Binding of Fibronectin by the Acute Phase Reactant C

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1984 by The American Society of Biological Chemists, Inc.

Binding of Fibronectin by the Acute Phase ReactantC-reactive Protein* (Received for publication, May 10, 1983)

Eeva-Marjatta Salonen, Tapio Vartio, Klaus Hedman, and Antti Vaheri From the Departmentof Virology and the Department of Pathology, University of Helsinki, SF-00290 Helsinki,Finland

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Fibronectin is an adhesive glycoprotein characteristically present both in soluble form in plasma and other body fluids and in insoluble form in interstitialconnective tissues and in association with basement membranes. Fibronectin is a macromolecular dimer of disulfide-bonded subunit polypeptides each with M, 220,000. Other distinctive features of fibronectin include its domain structure, susceptibility to proteolytic fragmentation, and itsmultiple molecular and biological interactionsthought to be involved in cell migration and anchorage, elaboration of the extracellular matrix, chemotaxis, and opsonization (1-5). Many of the binding sites, such as those for fibrin, Staphylococcus aureus, heparin, gelatin, and polyamines, as well as the cell-binding site, have been mapped to theinteraction domainsof the fibronectin molecule (1-5). The concentration of fibronectin in human plasma, about 300 pg/ml (6), is decreased for 6-24 h after extensive tissue destruction such as occurs following major surgery, burns, or other trauma(7,8). This decrease has been generally thought

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*This work was supported by grants from the Finnish Medical Research Council and the Finnish Cancer Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

CRP may be incorporated into lipid bilayers (26) and has been found in close association with membrane structures of altered or necrotic but not of normal cells (15). Attempts to detect a possible link between the acute phase response and fibronectin led us to study whether fibronectin and CRPmay interact. MATERIALS AND METHODS AND RESULTS*

Binding of Fibronectin by Immobilized CRP-After the binding had been discovered, toquantitatethe optimal amount of immobilized CRP for the binding reaction, different amounts of ’251-labeled CRP (up to 16 pg/ml) were used for immobilization from a volume of 200 pl/tube, and the tubes were exposed to a constant amount of fibronectin (12.5 pg/ml) diluted in NaC1/Pi-Tween (overnight at room temperature) supplemented in some experiments with 4% (w/v)

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The abbreviations used are: CRP, C-reactive protein; EIA, enzyme immunoassay; NaDodSO., sodium dodecyl sulfate; SAP, serum amyloid P component; PEG, polyethylene glycol. *Portions of this paper (including “Materials and Methods,” part of “Results,” and Figs. 1, 4, and 7) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 83M-1274, cite the authors, and include a check or money order for $4.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

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Following tissue injury, the concentration of C-re- to be due to increased consumption of soluble plasma fibroactive protein (CRP) is known to increase in plasma nectin in opsonization of particulate and soluble debris from rapidly, while that of fibronectin often decreases.We circulation by the reticuloendothelial system, Fibronectin rapnowreportthatCRPimmobilizedonto polystyrene idly appears in injured areas, in experimentally induced skin surfaces binds soluble plasma fibronectin (Kd = 1.5 X blisters (91, wounded tissues, and epithelia (10,11). Fibroneclo-’ M). The binding of fibronectinby CRP was rela- tin added to circulation has been shown to be incorporated tively sensitive to ionic conditions, being maximal at rapidly into various tissues in matrix form (12), suggesting physiological NaCl concentrations. A decrease of pH that the soluble and matrix forms of fibronectin maybe from neutral to 5-6 greatly enhanced the binding of interchangeable, in one direction at least. fibronectinby CRP. Ca2+ ions >1 at mM inhibited binding. No binding was observed between fibronectin and CRP,’ on the contrary, is a prompt acute phase reactant that elevates within 6-24 h in plasma from t,race levels up to CRPin solublephase. CRP was found also tobind fibrinogen, which competed with fibronectin for CRP- 1,000-foldin concentration during reactions of inflammation and tissue destruction (13,14) and is deposited at these lesions binding sites. This was shown to explain why fibronecin uiuo (15-18). CRP (Mr 120,000) is a pentamerof identical tin was effectively boundfromserumbutnotfrom plasma by immobilized CRP. The amount of CRP im- nonglycosylated polypeptide subunits, each with a molecular size of approximately 21,000 daltons (19, 20). It has about mobilizedwas critical inbindingfibronectin;atoo dense molecular layer of CRP inhibited the binding, as 60% sequence homology with amyloid P component, another did the postsaturation of free surfaces with albumin, protein found both in plasma and as a constituent of the which itself was notbound by CRP.Soluble fibronectin extracellular matrix of connective tissues (21). Biological efagglutinated CRP-coated latex particles. Most or all of fects of CRP include its interaction with the pneumococcal C the CRP-binding activity in the fibronectin molecule polysaccharide (22), with the phospholipids, lecithin and waslocalizedtothe 120-140-kilodalton fragment, sphingomyelin (23), and with various polyionic compounds which also contains cell-binding and heparin-binding including leukocyte cationic protein, myelin basic protein, domains of fibronectin. The results provide a link be- heparin, and dextran sulfate (24). Such binding of CRP has tween acute phase response and tissue repair. been suggested to initiate complement activation (24, 25).

Fibronectin Binds toProtein C-reactive

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FIG. 3. Effect of fibrinogen on the binding to solid phase CRP. Constant amounts (5 pg/ml) of fibronectin ( F N ) were incubated for 60 min at room temperature with different amounts of fibrinogen in soluble phase in an end-over-end mixer using low adsorption tubes, and 200 pl of the mixtures were then transferred to CRP-coated tubes. The curve (0)shows the effect of increasing amounts of fibrinogen on the binding of fibronectin to solid phase CRP as studied by anti-fibronectin antibodies (1:1000 dilution) and enzyme immunoassay. The broken line (-----) indicates the level of fibronectin binding with no fibrinogen present. The triangles show the binding of fibrinogen to solid phase CRP from the above soluble phase incubation mixtures containing 5 pg/ml of fibronectin and the indicated amounts of fibrinogen. The black circles show the binding of purified fibrinogen to solid phase CRP with no fibronectin in the assay.

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FIG. 5. Scatchard plot of the binding of fibronectin tosolid phase CRP.Different amounts (upto 2.5 pg) of3H-labeIedfibronectin were incubated at room temperature (overnight in NaCl/Pi-Tween buffer) with a constant amount of solid phase CRP (105 ng). The amount of CRP-bound fibronectinand theConcentration of unbound fibronectin were determined as indicated under "Materials and Methods."

the final arginine-Sepharose step was omitted and only gelatin-Sepharose was used, the binding was somewhat less. In W/" FIG. 2. Interaction between fibronectin and CRP as studied the reversed reaction, soluble CRP was bound considerably using different concentrations of proteins in soluble phase less efficiently by immobilized fibronectin (Fig. 2). To mimic the possible in uioo situation, we studied whether and a constant amountof the otherin solid phase. As indicated, either purified CRP or fibronectin (FN) was immobilized onto a fibronectin was able to be bound also by immobilized CRP polystyrene surface (solidphase) from a concentrationof 2 pg of CRP from serum or plasma. Binding of fibronectin occurred from and 4 pg of fibronectin/ml of NaC1/Pi, pH 7.5. The coated wells were serum, but, unexpectedly, only to a minimal degree from treated with different concentrations of either purified fibronectin, purified CRP, or serum or plasma with predetermined concentrations plasma (Fig. 2). As it was known that fibronectin interacts of fibronectin. The binding of the protein from the soluble phase to with fibrin and insolubilized fibrinogen (34,43), theeffect of the solid phase was quantitated using rabbit antibodies to fibronectin purified fibrinogen (see Fig. 1) on the fibronectin-CRP interor CRP in enzyme immunoassay. action was investigated. As shown in Fig. 3, fibrinogen inter-

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polyethylene glycol (2h at room temperature). Maximal binding of fibronectin,detected by enzyme immunoassay, was obtained with an exposure concentration of 400 ng of CRP/ 200 pl. From this 122 ng of ?-CRP (30.3%) initially bound to the tube surface, 105 ng (0.875 pmol) of'251-CRPwere present during the binding reaction and 92% of this amount remained immoblized throughout the EIA procedure. Thus, the concentration of 2 pgof CRP/ml was used in further studies for the immobilization of CRP. The amountof CRP bound from this concentration was far lower than could be immobilized, but higher amounts of CRP did not increase fibronectin binding. The density of the immobilized proteins thus seemed to be critical in the binding reaction, and a too dense molecule layer resulted in binding site inhibition, e.g. when 8 pg/ml of CRP were used, a 25% inhibition was seen. The effect of postsaturation of the CRP-coated tubes with 1%(w/v) bovine serum albumin before incubation with fibronectin was studied. CRP immobilized onto solid phase (105 ng/1.73 cm') bound fibronectin in the presence of detergent from solutions as dilute as 400 ng/ml. When the CRP-coated tubes were incubated with 1%bovine serum albumin inNaCl/ Pi buffer before binding experiments, the same fibronectin binding by solid phase CRP, as without post-treatment, was obtained with about four times higher concentration of fibronectin. The polystyrene tubes without CRP or with bovine serum albumin only did not bind fibronectin in the presence of 0.02% (v/v) Tween 20 in EIA, furthermore, bovine serum albumin was not bound by immobilized CRP as studied by anti-albumin serum in EIA and did not elute the bound CRP. Thus, the inhibitory effect of postsaturation with albumin seemed to be due to steric hindrance of the binding of fibronectin by immobilized CRP molecules. Inthe next set of experiments,a constantamount of immobilized CRP was treated with different concentrations of purified fibronectin (up to 100 pg/ml). Fibronectin was bound by the solid phase CRP in a dose-dependent manner as detected by enzyme immunoassay. Binding curves similar to that of Fig. 2 were obtained with eight different preparations of purified fibronectin. If, in the purification procedure

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Fibronectin to Binds

characterize which portion of the fibronectin molecule is involved in the binding by immobilized CRP, the interaction of fibronectin fragments (NHn-terminal30-kDa and gelatinbinding 40-kDa fragment or total cathepsin G digest of fibronectin) with CRP was studied. In order to equalize the detection system, the purified fragments (see Fig. 1C) and the total test digest (all used at 2 pg/ml) were separately immobilized to polystyrene tubes, and thereactivity of the polyclonal antifibronectin in enzyme immunoassay was determined. An equal antibody binding was obtained for the 40 kDa fragment at about 1:500 dilution of the anti-fibronectin, for the 30-kDa fragment at 1:2,000, for the total digest at 1:10,000, and for intact fibronectin at 1:50,000. In the first set of experiments, tubes coated with a constant amount of CRP (105 ng) were treated with increasing amounts (up to 50 pg/ml) of purified proteolytic fragments of fibronectin. Maximal binding by immobilized CRP was obtained with the fibronectin digest. The 30-kDa fragment bound slightly, but about 10 times more of it was needed to yield the same binding as with the total digest. No binding occurred with the 40-kDa fragment. This suggested that the binding was not to the NHp-terminal or gelatin-binding domains of fibronectin. A 120-140kDa fragment corresponding to a more COOH-terminally located region was purified and detected when immobilized to polystyrene with a1:10,000 dilution of the polyclonal antibodies roughly equally to the total digest. As seen in Fig. 6, the purified 120-140-kDa fragment was bound by immobilized CRP (105 ng) in a dose-dependent manner as determined by enzyme immunoassay using a 1:1,000 dilution of anti-fibronectin antibodies, and thebinding was far superior to thatof the other fragments. The binding of 120-140 kDa was equal or slightly better than that of the total digest and could thus account for the major binding capacity of the digest. DISCUSSION

This work demonstrates that soluble plasma fibronectin binds to substratum-attached (solid phase) C-reactive protein. This conclusion is based on the use of purified proteins as well as demonstration of binding of fibronectin from serum by immobilized CRP. The interaction was seen using radiolabeled proteins, enzyme immunoassay, or latex bead agglutination. The equilibrium dissociation constant, K d = 1.5 X IO-' M, indicated strong binding of soluble fibronectin by CRP immobilized onto polystyrene surfaces. Notably, no interaction was observed between these two proteins, fibronectin and CRP,in soluble phase. The binding of fibronectin to immobilized CRP was relatively sensitive to ionic influences. The binding was maximal at physiological NaCl concentrations, and Ca" ions at 21 mM as well as lysine at 2 2 mM inhibited the binding. A decrease of pH from 7.4 to 5-6 greatly enhanced the interaction between fibronectin and CRP. The nonionic detergent Tween 20 had no detectable inhibitory effect. The neutral polymer polyethylene glycol increased the speed of the reaction similar to its effect on solid phase antigen-antibodyinteractions(42). Thus,the fibronectinCRP binding has characteristics of an ionic interaction. Studies with purified proteolytic fragments of fibronectin showed that all or most of the binding capacity was localized pQ/m' FIG. 6. Localization of the CRP-bindingsite in fibronectin. to the 120-140-kDa fragment known to contain cell-binding Total cathepsin G digest of fibronectin (O), 120-140-kDa fragments and heparin-binding regions. The latex agglutination experi(O),40-kDa fragment (A), and 30-kDa fragment (A)of fibronectin ments using CRP-coated latex beads suggested that each were incubated with solid phase CRP (105 ng) a t the indicated plasma fibronectin molecule has at least two CRP-binding concentrations for 2 h at room temperature, and the binding was determined using a 1:lOOO dilution of antifibronectin serum and sites. In preliminary experiments, we found that intact dimeric fibronectin bound by immobilized CRP retains its caenzyme immunoassay.

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fered with the binding of fibronectin by solid phase CRP. In fact, purified fibrinogen was itself bound by immobilized CRP (Fig. 3, black circles). Solid phase bound CRP also absorbed fibrinogen effectively from citrated plasma (not shown). The binding of fibrinogen by CRP was not significantly affected by soluble fibronectin as detected by antibodies to fibrinogen (compare binding curves in Fig. 3) in EIA. Quantitation of the Binding-To quantitate the binding, different amounts (up to 2500 ng) of fibronectin labeled with the periodate-NaB3H4procedure were incubated with a constant amount of immobilized CRP (105 ng). The unbound fibronectin was removed by washing the tubes once with NaCl/Pi-Tween buffer and then twice with distilled water. The bound fibronectin was dissolved in 200plof 1%(w/v) NaDodS04 using mechanical scraping. The treatment was repeated twice, and theradioactivity of the combined extracts was determined. The Scatchardplot (44) of binding of fibronectin is shown in Fig. 5. The linearity of the graph indicates that a single class of CRP acceptor sites is involved in the binding. When, however, higher concentrations (>5000 ng/ 200 pl) of fibronectin were used, a biphasic line was obtained, implying the possible presence of lowaffinity sites. Maximally 45 ng of fibronectin were bound by the 105 ng of immobilized CRP. Theslope of the line gives the reciprocal of the equilibrium dissociation constant, K d , and its intercept at the abscissa gives the number of CRP acceptor sites. The datayield a value of 1.5 X lo-' M for K d and 0.15 pmol (0.875 pmol of CRP) as the number of binding sites inthe CRPwhich equals a molar ratio of1:5.8. When radioiodinated lZ5I-fibronectin was used in similar analyses, less binding by the immobilized CRP (35-50% of the amountof 3H-labeled fibronectin bound) was observed. Since we knew that in the internal homologies (type I inbovine fibronectin), tyrosineresidues (targets in the iodination) are conserved (45), the effect of tyrosine on fibronectin-CRP interaction was studied using pH 9.6 to dissolve L-tyrosine (Merck, Darmstadt, Federal Republic of Germany). At this pH, a concentration of 500 ~ L Mtyrosine inhibited the interaction by 70%, 100 PM by 50%, 10 p M by 40%, and 2 p M by 20%. Localization of the CRP-binding Site in Fibronectin-To

C-reactive Protein

Fibronectin Binds toProtein C-reactive

E.". Salonen, T. Vartio, K. Hedman, and A. Vaheri, unpublished observations.

18). Purified CRP has been demonstrated to bind to artificially modified lipid bilayers (26) and liposomes (51, 52). Interestingly, CRP liposomes when injected into tumor-bearing mice appeared to inhibit metastatic spreadinto lungs (53). The present studies suggest that the CRPdeposits may trap at the siteof injury circulatingfibronectin, well known for its role in tissue repair (54). It is also well known that ininjured tissue the pHdecreases; in thepresent experiments, fibronectin-CRP interaction was optimal at pH5-6. We found fibrinogen to interfere with the fibronectin-CRP interaction; this may occur in plasma but not wounded in areas once fibrinogen is clotted. There is more direct evidence that plasma fibronectin can be deposited in tissue matrices (12). The interactions of CRP with polycations can activate the classical pathway of complement (14); whether this is true for the fibronectin interaction is not known. CRP and fibronectin are among the few proteins found both in soluble form in circulation and in tissue deposits. Our studies demonstrated a preferentially one-way interaction between soluble fibronectin and deposited CRP. It is possible that CRP acts as a fibronectin acceptor in damaged cells. Acknowledgments-We thank Liisa Larjo and Leena Kostamovaarafor technical assistance and Virpi Tiilikainen forsecretarial assistance. REFERENCES 1. Mosesson, M. W., and Amrani, D. L. (1980) Blood 56,145-158 2. Vaheri, A., Keski-Oja,J., Vartio, T., Alitalo, K., Hedman, K., and Kurkinen, M. (1980) Deu. Biochem. 1 5 , 161-178 3. Mosher, D. F., and Blout, E. R. (1981) J. Inuest.Dermatol. 77,175-180 4. Ruoslahti, E., Engvall, E., and Hayman, E. G. (1981) Collagen Relat. Res. 1, 95-128 5. Hynes, R. O., and Yamada, K. M. (1982) J. Cell Biol. 9 5 , 369377

6. Mosesson, M. W., and Umfleet, R. A. (1970) J. Biol. Chem. 2 4 5 , 5728-5736 7. Saba, T. M., Albert, W. H., Blumenstock, F. A., Evanega, G., Staehler, F., and Cho, E. (1981) J. Lab. Clin. Med. 98,482-491 8. Scovill, W. A,, Annest, S. J., Saba, T. M., Blumenstock, F. A., Newell, J. C., Straton, H. H., and Powers, S. R. (1979) Surgery (St. Louis) 86,284-293 9. Saksela, O., Alitalo, K., Kiistala, U., andVaheri, A. (1981) J. Invest. Dermntol. 77, 283-286 10. Kurkinen, M., Vaheri, A., Roberts, P. J., Stenman, S., and Saxen, L. (1979) Deu. Biol. 69, 589-600 11. Fujikawa, L. S., Foster, C. S., Harrist, T. J., Lanigan, J. M.,and Colvin, R. B. (1981) Lab. Inuest. 4 5 , 120-129 12. Oh, E., Pierschbacher, M., and Ruoslahti, E. (1981) Proc. Natl. Acad. Sci. U. S. A. 78,3218-3221 13. Pepys, M. B. (1981) Lancet i, 653-657 14. Kushner, I., Gewury, H., and Benson, M. D. (1981) J. Lab. Clin. Med. 97,739-749 15. Kushner, I., and Kaplan, M. H. (1961) J. Exp. Med. 114, 961973 16. Kushner, I., Rakita, L. and Kaplan, M. H. (1963) J. Clin. Inuest. 42,286-292 17. Parish, W. E. (1976) Clin. Allergy 6,543-550 18. Du Clos, R.W., Mold, C., Paterson, P. Y., Alroy, J., and Geuruz, H. (1981) Clin. Exp. Immunol. 4 3 , 565-573 19. Kushner, I., and Somerville, J. A. (1970) Biochim. Biophys. Acta 207,105-114 20. Oliveira, E. B., Gotschlich, E. C., and Liu, T. (1979) J. Biol. Chem. 254,489-502 21. Breathnach, S. M., Melrose, S. M., Bhogal, B., de Beer, F. C., Dyck, R. F., Tennet,G., Black, M. M., and Pepys, M. B. (1981) Nature (Lord.)293,652-654 22. Tillett, W. S., and Francis, T. Jr. (1930) J. Exp. Med. 52, 561571 23. Kaplan, M. H., and Volanakis, J. E. (1974) J. Immunol. 112, 2135-2147 24. Siegel, J., Osmand, A. P., Wilson, M. F. and Gewurz, H. (1975) J. Exp. Med. 1 4 2 , 709-721

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pacity to promote adhesion and spreading of human fibroblast~.~ The amountof immobilized CRP appeared to be criticalfor fibronectin binding. Increase of molecular densities of CRP above 60 ng/cm2 (500 pmol/cm2) inhibited fibronectin binding, suggesting a requirementfor proper molecular orientation for the binding. This view is supported by the observations that postsaturation of free protein-binding sites on polystyrene by bovine serum albumin, which itself is not bound by either CRP or fibronectin and does not release immobilized CRP, inhibited binding of fibronectin. In this experiment, freely oriented CRP molecules may be sterically hindered by albumin molecules in their interaction with fibronectin. An unexpected finding was that radioiodination, unlike the periodate NaB3H4 procedure, which labels sialic acids (331, yielded fibronectin with drasticallydecreased binding capacity to CRP. One possible explanation is that the chloramine-T method used, known to iodinate preferentially tyrosine residues, critically modifies the fibronectin molecules. It is known from the partialprimary structure of bovine plasma fibronectin (45) that in the 12 internal homologies of type I, only tyrosine and half-cystine residues are fully conserved. The possibility that tyrosine residues are involved in the fibronectin-CRP interaction receives some support from the inhibitory effect of tyrosine on theinteraction. It was recently reported that SAP, which has about 60% sequence homology with CRP (19), also interacts with fibronectin (46). Both CRP and SAPare composed of five noncovalently associated subunits in cyclic pentameric configuration, but unlike CRP, SAP is glycosylated. It is of interest that this fibronectin-SAPinteraction only occurred when aggregated SAP coupled to Sepharose was used. When fibronectin-Sepharose was used or when both proteins were in soluble phase,nointeraction was seen. According tothe Scatchard plot analysis 1 mol of fibronectin was bound by 5.8 molof CRP;the significance of this value is difficult to interpret since it is not known which proportion of polystyrene-bound CRP molecules are operationally active. The fibronectin-SAP binding is strictlydependent on Ca2+ ions (46), while increasing amounts of Ca2+inhibited the fibronectin-CRP interaction (Fig. 4).We have no explanation for the possible in viuo relevance of the inhibitory effects of Ca2+ ions. The ligand-binding activitiesof CRP aregenerally classified into two major categories according to requirement for calcium ions. Calcium-independent interactions include those with the polyionic components, poly-L-lysine, heparin, myelin basic protein, and leukocyte cationic proteins (14). In these interactions, as in the presentcase, Ca2+ions are inhibitory. Our results suggested that polycationic binding sites of CRP are binding targets for fibronectin. The interactionsof fibronectin with CRP andSAP are not the only ones that are conditional (47). It is well established that soluble fibronectin will not bind to suspended cells but will promote cell anchorage and spreading if substrate-attached (1-5). An apparently related findingis that aggregated cellular fibronectin binds effectively to hyaluronic acid, while soluble plasma fibronectin does not (48). Some of the interactions of fibronectin are only obtained with its fragments (49, 50). In considering the possible in vivo relevance of the fibronectin-CRP interaction, the followingfindings seem pertinent. Following injury, CRP, a prompt acute phase reactant, is found deposited in close association with membrane structures of cultured or necrotic cells but not of normal cells (15-

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Fibronectin Protein C-reactive Binds to

Supplementary material to: BINDING OF FIBRONECTIN BY THE ACUTE-PHASE KSACTANT C-REACTIVE PROTEIN

41. Fairbanks, G., Steck, T. L., and Wallach, D. F. H. (1971) Biochemistry 10,2606-2617 42. Salonen, E.”., and Vaheri, A. (1981) J. Zmmunol. Methods 4 1 , 95-103 43. Stathakis, N. E., and Mosesson, M. W. (1977) J. Clin. Inuest. 60, 855-865 44. Scatchard, G. (1949) Ann. N. Y. Acud. Sci. 5 1 , 660-672 45. Petersen, T. E., Thogersen, H. C., Skorstengaard, K., Vibe46. 47.

48. 49. 50. 51.

Pedersen, K., Sahl, P., Sotrup-Jensen, L., and Magnusson, s. (1983) Proc. Natl. Acud. Sci. U.S. A. 80, 137-141 de Beer, F. C., Baltz, M. L., Holford, S., Feinstein, A., and Pepys, M. B. (1981) J. Erp. Med. 1 5 4 , 1134-1149 Vaheri, A., Vartio, T., Salonen, E.”., Hedman, K., De Petro, G., and Barlati, S. (1983) in Factor XZZZ and Fibronectin (Egbring, R., and Klingemann, H.-G., eds) pp. 195-204, Die Medizinische Verlagsgesellschaft,Marburg, Federal Republic of Germany Laterra, J., and Culp, L. A. (1982) J. Biol. Chem. 257, 719-726 De Petro, G., Barlati, S., Vartio, T., and Vaheri, A. (1981) Proc. Natl. Acud. Sci. U. S. A. 7 8 , 4965-4969 Czop, J. K., Kadish, J. L., and Austen, K. F. (1981) Proc. Natl. Acud. Sci. U.S. A. 78,3649-3653 Volanakis, J. E., and Narkates, A. J. (1981) J. Zmmunol. 126,

1820-1825 52. Mold, C., Rodgers, C.P., Richards, R.L., Alving, C . R., and Gequrz, H. (1981) J. Zmmunol. 126,856-860 53. Depdhar, S. D., James, K., Chiang, T., Edlinger, M., and Barna, B. P. (1982) Cancer Res. 42,5084-5088 54. Vaheri, A., Salonen, E.”., Vartio, T., Hedman, K., and Stenman, S. (1983) in Biology and Pathology of the Vessel Wall (Woolf, N., ed) pp. 161-171, Holt-Saunders, Eastbourne, United King-

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fluoride (PhCH so F I to give a final concentration of 1 m. The individual fragments (the2NH2terminal I4 r 3 0 000 the gelatin-binding n,=40 000. and the gelatin non-bindi?ci COQH-terbnal HrL120-140 000 framnentsl were isolated by sequential aff1nity’~hromatographyusing gelatin-Sepharose, spermine-Sepharo8e and heparin-Sepharose ab described 1 3 0 . 1 1 ) . The fragments were ) 9 5 % pure when analvzed in N a W S O d - P A G E as shorn in Figure 1. Radiolabelin of CRP and fibronectin. Radioiodination of proteins vas carried OUt acczrding to Xrohn et al. 7 3 2 ) using 1251 (The Radiochemical Centre llmersham u . K . 1 in a modification Of the chloramln -T method. The specifi5 radioac;ivity was about 2.5 uC11pgl1 Ci * 3.7X101$ becquerelal. Periodate- H labeling Of fibronectin was carried OYt according to Gahmberg and Andrjrsson 1331 using a final concentration Of 1 d4 periodate and 5 mCi of Ha6 H (The Radiocheytyl Centre). The (Ipcific radioactfity wa8 0.1 uCi/YB. The pteparations Of I-labeled fibronectin and CRP and H-labeled fibzonectin gave in NaDodSOd-PAGE under redvcing conditions single bands at the pOaitionS of the subunits of the unlabeled proteins. G e l filtration experiments. A c o l w 1 1 . 5 cm x 1 1 0 cml Of Bio-Gel 1.5 A (BiO-Rad, R ~ C n , CA equilibrated with NaCl/P 10.01 n sodium phosphate/ 0 . 1 4 I( NaCl/ps.:l an: presaturated with 0.5 % k / v ) bovine serum albumin was used at a f l w rare Of 7.5 ml per hour at rwm temperature. The samples preincubated for 120 min at room temperature in an end-over-end mixer contained 200 p g Of fibronectln or CRP in one ml, radioiodinated protein a s marker and an eaual amount 1200 y q l of the ocherprotein unlabeled. ””

MATERIALS AND METHODS Purified proteins. FibroMCtin was purified from feemh citrated human plasma by a tvo-step affinity chromatography procedure using gelatin-Sepharose and arginine-Seeharose as deecribed 127). The purifhed protein wa.8 dissolved in 0.1 I4 NaC110.05 M Ti-iB-HCl, pH 7.5, stored at -70 C at a concentration Of 1-3 mg/ml if nor need immediately. and wae b-emus by the following criThirty niorograma Of chemically reduced plasma fibronaotin migrated on dodecyl sulfate ( N n ~ d S O 4 l - p o l y a ~ r y l o n l d gel s electrophoresis IPAGEI as a single -)or polypeptide band of +-220 000 (Pig. 11. It gave in t M dimensional immunoelectrophoreals against pOlyYalent antibodlee to h U B n plasma proteins (Orion Diagnostlca, Helsinki. Finland) only one immunoprecipitate arc, identical with that obtained with anti-fibron& mtlbcdies. The purified flbronectin did not s h w any contamination with fibrinogen as jUased by *unoelectroohoresis aaainst anti-fibrinwen antiserum IDOiko, Copenhagen.Denrarkl. to

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Imunochemically pure fibrinogen was a gift f r m Dr. Birger Blorhllck, Department of Coagulation Research, Karolinska Institutet, Stockholm, Sweden, and gayem N a W S O PAGE three major polypeptide bande at K f . 7 0 000. 5 5 0 0 0 . and 50 000 IFig. I f : corresponding to the M, 88, and ‘/=ha ns of fibrinogen.

Isolation of fragments of fibronectin. Purified human p1oi-a fibronectin 11500-3000 ug/mll wae digested with cathepsin G la gift f r w Dr. Jeremy Saklatvnla Stranaeways Researoh laboratory. Cambridge, U.X.1 in 5 0 111 TrisH C 1 , pH 7.5 at 37 C for 10 seconds using a e n z w - m h s t r a t e ratio of 1:200 (wlwl. The digestion was terminated by the addition of phenylmetbanesulfonyl

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25. Narkates, A. J., and Volanakis, J. E. (1982) Ann. N.Y. Acud. Sci. 389,172-182 26. Volanakis, J. E., and Wirtz, K. W. A. (1979) Nature ( L o n d . ) 2 8 1 , 155-157 27. Vuento, M., and Vaheri, A. (1979) Biochem. J. 183,331-337 28. Hokama, Y., and Riley, R. F. (1963) Biochim. Biophys. Acta 7 4 , 305-308 29. Lowry, 0.H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Bwl. Chem. 193,265-275 30. Vartio, T. (1982) Eur. J. Bwchem. 123, 223-233 31. Vartio, T., Salonen, E.”., DePetro, G., Barlati, S., Miggiano, V., Stahli, C., Virgallita, G., Takacs, B., and Vaheri, A. (1983) Bwchem. J. 2 1 5 , 147-151 32. Krohn, K., Sherman, L., and Welch, M. (1972) Biochim. Biophys. Acta 285,404-413 33. Gahmberg, C.G., and Anderson, L. C. (1977) J. Biol. Chem. 252,5888-5894 34. Ruoslahti, E., and Vaheri, A. (1975) J. Exp. Med. 141,497-501 35. Mosher, D. F., and Vaheri, A. (1978) Erp. Cell Res. 112, 323334 36. Salonen, E.”. (1982) J. Zmmunol. Methods 4 8 , 45-50 37. Vaheri, A., Kurkinen, M., Lehto, V. P., Linder, E., and Timpl, R. (1978) Proc. Natl. Acad.Sci. U. S. A. 75, 4944-4948 38. Stenman, S., Wartiovaara, J., and Vaheri, A. (1977) J. Cell Biol. 74,453-467 39. Hedman, K., Vaheri, A., and Wartiovaara, J. (1978) J. Cell Biol. 76,748-760 40. Laemmli, U.K. (1970) Nature (Lond.)2 2 7 , 680-685

Fibronectin Binds to C-reactive Protein Sodlum dodecyl sulfate polyacrylamlde gel electrophoreslslNaDodSO4-PAGEl. Thls was performed acccrdlnq t o the method of Laiemll I401 uslnq v e r t l m s l a b gels. The acrylamlde m n c e n t r a t l o n was 3 . 3 8 In the Spacer qel and 5, 8 Or 10 8 In the ssparatlnq qel, as Indlcated. The samples were reduced wlth 10 % I v l v l 8-mercaptoethanol In Laemll's sample buffer. After electrophocesls, the qels were eralned wlrh Coomasale Brllllant Blue R-250 1411. Commercially Obtalned low wlecular welqht markere IPhamacla, Uppnala, Sweden1 were used. I-blllzatlon Of CRP onto solld phame. Purlfled 1251-labeled or unlabeled CRP was I m b l l l r e d onto slnqle syllndrlcal flat-bottomed polystyrene tubes with Inner bottom surface area of 0.16 cm2 (removable tubes Of fP-9 cuvette blocka: Idbsystems, Helslnkl, Flnlondl. Trlpllcate samples of CRP 0-16 qlm1 In 200 Y 1 each lcorrespondlnq to an actlve I ~ b I l l z a t I o nares of i.13 cm31 of' NaCl/PI. were Incubated Overnlqht 116-20 hl a t rwm temperature 122-24°Clus1nq tlqht adhesion plastlc tape t o cover the tubes durlnq adsorption. The 1110blllzatton was terrnlnated by lncubatlnq the tubes wlth NaCllP contslnlnq 0.02 8 I V l V I h e e n 2 0 lpolyoxyethylene sorbitan monolaureate;'NaCl/PI-heen). P.fter 10 mln at roo.1 temmrature the tubes r e r e washed twlce wlth dlstllled water, 400 ul each, and blr-dried. If not used Imedlately for blndlnq assays, they were stored a t .4OC. The I-blllred CRP malntalned Its blndlnq properties for flbronecrln. flbrlnoqen and antl-CW antlbodlea for elqht m n t h a & * studled by enzyme I m u n ~ a s s d y . The radloactlvlty bound to the solld phase wae measured In an LKB Wallac 80 000 G-a Sample Counter. The blndlnq experiments and e n z w I&unoas~av IEIAl The binding of flbrcnectln to the predetemlned amount of solld-phase CRP was quantlfled by addlnq a volume of 2 0 0 ul Of labeled or unlabeled fIbronectln or It. fraqments (UP to 12.5 uq/mlI. The Incubation solutlon contslned 0.02 8 l Y / V I ?Ween 20

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Analysis of the purlfled protslnpreparations by NaDodSO4-PAGE uslnq 8 (81 , and 10 8 IC1 polyacrylamlde slab gel. In the presence of 8-mercaptoethanol and . t a l n l n q wlth Cwmas.Ie Brllllant Blue. The flqure shows 5 Y q Of CRP llsne All, 30 uq of fibrinogen IBll, 30 u q Of flbronectln 1821. 5 y q of flbronectln (Cll, 40 u q of total cathepsln G digest Of flbronectln IC2I. 10 y q of the NR2-termInal 30 kd fra-nt IC)), 2 uq Of gelatinblndlnq 4 0 kd fraqment ICII, and 15 y q of the 120-140 kd frament of flbronectln IC51. The apparent molecular weightsare expressed In kllodaltons.

%ik, 5 unlabeled fIbrbnecrln.~serum flbronectln. p l a m a flbronectln & flbrlnoqsn was. assayed uslnq a volume of 200 u1 of 1:lOOO dllutlon. of rabblt mtlbodles t o flbronectln or flbcinoqen In NaCl/P contslnlnq 4 8 lw/vl PEG (PEG-buffer1 for one hour at r m temperature and wathed. PEG was Included In order t o enhance the speclflc antlqen-antlbody lnteraetlon 1421. Thls was followed by a 60 mln lncubstlon a t room temperature wlth a 1:lOO dllutlon of alkallnephosphataselabeled antlbodlea to rabblt IqG IOrlon DIaqnostlCal In PEG-buffer and 0 . 2 8

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The effect of solslum. lonlc strength and pH on the blndlnq of flbro.olld-phama CRP. IAI Effect of calclum. Flbronectln 15 uqlmll Was dlluted In N s C l / P ~ - w e nbuffer containing the Indicated amounts of CaCl and Incubated In CRP-coated tubes for 1 h at room temperature. The m u n t of flbronectin bound wan detected unlnq 1:lOOO dllutlon Of antl-flbronectln serum and enrImmunoassay. lBl Effect Of Ionlc strength. Plbronectln 15 Uqlmll was added to eolld-phane CRP for 2 hoursa t r w m temperature In NaCl/P Ween buffer contalnlnq the Indicated COnCentrstlons of NaC1 and the blndlnql;as detsrmlned by enzyme Imunoassay. IC1 Effect o f pH. Flbronectln 11.5 uq/nll was added to solid-phase CRP for 2 hours at r o o m temperature In NaC1/Pl-Neen buffer at the lndlcatcd pH and blndlnq was detenlned by enzyme Immunoassay.

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maxlmbl O C b . 0 5 - 0 . 3 0 M NaC1, b u t ~ w a ndekriaaed CO about 50 8 at 1 II NaCl. As seen In Fig. 40, about 30 8 of the maxlaal blndlnq occurredIn the abmsnss of NaCl. L-lyslne 12 MI lnhlblted the Interactlon betreen solld-phase CRP and flbronectln by 2 5 8. Polyethylene qlycol I 4 w / v l wa. found to speed up the flbronecrln-CRP Interactlon by a factor Of about 3.5, smlmllar to Its k n o w effects on antigen-mtlbody lnteractlon 1421. Polyclonal ontl-flbronectln rabbit antlbodles lnhlblced the Interactlon by 50 8, when used at 1:32 000 dlluclon and totally at 1:4000. The lnteractlon appeared to be relatively sensltlve to pH over the range 4-9. The blndlnq was Increased 3-fold by decreaslnq the pH f r a 1 to 5.0-6.0 lflq. 4CI.

Was

Latex particle aqqlutlnatlon experiment.. To verlfy the blndlnq by a very dlfferent technlque, CRP-Coated Latex particle. were Incubated wlth an q u a l vel- 110 "11 of flbmneotln dissolved In NaCl/P h e e n . Pibranrctln. but nor albumln, aqqlutlnated the CRP-Latex ParCIClesi;F19. 1 of flbronectln-;as rcqulred t o detect the aqqluclnstl& vlsually. Ylth albumln-coated control Latex psrtlcles no aqqlutlnatlon occurred. ThIs experI m n t suqqested that the flbmnectln molecule hasat leaet tblndlnq sites for CRP, posslbly the same reqlon In the 120-140 kd region (see above1 In the two subunlt polypeptldes. TO study thls further. CRP-coated Latex partlcles I 5 "11 were Incubated wlth 1 5 0 nq I5 u l l of purlfled 120-140 kd fragment. Thls did not result In aqqlutlnatlOn. but after addlrlon of palyclonal rabblt antlflbronectln serum I 5 y l o f 1:lOO dllutlonl, Imedlate aqqlutlnatlon Occurred. Without the fragment the antlbodles had no effect. These experlments support the conclusion that the 120-140 kd fragment. are monovalent In the CRP blndlnq.

Blndlnq o f flbroneetln t o CRP-coated Latex partlcles. A volume of 10 u of flbronectln (10 uq1 diluted In NaClIP - h e e n was Incubated wlth an equal volume of CRP-coated Latex partlcles andilmedlately photographed Ileftl. An m u n t of 5 uq of flbmnectln was Incubated wlth CRP-coated Latex particle). under the same condltlons lmlddlel. AS a control 10 Y q bovine serum albumln was Incubated wlth CRP-Coated Latex parrlcles under the 8 condltlons as for flbronectln lrlqhtl.

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.

Coated Latex Dartlcles. A volume of 500 Y1 of 2 8 Iwlvl Dar-Latex particle. ldlameter 1.101 urn; Serva. Heldelberq, FRGI were Incubated wlth an equal II Na-qlyclne buffer volume Of 100 Y q CRP or bovlne serum albumln In contalnlnq 0.15 II NaCI. pH 8.2 for 2 hoursat 37 c In an end-over-end mlxer. After CentrIfuQ~tion at 4000 rn for lo mln, the preclpltate was washed twice wlth the-Incubation buff& and the coated paitlsle. were stored at 4% ln an eaual -1of N.".~YCI..~ buffer contslnlnq 0.1 8 ( w l v l Ovalbumin and