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A recombinant human aggrecan G1-G2 fragment comprising amino acids Val(1)-Arg(656) has been expressed in Sf21 cells using a baculovirus expression system. The recombinant G1-G2 (rG1-G2) was purified to homogeneity by hyaluronan-Sepharose affinity chromatography followed by high performance liquid chromatography gel filtration, and gave a single band of M(r) 90,000-95,000 by silver stain or immunoblotting with monoclonal antibody 1-C-6. The expressed G1-G2 bound to both hyaluronan and link protein indicating that the immunoglobulin-fold motif and proteoglycan tandem repeat loops of the G1 domain were correctly folded. Further analysis of secondary structure by rotary shadowing electron microscopy confirmed a double globe appearance, but revealed that the rG1-G2 was more compact than its native counterpart. The size of rG1-G2 by SDS-polyacrylamide gel electorphoresis was unchanged following digestion with keratanase and keratanase II and reduced by only 2-5 kDa following digestion with either O-glycosidase or N-glycosidase F. Recombinant G1-G2 was digested with purified matrix metalloproteinases (MMP), isolated aggrecanase, purified atrolysin C, or proteinases present in conditioned medium from cartilage explant cultures, and the products analyzed on SDS gels by silver stain and immunoblotting. Neoepitope antibodies recognizing the N-terminal F(342)FGVG or C-terminal DIPEN(341) sequences were used to confirm MMP cleavage at the Asn(341) downward arrow Phe bond, while neoepitope antibodies recognizing the N-terminal A(374)RGSV or C-terminal ITEGE(373) sequences were used to confirm aggrecanase cleavage at the Glu(373) downward arrow Ala bond. Cleavage at the authentic MMP and aggrecanase sites revealed that these proteinases have the same specificity for rG1-G2 as for native aggrecan. Incubation of rG1-G2 with conditioned medium from porcine cartilage cultures revealed that active soluble aggrecanase but no active MMPs, was released following stimulation with interleukin-1alpha or retinoic acid. Atrolysin C, which cleaves native bovine aggrecan at both the aggrecanase and MMP sites, efficiently cleaved rG1-G2 at the aggrecanase site but failed to cleave at the MMP site. In contrast, native glycosylated G1-G2 with or without keratanase treatment was cleaved by atrolysin C at both the aggrecanase and MMP sites. The results suggest that the presence or absence per se of keratan sulfate on native G1-G2 does not affect the activity of atrolysin C toward the two sites.

A method was developed for generating soluble, active "aggrecanase" in conditioned media from interleukin-1-stimulated bovine nasal cartilage cultures. Using bovine nasal cartilage conditioned media as a source of the aggrecanase enzyme, an enzymatic assay was established employing purified aggrecan monomers as a substrate and monitoring specific aggrecanase-mediated cleavage products by Western analysis using the monoclonal antibody, BC-3 (which recognizes the new N terminus, ARGS, on fragments produced by cleavage between amino acid residues Glu373 and Ala374). Using this assay we have characterized cartilage aggrecanase with respect to assay kinetics, pH and salt optima, heat sensitivity, and stability upon storage. Aggrecanase activity was inhibited by the metalloprotease inhibitor, EDTA, while a panel of inhibitors of serine, cysteine, and aspartic proteinases had no effect, suggesting that aggrecanase is a metalloproteinase. Sensitivity to known matrix metalloproteinase inhibitors as well as to the endogenous tissue inhibitor of metalloproteinases, TIMP-1, further support the notion that aggrecanase is a metalloproteinase potentially related to the ADAM family or MMP family of proteases previously implicated in the catabolism of the extracellular matrix.

Cleavage of aggrecan core protein at the Glu373-Ala374 site by the unidentified enzyme, "aggrecanase," is thought to play an important role in cartilage degradation. To examine aggrecan cleavage by MMP-8 at this aggrecanase site, we evaluated the release of fragments with the N terminus ARGSVIL from freeze-thawed bovine nasal cartilage using the monoclonal antibody BC-3. Recombinant human MMP-8 catalytic domain cleaved native aggrecan in a concentration-related manner between 0.2 and 2 microg/ml, with complete release of glycosaminoglycan at 2 microg/ml or greater. Cleavage at the aggrecanase site was observed only at MMP-8 concentrations resulting in complete release of glycosaminoglycan from the cartilage, suggesting that preferential cleavage occurs at a different site. Time course studies indicated that only following depletion of substrate containing the preferred clip site did MMP-8 rapidly cleave at the aggrecanase site. Finally, MMP-8 resulted in a different pattern of BC-3-reactive fragments from that produced by endogenous aggrecanase in live cartilage, and SA751(N-(1(R)-carboxyethyl) -alpha-(S)-(4-phenyl-3-butynyl)glycyl-L-O-methyltyrosine, N-methylamide), a potent inhibitor of MMP-8 (Ki = 2 nM) which was effective in blocking cleavage by MMP-8 at the aggrecanase site with an IC50 in the nanomolar range, did not prevent aggrecan degradation or specific cleavage at this site by endogenously generated aggrecanase at concentrations up to 100 microM. Taken together these data suggest that MMP-8 does not represent cartilage aggrecanase.

Two primary cleavage sites have been identified within the interglobular domain of the cartilage aggrecan core protein: one is between amino acid residues Asn 341 and Phe342, where many matrix metalloproteinases (MMP) have been shown to cleave; and the other is between amino acid residues Glu373 and Ala374. Although cleavage at the Glu373-Ala374 site is believed to play a critical role in cartilage aggrecan degradation in arthritic diseases, the enzyme responsible for cleavage at this site, "aggrecanase," has not been identified. Members of the ADAM (a disintegrin and metalloproteinase) family of proteins, which shows structural homology to the snake venom hemorrhagic metalloproteinases (reprolysins), have recently been demonstrated to be expressed in articular chondrocytes. Because many ADAM family members have a putative proteinase function, this raises the possibility that aggrecanase may be a member of this family of proteases. To examine whether reprolysins have the ability to cleave aggrecan at either the aggrecanase site or the MMP site, the snake venom hemorrhagic toxin metalloproteinase HT-d (atrolysin C) was tested for its ability to cleave bovine aggrecan monomer. Cleavage was monitored using the BC-3 antibody, which recognizes aggrecan fragments with the new NH2 terminus ARGSV generated by cleavage at the aggrecanase site, and with the AF-28 antibody, which recognizes aggrecan fragments with the new NH2 terminus FFGVG generated by cleavage at the MMP site. Cleavage at both the aggrecanase and MMP sites occurred in a concentration-dependent manner with 100 nM atrolysin C or greater. AF-28-reactive fragments were generated by 30 min of incubation, and levels were maximal by 8 h; BC-3-reactive fragments were detected at 2 h and continued to increase through 48 h, thus suggesting that atrolysin C can cleave at the MMP and aggrecanase sites. NH2-terminal aggrecan fragments generated by cleavage at the aggrecanase site were also detected using antisera recognizing the new COOH terminus, NITEGE, formed by cleavage at the Glu373-Ala374 bond, indicating that cleavage at this site does not require prior cleavage at the MMP site. These data provide the first demonstration that a reprolysin can cleave the core protein of aggrecan and the first example of a specific protease that can cleave at the aggrecanase site independent of cleavage at the MMP cleavage site.