Involvement of proteoglycans in tendinopathy.

A major feature of chronic tendinopathy is a change in the nature and organisation of the extracellular matrix of tendon. Increased levels of proteoglycans have been shown in the extracellular matrix of tendinopathic tendons and these appear to influence the increased hydration and swelling of the tissue that is a feature of this condition. There is a paucity of knowledge about proteoglycans in normal and tendinopathic tendons. This review sets out to describe the nature, function and metabolism of proteoglycans present in normal tendon and in tendinopathy and outlines how changes in proteoglycan metabolism may contribute to the development and progression of this disease.

Effects of glucosamine on proteoglycan loss by tendon, ligament and joint capsule explant cultures.

OBJECTIVE: To investigate the effect of glucosamine on the loss of newly synthesized radiolabeled large and small proteoglycans by bovine tendon, ligament and joint capsule. DESIGN: The kinetics of loss of (35)S-labeled large and small proteoglycans from explant cultures of tendon, ligament and joint capsule treated with 10mM glucosamine was investigated over a 10-day culture period. The kinetics of loss of (35)S-labeled small proteoglycans and the formation of free [(35)S]sulfate were determined for the last 10 days of a 15-day culture period. The proteoglycan core proteins were analyzed by gel electrophoresis followed by fluorography. The metabolism of tendon, ligament and joint capsule explants exposed to 10mM glucosamine was evaluated by incorporation of [(3)H]serine and [(35)S]sulfate into protein and glycosaminoglycans, respectively. RESULTS: Glucosamine at 10mM stimulated the loss of small proteoglycans from ligament explant cultures. This was due to the increased loss of both macromolecular and free [(35)S]sulfate to the medium indicating that glucosamine affected the release of small proteoglycans as well as their intracellular degradation. The degradation pattern of small proteoglycans in ligament was not affected by glucosamine. In contrast, glucosamine did not have an effect on the loss of large or small proteoglycans from tendon and joint capsule or large proteoglycans from ligament explant cultures. The metabolism of cells in tendon, ligament and joint capsule was not impaired by the presence of 10mM glucosamine. CONCLUSIONS: Glucosamine stimulated the loss of small proteoglycans from ligament but did not have an effect on small proteoglycan catabolism in joint capsule and tendon or large proteoglycan catabolism in ligament, tendon or synovial capsule. The consequences of glucosamine therapy at clinically relevant concentrations on proteoglycan catabolism in joint fibrous connective tissues need to be further assessed in an animal model.

Increased versican content is associated with tendinosis pathology in the patellar tendon of athletes with jumper's knee.

Expansion of the extracellular matrix is a prominent but poorly characterized feature of tendinosis. The present study aimed to characterize the extent and distribution of the large aggregating proteoglycan versican in patients with patellar tendinosis. We obtained tendon from tendinopathy patients undergoing debridement of the patellar tendon and from controls undergoing intramedullary tibial nailing. Versican content was investigated by Western blotting and immunohistochemistry. Microvessel thickness and density were determined using computer-assisted image analysis. Markers for smooth muscle actin, endothelial cells (CD31) and proliferating cells (Ki67) were examined immunohistochemically. Western blot analysis and immunohistochemical staining revealed elevated versican content in the proximal patellar tendon of tendinosis patients (P=0.042). Versican content was enriched in regions of fibrocartilage metaplasia and fibroblast proliferation, as well as in the perivascular matrix of proliferating microvessels and within the media and intima of arterioles. Microvessel density was higher in tendinosis tissue compared with control tissue. Versican deposition is a prominent feature of patellar tendinosis. Because this molecule is not only a component of normal fibrocartilagenous matrices but also implicated in a variety of soft tissue pathologies, future studies should further detail both pathological and adaptive roles of versican in tendons.

Effects of long-term exposure to glucosamine and mannosamine on aggrecan degradation in articular cartilage.

OBJECTIVE: To investigate the effect of long-term exposure to glucosamine or mannosamine on the catabolism of aggrecan by explant cultures of bovine articular cartilage maintained in the presence of retinoic acid. DESIGN: The kinetics of loss of 35S-labeled and total aggrecan from explant cultures of bovine articular cartilage maintained in the presence of 1 micro M retinoic acid and exposed to varying concentrations of glucosamine or mannosamine was investigated over a 9-day culture period. In other experiments, the reversibility of the inhibition of aggrecan catabolism by glucosamine or mannosamine was investigated in cultures exposed to these amino sugars for the first 5 days of a 15-day culture period. The metabolism of chondrocytes exposed to these amino sugars was evaluated by measurement of lactate production or 3H-serine and 35S-sulfate incorporation into protein and glycosaminoglycans, respectively. The direct effect of these amino sugars on soluble aggrecanase activity was determined from immunoblots of aggrecan digests. RESULTS: Glucosamine at 5mM concentration and mannosamine at 2mM concentration inhibited degradation of radiolabeled and chemical levels of aggrecan. At concentrations of up to 10mM amino sugars, the metabolism of chondrocytes was not impaired, as determined by lactate production, protein synthesis and the incorporation of 35S-sulfate into proteoglycans. These amino sugars did not inhibit soluble aggrecanase activity. The exposure of articular cartilage explants to 5mM glucosamine or mannosamine for 5 days in culture in the presence or absence of retinoic acid did not provide long-term suppression of stimulated aggrecan loss. CONCLUSIONS: This study indicates that continuous presence of amino sugars is required to protect cartilage from stimulated loss of aggrecan.

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.

Expression and activity of ADAMTS-5 in synovium.

ADAMTS proteinases, belonging to the adamalysin subfamily of metalloproteinases, have been implicated in a variety of cellular events such as morphogenesis, cell migration, angiogenesis, ovulation and extracellular matrix breakdown. Aggrecanase-1 (ADAMTS-4) and aggrecanase-2 (ADAMTS-5) have been identified in cartilage and are largely responsible for cartilage aggrecan breakdown. We have shown previously that synovium, the membrane lining diarthrodial joints, generates soluble aggrecanase activity. We report here the expression, localization and activity of ADAMTS-5 from human arthritic and bovine synovium. ADAMTS-5 was expressed constitutively in synovium with little or no transcriptional regulation by recombinant human interleukin-1 alpha or all-trans-retinoate, factors previously shown to upregulate aggrecanase activity in cartilage. Aggrecanase activity generated by synovium in vitro and recombinant ADAMTS-5 cleaved aggrecan extensively, resulting in aggrecan fragments similar to those generated by chondrocyte-derived aggrecanases, and the activity was inhibited by heparin. ADAMTS-5 was immunolocalized in human arthritic synovium, where staining was mostly pericellular, particularly in the synovial lining and around blood vessels; some matrix staining was also seen. The possibility that synovium-derived ADAMTS-5 may play a role in cartilage aggrecan breakdown is discussed.

Calcium pentosan polysulfate inhibits the catabolism of aggrecan in articular cartilage explant cultures.

OBJECTIVE: The catabolism of aggrecan and loss of aggrecan fragments from articular cartilage is a key event in the pathogenesis of arthritic diseases such as osteoarthritis. The catabolism of aggrecan is mediated by the specific proteolytic activity termed aggrecanase. The aim of this study was to investigate the effect of the chondroprotective agent calcium pentosan polysulfate (CaPPS) on the aggrecanase-mediated catabolism of aggrecan. METHODS: The catabolism of 35S-labeled aggrecan and loss of tissue glycosaminoglycans (GAGs) were investigated using bovine articular cartilage explant cultures maintained in medium containing varying concentrations of CaPPS (1-100 microg/ml) in the presence or absence of 10(-6)M retinoic acid or 7 ng/ml recombinant human interleukin-1alpha (rHuIL-1alpha). In addition, the effect of CaPPS on the degradation of aggrecan monomers by aggrecanase activity present in conditioned medium from joint capsule explant cultures was investigated. RESULTS: CaPPS inhibited the catabolism of 35S-labeled aggrecan in a dose-dependent manner, particularly when retinoic acid or rHuIL-1alpha was used to stimulate aggrecan catabolism. These effects were reflected in the tissue levels of GAG remaining in these cultures at the end of the experiment. CaPPS inhibited the degradation of aggrecan monomers by soluble aggrecanase activity. CONCLUSION: CaPPS inhibits the catabolism of aggrecan by articular cartilage in a dose-dependent manner, particularly when the processes responsible for aggrecan loss are stimulated. This effect occurs, at least in part, through direct inhibition of aggrecanase activity. CaPPS did not adversely affect overall chondrocyte metabolism, as shown by the incorporation of 35S-sulfate and 3H-leucine into macromolecules and by lactate production in cartilage explant cultures.

Bovine joint capsule and fibroblasts derived from joint capsule express aggrecanase activity.

Bovine joint capsule was maintained in explant culture in the presence of bovine aggrecan monomer and it was shown that the aggrecan monomer was degraded. Amino-terminal sequence analysis of the resulting aggrecan core protein fragments revealed that the core protein was cleaved at five specific sites attributed to glutamyl endopeptidases referred to as aggrecanase activity. Fibroblast cultures were established from explant cultures of joint capsule and when these cells were exposed to aggrecan, cleavage of the core protein of aggrecan at the aggrecanase sites was observed. Inclusion of either retinoic acid or interleukin-1alpha in medium of either joint capsule explant cultures or fibroblast cultures did not increase the rate of cleavage of exogenous aggrecan present in the culture medium. When aggrecan monomer was incubated with conditioned medium from explant cultures of joint capsule maintained in medium, degradation could be detected after 10 min. After a 6-h incubation period the same fragments of aggrecan core protein were observed as those for tissue or cells incubated directly with aggrecan monomer. RT-PCR analysis of mRNA extracted from joint capsule fibroblasts showed that these cells express both aggrecanase-1 and -2 [ADAMTS-2 (Tang) and ADAMTS-5].

Metabolic processing of newly synthesized link protein in bovine articular cartilage explant cultures.

In explant cultures of articular cartilage from cattle of different ages radiolabeled leucine was shown to be incorporated into link proteins 1, 2 and 3. The newly synthesized link proteins were incorporated into and lost from the cartilage extracellular matrix with time. The levels of radiolabeled link proteins 1 and 2 remaining in the matrix declined over the culture period, but there was an initial increase in the amount of radiolabeled link protein 3, before its level declined. The turnover time of the radiolabeled link proteins 1 and 2 were similar, indicating that neither link protein was preferentially processed to generate link protein 3, nor lost from the extracellular matrix. The majority of the radiolabeled link protein lost from the cartilage matrix could not be recovered from the culture medium, suggesting that turnover of the radiolabeled aggrecan complexes involves the newly synthesized link protein being internalized by the chondrocytes. Inclusion of cytotoxic proteinase inhibitors to the culture medium resulted in a marked decrease in the rate of loss of link protein from the cartilage, suggesting that the catabolism of link protein is cell-mediated and dependent on metabolically active cells.

Coincubation of bovine synovial or capsular tissue with cartilage generates a soluble "Aggrecanase" activity.

The culture of bovine synovial or capsular tissue generated proteoglycan-degrading activity. When these tissues were incubated with living or dead bovine articular cartilage significantly more proteoglycan-degrading activity was revealed. The activity was present in a soluble form and required protein synthesis for its generation. The conditioned medium did not contain matrixin activity, although experiments with proteinase inhibitors suggested that the activity was due to a metalloproteinase. Western blotting of the aggrecan fragments suggested cleavage of aggrecan within the interglobular domain at the "aggrecanase" site, but not at the major matrixin site. N-terminal sequencing confirmed cleavage of aggrecan at a number of glutamyl bonds, including the aggrecanase site in the interglobular domain. We conclude that cultured synovial or capsular tissue produces soluble aggrecanase and an enzyme which releases aggrecanase from cartilage, possibly by cleavage of a chondrocyte membrane-bound form of aggrecanase.

Identification of distinct metabolic pools of aggrecan and their relationship to type VI collagen in the chondrons of mature bovine articular cartilage explants.

The metabolism and distribution of newly synthesized aggrecan present in the extracellular matrix of intact explant cultures of mature articular cartilage was investigated with respect to type VI collagen-stained chondrons. Using biochemical, autoradiographical and novel confocal immunohistochemical techniques it was shown that aggrecan exists as a number of distinct pools that are located within the extracellular matrix of the tissue. The first was identified as a pool of high specific radioactivity, much of which appeared in the medium one day after incubation with radiolabeled sulfate. Of the radiolabeled aggrecan remaining within the extracellular matrix, three pools were differentiated on the basis of time and location within the extracellular matrix. One pool was resident within the pericellular microenvironment associated with the chondron, one migrated into the territorial matrix adjacent to the chondron and one was sequestered long term in the interterritorial matrix. Analysis of the kinetics of loss of radiolabeled aggrecan macromolecules present in the region of matrix defined by the chondron suggests that this pool rapidly turns over and is a precursor to the pools of aggrecan present in the territorial and interterritorial matrix. There were marked differences in the distribution of newly synthesized aggrecan present in these regions of the extracellular matrix in explant cultures maintained with or without fetal calf serum. In the absence of serum, more of the newly synthesized aggrecan moved into the territorial and interterritorial matrix indicating that the presence of serum in the culture medium influenced the tissue distribution of aggrecan. This work indicates that the pericellular microenvironment of the chondron plays an important role in the retention and maturation of aggrecan prior to the sequestration of aggrecan complexes into the functional load bearing matrices of adult articular cartilage.

Characterization of aggrecan retained and lost from the extracellular matrix of articular cartilage. Involvement of carboxyl-terminal processing in the catabolism of aggrecan.

The catabolism of aggrecan in bovine articular cartilage explants is characterized by the release into the culture medium of high molecular weight aggrecan fragments, generated by the proteolytic cleavage of the core protein between residues Glu373 and Ala374 within the interglobular domain. In this study, the position of the carboxyl-terminus of these aggrecan fragments, as well as a major proteolytically shortened aggrecan core protein present in cartilage matrix, have been deduced by characterizing the peptides generated by the reaction of aggrecan core protein peptides with cyanogen bromide. It was shown that two out of three such peptide fragments having an amino terminus starting at Ala374 have their carboxyl terminus located within the chondroitin sulfate 1 domain. The third and largest aggrecan core protein peptide, with an amino terminus starting at Ala374, has a carboxyl terminus in a region of core protein between the chondroitin sulfate 1 domain and the chondroitin sulfate 2 domain. The carboxyl terminus of this peptide appeared to be the same as that of the proteolytically degraded aggrecan core protein, which is retained within the extracellular matrix of the tissue. Another two aggrecan fragments recovered from the medium of explant cultures with amino-terminal sequences in the chondroitin sulfate 2 domain at Ala1772 and Leu1872 were shown to have their carboxyl termini within the G3 globular domain. These results suggest that the catabolism of aggrecan between residues Glu373 and Ala374 in the interglobular domain by the putative proteinase, aggrecanase, may be dependent on prior proteolytic processing within the carboxyl-terminal region of the core protein.

"Aggrecanase" activity is implicated in tumour necrosis factor alpha mediated cartilage aggrecan breakdown but is not detected by an in vitro assay.

AIMS: To develop an in vitro assay for the putative glutamyl endopeptidase, "aggrecanase", which is thought to degrade cartilage aggrecan, and to examine the role of the enzyme in tumour necrosis factor stimulated aggrecan cleavage. METHODS: Aggrecan fragments released by bovine nasal cartilage explants, with and without exposure to tumour necrosis factor alpha, were purified and analysed by western blotting and N-terminal sequencing. Intact bovine aggrecan was incubated with extracts of cartilage, lysed chondrocytes, or cartilage explant conditioned culture medium under a variety of conditions. Deglycosylated aggrecan was incubated with nasal cartilage explants. Proteoglycan breakdown was assessed by metachromatic assay of fragments in culture media, and cleavage of the substrate at the aggrecanase cleavage site was detected and measured using the antibody BC3, which recognises a neoepitope produced by aggrecanase cleavage of aggrecan. RESULTS: Aggrecan fragments generated from explants treated with tumour necrosis factor had N-terminal sequences consistent with cleavage of aggrecan at a restricted number of glutamyl bonds. Aggrecanase generated fragments were found in cartilage explant culture medium and chondrocyte monolayers. However, no aggrecanase activity could be detected in extracts of cartilage, or chondrocytes from which endogenous aggrecan fragments had been removed, under a variety of assay conditions. Deglycosylated aggrecan, added to explant cultures, efficiently inhibited endogenous aggrecan breakdown. CONCLUSIONS: Aggrecanase is active in cartilage and in chondrocyte monolayers, and its action is stimulated by tumour necrosis factor alpha. However, activity due to this enzyme could not be detected in vitro under our assay conditions, although a deglycosylated version of the substrate inhibited aggrecan breakdown in explant cultures.

Presence of antibodies to native G1 domain of aggrecan core protein in synovial fluids from patients with various joint diseases.

OBJECTIVE: To investigate the occurrence of IgG antibodies to aggrecan in synovial fluids (SF) from patients with arthritis and various articular diseases, and to determine the nature of epitopes present within aggrecan that react with these antibodies. METHODS: SF samples were reacted with native aggrecan, reduced and alkylated aggrecan, chondroitin sulfate, and keratan sulfate, using dot-blots and a novel enzyme-linked immunosorbent assay (ELISA). The nature of the epitopes present on aggrecan was elucidated using Western blots and a competitive inhibition ELISA. RESULTS: IgG antibodies to aggrecan were found in > 50% of the SF samples tested. No IgG antibody reactivity was observed in serum from the same patients. The antibodies appeared to react predominantly with native aggrecan, and there was no disease specificity. It was shown that the epitopes to these antibodies were located within the N-terminal region of the core protein. CONCLUSION: This study demonstrates the frequent occurrence of IgG antibodies to aggrecan in human SF. The major epitope is located in the G1 domain of the aggrecan core protein. These IgG antibodies appear to be produced locally within the synovial cavity, probably in response to various articular diseases, resulting in the loss of native aggrecan from articular cartilage.

Catabolism and loss of proteoglycans from cultures of bovine collateral ligament.

This paper investigates the kinetics and mechanism of loss of the two major proteoglycan species from cultures of bovine collateral ligament. Following incubation of ligament with [35S]sulfate after 6 days in culture, the rate of loss of the predominant proteoglycan species, decorin, from the matrix was shown to be much slower (t1/2 approximately 18 days) than that of the large chondroitin sulfate proteoglycan (t1/2 approximately 1.4 days). Analysis of 35S-labeled proteoglycans released into the medium between Days 11 and 15 of the culture period on a column of Sepharose CL-4B revealed that these macromolecules constituted mainly decorin of similar hydrodynamic size to that present in the matrix. Furthermore, analysis of core proteins using gel electrophoresis followed by fluorography or immunodetection with LF-94, an antibody directed against the amino-terminal region of decorin, indicated that the core proteins of decorin released into the medium and those remaining in the matrix of ligament cultures had a similar molecular mass (approximately 49 kDa). Analysis of both the 35S-labeled and endogenous macromolecules using 5/6/3-B-3, an antibody directed against terminal unsaturated chondroitin-6-sulfate disaccharides, revealed that three core proteins with molecular masses greater than approximately 200 kDa were present in the matrix. Four additional core proteins (range approximately 80-200 kDa) derived from the large proteoglycan were detected in the medium of ligament cultures. These findings indicate that, unlike decorin, the loss of the large chondroitin sulfate proteoglycan from the matrix of ligament cultures involved proteolytic cleavage of its core protein. No difference in the hydrodynamic size of the 35S-labeled glycosaminoglycan chains derived from either proteoglycan species remaining in the matrix or released into the medium of ligament cultures was observed.

Catabolism of aggrecan by explant cultures of human articular cartilage in the presence of retinoic acid.

The N-terminal amino acid sequence of human aggrecan was determined and it was shown that two sequences were present. The major sequence, AVTVE-, accounted for 60% of the aggrecan and started at alanine residue 17 of the human aggrecan core protein cDNA sequence (K. Doege et al. (1991) J. Biol. Chem. 266, 894-920). The other N-terminal sequence, VETX-, started at valine residue 20. Characterization of aggrecan core protein peptides present in the matrix of adult human articular cartilage showed that at least 11 aggrecan core proteins were present with approximate M(r) between 300,000 and 43,000. All these core proteins were found to have the same N-terminal sequences as that observed in human aggrecan. When articular cartilage was placed in explant culture in medium containing 10(-6) M retinoic acid there was a 3.5-fold increase in the loss of aggrecan into the culture medium compared to tissue maintained in medium alone or medium containing 20% (v/v) newborn calf serum. Analysis of the aggrecan core protein fragments that were released to the culture medium containing 10(-6) M retinoic acid showed the presence of 13 core protein peptides of M(r) between 300,000 and 43,000. The 11 smaller peptides of M(r) 230,000 to 43,000 were shown to have the N-terminal sequence ARGS-. This sequence which starts at residue 393 of the human aggrecan core protein is located within the interglobular region between the G1 and G2 domains and is the site of aggrecan catabolism by the putative protease aggrecanase. The presence of core proteins of varying sizes but with the same N-terminal sequence reflects proteolytic processing from the C-terminal end of the core protein that was also observed in the aggrecan macromolecules extracted from the matrix of human articular cartilage. This proteolytic processing was also evident but to a lesser extent in newly synthesized 35S-labeled aggrecan macromolecules.

Inhibition of cartilage proteoglycan release by a specific inactivator of cathepsin B and an inhibitor of matrix metalloproteinases. Evidence for two converging pathways of chondrocyte-mediated proteoglycan degradation.

OBJECTIVE: To investigate mechanisms of cartilage matrix destruction by a study of the effects of a specific inactivator of cathepsin B and an inhibitor of several matrix metalloproteinases (MMP) on cartilage proteoglycan release. METHODS: Cartilage explants were treated with either recombinant human interleukin-1 alpha (rHuIL-1 alpha) or retinoic acid in the presence or absence of the inhibitors, and proteoglycan release was quantitated. Tests for nonspecific effects of the inhibitors included reversibility, rates of protein synthesis and glycolysis, and effects on other rHuIL-1 alpha-mediated events. RESULTS: The cathepsin B inactivator inhibited rHuIL-1 alpha-stimulated proteoglycan release at nanomolar concentrations, but failed to significantly inhibit retinoic acid-stimulated proteoglycan release. An inhibitor of MMP was inhibitory to both rHuIL-1 alpha-stimulated release and retinoic acid-stimulated release. CONCLUSION: Cathepsin B is implicated in rHuIL-1 alpha-stimulated loss of cartilage proteoglycan. Its lack of involvement in retinoic acid-stimulated proteoglycan release suggests the existence of at least 2 pathways of cartilage proteoglycan breakdown, which may converge at the activation of a matrix prometalloproteinase.

Mechanism of catabolism of aggrecan by articular cartilage.

Characterization of aggrecan core protein peptides appearing in the medium of adult articular cartilage maintained in tissue culture showed that eight major peptides could be detected. The two largest peptides had the same N-terminal sequence as bovine aggrecan core protein and probably represent partly degraded aggrecan lost to the medium in the form of the proteoglycan aggregate. The three next smallest peptides were all shown to have another N-terminal sequence which corresponded to a sequence in the interglobular domain starting at alanine residue 393 of the human aggrecan core protein (K. Doege et al., 1991, J. Biol. Chem. 266, 894-902). Two other peptides were isolated and shown to have two different N-terminal amino sequences corresponding to sequences in the chondroitin sulfate attachment domain 2 of the core protein starting at alanine residue 1839 and leucine residue 1939 of human aggrecan. This suggests that the catabolism of aggrecan by adult articular cartilage occurs by the proteolytic cleavage of the core protein of this proteoglycan at three separate sites. Examination of the amino acid sequences around each of these cleavage sites showed a similar pattern TEGE decreases ARGS, TAQE decreases AGEG, and VSQE decreases LGQR, suggesting that a single proteinase may be involved in the catabolism of aggrecan. Analysis of synovial fluids and serum of age-matched animals revealed the presence of aggrecan core protein peptides corresponding in size to those detected in vitro, thus indicating the cleavage observed in explant culture is the same as that which occurs in vivo.

Cleavage of proteoglycan aggregate by leucocyte elastase.

The partial degradation of proteoglycan aggregate by human leucocyte elastase yielded products that banded with Mr 190,000, 140,000, 88,000, and 71,000 when analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis. Analysis of these bands revealed that the 190,000- and 140,000-Da bands contained chondroitin and keratan sulfate stubs and had N-terminal amino acid sequences corresponding to a sequence starting at residue 398 of the core protein of rat or human aggrecan. With increased time of digestion, the staining intensities of the 190,000-, 140,000-, and 88,000-Da bands decreased relative to the 71,000-Da band. Analysis of the 88,000- and 71,000-Da bands showed that they contained peptides substituted only with keratan sulfate stubs and that each band contained two peptides with different N-terminal sequences. One of these corresponded to a sequence that started at residue 398 of rat or human aggrecan and the other to the N-terminal sequence of bovine aggrecan. Under conditions of complete digestion, bands of 71,000 and 56,000 Da which contained only keratan sulfate stubs were observed on SDS-polyacrylamide electrophoresis. The 71,000-Da band was shown to have a single sequence similar to that starting at residue 398 of human and rat aggrecan and thus represents the globular domain 2 (G2) of the core protein of aggrecan. The 56,000-Da band was shown to have a sequence similar to that of the N-terminal sequence of bovine aggrecan indicating that this peptide corresponds to the globular domain 1 (G1) of the molecule. These results suggest that leucocyte elastase cleaves the core protein of aggrecan between valine 397 and isoleucine 398, which are located in the interglobular domain linking the G1 and G2 domains of the core protein of aggrecan. Further digestion of the proteoglycan aggregate with elastase resulted in the cleavage of the core protein within the chondroitin sulfate attachment domains.