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• W2017608841 abstract "MARCO is a class A scavenger receptor capable of binding both Gram-negative and -positive bacteria. Using the surface plasmon resonance technique, we show here that a recombinant, soluble form of MARCO, sMARCO, binds the major Gram-negative and -positive bacterial surface components, lipopolysaccharide and lipoteichoic acid. Yet, the interaction of these two polyanions with sMARCO is of much lower affinity than that of polyinosinic acid, a polyanionic inhibitor of bacterial binding to MARCO. To further elucidate the ligand-binding functions of MARCO, we performed a phage display screen with sMARCO. The screening resulted in the enrichment of only a handful of phage clones. Contrary to expectations, no polyanionic peptides, but only those with a predominantly hydrophobic nature, were enriched. One peptide, VRWGSFAAWL, was displayed on two-thirds of the phages recovered after four rounds of screening. The VRWGSFAAWL phage-sMARCO interaction had significantly slower dissociation kinetics than that between sMARCO and lipopolysaccharide or lipoteichoic acid. Further work with this phage, and the second most enriched phage, displaying the peptide RLNWAWWLSY, demonstrated that both peptides bind to the SRCR domain of MARCO, and that they probably bind to the same site. Data base searches suggested that the VRWGSFAAWL peptide represents complement component C4, but we could not convincingly confirm this suggestion. A study with chimeric scavenger receptors indicated that even minor sequence changes in the MARCO scavenger receptor cysteine-rich (SRCR) domain can have profound effects on the binding of the prototypic scavenger receptor ligand, acetylated low density lipoprotein. As shown by differential binding of glutathione S-transferase-VR-WGSFAAWL, these differences were very likely due to conformational changes. These findings led to experiments that demonstrated a crucial role of the SRCR domain for acetylated low density lipoprotein binding in MARCO. Thus, our results strengthen the notion that the SRCR domain is the major ligand-binding domain in MARCO. Furthermore, they suggest that the domain may contain multiple ligand-binding sites. MARCO is a class A scavenger receptor capable of binding both Gram-negative and -positive bacteria. Using the surface plasmon resonance technique, we show here that a recombinant, soluble form of MARCO, sMARCO, binds the major Gram-negative and -positive bacterial surface components, lipopolysaccharide and lipoteichoic acid. Yet, the interaction of these two polyanions with sMARCO is of much lower affinity than that of polyinosinic acid, a polyanionic inhibitor of bacterial binding to MARCO. To further elucidate the ligand-binding functions of MARCO, we performed a phage display screen with sMARCO. The screening resulted in the enrichment of only a handful of phage clones. Contrary to expectations, no polyanionic peptides, but only those with a predominantly hydrophobic nature, were enriched. One peptide, VRWGSFAAWL, was displayed on two-thirds of the phages recovered after four rounds of screening. The VRWGSFAAWL phage-sMARCO interaction had significantly slower dissociation kinetics than that between sMARCO and lipopolysaccharide or lipoteichoic acid. Further work with this phage, and the second most enriched phage, displaying the peptide RLNWAWWLSY, demonstrated that both peptides bind to the SRCR domain of MARCO, and that they probably bind to the same site. Data base searches suggested that the VRWGSFAAWL peptide represents complement component C4, but we could not convincingly confirm this suggestion. A study with chimeric scavenger receptors indicated that even minor sequence changes in the MARCO scavenger receptor cysteine-rich (SRCR) domain can have profound effects on the binding of the prototypic scavenger receptor ligand, acetylated low density lipoprotein. As shown by differential binding of glutathione S-transferase-VR-WGSFAAWL, these differences were very likely due to conformational changes. These findings led to experiments that demonstrated a crucial role of the SRCR domain for acetylated low density lipoprotein binding in MARCO. Thus, our results strengthen the notion that the SRCR domain is the major ligand-binding domain in MARCO. Furthermore, they suggest that the domain may contain multiple ligand-binding sites. MARCO, a close relative of scavenger receptor A (see Ref. 1Peiser L. Mukhopadhyay S. Gordon S. Curr. Opin. Immunol. 2002; 14: 123-128Crossref PubMed Scopus (381) Google Scholar), is a trimeric type II transmembrane protein with an N-terminal intracellular domain, a transmembrane domain, and an extracellular portion composed of a short spacer domain, a long triple-helical collagenous domain, and a C-terminal scavenger receptor cysteine-rich (SRCR) 3The abbreviations used are: SRCR, scavenger receptor cysteine-rich domain; AcLDL, acetylated low density lipoprotein; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; LPS, lipopolysaccharide; LTA, lipoteichoic acid; PBS, phosphate-buffered saline; poly(I), polyinosinic acid; recV, recombinant domain V; sMARCO, recombinant soluble MARCO; RU, resonance unit; GST, glutathione S-transferase; CHO, Chinese hamster ovary; DMEM, Dulbecco's modified Eagle's medium; NTA, nitrilotriacetic acid; TRITC, tetramethylrhodamine isothiocyanate. 3The abbreviations used are: SRCR, scavenger receptor cysteine-rich domain; AcLDL, acetylated low density lipoprotein; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; LPS, lipopolysaccharide; LTA, lipoteichoic acid; PBS, phosphate-buffered saline; poly(I), polyinosinic acid; recV, recombinant domain V; sMARCO, recombinant soluble MARCO; RU, resonance unit; GST, glutathione S-transferase; CHO, Chinese hamster ovary; DMEM, Dulbecco's modified Eagle's medium; NTA, nitrilotriacetic acid; TRITC, tetramethylrhodamine isothiocyanate. domain (2Elomaa O. Kangas M. Sahlberg C. Tuukkanen J. Sormunen R. Liakka A. Thesleff I. Kraal G. Tryggvason K. Cell. 1995; 80: 603-609Abstract Full Text PDF PubMed Scopus (406) Google Scholar). MARCO has a very restricted expression pattern in adult mice living under pathogen-free conditions. It is expressed at significant levels only in the marginal zone macrophages of the spleen, in macrophages of the medullary cord of lymph nodes, and in the peritoneal macrophages (2Elomaa O. Kangas M. Sahlberg C. Tuukkanen J. Sormunen R. Liakka A. Thesleff I. Kraal G. Tryggvason K. Cell. 1995; 80: 603-609Abstract Full Text PDF PubMed Scopus (406) Google Scholar). 4Y. Chen, unpublished observations. 4Y. Chen, unpublished observations. In bacterial infections MARCO expression is up-regulated in macrophages of most tissues (3Elomaa O. Sankala M. Pikkarainen T. Bergmann U. Tuuttila A. RaatikainenAhokas A. Sariola H. Tryggvason K. J. Biol. Chem. 1998; 273: 4530-4538Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 4van der Laan L.J. Kangas M. Dopp E.A. Broug-Holub E. Elomaa O. Tryggvason K. Kraal G. Immunol. Lett. 1997; 57: 203-208Crossref PubMed Scopus (77) Google Scholar, 5van der Laan L.J. Dopp E.A. Haworth R. Pikkarainen T. Kangas M. Elomaa O. Dijkstra C.D. Gordon S. Tryggvason K. Kraal G. J. Immunol. 1999; 162: 939-947PubMed Google Scholar, 6Ito S. Naito M. Kobayashi Y. Takatsuka H. Jiang S. Usuda H. Umezu H. Hasegawa G. Arakawa M. Shultz L.D. Elomaa O. Tryggvason K. Arch. Histol. Cytol. 1999; 62: 83-95Crossref PubMed Scopus (54) Google Scholar). Cells transfected with a plasmid encoding MARCO avidly bind both Gram-negative and -positive bacteria, but not yeast (2Elomaa O. Kangas M. Sahlberg C. Tuukkanen J. Sormunen R. Liakka A. Thesleff I. Kraal G. Tryggvason K. Cell. 1995; 80: 603-609Abstract Full Text PDF PubMed Scopus (406) Google Scholar, 3Elomaa O. Sankala M. Pikkarainen T. Bergmann U. Tuuttila A. RaatikainenAhokas A. Sariola H. Tryggvason K. J. Biol. Chem. 1998; 273: 4530-4538Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). MARCO also has other than microbial ligands, because it was identified as the main receptor for large environmental particles in the lung alveolar macrophages (7Palecanda A. Paulauskis J. Al-Mutairi E. Imrich A. Qin G. Suzuki H. Kodama T. Tryggvason K. Koziel H. Kobzik L. J. Exp. Med. 1999; 189: 1497-1506Crossref PubMed Scopus (178) Google Scholar). Recent studies indicate that MARCO has endogenous ligands, too. Indeed, the interaction between MARCO and a cell surface determinant on the marginal zone B cells was found to contribute to the retention of the marginal zone B cells within the marginal zone (8Karlsson M.C. Guinamard R. Bolland S. Sankala M. Steinman R.M. Ravetch J.V. J. Exp. Med. 2003; 198: 333-340Crossref PubMed Scopus (190) Google Scholar). In another recent study, the uteroglobin-related protein 1, UGRP1, was identified as an endogenous ligand of MARCO in the lung (9Bin L.H. Nielson L.D. Liu X. Mason R.J. Shu H.B. J. Immunol. 2003; 171: 924-930Crossref PubMed Scopus (49) Google Scholar). Studies with MARCO knock-out mice have demonstrated a role for MARCO in lung defense against pneumococcal pneumonia and inhaled particles (10Arredouani M. Yang Z. Ning Y. Qin G. Soininen R. Tryggvason K. Kobzik L. J. Exp. Med. 2004; 200: 267-272Crossref PubMed Scopus (259) Google Scholar). MARCO may also have a role in the macrophage adhesion/spreading processes, because ectopic MARCO expression induces the formation of long dendritic processes (11Pikkarainen T. Brannstrom A. Tryggvason K. J. Biol. Chem. 1999; 274: 10975-10982Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The SRCR domain is crucial for this activity of MARCO (11Pikkarainen T. Brannstrom A. Tryggvason K. J. Biol. Chem. 1999; 274: 10975-10982Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Similarly, this domain is the major bacteria-binding domain in MARCO (3Elomaa O. Sankala M. Pikkarainen T. Bergmann U. Tuuttila A. RaatikainenAhokas A. Sariola H. Tryggvason K. J. Biol. Chem. 1998; 273: 4530-4538Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 12Brannstrom A. Sankala M. Tryggvason K. Pikkarainen T. Biochem. Biophys. Res. Commun. 2002; 290: 1462-1469Crossref PubMed Scopus (59) Google Scholar). We have recently succeeded in establishing a production system for the recombinant soluble MARCO (sMARCO), the extracellular part of this scavenger receptor. The protein was found to have assembled into a collagenous triple-helix, and bind both heat-killed and living Escherichia coli (13Sankala M. Brannstrom A. Schulthess T. Bergmann U. Morgunova E. Engel J. Tryggvason K. Pikkarainen T. J. Biol. Chem. 2002; 277: 33378-33385Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Furthermore, purified lipopolysaccharide (LPS), a surface component of Gram-negative bacteria, was found to interact with the polyhistidine-tagged sMARCO conjugated to nickel-nitrilotriacetic acid beads, but not with beads containing a polyhistidine-tagged control protein. Here, we have extended our studies on sMARCO, and first examined the interaction of LPS, lipoteichoic acid (LTA), a surface component of Gram-positive bacteria and a putative ligand of MARCO, as well as that of the polyribonucleotide poly(I) with the protein using surface plasmon resonance. We show that all these molecules bind MARCO, but poly(I), an inhibitor of bacterial binding to MARCO, has clearly the highest affinity. We then performed a phage display screen with immobilized sMARCO, as well as AcLDL binding studies with transfected cells, and both studies provide further support for the notion that the SRCR domain has a major role in the ligand-binding function of MARCO. Materials—The rat anti-mouse MARCO monoclonal antibody ED31 was kindly provided by Dr. G. Kraal (Free University, Amsterdam, The Netherlands). Cell culture reagents were obtained from Invitrogen. Polyinosinic acid (poly(I)), LPS (from E. coli serotype 0111:B4), and LTA (from Staphylococcus aureus) were from Sigma. The LPS was phenolextracted and purified by ion-exchange chromatography (L-3024). Construction of the random decapeptide phage library displayed on the minor M13 coat protein P3 has been described (14Koivunen E. Restel B.H. Rajotte D. Lahdenranta J. Hagedorn M. Arap W. Pasqualini R. Methods Mol. Biol. 1999; 129: 3-17PubMed Google Scholar). Human complement components C4 and C4b were from Advanced Research Technologies (>95% pure by SDS-PAGE). Recombinant human C4d of the B isotype corresponding in length to the physiological degradation fragment of C4b was expressed in bacteria, and purified to the end of the DEAE-Sephacel step as described previously (15van den Elsen J.M. Martin A. Wong V. Clemenza L. Rose D.R. Isenman D.E. J. Mol. Biol. 2002; 322: 1103-1115Crossref PubMed Scopus (47) Google Scholar). SDS-PAGE analysis indicated that the preparation was ∼90% pure. Mouse anti-M13 monoclonal antibody and goat anti-GST polyclonal antibodies were from Amersham Biosciences. Affinity-purified rabbit anti-GST polyclonal antibodies were generated in our laboratory. 5J. Ojala, unpublished data. Rabbit anti-human complement component C4 antibodies were from Sigma, or obtained from Dr. A. Blom (Lund University, Sweden). Secondary antibodies were purchased from Dako or Molecular Probes. Sulfo-NHS-biotin was from Pierce. Acetylated LDL and FITC-labeled E. coli were from Molecular Probes. The synthetic peptide and its keyhole limpet hemocyanin conjugate were purchased from Anaspec. Surface Plasmon Resonance Experiments—All experiments were run at 25 °C at a flow rate of 5 μl/min in PBS or 10 mm Hepes, pH 7.4, 150 mm NaCl, using a BIAcore 3000 instrument and NTA sensor chips (BIAcore AB). Buffers were degassed and filtered through 0.2-μm cut-off filters. Soluble MARCO was purified as described previously (13Sankala M. Brannstrom A. Schulthess T. Bergmann U. Morgunova E. Engel J. Tryggvason K. Pikkarainen T. J. Biol. Chem. 2002; 277: 33378-33385Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The NTA sensor chips were used according to the manufacturer's instructions. Prior to an experiment, purified sMARCO (ligand) in the eluent buffer (PBS) was coupled to the flow cells at densities ranging from 1000 to 3000 RU. Flow cells without immobilized sMARCO were used as reference cells. Analytes were injected over the flow cell surfaces at the following concentrations: LPS and LTA, 25-100 μg/ml; poly(I) and heparin, 5-15 μg/ml. The LPS and LTA solutions were sonicated 3 times for 15 s before the use. The injection time was 7 min. A fresh ligand was applied to the flow cells before each run. Thus, after a run, the chip was washed with 250 mm EDTA, followed with 100 mm NaOH. Thereafter, the NTA surface was first recharged with nickel ions before applying sMARCO. The control flow cell was not loaded with sMARCO. This reloading procedure was chosen, because we observed that a fraction of sMARCO was stripped away from the nickel-nitrilotriacetic acid surface when LPS and LTA were passed over the chip (see “Results”). Data Processing—Prior to analysis, the data were zero-adjusted and the reference cell signal was subtracted. Sensorgrams exhibiting instrumental artifacts were excluded from the analysis. The association and dissociation rate constants (Ka and Kd) were calculated according to the BIAcore evaluation program. The ratio of Kd and Ka yields the value of the apparent equilibrium dissociation constant (KD). A simple 1:1 Langmuir model was used to fit the data. The molar concentrations of the injected analytes were calculated using the following molecular weight values: E. coli 0111:B4 LPS, 10,000 (16Aurell C.A. Wistrom A.O. Biochem. Biophys. Res. Commun. 1998; 253: 119-123Crossref PubMed Scopus (105) Google Scholar); S. aureus LTA, 7,000 (17Greenberg J.W. Fischer W. Joiner K.A. Infect. Immun. 1996; 64: 3318-3325Crossref PubMed Google Scholar); poly(I), 500,000 (manufacturer's information). Selection of sMARCO-binding Phages—Selection of phages binding to sMARCO was done essentially as described by Koivunen et al. (14Koivunen E. Restel B.H. Rajotte D. Lahdenranta J. Hagedorn M. Arap W. Pasqualini R. Methods Mol. Biol. 1999; 129: 3-17PubMed Google Scholar). Briefly, sMARCO (100 μg/ml PBS) was first coated onto a Nunc Maxisorp microtiter well overnight at 4 °C. After blocking 1-2 h with 2% BSA/PBS at room temperature, the phage library solution (1 × 109 transducing units in 2% BSA/PBS) was added, and the plate was incubated for 2 h at room temperature. The well was washed with PBS containing Tween 20 to remove unbound phages. Bound phages were eluted with a low pH buffer, neutralized, and used to infect competent K91kan E. coli. Three more rounds of panning were carried out in the same manner, except that less sMARCO was coated onto the microtiter plates for rounds three and four (50 and 500 ng/well), and the phage solution was incubated with the immobilized sMARCO for 1 h only. During these rounds, enrichment was verified by comparing the phage binding onto sMARCO- and BSA-coated surfaces. Randomly selected clones were sequenced as described (18Koivunen E. Ranta T.M. Annila A. Taube S. Uppala A. Jokinen M. van Willigen G. Ihanus E. Gahmberg C.G. J. Cell Biol. 2001; 153: 905-916Crossref PubMed Scopus (61) Google Scholar). Binding of individual phages to surfaces coated with sMARCO, recombinant SRCR domain of MARCO, recV (13Sankala M. Brannstrom A. Schulthess T. Bergmann U. Morgunova E. Engel J. Tryggvason K. Pikkarainen T. J. Biol. Chem. 2002; 277: 33378-33385Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), or recombinant nephrin encompassing the first two IgG domains (rNephrin) was tested in the same manner (1 × 108 transducing units of phages added per well). Proteins were coated at the concentration of 10 μg/ml overnight at 4 °C (100 μl/well). Two wells were coated with each protein. Control wells were coated with a similar concentration of BSA. The assay was repeated three times. Production of the GST-VRWGSFAAWL Peptide Fusion Protein—A construct encoding a GST protein with the VRWGSFAAWL decapeptide extension was generated as follows. A fragment encoding the phage insert was first produced by PCR with the forward and reverse primers containing, respectively, BamHI and EcoRI recognition sites (the forward primer: 5′-AGGCTCGAGGATCCTCGGCCGACGGGGCT-3′, the reverse primer: 5′-AGGTCTAGAATTCGCCCCAGCGGCCCC-3′) using phage DNA as a template. The fragment was gel-purified, digested with BamHI and EcoRI, and cloned into the BamHI-EcoRI-digested pGEX-2TK. The correctness of the construct was verified by DNA sequencing. The GST fusion protein and GST alone were expressed in E. coli BL21 strain. The proteins were produced and purified according to the manufacturer's instructions (Amersham Biosciences). In Vitro Phage-binding Assays in the Presence of the GST Proteins or the Synthetic VRWGSFAAWL Peptide—Some of the in vitro phage-binding assays were carried out in the presence of 250 μg/ml GST or GST-VRWGSFAAWL. Also, before adding the test solution to wells coated with the different proteins, the wells were preincubated in the blocking solution containing a GST protein for 45 min. Some of the assays were carried out in the presence of the synthetic VRWGS-FAAWL peptide. The peptide was dissolved in Me2SO to obtain a stock solution of 4 mg/ml, and then diluted to the concentration of 200 μg/ml in the blocking solution. A fraction of the peptide precipitated in this solution, and therefore the solution was cleared by centrifugation before applying into the wells. Control binding assays were carried out in the presence of a similar vehicle concentration. Binding Assays with Transfected Cells—CHO cells transfected with the various MARCO expression constructs were tested for the binding of the VRWGSFAAWL phage, the RLNWAWWLSY phage, and a randomly picked control phage. Cells were prepared for the assay as described previously (12Brannstrom A. Sankala M. Tryggvason K. Pikkarainen T. Biochem. Biophys. Res. Commun. 2002; 290: 1462-1469Crossref PubMed Scopus (59) Google Scholar). At the time of assay, the culture medium was removed, and the cells were first incubated for 10 min in ice-cold DMEM containing 10 mm Hepes, pH 7.5, and 2% BSA (incubation medium). This solution was then removed, and the phage-containing solution was added (1 × 109 transducing units of phage in the ice-cold incubation medium). After 45 min incubation on ice, the cells were washed five times with ice-cold PBS. The cells were fixed with 4% paraformaldehyde, permeabilized for 5 min with 0.1% Triton X-100/PBS, and incubated in PBS containing 2% BSA (blocking solution) before staining for M13 phage and MARCO. All antibodies were diluted in the blocking solution. To detect the M13 phage, the cells were first incubated overnight at 4 °C with a mouse anti-M13 monoclonal antibody (10 μg/ml), then rinsed several times with PBS, and incubated with FITC-conjugated F(ab′)2 fragment of goat anti-mouse IgG (10 μg/ml) for 45 min. After rinsing several times in PBS, the cells were fixed again in the paraformaldehyde solution, and stained for MARCO using polyclonal rabbit anti-mouse MARCO antibodies recognizing the intracellular domain of mouse MARCO (2Elomaa O. Kangas M. Sahlberg C. Tuukkanen J. Sormunen R. Liakka A. Thesleff I. Kraal G. Tryggvason K. Cell. 1995; 80: 603-609Abstract Full Text PDF PubMed Scopus (406) Google Scholar). TRITC-conjugated swine antirabbit IgG was used as a secondary antibody. The binding of the GST proteins was tested in the same manner. The cells were incubated on ice with 100 μg/ml of a GST protein in DMEM/Hepes with or without 2% BSA for 45 min. After fixation, the permeabilization step was omitted, and the cells were directly stained for GST using either polyclonal goat or rabbit anti-GST antibodies. The antibodies were used, respectively, at the concentration of 25 and 10 μg/ml blocking solution. FITC-conjugated secondary antibodies were used to detect the bound GST proteins. We did not double stain for GST and MARCO in these assays. It was separately verified that the transfections were successful. In some experiments, we tested the binding of biotinylated GST proteins. The proteins (in PBS) were biotinylated by incubating2honice with sulfo-NHS-biotin (25:1 molar ratio of sulfo-NHS-biotin to protein). Unreacted biotin was then quenched by adding Tris-HCl, pH 7.5, to the final concentration of 20 mm, and the reaction mixtures were extensively dialyzed against PBS at 4 °C. Biotinylation was confirmed by Western blotting with a streptavidin-horseradish peroxidase conjugate. In some assays, the binding of AcLDL and FITC-labeled heat-killed E. coli was tested. Briefly, the cells were first washed once with DMEM, then incubated in DMEM containing either 2.5 μg/ml AcLDL or different concentrations of FITC-labeled E. coli for 1 h in a humidified atmosphere with 5% CO2 at 37 °C, after which the cells were washed two times with DMEM, 20 mm Hepes, pH 7.5, and two times with PBS before fixation with 4% paraformaldehyde. When testing the binding of human complement proteins C4 and C4b, the proteins (50 μg/ml in DMEM/Hepes) were incubated with the cells on ice for 1 h. Thereafter, the cells were processed as described above, and stained with rabbit anti-human complement C4 antibodies. The binding of biotinylated C4d was tested in a similar manner, except that the bound protein was detected with a streptavidin-horseradish peroxidase conjugate. We used the biotinylated form of C4d, because the anti-C4 antibodies did not recognize this fragment. Biotinylation of C4d was performed as described above. Anti-VRWGSFAAWL Peptide Antibodies—Antibodies were raised against the VRWGSFAAWLC peptide-keyhole limpet hemocyanin conjugate. A cysteine residue was added to the C terminus of the peptide to facilitate coupling to keyhole limpet hemocyanin. Immunoglobulins were purified from the antiserum on a Gamma-bind Sepharose column. Alternatively, anti-peptide antibodies were affinity purified from the antiserum using the GST-VRWGSFAAWL peptide fusion protein coupled to CNBr-activated Sepharose (10 mg of the fusion protein conjugated to 2 ml of the beads), or on a peptide affinity matrix (10 mg of the synthetic peptide coupled via its C-terminal cysteine residue to 2 ml of thiopropyl-Sepharose). 5 ml of antiserum, diluted to 25 ml with 10 mm Tris, pH 7.5, 50 mm NaCl (column buffer), was passed over the columns three to four times. After washing with 20 volumes of the column buffer, and 10 volumes of 10 mm Tris, pH 7.5, 0.4 m NaCl, the antibodies were eluted with 0.2 m glycine, pH 2.5, 150 mm NaCl. Eluted antibodies were immediately neutralized with 1 m Tris, pH 8.3, and dialyzed against PBS. Characterization of the Binding Properties of sMARCO Using the BIA-core System—We have previously described initial characterization of the binding properties of recombinant sMARCO (13Sankala M. Brannstrom A. Schulthess T. Bergmann U. Morgunova E. Engel J. Tryggvason K. Pikkarainen T. J. Biol. Chem. 2002; 277: 33378-33385Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Although the main aim of this study was to use sMARCO in a phage display screen to identify potential novel ligands for this scavenger receptor, we first wanted to further characterize sMARCO, and studied its binding properties using the BIAcore system. Another motive for setting up this system was that if obtaining enrichment in the phage display screen, the BIAcore system would provide a method for comparing the binding kinetics of the phage-sMARCO interaction to those between sMARCO and its other ligands. Polyhistidine-tagged sMARCO was bound onto NTA chips. Binding to the chip was very stable and there was no detectable dissociation (Fig. 1A). This was probably due to the fact that each sMARCO-molecule contains three polyhistidine tails. Interaction between sMARCO and LPS, LTA, poly(I), or heparin was then examined. Of these polyanionic compounds, LPS, LTA, and poly(I) are ligands of scavenger receptor A (19Hampton R.Y. Golenbock D.T. Penman M. Krieger M. Raetz C.R. Nature. 1991; 352: 342-344Crossref PubMed Scopus (439) Google Scholar, 20Ashkenas J. Penman M. Vasile E. Acton S. Freeman M. Krieger M. J. Lipid Res. 1993; 34: 983-1000Abstract Full Text PDF PubMed Google Scholar, 21Pearson A.M. Rich A. Krieger M. J. Biol. Chem. 1993; 268: 3546-3554Abstract Full Text PDF PubMed Google Scholar, 22Dunne D.W. Resnick D. Greenberg J. Krieger M. Joiner K.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1863-1867Crossref PubMed Scopus (303) Google Scholar). Both LPS and LTA have a tendency to form micelles in an aqueous environment (16Aurell C.A. Wistrom A.O. Biochem. Biophys. Res. Commun. 1998; 253: 119-123Crossref PubMed Scopus (105) Google Scholar, 23Santos N.C. Silva A.C. Castanho M.A. Martins-Silva J. Saldanha C. Chembiochem. 2003; 4: 96-100Crossref PubMed Scopus (114) Google Scholar, 24Labischinski H. Naumann D. Fischer W. Eur. J. Biochem. 1991; 202: 1269-1274Crossref PubMed Scopus (31) Google Scholar). As mentioned in the Introduction, we have found previously that sMARCO interacts with LPS in a test tube assay (13Sankala M. Brannstrom A. Schulthess T. Bergmann U. Morgunova E. Engel J. Tryggvason K. Pikkarainen T. J. Biol. Chem. 2002; 277: 33378-33385Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). MARCO-transfected cells have been shown to bind not only Gram-negative, but also Gram-positive bacteria, suggesting that LTA, the major surface component of this latter class of bacteria, might also be a ligand of MARCO (2Elomaa O. Kangas M. Sahlberg C. Tuukkanen J. Sormunen R. Liakka A. Thesleff I. Kraal G. Tryggvason K. Cell. 1995; 80: 603-609Abstract Full Text PDF PubMed Scopus (406) Google Scholar). The polyribonucleotide poly(I) is clearly a ligand of MARCO, because it can be used to block bacterial binding to MARCO-transfected cells. As shown in Fig. 1B, sMARCO binds LPS in the BIAcore system, too, but the binding is followed by rapid dissociation. Curiously, the curve falls below the baseline that might be due to stripping of sMARCO from the NTA surface. A survey of the literature indicates that LPS can interact with nickel (25Wellinghausen N. Schromm A.B. Seydel U. Brandenburg K. Luhm J. Kirchner H. Rink L. J. Immunol. 1996; 157: 3139-3145PubMed Google Scholar). Thus, if a fraction of NTA-chelated nickel is removed when LPS is passed over the flow cell surface, the amount of bound sMARCO also decreases. Fig. 1C shows that sMARCO binds LTA in a similar manner as it binds LPS. Interestingly, no interaction between immobilized sMARCO and LPS or LTA was detected, if sonicated solutions were left to stand for some hours before the BIAcore assay. Because it is known that sonication causes dispersion of large aggregates into smaller and more uniform micelles (26Risco C. Carrascosa J.L. Bosch M.A. J. Histochem. Cytochem. 1991; 39: 607-615Crossref PubMed Scopus (18) Google Scholar), micelle size appears to be a parameter that affects the interaction of LPS and LTA with sMARCO in this system. Analysis of the binding curves indicated variation in the KD values ranging from the high nanomolar to low micromolar level for both interactions. This variation is very likely due to the two above described potential sources of variability. As shown in Fig. 1D, poly(I) was found to interact with sMARCO with very high affinity. Determination of the binding constants indicated that this polyanionic macromolecule bound immobilized sMARCO with the association(Ka) and dissociation rate(Kd)constants of 4.4 (±2.9) × 105 m-1 s-1 and 1.6 (±0.9) × 10-4 s-1, respectively, yielding a KD value of 4.3 (±3.0) × 10-10 m (n = 4). Heparin, instead, did not interact with sMARCO coated onto the chip surface (data not shown). This finding is in line with the observation that heparin does not affect bacterial binding to MARCO. Isol" @default.
• W2017608841 created "2016-06-24" @default.
• W2017608841 creator A5000201491 @default.
• W2017608841 creator A5000224317 @default.
• W2017608841 creator A5047706639 @default.
• W2017608841 creator A5051826848 @default.
• W2017608841 creator A5064287830 @default.
• W2017608841 creator A5066234527 @default.
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• W2017608841 creator A5070647738 @default.
• W2017608841 date "2006-05-01" @default.
• W2017608841 modified "2023-10-03" @default.
• W2017608841 title "A Phage Display Screen and Binding Studies with Acetylated Low Density Lipoprotein Provide Evidence for the Importance of the Scavenger Receptor Cysteine-rich (SRCR) Domain in the Ligand-binding Function of MARCO" @default.
• W2017608841 cites W1480188565 @default.
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