Generation and Characterization of Aggrecanase
1999; Elsevier BV; Volume: 274; Issue: 10 Linguagem: Inglês
10.1074/jbc.274.10.6594
ISSN1083-351X
AutoresElizabeth C. Arner, Michael A. Pratta, James M. Trzăskos, Carl P. Decicco, Micky D. Tortorella,
Tópico(s)Chemical Synthesis and Analysis
ResumoA method was developed for generating soluble, active "aggrecanase" in conditioned media from interleukin-1-stimulated bovine nasal cartilage cultures. Using bovine nasal cartilage conditioned media as a source of the aggrecanase enzyme, an enzymatic assay was established employing purified aggrecan monomers as a substrate and monitoring specific aggrecanase-mediated cleavage products by Western analysis using the monoclonal antibody, BC-3 (which recognizes the new N terminus, ARGS, on fragments produced by cleavage between amino acid residues Glu373 and Ala374). Using this assay we have characterized cartilage aggrecanase with respect to assay kinetics, pH and salt optima, heat sensitivity, and stability upon storage. Aggrecanase activity was inhibited by the metalloprotease inhibitor, EDTA, while a panel of inhibitors of serine, cysteine, and aspartic proteinases had no effect, suggesting that aggrecanase is a metalloproteinase. Sensitivity to known matrix metalloproteinase inhibitors as well as to the endogenous tissue inhibitor of metalloproteinases, TIMP-1, further support the notion that aggrecanase is a metalloproteinase potentially related to the ADAM family or MMP family of proteases previously implicated in the catabolism of the extracellular matrix. A method was developed for generating soluble, active "aggrecanase" in conditioned media from interleukin-1-stimulated bovine nasal cartilage cultures. Using bovine nasal cartilage conditioned media as a source of the aggrecanase enzyme, an enzymatic assay was established employing purified aggrecan monomers as a substrate and monitoring specific aggrecanase-mediated cleavage products by Western analysis using the monoclonal antibody, BC-3 (which recognizes the new N terminus, ARGS, on fragments produced by cleavage between amino acid residues Glu373 and Ala374). Using this assay we have characterized cartilage aggrecanase with respect to assay kinetics, pH and salt optima, heat sensitivity, and stability upon storage. Aggrecanase activity was inhibited by the metalloprotease inhibitor, EDTA, while a panel of inhibitors of serine, cysteine, and aspartic proteinases had no effect, suggesting that aggrecanase is a metalloproteinase. Sensitivity to known matrix metalloproteinase inhibitors as well as to the endogenous tissue inhibitor of metalloproteinases, TIMP-1, further support the notion that aggrecanase is a metalloproteinase potentially related to the ADAM family or MMP family of proteases previously implicated in the catabolism of the extracellular matrix. The aggregating cartilage proteoglycan, aggrecan, along with type II collagen, is responsible for the mechanical properties of articular cartilage. Aggrecan molecules are composed of two N-terminal globular domains, G1 and G2, which are separated by an interglobular domain (IGD), 1The abbreviations used are: IGD, interglobular domain; GAG, glycosaminoglycan; MMP, matrix metalloproteinase; PAGE, polyacrylamide gel electrophoresis; IL, interleukin; TIMP-1, tissue inhibitor of metalloproteinases; APMA, 4-aminophenylmercuric acetate; MES, 4-morpholineethanesulfonic acid.followed by a long central glycosaminoglycan (GAG) attachment region and a C-terminal globular domain, G3(1Hardingham T.E. Fosang A.J. Dudhia J. Kuettner K.E. Schleyerbach R. Peyton J.G. Hascall V.C. Articular Cartilage and Osteoarthritis. Raven Press, New York1992: 5-20Google Scholar, 2Paulson M. Morgolin M. Wiedemann H. Beardmore-Gray M. Dunham D. Hardingham T.E. Heinegard D. Biochem. J. 1987; 245: 763-772Crossref PubMed Scopus (90) Google Scholar). These aggrecan monomers interact through the G1domain with hyaluronic acid and link protein to form large molecular weight aggregates which are trapped within the cartilage matrix (3Hardingham T.E. Muir H. Biochim. Biophys. Acta. 1972; 279: 401-405Crossref PubMed Scopus (419) Google Scholar, 4Heinegard D. Hascall V.C. J. Biol. Chem. 1974; 249: 4250-4256Abstract Full Text PDF PubMed Google Scholar, 5Hardingham T.E. Biochem. J. 1979; 177: 237-247Crossref PubMed Scopus (232) Google Scholar). Aggrecan provides normal cartilage with it properties of compressibility and resilience, and is one of the first matrix components to undergo measurable loss in arthritis. This loss appears to be due to an increased rate of aggrecan degradation that can be attributed to proteolytic cleavage within the IGD of the core protein. Cleavage within this region generates large C-terminal, GAG-containing aggrecan fragments lacking the G1 domain which are unable to bind to hyaluronic acid and thus diffuse out of the cartilage matrix. Two major sites of proteolytic cleavage have been identified within the IGD: one between amino acid residues Asn341 and Phe342 and the other between amino acid residues Glu373 and Ala374. Matrix metalloproteinases (MMP-1, -2, -3, -7, -8, -9, and 13) have been shown in vitroto cleave within the IGD predominately at the Asn341-Phe342 site (6Fosang A.J. Neame P.J. Hardingham T.E. Murphy G. Hamilton J.A. J. Biol. Chem. 1991; 266: 15579-15582Abstract Full Text PDF PubMed Google Scholar, 7Flannery C.R. Lark M.W. Sandy J.D. J. Biol. Chem. 1992; 267: 1008-1014Abstract Full Text PDF PubMed Google Scholar, 8Fosang A.J. Last K. Knauper V. Neame P.J. Murphy G. Hardingham T.E. Tschesche H. Hamilton J.A. Biochem. J. 1993; 295: 273-276Crossref PubMed Scopus (139) Google Scholar, 9Fosang A.J. Neame P.J. Last K. Hardingham T.E. Murphy G. Hamilton J.A. J. Biol. Chem. 1992; 267: 19470-19474Abstract Full Text PDF PubMed Google Scholar, 10Fosang A.J. Last K. Knauper V. Murphy G. Neame P.J. FEBS Lett. 1996; 380: 17-20Crossref PubMed Scopus (332) Google Scholar). Identification of G1 fragments formed by cleavage at the Asn341-Phe342 site within human articular cartilage (7Flannery C.R. Lark M.W. Sandy J.D. J. Biol. Chem. 1992; 267: 1008-1014Abstract Full Text PDF PubMed Google Scholar, 11Lark M.W. Bayne E.K. Flanagan J. Harper C.F. Hoerrner L.A. Hutchinson N.I. Singer I.I. Donatilli S.A. Weidner J.R. Williams H.R. Mumford R.A. Lohmander L.S. J. Clin. Invest. 1997; 100: 93-106Crossref PubMed Scopus (393) Google Scholar) and in synovial fluids (12Fosang A.J. Last K. Maciewicz R.A. J. Clin. Invest. 1996; 98: 2292-2299Crossref PubMed Scopus (178) Google Scholar) suggest a role for MMPs in proteoglycan degradation in vivo. The second cleavage site was first described a number of years ago based upon identification of aggrecan fragments with an ARGS N terminus (13Sandy J.D. Neame P.J. Boynton P.L. Flannery C.R. J. Biol. Chem. 1991; 266: 8683-8685Abstract Full Text PDF PubMed Google Scholar, 14Leulakis P. Shirkhanda A.V. Davis G. Maniglia C.A. Biochem. J. 1992; 264: 589-593Crossref Scopus (122) Google Scholar, 15Ilic M.Z. Handley C.J. Robinson H.C. Mok M.T. Arch. Biochem. Biophys. 1992; 294: 115-122Crossref PubMed Scopus (163) Google Scholar), however, the enzyme responsible for cleavage at the Glu373-Ala374 bond has not yet been identified. This uncharacterized activity has been given the name "aggrecanase" based on its ability to cleave the aggrecan core protein. Four other potential aggrecanase sites have been identified within the C-terminal region of aggrecan between G2 and G3 (14Leulakis P. Shirkhanda A.V. Davis G. Maniglia C.A. Biochem. J. 1992; 264: 589-593Crossref Scopus (122) Google Scholar, 16Sandy J.D. Plaas A.H.K. Koob T.J. Acta Orthop. Scand. 1995; 66 Suppl. 266: 26-32Crossref Scopus (57) Google Scholar), although the Glu373-Ala374 cleavage site within the IGD has been most widely studied. C-terminal fragments with the N terminus, ARGSV … , formed by cleavage between amino acid residues Glu373 and Ala374 have been identified in media from chondrocyte monolayer and cartilage explant cultures undergoing matrix degradation (13Sandy J.D. Neame P.J. Boynton P.L. Flannery C.R. J. Biol. Chem. 1991; 266: 8683-8685Abstract Full Text PDF PubMed Google Scholar, 14Leulakis P. Shirkhanda A.V. Davis G. Maniglia C.A. Biochem. J. 1992; 264: 589-593Crossref Scopus (122) Google Scholar, 15Ilic M.Z. Handley C.J. Robinson H.C. Mok M.T. Arch. Biochem. Biophys. 1992; 294: 115-122Crossref PubMed Scopus (163) Google Scholar, 16Sandy J.D. Plaas A.H.K. Koob T.J. Acta Orthop. Scand. 1995; 66 Suppl. 266: 26-32Crossref Scopus (57) Google Scholar, 17Sandy J.D. Boynton P.L. Flannery C.R. J. Biol. Chem. 1991; 266: 8198-8205Abstract Full Text PDF PubMed Google Scholar, 18Lark M.W. Gordy J.T. Weidner J.R. Ayala J. Kimura J.H. Williams H.R. Mumford R.A. Flannery C.R. Carisoni S.S. Iwatai M. Sandy J.D. J. Biol. Chem. 1995; 270: 2550-2556Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 19Ilic M.Z. Mok M.T. Williamson O.D. Campbell M.A. Hughes C.E. Handley C.J. Arch. Biochem. Biophys. 1995; 322: 22-30Crossref PubMed Scopus (26) Google Scholar). This sequence was found to be present on a number of different size fragments, indicating that aggrecanase was cleaving at the Glu373-Ala374bond within the IGD to generate products with a single N terminus which possessed variable C termini. N-terminal sequence analyses have identified aggrecan fragments in synovial fluids of patients with osteoarthritis, inflammatory joint disease, and joint injury (20Sandy J.D. Flannery C.R. Neame P.J. Lohmander L.S. J. Clin. Invest. 1992; 89: 1512-1516Crossref PubMed Scopus (386) Google Scholar, 21Lohmander L.S. Neame P.J. Sandy J.D. Arthritis Rheum. 1993; 36: 1214-1222Crossref PubMed Scopus (382) Google Scholar) which have the ARGSV N terminus, and G1 fragments have also been identified within the cartilage matrix with the NITEGE C terminus (11Lark M.W. Bayne E.K. Flanagan J. Harper C.F. Hoerrner L.A. Hutchinson N.I. Singer I.I. Donatilli S.A. Weidner J.R. Williams H.R. Mumford R.A. Lohmander L.S. J. Clin. Invest. 1997; 100: 93-106Crossref PubMed Scopus (393) Google Scholar) indicating that aggrecanase plays an important role in human aggrecan catabolism. Although evidence for induction of cleavage at the aggrecanase site within the IGD has been demonstrated in a number of in vitrotissue and cell culture studies (13Sandy J.D. Neame P.J. Boynton P.L. Flannery C.R. J. Biol. Chem. 1991; 266: 8683-8685Abstract Full Text PDF PubMed Google Scholar, 14Leulakis P. Shirkhanda A.V. Davis G. Maniglia C.A. Biochem. J. 1992; 264: 589-593Crossref Scopus (122) Google Scholar, 15Ilic M.Z. Handley C.J. Robinson H.C. Mok M.T. Arch. Biochem. Biophys. 1992; 294: 115-122Crossref PubMed Scopus (163) Google Scholar, 16Sandy J.D. Plaas A.H.K. Koob T.J. Acta Orthop. Scand. 1995; 66 Suppl. 266: 26-32Crossref Scopus (57) Google Scholar, 17Sandy J.D. Boynton P.L. Flannery C.R. J. Biol. Chem. 1991; 266: 8198-8205Abstract Full Text PDF PubMed Google Scholar, 18Lark M.W. Gordy J.T. Weidner J.R. Ayala J. Kimura J.H. Williams H.R. Mumford R.A. Flannery C.R. Carisoni S.S. Iwatai M. Sandy J.D. J. Biol. Chem. 1995; 270: 2550-2556Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 19Ilic M.Z. Mok M.T. Williamson O.D. Campbell M.A. Hughes C.E. Handley C.J. Arch. Biochem. Biophys. 1995; 322: 22-30Crossref PubMed Scopus (26) Google Scholar), attempts to identify aggrecanase proteolytic activity in culture media or cell/tissue extracts from these models have been largely unsuccessful. Recent work using an artificial recombinant protein comprising the IGD sequence of aggrecan, rAgg1, as a substrate, has identified activity in media from retinoic acid-stimulated rat chondrosarcoma cell cultures which cleaves at the Glu373-Ala374 bond (22Hughes C.E. Buttner F.H. Eidenmuller B. Caterson B. Bartnik E. J. Biol. Chem. 1997; 272: 20269-20274Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). However, the protease responsible for this cleavage remains unidentified and uncharacterized. In this paper we describe the generation of soluble, active aggrecanase activity in conditioned media from interleukin-1 (IL-1)-stimulated bovine nasal cartilage. Using this source of aggrecanase, we have developed an enzymatic assay employing purified native aggrecan monomers as the substrate and detection of specific products of aggrecanase-mediated cleavage by Western analysis with the monoclonal antibody, BC-3 (23Hughes C.E. Caterson B. Fosang A.J. Roughley P.J. Mort J.S. Biochem. J. 1995; 305: 799-804Crossref PubMed Scopus (195) Google Scholar), which recognizes the new N terminus ARGSV on fragments produced by cleavage at the aggrecanase site. We have performed initial enzymatic characterization of the soluble cartilage aggrecanase activity generated in response to IL-1. The work described herein provides a method for generating soluble aggrecanase activity and an assay for monitoring this activity which should serve as important tools to enable the isolation, purification, and molecular characterization of the enzyme. Dulbecco's modified Eagle's medium and fetal bovine serum were from Life Technologies, Inc. (Grand Island, NY). The IL-1 used was a soluble, fully active recombinant human IL-1β produced as described previously (24Huang J.J. Newton R.C. Pezzella K. Covington M. Tamblyn T. Rutledge S.J. Kelley M. Gray J. Lin Y. Mol. Biol. Med. 1987; 4: 169-181PubMed Google Scholar). The specific activity was 1 × 107 units/mg of protein, with 1 unit being defined as the amount of IL-1 that generated half-maximal activity in the thymocyte proliferation assay. Antibody BC-3 (23Hughes C.E. Caterson B. Fosang A.J. Roughley P.J. Mort J.S. Biochem. J. 1995; 305: 799-804Crossref PubMed Scopus (195) Google Scholar) which recognizes the new N terminus ARGSV … on aggrecan degradation produced by aggrecanase was provided by Dr. Bruce Caterson (University of Wales, Cardiff, United Kingdom). Chondroitinase ABC lyase (Proteus vulgaris) (EC 4.2.2.4), keratanase (Pseudomonas sp.) (EC 3.2.1.103), and keratanase II (Bacillus sp.) were from Seikuguku (Kogyo, Japan). Full-length bovine tissue inhibitor of metalloproteases-1 (TIMP-1) was from Calbiochem (Cambridge, MA). The protease inhibitors, antipain dihydrochloride, aprotinin, bestatin, chymotrypsin, E-64, EDTA, leupeptin, Pefabloc, pepstatin, and phosphoramidon were from Boehringer-Mannheim (Indianapolis, IN). The hydroxamic acid MMP inhibitors, BB-16 ((2S,3R)-2-methyl-3-(2-methylpropyl)-1-(N-hydroxy)-4-(o-methyl)-l-tyrosine-N-methyl amide), XS309 ([3S-[3R*, 2-[2R*,2-(R*,S*)]-hexahydro-2-[2-[2-(hydroxyamino)-1-methyl-2-oxoethyl]-4-methyl-1-oxopentyl]-N-methyl-3-pyridazinecarboxamide), and SA751 (N-[1(R)-Carboxyethyl]-a-(S)-(4-phenyl-3-butynyl)glycyl-l-O-methyltyrosine,N-methylamide) were synthesized at DuPont Pharmaceuticals as described previously (25.Campion, C., Davidson, A. H., Dickens, J. P., and Crimmins, M. J. (1989) PCT/GB89/01399, November 23, 1989.Google Scholar, 26.Xue C-B., Cherney, R. J., Decicco, C. P., DeGrado, W. F., He, X., Hodge, C. N., Jacobson, I. C., Magolda, R. L., and Arner, E. C. (1997) WO 9718207 A2, May 22, 1997.Google Scholar). BB-16 and XS309 are potent nanomolar inhibitors of a number of MMPs, including MMP-1, -2, -3, -8, and -9, while SA751 is a selective MMP-8 inhibitor with a K iof 2 nm against MMP-8 and a K i of >10,000 nm against MMP-3 and MMP-1. Bovine nasal cartilage septa were removed from bovine noses obtained fresh at the time of slaughter. Uniform cartilage disks (1 mm thick, 8 mm in diameter) were prepared. Prior to organ culture studies, disks were equilibrated in tissue culture for at least 3 days in Dulbecco's modified Eagle's medium supplemented with 5% heat-inactivated fetal calf serum, penicillin, streptomycin, amphotericin B, and neomycin (100 IU/ml, 100 μg/ml, 0.25 μg/ml, and 50 μg/ml, respectively). Cartilage slices were incubated in serum-free Dulbecco's modified Eagle's medium supplemented with antibiotics as above at 37 °C in an atmosphere of 95% air, 5% CO2 in the presence of 500 ng/ml IL-1β. Media were replaced every 2 days for the first 6 days of culture and saved at −70 °C for analysis. Cartilage was then incubated with IL-1 (500 ng/ml) from days 6 to 18 without media change to allow accumulation of aggrecanase activity in the media. Media were sampled every 2 days and saved at −70 °C for analysis. In some cases, at the end of incubation cartilage was digested with papain and the GAG content of the digest determined. Sulfated GAG in culture media or cartilage digests were monitored by the amount of polyanionic material reacting with 1,9-dimethylmethylene blue (27Farndale R.W. Sayers C.A. Barrett A.J. Connect. Tissue Res. 1982; 9: 247-248Crossref PubMed Scopus (1159) Google Scholar, 28.Deleted in proof.Google Scholar), using shark chondroitin sulfate as a standard. GAG release is reported as micrograms of GAG per mg wet weight cartilage or as percent of total GAG. Culture media were diluted 1:20 with water and 15 μl mixed with an equal volume of sample buffer (0.5m Tris-HCl, pH 6.8, 4% SDS, 0.005% bromphenol blue, 20% glycerol) (Novex, San Diego, CA), incubated at room temperature for 10 min, and run on a 10% SDS-PAGE gels containing 0.1% gelatin in running buffer (0.24 m Tris, 2 m glycine, 35 mm SDS, pH 8.3) for 90 min at 125 volts. The gels were renatured in 2.5% Triton X-100 in water for 30 min at room temperature and then incubated at 37 °C overnight in developing buffer (50 mm Tris, 0.2 m NaCl, 5 mmCaCl2, 0.02% Brij 35, pH 7.6). After incubation, gels were stained in 0.25% Coomassie Brilliant Blue R-250 for 4 h at room temperature and destained in distilled water containing 30% methanol and 10% glacial acetic acid to reveal zones of lysis within the gelatin matrix. EDTA (5 mm), E64 (10 μg/ml), pepstatin (1 μg/ml), or benzamidine HCl (10 mm) or XS309 (1 μm) were added to the developing buffer to identify which classes of proteinases were responsible for lysis of the gelatin. Casein labeled with resorufin (43.5 μm) was incubated with protease-containing media in a final volume of 200 μl of 50 mm Tris-HCl, 5 mm CaCl2 at pH 7.8 at 37 °C as described previously (29Tortorella M.D. Arner E.C. Inflamm. Res. 1997; 46 Suppl. 2: S122-S123Crossref PubMed Scopus (7) Google Scholar). The reactions were stopped and unclipped substrate precipitated by adding trichloroacetic acid to a final concentration of 5%. Samples were filtered through a 96-well filtration plate (Millipore Co., Bedford, MA) and filtrates collected and neutralized by addition of 2 m Tris to each well. The absorbance was then read at 575 nm. The concentration of the resorufin-labeled peptides in the filtrate, as determined from a standard curve, was used as a measure of proteolytic activity. Some conditioned medium samples were assayed in the presence of 2 mm 4-aminophenylmercuric acetate (APMA) to activate latent matrix metalloproteinases. With some samples EDTA was included to inhibit metalloproteinase activity or XS309 was included to inhibit matrix metalloproteinase activity. For analysis of aggrecan fragments generated by specific cleavage at the Glu373-Ala374 site, proteoglycans and proteoglycan metabolites were enzymatically deglycosylated with chondroitinase ABC (0.1 units/10 μg of GAG) for 2 h at 37 °C and then with keratanase (0.1 units/10 μg of GAG) and keratanase II (0.002 units/10 μg of GAG) for 2 h at 37 °C in buffer containing 50 mm sodium acetate, 0.1 mTris/HCl, pH 6.5 (23Hughes C.E. Caterson B. Fosang A.J. Roughley P.J. Mort J.S. Biochem. J. 1995; 305: 799-804Crossref PubMed Scopus (195) Google Scholar). The digests were monitored by measuring the decrease in dimethylmethylene blue reactivity. After digestion, the samples were precipitated with 5 volumes of acetone and reconstituted in an appropriate volume of SDS-PAGE sample buffer. Equivalent amounts of GAG from each sample were loaded on 4–12% gradient gels and then separated by SDS-PAGE under nonreducing conditions, transferred overnight to nitrocellulose, and immunolocated with 1:1000 dilution of the monoclonal antibody BC-3 (22Hughes C.E. Buttner F.H. Eidenmuller B. Caterson B. Bartnik E. J. Biol. Chem. 1997; 272: 20269-20274Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Overnight transfer resulted in complete transfer of low and high molecular weight standards and samples as determined by evaluating the gel by colloidal Coomassie analysis (Novex, San Diego, CA) following protein transfer; no detectable levels of protein remained in the gel. Subsequently, membranes were incubated with goat anti-mouse IgG alkaline phosphatase conjugate and aggrecan catabolites visualized by incubation with the appropriate substrate (Promega Western blot alkaline phosphatase system) for 10–30 min to achieve optimal color development. BC-3-reactive aggrecan fragments were then quantified by scanning densitometry. For quantitation, the stained blots were captured on a UVP Imagestore 7500 Camera System (Cambridge, UK) with GelBase Pro system software. The integrated pixel density of the bands was quantified using IPLAB GEL software and data obtained only in the 10-fold linear detection range and reported as sum total pixels. Bovine nasal cartilage septa were removed from bovine noses obtained fresh at slaughter. The aggrecan was isolated from the cartilage by extraction at 4 °C for 48 h with 4 m guanidine-HCl, 0.05 m sodium acetate, pH 5.8, containing the protease inhibitors disodium EDTA, 6-aminohexanoic acid, phenymethanesulfonyl fluoride, and benzamidine HCl. The aggrecan monomers were isolated by equilibrium density gradient centrifugation in cesium chloride (30Hascall V.C. Sajdera S.W. J. Biol. Chem. 1969; 244: 2384-2396Abstract Full Text PDF PubMed Google Scholar) and the bottom of this gradient (d > 1.54 g/ml) containing the monomers was dialyzed at 4 °C against water using M r10,000 cutoff membranes, lyophilized, and stored at −20 °C. Aggrecan substrate was prepared by dissolving lyophilized aggrecan monomers in buffer at a concentration based on the assumption of a molecular weight of 1 × 10−6. Conditioned medium from bovine nasal cartilage stimulated with IL-1 were filtered to remove any particulate matter using a Corning 0.45-micron filter prior to use in the enzymatic assay. Medium was then incubated with purified native bovine aggrecan monomers with intact GAG chains in a final volume of 200 μl in 20 mm Tris, 100 mm NaCl, 10 mm CaCl2 buffer at pH 7.5. At the end of the incubation the reaction was quenched with 20 mm EDTA, the aggrecan was deglycosylated with chondroitinase ABC, keratanase, and keratanase II, and the aggrecanase-generated products were detected by BC-3 Western blot analysis as described above. In initial enzymatic assays, product generation was quantitated by scanning all BC-3-reactive bands. However, under the conditions used for the enzymatic assay, quantitation of the predominant 250-kDa band, which represents the initial product detected, gave equivalent results to quantitation of both this band and the minor lower molecular mass bands. Therefore, product generation was routinely assessed by quantitation of the 250-kDa band. Based on kinetic analysis, the following conditions were established for the assay: 50 μl of aggrecanase-containing medium was incubated with 500 nmaggrecan substrate in a final volume of 200 μl at 37 °C for 4 h as indicated above. pH activity profiles were performed in 50 mm MES, 100 mm NaCl, 10 mmCaCl2 buffer and pH was adjusted using 5 n HCl. Inhibitors were dissolved in dimethyl sulfoxide as a 10 mmstock and added to the reaction mixture immediately prior to enzyme addition. Dimethyl sulfoxide concentrations in the enzyme assay never exceeded 1%; this concentration of dimethyl sulfoxide had no effect on aggrecanase activity. Incubation of bovine nasal cartilage with 500 ng/ml IL-1 for 6 days, with media replaced every 2 days, induced the degradation and release of aggrecan from the cartilage (Fig.1). By monitoring the amount of GAG remaining in the cartilage at the end of the incubation, percent release of GAG from the tissue was determined at each time period; greater than 95% release of GAG was achieved by day 6. Cartilage was then incubated with IL-1 for an additional 10–12 days without media change to allow aggrecanase to accumulate in the media. To evaluate the time course of aggrecanase activity generation, samples of conditioned media were taken from cultures at various times during incubation with IL-1 and evaluated for aggrecanase activity. Incubation of this conditioned media with aggrecan substrate for 4 h resulted in the generation of fragments cleaved at the Glu373-Ala374 site as detected by BC-3 Western analysis (Fig. 2). Media quenched with EDTA prior to incubation with aggrecan served as assay blanks to control for fragments present in the media prior to enzymatic assay. Incubation of the aggrecan substrate alone did not result in the generation of BC-3-reactive fragments (data not shown).Figure 2Time course of aggrecanase generation in media from IL-1-stimulated bovine nasal cartilage. Bovine nasal cartilage slices were incubated with 500 ng/ml IL-1 for 18 days with media being replaced every 2 days for the first 6 days of culture. Samples of media taken at various times during culture were assayed for aggrecanase activity by incubating with aggrecan substrate (500 nm) for 4 h at 37 °C and evaluating products by BC-3 Western blot analysis. Media quenched with EDTA prior to incubation with substrate served as assay blanks (lanes labeledB at each time point).View Large Image Figure ViewerDownload (PPT) Although BC-3 Western blot analysis was routinely used to monitor aggrecan fragments produced by cleavage at the Glu373-Ala374 bond, initially digests generated by aggrecanase cleavage of exogenous aggrecan monomers were also analyzed by NITEGE Western blot and by biotinylated hyaluronic acid binding to confirm results with the BC-3 antibody. Time course analyses showed that the G1-NITEGE fragment was generated simultaneously with the BC-3 epitope supporting cleavage at the Glu373-Ala374 bond by aggrecanase. 2M. D. Tortorella, M. A. Pratta, and E. C. Arner, unpublished data. The band of aggrecan fragments expressing the NITEGE epitope appeared as a doublet between 64 and 70 kDa, consistent with the G1-NITEGE products previously obtained following IL-1β treatment of bovine chondrocytes (18Lark M.W. Gordy J.T. Weidner J.R. Ayala J. Kimura J.H. Williams H.R. Mumford R.A. Flannery C.R. Carisoni S.S. Iwatai M. Sandy J.D. J. Biol. Chem. 1995; 270: 2550-2556Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). High background levels of BC-3-reactive fragments produced in response to IL-1-induced degradation of the cartilage matrix were observed in media on day 2 and day 4 (Fig. 2). However, subtraction of background levels of BC-3 from the levels present following the enzymatic assay indicated that aggrecanase activity was present even at these early time periods. By day 6 of culture, BC-3-reactive fragments were no longer detected in conditioned media. Incubation of day 6 media with 500 nm aggrecan substrate for 4 h resulted in the formation of a BC-3-reactive, aggrecanase-generated product of ∼250 kDa. Aggrecanase activity accumulated in the media as the enzyme activity was monitored from days 6 to 16. In media from cultures stimulated for 10 days or greater, additional minor BC-3- reactive bands that migrated between 100 and 250 kDa were detected. In order to establish appropriate assay conditions and evaluate aggrecanase kinetic properties, the effect of incubation time, enzyme concentration, and substrate concentration on product formation were evaluated. Linearity of product formation was observed up to 4 h (Fig.3 A), and over enzyme concentrations between 25 and 80 μl of conditioned media (Fig.3 B). Aggrecanase activity appeared to approach saturation with respect to aggrecan substrate at 500–1000 nM (Fig.3 C). However, increasing viscosity precluded use of higher substrate concentrations. Aggrecanase activity was found to be stable during storage at −70, −20, or 4 °C in culture media; greater than 90% of the activity was recovered after 14 days or longer storage at −70, −20, and 4 °C. Activity was also stable to repeated freeze-thaw cycles of −70 to 4 °C. Heating at 42 °C for 15 min did not affect activity, but activity was completely lost following 15 min heating at 56 °C or above. Using the assay conditions defined above, we studied the salt and pH optimum of crude aggrecanase present in conditioned medium. Optimal activity was achieved with 100 mm NaCl. At 250 mm NaCl, activity was decreased by ∼50% and was completely lost at 500 mm NaCl or higher (Fig. 4). The pH optimum for aggrecanase was found to be at 7.5 (Fig.5). However, a rather broad pH range between 6.5 and 9.5 supported greater than 75% of the activity seen at the pH optimum.Figure 5Affect of pH on aggrecanase activity.Aggrecanase-containing media were incubated with 500 nmaggrecan substrate for 4 h in 50 mm MES, 100 mm NaCl, 10 mm CaCl2 buffer with pH adjusted using 5 n HCl. Product was monitored by BC-3 Western blot analysis and activity plotted as a percent of maximal activity versus pH.View Large Image Figure ViewerDownload (PPT) Aggrecanase activity was inhibited by the metalloprotease inhibitor, EDTA, while a panel of inhibitors of serine, cysteine, and aspartic proteinases had no effect (Table I). The ability of the endogenous inhibitor of matrix metalloproteinase, TIMP-1, to block aggrecanase activity was investigated using full-length recombinant bovine TIMP-1. TIMP-1 caused a concentration-dependent inhibition of aggrecanase activity (Fig. 6). The IC50 was estimated from the concentration-response curve to be 210 nm. However, TIMP-2 at concentrations up to 1 μm was inactive against aggrecanase (data not shown).Table IEffect of protease inhibitors on aggrecanase activityInhibitorConcentrationaProtease inhibitors were used at the
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