Fibronectin binds to the Clq component of complement

Proc. NatL Acad. Sci. USA Vol. 79, pp. 4198-4201, July 1982

Medical Sciences

Fibronectin binds to the Clq component of complement (binding affinity/cell receptors)

DAVID H. BING*, SHERI ALMEDA*, HENRI ISLIKERt, JUDITH LAHAVO, AND RICHARD 0. HYNES* *Center for Blood Research, Boston, Massachusetts 02115; tInstitut de Biochimie, Universite de Lausanne, Lausanne, Switzerland; and *Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Communicated by John T. Edsall, April 19, 1982

of 1-2 mg/ml. Reduced plasma fibronectin migrated with Mr = 220,000 on 5% NaDodSO4polyacrylamide gel electrophoresis. No Clq was detected inthe fibronectin with monospecific anti-Clq antiserum (Atlantic Antibodies, Westbrook, ME). Fibronectin prepared and stored in this way retains biological activity for months. Clq was purified from Cohn fiaction I paste as described by Bing et aL (17) and was further treated with gelatin-Sepharose (1 ml of resin per 5 mg of protein) prior to use. It was stored in 500 mM Na acetate buffer (pH 5.5) until used. Analysis of reduced and alkylated Clq (20 mM dithiothreitol, 30 min, 370C; 40 mM iodoacetamide, 30 min, 300C in 100 mM NaPO4/1% NaDodSO4, pH 7.4) revealed only the three chains A, B,; and C. Immunoelectrophoretic analysis of 0.1% Clq with antiserum to whole human serum (Atlantic Antibodies), complement proteins C19, Clf, C4, fibronectin, and IgG failed to detect any of these proteins. The Clq also was determined to contain 1 X 1013 effective molecules per mg in the functional complement assay which used EAC4, C2, and C3-9 (17). 125ILabeled Clq lost less than 10% activity in this assay. Gelatin was prepared from collagen by heating at 560C for 2 hr in 1.2 M HCL. To determine protein concentrations, the following es were used: Clq, 6.8; Clq globular region (Clqg), 7.0; fibronectin, 12.8. The collagenous portion of Clq (Clqc) was prepared by pepsin (Worthington) digestion and purified by gel filtration as described by Reid (18). The protein concentration was determined by the method of Lowry et aL (19). Clqg was prepared by digestion with collagenase and isolated by gel filtration chromatography on Sephacryl 200 as described by Hughes-Jones and Gardner (20). Gelatin concentrations were determined by measuring Am - A215 and reference to a standard curve made with serum albumin. The following Mr were used to calculate molarity: Clq, 400,000; Clq-c, 176,000; Clqg, 39,000; fibronectin, 440,000; gelatin, 95,000. Assays. The binding assay of Clq for solid-phase fibronectin used fibronectin-coated plastic tubes (Falcon, 12 X 75 mm polystyrene clear tubes). Briefly, the tubes were incubated overnight at 40C with 1 ml of fibronectin (5 gg/ml in 150 mM NaCl/10 mM NaPO4, pH 7.4), the excess fibronectin was removed by aspiration from the bottom, and the tubes were washed three times with the same buffer, incubated 30 min at room temperature with 1% human albumin (American Red Cross Blood Services, Northeast Region, Boston, MA) diluted in the same buffer, and washed three times more. If the tubes were not to be used immediately, they were stored containing 1 ml of 1% albumin/0.01% sodium azide at 40C. Solid-phase fibronectin prepared in this way maintained binding properties for C lq for up to 2 weeks. For example, the Kd of C lq for a single lot of solid-phase fibronectin was found to be 81.5, 78.2, 84.5,

ABSTRACT Fibronectin immobilized to plastic tubes binds soluble Clq with a Kd of 82 ± 2.6 nM. The binding of fibronectin to Clq is relatively insensitive to pH but is sensitive to ionic conditions. Clq covalently bound to Sepharose selectively binds cellular fibronectin produced by a hamster fibroblast cell line. The globular head regions of Clq have no effect on the binding of Clq to fibronectin but the collagenous tails of Clq interfere competitively with a K; of 59 nM. We conclude that fibronectin binds Clq via its collagen-like tail region and thus the process resembles the binding offibronectin to gelatin. This is further emphasized by our observation that gelatin binds to fibronectin immobilized on plastic tubes with a Kd of 131 nM. Because fibronectin stimulates endocytosis in several systems and promotes the clearance of particulate material from the circulation, these results suggest the possibility that fibronectin could function in the clearance of Clqcoated material such as immune complexes or cellular debris.

Fibronectins are large glycoproteins that are found at cell surfaces, in extracellular matrices, and in blood plasma (reviewed in refs. 1-6). Both cellular and plasma fibronectins have binding sites for a number of other macromolecules including gelatin, collagen, fibrin, fibrinogen, heparin, and proteoglycans as well as for cells and bacteria (see reviews). A major function of fibronectins is in the adhesion of cells to extracellular materials such as solid substrata and matrices. Fibronectins have also been implicated in various more complex cellular functions which involve adhesion. One of the putative functions that may be pertinent is as an opsonin to promote the clearance of particles from the circulation (7, 8). Because fibronectins bind gelatin and collagens and also those forms of acetylcholinesterase that bear collagenous tails (9), we thought that they might also bind to the complement protein Clq which consists of globular regions attached to collagen-like tails (10, 11). Clq interacts with Cir and Cis through this collagenous region and with immunoglobulin complexes through the globular regions (10-13). In a preliminary report (14), we described an interaction between Clq and fibronectin, both of which were derived from human plasma or serum. We now report quantitative data on the interaction between these two proteins as well as results demonstrating that fibronectin binds to the collagenous regions of Clq and that both plasma and cellular fibronectins interact with Clq. MATERIALS AND METHODS Proteins. Plasma fibronectin was purified from fresh citrated human plasma as described (15, 16). It was dialyzed against and stored in 150 mM NaCl/10 mM cyclohexylaminopropanesulfonic acid/1 mM EDTA, pH 11, at -800C at a concentration The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Abbreviations: Clq-g, globular region of Clq; Clq-c, collagenous portion of Clq. 4198

Proc. Natl. Acad. Sci. USA 79 (1982)

Medical Sciences: Bing et aL and 82.1 nM in four separate experiments performed over a 2week period. The radiobinding assay of Clq and gelatin for solid-phase fibronectin used Clq and gelatin radiolabeled with Na125I (New England Nuclear) to a specific activity of 3.2 mCi/mg (1 Ci = 3.7 x 1010 becquerels) with Bio-Rad Enzymobeads according to directions provided by the manufacturer. Gelatin was labeled by the chloramine-T method to a specific activity of 1.3 JXCi/ mg (21). To avoid having to detect low levels of radioactivity in diluted Clq samples, a constant amount of '"I-labeled Clq (125I-Clq) (105_106 cpm) was added to dilutions of unlabeled Clq. The binding assay was done as follows. Samples of Clq (1 ml; 100-10 nM) diluted in 100 mM Na acetate (pH 7.4) were incubated in duplicate with solid-phase fibronectin for 30 min at 320C with shaking. The unbound Clq was removed by aspiration and the tubes were washed twice with 100 mM Na acetate (pH 7.4). Bound radioactivity was measured in a Searle gamma scintillation counter. The percentage bound was based on assays of 100-,ul aliquots of the initial 125I-Clq dilution. In inhibition experiments, the 125I-Clq dilutions were preincubated with competitor for 30 min at 30'C in glass tubes prior to addition to the solid-phase fibronectin. Intrinsically radiolabeled fibronectin was prepared by metabolic labeling of hamster NIL8 cells (8). Cultures near confluency were incubated overnight with [35S]methionine (25 ,uCi/ ml; 500 Ci/mmol; New England Nuclear), and the conditioned medium was collected into 2 mM phenylmethylsulfonyl fluoride. Five milliliters of labeled conditioned medium was applied to 2-ml columns of Sepharose 4B to which ovalbumin, Clq, or gelatin had been conjugated by cyanogen bromide activation (22). After extensive washing with 150 mM NaCVl10 mM. NaPi, pH 7.4, the columns were eluted with 8 M urea in cyclohexylaminopropanesulfonic acid buffer. Both flowthroughs and eluates were denatured and reduced with NaDodSO4 and dithiothreitol at final concentrations of2% and 0.1 M, respectively, and the samples were analyzed on 5% polyacrylamide slab gels according to Laemmli and Favre (23). Radioactivity was detected by fluorography using EN3HANCE (New England Nuclear) and preflashed Kodak x-ray film (XAR5). RESULTS Isliker et aL (14, 24) had noted that Clq binding to solid-phase plasma fibronectin can be detected readily by the enzymeWe confirmed that the linked immunosorbent assay technique. solid-phase fibronectin bound 1 I-Clq in a typical dose-response relationship, whereas the binding of 125I-Clq to albumin-coated tubes was minimal (Fig. 1A). Graphical analysis in a double-reciprocal plot of 1/[Clq]fr,, vs. 1/[Clq]bund permitted determination of a Kd of Clq for solid-phase fibronectin of 82 nM (Fig. 1B). The effect of pH and ionic strength on the interaction was investigated. 125I-Clq (20 nM) was added to solid-phase fibronectin in 100 mM Na acetate buffers between pH 5.5 and 8.0. There was a slight increase in binding at pH 6.0, but the amount of binding varied very little over the pH range tested (Fig. 2A). In contrast, the interaction of 125I-Clq with solid-phase fibronectin was very sensitive to increasing ionic strength. The interaction was 80% inhibited in 100 mM Na acetate (pH 7.0) containing 100 mM NaCl; at 250 mM NaCl, the interaction was 95% inhibited (Fig. 2B). Clq is a multidomain protein containing both globular regions arranged in a "flower-type" arrangement and collagenouslike sequences organized into a bundle. To ascertain what portion of the Clq molecule is involved in binding to fibronectin,

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FIG. 1. Binding of 125I-Clq to solid-phase fibronectin. (A) Binding of '"I-Clq to solid-phase fibronectin treated with albumin (0) and to tubes treated with albumin alone (o). (B) Double-reciprocal plot. y intercept = 31.6; slope = 2.6; r2 = 0.99. Calculated Kd, 82 nM.

inhibitions of binding by Clq-c and Clq-g of Clq were determined. In these experiments, Clq-c (50 nM) or Clq-g (100 nM) were added to the solid-phase fibronectin for 30 min prior to addition of '"I-Clq and then the binding assay was performed as above. Clq-c competitively inhibited the binding of '"I-Clq to solid-phase fibronectin; k1, calculated as described by Segal (25), was 59 nM (Fig. 3A). In contrast, Clq-g caused no inhibition of binding of 125I-Clq to solid-phase fibronectin (Fig. 3B). These results indicate that the collagenous region of Clq is involved in the binding to fibronectin. The reproducibility of the results is emphasized by the data in Fig. 3. Although the experiments were done 6 months apart with different lots of Clq and fibronectin, they gave similar results, Kd values of 84 and 64 nM. For comparative purposes we determined the binding affinity of gelatin for solid-phase fibronectin; "2I-labeled gelatin was used and the binding assay was as for '"I-Clq. Graphical analysis in a double-reciprocal plot yielded a Kd of gelatin for solid-phase fibronectin of 131 nM. To investigate the system by using a different technique and to ascertain the specificity of the interaction between Clq and fibronectin, we analyzed binding of cellular fibronectin to solidphase Clq. Culture medium containing several metabolically labeled secreted proteins including fibronectin and C3 (26) was prepared. This medium was fractionated on Clq, gelatin, or ovalbumin affinity resin. The proteins that did not bind to the B

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FIG. 2. Effect of pH and ionic strength on binding of '25I-Clq to solid-phase fibronectin. (A) Effect of pH. 125I-Clq (25 nM) was added to solid-phase fibronectin in 100 mM Na acetate buffer at indicated pH and binding was determined. (B) Effect of ionic strength. '25I-Clq (25 nM) was added to solid-phase fibronectin in 100 mM Na acetate (pH 7.0) containing the indicated concentrations of NaCl and binding was determined.

4200

Medical Sciences: Bing et aPProc. Nad Acad. Sci. USA 79 (1982) 300

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FIG. 4. Specificity of binding of M5Slabeled hamster cellular fibronectin to C1q. Culture medium containing metabolically labeled secreted proteins was chromatographed on affinity resins of C1q, gelatin, or ovalbumin. The flow-through (Left) and the 8 M urea eluates (Right) were analyzed on NaDodSO4 gels in the presence of 0.1 M dithiothreitol. (Middke) Conditioned culture medium not treated with affinity resin. Arrows, migration positions of cellular fibronectin (220,000 daltons) and C3a chain (130,000 daltons), determined in separate experiments (see ref. 26).

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1/Clqfm, pM-' FIG. 3. Inhibition of binding of 15I-C1q to solid-phase fibronectin by Clq-c and Clq-g. (A) Inhibition by Clq-c. e, 125I-Clq alone: y intercept = 38.6; slope = 3.26; r2 = 0.98; Kd = 84 nM. o, 50 nM Clq-c + 125I-Clq: y intercept = 38.4; slope = 5.98; r2 = 0.99; Ki = 59 nM. (B) Inhibition by Clq-g. 125I-Clq alone: y intercept = 68.7; slope = 4.13; r2 = 0.99; Kd = 64 nM. 0, 50 nM Clq-g + 125I-Clq. Experiments in A and B were done with two separate lots of C1q and fibronectin 6 months apart. e,

resins and those that were eluted by 8 M urea were analyzed by NaDodSO4/polyacrylamide gel electrophoresis (Fig. 4). Fibronectin was specifically retained both by the gelatin and by the Clq affinity resins.

DISCUSSION We previously used an enzyme-linked immunosorbent assay to

show that human plasma fibronectin binds to solid-phase human C1q and that fluid phase Clq is also taken up by solid-phase plasma fibronectin (14, 24). These results have now been confirmed in this study in which we used a radiobinding assay (Fig. 1) and metabolically labeled hamster fibronectin (Fig. 4). In both cases the binding appears to be specific and does not occur when irrelevant proteins are substituted for the solid-phase reactant. The binding of Clq to fibronectin has a Kd of 82 2.6 nM for one lot of fibronectin and C1q, and 64 5 nM for a second lot of fibronectin and Clq prepared 6 months later, showing an affinity similar to or slightly higher than that observed for the interaction between gelatin and solid-phase fibronectin under similar conditions (Kd = 131 nM). The interaction appears to be relatively insensitive to pH over the range 5.5 to 8.0 but is inhibited by increasing salt concentrations (Fig. 2). The binding does occur under physiological salt conditions (Fig. 4). Finally, whereas Clq-g has no effect on the binding, ±

±

the collagenous tails act as competitive inhibitors (Fig. 3), consistent with the hypothesis that fibronectin may interact with Clq by virtue of its affinity for collagen-like molecules. To confirm this supposition, it will be necessary to demonstrate competition between Clq and collagen or gelatin and the binding of Clq by fragments of fibronectin known to contain the collagen/gelatin-binding site. Although the experiments reported here do not demonstrate an interaction between fibronectins and Clq in vivo, they provide a prima facie case for such an interaction. The sequence of events in vivo that would lead

to

an

interaction offibronectins

with Clq is based on recent data that have demonstrated that the collagenous domain of Clq becomes exposed and accessible during activation of the complement cascade. Under nonactivating conditions, Clq circulates in blood as part ofthe zymogen C1 complex which binds plasma fibronectin poorly (14, 24). The C1 complex consists of a Ca2-dependent complex of two molecules each of the zymogen serine proteinases Clr and Cls bound to one molecule of Clq (11). The Clr2Cls2 binds as a tetrameric unit to the collagenous region of Clq with a Kd of 14 nM (27, 28). When C1 binds via the Clq-g domain to immune complexes, both Cir and Cls are converted, via conformational changes and limited proteolysis, to active serine proteinases and the Kd of the Cl?2Cs12 complex increases 10-fold to 140 nM (28). We observed that the Kd of solid-phase fibronectin for Clq is 82 2.6 to 64 5 nM, values that suggest that at this point in the complement-activation process fibronectin could compete with C1?2C1§2 for available Clq. In circulating blood, the interaction of fibronectin with Clq would be further enhanced because Clf2ClH2 rapidly dissociates from the C1 complex via an irreversible reaction with C1 inhibitor, a circulating protein serine proteinase inhibitor (29, ±

±

Proc. Natl Acad. Sci. USA 79 (1982)

Medical Sciences: Bing et aL 30). The C1q now remains in the circulation with the Clq-c domain free in a complex which as a minimum contains one molecule of C1q per two molecules of immunoglobulin (11). Depending on accessibility of the Clq-c domain, the binding affinity for fibronectin would increase logarithmically as a function of number of C1q binding sites. Thus, a likely form of interaction would be between fibronectin and C1q bound to circulating immune complexes (11), to cellular debris derived from complement lysed cells that previously had reacted with antibodies directed against antigenic components on the cell surface (10-13), or to subcellular particles such as mitochondria which can combine with and activate C1 in an antibody-independent fashion (31). There is evidence that fibronectin plays a role in the clearance of particulate material both in vivo (7, 8) and in defined systems in vitro (32-34). The interaction of fibronectin (whether free or cell bound) with C1q could play a prominent role in clearance of particulate debris coated with C1q, analogous with the clearance mechanisms that exist for removal of material bearing exposed C3b and Fc regions ofimmunoglobulins. We are grateful to Richard Black and Rachelle Rosenbaum for excellent secretarial assistance. This work was supported by U.S. Public Health Service Grants AM17351 (to D. H. B.) and CA17007 (to R.O. H.). D.H.B. was an Established Investigator of the American Heart Association and R.O.H. was the recipient of a National Cancer Institute Research Career Development Award. 1. Yamada, K. M. & Olden, K. (1978) Nature (London) 275,

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