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.     

Identification of a stromelysin cleavage site within the interglobular domain of human aggrecan. Evidence for proteolysis at this site in vivo in human articular cartilage.

Products generated by the digestion of human aggrecan with recombinant human stromelysin have been purified and analyzed by N-terminal sequencing and C-terminal peptide isolation. N-terminal analysis of chondroitin sulfate-bearing fragments revealed a clearly identifiable sequence initiating at residue Phe342 of human aggrecan, providing evidence for a cleavage site at the Asn341-Phe342 bond located within the interglobular domain. This cleavage site, which separates the G1 domain from the remainder of the molecule, was confirmed by isolation from the liberated G1 domain of a C-terminal tryptic peptide with the sequence YDAICYTGEDFVDIPEN (in which the C-terminal residue is Asn341). This peptide was also isolated from tryptic digests of hyaluronan-binding proteins (A1D4 samples) prepared by CsCl gradient centrifugation of extracts of mature human articular cartilages. Since these A1D4 samples contain G1 domain which accumulates as a result of aggrecan catabolism in vivo, these results clearly indicate that stromelysin cleaves the Asn341-Phe342 bond of human aggrecan in situ.     

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

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

MMP proteolysis of the human extracellular matrix protein aggrecan is mainly a process of normal turnover

Although it has been shown that aggrecanases are involved in aggrecan degradation, the role of MMP (matrix metalloproteinase) aggrecanolysis is less well studied. To investigate MMP proteolysis of human aggrecan, in the present study we used neoepitope antibodies against MMP cleavage sites and Western blot analysis to identify MMP-generated fragments in normal and OA (osteoarthritis/osteoarthritic) cartilage, and in normal, knee injury and OA and SF (synovial fluid) samples. MMP-3 in vitro digestion showed that aggrecan contains six MMP cleavage sites, in the IGD (interglobular domain), the KS (keratan sulfate) region, the border between the KS region and CS (chondroitin sulfate) region 1, the CS1 region, and the border between the CS2 and the G3 domain, and kinetic studies showed a specific order of digestion where the cleavage between CS2 and the G3 domain was the most preferred. In vivo studies showed that OA cartilage contained (per dry weight) 3.4-fold more MMP-generated FFGV fragments compared with normal cartilage, and although aggrecanase-generated SF-ARGS concentrations were increased 14-fold in OA and knee-injured patients compared with levels in knee-healthy reference subjects, the SF-FFGV concentrations did not notably change. The results of the present study suggest that MMPs are mainly involved in normal aggrecan turnover and might have a less-active role in aggrecan degradation during knee injury and OA.     

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.     

Identification of aggrecanase activity in medium of cartilage culture.

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

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.     

Cleavage of proteoglycan aggregate by leucocyte elastase.

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

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.     

Gene structure of chick cartilage chondroitin sulfate proteoglycan (aggrecan) core protein

Large aggregating chondroitin sulfate proteoglycan (CSPG/aggrecan) is one of the major extracellular matrix components in cartilage. The core protein is also large, over 200 kDa, and modular with a distinct correspondence between protein structural domains and the encoding exons. Here we report the isolation, using chick CSPG cDNA probes and the ensuing sequencing, of genomic clones containing exons encoding the chick CSPG core protein. The 5 two globular domains, G1 and G2, are encoded by four and three exons, respectively, and the interglobular domain is encoded by a single exon. The chondroitin sulfate attachment domain is encoded by the largest exon, 3,216 bp, which is approximately 50% of the total coding sequence. Combined with the previous report (Tanaka, T., Har-el, R. Tanzer, M.L. 1988 J. Biol. Chem. 263, 15831–15835), these data reveal that the chick CSPG gene contains at least 18 exons spanning a genome which is greater than 30 kb. No evidence was obtained for multiple genes for aggrecan in the chick genome. Elucidation of the chick genomic structure allows comparison of the avian and mammalian link protein genes to the homologous portions of avian and mammalian core protein genes (hyaluronate binding domain) with respect to their origins and paths of duplication and divergence. Correspondence to: N.B. Schwartz     

Mechanism of catabolism of aggrecan by articular cartilage.

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

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.     

MMPs are less efficient than ADAMTS5 in cleaving aggrecan core protein

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

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

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

Identification of proteoglycan from salmon nasal cartilage

There has been no structural information about the core protein of salmon nasal cartilage proteoglycan although its physiological activities have been investigated. Internal amino acid sequencing using nano-LC/MS/MS revealed that the salmon proteoglycan was aggrecan. Primer walk sequencing based on the amino acid information determined that the salmon aggrecan cDNA is comprised of 4207 bp nucleotides predicted to encode 1324 amino acids with a molecular mass of 143,276. It exhibited significant similarities to predicted pufferfish aggrecan, zebrafish similar to aggrecan, zebrafish aggrecan, bovine aggrecan and human aggrecan isoform 2 precursor; whose amino acid identities were 56%, 55%, 49%, 31% and 30%, respectively. Salmon cartilage aggrecan had globular domains G1, G2 and G3 as in mammalian aggrecans. Neither the putative keratan sulfate attachment domain enriched with serine, glutamic acid and proline, nor the putative chondroitin sulfate attachment domain with repeating amino acid sequence containing serine–glycine, found in mammalian aggrecans were observed in salmon, however, random serine–glycine (or glycine–serine) sequences predicted to the sugar chain attachment sites were observed. Based on cDNA analysis and amino acid analysis after β-elimination, the ratio of serine attached to sugar chains was calculated to be approximately 37.7% of total serine, that is, 46 of 123 serine residues.     

Sites of aggrecan cleavage by recombinant human aggrecanase-1 (ADAMTS-4)

Aggrecan, the major proteoglycan of cartilage that provides its mechanical properties of compressibility and elasticity, is one of the first matrix components to undergo measurable loss in arthritic diseases. Two major sites of proteolytic cleavage have been identified within the interglobular domain (IGD) of the aggrecan core protein, one between amino acids Asn(341)-Phe(342) which is cleaved by matrix metalloproteinases and the other between Glu(373)-Ala(374) that is attributed to aggrecanase. Although several potential aggrecanase-sensitive sites had been identified within the COOH terminus of aggrecan, demonstration that aggrecanase cleaved at these sites awaited isolation and purification of this protease. We have recently cloned human aggrecanase-1 (ADAMTS-4) (Tortorella, M. D., Burn, T. C., Pratta, M. A., Abbaszade, I., Hollis, J. M., Liu, R., Rosenfeld, S. A., Copeland, R. A., Decicco, C. P., Wynn, R., Rockwell, A., Yang, F., Duke, J. L., Solomon, K., George, H., Bruckner, R., Nagase, H., Itoh, Y., Ellis, D. M., Ross, H., Wiswall, B. H., Murphy, K., Hillman, M. C., Jr., Hollis, G. F., Newton, R. C., Magolda, R. L., Trzaskos, J. M., and Arner, E. C. (1999) Science 284, 1664-1666) and herein demonstrate that in addition to cleavage at the Glu(373)-Ala(374) bond, this protease cleaves at four sites within the chondroitin-sulfate rich region of the aggrecan core protein, between G2 and G3 globular domains. Importantly, we show that this cleavage occurs more efficiently than cleavage within the IGD at the Glu(373)-Ala(374) bond. Cleavage occurred preferentially at the KEEE(1667-1668)GLGS bond to produce both a 140-kDa COOH-terminal fragment and a 375-kDa fragment that retains an intact G1. Cleavage also occurred at the GELE(1480-1481)GRGT bond to produce a 55-kDa COOH-terminal fragment and a G1-containing fragment of 320 kDa. Cleavage of this 320-kDa fragment within the IGD at the Glu(373)-Ala(374) bond then occurred to release the 250-kDa BC-3-reactive fragment from the G1 domain. The 140-kDa GLGS-reactive fragment resulting from the preferential cleavage was further processed at two additional cleavage sites, at TAQE(1771)-(1772)AGEG and at VSQE(1871-1872)LGQR resulting in the formation of a 98-kDa fragment with an intact G3 domain and two small fragments of approximately 20 kDa. These data elucidate the sites and efficiency of cleavage during aggrecan degradation by aggrecanase and suggest potential tools for monitoring aggrecan cleavage in arthritis.     

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.     

Two structural genes on different chromosomes are required for encoding the major subunit of human red cell glucose-6-phosphate dehydrogenase

Structural analysis revealed the existence of two types of subunits in human red cell glucose-6-phosphate dehydrogenase. The two subunits have the same COOH region consisting of 479 amino acid residues, but their NH2-terminal regions are different in size and sequence. The minor subunit can be fully encoded by the X-linked G6PD cDNA, but the NH2-terminal region of the major subunit cannot. The cDNA and the gene for the NH2-terminal region of the major subunit were cloned and characterized. Southern blot hybridization indicated that the gene for the NH2-terminal region is on chromosome 6, not on the X chromosome. Northern blot hybridization demonstrated an existence of two separate mRNA components, one for the COOH-terminal region and the other for the NH2-terminal region. Two separate structural genes, the X-linked and chromosome 6-linked genes, must be coresponsible for encoding the single chain subunit. Either cross-translation of two mRNAs, or transpeptidation, or some other mechanism must be involved in the synthesis of human red cell G6PD.     

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

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

G3 domains of aggrecan and PG-M/versican form intermolecular disulfide bonds that stabilize cell-matrix interaction

The extracellular matrix plays a critical role in maintaining tissue integrity. Among the matrix molecules, the large aggregating chondroitin sulfate proteoglycans are the major structural molecules and are the primary contributors to the stability for some tissues such as cartilage. The notable exceptions are nanomelic cartilage and arthritic cartilage: the former contains a point mutation leading to a stop codon before translating to the C-terminal G3 domain; the latter contains a large proportion of aggrecan from which the G3 domain has been cleaved. These phenomena suggest that the G3 domain may be important in cartilage stability. Here, we demonstrated for the first time that the G3 domains of aggrecan and another proteoglycan, PG-M/versican, formed intermolecular disulfide bonds, and all subdomains were involved. Further studies indicated that each of the 10 cysteine residues of the aggrecan G3 domain could potentially form intermolecular disulfide bonds in vitro. The disulfide bonds were disrupted in the presence of reducing reagent beta-mercaptoethanol and dithiothreitol. As a result, normal chondrocyte-matrix interaction was disrupted, and the structure of the extracellular matrix was altered. Furthermore, disruption of disulfide bonds also reduced the role of PG-M/versican G3 domain in mediating cell adhesion. Our study provides strong evidence of the importance of proteoglycan interactions through intermolecular disulfide bonds in cartilage firmness and cell-matrix stability.