Extracellular Matrix Binding Properties of Recombinant Fibronectin Type II-like Modules of Human 72-kDa Gelatinase/Type IV Collagenase
1995; Elsevier BV; Volume: 270; Issue: 19 Linguagem: Inglês
10.1074/jbc.270.19.11555
ISSN1083-351X
AutoresBjorn Steffensen, U. Margaretha Wallon, Christopher M. Overall,
Tópico(s)Blood Coagulation and Thrombosis Mechanisms
Resumo72-kDa gelatinase/type IV collagenase is an important matrix metalloproteinase in the degradation of basement membranes and denatured collagens (gelatin). These proteolytic processes are required for pathologic tissue destruction and physiologic tissue remodeling. To investigate the molecular determinants of substrate specificity of this enzyme, a 21-kDa domain of 72-kDa gelatinase, consisting of three tandem fibronectin type II-like modules, was expressed in Escherichia coli. Similar to full-length 72-kDa gelatinase and the type H modules in fibronectin, the recombinant (r) fibronectin-like domain of this proteinase bound denatured type I collagen with an apparent Kd in the micromolar range. This domain, designated the collagen-binding domain (rCBD123), possesses at least two collagen-binding sites that can each be simultaneously occupied. rCBD123 also avidly bound elastin and denatured types IV and V collagens, but neither native types IV and V collagens nor fibronectin, all of which are substrates of the enzyme. Although 72-kDa gelatinase is involved in basement membrane degradation, rCBD123 also did not bind reconstituted basement membrane, laminin, or SPARC. Native type I collagen, which is not degraded by 72-kDa gelatinase, competed with gelatin for a shared binding site on rCBD123. rCBD123 also displaced full-length 72-kDa gelatinase bound to native type I collagen, further demonstrating that the collagen binding properties of the recombinant domain closely mimicked those of the full-length enzyme. Since rCBD123 showed reduced binding to pepsin-cleaved type I collagen, either or both of the collagen telopeptide ends contain recognition sites for the 72-kDa gelatinase fibronectin-like domain. This was confirmed by the avid binding of rCBD123 to the α1(I) collagen cyanogen bromide fragment CB2 from the NH2-terminal telopeptide. rCBD123 also bound α1(I)-CB7, which encompasses the fibronectin-binding site, and to α1(I)-CB8, a fragment not bound by fibronectin. Thus, type I collagen contains multiple binding sites for rCBD123 which are partially masked by the triple helical conformation of native collagen and fully exposed upon unfolding of the triple helix. The potential of the fibronectin-like collagen binding domain of 72-kDa gelatinase to bind extracellular matrix proteins may facilitate enzyme localization in connective tissue matrices. 72-kDa gelatinase/type IV collagenase is an important matrix metalloproteinase in the degradation of basement membranes and denatured collagens (gelatin). These proteolytic processes are required for pathologic tissue destruction and physiologic tissue remodeling. To investigate the molecular determinants of substrate specificity of this enzyme, a 21-kDa domain of 72-kDa gelatinase, consisting of three tandem fibronectin type II-like modules, was expressed in Escherichia coli. Similar to full-length 72-kDa gelatinase and the type H modules in fibronectin, the recombinant (r) fibronectin-like domain of this proteinase bound denatured type I collagen with an apparent Kd in the micromolar range. This domain, designated the collagen-binding domain (rCBD123), possesses at least two collagen-binding sites that can each be simultaneously occupied. rCBD123 also avidly bound elastin and denatured types IV and V collagens, but neither native types IV and V collagens nor fibronectin, all of which are substrates of the enzyme. Although 72-kDa gelatinase is involved in basement membrane degradation, rCBD123 also did not bind reconstituted basement membrane, laminin, or SPARC. Native type I collagen, which is not degraded by 72-kDa gelatinase, competed with gelatin for a shared binding site on rCBD123. rCBD123 also displaced full-length 72-kDa gelatinase bound to native type I collagen, further demonstrating that the collagen binding properties of the recombinant domain closely mimicked those of the full-length enzyme. Since rCBD123 showed reduced binding to pepsin-cleaved type I collagen, either or both of the collagen telopeptide ends contain recognition sites for the 72-kDa gelatinase fibronectin-like domain. This was confirmed by the avid binding of rCBD123 to the α1(I) collagen cyanogen bromide fragment CB2 from the NH2-terminal telopeptide. rCBD123 also bound α1(I)-CB7, which encompasses the fibronectin-binding site, and to α1(I)-CB8, a fragment not bound by fibronectin. Thus, type I collagen contains multiple binding sites for rCBD123 which are partially masked by the triple helical conformation of native collagen and fully exposed upon unfolding of the triple helix. The potential of the fibronectin-like collagen binding domain of 72-kDa gelatinase to bind extracellular matrix proteins may facilitate enzyme localization in connective tissue matrices. A central characteristic of metastatic tumor cells is their ability to degrade and penetrate basement membranes. Considerable evidence has linked elevated matrix metalloproteinase (MMP) 1The abbreviations used are: MMP, matrix metalloproteinase; BSA, bovine serum albumin; CB, cyanogen bromide; DTT, dithioerythreitol; gelatin, denatured collagen (denatured type I collagen consists of two α1(I) chains and one α2(I) chain); ME2SO, dimethyl sulfoxide; PAGE, polyacrylamide gel electrophoresis; rCBD123, recombinant collagen-binding domain consisting of fibronectin type II-like modules 1, 2 and 3; SDS, sodium dodecyl sulfate; SPARC, secreted protein which is acidic and rich in cysteine; TIMP-1, tissue inhibitor of matrix metalloproteinases-1. expression by many tumor cells with these processes (1Liotta L.A. Tryggvason K. Garbisa S. Hart I. Folts C.M. Shafie S. Nature. 1980; 284: 67-68Crossref PubMed Scopus (1584) Google Scholar). Type IV collagen is the major structural component of basement membranes (2Timpl R. Martin G.R. Bruckner P. Wick G. Wiedemann H. Eur. J. 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Gustav Fischer, Stuttgart1992: 209-211Google Scholar, 21Overall C.M. Sodek J. J. Biol. Chem. 1990; 265: 21141-21151Abstract Full Text PDF PubMed Google Scholar). The hemopexin/vitronectin-like carboxyl domains of collagenase and stromelysin also bind native type I collagen (15Murphy G. Allan J.A. Willenbrock F. Cockett M.I. O'Commell J.P. Docherty A.J.P. J. Biol. Chem. 1992; 267: 9612-9618Abstract Full Text PDF PubMed Google Scholar, 22Clark I.M. Cawston T.E. Biochem. J. 1989; 263: 201-206Crossref PubMed Scopus (171) Google Scholar, 23Windsor L.J. Birkedal-Hansen H. Birkedal-Hansen B. Engler J.A. Biochemistry. 1991; 30: 641-647Crossref PubMed Scopus (77) Google Scholar). Removal of this domain from collagenase ablates collagenolysis but not catalytic competence, that is, the truncated collagenase lacking the carboxyl domain still degrades synthetic peptide substrates and casein, but not native type I collagen (22Clark I.M. Cawston T.E. Biochem. 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Sipos G. Patthy L. Eur. J. Biochem. 1990; 193: 801-806Crossref PubMed Scopus (58) Google Scholar) reported that a recombinant type II module from fibronectin and a type II-like module in bovine seminal fluid protein PDC-109 binds gelatin. Subsequent work demonstrated that the type II-like modules in both the 72- (32Banyai L. Patthy L. FEBS Lett. 1991; 282: 23-25Crossref PubMed Scopus (64) Google Scholar, 33Banyai L. Tordai H. Patthy L. Biochem. J. 1994; 298: 403-407Crossref PubMed Scopus (70) Google Scholar) and the 92-kDa (34Collier E.E. Krasnov P.A. Strongin A.Y. Birkedahl-Hansen H. Goldberg G.I. J. Biol. Chem. 1992; 267: 6776-6781Abstract Full Text PDF PubMed Google Scholar) gelatinases also bind denatured type I collagen. Binding specialization of the different fibronectin type II-like modules in the 72-kDa gelatinase may have occurred to generate exosites specific for the other collagens and extracellular matrix molecules degraded by the enzyme including native types IV, V, VII, and X collagens, elastin, and fibronectin (25Collier I.E. Wilhelm S.M. Eisen A.Z. Marmer B.L. Grant G.A. Seltzer J.L. Kronberger A. He C. Bauer E.A. Goldberg G.I. J. Biol. Chem. 1988; 263: 6579-6587Abstract Full Text PDF PubMed Google Scholar, 35Seltzer J.L. Akers K.T. Weingarten H. Grant G.A. McCourt D.W. Eisen A.Z. J. Biol. Chem. 1990; 265: 20409-20413Abstract Full Text PDF PubMed Google Scholar). To further understand the function of the structural elements of the 72-kDa gelatinase, we have characterized the binding properties of the fibronectin-like domain of human 72-kDa gelatinase to a number of the enzymes substrates, reconstituted basement membrane, TIMP-1, and other extracellular matrix proteins. Reported here are experiments which establish that, in addition to binding denatured type I collagen, a recombinant fibronectin-like domain from human 72-kDa gelatinase, encompassing all three type II-like modules, binds with high affinity to denatured types IV and V collagens and elastin. Although human 72-kDa gelatinase cleaves native type IV collagen but not native type I collagen, surprisingly, the fibronectin-like domain avidly binds native type I collagen but neither native type IV collagen nor other basement membrane components. Thus, in addition to fulfilling the criteria as an exosite for a number of substrates, the fibronectin-like domain of 72-kDa gelatinase may have an ancillary role as an extracellular matrix localization domain by virtue, in particular, of its native type I collagen binding properties. Acid-soluble native type I collagen was prepared from rat tail tendons as described by Piez (36Piez K.A. Ramachandran G.N. Treatise on Collagen. Academic Press, London1967: 207-252Google Scholar) by extraction with 0.5 m acetic acid and differential precipitation with 1.7 m NaCl. Pepsin-treated type I collagen was prepared by digestion of the acid-soluble type I collagen with pepsin (Sigma) at pH 2.0, 4 °C for 20 h, then precipitated with 1.7 m NaCl, redissolved in 0.15 m acetic acid, and lyophilized. Gelatin was prepared from acid-soluble type I collagen (non-pepsin treated) by heat denaturation at 56 °C for 30 min. [14C]Glycine-labeled type I collagen, with a specific activity of 3.5 × 108 disintegrations/min/mg, was prepared by metabolic labeling and purified from conditioned cell medium by pepsin digestion and NaCl precipitation as described previously (37Overall C.M. Sodek J. J. Dent. Res. 1987; 66: 1271-1282Crossref PubMed Scopus (63) Google Scholar). To confirm the native collagen content of the metabolically labeled preparation, the labeled type I collagen was incubated with 0.1 or 0.01 µg/ml trypsin (type XII bovine pancreas, Sigma) (enzyme to substrate ratio ∼1:2 and 1:20) for 19 h at 20 °C. Intact protein was then precipitated in 10% (w/v) trichloroacetic acid, 1% (w/v) tannic acid for 2 h at 0 °C and the pellets collected by centrifugation at 10,000 × g for 20 min at 0 °C (37Overall C.M. Sodek J. J. Dent. Res. 1987; 66: 1271-1282Crossref PubMed Scopus (63) Google Scholar). The trypsin-digested denatured type I collagen content was determined by scintillation counting of the trichloroacetic acid/tannic acid-soluble protein fraction and calculated to constitute 100-fold mm excess/disulfide bond) for 30 min at 20 °C. This concentration of DTT was maintained in all buffers in column assays. rCBD123 was also reduced and carboxymethylated according to Creighton (1990) (47Creighton T.E. Creighton T.E. Protein Structure: a Practical Approach. Oxford University Press, Oxford1990: 155-167Google Scholar) and Hollecker (1990) (48Hollecker M. Creighton T.E. Protein Structure: A Practical Approach. Oxford University Press, Oxford1990: 145-153Google Scholar), modified as follows. rCBD123 was first equilibrated in denaturation buffer (8.0 m urea, 0.5 m Tris-HCl, 2 mm EDTA, pH 8.1) by gel filtration on a 10 DG column (Vt 10 ml) (Bio-Rad) and then reduced by addition of 100-fold molar excess of DTT (65 mm) over the estimated disulfide bond content and incubated at 50 °C for 1 h. After cooling to 20 °C, the alkylating agent, iodoacetic acid, was added to a 2-fold molar excess over DTT (130 mm) and reacted at 20 °C for 30 min. The reduced and carboxymethylated protein was then equilibrated in 50 mm Tris, pH 7.4, by chromatography over a 10 DG column and assessed by SDS-PAGE. Column assays with reduced and carboxymethylated rCBD123 were performed in the absence of reductant under buffer conditions identical to those described for non-reduced rCBD123. Proteins were separated by SDS-polyacrylamide gel electrophoresis according to Laemmli (1970) (49Laemmli U.U. Nature. 1970; 227: 680-685Crossref PubMed Scopus (214196) Google Scholar). Protein samples were analyzed without reduction or with the addition of 65 mm DTT and heating at 95 °C for 5 min. Gels were stained with Coomassie Brilliant Blue R-250 at 42 °C, and protein bands were quantitated by laser densitometry at 633 nm (LKB Ultrascan XL). For enzymography, non-reduced protein samples were electrophoresed on 10% (w/v) polyacrylamide gels containing 100 µg/ml heat-denatured acid-soluble type I collagen. Gels were processed as described previously (8Overall C.M. Limeback H. Biochem. J. 1988; 256: 965-972Crossref PubMed Scopus (97) Google Scholar). Briefly, after electrophoresis gels were equilibrated in 5% (v/v) Triton X-100, incubated in assay buffer (50 mm Tris, 200 mm NaCl, 5 mm CaCl2) for 2–4 h at 37 °C, and the cleared bands, identifying the position of 72-kDa gelatinase, revealed by counterstaining of the gelatin in the gels by Coomassie Brilliant Blue R-250. Reduced molecular mass markers used were rabbit muscle phosphorylase b (97 kDa), bovine serum albumin (BSA) (67 kDa), chicken egg ovalbumin (43 kDa), bovine carbonic anhydrase (29 kDa), horse heart myoglobin (18.8 kDa), chicken egg-white lysozyme (14.4 kDa), and bovine insulin (6.2 kDa) (Sigma). Proteins were transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore) after separation by SDS-PAGE. Transferred rCBD123 was then reacted with a polyclonal anti-72-kDa gelatinase antibody diluted 1:1,000 in TBS/Tween with 1% (w/v) BSA for 1 h, washed, and conjugates detected using enhanced chemiluminescence (ECL) reagents and Hyperfilm (Amersham Corp.). To screen for potential interaction with rCBD123, a number of known substrates of 72-kDa gelatinase as well as other extracellular matrix proteins were coated as films in 96-microwell plates. Proteins included native type I collagen, pepsin-treated native type I collagen (telopeptide-free), and heat-denatured type I collagen (gelatin); collagen α1(I) cyanogen bromide fragments 2, 7, and 8; native and heat-denatured types IV and V collagens; elastin, Matrigel®, laminin, fibronectin, SPARC, and TIMP-1. Myoglobin and BSA served as negative control proteins for the assays. Microtiter plates were coated overnight at 4 °C with 10 pmol protein/well (typically 1–5 µg) in coating buffer (15 mm Na2CO3, 35 mm NaHCO3, 0.02% (w/v) NaN3, pH 9.6). Consistent and equal binding of protein coate
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