Title: Age-related Changes in Aggrecan Glycosylation Affect Cleavage by Aggrecanase
Abstract: Aggrecan degradation involves proteolytic cleavage of the core protein within the interglobular domain. Because aggrecan is highly glycosylated with chondroitin sulfate (CS) and keratan sulfate (KS), we investigated whether glycosylation affects digestion by aggrecanase at the Glu373–Ala374 bond. Treatment of bovine aggrecan monomers to remove CS and KS resulted in loss of cleavage at this site, suggesting that glycosaminoglycans (GAGs) play a role in cleavage at the Glu373–Ala374 bond. In contrast, MMP-3 cleavage at the Ser341–Phe342bond was not affected by glycosidase treatment of aggrecan. Removal of KS, but not CS, prevented cleavage at the Glu373–Ala374 bond. Thus, KS residues may be important for recognition of this cleavage site by aggrecanase. KS glycosylation has been observed at sites adjacent to the Glu373-Ala374 bond in steer aggrecan, but not in calf aggrecan (Barry, F. P., Rosenberg, L. C., Gaw, J. U., Gaw, J. U., Koob, T. J., and Neame, P. J. (1995) J. Biol. Chem. 270, 20516–20524). Interestingly, although we found that aggrecanase degraded both calf and steer cartilage aggrecan, the proportion of fragments generated by cleavage at the Glu373–Ala374 bond was higher in steer than in calf, consistent with our observations using aggrecan treated to remove KS. We conclude that the GAG content of aggrecan influences the specificity of aggrecanase for cleavage at the Glu373–Ala374 bond and suggest that age may be a factor in aggrecanase degradation of cartilage. Aggrecan degradation involves proteolytic cleavage of the core protein within the interglobular domain. Because aggrecan is highly glycosylated with chondroitin sulfate (CS) and keratan sulfate (KS), we investigated whether glycosylation affects digestion by aggrecanase at the Glu373–Ala374 bond. Treatment of bovine aggrecan monomers to remove CS and KS resulted in loss of cleavage at this site, suggesting that glycosaminoglycans (GAGs) play a role in cleavage at the Glu373–Ala374 bond. In contrast, MMP-3 cleavage at the Ser341–Phe342bond was not affected by glycosidase treatment of aggrecan. Removal of KS, but not CS, prevented cleavage at the Glu373–Ala374 bond. Thus, KS residues may be important for recognition of this cleavage site by aggrecanase. KS glycosylation has been observed at sites adjacent to the Glu373-Ala374 bond in steer aggrecan, but not in calf aggrecan (Barry, F. P., Rosenberg, L. C., Gaw, J. U., Gaw, J. U., Koob, T. J., and Neame, P. J. (1995) J. Biol. Chem. 270, 20516–20524). Interestingly, although we found that aggrecanase degraded both calf and steer cartilage aggrecan, the proportion of fragments generated by cleavage at the Glu373–Ala374 bond was higher in steer than in calf, consistent with our observations using aggrecan treated to remove KS. We conclude that the GAG content of aggrecan influences the specificity of aggrecanase for cleavage at the Glu373–Ala374 bond and suggest that age may be a factor in aggrecanase degradation of cartilage. glycosaminoglycan chondroitin sulfate keratan sulfate proteoglycan interglobular domain matrix metalloproteinase interleukin-1 tumor necrosis factor alpha dimethylmethylene blue bovine serum albumin Tris-buffered saline CSPG, chondroitinsulfate proteoglycan The articular cartilage matrix consists primarily of collagen, which provides strength and support, and proteoglycan, which contributes qualities of compressibility and elasticity. The major type of proteoglycan present in cartilage is aggrecan, which is composed of a protein core that contains a high level of sulfated glycosaminoglycans (GAGs),1both CS and KS. The sulfate groups of these sugar molecules impart a net negative charge to aggrecan thus providing the attractive forces that incorporate water into the matrix and endow the tissue with its shock-absorbing quality. The aggrecan core protein contains three globular domains; G1, through which the molecule binds to hyaluronic acid, G2, and 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 (96) Google Scholar). In diseased tissue, the matrix is lost, and this loss is associated with degradation of the aggrecan monomers (3Mankin HJ Lippiello L. J. Bone Jt. Surg. Am. Vol. 1970; 52: 424-434Crossref PubMed Scopus (341) Google Scholar). The interglobular domain (IGD) of the aggrecan core protein, which is located between G1 and G2, contains proteolytic cleavage sites that are believed to be critical to the overall loss of aggrecan function. Two major sites of digestion have been identified within the IGD between residues Asn341and Phe342 and between Glu373 and Ala374. The first site has been shown to be cleaved by a variety of matrix metalloproteinases (MMPs) (4Fosang 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, 5Flannery C.R. Lark M.W. Sandy J.D. J. Biol. Chem. 1992; 267: 1008-1014Abstract Full Text PDF PubMed Google Scholar, 6Fosang 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 (140) Google Scholar, 7Fosang 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, 8Fosang A.J. Last K. Knauper V. Murphy G. Neame P.J. FEBS Lett. 1996; 380: 17-20Crossref PubMed Scopus (335) Google Scholar), whereas cleavage at the second site is catalyzed by aggrecanase. The contribution of aggrecanase to aggrecan cleavage has been investigated based on the generation of products that terminate with Glu373 or begin with Ala374. Several reports have shown that the majority of the aggrecan fragments found both in vitro in response to stimulated cartilage degradation (9Sandy J.D. Boynton R.E. Flannery C.R. J. Biol. Chem. 1991; 266: 8198-8205Abstract Full Text PDF PubMed Google Scholar, 10Sandy J.D. Neame P.J. Boynton R.E. Flannery C.R. J. Biol. Chem. 1991; 266: 8683-8685Abstract Full Text PDF PubMed Google Scholar, 11Loulakis P. Shrikhande A. Davis G. Maniglia C.A. Biochem. J. 1992; 284: 589-593Crossref PubMed Scopus (122) Google Scholar, 12Ilic M.Z. Handley C.J. Robinson H.C. Mok M.T. Arch. Biochem. Biophys. 1992; 294: 115-122Crossref PubMed Scopus (166) Google Scholar, 13Lark M.W. Gordy J.T. Weidner J.R. Ayala J. Kimura J.H. Williams H.R. Mumford R.A. Flannery C.R. Carlson S.S. Iwata M. Sandy J.D. J. Biol. Chem. 1995; 270: 2550-2556Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar) and in vivo in arthritic synovial fluids (14Sandy J.D. Flannery C.R. Neame P.J. Lohmander L.S. J. Clin. Invest. 1992; 89: 1512-1516Crossref PubMed Scopus (391) Google Scholar, 15Lohmander L.S. Neame P.J. Sandy J.D. Arthritis Rheum. 1993; 36: 1214-1222Crossref PubMed Scopus (383) Google Scholar) are generated by cleavage at the aggrecanase site. Recent in vitro studies from our laboratory (16Arner E.C. Hughes C.E. Decicco C.P. Caterson B. Tortorella M.D. Osteoarthritis Cartilage. 1998; 6: 214-228Abstract Full Text PDF PubMed Scopus (127) Google Scholar) show that there is a strong correlation between specific cleavage at the Glu373–Ala374 bond and the release of aggrecan catabolites in response to cytokine stimulation. In addition, the ability of inhibitors to block the release of aggrecan catabolites correlates with their ability to block specific cleavage at the aggrecanase site. Taken together these data suggest that aggrecanase plays a key role in aggrecan degradation. Thus, identifying factors that influence the ability of aggrecanase to cleave cartilage aggrecan is important for understanding the regulation of aggrecan catabolism by this enzyme in both normal matrix turnover and in arthritic disease. Because the aggrecan core protein is heavily glycosylated, it is possible that the glycosaminoglycans on the aggrecan molecule may affect the ability of either MMPs or aggrecanase to cleave the core protein. This notion is especially intriguing based on the finding by Barry et al. (17Barry F.P. Rosenberg L.C. Gaw J.U. Gaw J.U. Koob T.J. Neame P.J. J. Biol. Chem. 1995; 270: 20516-20524Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) that glycosylation of aggrecan in articular cartilage is altered with age and thus could potentially play a role in regulating the susceptibility of aggrecan to degradation during aging. These authors reported that aggrecan isolated from steer (age of 1.5–2 years) articular cartilage is post-translationally modified with a keratan sulfate within the sequence368NITEGEAR375 whereas aggrecan from calf (age of 1 week) articular cartilage lacks glycosylation in this region. Because this sequence contains the aggrecanase cleavage site, the presence of glycosylation may play a role in regulating aggrecanase-mediated degradation. Studies by Bayliss and co-workers (18Hutton S.E. Haward J. Maciewicz R. Bayliss M.T. Trans. Orthop. Res. Soc. 1996; 21: 150Google Scholar) detected aggrecan G1 fragments with the C terminus (ITEGE), indicating that they had been cleaved at the aggrecanase site in normal adult human articular cartilage but not in immature articular cartilage, and the amount of these aggrecanase-generated fragments increased with age. These data are consistent with the possibility that aggrecanase can cleave adult aggrecan more readily than the immature aggrecan, which lacks glycosylation in the region surrounding the aggrecanase cleavage site. Therefore, we investigated the influence of glycosylation on aggrecan digestion by aggrecanase. We have recently generated soluble aggrecanase activity in media from IL-1 stimulated bovine nasal cartilage and developed an enzymatic assay using purified bovine aggrecan monomers as substrate (19Arner E.C. Pratta M.A. Trzaskos J.M. Decicco C.P. Tortorella M.D. J. Biol. Chem. 1998; 274: 6594-6601Abstract Full Text Full Text PDF Scopus (129) Google Scholar). Products were monitored by Western analysis using the monoclonal antibody, BC-3, which recognizes the N terminus of the aggrecanase-generated products with the sequence 374ARGSVIL. In this report, we demonstrate that deglycosylation of the aggrecan substrate results in total inhibition of BC-3-reactive product generation by aggrecanase, suggesting that GAGs play a role in aggrecanase-mediated cleavage at the Glu373–Ala374 bond. Further, we show that aggrecanase differs in the ability to cleave at the Glu373–Ala374 bond in steer versuscalf aggrecan, suggesting that the changes in glycosylation with age may influence the ability of aggrecan to be cleaved at the Glu373–Ala374 site by aggrecanase. Fresh bovine nasal and articular cartilage were obtained from Covance Inc., (Denver, PA). Guanidine hydrochloride was from Pierce Chemical Co. (Rockford, IL). Cesium chloride and β-mercaptoethanol were from Sigma (St. Louis, MO). Dulbecco's modified Eagle's medium, Antibiotic-Antimycotic and neomycin sulfate 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 (20Huang J.J. Newton R.C. Pezzella K. Covington M. Tamblyn T. Rutlege S.J. Gray J. Kelley M. Lin Y. Mol. Biol. Med. 1987; 4: 169-181PubMed Google Scholar). The specific activity was 1 × 107 units/mg, with 1 unit being defined as the amount of IL-1 that generated half-maximal activity in the thymocyte proliferation assay. XS309 ([3S-[3R*,2-[2R*,2-(R*,S*)]-hexahydro-2-[2-[2-(hydroxyamino)-1-methyl-2-oxo ethyl]-4-methyl-1-oxopentyl]-N-methyl-3-pyridazine-carboxamide), which is a potent nanomolar inhibitor of a number of MMPs, including MMP-1, MMP-2, MMP-3, MMP-8, and MMP-9, was synthesized at DuPont as described previously (19Arner E.C. Pratta M.A. Trzaskos J.M. Decicco C.P. Tortorella M.D. J. Biol. Chem. 1998; 274: 6594-6601Abstract Full Text Full Text PDF Scopus (129) Google Scholar, 26Xue, C-B., Cherney, R. J., DeCicco, C. P., DeGrado, W. F., He, X., Hodge, C. N., Jacobsen, I. C., Magolda, R. L., and Arner, E. C. (May 22, 1997) WO 9718207 A2.Google Scholar). GAG digestion enzymes 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 Seikagaku (Rockville, MD). SDS-polyacrylamide gel electrophoresis loading and running buffers, 4–12% gradient gels, See Blue prestained standards, and transfer buffer were purchased from NOVEX (San Diego, CA). polyvinylidene difluoride membrane was obtained from PerkinElmer Life Sciences. A goat anti-mouse IgG linked to alkaline phosphatase and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) substrate solution were from Promega (Madison, WI). Mab 2035 (mouse anti-chondroitin-6-sulfate) was obtained from Chemicon (Temecula, CA). A protease inhibitor mixture (COMPLETETM) was purchased from Roche Molecular Biochemicals (Indianapolis, IN). All other reagents that were used were of the highest quality available. Antibody BC-3 (21Hughes C.E. Caterson B. Fosang A.J. Roughley P.J. Mort J.S. Biochem. J. 1995; 305: 799-804Crossref PubMed Scopus (199) Google Scholar), which recognizes the new N terminus374ARGSVIL of the product(s) generated by aggrecanase cleavage at the Glu373–Ala374 bond within the IGD was licensed from Dr. Bruce Caterson (University of Wales, Cardiff, UK). Antibody AF-28 (22Fosang A.J. Last K. Gardiner P. Jackson D.C. Brown L. Biochem. J. 1995; 310: 337-343Crossref PubMed Scopus (85) Google Scholar), which recognizes the new N terminus342FFGVGG of the product(s) generated by cleavage at the Asn341–Phe342 bond (Ser341–Phe342 in bovine) within the IGD was a gift from Dr. Amanda Fosang (University of Melbourne, Parkville, Australia). Antibody NITEGE (13Lark M.W. Gordy J.T. Weidner J.R. Ayala J. Kimura J.H. Williams H.R. Mumford R.A. Flannery C.R. Carlson S.S. Iwata M. Sandy J.D. J. Biol. Chem. 1995; 270: 2550-2556Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar), which recognizes the new C terminus of the product(s) generated by cleavage at the Glu373–Ala374 bond was developed by Dr. Michael Lark and was a gift from Dr. Denise Visco (Merck Research Laboratories, Rahway, NJ). An anti-G1 rabbit polyclonal was a kind gift from Dr. John Sandy (Shriners Hospital for Children, Tampa, FL). A recombinant, C-terminally truncated form of stromelysin-1 (t-MMP-3), representing the catalytic domain, was cloned, expressed, and purified at DuPont (24Marcy A.I. Eiberger L.I. Harrison R. Chan H.K. Hutchinson N.I. Hagmann W.K. Cameron P.M. Boulton D.A. Hermes J.D. Biochemistry. 1991; 30: 6476-6483Crossref PubMed Scopus (99) Google Scholar). BSA, coupled to peptides containing the sequence ARGSVIL, was prepared by Quality Controlled Biochemicals (Hopkinton, MA). Articular cartilage was peeled from the metacarpalphalangeal joint from calf (<3 months) or steer (>9 months), and rinsed several times in sterile saline. The tissue was rendered non-viable by freezing on dry ice, followed by thawing at 37 °C, and this cycle was repeated at least three times. The freeze/thawed cartilage was then used as an aggrecan substrate. Aggrecan was isolated from calf (<3 months) or steer (>9 months) nasal or articular cartilage by extraction in 4 m guanidine hydrochloride and separation by cesium chloride density gradient centrifugation (25Hascall V.C. Kimura J.H. Methods Enzymol. 1982; 82: 769-800Crossref PubMed Scopus (224) Google Scholar). Gradient fractions with a density greater than 1.5 g/ml containing aggrecan monomers were dialyzed into water and lyophilized. Prior to enzymatic assay, aggrecan was resuspended in water at a concentration of 2 mg/ml (w/v). Calf and steer articular aggrecan preparations were found to be similar with respect to GAG concentration (steer: 1630 ± 71 μg/ml; calf: 1599 ± 51 μg/ml) as well as to staining in an anti-G1 Western analysis (described below). Deglycosylated aggrecan substrate was prepared by incubating bovine aggrecan monomers with chondroitinase ABC (0.1 units/10 μg), keratanase (0.1 units/10 μg), and keratanase II (0.002 units/10 μg) for 4 h at 37 °C in 100 mmTris/HCl with 100 mm sodium acetate, pH 6.5. Deglycosylation was confirmed by monitoring GAG concentration using the dimethylmethylene blue (DMMB) assay (27Farndale R.W. Sayers C.A. Barrett A.J. Connect. Tissue Res. 1992; 9: 247-248Crossref Scopus (1183) Google Scholar). Aggrecan incubated with heat-inactivated glycosidases served as a control. There was no difference in data obtained using fully glycosylated substrate, and this same substrate incubated with boiled deglycosylation enzymes (data not shown). Isolation of aggrecan core from digestion products by acetone precipitation did not alter results (data not shown), suggesting that the GAG fragments generated do not affect digestion by aggrecanase. A crude preparation of aggrecanase was generated in conditioned media from bovine nasal cartilage stimulated over a 14-day culture period with human recombinant IL-1β (500 ng/ml) (19Arner E.C. Pratta M.A. Trzaskos J.M. Decicco C.P. Tortorella M.D. J. Biol. Chem. 1998; 274: 6594-6601Abstract Full Text Full Text PDF Scopus (129) Google Scholar, 26Xue, C-B., Cherney, R. J., DeCicco, C. P., DeGrado, W. F., He, X., Hodge, C. N., Jacobsen, I. C., Magolda, R. L., and Arner, E. C. (May 22, 1997) WO 9718207 A2.Google Scholar). Because this preparation of aggrecanase also contained MMPs, which could potentially interfere with evaluation of aggrecanase activity, 0.1–1 μm XS309 (26Xue, C-B., Cherney, R. J., DeCicco, C. P., DeGrado, W. F., He, X., Hodge, C. N., Jacobsen, I. C., Magolda, R. L., and Arner, E. C. (May 22, 1997) WO 9718207 A2.Google Scholar), a potent broad-spectrum MMP inhibitor that is a weak inhibitor of aggrecanase (IC50 > 10 μm), was included during aggrecanase digestion. Aggrecanase was incubated with native or deglycosylated substrate (50 μg, 300 nm) for up to 4 h or freeze/thawed calf or steer articular cartilage (10–20 mg) for up to 48 h at 37 °C in 50 mm Tris, 100 mmNaCl, 10 mm CaCl2, pH 7.5, and the reaction was quenched with EDTA (20 mm). Aggrecan fragments released from the freeze/thawed tissue were determined colorimetrically by glycosaminoglycan assay (27Farndale R.W. Sayers C.A. Barrett A.J. Connect. Tissue Res. 1992; 9: 247-248Crossref Scopus (1183) Google Scholar). Products of the enzymatic reaction were detected by Western analysis (19Arner E.C. Pratta M.A. Trzaskos J.M. Decicco C.P. Tortorella M.D. J. Biol. Chem. 1998; 274: 6594-6601Abstract Full Text Full Text PDF Scopus (129) Google Scholar, 26Xue, C-B., Cherney, R. J., DeCicco, C. P., DeGrado, W. F., He, X., Hodge, C. N., Jacobsen, I. C., Magolda, R. L., and Arner, E. C. (May 22, 1997) WO 9718207 A2.Google Scholar). A pre-quenched sample in which aggrecanase was treated with EDTA (20 mm) prior to incubation with substrate represents background levels of the BC-3-reactive fragments present in the aggrecanase preparation. Aggrecan (either native or deglycosylated) incubated in buffer alone for up to 4 h at 37 C had similar staining to the pre-quenched sample (data not shown). In previous studies, buffer from freeze/thawed steer cartilage incubated for 0–48 h was analyzed by BC-3 and AF-28 Western analysis, as well as for GAG release (31Arner E.C. Pratta M.A. Decicco C.P. Xue C.B. Newton R.C. Trzaskos J.M. Magolda R.L. Tortorella M.D. Ann. N. Y. Acad. Sci. 1999; 878: 92-107Crossref PubMed Scopus (51) Google Scholar). Although low levels of GAG release occurred over this time period, it was likely because of non-enzymatic diffusion of aggrecan from the cut surfaces of the cartilage as no BC-3 or AF-28 reactive fragments were detected. Recombinant C-terminally truncated pro-MMP-3 was activated for 24 h at 37 °C in the presence of 1 mm 4-aminophenylmercuric acetate (APMA) and dialyzed overnight at 4 °C into 50 mm Tris, 10 mmCaCl2, 400 mm NaCl, 0.05% Brij 35, and 0.02% NaN3, pH 7.5. Aggrecan was digested with tMMP-3 as described above using 50 nm tMMP-3. Following digestion by aggrecanase or tMMP-3, aggrecan and aggrecan fragments in the reaction mixture were deglycosylated with chondroitinase ABC, keratanase, and keratanase II as described above, with the addition of a protease inhibitor mixture (COMPLETETM) based on conditions recommended by the manufacturer (1 tablet/50 ml). The mixture was shown to have no effect on the activity of the GAG digestion enzymes (data not shown). The samples were then precipitated with ice-cold acetone for 15 min, centrifuged (14000 × g) for 5 min at 4 °C, and the supernatant was removed by aspiration. The pellet was dried under nitrogen and solubilized in SDS-polyacrylamide gel electrophoresis loading buffer containing 2.5% β-mercaptoethanol. Proteins were separated by SDS-polyacrylamide gel electrophoresis using a 4–12% gradient gel and then transferred to polyvinylidene difluoride overnight at 30 V in Tris/glycine, pH 8.3, transfer buffer containing 20% methanol, which resulted in the complete transfer of both low and high molecular weight aggrecan fragments (19Arner E.C. Pratta M.A. Trzaskos J.M. Decicco C.P. Tortorella M.D. J. Biol. Chem. 1998; 274: 6594-6601Abstract Full Text Full Text PDF Scopus (129) Google Scholar). Membranes were blocked with 5% BSA and then probed with various antibodies. Membranes were probed with BC-3 antibody (0.8 μg/ml) or AF-28 antibody (1:2000) in 1% BSA/TBS, and immunoreactive proteins were detected with goat anti-mouse IgG linked to alkaline phosphatase used at a 1:5000 dilution in 1% BSA/TBS, followed by the color reagent, NBT/BCIP substrate solution. Monoclonal antibody Mab 2035 (mouse anti-chondroitin-6-sulfate), which recognizes the chondroitin sulfate stubs on the core protein following deglycosylation, was diluted 1:1000 in 1% BSA/TBS. Western analysis was performed by the procedure described above. Anti-NITEGE and anti-G1 Western analyses were performed as described previously (13Lark M.W. Gordy J.T. Weidner J.R. Ayala J. Kimura J.H. Williams H.R. Mumford R.A. Flannery C.R. Carlson S.S. Iwata M. Sandy J.D. J. Biol. Chem. 1995; 270: 2550-2556Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 29Sandy J.D. Plaas A.H.K. Koob T.J. Acta Orthop. Scand. Suppl. 1995; 266: 26-32Crossref PubMed Scopus (57) Google Scholar). The intensity of staining was quantitated by scanning densitometry. BC-3 Western blots were standardized by the inclusion of ARGSVIL-BSA protein on each blot to account for differences in color development time. Densitometry values were corrected between BC-3 Western blots based on staining of the ARGSVIL-BSA standard. Molecular mass standards are indicated in kDa. GAG levels were determined based on the amount of polyanionic material reacting with DMMB using shark chondroitin sulfate as standard (27Farndale R.W. Sayers C.A. Barrett A.J. Connect. Tissue Res. 1992; 9: 247-248Crossref Scopus (1183) Google Scholar). Aggrecanase digestion of native steer aggrecan resulted in the generation of several BC-3-reactive products (Fig. 1 A, lane 4), whereas deglycosylation of the aggrecan substrate resulted in a complete loss of BC-3 reactive fragment generation (Fig. 1 A,lane 2). However, evaluation of these same samples by CSPG Western analysis (Fig. 1 B) indicated depletion of intact aggrecan based on the loss of staining of high molecular mass proteins (>250 kDa; Fig. 1 B, lane 2). These data suggest that the deglycosylated aggrecan substrate is cleaved by aggrecanase at an alternative site. This conclusion is supported by the observation that the products formed are different when compared with the fully glycosylated substrate (Fig. 1 B, lane 4). For example, an aggrecan fragment produced upon cleavage of glycosylated aggrecan (lane 4, arrowhead a) was not detected when substrate was deglycosylated (lane 2). In contrast, a fragment produced by cleavage of deglycosylated aggrecan (lane 2, arrowhead b) was absent when glycosylated aggrecan was used. No AF-28 reactive fragments were detected with either native or deglycosylated substrate (data not shown), indicating that deglycosylation does not facilitate aggrecanase cleavage at the Ser341–Phe342 MMP site. Next, the effect of glycosylation on cleavage of bovine aggrecan at the MMP cleavage site (Ser341–Phe342) was assessed using tMMP-3. No difference in the generation of AF-28-reactive products was detected using deglycosylated as compared with fully glycosylated aggrecan (Fig. 2). Consistent with these findings, CSPG Western analysis showed no difference with deglycosylation of the aggrecan substrate (data not shown). Finally, MMP-3 did not cleave at the aggrecanase site (Glu373–Ala374) using fully glycosylated or deglycosylated aggrecan as substrate (data not shown).Figure 2Effect of aggrecan glycosylation on cleavage at the Ser341–Phe342 bond by MMP-3.Aggrecan substrates prepared as described in the legend to Fig. 1 were incubated with 50 nm t-MMP-3 for 0, 1, and 4 h at 37 °C, and products generated by cleavage at the Ser341–Phe342 bond were identified by AF-28 Western analysis. The total amount of aggrecan loaded in each lane was 0.2 μg. De-GAG represents deglycosylated substrate, andNative represents substrate treated with heat-denatured GAG digestion enzymes.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To study the individual roles of keratan sulfate and chondroitin sulfate in cleavage at the Glu373–Ala374 bond, we treated aggrecan from articular cartilage individually either with chondroitinase ABC or with keratanase and keratanase II. The use of keratanase-treated aggrecan as substrate resulted in complete loss of cleavage at the Glu373–Ala374 bond similar to fully deglycosylated aggrecan, suggesting a role for keratan sulfate in the recognition of the Glu373–Ala374 cleavage site by aggrecanase (Fig. 3 A). When chondroitinase ABC-treated aggrecan was used as substrate, products were generated by cleavage at the Glu373–Ala374 bond (Fig. 3 B), although several higher molecular mass products (>250 kDa; indicated by arrowheads at the 4-h time point) were also generated. Similar results were obtained using aggrecan isolated from nasal cartilage (data not shown). Barry et al. (17Barry F.P. Rosenberg L.C. Gaw J.U. Gaw J.U. Koob T.J. Neame P.J. J. Biol. Chem. 1995; 270: 20516-20524Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) demonstrated that aggrecan isolated from steer articular cartilage is post-translationally modified with a keratan sulfate within the sequence368NITEGEAR375 whereas calf aggrecan lacks glycosylation in this area. Because this region contains the aggrecanase cleavage site, we evaluated digestion of calf and steer cartilage by aggrecanase. Freeze-thawed articular cartilage from the metacarpalphalangeal joint of calf (<3 months) or steer (>9 months) was used as substrate for aggrecanase. Overall aggrecan cleavage as measured by total GAG release over time was similar from calf and steer articular cartilage (Fig. 4 A). Because the aggrecanase preparation also contains MMPs (19Arner E.C. Pratta M.A. Trzaskos J.M. Decicco C.P. Tortorella M.D. J. Biol. Chem. 1998; 274: 6594-6601Abstract Full Text Full Text PDF Scopus (129) Google Scholar), which could contribute to the GAG release shown in Fig. 4 A, the effect of aggrecanase digestion of calf and steer cartilage was evaluated in the presence of the MMP inhibitor, XS309 (100 nm), and results are shown in Fig. 4 B. Aggrecanase digestion of steer cartilage was not affected by the inclusion of XS309, consistent with the hypothesis that the enzyme responsible for the majority of GAG release from steer cartilage is aggrecanase. In contrast, inclusion of XS309 in the aggrecanase digestion of calf cartilage resulted in a significant decrease in GAG release suggesting that MMPs are playing a prominent role in the GAG release from calf cartilage. Evaluation of aggrecan fragments from both calf and steer cartilage in an AF-28 Western blot showed that a larger portion of the GAG release was derived from MMP digestion in calf than in steer and that the generation of the AF-28-reactive fragments was blocked by XS309 in both tissues (Fig. 4 C). Evaluation of digests by BC-3 Western analysis indicated that there was a striking difference in the amount of BC-3-reactive products generated (Fig. 4 D). Although the amount of GAG loaded in each lane was similar (5 μg/lane), the amount of BC-3 reactive products released from steer cartilage was several-fold greater than that released from calf cartilage. Thus, when BC-3-reactive products were quantitated and expressed per μg of GAG, levels at the 16-h time point were found to be at least 4-fold greater for steer than for calf cartilage. Taken together, these data suggest that both calf and steer aggrecan are digested by aggrecanase, but steer aggrecan is cleaved more efficiently at the Glu373–Ala374bond. Because the studies in Fig. 4 were performed using intact cartilage, diffusion of aggrecanase into the cartilage, as well as the release of aggrecan fragments from the cartilage, may influence the results. To eliminate the issue of diffusion and to further evaluate aggrecanase-mediated cleavage of calf and steer aggrecan, we isolated aggrecan monomers from calf and steer articular cartilage and digested them with aggrecanase. Digests were then evaluated with an antibody to the NITEGE C terminus as well as with the BC-3 antibody to the ARGSV N terminus (Fig. 5). 3- to 5-fold higher levels of BC-3-reactive fragments were produced by aggreca