Analysis of aggrecan in human knee cartilage and synovial fluid indicates that aggrecanase (ADAMTS) activity is responsible for the catabolic turnover and loss of whole aggrecan whereas other protease activity is required for C-terminal processing in vivo.

Studies of aggrecan proteolysis in human joints have implicated both the aggrecanase [ADAMTS, a disintegrin-like and metalloprotease (reprolysin-type) with thrombospondin type 1 motif] and matrix metalloproteinase (MMP) families. We have analysed the aggrecan core protein species present in vivo in both articular cartilage and synovial fluids from normal, acutely injured and osteoarthritic joints. Normal cartilage contains at least seven major G1 domain (the N-terminal globular domain of aggrecan)-bearing species, of which three (full-length core, G1-NITEGE(373) and G1-VDIPEN(341)) have been identified. The C-terminals of the others are unknown but digestion of fetal human aggrecan with MMP-3 and crude aggrecanase suggests that they are products of MMP-like activity in vivo. Normal synovial fluids contain at least 10 species, of which nine result from ADAMTS-dependent cleavage, and this cleavage occurs at all of the five known aggrecanase sites. Aggrecan fragments in the cartilage and synovial fluids of acutely injured joints are generally similar to normal, but all contain a markedly increased ratio of G1-NITEGE to G1-VDIPEN. Aggrecan from the cartilage of late-stage osteoarthritis patients is remarkably similar to normal, whereas the synovial fluid aggrecan is more fragmented than that from normal or injured knees. The analyses suggest that the role of the ADAMTS and these MMP-like activities in human cartilage are distinctly different. Excessive ADAMTS activity in vivo is destructive to cartilage matrix, since the bulk of the glycosaminoglycan (GAG)-bearing products are released from the tissue into the synovial fluid following cleavage of the Glu(373)-Ala(374) bond. In contrast, the MMP-like activity appears to be essentially non-destructive, since much of the GAG-bearing product is retained in the tissue following cleavages that are in the more C-terminal regions of the molecule.

Versican V1 proteolysis in human aorta in vivo occurs at the Glu441-Ala442 bond, a site that is cleaved by recombinant ADAMTS-1 and ADAMTS-4.

Mature human aorta contains a 70-kDa versican fragment, which reacts with a neoepitope antiserum to the C-terminal peptide sequence DPEAAE. This protein therefore appears to represent the G1 domain of versican V1 (G1-DPEAAE(441)), which has been generated in vivo by proteolytic cleavage at the Glu(441)-Ala(442) bond, within the sequence DPEAAE(441)-A(442)RRGQ. Because the equivalent aggrecan product (G1-NITEGE(341)) and brevican product (G1-EAVESE(395)) are generated by ADAMTS-mediated cleavage of the respective proteoglycans, we tested the capacity of recombinant ADAMTS-1 and ADAMTS-4 to cleave versican at Glu(441)-Ala(442). Both enzymes cleaved a recombinant versican substrate and native human versican at the Glu(441)-Ala(442) bond and the mature form of ADAMTS-4 was detected by Western analysis of extracts of aortic intima. We conclude that versican V1 proteolysis in vivo can be catalyzed by one or more members of the ADAMTS family of metalloproteinases.

The intermediates of aggrecanase-dependent cleavage of aggrecan in rat chondrosarcoma cells treated with interleukin-1.

We have examined the abundance and structure of intermediates in the chondrocyte-mediated degradation of aggrecan by aggrecanase(s). Degradation products were identified by Western-blot analysis with antibodies to cleavage-site neoepitopes and to peptides within the globular domains. Rat chondrosarcoma tumour contained full-length aggrecan and all of the individual peptides expected from single independent cleavages at each of the four aggrecanase sites in the chondroitin sulphate (CS) domain. Kinetic analysis of the products present in rat chondrosarcoma cell cultures treated with interleukin-1b showed that the first aggrecanase-mediated cleavages occurred at the four sites within the CS attachment region to generate two stable intermediates, Val(1)-Glu(1459) and Val(1)-Glu(1274). These species were subsequently cleaved at the Glu(373) site in the interglobular domain to form the terminal products, Val(1)-Glu(373), Ala(374)-Glu(1274) and Ala(374)-Glu(1459). It therefore appears that the aggrecanase-mediated processing of native aggrecan by chondrocytes in situ is initiated within the CS-attachment region and completed by cleavage within the interglobular domain. Since it has been shown that digestion of aggrecan monomer in solution with recombinant ADAMTS-4 [Tortorella, Pratta, Liu, Austin, Ross, Abbaszade, Burn and Arner (2000) Sites of aggrecan cleavage by recombinant human aggrecanase-1 (ADAMTS-4). J. Biol. Chem. 275, 18566-18573] exhibits similar kinetics, it appears that preferential proteinase cleavage in the CS-rich region is determined by properties inherent in the aggrecan monomer itself, such as preferred peptide sequences for enzyme binding or enhanced accessibility to the core protein at these sites.

Chondrocyte-mediated catabolism of aggrecan: evidence for a glycosylphosphatidylinositol-linked protein in the aggrecanase response to interleukin-1 or retinoic acid.

The control of chondrocyte-mediated degradation of aggrecan has been studied in rat chondrosarcoma cells and bovine cartilage explants treated with either IL-1 or retinoic acid. The capacity of glucosamine to inhibit the aggrecanase-mediated response (J. D. Sandy, D. Gamett, V. Thompson, and C. Verscharen (1998) Biochem. J. 335, 59-66) has been extended to an investigation of the effect of other hexosamines. Mannosamine inhibits the aggrecanase response to both IL-1 and RA at about one-tenth the concentration of glucosamine in both rat cell and bovine explant systems. This effect of mannosamine appears to be due to its capacity to inhibit the synthesis of glycosylphosphatidylinositol (GPI)-linked proteins by chondrocytes since the GPI synthesis inhibitor 2-deoxyfluoroglucose (2-DFG) also inhibited the aggrecanase response to IL-1b and RA in rat cells. Moreover, phosphatidylinositol-specific phospholipase C (PIPLC) treatment of rat cells markedly inhibited the aggrecanase response to IL-1b and RA. These inhibitory effects of mannosamine, 2-DFG, and PIPLC in rat cells did not appear to be due to an interference with general biosynthetic activity of the cells as measured by [3H]proline incorporation into secreted proteins. We suggest that the aggrecanase response by chondrocytes to IL-1 and RA is dependent on the activity of a GPI-anchored protein on the chondrocyte cell surface.

Chondrocyte-mediated catabolism of aggrecan: aggrecanase-dependent cleavage induced by interleukin-1 or retinoic acid can be inhibited by glucosamine.

A rat chondrosarcoma cell line and bovine cartilage explants have been used to study the control of aggrecan degradation by chondrocytes treated with interleukin-1 (IL-1) or retinoic acid (RA). Aggrecan fragment analysis with anti-neo-epitope antibodies suggests that aggrecanase (an as yet unidentified enzyme) is the only aggrecan-degrading proteinase active in these cultures. With rat cells, aggrecanase converts the aggrecan core protein into two major G1-domain-bearing products (60 kDa with a C-terminal Glu-373, and 220 kDa with a C-terminal Glu-1459). Both products were quantified on a standardized Western analysis system with a G1-specific antibody. Immunoblots were analysed by scanning densitometry and the sensitivity, linearity and reproducibility of the assay were established. With rat cells the aggrecanase response to IL-1 was optimal at about 2 mM glutamine, but was progressively inhibited at higher concentrations, with about 90% inhibition at 10 mM glutamine. Such inhibition by glutamine was not, however, observed with bovine explants. On the other hand, marked inhibition of aggrecanase-dependent cleavage was observed with both rat cells and bovine explants when d(+)-glucosamine was included at concentrations above 2 mM. Inhibition was apparently not due to cytotoxicity or interference with IL-1 signalling, since biosynthetic activity was not inhibited and inhibition of the aggrecanase response was also obtained when RA was used as the catabolic stimulator. Possible mechanisms for the inhibition of the aggrecanase response by glucosamine in chondrocytes treated with IL-1 or RA are discussed.