous for the polymorphism exhibit an additional melting peak at 63.81C. Melting curves from three wild-type patients and the patient carrying the mutation are shown in Figure 2. Analyzing DNA samples from a further 41 patients failed to give rise to any additional polymorphism carrier, indicating the Thr35Ala mutation to be a rare mutation. Thr35Ala mutation results in decreased LPS- induced N-jB-activation Next we investigated whether the Thr35Ala mutation may potentially influence TLR-4/MD-2-mediated LPSinduced signaling. The A-G transition at position 103 was introduced into an MD-2 expression vector by sitedirected mutagenesis. As shown in Figure 3, transfection of HEK/CD14 cells with TLR-4 and wild-type MD-2 resulted in strong LPS-induced NF-kB-activation. Transfection of the mutated MD-2 together with TLR-4 gave rise to a reduced activation of NF-kB, representing approximately 70% of the activation of cells transfected with wild-type MD-2.


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Figure 3 Reduced cellular activation by mutant MD-2. HEK293 cells stably transfected with CD14 were transiently co-transfected with wild-type or mutant MD-2, an NF-kB-dependent luciferase vector and a constitutive b-Gal reporter construct. Cells were stimulated with 10 ng/ml Re 595 LPS for 20 h and luciferase activity was determined.

Figure 4 Mutant MD-2 binds to TLR-4 and is secreted to similar amounts as compared to wild-type MD-2. HEK/293 cells stably transfected with a TLR-4/YFP fusion construct were transiently transfected with wild-type and mutant MD-2. TLR-4 was immunoprecipitated from cell lysates using an anti-GFP pAb and the immunocomplexes were resolved by SDS–PAGE. After blotting, the upper part of the membrane was probed with anti–GFP mAb to detect TLR-4 (a) and the lower part with anti-FLAG mAb to detect TLR-4-associated MD-2 (b). The level of secreted MD-2 was determined by immunoprecipitation with anti-FLAG mAb from culture supernatants followed by Western blot analysis using the same mAb (c).

Mutant MD-2 behaves like wild-type MD-2 in terms of secretion and TLR-4 association Since MD-2 is associated with TLR-4 on the cell surface and secreted by MD-2-expressing cells, it was reasonable to speculate that the impaired phenotype of MD-2 Thr35Ala might be due to a reduced ability to bind TLR-4 or to reduced protein synthesis. In order to test this hypothesis, we performed co-immunoprecipitation experiments using HEK293 cells stably expressing a TLR-4YFP chimeric construct,20 transiently transfected with wild-type and mutant MD-2. As shown in Figure 4b, anti-GFP immunoprecipitation from these cells revealed that identical amounts of MD-2 for both the wild type and the mutant forms were co-precipitated by YFPtagged TLR-4. This finding suggests that the ability of MD-2 to bind to TLR-4 is not impaired in MD-2 Thr35Ala. TLR-4 was equally represented in the samples (Figure 4a). Next, we immunoprecipitated FLAG-tagged MD-2 from the conditioned media of cells used for coprecipitation studies to test for secreted protein levels. As shown in the anti-FLAG immunoprecipitation (Figure 4c), almost identical quantities of MD-2 were found in the conditioned medium of MD-2-expressing cells. The staining pattern of the mutant MD-2 was indistinguishable from wild-type MD-2.

Figure 5 TNF-a levels with and without LPS stimulation. TNF-a levels were determined with and without LPS stimulation in a whole-blood assay from control patients without SIRS (n ¼ 12) and the patient carrying the MD-2 mutation at day 0 preoperatively and day 3 postoperatively. One patient of the control group showing the same genotype as the patient carrying the MD-2 mutation regarding the TNF-a 308 polymorphism is marked by an asterisk.

Decreased TNF-a levels in the patient expressing Thr35Ala MD-2 Serum levels of TNF-a in all patients were determined pre and postoperatively at days 0 and 3. In addition, a whole-blood ex vivo LPS stimulation was performed followed by TNF-a assessment. TNF-a levels were unchanged in the patient carrying the mutation as compared with a wild-type group of patients after uneventful surgical therapy (data not shown). Interestingly, at day 3 postoperatively a decrease in TNF-a secretion after in vitro stimulation with LPS was found as

compared to control group of patients (n ¼ 12) also without SIRS (Figure 5). Since TNF-a gene transcription seems to be affected by a polymorphism at position -308 within the TNF-a gene promotor,21 we additionally genotyped our patients for this polymorphism as described elsewhere,22 finding the patient carrying the MD-2 mutation and one patient from the control group being homozygous for the TNF-a polymorphism. Comparison of these two patients showed a decreased TNF-a production after ‘in vitro’ LPS stimulation pre- and Genes and Immunity


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postoperatively by the patient carrying the MD-2 mutation (Figure 5).

Discussion The TLR system is one of the most important pathogen sensors of the host initiating an innate immune response against microbes and their products.2,23 Mutations within TLRs have been shown to attenuate innate immune response in mice and Drosophila.11,24 For sensing minute concentrations of microbial cell wall components such as peptidoglycan, lipoteichoic acid, lipoproteins or LPS, TLR-2 and/or TLR-4 are required.7,10 For TLR-4 the accessory protein MD-2 is crucial, which has been shown to bind LPS and to confer responsiveness.12,25 MD-2 knockout mice have been shown to be resistant against LPS-induced septic shock.13 Within the expanding field of the TLR family and their interactions with microbial ligands, currently the LPS–MD-2 interaction is the only one proven by binding experiments.25 Thus, we speculated that genetic variants of the MD-2 gene would be of major importance for LPS responsiveness and disease susceptibility. In both TLR-2 and TLR-4, polymorphisms have been found causing a loss of function mutation: the intracellular Arg753Gln polymorphism within the TLR-2 gene renders TLR-2 nonfunctional and has been suggested to be associated with a higher risk for staphylococcal septic shock.26 The extracellular Asp299Gly polymorphism of the TLR-4 gene also leading to dysfunctional TLR has been shown to be associated with a higher risk for Gramnegative sepsis;17 furthermore, this polymorphism was postulated to be associated with a lower risk for carotid atherosclerosis.18 Here, we report on a novel mutation within the MD-2 gene that may influence innate immune responses against Gram-negative pathogens. We established a real-time PCR method employing the LightCyclert instrument allowing for fast and reliable genotyping of this Thr35Ala mutation. Genotyping of 61 patients results in one polymorphism carrier only, suggesting that this is a rare mutation. However, the method provided by us allows high-throughput analysis to screen larger sample numbers, which is necessary to determine the frequency of this mutation among different populations. Recently, murine MD-2 expression has been also found in the embryonic and adult hematopoietic system.27 This report suggests a function of MD-2 in the absence of TLR-4 in the early development of hematopoietic, nervous and reproductive systems similar to the function of Toll for development in Drosophila. Expression of soluble MD-2 by human immature dendritic cells that do not express TLR-4 has also been shown.14,28 Therefore, further investigation to unravel the complete function of MD-2 is needed to determine the whole impact of mutations within the MD-2 gene. The A-G transition at position 103 causes a Thr-Ala substitution at position 35 in a highly conserved region of polar residues adjacent to cysteine residue 37. This domain of MD-2 has been suggested to be involved in stabilization of the protein by the formation of disulfide bridges.29 Furthermore, substitution of this cysteine by an alanine brought about by mutagenesis resulted in a Genes and Immunity

three-fold reduced LPS-induced NF-kB-activation,29 indicating that this region is important for MD-2-function. The new mutation reported here results in a moderately reduced MD-2 activity as determined by a reduced LPSinduced NF-kB-activation as compared to the wild-type molecule. In mouse MD-2, the same reduction in NF-kB activity was found for this mutation employing a single alanine substitution analysis.30 Interestingly, the patient carrying this mutation also exhibited a reduced TNF-asecretion after LPS stimulation of whole blood. Secretion of MD-2 and binding of MD-2 to TLR-4 is not affected by the mutation. Whether the reduced signaling by the mutant MD-2 is due to an impaired interaction with LPS remains to be investigated.

Material and Methods Reagents and materials The human embryonic kidney cell line HEK293 (CLR1573) was obtained by American Type Culture Collection (Rockville, MD). Salmonella minnesota LPS Re 595 was obtained by Sigma (Deisenhofen, Germany). Expression vectors pFLAG-TLR-4, phuEFBos-MD-2, pCMV-b-galactosidase (b-Gal) and NF-kB reporter construct pELAMluciferase were kindly provided by CJ Kirschning, Technical University Munich, Munich, Germany. Patient recruitment The study included 61 European Caucasian patients that underwent surgical cancer therapy. These patients were part of a prospective cohort study examining the role of the TLR-4 polymorphism and other genetic risk factors on morbidity and mortality after major surgical procedures with a high risk for developing postoperative infections. All patients were treated at the Department of Surgery and Surgical Oncology, Charite´-University-Hospital, Campus Berlin-Buch, Robert-Ro¨ssle-Clinic. The study protocol was approved by the ethics committee and all patients gave written informed consent for the genetic analyses. Genomic DNA was prepared by Agowa (Berlin, Germany) from blood cells. Single-strand conformation polymorphism assay All five exons of the MD-2 gene were amplified by PCR with primers binding to adjacent intron sequences. Exon 1: sense, 50 -CTGATCCTCTTTGCATTTGT-30 ; antisense, 50 -ACAA-TTATTTGTTTTGCGGT; exon 2: sense, 50 -ATT GACATATCTTTATTGCT-30 ; antisense, 50 -AGCAAAAAT TTGAACTTAC-30 ; exon 3: sense, 50 -TTTGAGGGCC TAATGG-30 ; antisense, 50 -TCCTATTCTAAAAGTAATAT GAAC-30 ; exon 4: sense, 50 -TATCACCT-AACCGTGAC30 ; antisense, 50 -GCTTGTTTCAAATGCT-30 , exon 5: sense, 50 -TGTATTACTTGTATTTCTTATACT-30 ; antisense, 50 -AGGAACAATACCTTTA. PCR conditions were optimized for each primer pair. PCR fragments were purified employing a Qiaquick Kit (Qiagen, Hilden, Germany) and subjected to SSCP analysis. In all, 10–15 ml of PCR product as supplemented with 2 ml denaturation buffer containing 1% SDS, 10 mM EDTA/pH 8.0 and 2 ml of stop solution (50% glycerol and bromophenol blue). Probes were heated for 10 min. at 951C and chilled on ice. Fragments were separated by 6% PAGE using MDE Hydrolink Gelsolution (FMC, USA) for 5 h at 50 w. Bands were visualized by silver staining.


Sequencing PCR fragments encompassing the first exon from one wild-type sample (patient 6) and the polymorphism candidate sample (patient 7) were cloned into pCR3.1. (Invitrogen, Karlsruhe, Germany) and eight different clones were sequenced (MWG-Biotech, Ebersberg, Germany), respectively. Genotyping by Lightcycler/FRET method DNA was extracted from whole blood and PCR was performed in a volume of 20 ml containing 2 ml DNA (10– 50 ng/ml), primers (sense: 50 -TGCATTTGTAAAGCTTTG GAGA-TA-30 ; antisense: 50 -TGGAACACACTGATGTT TCGT-30 ) at 0.25 mmol/l,. 4 mmol/l MgCl2, 2 ml of 10 Lightcycler DNA master hybridization probes (Roche Diagnostic), and 0.2 mmol/l of each fluorescence probe. The sensor probe (50 -ACTTACCACAGTAGGCGTAT GAAATAC-30 ) covering the polymorphism was labeled with fluorescin 30 . The anchor probe (50 -GCATCGGAT GAGTTGCAGACCCAATA-30 ) was labeled with Lightcycler Red 640 50 . The PCR was run using the Lightcyclert (Roche Diagnostic) as follows: initial denaturation at 951C for 10 min, 45 cycles of denaturation (951C for 0 s, 201C/s), annealing (501C for 10 s, 201C/s), and extension (721C for 12 s, 201C/s). Melting curve analysis: 1 cycle at 951C for 0 s, 531C for 30 s, followed by an increase of temperature to 801C at a slope of 0.11C/s. The fluorescence signal was monitored continuously and plotted against the temperature. Melting curves were converted to melting peaks by plotting the negative derivative of fluorescence with respect to temperature. Mutagenesis pHuMD-2-EFBos was used for mutagenesis employing a mutagenesis kit (Stratagene, La Jolla, USA). Mutagenesis was performed according to the manufacturer’s instructions using two mutagenesis primers that introduce the new mutation and a unique noncoding MluI restriction site. Sense primer: 50 -CCGATGCAAGTATTTCATACG CGTACTGTGATAAA-ATGCAATACC-30 ; antisense primer: 50 -GGTATTGCATTTTATCACAGTACGCGTA-TGA AATACTTGCATCGG-30 . MluI positive clones were sequenced (MWG-Biotech, Germany) to confirm the MD-2 coding sequence. Reporter gene assay Human embryonic kidney HEK293/CD14 cells31 were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Eggenstein, Germany) supplemented with 10% FCS (Bio Whittaker, Europe), 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 400 mg/ ml G418 (Gibco, Eggenstein, Germany). For transient transfection 105 HEK/CD14 cells were seeded into 12well plates and cultured overnight. After washing, cells were transfected with 1 ng pFlag-TLR-4, 200 ng pCMVLuciferase, 200 ng pCMV-b-Gal, and 10 ng phuEFBosMD-2wt or 10 ng phuEFBos-MD-2mu, respectively, employing Fugene Reagent (Roche) according to the manufacturer’s instructions. At 1 day after transfection, cells were stimulated with 10 ng/ml Re 595 LPS in the presence of 2% human serum. Cells were lysed and bGal and luciferase activity was measured after 20 h using luciferase and b-gal reporter gene assays (Roche, Mannheim, Germany). Experiments were repeated two

times in triplicate. Mean values and SD of one representative experiment was shown.

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Immunprecipitation Co-immunoprecipitation of TLR-4 and MD-2 was performed as previously described.32 Briefly, cells stably expressing TLR-4YFP20 were plated on 10 cm culture dishes and transiently transfected with MD-2 wt and MD-2 mutant by lipofection, according to the manufacturer’s recommendations (Novagen, La Jolla, CA). After 48 h, MD-2 conditioned medium (10 ml) was collected and the cells were lysed in lysis buffer (10% glycerol, 137 mM NaCl, 20 mM Tris pH 8, 1% Triton X-100, 2 mM EDTA) plus protease inhibitors. Lysates were precleared by centrifugation and postnuclear supernatants were incubated overnight with 2 mg/sample of anti-GFP polyclonal antibody (Molecular Probes, Eugene, OR) and 20 ml of packed protein A sepharose (PAS, Amersham, Uppsala, Sweden) at 41C. Immunoconjugates were washed three times in lysis buffer, subjected to SDS– PAGE electrophoresis under reducing conditions (4–15% gradient precast minigels; BioRad Ercules, CA) and Western blotted for the GFP (upper part of the membrane; TLR-4/YFP) and the FLAG (lower portion, MD-2) epitopes. MD-2 from supernatants was immunoprecipitated using 4 mg anti-FLAG (Sigma Chemicals, St. Louis, MO) and 20 ml of PAS/10 ml. Immunocomplexes were treated as described before and Western blotted for the FLAG epitope. The anti-GFP mAb and the HRPconjugated anti-FLAG used in the Western-blotting procedures were from Clontech and Sigma, respectively. Blots were developed by enhanced chemiluminescence according to the manufacturer’s instructions (Amersham). LPS stimulation and cytokine analysis Blood from patients was drawn from indwelling arterial or central-venous catheters, that were part of the standard care of the patients, into endotoxin-free sample tubes (Endo-Tubess, Chromogenix, Sweden). LPS-stimulation was performed with the TNF-ex vivo stimulation kit from Milenia Biotec, Germany. In all, 50 pg LPS was used for the stimulation of 50 ml blood as described by the manufacturer. TNF-a levels were measured with ELISA using the Immulites System from DPC-Biermann, Germany.

Acknowledgements This work was supported by the Bundesministerium fu¨r Bildung und Forschung (BMBF, CAPNetz, Project C5) and the Deutsche Forschungsgemeinschaft (DFG) to RRS (Schr 726/1-1). We would like to thank Olfert Landt (TIB MOLBIOL, Berlin, Germany) for designing primers and fluorescent probes for Lightcycler/FRET genotyping.

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