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.     

The Different Roles of Aggrecan Interaction Domains

The aggregating proteoglycans of the lectican family are important components of extracellular matrices. Aggrecan is the most well studied of these and is central to cartilage biomechanical properties and skeletal development. Key to its biological function is the fixed charge of the many glycosaminoglycan chains, that provide the basis for the viscoelastic properties necessary for load distribution over the articular surface. This review is focused on the globular domains of aggrecan and their role in anchoring the proteoglycans to other extracellular matrix components. The N-terminal G1 domain is vital in that it binds the proteoglycan to hyaluronan in ternary complex with link protein, retaining the proteoglycan in the tissue. The importance of the C-terminal G3 domain interactions has recently been emphasized by two different human hereditary disorders: autosomal recessive aggrecan-type spondyloepimetaphyseal dysplasia and autosomal dominant familial osteochondritis dissecans. In these two conditions, different missense mutations in the aggrecan C-type lectin repeat have been described. The resulting amino acid replacements affect the ligand interactions of the G3 domain, albeit with widely different phenotypic outcomes.     

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.     

The folded modules of aggrecan G3 domain exert two separable functions in glycosaminoglycan modification and product secretion.

Aggrecan is the major proteoglycan in the extracellular matrix of cartilage. A notable exception is nanomelic cartilage, which lacks aggrecan in its matrix. The example of nanomelia and other evidence leads us to believe that the G3 domain plays an important role in aggrecan processing, and it has indeed been confirmed that G3 allows glycosaminoglycan (GAG) chain attachment and product secretion. However, it is not clear how G3, which contains at least a carbohydrate recognition domain (CRD) and a complement binding protein (CBP) motif, plays these two functional roles. The present study was designed to dissect the mechanisms of this phenomenon and specially 1) to determine the effects of various cysteine residues in GAG modification and product secretion as well as 2) to investigate which of the two processing events is the critical step in the product processing. Our studies demonstrated that removal of the two amino-terminal cysteines in the CRD motif and the single cysteine in the amino terminus of CBP inhibited secretion of CRD and CBP. Use of the double mutant CRD construct also allowed us to observe a deviation from the usual strict coupling of GAG modification and product secretion steps. The presence of a small chondroitin sulfate fragment overcame the secretion-inhibitory effects once the small chondroitin sulfate fragment was modified by GAG.     

Link protein has greater affinity for versican than aggrecan

The function of link protein in stabilizing the interaction between aggrecan and hyaluronan to form aggrecan aggregates, via the binding of link protein to the aggrecan G1 domain and hyaluronan, is well established. However, it is not known whether link protein can function with similar avidity with versican, another member of the large hyaluronan-binding proteoglycan family that also binds to hyaluronan via its G1 domain. To address this issue, we have compared the interaction of the versican and aggrecan G1 domains with link protein and hyaluronan using recombinant proteins expressed in insect cells and BIAcore analysis. The results showed that link protein could significantly improve the binding of both G1 domains to hyaluronan and that its interaction with VG1 is of a higher affinity than that with AG1. These observations suggest that link protein may function as a stabilizer of the interaction, not only between aggrecan and hyaluronan in cartilage, but also between versican and hyaluronan in many tissues.     

Analysis of the catabolism of aggrecan in cartilage explants by quantitation of peptides from the three globular domains

A method has been developed for the production, isolation, and quantitation of 15 marker peptides from the three globular domains (G1, G2, and G3) and the interglobular domain of bovine aggrecan (aggregating cartilage proteoglycan). Three of the peptides are from G1, two are from the interglobular domain, four are from G2, and six are from G3. The method involves separation of tryptic peptides by sequential anion-exchange, cation-exchange, and reversed-phase high performance liquid chromatography and quantitation by absorbance at 220 nm. The values obtained (peak area per microgram of core protein) were a function of the molar yield and also the size and aromatic residue content of individual peptides. This procedure has been applied to aggrecan purified from fresh calf articular cartilage and to aggrecan isolated from the medium and tissue compartments of cartilage explant cultures, maintained in basal medium for 15 days without and with interleukin-1 alpha. These analyses indicate that aggrecan which is released into explant medium has a reduced content of the G1 domain, but has a normal content of the G2 domain, the COOH-terminal region of the interglobular domain, and also the G3 domain. On the other hand, aggrecan which is retained by the cartilage during 15 days of culture has a normal content of G1, interglobular domain, and G2 domains, but, in the presence of interleukin-1 alpha, it has a reduced content of the G3 domain. The percentage of medium molecules which retained the G1 domain was higher in control cultures (about 35%) than in interleukin cultures (about 20%), and this was consistent with the relative aggregability of these samples. Taken together these results suggest that catabolism of aggrecan in articular cartilage involves a specific proteolysis of the core protein at a site which is within the interglobular domain and NH2-terminal to the sequence LPGG. This process occurs in control cultures but is accelerated by the addition of interleukin-1 alpha. Degraded molecules which lack the G1 domain are released preferentially into the medium; however, these molecules carry both the G2 and G3 domains, indicating that these domains do not confer strong matrix binding properties on aggrecan. The method described here for the isolation of peptides from bovine aggrecan should have wide application to structural and biosynthetic studies on this molecule in species such as human and rat, since many of the marker peptides are from highly conserved regions of the aggrecan core protein.     

Force-mediated dissociation of proteoglycan aggregate in articular cartilage

Proteoglycan aggregate is the primary component in articular cartilage responsible for resisting compressive loading. It consists of a core molecule of hyaluronan and a number of side chains of aggrecan bound to hyaluronan non-covalently. The loss of aggrecan from articular cartilage is considered to be a major factor in the development of osteoarthritis. Though enzymatic digestion of aggrecan is believed to be responsible for the release of aggrecan from osteoarthritic cartilage, other mechanisms, such as direct force-mediated detachment of aggrecan from hyaluronan may also be involved. In this study, the rupture force of the single bond between hyaluronan and aggrecan in articular cartilage was directly quantified using experimental measurement and Monte Carlo simulation. Low rupture force of this bond, as determined in this study suggested a possible direct force-mediated detachment of aggrecan from proteoglycan aggregate in osteoarthritic cartilage.     

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

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

Isolation and characterization of disulfide-bonded peptides from the three globular domains of aggregating cartilage proteoglycan

The aggregating cartilage proteoglycan core protein contains two globular domains near the N terminus (G1 and G2) and one near the C terminus (G3). The G1-G3 domains contain 10, 8, and 10 cysteine residues, respectively. The disulfide assignments of the G1 domain have previously been deduced (Neame, P. J., Christner, J. E., and Baker, J. R. (1987) J. Biol. Chem. 262, 17768-17778) as Cys1-Cys2, Cys3-Cys6, Cys4-Cys5, Cys7-Cys10, and Cys8-Cys9, in which the numbers cited after the half-cystine residues are their relative positions from the N terminus. Here we describe a method for the isolation of disulfide-bonded peptides from tryptic digests of bovine nasal cartilage monomer. Sequence analysis of these peptides has allowed us to confirm the pairings previously determined for the G1 domain and to assign a disulfide pattern for the G2 domain of Cys11-Cys14, Cys12-Cys13, Cys15-Cys18, and Cys16-Cys17, in which the Cys15-Cys18 pairing was deduced indirectly. Similarly, for the G3 domain, a pattern of Cys19-Cys20, Cys21-Cys24, Cys22-Cys23, Cys25-Cys27, and Cys26-Cys28 was assigned, in which the Cys22-Cys23 pair was deduced indirectly. The G2 domain therefore contains disulfide bonding which is characteristic of the tandem repeat structures found in the G1 domain and link protein, and the G3 domain contains the three disulfide linkages previously assigned to the family of C-type animal lectins. The method described here, which combines anion-exchange, cation-exchange, and reversed-phase chromatography, should have broad application to the isolation of disulfide-bonded peptides from other heavily glycosylated proteins and proteoglycans.     

Variability in the G3 domain content of bovine aggrecan from cartilage extracts and chondrocyte cultures.

The content of the globular domains G1, G2 and G3 on the core protein of high-density (A1D1) aggrecan isolated from newborn and mature bovine cartilage and from cultures of bovine chondrocytes was examined. Quantitation based on the 220 nm absorbance of tryptic marker peptides from each domain isolated by reversed-phase HPLC showed that while the content of G1 and G2 was essentially the same for all samples, the content of G3 varied markedly. The molar yield of G3 and G1 marker peptides indicated that approximately 55% of the G1-bearing aggrecan from immature cartilage carried the G3 domain, while for mature cartilage this figure was markedly reduced, at about 35%. Aggrecan prepared from the cell layer matrix of calf chondrocyte cultures had an apparent G3 content similar to newborn cartilage (55%), whereas aggrecan prepared from the medium of these cultures had a markedly higher G3 content, at about 80%. The high content of G3 in cell medium samples compared to cartilage extracts was supported by electron microscopic analysis of A1D1 preparations. The G3 content of the two subpopulations of aggrecan present in mature cartilage and separable by flat bed agarose gel electrophoresis was also determined at about 45% (Band I) and 20% (Band II) respectively. These results are discussed in terms of the likely origin of the marked variability in the G3 domain content of aggrecan.     

Ultrastructural characterization of embryonic chick cartilage proteoglycan core protein and the mapping of a monoclonal antibody epitope.

The ultrastructure of embryonic chick cartilage proteoglycan core protein was investigated by electron microscopy of specimens prepared by low angle shadowing. The molecular images demonstrated a morphological substructural arrangement of three globular and two linear regions within each core protein. The internal globular region (G2) was separated from two terminally located globular regions (G1 and G3) by two elongated strands with lengths of 21 +/- 3 nm (E1) and 105 +/- 22 nm (E2). The two N-terminal globular regions, separated by the 21-nm segment, were consistently visualized in well spread molecules and showed little variation in the length of the linear segment connecting them. The E2 segment, however, was quite variable in length, and the C-terminal globular region (G3) was detected in only 53% of the molecules. The G1, G2, and G3 regions in chick core protein were 10.1 +/- 1.7 nm, 9.7 +/- 1.3 nm, and 8.3 +/- 1.3 nm in diameter, respectively. These results are similar to those described previously for proteoglycan core proteins isolated from rat chondrosarcoma, bovine nasal cartilage, and pig laryngeal cartilage (Paulsson, M., Morgelin, M., Wiedemann, H., Beardmore-Gray, M., Dunham, D., Hardingham, T., Heinegard, D., Timpl, R., and Engel, J. (1987) Biochem. J. 245, 763-772). However, a significant difference was detected between the length of the elongated strand (E2) of core proteins isolated from chick cartilage, E2 length = 105 +/- 22 nm, compared to bovine nasal cartilage, E2 length = 260 +/- 39 nm. The epitope of the proteoglycan core protein-specific monoclonal antibody, S103L, was visualized by electron microscopy, and the distance from the core protein N terminus to the S103L binding site was measured. The S103L binding site was localized to the E2 region, 111 +/- 20 nm from the G1 (N terminus) domain and 34 nm from the G3 (C terminus) domain. cDNA clones selected from an expression vector library of chicken cartilage mRNA also show this epitope to be located near the C-terminal region (R. C. Krueger, T. A. Fields, J. Mensch, and B. Schwartz (1990) J. Biol. Chem. 265, 12088-12097).     

Direct quantification of the rupture force of single hyaluronan/hyaluronan binding protein bonds

The non-covalent bond between aggrecan and hyaluronan is critical for maintaining the normal structure and function of the extracellular matrix in articular cartilage. The failure of this bond can cause the loss of aggrecan and destruction of the extracellular matrix of articular cartilage. In this study, the rupture force of the single bond between hyaluronan and hyaluronan binding protein - the complex of the hyaluronan binding region of aggrecan and link protein - was directly measured with a nanomechanical testing system as 40+/-11 pN. The results were compared to a theoretical prediction based on a smart version of the Monte Carlo simulation.     

The macromolecular characteristics of cartilage proteoglycans do not change when synthesis is up-regulated by link protein peptide.

Previous studies have shown that a synthetic, unglycosylated analogue of the N-terminal peptide from link protein can function as a growth factor and up-regulate proteoglycan biosynthesis in explant cultures of normal human articular cartilage from a wide age range of subjects (McKenna et al., Arthritis Rheum. 41 (1998) 157-162). The present work further shows that link peptide increased proteoglycan synthesis by cartilage cultured in both the presence and absence of serum, suggesting that the mechanism of up-regulation may be different from that of insulin-like growth factors. The proteoglycans synthesised during stimulation with link peptide were of normal hydrodynamic size and the ratio of core protein to glycosaminoglycan side chains and the proportions of the large proteoglycan aggrecan to the small proteoglycans, decorin and biglycan, remained constant. Aggrecan molecules were equally capable of forming aggregates as those from control tissues and the relative proportions of decorin and biglycan were unchanged showing that both were co-ordinately up-regulated. These results confirmed that this novel peptide is a potent stimulator of proteoglycan synthesis by articular cartilage and showed that the newly synthesised proteoglycans were of normal composition.     

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

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

Isolation of the N-terminal globular protein domains from cartilage proteoglycans. Identification of G2 domain and its lack of interaction with hyaluronate and link protein.

The N-terminal fragment (G1-G2) of cartilage proteoglycan protein core contains two globular domains, binding region (G1) and a second globular domain (G2), G1-G2 was isolated after mild trypsin digestion of purified proteoglycan aggregates followed by chromatography first on Sepharose CL-2B under associative conditions and then on a TSK-4000 column in 4 M-guanidinium chloride. It migrated as a single band (apparent Mr 150,000) on SDS/polyacrylamide-gel electrophoresis. G2 was isolated by V8-proteinase digestion of G1-G2 followed by aggregation of the G1-containing fragments with hyaluronate and chromatography on TSK-4000. It migrated as a single band on SDS/polyacrylamide-gel electrophoresis of apparent Mr 66,000 after digestion with keratanase. G2 did not interact with proteoglycan monomer, hyaluronate, link protein or other extractable cartilage matrix proteins. A polyclonal antibody raised against G2 did not cross-react with G1 or link protein. These data show that, despite a high degree of sequence similarity, G1 and G2 do not share any functional properties nor have major antigenic sites in common.     

Cartilage link protein interacts with neurocan, which shows hyaluronan binding characteristics different from CD44 and TSG-6

The interaction of neurocan with hyaluronan was qualitatively characterized with alkaline phosphatase fusion proteins secreted by mammalian cells. The wild type neurocan hyaluronan binding domain fused to alkaline phosphatase bound to immobilized hyaluronan under physiological as well as moderately hypertonic conditions, whereas its ability to bind to immobilized chondroitin sulfate dropped rapidly with increasing salt concentration. Strong hyaluronan binding ability was still evident when in both link modules within the hyaluronan binding domain a basic amino acid was mutated, which is well conserved among link modules of hyaluronan binding proteins. A strong enhancement of the binding of neurocan to immobilized hyaluronan was observed after preincubation of the immobilized hyaluronan with cartilage link protein. Moreover, this preincubation mediated also the binding of a fusion protein representing only the immunoglobulin module of neurocan linked to alkaline phosphatase, which showed no binding to immobilized hyaluronan alone. The interaction of the neurocan immunoglobulin module with link protein could also be shown by overlay blot analysis. These observations suggest that the hyaluronan binding characteristics of paired link modules are different from those of single link modules, and that the reported temporal co-expression of cartilage link protein and of neurocan in developing brain implicates the possibility of a cooperative function of these molecules.     

Aggrecan: Beyond cartilage and into the brain

Aggrecan is well-studied in cartilage but its expression and function in the central nervous system has only recently begun to be appreciated. Aggrecan plays an important role in the organization of the neural extracellular space by binding and organizing hyaluronan to the cell surface through interactions with link protein and tenascins forming a large aggregated quaternary complex. While all members of the lectican family to which aggrecan belongs are thought to mediate similar roles in organizing the neural matrix, aggrecan is unique in that it is the only family member found almost exclusively in an enigmatic matrix substructure called the perineuronal net. Current work has established a critical role for perineuronal nets and aggrecan in regulating developmental neural plasticity and in the recover from injury. In this review we focus on the structure, expression and function of aggrecan in the central nervous system.     

The Sad1-UNC-84 homology domain in Mps3 interacts with Mps2 to connect the spindle pole body with the nuclear envelope

The spindle pole body (SPB) is the sole site of microtubule nucleation in Saccharomyces cerevisiae; yet, details of its assembly are poorly understood. Integral membrane proteins including Mps2 anchor the soluble core SPB in the nuclear envelope. Adjacent to the core SPB is a membrane-associated SPB substructure known as the half-bridge, where SPB duplication and microtubule nucleation during G1 occurs. We found that the half-bridge component Mps3 is the budding yeast member of the SUN protein family (Sad1-UNC-84 homology) and provide evidence that it interacts with the Mps2 C terminus to tether the half-bridge to the core SPB. Mutants in the Mps3 SUN domain or Mps2 C terminus have SPB duplication and karyogamy defects that are consistent with the aberrant half-bridge structures we observe cytologically. The interaction between the Mps3 SUN domain and Mps2 C terminus is the first biochemical link known to connect the half-bridge with the core SPB. Association with Mps3 also defines a novel function for Mps2 during SPB duplication.     

Isolation and primary structure of PARP, a 24-kDa proline- and arginine-rich protein from bovine cartilage closely related to the NH2-terminal domain in collagen alpha 1 (XI)

A protein rich in proline and arginine (proline/arginine-rich protein (PARP] has been isolated from dissociative extracts of bovine nasal and articular cartilage, and its primary structure has been determined. The protein has 218 amino acids, giving a calculated protein Mr of 24,075. In nasal cartilage, this protein is in molar concentrations equivalent to 1/20-1/10 that of the link protein of cartilage proteoglycan aggregates. PARP has also been isolated from bovine articular cartilage, bovine fetal epiphysis, and nonossified human tarsal bones. PARP is similar to various collagen NH2-terminal domains. It is 49% identical to the NH2-terminal end of collagen alpha 1 (XI), 17% identical to the NC4 domain of collagen alpha 1 (IX), and 14% identical to the NC3 domain of collagen alpha 1 (XII). Four cysteines are conserved between type XI collagen and PARP, and these form two disulfide bonds. Two of the cysteines are also conserved between PARP and collagens IX and XII. The homology between the collagens and PARP makes it possible to speculate on the likely disulfide bond pattern in the collagen NH2-terminal domains. It is probable that PARP is a collagen fragment removed during processing in a manner analogous to chondrocalcin (the C-terminal propeptide of type II collagen).