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J. Biol. Chem., Vol. 275, Issue 24, 18566-18573, June 16, 2000

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Sites of Aggrecan Cleavage by Recombinant Human Aggrecanase-1 (ADAMTS-4)^*

Micky D. Tortorella^§, Michael Pratta, Rui-Qin Liu, Julian Austin, O. Harold Ross^¶, Ilgar Abbaszade^¶, Tim Burn^¶, and Elizabeth Arner

From the Departments of  Inflammatory Diseases Research and ^¶ Applied Biotechnology, DuPont Pharmaceuticals Company, Wilmington, Delaware 19880-0400

Received for publication, November 19, 1999, and in revised form, March 30, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Aggrecan, the major proteoglycan of cartilage that provides its mechanical properties of compressibility and elasticity, is one of the first matrix components to undergo measurable loss in arthritic diseases. Two major sites of proteolytic cleavage have been identified within the interglobular domain (IGD) of the aggrecan core protein, one between amino acids Asn³⁴¹-Phe³⁴² which is cleaved by matrix metalloproteinases and the other between Glu³⁷³-Ala³⁷⁴ that is attributed to aggrecanase. Although several potential aggrecanase-sensitive sites had been identified within the COOH terminus of aggrecan, demonstration that aggrecanase cleaved at these sites awaited isolation and purification of this protease. We have recently cloned human aggrecanase-1 (ADAMTS-4) (Tortorella, M. D., Burn, T. C., Pratta, M. A., Abbaszade, I., Hollis, J. M., Liu, R., Rosenfeld, S. A., Copeland, R. A., Decicco, C. P., Wynn, R., Rockwell, A., Yang, F., Duke, J. L., Solomon, K., George, H., Bruckner, R., Nagase, H., Itoh, Y., Ellis, D. M., Ross, H., Wiswall, B. H., Murphy, K., Hillman, M. C., Jr., Hollis, G. F., Newton, R. C., Magolda, R. L., Trzaskos, J. M., and Arner, E. C. (1999) Science 284, 1664-1666) and herein demonstrate that in addition to cleavage at the Glu³⁷³-Ala³⁷⁴ bond, this protease cleaves at four sites within the chondroitin-sulfate rich region of the aggrecan core protein, between G2 and G3 globular domains. Importantly, we show that this cleavage occurs more efficiently than cleavage within the IGD at the Glu³⁷³-Ala³⁷⁴ bond. Cleavage occurred preferentially at the KEEE^1667-1668GLGS bond to produce both a 140-kDa COOH-terminal fragment and a 375-kDa fragment that retains an intact G1. Cleavage also occurred at the GELE^1480-1481GRGT bond to produce a 55-kDa COOH-terminal fragment and a G1-containing fragment of 320 kDa. Cleavage of this 320-kDa fragment within the IGD at the Glu³⁷³-Ala³⁷⁴ bond then occurred to release the 250-kDa BC-3-reactive fragment from the G1 domain. The 140-kDa GLGS-reactive fragment resulting from the preferential cleavage was further processed at two additional cleavage sites, at TAQE¹⁷⁷¹-¹⁷⁷²AGEG and at VSQE^1871-1872LGQR resulting in the formation of a 98-kDa fragment with an intact G3 domain and two small fragments of ~20 kDa. These data elucidate the sites and efficiency of cleavage during aggrecan degradation by aggrecanase and suggest potential tools for monitoring aggrecan cleavage in arthritis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Aggrecan is the major proteoglycan of cartilage and provides this tissue with its mechanical properties of compressibility and elasticity. Aggrecan monomers interact with hyaluronan and are usually found as part of a large aggregate containing 10-100 monomers per hyaluronan molecule. The primary role of aggrecan is to swell and hydrate the framework of cartilage collagen fibrils thus providing cartilage with its properties of compressibility and elasticity. The NH₂ terminus of the aggrecan monomer core protein is comprised of two globular domains called G1 and G2 that are separated by an interglobular domain (IGD)¹ that spans about 150 residues in length. The G2 region is followed by a long central glycosaminoglycan (GAG) attachment region and by a COOH-terminal globular domain, G3 (2, 3).

In cartilage degradation associated with diseases such as osteoarthritis and rheumatoid arthritis, aggrecan is one of the first matrix components to undergo measurable loss that ultimately leads to a loss of cartilage function. Proteolytic cleavage within the IGD is believed to be responsible for the loss of aggrecan from cartilage. Two major sites of proteolytic cleavage have been identified within the IGD, one between amino acids Asn³⁴¹ and Phe³⁴² and the other between Glu³⁷³ and Ala³⁷⁴. Matrix metalloproteinases, including MMP-1, -2, -3, -7, -8, -9, -13 and MT1-MMP have been shown to cleave aggrecan at the Asn³⁴¹-Phe³⁴² site (4-8), whereas cleavage at the Glu³⁷³-Ala³⁷⁴ site has been attributed to aggrecanase (9-13).

Cleavage within the sequence TEGE^373-374ARGS is the hallmark of aggrecanase activity. However, homology comparison of this site with other regions within the aggrecan molecule indicates that there may be four additional aggrecanase-sensitive sites located in the chondroitin sulfate-rich region between the G2 and G3 globular domains. These sites all have a glutamic acid in the P₁ position and a non-polar or uncharged polar residue (Ala, Leu, and Gly) residue in the P₁' position (10, 14). They include KEEE^1667-1668GLGS, GELE^1480-1481GRGT, TAQE^1771-1772AGEG, VSQE^1871-1872LGQR, and are highly conserved in aggrecan from various species. Homology is illustrated for bovine and human aggrecan in Fig. 1. Consistent with this hypothesis, in bovine articular cartilage organ cultures and rat chondrosarcoma cells stimulated with either retinoic acid or IL-1 (10, 14, 15), aggrecan fragments were identified having NH₂ or COOH termini indicating that they had been generated by cleavage at some of these sites. However, confirmation that this cleavage was due to the same enzyme, aggrecanase, awaited its isolation.

View larger version (39K): [in this window] [in a new window]   Fig. 1.   Comparison of putative aggrecanase-sensitive sites in bovine and human aggrecan.

Recently, our laboratory purified aggrecanase-1 (ADAMTS-4) and aggrecanase-2 (ADAMTS-11/5) from bovine nasal cartilage-conditioned media and cloned and expressed the human orthologs of these proteases (1, 16). Aggrecanase-1 and -2 are members of the adamalysin family of zinc-binding metalloproteases that exhibit a catalytic domain with the zinc-binding motif HEXXHXXNXXH, similar to that found in several MMPs, and cleave aggrecan at the Glu³⁷³-Ala³⁷⁴ bond. In the work reported herein, we identify the sites of cleavage and order of cleavage of aggrecan by recombinant human aggrecanase-1 using NH₂-terminal sequence analysis and neo-epitope antibodies that recognize either the new COOH or NH₂ terminus generated by cleavage at the putative aggrecanase-sensitive sites.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Aggrecanase-1 was cloned and expressed in Drosophila S2 cells at DuPont Pharmaceuticals as described (1). Antibody BC-3 (17) that recognizes the new NH₂ terminus, ARGS, on aggrecan fragments produced by aggrecanase cleavage at the Glu³⁷³-Ala³⁷⁴ bond, was licensed from Dr. Bruce Caterson (University of Wales, Cardiff, UK). Antibody AF-28 (18) that recognizes the new NH₂ terminus, FFGVG, on aggrecan fragments produced by MMP cleavage at the Asn³⁴¹-Phe³⁴² bond, was a gift from Dr. Amanda Fosang (University of Melbourne, Parkville, Australia). The NITEGE antibody (12) that recognizes the new COOH terminus, NITEGE, on aggrecan fragments produced by aggrecanase cleavage, was developed by Dr. Michael Lark and was a gift from Dr. Denise Visco (Merck Research Laboratories, Rahway, NJ). The monoclonal antibody, MAB2035, which recognizes chondroitin sulfate stubs on the core protein of aggrecan following deglycosylation, was purchased from Chemicon International (Temecula, CA).

Neo-epitope Antibodies-- Polyclonal neo-epitope antibodies were prepared (Quality Controlled Biochemicals, Hopkinton, MA) to the peptide sequences ATTAGELE and PTTFKEEE, that are present on the COOH terminus of aggrecan fragments generated by cleavage at the Glu¹⁴⁸⁰-Gly¹⁴⁸¹ bond and at the Glu¹⁶⁶⁷-Gly¹⁶⁶⁸ bond, respectively. Polyclonal neo-epitope antibodies were also prepared to the peptide sequences GLGSVELS, AGEGPSGI, and LGQRPPVT that are present on the NH₂ terminus of aggrecan fragments generated by cleavage at the Glu¹⁶⁶⁷-Gly¹⁶⁶⁸, Glu¹⁷⁷¹-Ala¹⁷⁷², and the Glu¹⁸⁷¹-Leu¹⁸⁷² bond, respectively. Intact, undigested aggrecan was not detected with any of the antibodies. Specificity of the antibodies was determined in competitive ELISA using synthetic peptide antigens as inhibitors (Table I). Only the immunizing peptide for each antibody blocked binding to the antigen, whereas the spanning peptide did not block binding indicating that the antibodies do not recognize the same sequence when present within the uncleaved protein. Equally important, peptides designed for one antibody did not block binding of the other antibodies, despite sequence similarity. In human (and rat) aggrecan, the GELE COOH-terminal neo-epitope of the bovine aggrecan fragment generated by cleavage at the Glu¹⁴⁸⁰-Gly¹⁴⁸¹ bond is SELE with a single amino acid substitution of Ser for Gly (Fig. 1). Interestingly, an antibody raised against the SELE neo-epitope does not recognize the GELE COOH terminus on the bovine aggrecan fragment, illustrating the high degree of specificity of these antibodies.

Aggrecan Isolation-- Nasal septa were removed from bovine noses obtained fresh from slaughter. The cartilage was sliced and aggrecan was extracted by stirring at 4 °C for 48 h in 10 volumes of 4 M guanidine-HCl in 0.05 M sodium acetate, pH 5.8, containing the protease inhibitors, 0.01 M EDTA, 0.1 M 6-aminohexanoic acid, 2 mM phenymethanesulfonyl fluoride, and 0.05 M benzamidine-HCl. Aggrecan monomers were isolated by equilibrium density gradient centrifugation in cesium chloride (20) and the bottom of this gradient (d > 1.54 g/ml) containing aggrecan monomers was dialyzed at 4 °C against water, lyophilized, and stored at 20 °C.

Enzyme Digestion-- Digestions were carried out in 100 µl of 50 mM Tris/HCl buffer, pH 7.5, containing 100 mM NaCl and 10 mM CaCl₂. Purified bovine aggrecan (500 nM) was incubated with recombinant human aggrecanase-1 at 37 °C for the indicated times. The reactions were quenched with EDTA and products analyzed by immunolocation in a Western blot analysis.

NH₂-terminal Sequencing-- Aggrecan fragments digested with aggrecanase-1 were NH₂ terminally sequenced at Argo Bioanalytical Inc. (Morristown, NJ). The NH₂-terminal sequence analyses were performed using a Hewlett-Packard G1005A NH₂-terminal sequencer. These analyses of polyvinylidene difluoride-blotted protein were carried out using a modification of the Hewlett-Packard Routine 3.0 Sequencing Methods (21).

Deglycosylation of Aggrecan Products-- For analysis of fragments by Western blot, aggrecan was enzymatically deglycosylated with chondroitinase ABC (0.1 units/10 µg of aggrecan) for 1 h at 37 °C and then with keratanase (0.1 units/10 µg of aggrecan) and keratanase II (0.002 units/10 µg of aggrecan) for 2 h at 37 °C in buffer containing 50 mM sodium acetate, 0.1 M Tris/HCl, pH 6.5. After digestion the aggrecan was precipitated with 5 volumes of acetone and reconstituted in 30 µl of Tris glycine-SDS sample buffer containing 2.5% -mercaptoethanol and heated for 3 min at 100 °C.

Western Blot Analysis-- 20 µg of glycosaminoglycan from each sample was loaded on a 4-12% Tris glycine gel and separated by SDS-polyacrylamide gel electrophoresis under reducing conditions. The separated proteins were transferred to polyvinylidene difluoride membranes and immunolocated with a 1:1000 dilution of one of the neo-epitope antibodies conditions or a 1:10,000 dilution of antibody mAB2035. Subsequently, the membranes were incubated with a 1:5000 dilution of goat anti-mouse or goat anti-rabbit IgG alkaline phosphatase conjugate as the secondary antibody. Products were visualized by developing the blots in nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate color developing reagent. NITEGE Western analysis was performed as described previously (12). Overnight transfer resulted in complete transfer of both low and high molecular fragments and the densitometric response was found to be linear over the density ranges required for the blots. The immunoreactive bands were quantified by scanning densitometry. For comparison of production of GELE- and KEEE-reactive fragments at early time points, conditions were adjusted to provide equal reactivity (i.e. intensity of staining) for equivalent amounts of antigen. The affinity of the antibodies was determined by ELISA. The immunizing peptide for each antibody was linked to biotin at the NH₂ terminus and at a concentration of 10 nM was immobilized on a 96-well streptavidin-coated plate. Varying concentrations of each antibody ranging from a 1:10 to a 1:100,000 dilution were added followed by a rabbit IgG second antibody conjugated to alkaline phosphatase. Binding was assessed using a p-nitrophenyl phosphate substrate and monitoring OD at 405 nM. The antibodies exhibited different affinities for their respective antigen based on OD. Therefore, in order to attain equal reactivity for the Western analysis, the dilution of each antibody that yielded an OD of 0.5 was used. For the KEEE antibody a 1:20000 dilution and for the GELE antibody a dilution of 1:10000 was used.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Three sites of cleavage by aggrecanase-1 were identified within bovine aggrecan (Fig. 2). We first examined the ability of aggrecanase-1 to cleave at the traditional aggrecanase site, between Glu³⁷³-Ala³⁷⁴. Aggrecanase-1 was incubated with bovine aggrecan monomers at 37 °C for various amounts of time ranging from 0 to 20 h. The products were then analyzed using both the anti-ARGS monoclonal antibody, BC-3, that recognizes the new NH₂ terminus and the anti-NITEGE monoclonal antibody that recognizes the new COOH terminus, on aggrecan fragments generated by cleavage at the Glu³⁷³-Ala³⁷⁴ site. Western analysis showed the appearance of a 250-kDa BC-3-reactive fragment at 1 h that increased in band intensity over time (Fig. 3A). As expected, a NITEGE-reactive fragment was generated simultaneously with the BC-3 epitope (Fig. 3B) supporting cleavage at the Glu³⁷³-Ala³⁷⁴ bond by aggrecanase. The band of aggrecan fragments expressing the NITEGE epitope appeared as a doublet between 64 and 70 kDa, consistent with the G1-NITEGE products previously obtained following IL-1 treatment of bovine chondrocytes (12). Although faint NITEGE-reactive bands were visible at 15 and 30 min whereas the BC-3 band was not detected until 1 h, this is likely due to differences in sensitivity of the two antibodies.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Aggrecanase-1 cleavage sites in the aggrecan core protein.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Identification of aggrecan cleavage at the TEGE373-374ARGS bond and the KEEE1667-1668GLGS bond. Human recombinant aggrecanase-1 was incubated at 37 °C with 500 nM bovine aggrecan for 0 to 20 h. Following incubation the product was analyzed for by Western blot analysis for (A) BC-3-reactive fragments, (B) NITEGE-reactive fragments, (C) MAB2035-reactive fragments containing chondroitin sulfate stubs, and (D) KEEE-reactive fragments. The 375-, 250-, and 140-kDa fragments were NH2 terminally sequenced, and the first 5 residues were determined for each band.

By Western analysis with the MAB2035 antibody, a 500-kDa band that represents intact bovine aggrecan was seen at time 0, prior to digestion (Fig. 3C). Within 15 min the 500-kDa fragment was converted to two major fragments, including a 375- and 140-kDa fragment. Over time the 375-kDa band was converted to a 250-kDa fragment corresponding with the BC-3-reactive 250-kDa band. By 20 h there was ~50% conversion of the 375-kDa fragment to the 250-kDa fragment. Interestingly, neither the 375- nor the 140-kDa bands that appeared before the 250-kDa band reacted with the BC-3 antibody. These data indicate that aggrecanase-1 preferentially cleaves aggrecan at an alternative site within the aggrecan molecule.

In order to determine the preferential site of cleavage, the three major fragments (375, 250, and 140 kDa) generated by aggrecanase-1 were assessed by NH₂-terminal sequencing. As expected, the intact 500-kDa band was found to have the NH₂ terminus VEVSE that corresponds to the NH₂ terminus of native, intact bovine aggrecan (Fig. 3C). The 375-kDa fragment was also found to have the NH₂ terminus VEVSE, indicating that this fragment has an intact NH₂ terminus, and thus the preferential site of cleavage by aggrecanase is within the COOH terminus of the aggrecan molecule. The 250-kDa fragment was found to have the NH₂ terminus ARGSV indicating that this fragment is the result of aggrecanase-1 cleavage at the Glu³⁷³-Ala³⁷⁴ site consistent with the BC-3 Western blot that shows the 250-kDa fragment reacts with the BC-3 antibody. Finally, the 140-kDa fragment, presumably generated by cleavage of aggrecan at the preferential site was found to have the NH₂ terminus GLGS. This sequence is located within the chondroitin sulfate-rich region between the G2 and G3 globular domains. These data suggest that the preferential site of cleavage is between Glu¹⁶⁶⁷-Gly¹⁶⁶⁸ which would result in the formation of two large fragments, a 375-kDa fragment with an intact NH₂ terminus and a new COOH terminus, KEEE, and a 140-kDa fragment with an intact COOH terminus and a new NH₂ terminus, GLGS.

In order to confirm cleavage at the Glu¹⁶⁶⁷-Gly¹⁶⁶⁸ site, a neo-epitope antibody was generated to recognize the KEEE COOH terminus. This KEEE antibody was then used in a Western analysis to follow the aggrecanase-1-mediated cleavage of bovine aggrecan over time. The KEEE Western blot showed the appearance of a 375-kDa fragment within 15 min (Fig. 3D) that corresponds to a 375-kDa fragment detected in the CSPG Western (Fig. 3C). However, over time the 375-kDa fragment began to undergo conversion to a 55-kDa fragment. By 2 h the 375-kDa KEEE-reactive fragment was completely converted to the 55-kDa band. The 55-kDa fragment did not react with the BC-3 antibody, suggesting that there is another cleavage in addition to that at Glu¹⁶⁶⁷-Gly¹⁶⁶⁸ that occurs faster than at the Glu³⁷³-Ala³⁷⁴ bond. Based on molecular mass and the fact that this fragment reacts with the KEEE antibody, suggests that the other cleavage site is downstream from the Glu¹⁶⁶⁷-Gly¹⁶⁶⁸ cleavage site. Based on sequence homology, the most likely site of aggrecanase-1 cleavage would be at Glu¹⁴⁸⁰-Gly¹⁴⁸¹.

To confirm that this was the site of cleavage resulting in the production of the 55-kDa fragment, a neo-epitope antibody was produced to recognize the COOH terminus GELE that would be produced by cleavage at the Glu¹⁴⁸⁰-Gly¹⁴⁸¹ bond. This antibody was then used in a Western blot analysis to follow cleavage of bovine aggrecan. The GELE Western blot showed the appearance of a 375-kDa fragment within 15 min (Fig. 4A) that corresponds to a 375-kDa fragment detected in the CSPG Western (Fig. 4B). Over time, the 375-kDa fragment began to convert to a 250-kDa fragment that reacted with the GELE antibody, confirming that this fragment had undergone cleavage at the Glu¹⁴⁸⁰-Gly¹⁴⁸¹ bond. By 8 h, 50% of the product represented the 375-kDa GELE-reactive fragment and 50% represented the 250-kDa GELE-reactive fragment. This 250-kDa fragment also reacted with the BC-3 antibody (Fig. 4C), indicating that the 250-kDa fragment has the NH₂ terminus ARGS and the COOH terminus GELE. Thus, based on these data, aggrecanase-1 cleaves aggrecan preferentially at two sites within the COOH terminus of aggrecan. Cleavage results in the formation of a 140-kDa fragment with an intact COOH terminus and the new NH₂ terminus, GLGS, a 55-kDa fragment with the new NH₂ terminus, GRGT, and a new COOH terminus, KEEE, and a 375-kDa fragment with an intact NH₂ terminus and a COOH terminus of either KEEE or GELE. Based on these data, it is clear that the 375-kDa fragment detected in the CSPG blot is a fine doublet containing two product species. The data also suggest that the 375-kDa KEEE fragment is a short-lived intermediate, based on its rapid conversion to the 55-kDa fragment (Fig. 3D). The persistence of the 375-kDa GELE fragment for longer times (Fig. 4A) suggests that it is a more stable intermediate, and most likely the major component of the bands detected with mAB 3025 in Figs. 3C and 4B.

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Identification of aggrecan cleavage at the GELE1480-1481GRGT bond. Human recombinant aggrecanase-1 was incubated at 37 °C with 500 nM bovine aggrecan for 0 to 8 h. Following incubation the product was analyzed by Western blot analysis for GELE-reactive fragments (A), MAB2035-reactive (B) fragments containing chondroitin sulfate stubs and BC-3 reactive fragments (C).

In order to determine whether the greater rate of cleavage within the CS-rich region occurred at Glu¹⁶⁶⁷-Gly¹⁶⁶⁸ or Glu¹⁴⁸⁰-Gly¹⁴⁸¹, aggrecanase-1-mediated cleavage was evaluated at early time periods. For these studies, antibody concentrations were used which resulted in equal reactivity of the KEEE and GELE antibodies with there respective antigens. The 375-kDa KEEE-reactive fragment appeared in less than 3 min (Fig. 5A). In contrast, the 375-kDa GELE-reactive fragment did not appear until 24 min (Fig. 5B), well after the appearance of the KEEE-reactive fragment. This was followed by cleavage of the 375-kDa fragment within the IGD at the Glu³⁷³-Ala³⁷⁴ site to form the 250-kDa GELE-reactive fragment (Fig. 5B). These data collectively demonstrate that aggrecanase-1 cleaves faster at the Glu¹⁶⁶⁷-Gly¹⁶⁶⁸ than at the Glu¹⁴⁸⁰-Gly¹⁴⁸¹ site (Fig. 2).

View larger version (47K): [in this window] [in a new window]   Fig. 5.   Comparison of efficiency of cleavage at the KEEE1667-1668GLGS bond and GELE1480-1481GRGT bond of aggrecan. Human recombinant aggrecanase-1 was incubated at 37 °C with 500 nM bovine aggrecan for 0 to 49 min. Following incubation the product was analyzed by Western blot analysis for: A, KEEE-reactive fragments; and B, GELE-reactive fragments. Amounts of reaction mixture were loaded which provided equivalent reactivity of KEEE and GELE antibodies.

We next evaluated processing of the 140-kDa G3 fragment with the NH₂ terminus GLGS, formed by the preferential cleavage at the Glu¹⁶⁶⁷-Gly¹⁶⁶⁸ site (Fig. 6). In order to determine the fate this fragment, a neo-epitope antibody to the NH₂ terminus GLGS was used to follow cleavage. A 140-kDa GLGS-reactive fragment was detectable following digestion for 5 min (Fig. 7A). Band intensity increased over time, peaking at 30 min. However, after 60 min this fragment began to decrease in intensity and by 6 h no band was detected. The disappearance of the 140-kDa band did not correspond with the appearance of smaller molecular fragments using 4-12% Tris glycine gels, which have a molecular mass range between 400 and 36 kDa, suggesting that the resulting fragment(s) were less than 36 kDa in mass. Thus, these data indicate that the 140-kDa fragment was further being cleaved by aggrecanase-1.

View larger version (6K): [in this window] [in a new window]   Fig. 6.   Cleavage sites within the COOH-terminal GLGS-reactive G3 fragment of aggrecan.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Identification of the cleavage of the GLGS-G3 aggrecan fragment at the TAQE1771-1772AGEG bond and the VSQE1871-1872LGQR bond. Human recombinant aggrecanase-1 was incubated at 37 °C with 500 nM bovine aggrecan for 0 to 360 min. Following incubation the reaction mixture was analyzed by Western blot for: A, GLGS-reactive fragments; B, AGEG-reactive fragments; and C, LGQR-reactive fragments.

Based on putative aggrecanase-sensitive sites within the COOH terminus and the formation of a fragment smaller than 36 kDa, the most likely site of cleavage was predicted to be between residues Glu¹⁷⁷¹-Ala¹⁷⁷². In order to determine whether aggrecanase-1 cleaves at this site, a neo-epitope antibody to the new NH₂ terminus AGEG was used in a Western analysis to evaluate aggrecan digests. An AGEG-reactive, 120-kDa fragment was detected within 5 min, and increased in band intensity up to 30 min (Fig. 7B). At 30 min the band intensity peaked and was maintained through 4 h. However, at 6 h the band intensity began to decrease and by 24 h the band was no longer detectable. The loss of the 120-kDa AGEG-reactive fragment suggested yet another site of cleavage downstream from the Glu¹⁷⁷¹-Ala¹⁷⁷² cleavage site. The most likely site of cleavage was predicted to be between residues Glu¹⁸⁷¹-Leu¹⁸⁷². To determine whether aggrecanase-1 was in fact cleaving at this site, a neo-epitope antibody to the new NH₂ terminus, LGQR was used to evaluate digests. An LGQR-reactive, 98-kDa fragment was observed by 5 min that increased in band intensity up to 60 min (Fig. 7C). The band intensity was maintained throughout the remainder of the time course, indicating no further processing of the 98-kDa reactive fragment.

Taken together, these experiments indicate that aggrecanase-1 cleaves the 140-kDa GLGS-reactive G3 aggrecan fragment at the Glu¹⁷⁷¹-Ala¹⁷⁷² and residues Glu¹⁸⁷¹-Leu¹⁸⁷² bonds. This cleavage results in the generation of a 98-kDa fragment with an intact COOH terminus and the new NH₂ terminus LGQR and two small fragments of ~20 kDa mass, one with the NH₂ terminus AGEG and the COOH terminus VSQE and a second with the NH₂ terminus GLGS and the COOH terminus TAQE (Fig. 6).

Finally, we looked at the ability of aggrecanase-1 to cleave at the MMP site, between residues Asn³⁴¹-Phe³⁴² using the monoclonal antibody AF-28 that recognizes the new NH₂ terminus FFGVG on aggrecan fragments generated upon cleavage by MMPs at Asn³⁴¹-Phe³⁴². Western blot analysis demonstrated that, as expected, aggrecanase-1 did not cleave at the MMP site (data not shown), consistent with the enzyme profile for aggrecanase in bovine cartilage explant cultures and rat chondrosarcoma cell lines (9-15).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

These studies provide the first demonstration of the ability of a protease to cleave aggrecan at the putative aggrecanase-sensitive sites within the COOH terminus of the molecule. Identification of aggrecan fragments with the NH₂ terminus AGEG, GLGS, and LGQR in media from cartilage explants stimulated with IL-1 or retinoic acid indicated that cleavage was occurring within the COOH terminus of aggrecan (11, 14, 15). Sequence alignment of the putative degradation sites with the site of aggrecanase-mediated cleavage within the IGD showed that all sites contained a glutamic acid residue in the P₁ position and a non-polar or uncharged polar residue (alanine, leucine, or glycine) in the P₁' position. Thus, it was speculated that a single enzyme generated all these fragments. However, confirmation that this was the case awaited purification and identification of cartilage aggrecanase.

The studies reported herein demonstrate that aggrecanase-1 cleaves at each of the proposed sensitive sites and describes the relative efficiency of cleavage at each of the sites. As expected, aggrecanase-1 cleaves within the IGD of aggrecan at the Glu³⁷³-Ala³⁷⁴ bond and does not cleave at the Asn³⁴¹-Phe³⁴² MMP cleavage site. However, we have shown that this site within the IGD is not the preferential site of cleavage by aggrecanase-1. The enzyme most efficiently cleaves within the CS-rich G2-G3 domain at the Glu¹⁶⁶⁷-Gly¹⁶⁶⁸ bond to release the COOH terminus of the molecule. The G1 fragment is further trimmed by cleavage at the Glu¹⁴⁸⁰-Gly¹⁴⁸¹ bond to remove an additional small portion of the COOH terminus. Cleavage within the IGD at the Glu³⁷³-Ala³⁷⁴ bond occurs more slowly to generate the fragment with the ARGS NH₂ terminus that has been identified both in tissue culture medium during cytokine or retinoic acid-induced cartilage matrix degradation and in synovial fluids of patients with various types of arthritis. Furthermore, using the neo-epitope antibodies generated for the present studies, we have shown in IL-1-stimulated bovine articular cartilage explant cultures that cleavage of aggrecan in response to IL-1 generates the same cleavage fragments as demonstrated in these studies using soluble aggrecan and recombinant aggrecanase-1.²

Although these studies clearly demonstrate that cleavage occurs preferentially at certain sites within aggrecan, preferential could mean "first" or it could mean "faster" and the experiments conducted cannot completely resolve this point. The lack of detection of BC-3-reactive fragments prior to 1 h despite of the fact that there are weak bands at 250 kDa representing an ARGS fragment seen at earlier times in blots using the MAB3025 (Figs. 3C and 4B) and in blots using the GELE antibody (Figs. 4A and 5B) suggests that this is due to lower BC-3 antibody sensitivity. However, the data in Fig. 5B using the GELE antibody to detect both the G1-GELE fragment and the ARGS-GELE fragment, clearly support cleavage at the Glu¹⁴⁸⁰-Gly¹⁴⁸¹ bond to produce the G1-GELE fragment occurring more rapidly than the cleavage at the Glu³⁷³-Ala³⁷⁴ bond to produce the ARGS-GELE fragment. In this study where earlier time points were assessed, the 375-kDa GELE-reactive fragment was detected at 24 and 27 min when the 250-kDa GELE-reactive fragment was not present. A weak GELE-reactive band at 250 kDa, representing the ARGS fragment, was observed by 31 min that then increased in intensity over time.

In addition, in Fig. 5, where conditions resulting in equal reactivity for the GELE and KEEE epitopes were used, it appears that the G1-KEEE fragment is produced faster than the G1-GELE fragment as well as the ARGS-GELE fragment. However, because equal reactivity was determined based on antibody reactivity to the immunizing peptide by ELISA, there is the possibility that the orientated presentation of the peptides on the ELISA plate may be more or less favorable for antibody binding than the native epitope immobilized on a membrane. Therefore, it is difficult to unequivocally assure absolute equal reactivity in the Western analysis.

The GLGS-reactive G3 fragment generated by cleavage at Glu¹⁶⁶⁷-Gly¹⁶⁶⁸ was further processed at two additional sites at similar rates of cleavage. One clip occurred between residues Glu¹⁷⁷¹-Ala¹⁷⁷² and the other between residues Glu¹⁸⁷¹-Leu¹⁸⁷². This generated two small fragments, one with the NH₂ terminus ¹⁶⁶⁸GLGS and the new COOH terminus TAQE¹⁷⁷¹ and another with the new NH₂ terminus ¹⁷⁷²AGEG and the new COOH terminus VSQE¹⁸⁷¹, as well as a 98-kDa G3 fragment with the new NH₂ terminus ¹⁸⁷²LGQR. These data are also consistent with the involvement of aggrecanase in cartilage aggrecan degradation, as aggrecan G3 fragments with the NH₂ terminus AGEG and LGQR have been identified in media from IL-1-stimulated bovine articular cartilage explant cultures (15). However, since these cleavages serve only to further clip the GLGS-reactive G3 fragment following its release from the intact aggrecan core protein, they may be less critical to loss of aggrecan from the cartilage than the first three cleavages.

The five cleavage sites described appear to be the only major sites of cleavage for aggrecanase-1 within aggrecan. However, we have found using high concentrations of enzyme with long incubation times, that several very minor additional bands of BC-3-reactive fragments can be detected (data not shown). This suggests that aggrecanase has the capability of further trimming the ARGS to GELE fragment. The high concentrations of enzyme and long time required for these cleavages suggest that they are of minor importance. However, they may be responsible for the additional bands of BC-3-reactive fragments sometimes detected in cartilage cultures.

Although the current work focused on cleavage of aggrecan by aggrecanase-1, we have also followed the cleavage by aggrecanase-2 (also designated ADAMTS-11 or ADAMTS-5) using the KEEE, GELE, and BC-3 antibodies and find a similar pattern and order of cleavage (data not shown). In addition, like aggrecanase-1, aggrecanase-2 does not cleave at the Asn³⁴¹-Phe³⁴² bond. Our demonstration that aggrecanase-1 and aggrecanase-2 cleave between Glu³⁷³-Ala³⁷⁴ but do not cleave at the Asn³⁴¹-Phe³⁴² MMP site make these the only enzymes to date shown to cleave at the aggrecanase site within the IGD without cleaving at the MMP site. Prior to these studies, two proteases had been shown to cleave at the Glu³⁷³-Ala³⁷⁴ aggrecanase site, MMP-8 (22, 23) and atrolysin-C (24). MMP-8 had been shown to cleave at the Glu³⁷³-Ala³⁷⁴ bond, but only following complete cleavage at the Asn³⁴¹-Phe³⁴² MMP site. Although atrolysin-C did not require prior cleavage at the Asn³⁴¹-Phe³⁴² bond, it also cleaved aggrecan readily at this site. In addition, these enzymes were >100-fold less efficient than the aggrecanases in cleaving aggrecan at the Glu³⁷³-Ala³⁷⁴ bond.

Since aggrecanase(s) cleaves more efficiently at sites within the COOH terminus of the molecule than within the IGD, neo-epitope antibodies to fragments generated by cleavage at one or more of these sites may have application as early surrogate markers of aggrecanase activity. These markers could potentially detect increases in aggrecanase activity early on in the disease process, prior to the cleavage within the IGD that releases the entire COOH terminus of the molecule and leads to loss of cartilage mechanical properties. Studies evaluating media and cartilage extracts from IL-1-stimulated bovine articular cartilage explant cultures identified fragments containing the COOH terminus KEEE¹⁶⁶⁷ and GELE¹⁴⁸⁰ resulting from aggrecanase cleavage at Glu¹⁶⁶⁷-Gly¹⁶⁶⁸ and Glu¹⁴⁸⁰-Gly¹⁴⁸¹, respectively.³ In addition, the sites and efficiency of cleavage in these cartilage cultures was the same as that observed upon digestion of aggrecan by aggrecanase-1, suggesting that the cleavage of aggrecan in this system is due to aggrecanase and that these fragments may provide markers of aggrecanase activity. Current studies are focusing on evaluation of synovial fluid samples for products formed by aggrecanase cleavage within the COOH terminus of the molecule compared with those produced by cleavage within the IGD.

The preferential cleavage by aggrecanase within the COOH terminus of the aggrecan core protein raises the question of whether this cleavage is required for cleavage to occur within the IGD. The data presented in this study do not definitively provide an answer. Determining if one cleavage is dependent on the other will require cloning and expression of aggrecan with targeted mutations at these cleavage sites. Interestingly, however, in bovine articular cartilage stimulated with IL-1, Ilic et al. (15) detected shortened aggrecan fragments with the VEVS NH₂ terminus and no ARGS fragments extending into the G3 domain, suggesting that cleavage within the interglobular domain is not the primary event in aggrecan degradation. Our studies evaluating aggrecan cleavage by recombinant aggrecanase-1 are consistent with this work and, in addition, suggest that aggrecanase is responsible for cleavage within the COOH terminus of the core protein.

Regardless of whether prior cleavage within the COOH terminus is required for cleavage in the IGD, this preferential cleavage by aggrecanase may have implications for changes in the susceptibility of cartilage aggrecan to degradation with age. Work investigating the presence of an intact G3 on human articular cartilage aggrecan (19) has shown that, although the majority of the aggrecan monomers from young cartilage contain an intact G3, in mature and older cartilage a very small proportion of the total aggrecan molecules possess a G3. These data indicate that the COOH terminus of the molecule had been cleaved in older cartilage. Although the site of cleavage was not determined in these studies, it is feasible that aggrecanase may play a role in trimming the core protein with age by cleavage within the COOH terminus. Elimination of the preferred cleavage sites within the COOH terminus of aggrecan would be expected to lead to an increased rate of cleavage by aggrecanase within the IGD. Thus, aggrecan may become more susceptible to cleavage within the IGD and release of the entire COOH terminus of the molecule from the matrix with age.

These studies are the first to examine aggrecan degradation by human aggrecanase-1. Not only does this work elucidate the sites and efficiency of aggrecan cleavage by aggrecanase, but suggests potential tools for monitoring aggrecan degradation and approaches for therapy in arthritic diseases.

	ACKNOWLEDGEMENTS

We thank Dr. Amanda Fosang for the kind gift of the AF-28 monoclonal antibody. We are grateful to Dr. Ada Cole and Barbara L. Schumacher (Rush Presbyterian, St. Luke's Medical Center, Chicago, IL) for performing the NITEGE Western blot analysis.

	FOOTNOTES

^* 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: The Kennedy Institute of Rheumatology, 1 Aspenlea Rd., Hammersmith, London W6 8LH, United Kingdom. Tel.: 44-208-383-4446; Fax: 44-181-383-4499; E-mail: tortormd@aol.com.

Published, JBC Papers in Press, April 4, 2000, DOI 10.1074/jbc.M909383199

² M. T. Tortorella, M. Pratta, R-Q. Liu, J. Austin, O. H. Ross, I. Abbaszade, T. Burn, and E. Arner, unpublished data.

³ M. Tortorella, M. Pratta, and E. Arner, unpublished data.

	ABBREVIATIONS

The abbreviations used are: IGD, interglobular domain; MMP, matrix metalloproteinase; IL-1, interleukin-1; ELISA, enzyme-linked immunosorbent assay.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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