Age-related changes in aggrecan glycosylation affect cleavage by aggrecanase

Aggrecan degradation involves proteolytic cleavage of the core protein within the interglobular domain. Because aggrecan is highly glycosylated with chondroitin sulfate (CS) and keratan sulfate (KS), we investigated whether glycosylation affects digestion by aggrecanase at the Glu(373)-Ala(374) bond. Treatment of bovine aggrecan monomers to remove CS and KS resulted in loss of cleavage at this site, suggesting that glycosaminoglycans (GAGs) play a role in cleavage at the Glu(373)-Ala(374) bond. In contrast, MMP-3 cleavage at the Ser(341)-Phe(342) bond was not affected by glycosidase treatment of aggrecan. Removal of KS, but not CS, prevented cleavage at the Glu(373)-Ala(374) bond. Thus, KS residues may be important for recognition of this cleavage site by aggrecanase. KS glycosylation has been observed at sites adjacent to the Glu(373)-Ala(374) bond in steer aggrecan, but not in calf aggrecan (Barry, F. P., Rosenberg, L. C., Gaw, J. U., Gaw, J. U., Koob, T. J., and Neame, P. J. (1995) J. Biol. Chem. 270, 20516-20524). Interestingly, although we found that aggrecanase degraded both calf and steer cartilage aggrecan, the proportion of fragments generated by cleavage at the Glu(373)-Ala(374) bond was higher in steer than in calf, consistent with our observations using aggrecan treated to remove KS. We conclude that the GAG content of aggrecan influences the specificity of aggrecanase for cleavage at the Glu(373)-Ala(374) bond and suggest that age may be a factor in aggrecanase degradation of cartilage.     

Effects of covalently attached chondroitin sulfate on aggrecan cleavage by ADAMTS-4 and MMP-13

Aggrecan is degraded by several aggrecanase-1 (ADAMTS-4) isoforms differing in the number of sulfated glycosaminoglycan (sGAG)-binding motifs. ADAMTS-4 and MMPs cleave aggrecan more efficiently within the chondroitin sulfate (CS)-rich region than the interglobular domain (IGD). We investigated the influence of CS on aggrecan core protein cleavage by ADAMTS-4 (p68) and (p40) as well as MMP-13, which has no recognizable GAG-binding sites. Chondroitinase ABC-treated cartilage aggrecan was cleaved with ADAMTS-4 (p68) less efficiently than CS-substituted aggrecan within the CS-2 domain. Keratanase-treated aggrecan exhibited reduced IGD cleavage, but when both CS and KS were removed, the IGD cleavage was restored. This result suggests that KS in the IGD may compete with CS for ADAMTS-4 (p68) binding. In the absence of KS, however, p68 binding was shifted to the CS-2 domain. CS-deficient full-length recombinant aggrecan (rbAgg) was produced by chondroitinase ABC treatment, or by expression in the xylosyltransferase-deficient CHO-pgsA745 cell line. When digested with the ADAMTS-4 (p68), each of these preparations exhibited reduced CS-2 domain cleavage compared to CS-substituted CHO-K1 cell-derived aggrecan. Additionally, CS-deficient rbAgg showed increased IGD scission prior to cleavage within the CS-2 domain. ADAMTS-4 (p40) readily cleaved both rbAggs within the IGD, but cleaved poorly within the CS-2 domain, indicating little CS dependence. MMP-13, in contrast, cleaved the CS region and the IGD of both CS-substituted and CS-deficient rbAgg equally well. These data indicate that covalently bound CS enhances ADAMTS-4-mediated cleavage within the CS-rich region. MMP-13 also cleaves preferentially within the CS-region, but by an apparently CS-independent mechanism.     

Association of the aggrecan keratan sulfate-rich region with collagen in bovine articular cartilage

Aggrecan, the predominant large proteoglycan of cartilage, is a multidomain macromolecule with each domain contributing specific functional properties. One of the domains contains the majority of the keratan sulfate (KS) chain substituents and a protein segment with a proline-rich hexapeptide repeat sequence. The function of this domain is unknown but the primary structure suggests a potential for binding to collagen fibrils. We have examined binding of aggrecan fragments encompassing the KS-rich region in a solid-phase assay. A moderate affinity (apparent Kd = 1.1 microM) for isolated collagen II, as well as collagen I, was demonstrated. Enzymatic digestion of the KS chains did not alter the capacity of the peptide to bind to collagen, whereas cleavage of the protein core abolished the interaction. The distribution of the aggrecan KS-rich region in bovine tarsometatarsal joint cartilage was investigated using immunoelectron microscopy. Immunoreactivity was relatively low in the superficial zone and higher in the intermediate and deep zones of the uncalcified cartilage. Within the pericellular and territorial matrix compartments the epitopes representing the aggrecan KS-rich region were detected preferentially near or at collagen fibrils. Along the fibrils, epitope reactivity was non-randomly distributed, showing preference for the gap region within the D-period. Our data suggest that collagen fibrils interact with the KS-rich regions of several aggrecan monomers aligned within a proteoglycan aggregate. The fibril could therefore serve as a backbone in at least some of the aggrecan complexes.     

G1-G2 aggrecan product that can be generated by M-calpain on truncation at Ala709-Ala710 is present abundantly in human articular cartilage

To elucidate the specific function of m-calpain in the metabolism of aggrecan in human articular cartilage, the prevalence and localization of a large glycosaminoglycan-bearing aggrecan product generated by m-calpain in human osteoarthritis (OA) cartilage were investigated. Extracts of human OA articular cartilage were analysed by immunostaining using new polyclonal anti-VPGVA antiserum that detects the COOH terminal neoepitope IVTQVVPGVA(709) generated by m-calpain-related cleavage within the keratan sulphate rich region of human aggrecan. Immunoblotting analyses of aggrecan populations in guanidine hydrochloride-extracts showed that OA cartilages contained anti-VPGVA positive aggrecan products with the COOH terminal neoepitope ... VPGVA(709), resulting from truncation between the Ala(709)-Ala(710) m-calpain-related cleavage site. This aggrecan product consisted of two NH(2) terminal globular domain (G1 and G2) and KS side chains. Immunohistochemical staining showed that anti-VPGVA positive staining was localized within chondrocytes and spread to the surrounding interterritorial matrix. Confocal microscopic analysis showed subcellular colocalization of anti-VPGVA and anti m-calpain. These results indicate that the aggrecan product with the COOH terminal neoepitope VPGVA(709) is synthesized regularly by intracellular processing in chondrocytes, and is present abundantly as a limited form of aggrecan. M-calpain is the major candidate of the proteinase to generate this aggrecan product during the intracellular aggrecan processing.     

Mammalian expression of full-length bovine aggrecan and link protein: formation of recombinant proteoglycan aggregates and analysis of proteolytic cleavage by ADAMTS-4 and MMP-13

Aggrecan, a large chondroitin sulfate (CS) and keratan sulfate (KS) proteoglycan, has not previously been expressed as a full-length recombinant molecule. To facilitate structure/function analysis, we have characterized recombinant bovine aggrecan (rbAgg) and link protein expressed in COS-7 cells. We demonstrate that C-terminally truncated rbAgg was not secreted. Gel filtration chromatography of rbAgg and isolated glycosaminoglycan (GAG) chains, and their susceptibility to chondroitinase ABC digestion indicate that the GAG chains are predominantly CS, which likely occupy fewer serine residues than native aggrecan. To confirm functionality, we determined that rbAgg bound hyaluronan and recombinant link protein to form proteoglycan aggregates. In addition, cleavage of rbAgg by ADAMTS-4 revealed that the p68 form of ADAMTS-4 preferentially cleaves within the CS-2 domain, whereas the p40 form only effectively cleaves within the interglobular domain (IGD). MMP-13 cleaved rbAgg within the IGD, but cleaved more rapidly at a site within the CS domains, suggesting a role in C-terminal processing of aggrecan. Our results demonstrate that recombinant aggrecan can be used for in vitro analyses of matrix protease-dependent degradation of aggrecan in the IGD and CS domains, and both recombinant aggrecan and link protein can be used to study the assembly of proteoglycan aggregates with hyaluronan.     

MMPs are less efficient than ADAMTS5 in cleaving aggrecan core protein

Aggrecan degradation in articular cartilage occurs predominantly through proteolysis and has been attributed to the action of members of the matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) families. Both families of enzymes cleave aggrecan at specific sites within the aggrecan core protein. One cleavage site within the interglobular domain (IGD), between Glu^373-374Ala and five additional sites in the chondroitin sulfate-2 (CS-2) region of aggrecan were characterized as “aggrecanase” (ADAMTS) cleavage sites, while cleavage between Ser^341-342Phe within the IGD of bovine aggrecan is attributed to MMP action. The objective of this study was to assess the cleavage efficiency of MMPs relative to ADAMTS and their contribution to aggrecan proteolysis in vitro. The analysis of aggrecan IGD degradation in bovine articular cartilage explants treated with catabolic cytokines over a 19-day period showed that MMP-mediated degradation of aggrecan within the IGD can only be observed following day 12 of culture. This delay is associated with the lack of activation of proMMPs during the first 12 days of culture. Analysis of MMP1, 2, 3, 7, 8, 9, 12, 13 and ADAMTS5 efficiencies at cleaving within the aggrecan IGD and CS-2 region in vitro was carried out by the digestion of bovine aggrecan with the various enzymes and Western blot analysis using aggrecan anti-G1 and anti-G3 antibodies. Of these MMPs, MMP12 was the most efficient at cleaving within the aggrecan IGD. In addition to cleavage in the IGD, MMP, 3, 7, 8 and 12 were also able to degrade the aggrecan CS-2 region. MMP3 and MMP12 were able to degrade aggrecan at the very C-terminus of the CS-2 region, cleaving the Glu^2047-2048Ala bond which was previously shown to be cleaved by ADAMTS5. However, in comparison to ADAMTS5, MMP3 was about 100 times and 10 times less efficient at cleaving within the aggrecan IGD and CS-2 regions, respectively. Collectively, our results showed that the delayed activation of proMMPs and the relatively low cleavage efficiency of MMPs can explain the minor contribution of these enzymes to aggrecan catabolism in vivo. This study also uncovered a potential role for MMPs in the C-terminal truncation of aggrecan.     

Matrix metalloproteinases are involved in C-terminal and interglobular domain processing of cartilage aggrecan in late stage cartilage degradation.

Monoclonal antibody (MAb) technology was used to examine aggrecan metabolites and the role of aggrecanases and matrix metalloproteinases (MMPs) in proteolysis of the interglobular domain (IGD) and C-terminus of aggrecan. An in vitro model of progressive cartilage degradation characterized by early proteoglycan loss and late stage collagen catabolism was evaluated in conjunction with a broad-spectrum inhibitor of MMPs. We have for the first time demonstrated that IGD cleavage by MMPs occurs during this late stage cartilage degeneration, both as a primary event in association with glycosaminoglycan (GAG) release from the tissue and secondarily in trimming of aggrecanase-generated G1 metabolites. Additionally, we have shown that MMPs were responsible for C-terminal catabolism of aggrecan and generation of chondroitin sulfate (CS) deficient aggrecan monomers and that this aggrecan truncation occurred prior to detectable IGD cleavage by MMPs. The onset of this later stage MMP activity was also evident by the generation of MMP-specific link protein catabolites in this model culture system. Recombinant MMP-1, -3 and -13 were all capable of C-terminally truncating aggrecan with at least two cleavage sites N-terminal to the CS attachment domains of aggrecan. Through analysis of aggrecan metabolites in pathological synovial fluids from human, canine and equine sources, we have demonstrated the presence of aggrecan catabolites that appear to have resulted from similar C-terminal processing of aggrecan as that induced in our in vitro culture systems. Finally, by developing a new MAb recognizing a linear epitope in the IGD of aggrecan, we have identified two novel aggrecan metabolites generated by an as yet unidentified proteolytic event. Collectively, these results suggest that C-terminal processing of aggrecan by MMPs may contribute to the depletion of cartilage GAG that leads to loss of tissue function in aging and disease. Furthermore, analysis of aggrecan metabolites resulting from both C-terminal and IGD cleavage by MMPs may prove useful in monitoring different stages in the progression of cartilage degeneration.     

Identification of aggrecanase activity in medium of cartilage culture.

Erosion of cartilage is a major feature of joint diseases, i.e., osteoarthritis and rheumatoid arthritis, which leads with time to a loss of joint function. Proteolytic cleavage of the aggrecan core protein is a key event in the progress of these joint diseases. Aggrecan degradation has been believed to be mediated by a putative proteinase, aggrecanase. We identified aggrecanase activity in conditioned medium from explant culture of bovine nasal cartilage stimulated by retinoic acid. The activity was partially purified more than 10,000-fold. The enzyme cleaves at the aggrecanase site (Glu(373)-Ala(374)) but not at the MMP site (Asn(341)-Phe(342)) in the interglobular domain of the aggrecan. It also cleaves at Glu(1971)-Leu(1972), which is located in the gap region in the chondroitin sulfate attachment region prior to the aggrecanase site. The enzyme is a typical Ca(2+)-dependent metalloproteinase with a unique salt-dependency and is inhibited by several hydroxamate-based inhibitors for matrix metalloproteinases. Heparin and chondroitin sulfate inhibited the enzyme in a dose-dependent manner, suggesting that the large carbohydorate in aggrecan is important for substrate recognition by aggrecanase.     

Cathepsin D cleaves aggrecan at unique sites within the interglobular domain and chondroitin sulfate attachment regions that are also cleaved when cartilage is maintained at acid pH

Bovine aggrecan was digested with bovine cathepsin D at pH 5.2 under conditions of partial digestion and the resulting aggrecan core protein fragments were separated by electrophoresis on gradient polyacrylamide gels. The fragments were characterized by their reactivity to specific antibodies and by N-terminal amino acid sequencing. It was also demonstrated that cathepsin D cleaved bovine aggrecan at five sites within the core protein, between residues Phe(342)-Phe(343) in the interglobular domain, Leu(1462)-Val(1463) between the chondroitin sulfate attachment regions 1 and 2 and Leu(1654)-Val(1655), Phe(1754)-Val(1755) and Leu(1854)-Ile(1855) that are located within the chondroitin sulfate attachment region 2 of the core protein. The time course of digestion showed that there was a continued degradation of aggrecan and there was no preferential cleavage of the core protein at any one site. It was shown that cathepsin D digested aggrecan over the pH range 5.2-6.5 resulting in the same products. When bovine cartilage was maintained in explant culture at pH 5.2 there was a rapid loss of both radiolabeled and chemical pools of sulfated glycosaminoglycans into the culture medium and this loss was inhibited by the inclusion in the medium of the aspartic proteinase inhibitor, pepstatin A. The aggrecan core protein fragments appearing in the medium of cultures maintained at pH 5.2 were characterized and it was shown that the fragments had N-terminal sequences starting at Phe(343), Ile(1855), and Val(1755) or Val(1463). This work demonstrates that cathepsin D present within the extracellular matrix of articular cartilage has the potential to contribute to the proteolytic processing of the core protein of aggrecan in this tissue.     

ADAMTS-5 deficiency does not block aggrecanolysis at preferred cleavage sites in the chondroitin sulfate-rich region of aggrecan

In the mouse, proteolysis in the aggrecan interglobular domain is driven by ADAMTS-5, and mice deficient in ADAMTS-5 catalytic activity are protected against aggrecan loss and cartilage damage in experimental models of arthritis. Here we show that despite ablation of ADAMTS-5 activity, aggrecanolysis can still occur at two preferred sites in the chondroitin sulfate-rich region. Retinoic acid was more effective than interleukin-1alpha (IL) in promoting cleavage at these sites in ADAMTS-5-deficient cartilage. These results suggest that cleavage at preferred sites in the chondroitin sulfate-rich region is mediated by ADAMTS-4 or an aggrecanase other than ADAMTS-5. Following retinoic acid or IL-1alpha stimulation of cartilage explants, aggrecan fragments in medium and extracts contained SELE(1279) or FREEE(1467) C-terminal sequences. Some SELE(1279) and FREEE(1467) fragments were retained in the cartilage, with intact G1 domains. Other SELE(1279) fragments were released into the medium and co-migrated with the (374)ALGS neoepitope, indicating they were aggrecanase-derived fragments. In contrast none of the FREEE(1467) fragments released into the medium co-migrated with the (374)ALGS neoepitope, suggesting that, despite their size, these fragments were not products of aggrecanase cleavage in the interglobular domain. ADAMTS-5, but not ADAMTS-1, -4, or -9, was up-regulated 8-fold by retinoic acid and 17-fold by IL-1alpha treatment. The data show that whereas ADAMTS-5 is entirely responsible for cleavage in the interglobular domain, cleavage in the chondroitin sulfate-rich region is driven either by ADAMTS-4, which compensates for loss of ADAMTS-5 in this experimental system, or possibly by another aggrecanase. The data show that there are differential aggrecanase activities with preferences for separate regions of the core protein.     

Mannosamine inhibits aggrecanase-mediated changes in the physical properties and biochemical composition of articular cartilage

The enzymatic processes underlying the degradation of aggrecan in cartilage and the corresponding changes in the biomechanical properties of the tissue are an important part of the pathophysiology of osteoarthritis. Recent studies have demonstrated that the hexosamines glucosamine (GlcN) and mannosamine (ManN) can inhibit aggrecanase-mediated cleavage of aggrecan in IL-1-treated cartilage cultures. The term aggrecanase describes two or more members of the ADAMTS family of metalloproteinases whose glutamyl endopeptidase activity is known to be responsible for much of the aggrecan degradation seen in human arthritides. In this study we examined the effect of ManN and GlcN on aggrecanase-mediated degradation of aggrecan induced by IL-1alpha and the corresponding tissue mechanical properties in newborn bovine articular cartilage. After 6 days of culture in 10 ng/ml IL-1 plus ManN, mechanical testing of explants in confined compression demonstrated that ManN inhibited the IL-1alpha-induced degradation in tissue equilibrium modulus, dynamic stiffness, streaming potential, and hydraulic permeability, in a dose-dependent fashion, with peak inhibition ( approximately 75-100% inhibition) reached by a concentration of 1.35 mM. Aggrecan from explants cultured in IL-1 was found by Western analysis to be almost entirely processed down to the G1-NITEGE(373) end product. Addition of ManN or GlcN was found to produce 75-90% inhibition of this cleavage, but the proportion of aggrecan remaining in the tissue which was cleaved at aggrecanase sites in the chondroitin sulfate (CS)-rich region (Glu(1501) and Glu(1687)) was higher than with IL-1 alone. This result suggests that the preservation of mechanical properties by hexosamines in explants is primarily due to inhibition of cleavage at the Glu(373) site in the interglobular domain. While the precise mechanism by which hexosamines function in this system is unclear, the present analysis suggests that the mechanical properties examined may be predominantly a function of electrostatic repulsion due to the charged CS chains in the tightly packed repetitive sequences of the CS-1 region.     

Differential responses of chondrocytes from normal and osteoarthritic human articular cartilage to mechanical stimulation

Mechanical stimulation is critically important for the maintenance of normal articular cartilage integrity. Molecular events regulating responses of chondrocytes to mechanical forces are beginning to be defined. Chondrocytes from normal human knee joint articular cartilage show increased levels of aggrecan mRNA following 0.33 Hz mechanical stimulation whilst at the same time relative levels of MMP3 mRNA are decreased. This anabolic response, associated with membrane hyperpolarisation, is activated via an integrin-dependent interleukin (IL)-4 autocrine/paracrine loop. Work in our laboratory suggests that this chondroprotective response may be aberrant in osteoarthritis (OA). Chondrocytes from OA cartilage show no changes in aggrecan or MMP3 mRNA following 0.33 Hz mechanical stimulation. alpha5beta1 integrin is the mechanoreceptor in both normal and OA chondrocytes but downstream signalling pathways differ. OA chondrocytes show membrane depolarisation following 0.33 Hz mechanical stimulation consequent to activation of an IL1beta autocrine/paracrine loop. IL4 signalling in OA chondrocytes is preferentially through the type I (IL4alpha/cgamma) receptor rather than via the type II (IL4alpha/IL13R) receptor. Altered mechanotransduction and signalling in OA may contribute to changes in chondrocyte behaviour leading to increased cartilage breakdown and disease progression.     

Inhibition of ADAM-TS4 and ADAM-TS5 prevents aggrecan degradation in osteoarthritic cartilage

Osteoarthritis is a degenerative joint disorder characterized by breakdown of articular cartilage. Degradation of aggrecan, which together with type II collagen provides cartilage with its unique characteristics of compressibility and elasticity, is an early and sustained feature of osteoarthritis. The present work was set up to identify the enzyme(s) responsible for aggrecan breakdown in osteoarthritis. We found that the two cartilage aggrecanases, ADAM-TS4 and ADAM-TS5, are present in osteoarthritic cartilage and that they are responsible for aggrecan degradation without the participation of matrix metalloproteinases. This is based on 1) neoepitopes found on aggrecan fragments in osteoarthritis (OA) cartilage explants in vitro, 2) aggrecan fragments detected in synovial fluid of OA patients, 3) the observation that an aggrecanase inhibitor, BB-16, blocked aggrecan degradation in OA cartilage in vitro, whereas the matrix metalloproteinase inhibitor XS309 did not, and 4) the presence of mRNA and protein for ADAM-TS4 and ADAM-TS5 in OA cartilage. These results suggest that ADAM-TS4 and ADAM-TS5 represent a potential target for the treatment of osteoarthritis.     

Degradation of small leucine-rich repeat proteoglycans by matrix metalloprotease-13: identification of a new biglycan cleavage site

A major and early feature of cartilage degeneration is proteoglycan breakdown. Matrix metalloprotease (MMP)-13 plays an important role in cartilage degradation in osteoarthritis (OA). This MMP, in addition to initiating collagen fibre cleavage, acts on several proteoglycans. One of the proteoglycan families, termed small leucine-rich proteoglycans (SLRPs), was found to be involved in collagen fibril formation/interaction, with some members playing a role in the OA process. We investigated the ability of MMP-13 to cleave members of two classes of SLRPs: biglycan and decorin; and fibromodulin and lumican. SLRPs were isolated from human normal and OA cartilage using guanidinium chloride (4 mol/l) extraction. Digestion products were examined using Western blotting. The identities of the MMP-13 degradation products of biglycan and decorin (using specific substrates) were determined following electrophoresis and microsequencing. We found that the SLRPs studied were cleaved to differing extents by human MMP-13. Although only minimal cleavage of decorin and lumican was observed, cleavage of fibromodulin and biglycan was extensive, suggesting that both molecules are preferential substrates. In contrast to biglycan, decorin and lumican, which yielded a degradation pattern similar for both normal and OA cartilage, fibromodulin had a higher level of degradation with increased cartilage damage. Microsequencing revealed a novel major cleavage site (... G177/V178) for biglycan and a potential cleavage site for decorin upon exposure to MMP-13. We showed, for the first time, that MMP-13 can degrade members from two classes of the SLRP family, and identified the site at which biglycan is cleaved by MMP-13. MMP-13 induced SLRP degradation may represent an early critical event, which may in turn affect the collagen network by exposing the MMP-13 cleavage site in this macromolecule. Awareness of SLRP degradation products, especially those of biglycan and fibromodulin, may assist in early detection of OA cartilage degradation.     

Monoclonal antibodies directed against epitopes within the core protein structure of the large aggregating proteoglycan (aggrecan) from the swarm rat chondrosarcoma.

The core protein of the large hyaline cartilage proteoglycan, aggrecan, is composed of six distinct domains: globular 1 (G1), interglobular, globular 2 (G2), keratan sulfate attachment, chondroitin sulfate (CS) attachment, and globular 3 (G3). Monoclonal antibodies that recognize epitopes in these domains were raised against Swarm rat chondrosarcoma aggrecan that was either denatured through reduction and alkylation or partially deglycosylated through chondroitinase ABC digestion or alkali elimination, the latter with or without sulfite addition. Monoclonal antibodies were further characterized for reactivity to purified aggrecan substructures including rat chondrosarcoma G1 and CS attachment domains, a recombinant rat chondrosarcoma G3 domain fusion protein, bovine articular cartilage G2 domain, and rat chondrosarcoma link protein (LP). Biochemical characterization of the specificities of these monoclonal antibodies indicated that one (1C6) recognized an epitope shared by both the G1 and the G2 domains; one (5C4) recognized an epitope shared by both LP and the G1 domain; one (7D1) recognized an epitope shared by both the G1 and the CS attachment domains; two (14A1 and 15B2) recognized epitopes in the CS attachment domain; one (14B4) recognized an epitope in the G3 domain; and one (13D1) recognized a ubiquitous epitope shared by the G1, G2, G3, and CS attachment domains of aggrecan and also LP. Collectively the specificities of these antibodies confirm the occurrence of multiple repeated epitopes (both carbohydrate and protein in nature) throughout the different domain structures of aggrecan. These antibodies have been proven to be useful for identifying aggrecan-like molecules in several connective tissues other than cartilage.     

Degradation of small leucine-rich repeat proteoglycans by matrix metalloprotease-13: identification of a new biglycan cleavage site

A major and early feature of cartilage degeneration is proteoglycan breakdown. Matrix metalloprotease (MMP)-13 plays an important role in cartilage degradation in osteoarthritis (OA). This MMP, in addition to initiating collagen fibre cleavage, acts on several proteoglycans. One of the proteoglycan families, termed small leucine-rich proteoglycans (SLRPs), was found to be involved in collagen fibril formation/interaction, with some members playing a role in the OA process. We investigated the ability of MMP-13 to cleave members of two classes of SLRPs: biglycan and decorin; and fibromodulin and lumican. SLRPs were isolated from human normal and OA cartilage using guanidinium chloride (4 mol/l) extraction. Digestion products were examined using Western blotting. The identities of the MMP-13 degradation products of biglycan and decorin (using specific substrates) were determined following electrophoresis and microsequencing. We found that the SLRPs studied were cleaved to differing extents by human MMP-13. Although only minimal cleavage of decorin and lumican was observed, cleavage of fibromodulin and biglycan was extensive, suggesting that both molecules are preferential substrates. In contrast to biglycan, decorin and lumican, which yielded a degradation pattern similar for both normal and OA cartilage, fibromodulin had a higher level of degradation with increased cartilage damage. Microsequencing revealed a novel major cleavage site (... G₁₇₇/V₁₇₈) for biglycan and a potential cleavage site for decorin upon exposure to MMP-13. We showed, for the first time, that MMP-13 can degrade members from two classes of the SLRP family, and identified the site at which biglycan is cleaved by MMP-13. MMP-13 induced SLRP degradation may represent an early critical event, which may in turn affect the collagen network by exposing the MMP-13 cleavage site in this macromolecule. Awareness of SLRP degradation products, especially those of biglycan and fibromodulin, may assist in early detection of OA cartilage degradation.     

Cleavage site specificity of cathepsin K toward cartilage proteoglycans and protease complex formation

Cathepsin K is a potent extracellular matrix-degrading protease that requires interactions with soluble glycosaminolycans for its collagenolytic activity in bone and cartilage. The major sources of glycosaminoglycans in cartilage are aggrecan aggregates. Therefore, we investigated whether cathepsin K activity is capable to hydrolyze aggrecan into fragments allowing the formation of glycosaminoglycan-cathepsin K complexes and determined the cleavage site specificity of cathepsin K toward the cartilage-resident link protein and aggrecan. The cleavage site specificity was compared with those of cathepsins S and L. All three cathepsins released glycosaminoglycans from native bovine cartilage at lysosomal pH and to a lesser degree at neutral extracellular pH. Cathepsin-predigested aggrecan complexes and cartilage provided suitable glycosaminoglycan fragments that allowed the formation of collagenolytically active cathepsin K complexes. A detailed analysis of the degradation of aggrecan aggregates revealed two cathepsin K cleavage sites in the link protein and several sites in aggrecan, including one site within the interglobular domain E1. In summary, these results demonstrate that cathepsin K is capable to degrade aggrecan complexes at specific cleavage sites and that cathepsin K activity alone is sufficient to self-provide the glycosaminoglycan fragments required for the formation of its collagenolytically active complex.     

Isolation and characterization of matrix proteoglycans from human nasal cartilage. Compositional and structural comparison between normal and scoliotic tissues

The content, types and the fine structures of proteoglycans (PGs) present in human normal nasal cartilage (HNNC) were investigated and compared with those in human scoliotic nasal cartilage (HSNC). Three PG types were identified in both HNNC and HSNC; the large-sized high buoyant density aggrecan, which is the predominant PG population, and the small-sized low buoyant density biglycan and decorin. HSNC contained a significantly higher amount of keratan sulfate (KS)-rich aggrecan (30%) of smaller hydrodynamic size as compared to HNNC. The average molecular sizes (M(r)s) of aggecan-derived chondroitin sulfate (CS) chains in both HNNC and HSNC were identical (18 kDa), but they significantly differ in disaccharide composition, since CS isolated from HSNC contained higher proportions of 6-sulfated disaccharides as compared to those from HNNC. Scoliotic tissue contained also higher amounts (67%) of the small PGs, biglycan and decorin as compared to HNNC. It is worth noticing that both normal and scoliotic human nasal cartilage contain also non-glycanated forms of decorin and biglycan. Dermatan sulfate (DS) was the predominant glycosaminoglycan (GAG) present on biglycan and decorin in both tissues. The small PGs-derived CS chains in both normal and scoliotic cartilage had the same M(r) (20 kDa), whereas DS chains from scoliotic cartilage were of greater M(r) (32 kDa) than those from normal cartilage (24 kDa). Furthermore, scoliotic tissue-derived DS chains contained higher amounts of iduronate (20%) as compared to those of normal cartilage (12%). Disaccharide analysis of small PGs showed that both HNNC and HSNC were rich in 4-sulfated disaccharides and in each case, the small size PGs contained a considerably higher proportion of 4-sulfated disaccharides than the aggrecan of the same tissue. The higher amounts of matrix PGs identified in scoliotic tissue as well as the differences in fine chemical composition of their GAG chains may reflect the modified architecture and functional failure of scoliotic tissue.     

Keratan sulfate suppresses cartilage damage and ameliorates inflammation in an experimental mice arthritis model

Proteoglycans bearing keratan sulfate (KS), such as aggrecan, are components of the human cartilage extracellular matrix (ECM). However, the role of KS in influencing cartilage degradation associated with arthritis remains to be completely understood. KS side chains of the length found in human cartilage are not found in murine skeletal tissues. Using a murine model of inflammatory polyarthritis and cartilage explants exposed to interleukin-1α (IL-1α), we examined whether administering KS could influence intraarticular inflammation and cartilage degradation. Acute arthritis was induced by intravenous administration of an anti-type II collagen antibody cocktail, followed by an intraperitoneal injection of lipopolysaccharide. This treatment was followed by an intraperitoneal KS administration in half of the total mice to evaluate the therapeutic potential of KS for ameliorating arthritis. To investigate the therapeutic potential ex vivo, we examined cartilage fragility by measuring IL-1α-induced aggrecan release from cartilage explants treated with or without KS. Intraperitoneal KS administration ameliorated arthritis in DBA/1J mice. The aggrecan release induced by IL-1α was less in cartilage explants containing media with KS than in those without KS. Our data indicate that exogenous KS ameliorated arthritis in vivo and suppressed cartilage degradation ex vivo. KS may have important therapeutic potential in the treatment of inflammatory arthritis. The mechanism responsible for this requires further investigation, but KS may become a novel therapeutic agent for treating inflammatory diseases such as rheumatoid arthritis.     

Aggrecan protects cartilage collagen from proteolytic cleavage

The matrix components responsible for cartilage mechanical properties, type II collagen and aggrecan, are degraded in osteoarthritis through proteolytic cleavage by matrix metalloproteinases (MMPs) and aggrecanases, respectively. We now show that aggrecan may serve to protect cartilage collagen from degradation. Although collagen in freeze-thawed cartilage depleted of aggrecan was completely degraded following incubation with MMP-1, collagen in cartilage with intact aggrecan was not. Using interleukin-1-stimulated bovine nasal cartilage explants where aggrecan depletion occurs during the first week of culture, followed by collagen loss during the second week, we evaluated the effect of selective MMP and aggrecanase inhibitors on degradation. A selective MMP inhibitor did not block aggrecan degradation but caused complete inhibition of collagen breakdown. Similar inhibition was seen with inhibitor addition following aggrecan depletion on day 6-8, suggesting that MMPs are not causing significant collagen degradation prior to the second week of culture. Inclusion of a selective aggrecanase inhibitor blocked aggrecan degradation, and, in addition, inhibited collagen degradation. When the inhibitor was introduced following aggrecan depletion, it had no effect on collagen breakdown, ruling out a direct effect through inhibition of collagenase. These data suggest that aggrecan plays a protective role in preventing degradation of collagen fibrils, and that an aggrecanase inhibitor may impart overall cartilage protection.