Catabolism of aggrecan in cartilage explants. Identification of a major cleavage site within the interglobular domain

The catabolism of aggrecan has been studied in calf articular cartilage explant cultures. The chondroitin sulfate-rich, high buoyant density products that accumulate in culture medium have been purified, and NH2-terminal sequence data have been obtained. Aggrecan released from the tissue in the presence or absence of interleukin-1 alpha, whether analyzed before or after reduction and alkylation, exhibited only one major and one minor NH2-terminal sequence. The major sequence, ARGXVILXAKPDF, shows very high similarity to a region of the interglobular domain (between the G1 and G2 domains) of both human and rat aggrecan. The minor sequence, VEVS, was that previously described for the NH2 terminus of the intact core protein. These results indicate that catabolism of aggrecan in cartilage explants involves proteolytic cleavage within a conserved region of the interglobular domain and that this results in the separation of the G1 domain from the remainder of the molecule. A major product of this process is a large nonaggregating species that consists of an NH2-terminal sequence beginning with ARG (and composed of about 100 residues of the interglobular domain) that is attached to an intact G2 domain followed by an extended section of the chondroitin sulfate-bearing domain toward the COOH terminus.     

Mutations in the interglobular domain of aggrecan alter matrix metalloproteinase and aggrecanase cleavage patterns. Evidence that matrix metalloproteinase cleavage interferes with aggrecanase activity

We have expressed G1-G2 mutants with amino acid changes at the DIPEN(341) downward arrow(342)FFGVG and ITEGE(373) downward arrow(374)ARGSV cleavage sites, in order to investigate the relationship between matrix metalloproteinase (MMP) and aggrecanase activities in the interglobular domain (IGD) of aggrecan. The mutation DIPEN(341) to DIGSA(341) partially blocked cleavage by MMP-13 and MMP-8 at the MMP site, while the mutation (342)FFGVG to (342)GTRVG completely blocked cleavage at this site by MMP-1, -2, -3, -7, -8, -9, -13, -14. Each of the MMP cleavage site mutants, including a four-amino acid deletion mutant lacking residues ENFF(343), were efficiently cleaved by aggrecanase, suggesting that the primary sequence at the MMP site had no effect on aggrecanase activity in the IGD. The mutation (374)ARGSV to (374)NVYSV completely blocked cleavage at the aggrecanase site by aggrecanase, MMP-8 and atrolysin C but had no effect on the ability of MMP-8 and MMP-13 to cleave at the Asn(341) downward arrowPhe bond. Susceptibility to atrolysin C cleavage at the MMP site was conferred in the DIGSA(341) mutant but absent in the wild-type, (342)GTRVG, (374)NVYSV, and deletion mutants. To further explore the relationship between MMP and aggrecanase activities, sequential digest experiments were done in which MMP degradation products were subsequently digested with aggrecanase and vice versa. Aggrecanase-derived G1 domains with ITEGE(373) C termini were viable substrates for MMPs; however, MMP-derived G2 fragments were resistant to cleavage by aggrecanase. A 10-mer peptide FVDIPENFFG, which is a substrate analogue for the MMP cleavage site, inhibited aggrecanase cleavage at the Glu(373) downward arrowAla bond. This study demonstrates that MMPs and aggrecanase have unique substrate recognition in the IGD of aggrecan and suggests that sequences at the C terminus of the DIPEN(341) G1 domain may be important for regulating aggrecanase cleavage.     

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.     

Conserved sequence in the aggrecan interglobular domain modulates cleavage by ADAMTS-4 and ADAMTS-5

Background

Cleavage of aggrecan by ADAMTS proteinases at specific sites within highly conserved regions may be important to normal physiological enzyme functions, as well as pathological degradation.

Methods

To examine ADAMTS selectivity, we assayed ADAMTS-4 and -5 cleavage of recombinant bovine aggrecan mutated at amino acids N-terminal or C-terminal to the interglobular domain cleavage site.

Results

Mutations of conserved amino acids from P18 to P12 to increase hydrophilicity resulted in ADAMTS-4 cleavage inhibition. Mutation of Thr, but not Asn within the conserved N-glycosylation motif Asn-Ile-Thr from P6 to P4 enhanced cleavage. Mutation of conserved Thr residues from P22 to P17 to increase hydrophobicity enhanced ADAMTS-4 cleavage. A P4′ Ser377Gln mutant inhibited cleavage by ADAMTS-4 and -5, while a neutral Ser377Ala mutant and species mimicking mutants Ser377Thr, Ser377Asn, and Arg375Leu were cleaved normally by ADAMTS-4. The Ser377Thr mutant, however, was resistant to cleavage by ADAMTS-5.

Conclusion

We have identified multiple conserved amino acids within regions N- and C-terminal to the site of scission that may influence enzyme–substrate recognition, and may interact with exosites on ADAMTS-4 and ADAMTS-5.

General significance

Inhibition of the binding of ADAMTS-4 and ADAMTS-5 exosites to aggrecan should be explored as a therapeutic intervention for osteoarthritis.     

The interglobular domain of cartilage aggrecan is cleaved by PUMP, gelatinases, and cathepsin B.

The action of three matrix metalloproteinases (MMPs), 72- and 95-kDa gelatinases (MMP-2 and MMP-9) and PUMP (MMP-7), and a cysteine proteinase, cathepsin B, were investigated on aggrecan the major proteoglycan of cartilage. All the enzymes cleaved aggrecan although the activity of the 95-kDa gelatinase was very low. Specific cleavage sites were investigated following incubation with a purified aggrecan G1-G2 domain fragment (150 kDa). Both gelatinases produced 110-kDa G2 and 56-kDa G1 products by a single cleavage at an Asn-Phe bond within the interglobular domain close to the G1 domain. This was similar to the action of stromelysin (MMP-3) (Fosang, A. J., Neame, P. J., Hardingham, T. E., Murphy, G., and Hamilton, J. A. (1991) J. Biol. Chem. 266, 15579-15582). Cathepsin B also produced two fragments from a single cleavage at a Gly-Val bond only three amino acids C-terminal to the metalloproteinase cleavage site. PUMP cleaved at the metalloproteinase Asn-Phe site, but in addition produced a low yield of a smaller G2 fragment (56 kDa) corresponding to cleavage between Asp441 and Leu442 (human sequence), within the interglobular domain, close to the G2 domain. The apparent difference in size between the two G2 fragments released by PUMP (110 and 56 kDa) was much greater than predicted from the peptide length between the cleavage sites (100 amino acids). However, keratanase digestion greatly reduced the size of the 110-kDa G2 fragment, while producing only a small reduction in size of the 56-kDa product, showing that there was approximately 30-40 kDa of keratan sulfate attached to the interglobular domain between the PUMP cleavage sites. This new structural information on aggrecan may account for the previously observed stiffness of the interglobular domains when viewed by rotary shadowing electron microscopy (Paulsson, M., Morgelin, M., Wiedemann, H., Beardmore-Gray, M., Dunham, D. G., Hardingham, T. E., Heinegard, D., Timpl, R., and Engel, J. (1987) Biochem. J. 245, 763-772). These results show that in spite of a high keratan sulfate content the interglobular domain provides important sites for cleavage by different proteinases, including several members of the matrix metalloproteinase family.     

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.     

The organization of aggrecan in human articular cartilage. Evidence for age-related changes in the rate of aggregation of newly synthesized molecules

The effect of age on the incorporation of newly synthesized aggrecan into the extracellular matrix of human articular cartilage was investigated. This property was measured in a pulse-chase explant culture system by determining the distribution of radiolabeled molecules ([(35)S]sulfate-labeled) between a nondissociating extract (phosphate-buffered saline), which extracts mainly nonaggregated macromolecules, and a dissociating extract (4 M GnHCl) containing mainly aggrecan that was complexed in situ with hyaluronan. The rate of incorporation of aggrecan into aggregates was much slower in mature cartilage than in tissue obtained from younger individuals. Furthermore, autoradiography showed that in mature cartilage, newly synthesized aggrecan is not transported from the pericellular environment within the first 18 h of chase culture, whereas in immature cartilage, it moves into the intercellular space during the same period, i.e. aggrecan is processed in the extracellular space very differently in young and adult articular cartilage. Experiments were also performed to show that the interaction of link protein with newly synthesized aggrecan depends on the maturity of the G(1) domain of aggrecan. This investigation has shown that the extracellular aggregation of aggrecan in adult human articular cartilage involves a number of intermediate structures. These have not been identified in the very young cartilage obtained from laboratory animals or in porcine and bovine articular cartilage obtained from the abattoir.     

Membrane-type 1 MMP (MMP-14) cleaves at three sites in the aggrecan interglobular domain

An aggrecan G1-G2 substrate was used to determine sites within the interglobular domain that were susceptible to cleavage by MT1-MMP. Degradation products were identified by Western blotting with neo-epitope antibodies specific for MMP-derived N- and C-terminal sequences. The results showed that MT1-MMP cleaved at the N₃₄₁-F₃₄₂ and D₄₄₁-L₄₄₂ bonds, as shown for other MMPs, and also at a site 13 amino acids C-terminal to the N₃₄₁-F₃₄₂ site. The G2 product of this additional cleavage was identified by sequence analysis and revealed an N-terminus commencing T₃₅₅VxxPDVELPLP. The data are consistent with MT1-MMP cleavage at three sites in the aggrecan interglobular domain; one at N₃₄₂-F₃₄₂, a second at D₄₄₁-L₄₄₂ and a third at Q₃₅₄-T₃₅₅.     

Peptides of type II collagen can induce the cleavage of type II collagen and aggrecan in articular cartilage.

The objective of this study was to determine whether a fragment(s) of type II collagen can induce cartilage degradation. Fragments generated by cyanogen bromide (CB) cleavage of purified bovine type II collagen were separated by HPLC. These fragments together with selected overlapping synthetic peptides were first analysed for their capacity to induce cleavage of type II collagen by collagenases in chondrocyte and explant cultures of healthy adult bovine articular cartilage. Collagen cleavage was measured by immunoassay and degradation of proteoglycan (mainly aggrecan) was determined by analysis of cleavage products of core protein by Western blotting. Gene expression of matrix metalloproteinases MMP-13 and MMP-1 was measured using Real-time PCR. Induction of denaturation of type II collagen in situ in cartilage matrix with exposure of the CB domain was identified with a polyclonal and monoclonal antibodies that only react with this domain in denatured but not native type II collagen. As well as the mixture of CB fragments and peptide CB12, a single synthetic peptide CB12-II (residues 195-218), but not synthetic peptide CB12-IV (residues 231-254), potently and consistently induced in explant cultures at 10 microM and 25 microM, in a time, cell and dose dependent manner, collagenase-induced cleavage of type II collagen accompanied by upregulation of MMP-13 expression but not MMP-1. In isolated chondrocyte cultures CB12-II induced very limited upregulation of MMP-13 as well as MMP-1 expression. Although this was accompanied by concomitant induction of cleavage of type II collagen by collagenases, this was not associated by aggrecan cleavage. Peptide CB12-IV, which had no effect on collagen cleavage, clearly induced aggrecanase specific cleavage of the core protein of this proteoglycan. Thus these events involving matrix molecule cleavage can importantly occur independently of each other, contrary to popular belief. Denaturation of type II collagen with exposure of the CB12-II domain was also shown to be much increased in osteoarthritic human cartilage compared to non-arthritic cartilage. These observations reveal that peptides of type II collagen, to which there is increased exposure in osteoarthritic cartilage, can when present in sufficient concentration induce cleavage of type II collagen (CB12-II) and aggrecan (CB12-IV) accompanied by increased expression of collagenases. Such increased concentrations of denatured collagen are present in adult and osteoarthritic cartilages and the exposure of chondrocytes to the sequences they encode, either in soluble or more likely insoluble form, may therefore play a role in the excessive resorption of matrix molecules that is seen in arthritis and development.     

ADAMTS4 cleaves at the aggrecanase site (Glu373-Ala374) and secondarily at the matrix metalloproteinase site (Asn341-Phe342) in the aggrecan interglobular domain

Two major proteolytic cleavages, one at NITEGE(373)/A(374)RGSVI and the other at VDIPEN(341)/F(342)FGVGG, have been shown to occur in vivo within the interglobular domain of aggrecan. The Glu(373)-Ala(374) site is cleaved in vitro by aggrecanase-1 (ADAMTS4) and aggrecanase-2 (ADAMTS5), whereas the other site, at Asn(341)-Phe(342), is efficiently cleaved by matrix metalloproteinases (MMPs) and by cathepsin B at low pH. Accordingly, the presence of the cleavage products globular domain 1 (G1)-NITEGE(373) and G1-VDIPEN(341) in vivo has been widely interpreted as evidence for the specific involvement of ADAMTS enzymes and MMPs/cathepsin B, respectively, in aggrecan proteolysis in situ. We show here, in digests with native human aggrecan, that purified ADAMTS4 cleaves primarily at the Glu(373)-Ala(374) site, but also, albeit slowly and secondarily, at the Asn(341)-Phe(342) site. Cleavage at the Asn(341)-Phe(342) site in these incubations was due to bona fide ADAMTS4 activity (and not a contaminating MMP) because the cleavage was inhibited by TIMP-3 (a potent inhibitor of ADAMTS4), but not by TIMP-1 and TIMP-2, at concentrations that totally blocked MMP-3-mediated cleavage at this site. Digestion of recombinant human G1-G2 (wild-type and cleavage site mutants) confirmed the dual activity of ADAMTS4 and supported the idea that the enzyme cleaves primarily at the Glu(373)-Ala(374) site and secondarily generates G1-VDIPEN(341) by removal of the Phe(342)-Glu(373) peptide from G1-NITEGE(373). These results show that G1-VDIPEN(341) is a product of both MMP and ADAMTS4 activities and challenge the widely held assumption that this product represents a specific indicator of MMP- or cathepsin B-mediated aggrecan degradation.     

High pressure gel-permeation assay for the proteolysis of human aggrecan by human stromelysin-1: kinetic constants for aggrecan hydrolysis.

The adaptation of an analytical procedure for aggrecan based upon gel-permeation chromatography to an FPLC-based protocol has significantly sped up the analysis. The faster assay has permitted determination of the kinetic constants for digestion of human aggrecan by human stromelysin-1. Monomeric aggrecan appeared to be hydrolyzed by stromelysin-1 to multiple forms with lower molecular weight. The disappearance of high-molecular-weight aggrecan was first-order, showing Km much larger than 2 microM and kc/Km = 4000 M-1 s-1 at pH 7.5. The disappearance of high-molecular-weight aggrecan upon hydrolysis by stromelysin-1 at pH 5.5 was also first-order, with kc/Km = 10,700 M-1 s-1. The disappearance of high-molecular-weight aggrecan at pH 7.5 was first-order for digestion by human leukocyte elastase with kc/Km = 230,000 M-1 s-1, by human cathepsin G with kc/Km = 4200 M-1 S-1, and by human plasma plasmin with kc/Km = 2800 M-1 s-1, all with Km much larger than 2 microM.     

Soluble recombinant neprilysin induces aggrecanase-mediated cleavage of aggrecan in cartilage explant cultures.

Neprilysin (neutral endopeptidase, enkephalinase, CALLA, CD10, NEP) is a regulatory Zn metallopeptidase expressed in the brush border membranes of the kidney and has been found in porcine chondrocytes and rat articular cartilage as well as other cell types and tissues. Although its function in cartilage is not currently known, previous observations of high levels of NEP enzymatic activity in the synovial fluid of arthritic patients and on the chondrocyte membranes of human osteoarthritic cartilage have led to the hypothesis that NEP is involved in the inflammation or degradation pathways in articular cartilage. Our study localized endogenous NEP to the membranes of mature bovine articular chondrocytes in a tissue explant model and demonstrated that the addition of soluble recombinant NEP (sNEP) to the culture medium of bovine cartilage explants leads to the degradation of aggrecan through the action of aggrecanase. A 6-day exposure to sNEP was necessary to initiate the degradation, suggesting that the chondrocytes were responding in a delayed manner to an altered composition of regulatory peptides. This NEP-induced degradation was completely inhibited by the NEP inhibitors thiorphan and phosphoramidon. These results suggest that NEP is present as a transmembrane enzyme on articular chondrocytes where it can cleave regulatory peptides and lead to the induction of aggrecanase.     

Keratan sulphate in the interglobular domain has a microstructure that is distinct from keratan sulphate elsewhere on pig aggrecan

The microstructure of keratan sulphate purified from the interglobular domain, the keratan sulphate-rich region and total aggrecan was compared using fluorophore-assisted-carbohydrate-electrophoresis. Keratan sulphate in the interglobular domain was substantially less sulphated than keratan sulphate elsewhere on aggrecan, based on the ratio of unsulphated: monosulphated disaccharides generated by endo-β-galactosidase digestion, and the ratio of monosulphated: disulphated disaccharides generated by keratanase II digestion. The ratio of unsulphated: monosulphated: disulphated disaccharides was 1:4:5 for keratan sulphate from total aggrecan and the keratan sulphate-rich region, but only 1:0.9:0.8 for the interglobular domain. These results show that keratan sulphate in the interglobular domain of pig aggrecan has a microstructure that is distinct from keratan sulphate in the keratan sulphate-rich region.     

Metalloprotease-mediated GH receptor proteolysis and GHBP shedding. Determination of extracellular domain stem region cleavage site

Growth hormone-binding protein (GHBP) is complexed to a substantial fraction of circulating GH. In humans, rabbits, and other species, GHBP derives from proteolytic shedding of the GH receptor (GHR) extracellular domain. In cell culture studies, stimuli such as phorbol ester, platelet-derived growth factor, or serum induce GHR proteolysis, which concomitantly yields shed GHBP in cell supernatants and a cell-associated cytoplasmic domain-containing GHR remnant. This process is sensitive to metalloprotease inhibition, and genetic reconstitution studies identify tumor necrosis factor-alpha converting enzyme (TACE/ADAM-17), a transmembrane metalloprotease, as a GHR sheddase. Stimuli that induce GHR proteolysis render cells less responsive to GH, but the mechanism(s) of this desensitization is not yet understood. In this study, we mapped the rabbit (rb) GHR cleavage site. We adenovirally expressed a C-terminal epitope-tagged rbGHR lacking most of its cytoplasmic domain, purified the remnant protein induced by the phorbol ester, PMA, and derived the cleavage site by N-terminal sequencing of the purified remnant. The N-terminal sequence, (239)FTCEEDFR(246), matched perfectly the rbGHR and suggests that cleavage occurs eight residues from the membrane in the proximal extracellular domain stem region. Deletion and alanine substitution mutagenesis indicated that, similar to other TACE substrates, the spacing of residues in this region, more than their identity, influences GHR cleavage susceptibility. Further, we determined that PMA pretreatment desensitized a cleavage-sensitive GHR mutant, but not a cleavage-insensitive mutant, to GH-induced JAK2 activation. These results suggest that inducible GHR proteolysis can regulate GH signaling.     

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.     

Distribution of hyaluronan in articular cartilage as probed by a biotinylated binding region of aggrecan

The proportion of total tissue hyaluronan involved in interactions with aggrecan and link protein was estimated from extracts of canine knee articular cartilages using a biotinylated hyaluronan binding region-link protein complex (bHABC) of proteoglycan aggregate as a probe in an ELISA-like assay. Microscopic sections were stained with bHABC to reveal free hyaluronan in various sites and zones of the cartilages. Articular cartilage, cut into 20 m-thick sections, was extracted with 4 M guanidinium chloride (GuCl). Aliquots of the extract (after removing GuCl) were assayed for hyaluronan, before and after papain digestion. The GuCl extraction residues were analyzed after solubilization by papain. It was found that 47–51% of total hyaluronan remained in the GuCl extraction residue, in contrast to the 8–15% of total proteoglycans. Analysis of the extract revealed that 24–50% of its hyaluronan was directly detecable with the probe, while 50–76% became available only after protease digestion. The extracellular matrix in cartilage sections was stained with the bHABC probe only in the superficial zone and the periphery of the articular surfaces, both sites known to have a relatively low proteoglycan concentration. Trypsin pretreatment of the sections enhanced the staining of the intermediate and deep zones, presumably by removing the steric obstruction caused by the chondroitin sulfate binding region of aggrecans. Enhanced matrix staining in these zones was also obtained by a limited digestion with chondroitinase ABC. The results indicate that a part of cartilage hyaluronan is free from endogenous binding proteins, such as aggrecan and link protein, but that the chondroitin sulfate-rich region of aggrecan inhibits its probing in intact tissue sections. Therefore, hyaluronan staining was more intense in cartilage areas with lower aggrecan content. A large proportion of hyaluronan resists GuCl extraction, even from 20-m-thick tissue sections.     

Identification of a heparin binding domain in the N-terminal cleavage site of pro-islet amyloid polypeptide. Implications for islet amyloid formation

Islet amyloid deposits are a characteristic pathologic lesion of the pancreas in type 2 diabetes and are composed primarily of the islet beta cell peptide islet amyloid polypeptide (IAPP or amylin) as well as the basement membrane heparan sulfate proteoglycan perlecan. Impaired processing of the IAPP precursor has been implicated in the mechanism of islet amyloid formation. The N- and C-terminal cleavage sites where pro-IAPP is processed by prohormone convertases contain a series of basic amino acid residues that we hypothesized may interact with heparan sulfate proteoglycans. This possibility was tested using affinity chromatography by applying synthetic fragments of pro-IAPP to heparin-agarose and heparan sulfate-Sepharose. An N-terminal human pro-IAPP fragment (residues 1-30) was retained by both heparin-agarose and heparan sulfate-Sepharose, eluting at 0.18 m NaCl at pH 7.5. Substitution of alanine residues for two basic residues in the N-terminal cleavage site abolished heparin and heparan sulfate binding activity. At pH 5.5, the affinity of the wild-type peptide for heparin/heparan sulfate was increased, implying a role for histidine residues at positions 6 and 28 of pro-IAPP. A C-terminal pro-IAPP fragment (residues 41-67) had no specific affinity for either heparin or heparan sulfate, and the N- or C-terminal fragments had only weak affinity for chondroitin sulfate. These data suggest that monomeric N-terminal human pro-IAPP contains a heparin binding domain that is lost during normal processing of pro-IAPP.     

Monoclonal antibodies recognizing protease-generated neoepitopes from cartilage proteoglycan degradation. Application to studies of human link protein cleavage by stromelysin.

Monoclonal antibodies were raised that specifically recognize the NH2-terminal neoepitope sequence present in link protein cleavage products derived from stromelysin-degraded proteoglycan aggregate. Competitive enzyme-linked immunosorbent assay, using synthetic peptides as inhibitors, showed that one of these antibodies (CH-3) required, for antibody recognition, the free NH2-terminal amino acid isoleucine (residue 17 of the intact protein) in the sequence NH2-IQAENG at the stromelysin cleavage site of link protein 3. Human proteoglycan aggregate was digested with recombinant human stromelysin, bovine chymotrypsin, bovine trypsin, and porcine elastase, and their respective link protein degradation products were tested for immunoreactivity with antibody CH-3. Only stromelysin- and chymotrypsin-generated link protein 3 were recognized by antibody CH-3. Both of these enzymes generate link protein NH2 termini with the sequence 17IQAENG. . .; hence these studies indicated that monoclonal antibody CH-3 recognized this neoepitope sequence in only specific proteolytically modified link protein molecules. Since the occurrence of link protein 3 increases with aging, the incidence of CH-3 epitope in proteoglycans isolated from human knee articular cartilage of individuals of different ages was investigated. The prevalence of CH-3 epitope was found to be highest in newborn and adolescent articular cartilage samples. However, little CH-3 epitope was detected in older adult cartilage, although considerably more link protein 3 was present in these samples. These results suggest that additional proteolytic agents are responsible for the increased occurrence of link protein degradation products with aging.     

Identification and clonal characterisation of a progenitor cell sub-population in normal human articular cartilage

Background

Articular cartilage displays a poor repair capacity. The aim of cell-based therapies for cartilage defects is to repair damaged joint surfaces with a functional replacement tissue. Currently, chondrocytes removed from a healthy region of the cartilage are used but they are unable to retain their phenotype in expanded culture. The resulting repair tissue is fibrocartilaginous rather than hyaline, potentially compromising long-term repair. Mesenchymal stem cells, particularly bone marrow stromal cells (BMSC), are of interest for cartilage repair due to their inherent replicative potential. However, chondrocyte differentiated BMSCs display an endochondral phenotype, that is, can terminally differentiate and form a calcified matrix, leading to failure in long-term defect repair. Here, we investigate the isolation and characterisation of a human cartilage progenitor population that is resident within permanent adult articular cartilage.

Methods and Findings

Human articular cartilage samples were digested and clonal populations isolated using a differential adhesion assay to fibronectin. Clonal cell lines were expanded in growth media to high population doublings and karyotype analysis performed. We present data to show that this cell population demonstrates a restricted differential potential during chondrogenic induction in a 3D pellet culture system. Furthermore, evidence of high telomerase activity and maintenance of telomere length, characteristic of a mesenchymal stem cell population, were observed in this clonal cell population. Lastly, as proof of principle, we carried out a pilot repair study in a goat in vivo model demonstrating the ability of goat cartilage progenitors to form a cartilage-like repair tissue in a chondral defect.

Conclusions

In conclusion, we propose that we have identified and characterised a novel cartilage progenitor population resident in human articular cartilage which will greatly benefit future cell-based cartilage repair therapies due to its ability to maintain chondrogenicity upon extensive expansion unlike full-depth chondrocytes that lose this ability at only seven population doublings.