©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cell-mediated Catabolism of Aggrecan
EVIDENCE THAT CLEAVAGE AT THE ``AGGRECANASE'' SITE (Glu-Ala) IS A PRIMARY EVENT IN PROTEOLYSIS OF THE INTERGLOBULAR DOMAIN (*)

(Received for publication, August 18, 1994; and in revised form, November 3, 1994)

Michael W. Lark (1) John T. Gordy (4) Jeffrey R. Weidner (1) Julia Ayala (1) James H. Kimura (3) Hollis R. Williams (1) Richard A. Mumford (1) Carl R. Flannery (4) Steven S. Carlson (2) Mineo Iwata (2) John D. Sandy (4)(§)

From the  (1)Department of Immunology and Inflammation Research, Merck Research Laboratories, Rahway, New Jersey 07065, the (2)Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98915, the (3)Henry Ford Hospital-Bone and Joint Center, Detroit, Michigan 48202, and the (4)Shriners Hospital for Crippled Children and College of Medicine, University of South Florida, Tampa, Florida 33612

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A rat chondrosarcoma cell line and primary bovine chondrocytes have been used to study cell-mediated aggrecan catabolism. Addition of 1 µM retinoic acid to chondrosarcoma cultures resulted in aggrecan proteolysis with the release of greater than 90% of the cell layer aggrecan into the medium within 4 days. NH(2)-terminal sequencing of chondroitin sulfate-substituted catabolic products gave a single major NH(2)-terminal sequence of ARGNVILTXK, initiating at Ala. This showed that the proteinase, commonly referred to as ``aggrecanase,'' which cleaves the Glu-Ala bond of the interglobular domain of aggrecan (Sandy, J. D., Neame, P. J., Boynton, R. E., and Flannery, C. R.(1990) J. Biol. Chem. 266, 8683-8685), is active in this cell system.

Aggrecan G1 domain, generated by cleavage of the interglobular domain, was also liberated during catabolism and this was characterized with three antipeptide antisera. Anti-CDAGWL was used as a general probe for G1 domain. Anti-FVDIPEN was used to specifically detect G1 domain with COOH terminus of Asn, the form which is readily generated by cleavage of aggrecan by a wide range of matrix metalloproteinases. Anti-NITEGE antiserum was used to specifically detect G1 domain with COOH terminus of Glu, the form which is the expected product of ``aggrecanase''-mediated cleavage of aggrecan. Western blot analysis indicated that a single form of G1 domain of about 60 kDa was formed. G1 domain of this size reacted with both anti-CDAGWL and anti-NITEGE but not with anti-FVDIPEN. Similar experiments with primary bovine chondrocyte cultures, treated with either retinoic acid or interleukin 1, showed that two forms of catabolic G1 domain, of about 62 and 66 kDa, were formed. Both of these forms reacted on Western blots with anti-CDAGWL and also with anti-NITEGE.

It is suggested that cell-mediated catabolism of the aggrecan interglobular domain in these culture systems, whether promoted by retinoic acid or interleukin 1, primarily involves cleavage of the Glu-Ala bond by aggrecanase. The accumulation of G1 domain with a COOH-terminal of Glu shows that such aggrecanase-mediated cleavage can occur independent of the cleavage of the Asn-Phe bond by matrix metalloproteinases.


INTRODUCTION

Aggrecan is the major osmotically active component of cartilage matrix, and it confers on the tissue a capacity to resist compression under load. The aggrecan core protein has two globular domains near the amino terminus which are referred to as G1 and G2 and these are connected by a proteolytically sensitive segment known as the interglobular domain (IGD). (^1)The ternary complex formed by the interaction of the G1 domain with hyaluronan and link protein anchors the aggrecan within the tissue. Accelerated catabolism of matrix components in articular cartilage appears to be a hallmark of cartilage loss in joint diseases such as osteoarthritis; however, the specific proteinase(s) responsible for aggrecan catabolism have yet to be conclusively identified(1) .

Analysis of aggrecan fragments from a range of experimental systems has recently indicated that a novel proteinase (``aggrecanase''), which cleaves the Glu-Ala (see Footnote 2) bond of the IGD of aggrecan, plays a central role in the catabolic process. CS-substituted catabolic products which initiate at Ala are released from articular cartilage explants treated with RA or IL-1(2, 3, 4) , growth plate explants treated with RA (5) , and are present in human synovial fluids from patients with both osteoarthritis (6) and inflammatory joint diseases(7) . However, attempts to generate such cleavage products with a range of purified proteinases (8, 9, 10, 11) and with subcellular fractions from chondrocytes (8) have been unsuccessful, so that the identity of this activity remains obscure. The finding that aggrecan catabolism in cartilage explants can be slowed by addition of specific inhibitors of both cysteine and metalloproteinases has however lead to the suggestion that the activity forms part of a complex proteolytic cascade responsible for aggrecan catabolism in vivo(12) .

In all of these studies, evidence for this activity has been obtained by isolation of high molecular weight CS-bearing core protein products and detection of the new NH(2) terminus of Ala by automated sequence analysis. This approach has, however, not clarified the precise role of this proteinase in chondrocyte-mediated aggrecan catabolism. For example, it is not known whether cleavage of the Glu-Ala bond alone is sufficient for catabolism of the aggrecan IGD by chondrocytes in situ. This is an important point, since inhibitor studies (12) have suggested that a proteolytic cascade may be involved. In addition, many matrix metalloproteinases (MMP-1, -2, -3, -7, -8, and -9) have been shown to cleave the IGD at the Asn-Phe bond (8, 9, 10, 11) , which is 32 residues NH(2)-terminal of the aggrecanase site, and the G1 domain generated by this cleavage has been isolated from human cartilage(10) . Moreover, evidence has recently been presented to show that although neutrophil collagenase (MMP-8) preferentially cleaves the Asn -Phe bond (9) , cleavage of the Glu-Ala bond also occurs following prolonged incubation of an aggrecan G1-G2 fragment with high concentrations of MMP-8(13) .

In order to understand the precise cellular mechanisms of aggrecan catabolism, it is therefore critical to determine whether one or both of these cleavages, or indeed any other cleavages, occur in a cell-dependent catabolic system. Clarification of this issue can be obtained by examining the structure of the G1 domain liberated in this process. Thus, if the activity referred to as aggrecanase can act alone, and cleaves only the Glu-Ala bond, then the G1 domain generated should bear a COOH terminus of Glu and nonaggregating fragments should bear an NH(2)-terminal of Ala. On the other hand, if proteolysis at site(s) other than the aggrecanase site also occur, such as the Asn-Phe bond, then other separable forms of G1 domain should be generated. Therefore, in the present study we have used a chondrosarcoma culture system(14) , and primary bovine chondrocyte cultures, in which aggrecan catabolism is accompanied by the liberation of G1 domain. The nonaggregating fragments have been analyzed by NH(2)-terminal analysis and the structure of the G1 domain has been examined by Western analysis with antisera which are specific for a COOH terminus of either Asn or Glu.


EXPERIMENTAL PROCEDURES

All-trans-retinoic acid and insulin (from bovine pancreas) were from Sigma. Dulbecco's modified Eagle's medium culture medium (Cellgro) was from Mediatech, Washington, DC Fetal bovine serum (FBS) was from HyClone, Logan, UT. Human recombinant IL-1alpha was from Genzyme, Boston, MA. Goat-anti-rabbit IgG (peroxidase-conjugated) was from Calbiochem Corp. The ECL detection system, biotinylated molecular weight markers, and streptavidin-horseradish peroxidase conjugate were from Amersham Corp. and were used according to the manufacturers instructions. Materials and procedures used for the isolation, deglycosylation, and NH(2)-terminal sequencing of aggrecan fragments were as described previously(2, 6) . Aggrecan was assayed as CS equivalents by dimethylmethylene blue (15) with shark chondroitin sulfate as standard. Protein was assayed by the bicinchoninic acid kit from Pierce, with bovine serum albumin as standard. Purified samples of bovine G1 domain (Val^1-Arg) and link protein were a gift from Dr. L. Rosenberg, Montefiore Hospital, New York. Antibody 8-A-4 was obtained from the Developmental Studies Hybridoma Bank, Baltimore, MD.

Chondrocyte Culture

Cells from a rat chondrosarcoma cell line termed LTC (14) were plated at 1-2 times 10^6 cells/35-mm dish in 2 ml of medium and were maintained for 3-4 days in Dulbecco's modified Eagle's medium, 10% FBS, 25 µg/ml ascorbate (medium A) to establish an aggrecan-rich matrix. For catabolic experiments, cells were switched to medium A without ascorbate and 1 µM RA was added with daily medium changes. RA (1 mM stock in Me(2)SO) was added to achieve 1 µM in a final Me(2)SO concentration of 0.1% (v/v). Control cultures received Me(2)SO alone at this concentration. For experiments in which unfractionated medium was analyzed by SDS-PAGE, medium A was modified to contain 0.5% (v/v) FBS, 0.1 µg/ml insulin, 1 µM RA. In control experiments it was shown that the catabolic response to 1 µM RA in the modified medium was similar to that in medium with 10% FBS. Calf articular chondrocytes were maintained in Ham's F-12, 10% FBS, 25 µg/ml ascorbate for 3-7 days as described (8) and for catabolic experiments cells were switched to this medium with 1 µM RA or 50 units/ml IL-1alpha added with daily medium changes for 4 days.

Isolation of Aggrecan Fragments

Aggrecan core species and link protein were prepared from cell layers by digestion of the layer with chondroitinase ABC (0.1 unit/mg CS) in 50 mM Tris acetate, 10 mM EDTA, pH 7.6, for 1 h at 37 °C, followed by boiling for 5 min in 0.5% SDS, 20 mM dithiothreitol. Insoluble material was removed by centrifugation.

Aggrecan fragments present in medium samples were prepared for analysis by three methods. For NH(2)-terminal sequencing, D1 samples were prepared and processed as described previously(2) . For SDS-PAGE analysis of unfractionated medium, 25-µl portions containing about 5 µg of CS were dried, dissolved in 25 µl of 50 mM Tris, 50 mM sodium acetate, 10 mM EDTA, pH 7.6, and digested with chondroitinase ABC (0.1 unit/mg CS) for 1 h at 37 °C. Alternatively, medium samples containing about 1 mg of CS were taken for CsCl gradient centrifugation under associative conditions (starting density 1.45 g/ml), and the A1 through A4 fractions were dialyzed sequentially against water, 1 M NaCl/50 mM Tris acetate, pH 7.6, and water. The A1 fraction contained greater than 95% of the recovered aggrecan in each case. For SDS-PAGE, portions were deglycosylated with chondroitinase ABC and keratanase as described (2) and in some cases with stromelysin (MMP-3) as described(10) .

SDS-PAGE and Western Blotting

Deglycosylated samples (about 3 µg of protein) were electrophoresed for 90 min at 125 V on precast Tris/glycine 4-12% gradient gels from Novex Corp., San Diego, CA. Electroblotting onto nitrocellulose was for 80 min at 30 V using an XCELL transfer cell from Novex. Membranes were blocked with 1% (w/v) bovine serum albumin/Tris-buffered saline (0.5 M NaCl, 20 mM Tris, pH 7.5) for 16 h at 4 °C. Both first antibody (1:1000 of anti-CDAGWL or anti-NITEGE and 1:3000 of anti-VDIPEN) and second antibody (1:5000 of peroxidase-conjugated goat-anti-rabbit IgG) were diluted in 1% (w/v) bovine serum albumin/Tris-buffered saline and applied for 1 h. Wash cycles (3 times 10 min) were done with Tris-buffered saline, 0.05% (v/v) Tween 20, and ECL detection was with Kodak X-Omat film.

Peptide Synthesis, Purification, and Characterization

Peptides were synthesized via the Merrifield solid-phase technique on an Applied Biosystems 430A peptide synthesizer using standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) protected amino acids and the manufacturers suggested protocols for (2-(1-H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate)-mediated couplings on p-benzyloxybenzyl alcohol resins. Peptides were simultaneously deprotected and cleaved from the resin with 90% trifluoroacetic acid, 5% thioanisole, 3% ethanedithiol, 2% anisole at room temperature for 2 h. Crude peptides were precipitated with ethyl ether then dissolved in 10% acetic acid and lyophilized. The resulting crude peptides were purified by reversed phase HPLC on Waters C18 Deltapak columns with a 45-min gradient of 5-50% acetonitrile in aqueous 0.1% trifluoroacetic acid. Purity of the peptides was assessed by reversed phase HPLC on Brownlee Spheri-5 ODS column with a 45-min gradient of 5-50% acetonitrile in aqueous 0.1% trifluoroacetic acid. All peptides were >95% pure. Molecular ions were obtained by electrospray ionization-mass spectroscopy to confirm the structure of each peptide.

Radioiodination of Peptide

Peptide solution (50 µl of 220 µg/ml YPLPRNITEGE in phosphate-buffered saline) was added to 10 µl of 0.5 M potassium phosphate, pH 7.5, followed by 2 mCi of NaI and 10 µl of chloramine T (0.1 mg/ml in water). The reaction is stopped after 30 s with 10 µl of sodium iodide/sodium thiosulfate (1 mg/ml each in water). The labeled peptide was purified by reversed phase HPLC on a Supelco C-8 column (0.4 times 25 cm) with an acetonitrile gradient in 0.1% trifluoroacetic acid to isolate the monoiodinated species which had a specific activity of about 1500 Ci/mmol.

Radioimmunoassay Procedure

All assays were performed in a total of 300 µl of Dulbecco's calcium/magnesium-free phosphate-buffered saline supplemented with 0.1% (w/v) gelatin, 0.01% (w/v) thimerasol, and 1 mM EDTA. 100 µl of buffer (control) or competing peptide sample (test) was mixed with 100 µl of a 1:3000 dilution of the anti-NITEGE antiserum and incubated for 24 h at 4 °C. Iodinated peptide (30,000 cpm of I-YPLPRNITEGE) was then added to each sample and allowed to bind to antibody at 4 °C for 24 h. Unbound radiolabel was then removed by the addition of 1 ml of 0.3% (w/v) dextran-coated charcoal followed by incubation at 0 °C for 10 min. The sample was clarified by centrifugation at 3000 times g for 10 min and the supernatant counted to measure the antibody-bound probe. Data were plotted as a ratio of I-YPLPRNITEGE bound to antibody (test/control times 100) against the concentration (nM) of unlabeled competing peptide added to the assay.

Preparation of Peptide-specific Antisera

Anti-NITEGE serum was prepared as follows. The 6 residue carboxyl-terminal sequence of the G1 species generated by cleavage of the Glu-Ala bond of both the rat and human aggrecan core proteins is NITEGE(16, 17) . This peptide was synthesized with Cys-Nle at the amino terminus to give the peptide CNleNITEGE. The cysteine was added as a linker to attach the peptide to the immunogenic carrier, bovine thyroglobulin, by means of the heterobifunctional cross-linker, n-maleimidobenzoyl-N-hydroxysuccinimide ester (18) , as described(19) . The Nle was added as an internal marker to quantify the mol of peptide cross-linked to the carrier protein. New Zealand White rabbits were injected as described (18) to generate the anti-NITEGE serum and serum batch 1778 was used throughout this study.

Anti-FVDIPEN serum was prepared similarly but using the peptide conjugate thyroglobulin-Cys-NLeu-FVDIPEN(19) . The peptide FVDIPEN corresponds to the 7-residue carboxyl-terminal sequence of the G1 species generated by cleavage of the Asn-Phe bond of both the rat and human aggrecan core proteins(16, 17) . Anti-FVDIPEN serum batch 1776 was used throughout this study.

Anti-CDAGWL antiserum (previously termed anti-HAL) was raised against a keyhole limpet hemocyanin conjugate of the aggrecan G1 peptide (CDAGWLADQTVRYPI) which corresponds to residues 180 through 194 of both the human and rat sequences (16, 17) and was affinity-purified for use as described previously (20) .


RESULTS

Characterization of Antisera and Aggrecan Core Species

The reactivity and specificity of anti-CDAGWL, anti-NITEGE, and anti-FVDIPEN were examined by Western analysis of different aggrecan preparations from the Swarm rat chondrosarcoma tumor (Fig. 1). Anti-CDAGWL detected at least six separable species at about 350, 250, 180, 60, 55, and 46 kDa in an A1 fraction (lane 1). The high molecular weight species presumably represent G1-bearing rat aggrecan core proteins with different COOH termini, and this is supported by their presence in the A1D1 fraction (lane 4). Identification of the 46-kDa band as link protein was made on the basis of reactivity with antibody 8-A-4 at this position on a parallel blot (not shown). The size and anti-CDAGWL reactivity of the 60-kDa and 55-kDa species (lane 1) suggested that these were rat aggrecan G1 domain species with different COOH termini (see Fig. 2for schematic). Thus, when the same material was probed with anti-NITEGE, designed to react specifically with G1 bearing a COOH-terminal of Glu, the 60-kDa form alone was detected (lane 2). Likewise when this material was probed with anti-FVDIPEN, designed to react specifically with G1 with a COOH-terminal of Asn, the 55-kDa form alone was detected (lane 3).


Figure 1: Specificity of anti-CDAGWL, anti-FVDIPEN, and anti-NITEGE. Aggrecan preparations from the Swarm rat chondrosarcoma tumor were chondroitinase digested, electrophoresed on a 4-12% gradient gel, and blotted to nitrocellulose. The sample analyzed and the antiserum used for each lane were as follows: lane 1, A1 fraction, anti-CDAGWL; lane 2, A1 fraction, anti-NITEGE; lane 3, A1 fraction, anti-FVDIPEN; lane 4, A1D1 fraction, anti-CDAGWL; lane 5, A1D1 fraction, MMP-3-digested, anti-CDAGWL; lane 6, A1D4 fraction, anti-CDAGWL; lane 7, A1D4 fraction, MMP-3-digested, anti-CDAGWL.




Figure 2: Schematic of G1 domain species under investigation. The structure of the 55-kDa form of G1 (Glu^1-Asn) detected specifically by the anti-FVDIPEN serum, and the 60-kDa form of G1 (Glu^1-Glu) detected specifically by the anti-NITEGE serum are shown in relation to the position of the disulfide-bonded loops of the aggrecan G1 domain.



The reactivity of both forms of G1 with anti-CDAGWL was supported by studies with MMP-3 which cleaves the Asn-Phe bond and so converts all core species, including the 60-kDa G1 into the 55-kDa G1. Thus when rat A1D1 (lane 4) was treated with MMP-3, all high molecular weight species disappeared and the only detectable product was the 55 kDa G1 (lane 5). Likewise, when rat A1D4 (lane 6) was treated with MMP-3, high molecular weight species including the 60-kDa G1 disappeared, and the amount of 55-kDa G1 present was markedly increased (lane 7). Reactivity of anti-CDAGWL with these multiple size forms of aggrecan core (lane 1) is predicted from the presence of four regions of aggrecan (residues 180 through 194 and 278 through 292 in the G1 domain; residues 514 through 528 and 617 through 631 in the G2 domain) which show high sequence similarity to the immunizing peptide (residues 180 through 194 in the G1 domain). Reactivity of anti-CDAGWL with link protein (lanes 1, 6, and 7) is predicted from the presence of two regions of link protein (residues 259 through 273 and 358 through 372) which show high sequence similarity (21) to the immunizing peptide.

To examine the peptide structure required for optimal reactivity with anti-NITEGE, a radioimmunoassay was established using radioiodinated YPLPRNITEGE. The assay detected the unlabeled form of this peptide with a log-linear range of detection of about 0.3-8 nM. Next, a series of COOH-terminal truncation peptides including YPLPRNITEG, YPLPRNITE, YPLPRNIT, and YPLPRNI were prepared and assayed (Fig. 3A). Consistent with the peptide orientation of the immunogen, the anti-NITEGE antiserum clearly possessed a very high degree of specificity for the COOH-terminal Glu residue. Thus, when the COOH-terminal Glu was truncated to generate YPLPRNITEG, there was a greater than 10,000-fold loss in recognition by the antiserum. Indeed, none of the truncated peptides at concentrations up to about 500 nM were recognized by the antiserum, confirming the very strong requirement for the Glu residue at the carboxyl terminus of the NITEGE sequence. Thus YPLPRNITE was not recognized by the antiserum at any concentration tested, even though this closely related peptide contains a COOH-terminal Glu residue. The apparent immunoreactivity of YPLPRNIT at concentrations above 1000 nM was not further investigated. The effect of extension of the peptide with aggrecan core sequence beyond the carboxyl-terminal Glu residue was also examined with the peptides YPLPRNITEGEA, YPLPRNITEGEARG, and YPLPRNITEGEARGS (Fig. 3B). In each case there was at least a 1000-fold reduction in reactivity with the antiserum. When taken together, these data indicate that the COOH-terminal Glu residue bearing an unsubstituted alpha-carboxyl group is required for optimal recognition by the antiserum and that proteases cleaving NH(2)- or COOH-terminal to the Glu-Ala bond of the aggrecan IGD will generate fragments recognized 100-10,000-fold less well than those terminating in Glu. To further examine the importance of the carboxyl-terminal Glu residue, the effect of COOH-terminal substitutions on antiserum reactivity was also determined (data not shown). When the COOH-terminal L-Glu was replaced with the stereoisomer D-Glu, the amide of glutamic acid, glutamine, aspartic acid, or asparagine, the antiserum also recognized the peptides with markedly reduced sensitivity. The reduction in sensitivity was very marked (about 10,000-fold) on substitution with asparagine or aspartic acid, but somewhat less marked (100-1000-fold) on substitution with the glutamate analogues.


Figure 3: Peptide specificity of anti-NITEGE: the effect of carboxyl-terminal truncations and extensions. Peptides with COOH-terminal truncations and extensions were compared for their ability to compete with I-YPLPRNITEGE for antibody. Competition curves are shown in A (top) for the control peptide YPLPRNITEGE () and the peptides YPLPRNITEG (box), YPLPRNITE (bullet), YPLPRNIT (circle), and YPLPRNI (). Competition curves are shown in B (bottom) for the control peptide YPLPRNITEGE () and the peptides YPLPRNITEGEA (box),YPLPRNITEGEARG (bullet), and YPLPRNITEGEARGS (circle). Assay details and calculations are given under ``Experimental Procedures.''



The specificity of anti-FVDIPEN was examined in a similar set of experiments with COOH-terminal truncations extensions and modifications of the peptide YTGEDFVDIPEN(19) . These results indicated that the COOH-terminal Asn residue bearing an unsubstituted carboxyl group is required for optimal recognition by the antiserum and that proteases cleaving NH(2)- or COOH-terminal of the Asn-Phe bond of the aggrecan IGD will generate fragments recognized 40-10,000-fold less well than those terminating in Asn. Consistent with this high specificity for the respective COOH-terminal residues with an unsubstituted carboxyl group, neither anti-NITEGE nor anti-FVDIPEN antisera showed reactivity on Western blots with those forms of rat aggrecan core protein which are known to contain these sequences internally (see Fig. 1and Fig. 6-8).


Figure 6: Western blot of chondrosarcoma medium products with anti-NITEGE. Medium A1 samples from a CsCl gradient (see ``Experimental Procedures'') were prepared from cultures before RA addition (control) and after different periods of treatment (days 1-4). Portions were chondroitinase digested, electrophoresed on 4-12% gradient gels, and blotted to nitrocellulose for detection with anti-NITEGE. See text for description.



Aggrecan Catabolism by Rat Chondrosarcoma Cell Cultures

Each chondrosarcoma cell culture deposited about 0.85 mg of aggrecan in the cell layer matrix during the first 3-4 days of culture. Addition of 1 µM retinoic acid resulted in essentially complete inhibition of aggrecan synthesis and the release of greater than 90% of the preformed aggrecan into the medium over the following 4 days. In a typical experiment the cumulative product release for days 1, 2, 3, and 4 was 6.1, 21.8, 81.8, and 94.2%, respectively, of the total aggrecan present in the culture. The majority of the released aggrecan had been proteolytically degraded as indicated by hyaluronan aggregation studies; in one such experiment the product released on days 1, 2, and 3 after RA treatment was 56, 73, and 77% nonaggregating, respectively. This was in marked contrast to the medium product collected before RA addition which contained less than 20% nonaggregating material. In all samples the nonaggregating fragments were similar in size and eluted with a peak K of about 0.5 on Sepharose CL-2B, consistent with limited proteolysis of core protein.

To examine the structure of the fragments, aggrecan was prepared from a day 3 medium sample which contained the bulk of the released product. NH(2)-terminal analysis showed a major sequence of ARGNVILTXK with a minor sequence of EEVPXXXNS. The major sequence, which initiates at Ala, showed that aggrecan catabolism in this system clearly involves cleavage of aggrecan at the Glu-Ala bond of the aggrecan interglobular domain. Furthermore, the yield of the Ala NH(2) terminus (about 0.3 mol of NH(2) terminus/mol of core) was similar to that obtained from both bovine (2) and human (7) fragment preparations in which this product would appear to account for a high percentage of the degraded core protein present. Detection of the minor sequence, which initiates at Glu^1, was consistent with the presence of about 20% aggregatable species which would be expected to contain intact G1 domain and so initiate at the native NH(2)-terminal of Glu^1. Additionally, in two experiments with chondrocytes prepared from freshly excised Swarm rat chondrosarcoma tumor (details not shown), it was found that aggrecan catabolism could be induced by RA treatment and also that the major CS-bearing catabolic products released into the medium initiated at Ala.

Characterization of the G1 Domain Generated during Aggrecan Catabolism

Since aggrecan catabolism in these cultures clearly involved cleavage of the IGD, it seemed likely that G1 domain would be generated during this process and that this might be detectable on Western blots with the G1-reactive antisera described above. To investigate this, the structure of the aggrecan core species present in the cell layer and medium compartments both before catabolism and after different periods of RA treatment was examined with anti-CDAGWL. In control cell layers (Fig. 4) two major immunoreactive species were detected, a high molecular weight species (at about 350 kDa) which presumably represents intact aggrecan core protein and a sharp band at 46 kDa which was identified as link protein on the basis of size and strong immunoreactivity on Western blot with antibody 8-A-4 (data not shown). During RA-induced catabolism the high molecular weight core was progressively depleted from the cell layer and two new immunoreactive species (at about 250 and 60 kDa) appeared on day 2 and remained detectable in the cell layer until day 5. The properties of this 60-kDa species were consistent with the catabolic form of aggrecan G1 domain shown in Fig. 2.


Figure 4: Western blot of chondrosarcoma cell layer products with anti-CDAGWL. Cell layer extracts (see ``Experimental Procedures'') prepared from cultures before RA addition (control) and after different periods of treatment (days 1-5) were electrophoresed on 4-12% gradient gels and blotted to nitrocellulose for detection with anti-CDAGWL. See text for description.



When the corresponding medium samples were fractionated on a CsCl gradient and the A1 fractions were analyzed (Fig. 5) a very similar pattern of products to that seen in the cell layer was observed. In addition to the 350-kDa species, prominent immunoreactive species were also seen at about 250 and 125 kDa, and these probably represent aggrecan core species which have been generated from the 350-kDa species by cleavage within the CS attachment region. The precise structure of these products and the identity of the proteinase(s) involved in their generation was not investigated. In these medium samples, however, the major new immunoreactive species generated during the catabolic process was clearly the 60-kDa species (see days 2-4, Fig. 5). The size and immunoreactivity of this 60-kDa species, found in both the medium A1 samples (Fig. 5) and cell layer (Fig. 4), suggested that it was the discrete form of G1 domain (Fig. 2) liberated during RA-induced catabolism by these cells.


Figure 5: Western blot of chondrosarcoma medium products with anti-CDAGWL. Medium A1 samples from a CsCl gradient (see ``Experimental Procedures'') were prepared from cultures before RA addition (control) and after different periods of treatment (days 1-4). Portions were chondroitinase-digested, electrophoresed on 4-12% gradient gels, and blotted to nitrocellulose for detection with anti-CDAGWL. See text for description.



To characterize this material further the same medium A1 samples were electrophoresed and probed with anti-NITEGE (Fig. 6); a highly specific and strong reaction was also obtained with a 60-kDa species present in the day 2, 3, and 4 samples only. This suggested that the 60-kDa species detected by anti-CDAGWL ( Fig. 4and Fig. 5) was, in fact, G1 domain with a COOH terminus of Glu which therefore had been generated by aggrecanase-mediated cleavage of the Glu-Ala bond.

Further evidence to support the identification of G1 domain with a COOH terminus of Glu was obtained as follows. First, reactivity with anti-NITEGE (Fig. 7, lane 1) was completely eliminated (Fig. 7, lane 2) when the medium A1 sample was digested with MMP-3 before SDS-PAGE to remove the COOH-terminal extension of Phe-Glu (see Fig. 2for schematic). Second, reactivity with anti-NITEGE was completely blocked (Fig. 7, lane 3) by preincubation of the serum with 10 µg/ml of the immunizing peptide (YLPLPRNITEGE), but it was unaffected (Fig. 7, lane 4) by preincubation with the same concentration of the peptide (YLPLPRNITEGEARGS) which spans the cleavage site.


Figure 7: Western blot identification of chondrosarcoma medium products with specific antisera. Chondroitinase-digested products were electrophoresed on 4-12% gradient gels and blotted to nitrocellulose for detection with one of the three anti-peptide sera. The sample analyzed and the antiserum used for each lane were as follows: lane 1, day 3 medium, A1 fraction, anti-NITEGE; lane 2, day 3 medium, A1 fraction, MMP-3-digested, anti-NITEGE; lane 3, day 2 medium, A1 fraction, anti-NITEGE preadsorbed with YLPLPRNITEGE; lane 4, day 2 medium, A1 fraction, anti-NITEGE preadsorbed with YLPLPRNITEGEARGS; lane 5, day 3 medium, A1 fraction, anti-FVDIPEN; lane 6, day 3 medium, A1 fraction, MMP-3-digested, anti-FVDIPEN; lane 7, unfractionated day 3 medium, anti-CDAGWL.



Next, to examine these samples for the possible presence of G1 domain with a COOH terminus of Asn (see Fig. 2), Western blots of the same A1 preparations were probed with anti-FVDIPEN (Fig. 7, lane 5). In none of the medium A1 samples was immunoreactive protein detected. This absence of reactivity was not however due to poor sensitivity of detection with this antiserum, since when the same amount of A1 sample was digested with MMP-3 before SDS-PAGE, a single strongly immunoreactive product of about 55 kDa was detected in all samples with anti-FVDIPEN (Fig. 7, lane 6). Furthermore, this product was also detected by this method in A1 and A1D4 samples of rat chondrosarcoma tumor extracts (Fig. 1), and it therefore clearly represents the 55-kDa G1 with a COOH terminus of Asn shown schematically in Fig. 2. Finally, when unfractionated catabolic medium was electrophoresed and probed with anti-CDAGWL (Fig. 7, lane 7), the only G1 domain detected corresponded to the 60-kDa species, and there was no evidence for the presence of the 55-kDa species. This clearly indicated that the 60-kDa G1 species which was isolated in the medium A1 fractions ( Fig. 5and Fig. 6), and which was also apparently reactive with anti-NITEGE (Fig. 7), appeared to be the only G1 species released into the medium during RA-induced catabolism in this system.

The amount of 60-kDa G1 present in these samples was estimated by standardization of the anti-CDAGWL Western blot with variable loadings (0.1-1.0 µg) of purified bovine G1 domain (not shown). This visual comparison indicated that the amount of G1 present in the medium A1 samples run (Fig. 5) was about 1 µg. Since these A1 samples contained an estimated total of 3 µg of nonaggregating core protein, it appears that a major proportion of the G1 domain product expected to be generated by this catabolic process was indeed present in the culture medium.

Aggrecan Catabolism in Bovine Chondrocyte Cultures

To investigate the extent to which the same pattern of IGD cleavage occurs during the catabolism of aggrecan by primary chondrocytes, calf articular chondrocytes were grown until the cell layers contained about 1.2 mg of aggrecan, and catabolism was induced by addition of either RA or IL-1alpha. In a typical experiment daily cumulative release figures for RA-treated cultures were 56, 83, 91, and 95% of the total aggrecan on days 1, 2, 3, and 4 respectively. The equivalent figures for IL-1-treated cultures were 36, 55, 70, and 82%. Aggrecan fragments were prepared from the medium of RA-treated chondrocytes and on NH(2)-terminal analysis a major sequence of ARGXVIL was obtained, showing that cleavage of the Glu-Ala bond by aggrecanase is also a major catabolic pathway in these cultures. This is consistent with NH(2)-terminal analysis of fragments released from both IL-1-treated and RA-treated bovine cartilage explants (2, 5

To examine the structure of the G1 domain generated in these cultures, A1 preparations were made from the collected medium of control, RA-treated, and IL-1-treated cells and portions were taken for Western blot analysis with both anti-CDAGWL and anti-NITEGE (Fig. 6). In control medium (lane 1), the major components detected with anti-CDAGWL were two high molecular core species, at about 250 and 350 kDa, and a link protein doublet at about 46 kDa. Predictably, none of these species reacted with anti-NITEGE (lane 2). In the medium from RA-treated cells (lane 3), two new prominent species, at about 62 and 66 kDa were detected with anti-CDAGWL. Both of these species also appeared to react specifically and to the same relative intensity with anti-NITEGE (lane 4). The immunoreactivity patterns obtained with the medium from IL-1-treated cells (lanes 5 and 6) could not be distinguished from those obtained with the RA-treated cells.

It therefore appears that with primary bovine chondrocytes, under either RA or IL-1 treatment, the major G1 domain generated has a COOH-terminal of Glu, and it is therefore also a specific product of aggrecanase-mediated cleavage of the IGD. Interestingly, with bovine chondrocytes this catabolic G1 domain is itself generated in two discrete forms. Although the source of this heterogeneity has not been investigated here, it seems likely that it is a result of variable carbohydrate substitution, which is an established feature of bovine G1 domain structure. (^3)


DISCUSSION

The results presented clearly indicate that cleavage of the Glu-Ala bond is the primary proteolytic event in catabolism of the interglobular domain of aggrecan by both chondrosarcoma cells and primary bovine chondrocytes. This follows from the observation that G1 domain(s) with a COOH terminus of Glu and CS-bearing fragments with NH(2)-terminal Ala are the major catabolic products which can be detected in these systems. Furthermore, since products from both sides of the Glu-Ala bond have now been identified in two separate culture systems, it can be concluded that the activity responsible (aggrecanase) is an endopeptidase.

Identification of the 60-kDa product of the chondrosarcoma cell line as aggrecan G1 was based on the following observations. First, the protein appeared on day 2 of RA treatment in both the cell layer (Fig. 4) and medium (Fig. 5) and was therefore generated along with the bulk of the high molecular weight nonaggregating core species identified by Sepharose CL-2B chromatography. Second, the protein showed very similar electrophoretic properties to keratanase-treated bovine hyaluronan binding region (Val^1-Arg) which is detected on Western blots with anti-CDAGWL as a doublet in the 55-60 kDa range (not shown). Third, the protein could be isolated in the A1 fraction from a CsCl gradient fractionation of culture medium, consistent with the hyaluronan-binding properties of G1 domain.

The 60-kDa rat G1 detected by anti-CDAGWL (Fig. 5) appeared to be the same species detected by anti-NITEGE (Fig. 6), since the electrophoretic properties and relative abundance between samples of the material detected by the two antisera appeared to be identical. By the same reasoning, the two G1 species generated by bovine chondrocytes and detected by the anti-CDAGWL serum (Fig. 8, lanes 3 and 5) appeared to be the same species detected by the anti-NITEGE serum (Fig. 8, lanes 4 and 6). If this interpretation is correct, it would follow that all of the detectable G1 domain generated in these systems is a product of aggrecanase action.


Figure 8: Western blot identification of bovine chondrocyte medium products with specific antisera. Deglycosylated A1 medium aggrecan samples (in each case, day 3 plus day 4 combined) were electrophoresed on 4-12% gradient gels and blotted to nitrocellulose for detection with different antisera. The sample analyzed and the antiserum used for each lane were as follows: lane 1, control medium, anti-CDAGWL; lane 2, control medium, anti-NITEGE; lane 3, RA medium, anti-CDAGWL; lane 4, RA medium, anti-NITEGE; lane 5, IL-1 medium, anti-CDAGWL; lane 6, IL-1 medium, anti-NITEGE.



On the other hand, since the present data are qualitative only, it cannot be entirely excluded that a proportion of the G1 species detected by the anti-CDAGWL serum have a COOH-terminal other than Glu 373. Such a ``hidden'' product would necessarily have a COOH-terminal in close proximity to Glu, since this electrophoretic system ( Fig. 1and Fig. 7) can readily separate the 60- and 55-kDa forms of G1 domain which differ in size by only 32 amino acid residues (see Fig. 2). A definitive conclusion on this point will require the development of standardized immunoassays for G1 with the different antibodies used here.

The presence of aggrecanase-generated G1 in A1 fractions (density > 1.56 g/ml) of culture medium ( Fig. 6and Fig. 8) suggests that this protein is released from cell layers bound to hyaluronan in aggregate structures which contain one or more high buoyant density aggrecan molecules. This implies, much as previously indicated with IL-1-treated cartilage explants(22) , that aggrecanase-mediated cleavage of the IGD does not markedly interfere with the hyaluronan-binding properties of the G1 domain. Whereas aggregate turnover in steady-state cartilage explants appears to involve a co-ordinated process involving the removal of hyaluronan and release of aggrecan chondroitin sulfate(23) , the fate of the G1 domain generated under steady-state conditions is unknown. Since chondrocytes can exhibit receptor-mediated uptake of hyaluronan(24) , it seems possible that hyaluronan, along with catabolic G1 domain, might be endocytosed under steady state. The present study however suggests that in cell culture systems where rapid aggrecan catabolism has been induced with RA or IL-1, a sizable proportion of the catabolic G1 domain is released, along with the CS-bearing products, into the culture medium. The role of hyaluronan and its metabolic fate in the present systems remain to be investigated.

Although fragments with an NH(2) terminus of Ala appear to be the major CS-bearing products released into synovial fluid from articular cartilage in vivo(6, 7) , the enzyme responsible (aggrecanase) is not the only proteinase which cleaves aggrecan in vivo. Thus, our previous studies (10) on human articular cartilage showed that a proportion of the G1 domain found in this tissue is the product of metalloproteinase-mediated cleavage of the Asn-Phe bond (see Fig. 2). Furthermore, this 55-kDa form of G1 domain can be detected with anti-FVDIPEN on Western blots of human articular cartilage extracts. (^4)It is therefore possible that a proportion of the aggrecan catabolized in vivo undergoes an initiating cleavage of the Asn-Phe bond by a matrix metalloproteinase. On the other hand, the data in the present paper clearly shows that the activity (aggrecanase) which is responsible for aggrecan catabolism in these culture systems can cleave the Glu-Ala bond of the IGD without a prior cleavage of the Asn-Phe bond. Thus if cleavage of any IGD bond on the NH(2)-terminal side of the aggrecanase site was absolutely required to allow subsequent cleavage of the Glu-Ala bond then G1 domain with a COOH-terminal of Glu would not be generated in any catabolic system. In this regard it therefore seems likely that much of the aggrecan catabolized in vivo undergoes an initiating cleavage of the Glu-Ala bond and that the 55-kDa form of G1 domain present in human cartilage (10) results from secondary proteolysis of this 60-kDa form (see Fig. 2). Indeed this idea is supported by the finding that G1 domain with COOH terminus of Glu, as detected with anti-NITEGE on Western blots, is also present in rat chondrosarcoma extracts (Fig. 1), human articular cartilage extracts, and human synovial fluids.^5

Since the cell-mediated catabolism of the aggrecan interglobular domain described here apparently involves cleavage of the Glu-Ala bond alone, the activity responsible clearly exhibits a very high level of specificity for this cleavage site under these conditions. This specificity is unusual when compared with the multiple cleave sites within the interglobular domain generated on incubation of aggrecan in solution with a range of MMPs (8, 9, 10, 11) . In this regard, it is interesting that although MMP-1, -2, -3, -7, -8, and -9 have been shown to readily cleave the Asn-Phe bond, cleavage of the Glu-Ala bond also appears to occur following prolonged incubation of aggrecan with high concentrations of MMP-8(13) . It is therefore tempting to speculate that aggrecanase is a new member of the MMP family with an unusually narrow cleavage specificity for aggrecan. Alternatively, the site(s) of cleavage of aggrecan by such proteinases may be markedly influenced by the precise conditions of substrate structure or presentation which operate in cell culture and in vivo. Such conditions may be difficult to reproduce in solution incubations with purified components.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Shriners Hospital for Crippled Children, 12502 North Pine Dr., Tampa, FL 33612. Tel.: 813-972-2250; Fax: 813-975-7127.

(^1)
The abbreviations used are: IGD, interglobular domain; MMP, matrix metalloproteinase; CS, chondroitin sulfate; IL-1, interleukin 1; Nle, norleucine; FBS, fetal bovine serum.

(^2)
Residue numbers used in this paper are 19 less than the published numbering systems(16, 17) . This revised numbering has been widely adopted (5, 6, 7, 8, 13) since Val of the published human sequence (16) and Glu of the published rat sequence (17) in each case represent the NH(2)-terminal residue (residue 1) of the mature protein.

(^3)
L. Rosenberg, T. Johnson, L. Tang, and H. Choi, unpublished data.

(^4)
M. Lark and J. Sandy, unpublished data.

(^5)
J. Sandy, J. Gordy, and M. Lark, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Anna Plaas for helpful discussion and the insightful suggestion to use the chondrosarcoma cell line for these studies on aggrecan catabolism. We also wish to thank Dr. Peter Neame for NH(2)-terminal sequence analysis, Dr. Tony Calabro for providing Swarm rat chondrosarcoma cells, and Dr. Stefan Lohmander for critical discussion during preparation of the manuscript.


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