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W2022483292 abstract "Components that propagate inflammation in joint disease may be derived from cartilage since the inflammation resolves after joint replacement. We found that the cartilage component fibromodulin has the ability to activate an inflammatory cascade, i.e. complement. Fibromodulin and immunoglobulins cause comparable deposition of C1q, C4b, and C3b from human serum. Using C1q and factor B-deficient sera in combination with varying contents of metal ions, we established that fibromodulin activates both the classical and the alternative pathways of complement. Further studies revealed that fibromodulin binds directly to the globular heads of C1q, leading to activation of C1. However, deposition of the membrane attack complex and C5a release were lower in the presence of fibromodulin as compared with IgG. This can be explained by the fact that fibromodulin also binds complement inhibitor factor H. Factor H and C1q bind to non-overlapping sites on fibromodulin, but none of the interactions is mediated by the negatively charged keratan sulfate substituents of fibromodulin. C1q but not factor H binds to an N-terminal fragment of fibromodulin previously implicated to be affected in cartilage stimulated with the inflammatory cytokine interleukin 1. Taken together our observations indicate fibromodulin as one factor involved in the sustained inflammation of the joint. Components that propagate inflammation in joint disease may be derived from cartilage since the inflammation resolves after joint replacement. We found that the cartilage component fibromodulin has the ability to activate an inflammatory cascade, i.e. complement. Fibromodulin and immunoglobulins cause comparable deposition of C1q, C4b, and C3b from human serum. Using C1q and factor B-deficient sera in combination with varying contents of metal ions, we established that fibromodulin activates both the classical and the alternative pathways of complement. Further studies revealed that fibromodulin binds directly to the globular heads of C1q, leading to activation of C1. However, deposition of the membrane attack complex and C5a release were lower in the presence of fibromodulin as compared with IgG. This can be explained by the fact that fibromodulin also binds complement inhibitor factor H. Factor H and C1q bind to non-overlapping sites on fibromodulin, but none of the interactions is mediated by the negatively charged keratan sulfate substituents of fibromodulin. C1q but not factor H binds to an N-terminal fragment of fibromodulin previously implicated to be affected in cartilage stimulated with the inflammatory cytokine interleukin 1. Taken together our observations indicate fibromodulin as one factor involved in the sustained inflammation of the joint. The complement system forms the core of the innate immune system and is organized in three different routes depending on initiating agent such as antibodies (classical pathway) or certain carbohydrates (lectin pathway). The alternative pathway is started by autoactivation of unstable complement factor C3 (1Pillemer L. Blum L. Lepow I.H. Ross O.A. Todd E.W. Wardlaw A.C. Science. 1954; 120: 279-285Crossref PubMed Scopus (407) Google Scholar) and its subsequent binding to a surface lacking complement inhibitors, mainly factor H (FH). 2The abbreviations used are: FH, factor H; CRP, C-reactive protein; FM, fibromodulin;MAC, membrane attack complex; PRELP, proline-arginine-rich-end-leucine-rich repeat protein; ELISA, enzyme-linked immunosorbent assay. FH is able to protect self-surfaces where it is localized due to its ability to bind sialic acid and heparan sulfate (2Meri S. Pangburn M.K. Biochem. Biophys. Res. Commun. 1994; 198: 52-59Crossref PubMed Scopus (112) Google Scholar). The main event of complement activation is formation of enzymatic complexes such as C3 and C5 convertases, which leads to release of anaphylatoxins C5a and C3a (3Kohl J. Mol. Immunol. 2001; 38: 175-187Crossref PubMed Scopus (157) Google Scholar) and deposition of C3-fragments that are recognized by phagocytes (4Smith B.O. Mallin R.L. Krych-Goldberg M. Wang X. Hauhart R.E. Bromek K. Uhrin D. Atkinson J.P. Barlow P.N. Cell. 2002; 108: 769-780Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The final stage is the formation of the membrane attack complex (MAC). The control of this powerful system is maintained by a number of soluble or membrane-bound inhibitory proteins. Most inhibitors such as FH act on C3 and C5 convertases either by increasing the dissociation of enzyme complexes or by promoting degradation of C3b or C4b by the serine proteinase factor I. The physiological relevance of complement is demonstrated by recurrent infections when there are deficiencies of complement components and central involvement of the system in systemic lupus erythematosus and glomerulonephritis (5Morgan B.P. Walport M.J. Immunol. Today. 1991; 12: 301-306Abstract Full Text PDF PubMed Scopus (249) Google Scholar). However, complement resembles a double-edged sword as it also contributes to the pathogenesis of ischemic injury, rheumatoid arthritis, multiple sclerosis, and many more diseases (6Morgan B.P. Crit. Rev. Clin. Lab. Sci. 1995; 32: 265-298Crossref PubMed Scopus (126) Google Scholar). It appears that the undesirable side effects of complement are mainly due to excessive activation or activation initiated by erroneous molecules. For example, the complement system is involved in the pathology of Alzheimer disease and prion diseases due to the ability of the C1 complex to interact with abnormal protein structures (7Rogers J. Cooper N.R. Webster S. Schultz J. McGeer P.L. Styren S.D. Civin W.H. Brachova L. Bradt B. Ward P. Lieberburg I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10016-10020Crossref PubMed Scopus (762) Google Scholar, 8Mabbott N.A. Bruce M.E. Botto M. Walport M.J. Pepys M.B. Nat. Med. 2001; 7: 485-487Crossref PubMed Scopus (208) Google Scholar). C1, the major activator of the classical pathway, is a multimolecular protease formed by the association of a recognition protein, C1q, and a catalytic subunit, a calcium-dependent tetramer of the two proteases C1s and C1r (9Gaboriaud C. Thielens N.M. Gregory L.A. Rossi V. Fontecilla-Camps J.C. Arlaud G.J. Trends Immunol. 2004; 25: 368-373Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). C1q itself is composed of six subunits, each of which contains three polypeptide chains A, B, and C. The N-terminal part of each subunit is a collagen-like, 100-amino acid-long triple helical structure (stalk) that is followed by a globular domain (head) containing portions of all three chains and representing the part that binds to activating molecules. The classical activators of C1q are clustered IgG and IgM antibodies but also C-reactive protein (CRP), DNA-histone complexes, and lipopolysaccharide and surface molecules exposed on anoxic endothelial cells (for review, see Ref. 10Gewurz H. Ying S.C. Jiang H. Lint T.F. Behring Inst. Mitt. 1993; 93: 138-147PubMed Google Scholar). A well established clinical observation, pertinent to understanding of mechanisms in joint disease, is that joint replacement surgery is an effective treatment by e.g. ameliorating inflammation. We have now found that one of the cartilage molecules, fibromodulin, may have a role in propagating this inflammation by activating complement. Cartilage contains low numbers of cells separated by a predominating extracellular matrix with several distinct supramolecular assemblies. These include extremely negatively charged proteoglycans (aggrecan) in networks of collagen fibers. The latter contains many additional molecules bound to provide stability and interactions with surrounding structures. Among these molecules are members of the leucine-rich repeat protein family, such as decorin, biglycan, asporin, fibromodulin, lumican, PRELP (proline-arginine-rich-end-leucine-rich repeat protein), and chondroadherin (for review, see Ref. 11Heinegård D. Aspberg A. Franzén A. Lorenzo P. Royce P. Steinmann B. Connective Tissue and Its Heritable Disorders: Molecular, Genetic, and Medical Aspects. 2nd. Wiley-Liss, Inc., New York2002: 271-291Crossref Google Scholar). Some leucine-rich repeat proteins, present in several tissues, including cartilage, have previously been found to interact with complement factors. Thus, C1q binds to decorin (12Krumdieck R. Höök M. Rosenberg L.C. Volanakis J.E. J. Immunol. 1992; 149: 3695-3701PubMed Google Scholar), fibronectin (13Bing D.H. Almeda S. Isliker H. Lahav J. Hynes R.O. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 4198-4201Crossref PubMed Scopus (86) Google Scholar, 14Sorvillo J. Gigli I. Pearlstein E. Biochem. J. 1985; 226: 207-215Crossref PubMed Scopus (32) Google Scholar), and laminin (15Bohnsack J.F. Tenner A.J. Laurie G.W. Kleinman H.K. Martin G.R. Brown E.J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3824-3828Crossref PubMed Scopus (62) Google Scholar). However, in none of these examples does the interaction lead to activation of the complement cascade. Fibromodulin (FM) was first described as a 59-kDa protein (16Heinegård D. Larsson T. Sommarin Y. Franzen A. Paulsson M. Hedbom E. J. Biol. Chem. 1986; 261: 13866-13872Abstract Full Text PDF PubMed Google Scholar) that interacts with collagen types I and II (17Hedbom E. Heinegård D. J. Biol. Chem. 1989; 264: 6898-6905Abstract Full Text PDF PubMed Google Scholar) and is present on collagen fibers in cartilage (18Hedlund H. Mengarelli-Widholm S. Heinegard D. Reinholt F.P. Svensson O. Matrix Biol. 1994; 14: 227-232Crossref PubMed Scopus (107) Google Scholar). Its closest relatives based on gene organization and sequence are lumican, keratocan, and PRELP. FM often contains one or two keratan sulfate chains, distributed among the four potential substitution sites all present in the leucine-rich region (19Oldberg A. Antonsson P. Lindblom K. Heinegard D. EMBO J. 1989; 8: 2601-2604Crossref PubMed Scopus (228) Google Scholar), whereas the N-terminal region is anionic, containing up to nine tyrosine sulfate residues (20Antonsson P. Heinegård D. Oldberg A. J. Biol. Chem. 1991; 266: 16859-16861Abstract Full Text PDF PubMed Google Scholar, 21Önnerfjord P. Heathfield T.F. Heinegård D. J. Biol. Chem. 2004; 279: 26-33Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). FM is thought to play an important role in collagen fiber formation as shown by the observation that FM null mice form abnormal collagen fibrils in tendons (22Svensson L. Aszodi A. Reinholt F.P. Fassler R. Heinegård D. Oldberg A. J. Biol. Chem. 1999; 274: 9636-9647Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). In the present study we have investigated the interaction between FM and the globular heads of C1q which leads to activation of complement, suggesting that FM is one of the factors fueling joint inflammation. Proteins, Proteoglycans, and Sera—C1q was purified from human serum as described previously (23Tenner A.J. Lesavre P.H. Cooper N.R. J. Immunol. 1981; 27: 648-653Google Scholar). Serum depleted of C1q was obtained via the first step of this purification (Biorex 70 ion exchange chromatography, Bio-Rad) or purchased from Quidel. Pooled human serum was prepared from the blood of healthy volunteers. Factor B-deficient serum was from Quidel, FM was isolated from both bovine and human articular cartilage (16Heinegård D. Larsson T. Sommarin Y. Franzen A. Paulsson M. Hedbom E. J. Biol. Chem. 1986; 261: 13866-13872Abstract Full Text PDF PubMed Google Scholar), and FH was from human plasma (24Blom A.M. Kask L. Dahlbäck B. Mol. Immunol. 2003; 39: 547-556Crossref PubMed Scopus (117) Google Scholar). In most experiments described in this study we have used bovine FM, which is easier to obtain than human FM since cartilage is the starting material for the purification. However, all crucial experiments were also repeated with human FM with the same results. C3-met, which functionally corresponds to C3b, was prepared by treatment of C3 with methylamine according to the previously published protocol (25Blom A.M. Villoutreix B.O. Dahlbäck B. J. Biol. Chem. 2003; 278: 43437-43442Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The preparation of stalk and head portions of C1q was performed according to published protocols (26Paques E.P. Huber R. Preiss H. Wright J.K. Hoppe-Seyler's Z. Physiol. Chem. 1979; 360: 177-183Crossref PubMed Scopus (46) Google Scholar, 27Reid K.B. Porter R.R. Biochem. J. 1976; 155: 19-23Crossref PubMed Scopus (242) Google Scholar), where pepsin (Worthington) and collagenase (from Clostridium histolyticum, Worthington), respectively, were used for partial digestion of C1q. Keratan sulfate was prepared by papain digestion of cartilage as described (28Antonopoulos C.A. Fransson L.A. Gardell S. Heinegard D. Acta Chem. Scand. 1969; 23: 2616-2620Crossref PubMed Google Scholar), and heparin was from Leo. The 10-kDa fragment of FM was prepared by proteolytic digestion of the protein as described previously (21Önnerfjord P. Heathfield T.F. Heinegård D. J. Biol. Chem. 2004; 279: 26-33Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 29Heathfield T.F. Önnerfjord P. Dahlberg L. Heinegård D. J. Biol. Chem. 2004; 279: 6286-6295Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Complement Activation Assay—All incubation steps were made with 50 μl of solution for 1 h and in room temperature except when stated otherwise. Every step was followed by extensive washing with 50 mm Tris-HCl, 150 mm NaCl, 0.1% Tween, pH 7.5. Microtiter plates (Maxisorp, Nunc) were coated overnight at 4 °C with either FM (10 μg/ml), aggregated human IgG (5 μg/ml, Immuno), human IgM (2 μg/ml, Sigma, for C3b-deposition), mannan (100 μg/ml, Sigma, for lectin pathway) or zymosan (20 μg/ml, Sigma, for alternative pathway) diluted in 75 mm sodium carbonate, pH 9.6. The wells were blocked for 2 h with 200 μl of 1% bovine serum albumin (Sigma) in phosphate-buffered saline.Dilutions of human serum or sera deficient in C1q or factor B in GVB +2 (2.5 mm veronal buffer, pH 7.3, 150 mm NaCl, 0.1% gelatin, 1 mm MgCl2, and 0.15 mm CaCl2) or Mg-EGTA (2.5 mm veronal buffer, pH 7.3, 70 mm NaCl, 140 mm glucose, 0.1% gelatin, 7 mm MgCl2, and 10 mm EGTA) were added to the plates and incubated for 20 min (for detection of C3b and C4b) or 45 min (C1q, MAC components) at 37 °C followed by incubation with specific rabbit polyclonal antibodies against C1q (Dako), C3d (Dako), C4b (Dako), mouse monoclonal antibodies against iC3b (Quidel), C5b-9 (Quidel), or goat polyclonal antibodies against C7 (in-house) and C8 and C9 (both Advanced Research Technology) diluted 1:1000 in blocking solution. Horseradish peroxidase-labeled secondary antibodies against rabbit, mouse, or goat immunoglobulins (Dako, 1:1000 in blocking solution) were then allowed to bind. Bound enzyme was assayed using 1,2-phenylenediamine dihydrochloride tablets (Dako), and absorbance was measured at 490 nm. For C5a ELISA, microtiter plates were coated with FM or human IgG and blocked as described above for the complement activation assay. The wells were then incubated with serum dilutions in GVB2+ for 15 min at 37 °C. The samples were immediately assayed using a C5a-ELISA kit (IBL, Hamburg) according to the instructions of the manufacturer. Statistical significance of the observed differences was estimated using Student's t test. Direct Binding Assay—Coating, washing, blocking, and detection were performed as described for the complement activation assay. The proteins used for coating the plates were 5 μg/ml aggregated human IgG or 10 μg/ml FM, C1q, C3-met, and CRP (Calbiochem). After blocking, 50 mm Hepes, 100 mm NaCl, 2 mm CaCl2, pH 7.4, with increasing concentrations of proteins (FM, C1q, or FH) was added to the wells and left to incubate for 4 h or, alternatively, overnight at 4 °C. The plates were then incubated with rabbit antibodies against FM (16Heinegård D. Larsson T. Sommarin Y. Franzen A. Paulsson M. Hedbom E. J. Biol. Chem. 1986; 261: 13866-13872Abstract Full Text PDF PubMed Google Scholar), C1q (Dako), or monoclonal anti-FH antibodies (Quidel) followed by incubation with horseradish peroxidase-labeled secondary antibodies against rabbit or mouse immunoglobulins (Dako, 1:1000 in blocking solution) and development with 1,2-phenylenediamine dihydrochloride tablets. The binding buffer was supplemented with increasing NaCl concentrations to test the interactions for dependence on ionic strength. When further examining the interaction between FH and FM, heparin at 0.2 or 20 mg/ml was added in the binding step. For determination of metal ion dependence 5 mm EDTA was included in the binding buffer. When assessing the influence of keratan sulfate on the interactions, the standard protocol for direct binding was followed, except during the binding step where a fixed concentration of protein was used (5 μg/ml C1q or 15 μg/ml FH), and 10 or 50 μg/ml keratan sulfate was added to the binding buffer. C1q Head/Stalk Binding—Microtiter plates were coated with 5 μg/ml human IgG, 10 μg/ml FM, intact C1q, C1q heads, or C1q stalks as described for the complement activation assay. The blocking solutions used were 3% fish gelatin (Norland) in washing buffer for the direct binding experiments and 1% bovine serum albumin in phosphate-buffered saline for the assay testing blocking antibodies. In the latter case 3 μg/ml C1q was preincubated for 40 min at room temperature with a 50-fold molar excess of monoclonal antibodies against the head or stalk portions of C1q (a kind gift of Dr. E. Hack, Crucell, Leiden, The Netherlands (30Hoekzema R. Martens M. Brouwer M.C. Hack C.E. Mol. Immunol. 1988; 25: 485-494Crossref PubMed Scopus (46) Google Scholar)) immediately before binding of C1q and FM by incubation overnight at 4 °C. The bound proteins were detected using the antibodies and the procedure described for the complement activation and binding assays above. Competition ELISA—FM (10 μg/ml) was coated onto microtiter plates, and the wells were blocked as described above for the complement activation assay. Incubation with either 5 μg/ml C1q with increasing concentrations of FH (0 –20 μg/ml) or 15 μg/ml FH with the addition of C1q (0 – 60 μg/ml) in binding buffer followed. The proteins were allowed to interact for 4 h at room temperature after which bound proteins were detected with specific antibodies as described for the complement activation and binding assay. Binding to Deglycosylated FM—FM was incubated with N-glycosidase F (Roche Applied Science) in 50 mm Hepes, 100 mm NaCl, 2 mm CaCl2, pH 7.4, on a shaker overnight at 37 °C. The final FM concentration was 17 μg/ml, and the amount of enzyme used was 1 unit/μg of protein. To verify complete digestion, the protein was separated by electrophoresis on an SDS-polyacrylamide gel. Binding between deglycosylated FM and C1q/FH was assessed using the direct binding assay. Binding of C1q and FH to the 10-kDa N-terminal Fragment of FM— Streptavidin (Sigma) at a concentration of 10 μg/ml was coated onto microtiter plates at 4 °C overnight. After blocking with 1% bovine serum albumin in phosphate-buffered saline for 2 h, the biotinylated 10-kDa fragment, at a concentration of 5 μg/mlin50mm Hepes, 100 mm NaCl, 2mm CaCl2, pH 7.4, was added to the wells and allowed to bind overnight at 4 °C. Next, C1q or FH were added at 15 μg/ml in the same buffer and incubated for 4 h. Bound C1q and FH were detected with specific antibodies as described for the complement activation and binding assays. Surface Plasmon Resonance (Biacore)—The interaction between C1q and FM was analyzed using surface plasmon resonance (Biacore 2000, Biacore). Two flow cells of a CM5 sensor chip were activated, each with 20 μl of a mixture of 0.2 m 1-ethyl-3-(3 dimethylaminopropyl) carbodiimide and 0.05 m N-hydroxysulfosuccinimide at a flow rate of 5 μl/min, after which FM (20 μg/mlin10mm sodium acetate buffer, pH 4.5) was injected over flow cell 2 to reach 2000 resonance units. Unreacted groups were blocked with 20 μl of 1 m ethanolamine, pH 8.5. A negative control was prepared by activating and subsequently blocking the surface of flow cell 1. The association kinetics was studied for various concentrations of purified C1q in 10 mm Hepes, pH 7.4, supplemented with 150 mm NaCl, 0.005% Tween 20. Protein solutions were injected for 60 or 400 s during the association phase at a constant flow rate of 30 μl/min. The sample was injected first over the negative control surface and then over immobilized FM. The signal from the control surface was subtracted. The dissociation was followed for 200 s at the same flow rate. In all experiments, a 30-μl bolus of 2 m NaCl, 100 mm HCl,5mm EDTA was used to remove bound ligands during a regeneration step. The BiaEvaluation 3.0 software (Biacore) was used to analyze sensograms obtained and to calculate rate affinity constants. Electron Microscopy—The structure of C1q-FM complexes was analyzed by negative staining and electron microscopy as described previously (31Engel J. Furthmayr H. Methods Enzymol. 1987; 145: 3-78Crossref PubMed Scopus (137) Google Scholar). Protein concentrations were about 10 μg/mlin50mm Tris-HCl, 150 mm NaCl, pH 7.4, and 5-μl aliquots were adsorbed onto carbon-coated grids for 1 min, washed with two drops of water, and stained with two drops of 0.75% uranyl formate. The grids were rendered hydrophilic by glow discharge at low pressure in air. In some experiments the fibromodulin particles were identified by labeling with colloidal thiocyanate gold (32Baschong W. Lucocq J.M. Roth J. Histochemistry. 1985; 83: 409-411Crossref PubMed Scopus (102) Google Scholar). Specimens were observed in a Jeol JEM 1230 electron microscope operated at a 60-kV accelerating voltage. Images were recorded with a Gatan Multiscan 791 CCD camera. FM Activates Human Complement—The ability of FM to activate human complement was assessed using a microtiter plate-based assay in which FM or aggregated human IgG or IgM was bound to plastic and incubated with normal human serum followed by detection of various deposited complement factors with specific antibodies. FM induced strong deposition of C1q, C4b, and C3b at levels similar to the positive control of human immunoglobulins (Fig. 1). Throughout the experiments we used aggregated human IgG to activate complement, but when measuring the deposition of C3b, human IgM was the activator. This was due to a certain degree of cross-reactivity between the IgG preparation and anti-C3 antibodies causing a high background. Although IgG caused strong deposition of C9 (a component of MAC), less C9 was detected on plates coated with FM (Fig. 2A). We also compared the deposition levels of several other components of the terminal MAC triggered by FM and immunoglobulins. FM proved to give rise to a consistently significant but lower levels of MAC formation than IgM (Fig. 2C).FIGURE 2Formation of MAC and C5a in the presence of immunoglobulins and FM. A, the plates were coated with FM or IgG and incubated with dilutions of normal human serum in GVB2+. As a measure of MAC formation, deposited C9 was detected with specific antibodies. B, release of C5a in 3.3% human serum incubated with FM and IgG was measured with a specific ELISA kit. *** denotes statistically significant differences; p <0.001 in Student's t test. C, human serum (1%) in GVB2+ was allowed to react with coated FM and IgM, and MAC formation was detected with specific antibodies directed against C7, C8, C9, and a neo-epitope on C5b-9. Data are normalized and given as the means of triplicates with bars indicating S.D, except in C, which is the representation of data from two experiments performed each in duplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Using an ELISA assay for detection of released C5a we found that FM caused less than half the level as compared with IgG (Fig. 2B). Based on the concentrations of standard samples supplied in the ELISA kit, we estimate the FM-mediated release of C5a in the conditions used to be 1.5 ng/ml. Apparently FM is able to initiate the early steps of the complement cascade. In contrast to what is observed with IgG, the later steps appear to be inhibited to some extent when FM is used as the activator, and there is less activation of C5. FM Activates Complement via the Classical and the Alternative Pathways—When human serum depleted of C1q was used, we did not detect any complement activation measured as deposition of C4b neither on IgG nor FM (Figs. 3, A and B). The C1q-depleted serum that we used had an intact lectin pathway since C4b deposition could be achieved by coating mannan to the microtiter plates (Fig. 3B). To investigate if FM can directly activate the alternative pathway, we used human serum diluted in Mg-EGTA buffer lacking calcium. This solution does not allow activation of lectin and classical pathways. Under these conditions FM caused deposition of C9 in a manner similar to zymosan, which is a known activator of the alternative pathway, whereas IgG did not trigger significant activation (Fig. 3C). No FM-mediated C9 deposition could be detected in Mg-EGTA when using factor B-deficient serum, confirming that the activity was indeed a result of alternative pathway activation (Fig. 3C).However, when factor B-deficient serum was exposed to FM in a GVB2 buffer, there was a prominent complement activation via the classical pathway (not shown). Also, there was FM-mediated C3b deposition in C1q-deficient serum in Mg-EGTA buffer (not shown). We conclude that FM is able to activate directly both the classical and alternative pathways of complement. The kinetics of FM-mediated complement activation in GVB2+ buffer that allows activation of all complement pathways was rapid as compared with zymosan. Maximal complement activation by FM as measured by deposition of C3b was obtained already at 15 min of incubation with serum (5 min for 50% of activation) and resembled the result obtained with IgM (Fig. 3D). The observation suggests that in the presence of calcium FM will activate complement mainly via the classical pathway, which is rapid and requires lower concentrations of serum than the alternative pathway. FM Binds C1q—Because FM was able to activate the classical pathway of complement via C1 it seemed plausible to hypothesize that FM interacts directly with C1q. We used a microtiter plate-based binding assay to demonstrate direct interaction between C1q and FM (Fig. 4). The binding was concentration-dependent and readily detectable at physiological ionic strength. Interaction occurred irrespective of which protein was immobilized (Fig. 4). We have also tested if there was interaction between C1s, a component of the C1-complex, and FM, but no specific binding could be detected (data not shown). Visualization of FM-C1q Interaction by Electron Microscopy—The interaction between FM and C1q was investigated by electron microscopy after adsorption onto a mica grid and negative staining. In this analysis C1q displayed its known appearance of a “bouquet of tulips” (Fig. 5D), whereas FM appeared as a small globular protein with a bent structure (Fig. 5E). When C1q and FM were incubated together before analysis by electron microscopy, we observed that FM, distinguished on the electrographs as a small globule with (Figs. 5, A and B) or without a gold label (Fig. 5C), was detected bound only to the globular heads of C1q and never to the stalks. It is noteworthy that most ligands known to activate C1q bind to this domain. Binding of C1q to FM Is Sensitive to Salt and Is Mediated by the Globular Heads of C1q—We found that binding between C1q and FM was decreased with increasing NaCl concentration (Fig. 6A, left panel), indicating a role for ionic interactions. Ionic strength dependence was also observed for human IgG, but in this case higher concentrations of NaCl were required to disrupt the binding. Repeating the experiment using KCl as the ion strength provider gave the same result as for NaCl (not shown). The FM-C1q interaction was not dependent on the presence of calcium or other divalent ions as it did not decrease in the presence of 5 mm EDTA (Fig. 6A, left panel). To determine which part of C1q is responsible for the interaction with FM, isolated head and stalk portions of C1q were prepared using partial digestion with collagenase and pepsin, respectively. The preparations were analyzed by Tricine SDS-polyacrylamide gels, and the separated proteins were detected by staining with Coomassie Brilliant Blue (Fig. 6B). By immobilizing these isolated parts of C1q on microtiter plates we could demonstrate that the binding of FM is localized to the globular heads (Fig. 6A, right panel). This observation was supported by the fact that monoclonal antibodies directed against the heads of C1q were able to inhibit the interaction, whereas the antibodies against the collagenous stalks of C1q did not affect binding (Fig. 6A, middle panel). The antibody directed against the globular heads also inhibited interaction between C1q and IgG albeit to a much lower extent. This can either be due to a difference in affinity between the IgG-C1q and FM-C1q interactions or, more plausibly, slightly shifted binding sites for FM and immunoglobulins on the head domains of C1q. Furthermore, we found using a direct binding assay that FM in solution is able to inhibit binding of C1q to immobilized IgG (not shown). We also performed a ligand blot by which we wanted to assess the binding of FM to A, B, and C chains of C1q separated by electrophoresis. However, no selective binding to the individual C1q chains could be detected; FM interacted with all three cha" @default.
W2022483292 created "2016-06-24" @default.
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W2022483292 date "2005-09-01" @default.
W2022483292 modified "2023-10-16" @default.
W2022483292 title "The Extracellular Matrix and Inflammation" @default.
W2022483292 cites W1483340343 @default.
W2022483292 cites W1519059617 @default.
W2022483292 cites W1543853846 @default.
W2022483292 cites W1549299892 @default.
W2022483292 cites W1748510480 @default.
W2022483292 cites W1906912370 @default.
W2022483292 cites W1967024742 @default.
W2022483292 cites W1971914435 @default.
W2022483292 cites W1973178658 @default.
W2022483292 cites W1973284569 @default.
W2022483292 cites W1973289394 @default.
W2022483292 cites W1974205638 @default.
W2022483292 cites W197569723 @default.
W2022483292 cites W1975908451 @default.
W2022483292 cites W1979923548 @default.
W2022483292 cites W1985498833 @default.
W2022483292 cites W1987482147 @default.
W2022483292 cites W1991700466 @default.
W2022483292 cites W1993363980 @default.
W2022483292 cites W2013437439 @default.
W2022483292 cites W2017715466 @default.
W2022483292 cites W2018133348 @default.
W2022483292 cites W2029643968 @default.
W2022483292 cites W2037450568 @default.
W2022483292 cites W2041827523 @default.
W2022483292 cites W2043103968 @default.
W2022483292 cites W2056824071 @default.
W2022483292 cites W2070274942 @default.
W2022483292 cites W2084753274 @default.
W2022483292 cites W2087282233 @default.
W2022483292 cites W2108676263 @default.
W2022483292 cites W2138495171 @default.
W2022483292 cites W2161435735 @default.
W2022483292 cites W2164395407 @default.
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