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J. Biol. Chem., Vol. 275, Issue 42, 33038-33045, October 20, 2000

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Mutations in the Interglobular Domain of Aggrecan Alter Matrix Metalloproteinase and Aggrecanase Cleavage Patterns

EVIDENCE THAT MATRIX METALLOPROTEINASE CLEAVAGE INTERFERES WITH AGGRECANASE ACTIVITY^*

Francesca A. Mercuri, Rose A. Maciewicz^§, Jonathan Tart^§, Karena Last, and Amanda J. Fosang^¶

From the  University of Melbourne, Department of Paediatrics, Orthopaedic Molecular Biology Research Unit and Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, 3052, Australia and the ^§ Respiratory and Inflammation Research Department, AstraZeneca, Mereside, Alderley Park, Cheshire, SK10 4TG, United Kingdom

Received for publication, December 23, 1999, and in revised form, June 30, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have expressed G1-G2 mutants with amino acid changes at the DIPEN³⁴¹³⁴²FFGVG and ITEGE³⁷³³⁷⁴ARGSV cleavage sites, in order to investigate the relationship between matrix metalloproteinase (MMP) and aggrecanase activities in the interglobular domain (IGD) of aggrecan. The mutation DIPEN³⁴¹ to DIGSA³⁴¹ partially blocked cleavage by MMP-13 and MMP-8 at the MMP site, while the mutation ³⁴²FFGVG to ³⁴²GTRVG completely blocked cleavage at this site by MMP-1, -2, -3, -7, -8, -9, -13, -14. Each of the MMP cleavage site mutants, including a four-amino acid deletion mutant lacking residues ENFF₃₄₃, were efficiently cleaved by aggrecanase, suggesting that the primary sequence at the MMP site had no effect on aggrecanase activity in the IGD. The mutation ³⁷⁴ARGSV to ³⁷⁴NVYSV completely blocked cleavage at the aggrecanase site by aggrecanase, MMP-8 and atrolysin C but had no effect on the ability of MMP-8 and MMP-13 to cleave at the Asn³⁴¹Phe bond. Susceptibility to atrolysin C cleavage at the MMP site was conferred in the DIGSA³⁴¹ mutant but absent in the wild-type, ³⁴²GTRVG, ³⁷⁴NVYSV, and deletion mutants. To further explore the relationship between MMP and aggrecanase activities, sequential digest experiments were done in which MMP degradation products were subsequently digested with aggrecanase and vice versa. Aggrecanase-derived G1 domains with ITEGE³⁷³ C termini were viable substrates for MMPs; however, MMP-derived G2 fragments were resistant to cleavage by aggrecanase. A 10-mer peptide FVDIPENFFG, which is a substrate analogue for the MMP cleavage site, inhibited aggrecanase cleavage at the Glu³⁷³Ala bond. This study demonstrates that MMPs and aggrecanase have unique substrate recognition in the IGD of aggrecan and suggests that sequences at the C terminus of the DIPEN³⁴¹ G1 domain may be important for regulating aggrecanase cleavage.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The large proteoglycan aggrecan is removed from cartilage by the action of at least two families of zinc-dependent metalloenzymes, the matrix metalloproteinases (MMPs)¹ and the aggrecanases. The MMPs are a family of matrix-degrading enzymes, comprising collagenases, gelatinases, stromelysins, and the membrane-bound MT-MMPs (reviewed in Refs. 1 and 2). Aggrecanase-1 (3) and aggrecanase-2 (4, 5), which exhibit the characteristic activity of Glu³⁷³Ala³⁷⁴ hydrolysis in the IGD, have recently been identified as members of the ADAMTS family (6) of proteinases containing disintegrin, metalloproteinase, and thrombospondin motifs. Identification of MMP (7-12) and aggrecanase (13-15) cleavage sites in the aggrecan IGD has enabled monitoring of MMP activity via detection of DIPEN³⁴¹ and ³⁴²FFGVG terminal sequences, and aggrecanase activity via detection of ITEGE³⁷³ and ³⁷⁴ARGSV sequences, present on digestion products. Both MMP- and aggrecanase-derived fragments have been detected in vivo by amino acid sequence analysis (8, 16, 17) and more recently by the use of neoepitope antibodies (18-28).

Aggrecanase is the major mediator of aggrecanolysis in vitro (29-32); however, MMPs are also active and the relationship between these activities is not understood. Three potential models for cleavage in the IGD have been proposed (33, 34). In the first model, aggrecan is cleaved initially by aggrecanase, and the G1 fragments remaining in the tissue are subsequently cleaved by MMPs. Model 1 therefore generates aggrecanase and MMP G1 fragments and a 32-amino acid Phe³⁴²-Glu³⁷³ fragment but fails to account for the presence of ³⁴²FFGVG fragments in which the aggrecanase site remains intact (19). In the second model, aggrecan is cleaved initially by MMPs, and the released fragment containing the ³⁴²FFGVG neoepitope is subsequently cleaved by aggrecanase. Model 2 generates both aggrecanase (with ³⁷⁴ARGSV N terminus) and MMP (with ³⁴²FFGVG N terminus) fragments and the 32-amino acid Phe³⁴²-Glu³⁷³ fragment but fails to account for the presence of aggrecanase G1 fragments present in human (21, 22), bovine (35, 29), pig (29), rat (35), and mouse (23, 26) cartilage. Model 3, which proposes that aggrecanase and MMP activities are independent, is favored by us (19) and others (21, 22). We favor this model, because in studies investigating the validity of each model (34) (see accompanying article (55)) we have observed that both ITEGE³⁷³ G1 domains and large ³⁴²FFGVG fragments are generated during IL-1 treatment of pig articular cartilage in vitro. Since the ITEGE³⁷³ G1 domain and ³⁴²FFGVG fragments cannot be derived from the one aggrecan molecule, we concluded that in pig cartilage stimulated with IL-1, MMP, and aggrecanase activities were mutually exclusive (36).

The aim of this study was to resolve the mechanism by which MMP and aggrecanase activities could be mutually exclusive. We asked whether mutations at the MMP cleavage site would affect cleavage by aggrecanase or vice versa, and we asked whether the products of one activity were viable substrates for the other. The answers to these questions led us to test the hypothesis that sequences surrounding the MMP cleavage site may be important for aggrecanase activity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- A baculovirus expression system was from CLONTECH. SF 9000 II serum-free medium was from Life Technologies, Inc. Restriction endonucleases, human interleukin-1 (1-457-748), and chemiluminescence blotting kit were from Roche Molecular Biochemicals. The ECL-plus enhanced chemiluminescence kit was from Amersham Pharmacia Biotech. AmpliCycle Sequencing kit and Taq DNA polymerase were from PerkinElmer Life Sciences. Oligonucleotides were synthesized by Bresatec, Australia. The BioSil SEC-400 (600 × 21.5 mm) column was from Bio-Rad. Geneclean was from Bio 101 Inc. Hyaluronan-coupled Sepharose was kindly provided by Professor T. Hardingham (University of Manchester, United Kingdom). The following reagents were generously provided by Professor G. Murphy (University of East Anglia, Norwich, UK): recombinant TIMP-1 (37), recombinant human proMMP-1 (38), recombinant human proMMP-3 (39), recombinant human proMMP-7 (40), proMMP-2, and proMMP-9 purified from human gingival fibroblast conditioned medium (41). Recombinant human proMMP-13 (42) and recombinant human proMMP-8 were gifts from Dr. V. Knäuper and Professor G. Murphy (University of East Anglia). Recombinant MT1-MMP (MMP-14) (43) was a gift from Prof. M. Seiki and Prof. Y. Okada. The snake venom hemorrhagic toxin Ht-d (atrolysin C) was purified from rattlesnake venom (44) and kindly provided by Prof. J. Fox (University of Virginia, Charlottesville, VA). Monoclonal AF-28 specific for the N-terminal sequence ³⁴²FFGVG (45), polyclonal anti-ITEGE³⁷³ (46), and polyclonal anti-DIPEN³⁴¹ (46) rabbit sera were as described. Monoclonal antibody BC-3 (47) specific for the N-terminal sequence ³⁷⁴ARGSV was a gift from Prof. B. Caterson and Dr. C. Hughes (University of Wales, Cardiff, UK). All other reagents were of analytical grade.

Site-directed Mutagenesis of the MMP and Aggrecanase Cleavage Sites in the Aggrecan IGD-- Mutations in the human G1-G2 construct (46) were produced using splicing by overlap extension PCR (48). The primer sets and their relative positions are shown in Table I. Primers Aggr S12 and Aggr S13 introduced a 12-base deletion of GAAAACTTCTTT, resulting in deletion of four amino acids, ENFF³⁴³. Primers Aggr S8 and Aggr S9 were used to replace CCAGAAAAC with GGATCAGCC, changing amino acids PEN³⁴¹ to GSA³⁴¹. Primers Aggr S14 and Aggr S15 were used to replace TTCTTTGGA with GGCACTAGA, changing amino acids ³⁴²FFG to ³⁴²GTR. Primers Aggr S10 and Aggr S11 were used to replace GCCCGAGGC with AACGTATAC, changing amino acids ³⁷⁴ARG to ³⁷⁴NVY. Each mutation resulted in the introduction or removal of restriction enzyme sites to facilitate screening.

The polymerase chain reactions were done using a PerkinElmer Life Sciences thermal cycler model 2400 or 9600, over 30 cycles. Cycle one was performed at 94 °C for 120 s, annealing (Table I) for 90 s and 72 °C for 90 s, followed by 30 cycles of 94 °C for 30 s, annealing (Table I) for 45 s and 72 °C for 30 s. The reactions were terminated at 72 °C for 7 min. Two independent PCR products were first produced using Aggr 7 (sense) with primer A (antisense), and primer B (sense) with Aggr 6 (antisense) of the primer sets in the first round of PCR using 10 ng of pBsktG1-G2 (46) as a template. The amplified products were purified by agarose electrophoresis, recovered using Geneclean, and subjected to a second round of overlapping PCR with primers Aggr 6 and Aggr 7. The PCR fragments were purified and digested with SphI and BsmI restriction enzymes to release a 375-bp fragment, which was then subcloned into pBsktG1-G2, replacing the normal 375-bp cassette. The pBsktG1-G2 mutants were then digested with EcoRI and XbaI, and the G1-G2 mutant construct was subcloned into the pBacPAK8 transfer vector. Prior to production of recombinant virus, the mutant constructs were screened by restriction enzyme digestion and sequenced using Amplitaq Cycle sequencing. Wild-type and mutant rG1-G2 were expressed and purified as described (46).

Proteinase Digestions of Wild-type and Mutant rG1-G2-- Matrix metalloproteinase, aggrecanase, and atrolysin C digestions were done at 37 °C in buffer containing 10 mM calcium chloride, 100 mM sodium chloride, 50 mM Tris-HCl, pH 7.5. Digests were stopped either by boiling or by adding EDTA and 1,10-phenanthroline to final concentrations of 10 and 2 mM, respectively. Denatured samples were analyzed by Western blotting or silver stain after SDS-polyacrylamide gel electrophoresis. In sequential digest experiments, 10 µg of substrates digested with 3 µg/ml MMP-13 for 2 h were incubated with a 2-fold molar excess of TIMP-1 for 30 min on ice to inhibit further MMP-13 action and then incubated overnight with 3 µl of aggrecanase. Similarly, substrates digested overnight with 3 µl of aggrecanase were subsequently digested for 2 h with 3 µg/ml MMP-13.

Preparation of Bovine Nasal Aggrecanase-- Aggrecanase was purified from bovine nasal cartilage in a similar fashion to that previously reported (4). In brief, nasal cartilage was dissected from bovine noses obtained within 4 h after slaughter. The tissue was cut into 1.2-mm cubed pieces and cultured for 2 days in Dulbecco's modified Eagle's medium containing 5% fetal calf serum, followed by 1 day in Dulbecco's modified Eagle's medium containing 2.5% fetal calf serum and finally 1 day in Dulbecco's modified Eagle's medium without fetal calf serum. After the 1 day in serum-free medium, IL-1 was added at a concentration of 0.15 nM. The conditioned medium was collected 48 h later, and aggrecanase was purified by ion exchange, gel filtration, and wheat germ agglutinin chromatography. Several silver-stained bands were present on SDS gels; however, the preparation was "proteolytically pure," since it contained no MMP activity, and other classes of proteinase inhibitors failed to reduce the amount of ITEGE³⁷³ products formed. The aggrecanase preparation was not inhibitable by TIMP-1 when assessed using bovine aggrecan as substrate and anti-ITEGE³⁷³ neoepitope as the read-out. TIMP-1 at 2.2 µM failed to inhibit the aggrecanase activity but gave an IC₅₀ against MMP-1 and MMP-8 of 4 nM and <2 nM, respectively, using a synthetic substrate assay. Since TIMP-1 has an IC₅₀ of 210 nM against ADAMTS-4,² it is unlikely that the aggrecanase preparation used in the present work is identical to ADAMTS-4 (3).

Inhibition of Aggrecanase Activity by Synthetic Peptides-- The 7-mer peptides IPENFFG and TEGEARG were from Charing Cross Hospital (London), and the 10-mer peptide FVDIPENFFG was from Auspep. IPENFFG and FVDIPENFFG were dissolved in distilled water at 25 mg/ml, and TEGEARG was dissolved in 75 mM NaCO₃, pH 8.0, at the same concentration. The peptides were present at a 3000-fold molar excess over rG1-G2 substrate in aggrecanase digests. The addition of 75 mM NaCO₃ buffer alone to aggrecanase digests did not alter the pH, nor did it affect the generation of ITEGE³⁷³ epitope.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mutations at the DIPEN³⁴¹FFGVG and ITEGE³⁷³ARGSV cleavage sites in the aggrecan IGD were made (Fig. 1), and the mutant substrates were tested for their susceptibility to digestion by MMPs, aggrecanase, and atrolysin C. 

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Sequence and position of the amino acid substitutions at the MMP and aggrecanase cleavage sites in the IGD.

MMP-13 Digestion of Wild-type and Mutant rG1-G2-- Wild type and mutant G1-G2 substrates were digested with MMP-13, since this MMP is abundantly expressed in arthritic cartilage. Silver staining (Fig. 2, a, d, g, j, and m) was used to monitor 1) the extent of digestion and the approximate ratio of undigested:digested material and 2) uniform loading of samples on gels. Silver staining was not useful for specifically identifying G1 and G2 domains, since the fragments often migrated together. Western botting with neoepitope antibodies was used to determine whether cleavage was occurring at the MMP site. Mutation of the sequence DIPEN³⁴¹ to DIGSA³⁴¹ retarded, but did not block, cleavage at the MMP site, since a higher concentration of MMP-13 (10 µg/ml) was required to produce the maximum amount of ³⁴²FFGVG epitope in digests of the DIGSA³⁴¹ mutant (Fig. 2e) compared with the wild type (Fig. 2b). However, mutation of ³⁴²FFGVG to ³⁴²GTRVG completely abolished cleavage at the MMP site, since no DIPEN₃₄₁ epitope was detected in digests of the ³⁴²GTRVG mutant (Fig. 2i). This result is consistent with previous reports that most substitutions at the P'₁ position are detrimental for collagenase activity (49). Silver staining showed that the ³⁴²GTRVG mutant was cleaved by MMP-13 elsewhere in the IGD, most likely at the minor sites Pro³⁸⁴Val³⁸⁵ and Asp⁴⁴¹Leu⁴⁴², as shown for native G1-G2 (11). Generation in the ³⁷⁴NVYSV mutant of DIPEN³⁴¹ and ³⁴²FFGVG neoepitopes by MMP-13 (Fig. 2, m-o) was similar to the wild type (Fig. 2, a-c), indicating that the ³⁷⁴NVYSV mutation at the aggrecanase site had no affect on MMP-13 activity against rG1-G2. MMP-13 at 100 µg/ml appeared to "overdigest" the substrate, leading to loss of ³⁴²FFGVG (Fig. 2, b, e, and n) and DIPEN₃₄₁ (Fig. 2, c and o) neoepitopes as well as G1 domain epitopes detected with polyclonal antisera (data not shown). This is consistent with our previous observation that rG1-G2 is more sensitive to proteolysis than native glycosylated pig G1-G2 (46).

View larger version (39K): [in this window] [in a new window]   Fig. 2.   MMP-13 digestion of wild-type and mutant rG1-G2. Recombinant G1-G2 (5 µg) was digested for 2 h at 37 °C with the concentrations of MMP-13 shown, in a total volume of 20 µl. Aliquots of digested and undigested rG1-G2 were electrophoresed on SDS gels and analyzed by silver staining (a, d, g, j, and m) or Western blotting with monoclonal anti-342FFGVG (b, e, h, k, and n) or anti-DIPEN341 antisera (c, f, i, l, and o). The slashed boxes indicate that the epitope sequence was absent in a particular mutant.

MMP-8 Digestion of Wild-type and Mutant rG1-G2-- MMP-8 cleaves bovine aggrecan (50) and native pig G1-G2 (51) at both the MMP and aggrecanase sites. MMP-8 cleaves its substrate in a sequential manner, at the Asn³⁴¹Phe bond initially, and the Glu³⁷³Ala bond subsequently. Wild-type rG1-G2 was also cleaved in a sequential manner by MMP-8. ³⁴²FFGVG epitope was maximal at low concentrations of enzyme but decreased at higher concentrations (Fig. 3b), and the decrease in epitope was concomitant with an increase in ³⁷⁴ARGSV epitope (Fig. 3d). DIPEN³⁴¹ epitope on the other hand was unchanged (Fig. 3c). As with MMP-13, the DIGSA³⁴¹ mutant was less susceptible to MMP-8 cleavage at the MMP site. Higher concentrations of MMP-8 were required to achieve maximum ³⁴²FFGVG epitope in the DIGSA³⁴¹ digests, and epitope levels were not noticeably decreased at the highest concentration of enzyme (200 µg/ml) (Fig. 3f), although a small amount of ³⁷⁴ARGSV epitope was detected (Fig. 3h). MMP-8 digestion of the ³⁴²GTRVG mutant showed that hydrolysis of the Asn³⁴¹Phe bond was not an essential prerequisite for cleavage at Glu³⁷³Ala. ³⁷⁴ARGSV epitope was detected in digested ³⁴²GTRVG mutant at enzyme concentrations of 30 µg/ml and higher (Fig. 3l), in the absence of MMP site cleavage (Fig. 3k). The ³⁷⁴NVYSV mutation did not interfere with MMP-8 cleavage at DIPEN³⁴¹FFGVG but did block cleavage at ITEGE³⁷³ARGSV, since there was no loss of ³⁴²FFGVG epitope at high concentrations of enzyme (Fig. 3r).

View larger version (43K): [in this window] [in a new window]   Fig. 3.   MMP-8 digestion of wild-type and mutant rG1-G2. Recombinant G1-G2 (10 µg) was digested for 2 h at 37 °C with the concentrations of MMP-8 shown, in a total volume of 20 µl. Aliquots of digested and undigested rG1-G2 were electrophoresed on SDS gels and analyzed by silver staining (a, e, i, m, and q) or Western blotting with monoclonal anti-342FFGVG (b, f, j, n, and r), anti-DIPEN341 antisera (c, g, k, o, and s) or monoclonal anti-374ARGSV (d, h, l, p, and t).

To determine whether the ³⁴²GTRVG mutant resisted cleavage by other MMPs, wild-type and mutant rG1-G2 were next digested with a single concentration of MMP-1, -2, -3, -7, -9, and -14 (Fig. 4). Silver staining showed that MMP-1, MMP-7, and MMP-14 cleaved the GTR mutant (Fig. 4g); however, the lack of DIPEN³⁴¹ reactivity in the digests showed that none of the MMPs cleaved at the mutated MMP site (Fig. 4i). Some ³⁴²FFGVG epitope was present in DIGSA³⁴¹ mutants digested with MMP-2, MMP-3, and MMP-14 (Fig. 4e). The data show that mutation of DIPENF³⁴²FGVG to DIPENG³⁴²TRVG blocked cleavage at the major MMP site by three collagenases, two gelatinases, stromelysin-1, matrilysin, and MT1-MMP.

View larger version (42K): [in this window] [in a new window]   Fig. 4.   MMP digestion of wild-type and mutant rG1-G2. Recombinant G1-G2 (2.5 µg) was digested for 2 h at 37 °C with 125 µg/ml MMP-1, 100 µg/ml MMP-2, 125 µg/ml MMP-3, 86.7 µg/ml MMP-7, 130.5 µg/ml MMP-9, or 75 µg/ml MMP-14 in a total volume of 10 µl. Aliquots of digested and undigested rG1-G2 were electrophoresed on 5% SDS gels and analyzed by silver staining (a, d, g, and j) or Western blotting with monoclonal anti-342FFGVG (b, e, h, and k) or anti-DIPEN341 antisera (c, f, i, and l).

Aggrecanase Digestion of Wild-type and Mutant rG1-G2-- Wild-type and mutant rG1-G2 were incubated with increasing amounts of bovine aggrecanase, and the products were detected by Western blotting with anti-ITEGE³⁷³ and anti-³⁷⁴ARGSV antibodies. All of the rG1-G2 substrates except the ³⁷⁴NVYSV mutant were efficiently cleaved by aggrecanase at the ITEGE³⁷³ARGSV site (Fig. 5). Silver staining showed that no ³⁷⁴NVYSV degradation products were produced by aggrecanase (Fig. 5m), suggesting that, unlike the MMPs, aggrecanase had specificity for only one site in the IGD.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Aggrecanase digestion of wild-type and mutant rG1-G2. Recombinant G1-G2 (5 µg) was digested for 21 h at 37 °C with the volumes of purified aggrecanase shown, in a total volume of 20 µl. Aliquots of digested and undigested rG1-G2 were electrophoresed on SDS gels and analyzed by silver staining (a, d, g, j, and m) or Western blotting with anti-ITEGE373 (b, e, h, k, and n) or monoclonal anti-374ARGSV (c, f, i, l, and o).

Atrolysin C Digestion of Wild-type and Mutant rG1-G2-- Native glycosylated bovine aggrecan (52) and pig G1-G2 (46) are cleaved by atrolysin C at both the DIPEN³⁴¹FFGVG and ITEGE³⁷³ARGSV bonds, in an independent rather than sequential manner. However, we have recently found that wild- type rG1-G2, which is largely unglycosylated, is cleaved by atrolysin C only at ITEGE³⁷³ARGSV and not DIPEN³⁴¹FFGVG (46). In the present experiments, all of the rG1-G2 substrates except the ³⁷⁴NVYSV mutant (Fig. 6n) were cleaved by atrolysin C at ITEGE³⁷³ARGSV, as detected by anti-ITEGE³⁷³ immunoreactivity (Fig. 6, b, e, h, and k). No DIPEN³⁴¹ epitope was detected in any of the digests (data not shown), suggesting that atrolysin C was not able to cleave at DIPEN³⁴¹FFGVG in the wild-type, ³⁴²GTRVG, or ³⁷⁴NVYSV substrates. Surprisingly, ³⁴²FFGVG immunoreactivity was detected in digested DIGSA³⁴¹ mutant (Fig. 6f). These results suggest that the conformational shape of the substrate surrounding the sequence DIPEN³⁴¹ may dictate atrolysin C specificity for cleavage in the IGD.

View larger version (39K): [in this window] [in a new window]   Fig. 6.   Atrolysin C digestion of wild-type and mutant rG1-G2. Recombinant G1-G2 (5 µg) was overnight at 37 °C with concentrations of atrolysin C as shown, in a total volume of 20 µl. Aliquots of digested and undigested rG1-G2 were electrophoresed on SDS gels and analyzed by silver staining (a, d, g, j, and m) or Western blotting with anti-ITEGE373 (b, e, h, k, and n) or monoclonal anti-342FFGVG (c, f, i, l, and o).

Relationship between MMP and Aggrecanase Activities in the IGD-- In order to further examine the relationship between proteolysis at the Asn³⁴²Phe and Glu³⁷³Ala bonds, we designed experiments to test whether aggrecanase-derived G1 fragments (with ITEGE³⁷³ C terminus) were substrates for MMPs (Fig. 7) and, conversely, whether MMP-derived G2 fragments (with ³⁴²FFGVG N terminus) were substrates for aggrecanase (Fig. 8). We predicted that if aggrecanase-G1 fragments were digested by MMPs (Fig. 7c), we would create DIPEN³⁴¹ epitope, lose ITEGE³⁷³ epitope (present on the 3-kDa fragment) and observe no change in ³⁷⁴ARGSV epitope (Fig. 7, d and h). The Western blots revealed that DIPEN³⁴¹ epitope was indeed increased (Fig. 7e, lanes 3 and 4), while ITEGE³⁷³ epitope was completely destroyed following digestion of aggrecanase-G1 with MMP-13 (Fig. 7f, lanes 3 and 4). These results therefore confirm that the aggrecanase-derived G1 domain is a viable substrate for MMPs.

View larger version (35K): [in this window] [in a new window]   Fig. 7.   Aggrecanase followed by MMP-13 sequential digest. a-d, schematic diagram showing the predicted fragments and neoepitope sequences generated by MMP digestion of the aggrecanase-G1 domain. e-g, wild-type rG1-G2 was digested with 3 µg/ml MMP-13 for 2 h (lane 2), 3 µl of aggrecanase for 21 h (lane 3), or 3 µl of aggrecanase for 21 h followed by 3 µg/ml MMP-13 for 2 h (lane 4). Aliquots were electrophoresed on SDS gels and analyzed by Western blotting with anti-DIPEN341 (e), anti-ITEGE373 (f), or monoclonal anti-374ARGSV (g). h, the expected and observed outcome for the products obtained in d and lane 4 relative to b and lane 3 is shown.

View larger version (54K): [in this window] [in a new window]   Fig. 8.   MMP-13 followed by aggrecanase sequential digest. a-d, schematic diagram showing the predicted fragments and neoepitope sequences generated by aggrecanase digestion of the MMP-G2 domain. f-i, wild-type rG1-G2 was digested with 3 µg/ml MMP-13 for 2 h (lane 2), 3 µl of aggrecanase for 21 h (lane 3), or 3 µg/ml MMP-13 for 2 h followed by 3 µl of aggrecanase for 21 h (lane 4). e, the expected and observed outcome for the products obtained in d and lane 4 relative to b and lane 2 is shown. Aliquots were electrophoresed on SDS gels and analyzed by Western blotting with monoclonal anti-342FFGVG (f), anti-DIPEN341 (g), anti-ITEGE373 (h), or monoclonal anti-374ARGSV (i). GTRGV mutant G1-G2 was digested with 3 µg/ml MMP-13 for 2 h (lane 6), 3 µl of aggrecanase for 21 h (lane 7), or 3 µg/ml MMP-13 for 2 h followed by 3 µl of aggrecanase for 21 h (lane 8). Aliquots were electrophoresed on SDS gels and analyzed by Western blotting with monoclonal anti-374ARGSV (j).

In contrast, we found that the MMP-G2 domain was not digested by aggrecanase. We predicted that if MMP-G2 fragments were digested by aggrecanase (Fig. 8c), we would create ³⁷⁴ARGSV epitope, lose ³⁴²FFGVG epitope (present on the 3-kDa fragment), and observe no change in DIPEN³⁴¹ epitope (Fig. 8, d and e). However, Western blot analysis showed that ³⁴²FFGVG epitope was not destroyed (Fig. 8f, lanes 2 and 4) and that there was no gain in ³⁷⁴ARGSV epitope (Fig. 8i, lane 4). The small amount of anti-³⁷⁴ARGSV epitope present in Fig. 8i, lane 4, is most likely derived from cleavage of intact substrate rather than MMP-G2 domain, since some undigested substrate survives digestion with MMP-13 for 2 h at 3 µg/ml (Fig. 2a). Furthermore, pretreatment of rG1-G2 with MMP-13 (Fig. 8i, lane 4) markedly reduced the yield of ³⁷⁴ARGSV epitope by aggrecanase (compare Fig. 8i, lane 4, with Fig. 8i, lane 3). Aggrecanase was active in the presence of TIMP-inhibited MMP-13, since ³⁴²GTRVG mutants digested under the same conditions gave ³⁷⁴ARGSV epitope (Fig. 8j, lane 4). The results show that the MMP-G2 domain is not a viable substrate for aggrecanase.

Sequences in the MMP-G1 Domain Are Required for Aggrecanase Activity-- A previous study in our laboratory showed that a 7-mer peptide IPENFFG, a substrate analogue for the MMP cleavage site, was able to inhibit the release of ³⁷⁴ARGSV fragments from cartilage cultured with and without interleukin-1 (51). The results of this experiment suggested that the IPENFFG peptide was able to inhibit aggrecanase and now, in conjunction with the results presented in Fig. 8, raise the possibility that sequences present at the C terminus of the MMP-G1 domain may be necessary for aggrecanase activity. To test this hypothesis, we used several synthetic peptides as competitive substrates in aggrecanase digests of rG1-G2 (Fig. 9). In these experiments, the IPENFFG 7-mer peptide was unable to block aggrecanase cleavage at Glu³⁷³Ala (Fig. 9, b, lane 3, and c); however, cleavage was inhibited by 50% in the presence of a 10-mer peptide with sequence FVDIPENFFG (Fig. 9, b, lane 4, and c). A 7-mer peptide TEGEARG, a substrate analogue for the aggrecanase site, completely blocked aggrecanase cleavage.

View larger version (40K): [in this window] [in a new window]   Fig. 9.   Competitive inhibition of aggrecanase by MMP-site peptide analogues. Wild-type rG1-G2 was digested with aggrecanase (3 µl) for 17 h at 37 °C with or without synthetic peptides, in a 3000-fold molar excess over rG1-G2, as shown. Digested and undigested samples were electrophoresed on SDS gels and analyzed by silver staining (a) or Western blotting with anti-ITEGE373 antisera (b). The inhibition of aggrecanase activity was determined by densitometric analysis of the ITEGE373 fragments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Our finding that MMP-derived G2 fragments are resistant to aggrecanase cleavage in the IGD is novel and provides an explanation for our earlier observation (34, 36) that IL-1-induced loss of aggrecan from pig articular cartilage by MMPs and aggrecanase appeared to be mutually exclusive. The present findings are not limited to digestion of rG1-G2, since native glycosylated ³⁴²FFGVG fragments (see Fig. 7 of accompanying article (55)) and native deglycosylated MMP-1-digested aggrecan (29) also resist digestion by aggrecanase. To revisit the models outlined in the Introduction, model 1, in which aggrecan is cleaved initially by aggrecanase and the G1 fragments remaining in the tissue are subsequently cleaved by MMPs, appears viable. However, the subsequent MMP cleavage in this model represents processing of the G1 domain and has no effect on further loss of aggrecan from the matrix. Model 2, in which aggrecan is cleaved initially by MMPs and the released fragment containing the ³⁴²FFGVG neoepitope is subsequently cleaved by aggrecanase, seems unlikely. Our finding that recombinant ³⁴²FFGVG fragments and native pig ³⁴²FFGVG fragments (see accompanying article (55)) resist aggrecanase cleavage in the IGD thus extends the independent model, model 3. The results show that, in terms of aggrecan loss from tissue (as opposed to ITEGE₃₇₃ G1 domain processing), MMP and aggrecanase activities appear to be mutually exclusive in IL-1-stimulated aggrecan release from pig cartilage and aggrecanolysis of rG1-G2 in vitro. The results are also consistent with our finding that small ³⁴²FFGVG fragments detected in synovial fluids of osteoarthritis and inflammatory arthritis patients do not contain an ITEGE³⁷³ C terminus (19).

In principle, these results have implications for therapeutic strategies designed to limit aggrecan loss, since it appears that inhibition of both activities may be required. Our results with the mutant G1-G2 substrates also suggest that abrogation of one activity has no consequence for the other activity. A spectrum of sizes of degraded aggrecan fragments is present in synovial fluids of arthritis patients. The large, high buoyant density fragments that can be recovered from synovial fluids fractionated on cesium chloride density gradients are predominantly aggrecanase-derived and do not contain any fragments with ³⁴²FFGVG N termini (16, 17). Low buoyant density fragments fractionated on cesium chloride density gradients do contain small ³⁴²FFGVG fragments (19), and since these fragments do not carry the ITEGE³⁷³ C terminus, it is possible they are the products of more extensive MMP processing. Studies of aggrecanolysis in chondrocyte and cartilage explant cultures show that aggrecanase is the predominant activity in vitro (35, 29) (see accompanying article (55)); however, the detection (45) and quantitation (19) of ³⁴²FFGVG fragments in human synovial fluids or released from IL-1-treated human OA cartilage (29) suggests that MMPs may play a greater role in aggrecanolysis in human disease than in in vitro animal models. The relative involvement of MMPs and aggrecanase in arthritis remains unclear.

The processing of ITEGE³⁷³ G1 to DIPEN³⁴¹ G1 is likely to occur in vivo. Immunolocalization studies in mice with experimentally induced arthritis have shown that ITEGE³⁷³ neoepitopes were less prominent in areas showing advanced cartilage damage, compared with DIPEN₃₄₁ epitope, and that when intense DIPEN₃₄₁ staining appeared, ITEGE₃₇₃ epitope disappeared (26). These results suggest that either ITEGE³⁷³ G1 is cleared quickly from the tissue or the G1 fragment is rapidly cleaved by MMPs or other proteinases, destroying the epitope. Under conditions where the tissue pH is acidic, the increased DIPEN³⁴¹ epitope and concomitant loss of ITEGE³⁷³ epitope could arise from the endo- and exopeptidase activity of cathepsin B (53).

The peptide experiment (Fig. 9), together with our previous observation that the 7-mer peptide IPENFFG was able to inhibit the release of ³⁷⁴ARGSV fragments from cartilage in culture (51), suggests that sequences present at the C terminus of the FVDIPEN³⁴¹ G1 domain may be important for aggrecanase activity. One possibility is that these sequences provide a docking site for the enzyme. If the Pro³³⁹, Glu³⁴⁰, or Asn³⁴¹ residues were critical for aggrecanase docking or activity, we would expect that cleavage at the ITEGE³⁷³ARGSV site may have been reduced in the DIGSA³⁴¹ and deletion mutants. This was not the case, suggesting therefore that sequences other than Pro-Glu-Asn are involved. This is consistent with our observation that a longer peptide with sequence FVDIPENFFG was effective in inhibiting aggrecanase activity by 50%, while the shorter IPENFFG peptide had no effect in the present style of experiment. An alternative explanation is that the longer sequence allows the formation of a peptide with secondary or tertiary structure and that it is the conformational shape rather than the peptide sequence that is important. This possibility could be addressed by determining whether a peptidic hydroxamate inhibitor based on the Pro-Glu-Asn residues could inhibit aggrecanase.

Three MMP cleavage site mutants were made, and these partially or totally blocked cleavage at the DIPEN³⁴¹FFGVG site; however, none of the mutations conferred complete protection from degradation by MMP-8 or -13, as seen in the fragmentation pattern by silver stain. The enzymes were clearly able to cleave elsewhere in the IGD, possibly at the minor sites (10, 11). The ³⁴²GTRVG mutant resisted cleavage by stromelysin-1, two gelatinases, three collagenases, matrilysin, and MT1-MMP at the mutated MMP site. The DIGSA³⁴¹ mutant was partially resistant to some of these MMPs. In the future, it will be interesting to determine whether different glycosylation affects MMP or aggrecanase specificity for cleavage in the IGD.

In contrast to the MMP cleavage site mutants, the ³⁷⁴NVYSV mutant was not cleaved at all by aggrecanase, showing that aggrecanase has specificity for only a single site in the IGD. Our ongoing studies generating an ³⁷⁴NVYSV knock-in mouse will enable us to further explore the role of aggrecanase in normal growth and development and also in the initiation and progression of arthritis.

	ACKNOWLEDGEMENTS

We express gratitude to Prof. G. Murphy, Dr. V. Knäuper, Prof. J. Fox, Prof. M. Seiki, Prof. Y. Okada, Prof. T. Hardingham, Prof. B. Caterson, and Dr. C. Hughes for providing reagents.

	FOOTNOTES

^* This work was supported by the National Health and Medical Research Council (Australia), the Arthritis Foundation of Australia, and the Royal Children's Hospital Research Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Paediatrics, University of Melbourne, Orthopaedic Molecular Biology Research Unit and Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville 3052, Australia. Tel.: 61-3-9345-6628; Fax: 61-3-9345-7997; E-mail: fosang@cryptic.rch.unimelb.edu.au.

Published, JBC Papers in Press, July 5, 2000, DOI 10.1074/jbc.M910208200

² E. Arner, personal communication.

	ABBREVIATIONS

The abbreviations used are: MMP, matrix metalloproteinase; ADAMTS, A disintegrin and metalloproteinase with thrombospondin motif-containing family of proteins; IGD, interglobular domain of aggrecan; PCR, polymerase chain reaction; IL, interleukin; rG1-G2, recombinant G1-G2.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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