Degradation of human proteoglycan aggregate induced by hydrogen peroxide. Protein fragmentation, amino acid modification and hyaluronic acid cleavage.

We have previously shown that treatment of neonatal human articular-cartilage proteoglycan aggregates with H2O2 results in loss of the ability of the proteoglycan subunits to interact with hyaluronic acid and in fragmentation of the link proteins [Roberts, Mort & Roughley (1987) Biochem. J. 247, 349-357]. We now show the following. (1) Hyaluronic acid in proteoglycan aggregates is also fragmented by treatment with H2O2. (2) Although H2O2 treatment results in loss of the ability of the proteoglycan subunits to interact with hyaluronic acid, the loss of this function is not attributable to substantial cleavage of the hyaluronic acid-binding region of the proteoglycan subunits. (3) In contrast, link proteins retain the ability to bind to hyaluronic acid following treatment with H2O2. (4) The interaction between the proteoglycan subunit and link protein is, however, abolished. (5) N-Terminal sequence analysis of the first eight residues of the major product of link protein resulting from H2O2 treatment revealed that cleavage occurred between residues 13 and 14, so that the new N-terminal amino acid is alanine. (6) In addition, a histidine (residue 16) is converted into alanine and an asparagine (residue 21) is converted into aspartate by the action of H2O2. (7) Rat link protein showed no cleavage or modifications in similar positions under identical conditions. (8) This species variation may be related to the different availability of histidine residues required for the co-ordination of the transition metal ion involved in hydroxyl-radical generation from H2O2. (9) Changes in function of these structural macromolecules as a result of the action of H2O2 may be consequences of both fragmentation and chemical modification.     

Degradation of proteoglycan aggregate by a cartilage metalloproteinase. Evidence for the involvement of stromelysin in the generation of link protein heterogeneity in situ.

Cartilage proteoglycan aggregates were subjected to degradation by a metalloproteinase, capable of degrading proteoglycan, released from cartilage in culture. This proteinase was demonstrated to be immunologically identical with fibroblast stromelysin. An early release of hyaluronic acid-binding region and large glycosaminoglycan-attachment regions was observed. With increasing time the glycosaminoglycan-attachment regions were digested into smaller fragments and the hyaluronic acid-binding regions accumulated. The degradation of link proteins also occurred concomitantly with these events. Link proteins were converted into a component of similar size to that of the smallest native link protein component. N-Terminal sequence analysis of the three human link protein components indicated that they are all derived from the same protein core, which is closely homologous to that of the rat chondrosarcoma link protein. The two larger link proteins (Mr 48,000 and 44,000) contain the same N-terminal sequence, but they differ by the apparent presence of an N-linked oligosaccharide at residue 6 of the largest link protein component. The smallest link protein (Mr 41,000), however, has an N-terminal sequence equivalent to that commencing at residue 17 in the larger link proteins. It was found that the cartilage metalloproteinase cleaves link proteins in human neonatal cartilage proteoglycan aggregates at the His-16-Ile-17 bond, the same position at which the smallest link protein component appears to be derived naturally from the two larger link protein components. These results suggest that stromelysin secreted by chondrocytes can account for the increased accumulation of hyaluronic acid-binding regions and much of the degradation of link protein observed during aging within human articular cartilage.     

The role of link protein in mediating the interaction between hyaluronic acid and newly secreted proteoglycan subunits from adult human articular cartilage

Normal adult human articular cartilage in organ culture secretes proteoglycan subunits that cannot initially interact in a normal manner with hyaluronic acid unless the latter is present at high concentrations and a neutral pH is employed. However, if the newly secreted subunit is allowed to mature in the cartilage matrix for up to 12 h, then its ability to interact is indistinguishable from that of its more mature counterparts. This conversion does not take place if the proteoglycan subunits are incubated in dilute solutions in the absence of the cartilage, and it is prevented by culturing at low temperature. The newly secreted proteoglycan subunits can, however, be induced to interact with hyaluronic acid by the presence of link proteins. The complex formed by these three components cannot be dissociated in the presence of hyaluronic acid oligosaccharides, suggesting a normal aggregate configuration. It is thus possible that proteoglycan aggregate formation within the cartilage is initially mediated by the presence of link proteins, which induce a conformational change with the hyaluronic acid-binding region of the proteoglycan subunits, although additional modification may be necessary to render any such change irreversible.     

Identification of a hyaluronic acid-binding protein that interferes with the preparation of high-buoyant-density proteoglycan aggregates from adult human articular cartilage.

Adult human articular cartilage contains a hyaluronic acid-binding protein of Mr 60 000-75 000, which contains disulphide bonds essential for this interaction. The molecule can compete with proteoglycan subunits for binding sites on hyaluronic acid, and can also displace proteoglycan subunits from hyaluronic acid if their interaction is not stabilized by the presence of link proteins. The abundance of this protein in the adult accounts for the reported inability to prepare high-buoyant-density proteoglycan aggregates from extracts of adult human cartilage [Roughley, White, Poole & Mort (1984) Biochem. J. 221, 637-644], whereas the deficiency of the protein in newborn human cartilage allows the normal recovery of proteoglycan aggregates from this tissue. The protein shares many common features with a hyaluronic acid-binding region derived by proteolytic treatment of a proteoglycan aggregate preparation, and this may also represent its origin in the cartilage, with its production increasing during tissue maturation.     

The action of human articular-cartilage metalloproteinase on proteoglycan and link protein. Similarities between products of degradation in situ and in vitro.

Interleukin 1 stimulation of human articular cartilage in organ culture produced the concomitant release of proteoglycan fragments and latent metalloproteinase. The released fragments ranged in size from that of almost intact proteoglycan subunits to the product of limiting digestion generated by the activated metalloproteinase. None of the fragments possessed the ability to interact with hyaluronic acid. Analysis of proteoglycan aggregate digested with the activated metalloproteinase showed that isolated hyaluronic acid-binding regions were produced from the proteoglycan subunits, and that the two higher-Mr link-protein components (Mr 48,000 and 44,000) were converted into the lowest-Mr component (Mr 41,000). Link protein extracted from cartilage under stimulation with interleukin 1 showed a similar conversion. These results suggest that interleukin 1 stimulates the release of latent metalloproteinase from chondrocytes and that a proportion of the enzyme is activated in situ in the cartilage matrix. The mode of action of the activated enzyme is compatible with a role in the changes in proteoglycan structure seen in aging.     

Immunochemistry of cartilage proteoglycan. Immunodiffusion and gel-electrophoretic studies

Cartilage proteoglycan is thought to be composed of subunits, core proteins with covalently attached sulphated polysaccharide side chains, which form aggregates by non-covalent association with a link protein. The new technique of non-disruptive extraction followed by fractionation in caesium chloride gradients provides a useful means of preparing relatively pure proteoglycan aggregate, subunit and link fractions. Immunological studies of these fractions led to the identification of an antigen associated with the proteoglycan subunit which was common to several species and to the demonstration of additional species-specific antigens in aggregate and link fractions derived from bovine nasal cartilage. Polyacrylamide-gel electrophoresis with sodium dodecyl sulphate of bovine proteoglycan aggregate and link fractions gave two protein bands in the gels and a protein-polysaccharide band at the origin; subunit fractions gave only the band at the origin. These results are consistent with the current concept of cartilage proteoglycan structure.     

The inability to prepare high-buoyant-density proteoglycan aggregates from extracts of normal adult human articular cartilage.

High-buoyant-density proteoglycan aggregates could not be prepared from extracts of adult human cartilage by associative CsCl-density-gradient centrifugation with a starting density of 1.68 g/ml, even though proteoglycan subunits, hyaluronic acid and link proteins were all present. In contrast, aggregates could be prepared when extracts of neonatal human cartilage or bovine nasal cartilage were subjected to the same procedure. This phenomenon did not appear to be due to a defect within the hyaluronic acid-binding region of the adult proteoglycan subunit, but rather to an interference in the stability of the interaction between the proteoglycan subunit and hyaluronic acid towards centrifugation. The factor responsible for this instability was shown to reside within the low-density cartilage protein preparation obtained by direct dissociative CsCl-density-gradient centrifugation of the adult cartilage extract.     

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.     

Cartilage proteoglycan aggregate is degraded more extensively by cathepsin L than by cathepsin B.

The degradative actions of cathepsins L and B on human articular-cartilage proteoglycan aggregates were examined. Cathepsin L was found to be much more extensive than cathepsin B in degrading proteoglycan aggregates. It released products with size similar to that of single chondroitin sulphate chains, and a series of degraded link-protein fragments in the digestion mixtures. These proteolytically modified link-protein components (Mr 25,000 and 33,000) have similar Mr values to those of fragments observed in adult human cartilage. In contrast, cathepsin B exhibited a much more limited degradation on both proteoglycan subunits and link-protein components. Both cathepsins L and B generate multiple but distinct cleavage sites on human link proteins, and the hydrolysed bonds have been identified in the region between residues 18 and 29. Protein sequencing analysis of these modified link-protein components also provided evidence for the location of a second N-linked glycosylation site at residue 41 in human link proteins, in addition to that previously described at residue 6 on a proportion of the link proteins. Furthermore, it allows us to report the sequence of human link protein up to residue 65.     

A comparison of isometallothionein synthesis in rat liver after partial hepatectomy and parenteral zinc injection.

Proteoglycan aggregates free of non-aggregating proteoglycan have been prepared from the annuli fibrosi and nuclei pulposi of intervertebral discs of three human lumbar spines by extraction with 4M-guanidinium chloride, associative density gradient centrifugation, and chromatography on Sepharose CL-2B. The aggregate (A1-2B.V0) was subjected to dissociative density-gradient ultracentrifugation. Three proteins of Mr 38 900, 44 200 and 50 100 found in the fraction of low buoyant density (A1-2B.V0-D4) reacted with antibodies to link protein from newborn human articular cartilage. After reduction with mercaptoethanol, two proteins of Mr 43 000 and two of Mr 20 000 and 14 000 were seen. The A1-2B.V0-D4 fraction, labelled with 125I, coeluted with both hyaluronate and a hyaluronate oligosaccharide (HA14) on a Sepharose CL-2B column. HA10 and HA14 reduced the viscosity of A1 fractions; HA4, HA6 and HA8 did not. HA14 decreased the viscosity of disc proteoglycans less than it did that of bovine cartilage proteoglycans. Thus, although a link protein was present in human intervertebral disc, it stabilized proteoglycan aggregates less well than did the link protein from bovine nasal cartilage.     

Production and characterization of monoclonal antibodies directed against connective tissue proteoglycans

Monoclonal antibodies have been raised against determinants present in cartilage proteoglycan. Characterization of the specificity of these antibodies indicated that they recognize determinants present in the keratan sulfate glycosaminoglycan chain and on chondroitin sulfate oligosaccharide stubs attached to the proteoglycan core protein after chondroitinase digestion of the proteoglycan (i.e., delta-unsaturated 4- and 6-sulfated and unsulfated chondroitin sulfate on the proteoglycan core). The antibody recognizing keratan sulfate has been used to demonstrate the presence of a keratan sulfate-rich proteoglycan subpopulation that increases with increasing age of animal compared with chondroitin sulfate-rich proteoglycans. Monoclonal antibodies recognizing determinants on chondroitinase-treated proteoglycan have been used in immunohistochemical localization studies determining the differential distribution of 4- and 6-sulfated and unsulfated proteoglycans in tissue sections of cartilage and other noncartilaginous tissues. Digestion with chondroitinase ABC or ACII can be used to differentiate between chondroitin sulfate and dermatan sulfate proteoglycan in different connective tissues. In addition, the presence of a 6-sulfated chondroitin sulfate proteoglycan that is associated with membranes surrounding nerve and muscle fiber bundles is described. Monoclonal antibodies were also raised against the link protein(s) of cartilage proteoglycan aggregate. They have been used in peptide map analyses of link protein and in demonstrating the presence of a high-mannose oligosaccharide chain of the link proteins. The presence of high-mannose oligosaccharide structures on the link protein(s) accounts for the microheterogeneity of the link proteins (link proteins 1, 2, or 3) that is observed on sodium dodecyl sulfate-polyacrylamide gels.     

Proteoglycans from bovine nasal and articular cartilages. Fractionation of the link proteins by wheat germ agglutinin affinity chromatography

Two forms of link protein, 46 and 51 kDa, are present in proteoglycan aggregates from both bovine nasal and bovine articular cartilages. Studies reported here show that the link proteins bind to concanavalin A, Lens culinaris agglutinin, Ricinus communis agglutinin, soybean agglutinin, and wheat germ agglutinin lectins. When the link proteins are eluted from these lectins with appropriate competing sugars, the 46- and the 51-kDa link proteins elute together and no separation is achieved. However, when the link proteins bound to wheat germ agglutinin are eluted with a 0 to 4 M guanidine hydrochloride linear gradient, a good separation of the 46- and 51-kDa link proteins is achieved. Wheat germ agglutinin affinity chromatography has been used on a preparative scale to isolate the 51-kDa link protein from mature bovine articular cartilage to homogeneity, in amounts sufficient to examine its effect on proteoglycan aggregate size and stability in sedimentation velocity studies. Proteoglycan aggregates were reassembled from proteoglycan monomers and hyaluronate in the absence of link protein, in the presence of both 46- and 51-kDa link proteins, and in the presence of the individual 51-kDa link protein. The sizes of the aggregates were compared in terms of sedimentation coefficients (s(0)20). The stability of the aggregates was compared in terms of the per cent aggregate present at pH 7 and 5. At pH 7, the sedimentation coefficients (s(0)20) of link-free aggregates, aggregates formed with both link proteins, and aggregates formed with 51-kDa link protein were 72, 93, and 112 S, respectively. Thus, the 51-kDa link protein has a pronounced effect on aggregate size. The link-free aggregate was grossly unstable, and only 36% aggregate was present at pH 5. The aggregate formed with both link proteins was effectively stabilized against dissociation and 79% aggregate was present at pH 5. The aggregate formed with 51-kDa link protein was not effectively stabilized against dissociation, and only 60% aggregate was present at pH 5. Thus, despite its pronounced effect on aggregate size, the 51-kDa link protein does not effectively stabilize the proteoglycan aggregate against dissociation. These results suggest that the 51-kDa link protein may selectively increase aggregate size, while the 46-kDa link protein may be required to effectively stabilize the proteoglycan aggregate against dissociation.     

Monoclonal antibodies as probes for determining the microheterogeneity of the link proteins of cartilage proteoglycan

Monoclonal antibodies were raised against Swarm rat chondrosarcoma link protein 2. Two of the resultant hybridomas (9/30/6-A-1 and 9/30/8-A-4) were used in structural analyses of the link proteins. The 9/30/6-A-1 monoclonal antibody recognized an epitope which was only present on rat chondrosarcoma link protein 2. This epitope was absent in rat chondrosarcoma link protein 3 obtained after trypsin or clostripain treatment of rat chondrosarcoma proteoglycan aggregate, indicating that proteolytic digestion either removed or modified the epitope. Contrasting this, the 9/30/8-A-4 monoclonal antibody recognized an epitope present in link protein(s) 1, 2, or 3 isolated from cartilage of several animal species (rat, bovine, human, and chicken). Rat chondrosarcoma link protein 2 was digested with Staphylococcus aureus V8 protease, and the resulting peptides were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to immunolocation analyses. The 9/30/6-A-1 and 9/30/8-A-4 monoclonal antibodies recognized epitopes in two different halves of the link protein molecule. The 9/30/8-A-4 monoclonal antibody was used to identify proteolytic cleavage peptides common to the individual link proteins (1, 2, or 3) purified from cartilage proteoglycans of several animal species. Digestion of rat chondrosarcoma link protein 2 with endoglycosidase H or alpha-mannosidase increased its electrophoretic mobility to that of link protein 3 and removed or altered the determinant recognized by the 9/30/6-A-1 monoclonal antibody, indicating that a high-mannose oligosaccharide chain was part of the antigenic determinant. The 9/30/8-A-4 monoclonal recognition of epitope was unaffected by endo- or exoglycosidase treatment. Endo- and exoglycosidase treatment of bovine nasal cartilage link proteins also altered their electrophoretic mobility, indicating that high-mannose oligosaccharide structures on the various link proteins (1, 2, or 3) accounted for the microheterogeneity observed in sodium dodecyl sulfate-polyacrylamide gels.     

Persistence of cartilage proteoglycan and link protein during matrix-induced endochondral bone development: An immunofluorescent study

Monospecific antibodies to cartilage proteoglycan monomer and link protein were employed with immunofluorescence microscopy to determine the tissue distribution of these constituents during matrix-induced endochondral bone development. Subcutaneous implantation of demineralized diaphyseal bone matrix resulted in new endochondral bone formation. On Day 3, the implant consisted of mesenchymal tissue which did not contain any demonstrable cartilage-related proteoglycan or link protein. With the onset of early chondrogenesis on Day 5, cartilage proteoglycan monomer and link protein were first localized together in the cartilage matrix, particularly around chondrocytes in territorial sites. Progressively more staining around cells was observed at Days 7 and 9. On Day 9, when mineralization was first observed, there was no evidence of a net loss of these molecules prior to mineralization of the cartilage matrix. On Day 11 and thereafter, bone formation was observed by appositional growth on calcified cartilage spicules. Whereas the osteoblasts and bone matrix were devoid of any staining for cartilage proteoglycan and link components, the residual, partly mineralized cartilage spicules still reacted with antibodies to cartilage proteoglycan monomer and link protein in territorial sites, but in reduced amounts, indicating a loss of these molecules associated with a loss of hypertrophic chondrocytes. Since mineral prevented the access of Fab' antibody subunits, demineralization after fixation was routinely employed. The results reveal that cartilage proteoglycan monomer and link protein are present around chondrocytes in hyaline cartilage during the early stages of endochondral bone formation and that there is no net loss of these molecules prior to mineralization of this cartilage matrix as was previously thought.     

Proteoglycan aggregate formation by articular chondrocytes. Decrease in link-protein synthesis during culture.

The synthesis of link-stabilized proteoglycan aggregates by rabbit articular chondrocytes was investigated by [35S]sulphate labelling of primary monolayer cultures maintained for up to 21 days. (1) At all culture times the cells secreted a high-molecular-weight cartilage-type proteoglycan monomer of which 75%-80% formed aggregates with hyaluronic acid. (2) At 2 days of culture all of the aggregates were in link-stabilized form, but by 21 days only 5% were link-stabilized, as shown by displacement of monomers from the aggregate by hyaluronic acid oligosaccharides. (3) The addition of purified link protein to 21-day culture medium increased the proportion of link-stable aggregate from 5% to 70%. (4) Analysis of [3H]serine-labelled proteoglycan aggregates in the medium showed a marked decrease with culture time in the ratio of 3H-labelled link protein to 3H-labelled core protein present. The results suggest that the secretion of proteoglycan monomers and link protein by articular chondrocytes changes independently during prolonged monolayer culture.     

The isolation of collagen-associated proteoglycan from bovine nasal cartilage and its preferential interaction with alpha2 chains of type I collagen.

A collagen complex from bovine nasal cartilage was prepared by extraction of the tissue with 3M-MgCl2 solutions, by using two different procedures. When it was compared with calf skin acid-soluble tropocollagen by polyacrylamide-gel electrophoresis, the 3M-MgCl2-soluble cartilage collagen in the complex appeared to be predominantly type I in nature, consisting of both alpha1 and alpha2 chains. The soluble cartilage collagens were digested with purified bacterial collagenase, and the soluble digests were fractionated on Sepharose 4B. Hydroxyproline-free proteoglycan was isolated in the excluded volume of the column eluate, and this was found to be an aggregate which could be dissociated to link proteins and proteoglycan subunit by equilibrium-density-gradient centrifugation in a CsCl-4M-guanidinium chloride gradient. Interaction with calf skin-soluble tropocollagen was studied by CM-cellulose chromatography. The link-protein system did not interact, but proteoglycan from the bottom of the gradient did interact. In addition, when proteoglycan subunit was allowed to interact with collagen, there was a preferential binding to the alpha2 and beta12 components, and this effect was also observed with the proteoglycan material obtained from the collagenase digests of 3M-MgCl2-soluble cartilage collagen complexes. However, specificity for alpha2 and beta12 chains was not exhibited by chondroitin sulphate glycosaminoglycan, and it is therefore concluded that preference for alpha2 and beta12 chains is a function of the intact proteoglycan structure.     

Distribution of chondroitin sulfate in cartilage proteoglycans under associative conditions.

Proteoglycan aggregates and proteoglycan subunits were extracted from bovine articular cartilage with guanidine-HC1 folowed by fractionation by equilibrium centrifugation in cesium chloride density gradients. The distribution of chondroitin sulfates (CS) in the cartilage proteoglycans was studied at the disaccharide level by digestion with chondroitinases. In the proteoglycan aggregate fraction, it was observed that the proportion of 4-sulfated disaccharide units to total CS increased from the bottom to the top fractions, whereas that of 6-sulfated disaccharide units was in the reverse order. Thus, the ratio of 4-sulfated disaccharide units to 6-sulfated disaccharide units increased significantly with decreasing density. The proportion of non-sulfated disaccharide units to total CS tended to increase with increasing density. These data indicate a polydisperse distribution of CS chains, under the conditions used here, in proteoglycan aggregates from bovine articular cartilage.     

Immunological studies of bovine nasal cartilage proteoglycan "link proteins".

Bovine nasal cartilage proteoglycan aggregates are dissociated and separated by density gradient centrifugation in 4 M guanidine into proteoglycan subunit (PGS) and glycoprotein link (GPL) fractions, the latter containing hyaluronic acid and "link proteins" responsible for aggregate formation. It was previously concluded on the basis of immunodiffusion studies that GPL has two antigenic components, one in common with PGS and one specific for the link proteins. However, in the present study it was found that antisera to PGS, which should lack link proteins, reacted with both "subunit" and "link" components of GPL, and antisera to fragments of PGS derived from the hyaluronic acid-binding portion of the molecule reacted preferentially with the link component. Reduction and alkylation of GPL led to modification of the reactions of both anti-GPL and anti-PGS sera with its link component. These immunodiffusion results indicate that the proteoglycan subunit and the link proteins are immunologically related and suggest that the link proteins may be identical with and derived from the hyaluronic acid binding portion of the proteoglycan subunit.     

Cartilage proteoglycan aggregate formation. Role of link protein.

Cartilage proteoglycan aggregate formation was studied by zonal rate centrifugation in sucrose gradients. Proteoglycan aggregates, monomers and proteins could be resolved. It was shown that the optimal proportion of hyaluronic acid for proteoglycan aggregate formation was about 1% of proteoglycan dry weight. The reaggregation of dissociated proteoglycan aggregate A1 fraction was markedly concentration-dependent and even at 9 mg/ml only about 90% of the aggregates were reformed. The lowest proportion of link protein required for maximal formation of link-stabilized proteoglycan aggregates was 1.5% of proteoglycan dry weight. It was separately shown that link protein co-sedimented with the proteoglycan monomer. By competition with isolated hyaluronic acid-binding-region fragments, a proportion of the link proteins was removed from the proteoglycan monomers, indicating that the link protein binds to the hyaluronic acid-binding region of the proteoglycan monomer.     

Chondrodysplasia of gene knockout mice for aggrecan and link protein

The proteoglycan aggregate of the cartilage is composed of aggrecan, link protein, and hyaluronan and forms a unique gel-like moiety that provides resistance to compression in joints and a foundational cartilage structure critical for growth plate formation. Aggrecan, a large chondroitin sulfate proteoglycan, is one of the major structural macromolecules in cartilage and binds both hyaluronan and link protein through its N-terminal domain G1. Link protein, a small glycoprotein, is homologous to the G1 domain of aggrecan. Mouse cartilage matrix deficiency (cmd) is caused by a functional null mutation of the aggrecan gene and is characterized by perinatal lethal dwarfism and craniofacial abnormalities. Link protein knockout mice show chondrodysplasia similar to but milder than cmd mice, suggesting a supporting role of link protein for the aggregate structure. Analysis of these mice revealed that the proteoglycan aggregate plays an important role in cartilage development and maintenance of cartilage tissue and may provide a clue to the identification of human genetic disorders caused by mutations in these genes. Published in 2003.