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PubMed1994

Aggrecan in bovine tendon.

Large proteoglycans were purified by ion-exchange chromatography, gel filtration and CsCl gradient centrifugation from the compressed and tensional regions of adult bovine deep flexor tendon. Tryptic peptide maps of proteoglycan from the compressed region were very similar to maps of aggrecan from bovine articular cartilage, with evidence for the presence of all fifteen previously identified markers from the G1, G2 and G3 domains. The presence of aggrecan in these samples was confirmed by sequencing the G1 peptide YPIHTPR. The equivalent maps for large proteoglycan from tensional tendon were also consistent with the presence of aggrecan, and this was confirmed by sequencing three marker peptides from each of the G2 and G3 domains. However, G1 marker peptides were conspicuously absent from tensional samples. Northern blots for aggrecan mRNA showed high levels in cells from compressed tendon and articular cartilage. Extended exposure revealed a lower level of hybridization to RNA from tensional tendon as well. The results confirm that aggrecan, which is similar in core protein structure to articular cartilage aggrecan, is the predominant chondroitin sulfate-bearing large proteoglycan of compressed tendon. The results also indicate that aggrecan fragments lacking the G1 domain can account for the small amounts of chondroitin sulfate-bearing large proteoglycan in tensional regions of adult tendon.

Aggrecans↗
PubMed2005

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.

ADAM Proteins↗
PubMed2006

Expression of aggrecan(ases) during murine preadipocyte differentiation and adipose tissue development.

The expression and potential functional role of aggrecan in adipogenesis and adipose tissue development was investigated in murine models of obesity. Aggrecan, as well as the two aggrecanases ADAMTS-4 and ADAMTS-5 (A Disintegrin And Metalloproteinase with Thrombospondin motif) mRNAs, are expressed in subcutaneous (SC) and gonadal (GON) adipose tissues of mice. Their presence was confirmed by western blotting using adipose tissue extracts. In mice with nutritionally induced obesity (high fat diet) as well as in lean controls, aggrecan mRNA expression was downregulated whereas ADAMTS-4 and ADAMTS-5 were upregulated with time. In mice with genetically determined obesity (ob/ob), ADAMTS-5 mRNA was upregulated in both SC and GON adipose tissues, as compared to wild-type (WT) mice (p<0.001). Enhanced aggrecanase expression levels in these tissues were associated with significantly elevated levels of G1-NITEGE, a degradation product of aggrecan. Thus, aggrecan levels were high at the early stages of adipose tissue development in mice, whereas its production decreased and its degradation increased during development of obesity. A functional role of aggrecan in promoting early stages of adipogenesis is supported by the findings that it stimulated the in vitro differentiation of 3T3-F442A preadipocytes and the de novo in vivo accumulation of fat in Matrigel plaques injected into WT mice. Proteoglycans in the extracellular matrix of adipose tissue, such as aggrecan, may contribute to the regulation of lipid uptake and obesity in mice.

ADAM Proteins↗
PubMed2005

Focal cerebral ischemia induces changes in both MMP-13 and aggrecan around individual neurons.

INTRODUCTION: To test the hypothesis that matrix metalloprotease-13 (MMP-13) and aggrecan may play roles in post-ischemic neuronal pathophysiology, we examined the impact of middle cerebral artery occlusion/reperfusion (MCAO/R) on the abundance of these proteins in different regions of the infarct by immunohistochemistry (IHC) and Western blotting (WB). METHODS: The effect of MCAO/R on the abundance of MMP-13 and aggrecan was examined in 23 Wistar rats using antibodies against MMP-13 and aggrecan. BrdU was administered the last 2 days of the experiment. The cellular source of the respective antigens was examined with fluorescent double labeling using the neuronal marker NeuN. Sections were also stained for BrdU. The ischemic zone was defined by MRI on T2-weighted images and also on the tissue sections with the help of H and E counterstain. WB was performed for MMP-13. RESULTS: MMP-13 protein is highly induced in ischemic brain and is associated with neurons, whereas aggrecan is associated with the perineuronal matrix in non-ischemic brain. After 3 days of cerebral ischemia, the number of MMP-13 positive neurons in the periphery of the ischemic lesion increased compared to the respective area in the non-ischemic brain with a peak on day 7. A stronger staining for aggrecan was observed around MMP-13 positive neurons compared with other neurons. The majority of the MMP-13 positive neurons in normal non-ischemic brain were also NeuN positive. BrdU was incorporated into MMP-13 positive neurons in the periphery of the infarct. WB confirmed this results by detecting MMP-13 bands in ischemic brains and activated MMP-13 up to 14 days after ischemia. CONCLUSIONS: There is a close spatial association of MMP-13 and aggrecan around individual neurons. Both MMP-13 and aggrecan appear to be involved in perineuronal matrix remodeling suggesting a role in neuronal reorganization after cerebral ischemia.

Aggrecans↗
PubMed2004

The effects of intraarticularly injected sodium hyaluronate on levels of intact aggrecan and nitric oxide in the joint fluid of patients with knee osteoarthritis.

OBJECTIVE: Intraarticular injections of sodium hyaluronate (Na-HA) appear effective in reducing subjective symptoms of osteoarthritis (OA) and may also have protective effects on the cartilage matrix. The present study analyzed the suppressive effects of Na-HA on the release and degradation of aggrecan and on levels of nitric oxide (NO) in the joint fluid of patients with knee OA. DESIGN: Sixteen OA patients with knee joint effusion were treated by 5 weekly intraarticular injections of Na-HA. Prior to each Na-HA injection, joint fluid was collected to determine the levels of chondroitin 4-sulfate (C4S) and chondroitin 6-sulfate (C6S), intact aggrecan and NO. RESULTS: One week after the final injection, the joint fluid levels of C4S, C6S, and NO were significantly decreased. In contrast, the joint fluid level of intact aggrecan was stable during the series of Na-HA injections. A trend was seen for a positive correlation (P < 0.1) between the clinical score and C4S or C6S joint fluid levels, and for a negative correlation between the joint fluid levels of intact aggrecan and C4S or C6S. No significant correlations were observed between joint fluid levels of NO, the clinical score, and levels of C4S, C6S, and intact aggrecan. CONCLUSION: The results of this study suggest that intraarticularly injected Na-HA is able to improve the clinical symptoms of OA partially based on its ability to reduce the release and degradation of aggrecan and/or to enhance the synthesis of aggrecan in the joint tissues of the patients with knee OA. While Na-HA also reduces the NO level in the joint fluid of patients with knee OA, this effect may be independent from the other effects of Na-HA.

Adjuvants, Immunologic↗
PubMed1993

A cartilage explant system for studies on aggrecan structure, biosynthesis and catabolism in discrete zones of the mammalian growth plate.

The structure, biosynthesis and catabolism of aggrecan has been studied in the bovine fetal rib growth plate. Comparative analyses were made on six 1-mm transverse slices which represent the resting zone (slice 6), proliferative zone (slices 5 and 4), upper hypertrophic zone (slice 3), middle hypertrophic zone (slice 2) and lower hypertrophic zone (slice 1). Aggrecan was abundant and exhibited very high aggregability in all zones. The aggrecan monomer was similar in structure in the resting and proliferative zones but showed a marked increase in hydrodynamic size in the lower hypertrophic zone; this was apparently due to an increase in the size of substituent glycosaminoglycans and an increase in core protein size as indicated by peptide analysis for G3 domain abundance. Biosynthetic studies with [35S]-sulfate showed the rate of synthesis per cell to be highest in the upper hypertrophic zone, and the structure of the newly synthesised molecules to be similar to the resident population in all zones. During explant culture in basal medium both aggregating and non-aggregating forms of aggrecan were released slowly from all zones. Addition of 10 nM retinoic acid to explants stimulated the release of both these forms of aggrecan whereas higher concentrations of retinoic acid (100 nM and 1000 nM) preferentially stimulated the release of the degraded forms. In this regard hypertrophic cells were the most responsive and resting cells were the least responsive. Analysis of the degraded fragments by polyacrylamide gel electrophoresis and by N-terminal sequencing indicated that aggrecan catabolism in all zones of the growth plate is due to the action of aggrecanase, a novel cartilage proteinase which is also active in normal and osteoarthritic articular cartilages (Sandy et al., 1992). These observations are discussed in terms of the role of aggrecan in the extensive matrix remodelling which accompanies chondrocyte hypertrophy in the growth plate.

Aggrecans↗
PubMed2001

Cathepsin D cleaves aggrecan at unique sites within the interglobular domain and chondroitin sulfate attachment regions that are also cleaved when cartilage is maintained at acid pH.

Bovine aggrecan was digested with bovine cathepsin D at pH 5.2 under conditions of partial digestion and the resulting aggrecan core protein fragments were separated by electrophoresis on gradient polyacrylamide gels. The fragments were characterized by their reactivity to specific antibodies and by N-terminal amino acid sequencing. It was also demonstrated that cathepsin D cleaved bovine aggrecan at five sites within the core protein, between residues Phe(342)-Phe(343) in the interglobular domain, Leu(1462)-Val(1463) between the chondroitin sulfate attachment regions 1 and 2 and Leu(1654)-Val(1655), Phe(1754)-Val(1755) and Leu(1854)-Ile(1855) that are located within the chondroitin sulfate attachment region 2 of the core protein. The time course of digestion showed that there was a continued degradation of aggrecan and there was no preferential cleavage of the core protein at any one site. It was shown that cathepsin D digested aggrecan over the pH range 5.2-6.5 resulting in the same products. When bovine cartilage was maintained in explant culture at pH 5.2 there was a rapid loss of both radiolabeled and chemical pools of sulfated glycosaminoglycans into the culture medium and this loss was inhibited by the inclusion in the medium of the aspartic proteinase inhibitor, pepstatin A. The aggrecan core protein fragments appearing in the medium of cultures maintained at pH 5.2 were characterized and it was shown that the fragments had N-terminal sequences starting at Phe(343), Ile(1855), and Val(1755) or Val(1463). This work demonstrates that cathepsin D present within the extracellular matrix of articular cartilage has the potential to contribute to the proteolytic processing of the core protein of aggrecan in this tissue.

Aggrecans↗
PubMed2003

Glucosamine sulfate modulates the levels of aggrecan and matrix metalloproteinase-3 synthesized by cultured human osteoarthritis articular chondrocytes.

OBJECTIVE: The functional integrity of articular cartilage is determined by a balance between chondrocyte biosynthesis of extracellular matrix and its degradation. In osteoarthritis (OA), the balance is disturbed by an increase in matrix degradative enzymes and a decrease in biosynthesis of constitutive extracellular matrix molecules, such as collagen type II and aggrecan. In this study, we examined the effects of the sulfate salt of glucosamine (GS) on the mRNA and protein levels of the proteoglycan aggrecan and on the activity of matrix metalloproteinase (MMP)-3 in cultured human OA articular chondrocytes. DESIGN: Freshly isolated chondrocytes were obtained from knee cartilage of patients with OA. Levels of aggrecan and MMP-3 were determined in culture media by employing Western blots after incubation with GS at concentrations ranging from 0.2 to 200 microM. Zymography (casein) was performed to confirm that effects observed at the protein level were reflected at the level of enzymatic activity. Northern hybridizations were used to examine effects of GS on levels of aggrecan and MMP-3 mRNA. Glycosaminoglycan (GAG) assays were performed on the cell layers to determine levels of cell-associated GAG component of proteoglycans. RESULTS: Treatment of OA chondrocytes with GS (1.0-150 microM) resulted in a dose-dependent increase in aggrecan core protein levels, which reached 120% at 150 microM GS. These effects appeared to be due to increased expression of the corresponding gene as indicated by an increase in aggrecan mRNA levels in response to GS. MMP-3 levels decreased (18-65%) as determined by Western blots. Reduction of MMP-3 protein was accompanied by a parallel reduction in enzymatic activity. GS caused a dose-dependent increase (25-140%) in cell-associated GAG content. Chondrocytes obtained from 40% of OA patients failed to respond to GS. CONCLUSIONS: The results indicate that GS can stimulate mRNA and protein levels of aggrecan core protein and, at the same time, inhibit production and enzymatic activity of matrix-degrading MMP-3 in chondrocytes from OA articular cartilage. These results provide a cogent molecular mechanism to support clinical observations suggesting that GS may have a beneficial effect in the prevention of articular cartilage loss in some patients with OA.

Aged↗
PubMed2003

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

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

Aggrecans↗
PubMed2004

Mature bovine articular cartilage contains abundant aggrecan that is C-terminally truncated at Ala719-Ala720, a site which is readily cleaved by m-calpain.

Extracts of normal mature articular cartilage contain aggrecan molecules which bear the G1 domain (the N-terminal globular domain of aggrecan) and are C-terminally truncated by proteolysis at a number of sites. A proportion of these molecules are generated by an aggrecanase and/or matrix-metalloproteinase-mediated cleavage in the IGD (interglobular domain between the G1 and G2 domains of aggrecan). However, the proteinase(s) responsible for formation of the majority of the larger G1-G2 and glycosaminoglycan-bearing truncated species is (are) unknown. N-terminal sequencing of aggrecan core fragments generated by m-calpain digestion of bovine aggrecan has identified four novel cleavage sites: one within the CS (chondroitin sulphate)-1 domain (at one or more of the bonds Ser1229-Val1230, Ser1249-Val1250, Ser1287-Val1288, Gly1307-Val1308 and Ser1346-Val1347), two within the IGD (at bonds Ala474-Ala475 and Gly365-Gly366) and one within the KS (keratan sulphate) domain (at Ala719-Ala720). A new monoclonal antibody (SK-28) to the C-terminal neoepitope at M710VTQVGPGVA719 showed that aggrecan products generated by this cleavage are present in high abundance in mature bovine articular cartilage extracts. We conclude that m-calpain, or an unidentified proteinase with the capacity to cleave at the same site, is active during aggrecan biosynthesis/secretion by mature chondrocytes or in the matrix of mature bovine articular cartilage in vivo.

Aggrecans↗
PubMed1997

Chemical and immunological assay of the nonreducing terminal residues of chondroitin sulfate from human aggrecan.

Samples of aggrecan chondroitin sulfate, isolated from normal human knee cartilages of individuals from fetal to 72 years of age, were digested with chondroitin lyases. The products were analyzed by fluorescence-based anion exchange high performance liquid chromatography to separate and quantitate nonreducing terminal structures, in addition to internal unsaturated disaccharide products. The predominant terminal structures were the monosaccharides, GalNAc4S and GalNAc4,6S as they were present on 85-90% of all chains. The remaining chains terminated with the disaccharides GlcAbeta1,3GalNAc4S and GlcAbeta1,3GalNAc6S. Marked changes in the relative abundance of these terminals were identified in the transition from growth cartilage to adult articular cartilage. First, terminal GalNAc residues were almost exclusively 4-sulfated in aggrecan from fetal through 15 years of age, but were approximately 50% 4,6-disulfated in aggrecans from adults (22-72 years of age). Second, the terminal disaccharide GlcAbeta1,3GalNAc4S was on approximately 7% of chains on aggrecan from fetal through 15 years of age, but on only approximately 3% of chains on adult aggrecan. In contrast, the proportion of chains terminating in GlcAbeta1,3GalNAc6S, approximately 9%, was unchanged from fetal to 72 years of age. This terminal disaccharide is proposed to be recognized by the widely used monoclonal antibody 3B3. However, chemical quantitation of the structure together with solid phase 3B3(-) immunoassay of fetal and adult aggrecans showed that the content of the terminal disaccharide does not necessarily correlate with immunoreactivity of the proteoglycan, as chain density and presentation on the solid phase are critical factors for recognition of chain terminals by 3B3. The quantitative results obtained from chemical analyses of all nonreducing termini of aggrecan chondroitin sulfate chains revealed important changes in chain termination that occur when cellular activities are altered as adult articular cartilage is formed after removal of growth cartilage. These findings are discussed in relation to specific enzymatic steps that generate the nonreducing termini of chains in the biosynthesis pathway of chondroitin sulfate proteoglycans and their modulation in tissue development and pathology.

Adolescent↗
PubMed1997

Identification of hyaluronan-binding domains of aggrecan.

Aggrecan, a large cartilage proteoglycan, interacts with hyaluronan (HA), to form aggregates which function to resist compression in joints. The N-terminal region of aggrecan contains two structurally related globular domains, G1 and G2 separated by IGD domain. The G1 domain consists of three subdomains, A, B, and B', structural features characteristic to many other HA-binding proteoglycans. Here, we studied the interaction of aggrecan domains with HA using recombinant proteins expressed in 293 cells, an embryonal kidney cell line. Deglycosylation of the recombinant aggrecan fragment reduced the HA binding activity. We found that both the B and B' subdomains were required for HA binding and that a single module of A, B, or B' was unable to bind HA. The A subdomain increased the HA binding activity of the B-B' region. The G2 domain had no HA binding activity confirming previous reports. Studies of HA-binding properties using a BIAcoreTM biosensor system revealed that the KD of recombinant aggrecan fragment (AgW) consisting of G1, IGD, and G2 was 0.226 microM, whereas the KD of another HA-binding protein, native bovine link protein, is 0.089 microM. In contrast, AgMut11 which lacked subdomain A showed little HA binding activity. AgMut12 consisting of only B-B' had a 3.4-fold lower affinity and AgMut13 containing A-B-B' was 1.5-fold lower than AgW. These results suggest that carbohydrates are essential for high level aggrecan binding to HA and that the A subdomain of aggrecan functions in a cooperative manner with subdomains B and B'.

Aggrecans↗
PubMed1998

Regulatory activities of the 5'- and 3'-untranslated regions and promoter of the human aggrecan gene.

Identification and characterization of the regulatory elements of the human aggrecan gene are necessary first steps in addressing the molecular mechanisms through which the gene is regulated. Using luciferase reporter constructs driven by the human aggrecan promoter or the cytomegalovirus promoter, the 5'- and 3'-untranslated regions of the human aggrecan gene were found to regulate gene expression transcriptionally in a promoter- and/or cell type-specific manner. Independent of cell type, the 5'-untranslated region was inhibitory with respect to the cytomegalovirus promoter, but it was stimulatory to the human aggrecan promoter. The 5'-untranslated region inhibited the cytomegalovirus promoter by approximately 60% in both chondrocytes and NIH 3T3 cells, but it stimulated the activity of the human aggrecan promoter about 8-fold in chondrocytes and 40-fold in NIH 3T3 cells. In contrast, the 3'-untranslated region inhibited the activities of the human aggrecan promoter by 40-70% in both cell types, but it stimulated the cytomegalovirus promoter activities by 50-60% in NIH 3T3 cells and inhibited its activity by 70% in chondrocytes. The differential effects of the untranslated regions on the two types of promoters may be a reflection of differences in regulation of TATA-less promoters, such as the human aggrecan promoter, and TATA-containing promoters, such as the cytomegalovirus promoter.

Aggrecans↗
PubMed2000

SOX9 enhances aggrecan gene promoter/enhancer activity and is up-regulated by retinoic acid in a cartilage-derived cell line, TC6.

SOX9 is a transcription factor that plays a key role in chondrogenesis. Aggrecan is one of the major structural components in cartilage; however, the molecular mechanism of aggrecan gene regulation has not yet been fully elucidated. TC6 is a clonal chondrocytic cell line derived from articular cartilage. The purpose of this study was to examine whether SOX9 modulates aggrecan gene expression and to further identify molecules that regulate Sox9 expression in TC6 cells. SOX9 overexpression in TC6 cells enhanced by approximately 3-fold the transcriptional activity of the AgCAT-8 construct containing 8-kilobase (kb) promoter/first exon/first intron fragments of the aggrecan gene. SOX9 enhancement of aggrecan promoter activity was lost when we deleted a 4.5-kb fragment from the 3'-end of the 8-kb fragment corresponding to the region including the first intron. In TC6 cells, SOX9 enhanced the transcriptional activity of a reporter construct containing the Sry/Sox consensus sequence >10-fold. SOX9 enhancement of aggrecan gene promoter activity and SOX9 transactivation through the Sry/Sox consensus sequence were not observed in osteoblastic osteosarcoma cells (ROS17/2.8), indicating the dependence on the cellular background. Northern blot analysis indicated that TC6 cells constitutively express Sox9 mRNA at relatively low levels. To examine regulation of Sox9 gene expression, we investigated the effects of calciotropic hormones and cytokines. Among these, retinoic acid (RA) specifically enhanced Sox9 mRNA expression in TC6 cells. The basal levels of Sox9 expression and its enhancement by RA were observed similarly at both permissive (33 degrees C) and nonpermissive (39 degrees C) temperatures. Furthermore, RA treatment enhanced the transcriptional activity of a reporter construct containing the Sry/Sox consensus sequence in TC6 cells. Moreover, RA treatment also enhanced the transcriptional activity of another reporter construct containing the enhancer region of the type II procollagen gene in TC6 cells. These observations indicate that SOX9 enhances aggrecan promoter activity and that its expression is up-regulated by RA in TC6 cells.

Aggrecans↗
PubMed2000

The thrombospondin motif of aggrecanase-1 (ADAMTS-4) is critical for aggrecan substrate recognition and cleavage.

Aggrecanase-1 (ADAMTS-4) is a member of the a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS) protein family that was recently identified. Aggrecanase-1 is one of two ADAMTS cartilage-degrading enzymes purified from interleukin-1-stimulated bovine nasal cartilage (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., and Arner, E.C. (1999) Science 284, 1664-1666; 2 Abbaszade, I., Liu, R. Q., Yang, F., Rosenfeld, S. A., Ross, O. H., Link, J. R., Ellis, D. M., Tortorella, M. D., Pratta, M. A., Hollis, J. M., Wynn, R., Duke, J. L., George, H. J., Hillman, M. C., Jr., Murphy, K., Wiswall, B. H., Copeland, R. A., Decicco, C. P., Bruckner, R., Nagase, H., Itoh, Y., Newton, R. C., Magolda, R. L., Trzaskos, J. M., and Burn, T. C. (1999) J. Biol. Chem. 274, 23443-23450). The aggrecan products generated by this enzyme are found in cartilage cultures stimulated with cytokines and in synovial fluid from patients with arthritis, suggesting that aggrecanase-1 may be important in diseases involving cartilage destruction. Here we demonstrate that the thrombospondin type-1 (TSP-1) motif located within the C terminus of aggrecanase-1 binds to the glycosaminoglycans of aggrecan. Data from several studies indicate that this binding of aggrecanase-1 to aggrecan through the TSP-1 motif is necessary for enzymatic cleavage of aggrecan. 1) A truncated form of aggrecanase-1 lacking the TSP-1 motif was not effective in cleaving aggrecan. 2) Several peptides representing different regions of the TSP-1 motif effectively blocked aggrecanase-1 cleavage of aggrecan by preventing the enzyme from binding to the substrate. 3) Aggrecanase-1 was not effective in cleaving glycosaminoglycan-free aggrecan. Taken together, these data suggest that the TSP-1 motif of aggrecanase-1 is critical for substrate recognition and cleavage.

ADAM Proteins↗
PubMed2005

Increased expression of aggrecan and biglycan mRNA in Achilles tendinopathy.

OBJECTIVES: To determine the expression of mRNA encoding the proteoglycans aggrecan, versican, biglycan and decorin in mid-tendon samples of chronic painful Achilles tendinopathy and ruptured Achilles tendons, compared with normal tendons. METHODS: Total RNA isolated from frozen tendon samples (14 normal, 13 painful, 14 ruptured) was assayed by relative quantitative reverse transcription polymerase chain reaction for aggrecan, versican, biglycan and decorin mRNA, normalized using 18S rRNA. Differences between sample groups were tested by univariate analysis of variance with age as co-variate. RESULTS: In normal tendon samples expression of each of the proteoglycan mRNA decreased with increasing age. Decorin mRNA was the most highly-expressed of the proteoglycan mRNA, while versican mRNA expression was higher (3.8-fold) than that of aggrecan. In painful tendinopathy both aggrecan and biglycan mRNA expression increased (more than 10-fold and 5-fold, respectively) compared with normal tendon samples, but levels of versican and decorin mRNA were not significantly changed. In ruptured tendons the levels of aggrecan, biglycan and versican mRNA were not changed compared with normal tendon samples, but decorin mRNA decreased markedly. CONCLUSIONS: Increased aggrecan and biglycan mRNA expression in painful tendinopathy resembles the pattern in fibrocartilaginous regions of tendon, and may reflect an altered mechanical environment at the site of the lesion. Increased aggrecan mRNA expression may underlie the increase in glycosaminoglycan observed in painful tendinopathy.

Achilles Tendon↗
PubMed1999

Association between an aggrecan gene polymorphism and lumbar disc degeneration.

STUDY DESIGN: A case-control study using magnetic resonance imaging findings and a polymerase chain reaction assay to investigate the association between aggrecan gene polymorphism and lumbar disc degeneration. OBJECTIVE: To analyze whether the aggrecan gene polymorphism is related to lumbar disc disease in young women. SUMMARY OF BACKGROUND DATA: It has been suggested that a genetic factor or familial predisposition contributes to the development of lumbar disc herniation. However, the precise genetic component related to disc disease remains unclear. Recently, a polymorphism has been identified in the region of the human aggrecan gene. The expressed variable numbers of tandem repeat polymorphism occur in the highly conserved repeat region. METHODS: The participants were 64 young women with or without low back problems. Magnetic resonance imaging was used to evaluate the degeneration and herniation of the intervertebral disc. Genomic deoxyribonucleic acid was extracted from all participants. A polymerase chain reaction assay was carried out to detect the alleles of the aggrecan gene. The association of intervertebral disc degeneration and herniation with the distribution of the aggrecan gene alleles was analyzed. RESULTS: Findings showed an overrepresentation of alleles with small numbers of repeats in subjects with multilevel disc degeneration, thus indicating a significant distribution difference. There also was a significant difference between the distribution of alleles and the severity of disc degeneration. No significant association was found between any of the alleles either in number or type of disc herniation. CONCLUSIONS: The current study showed that multilevel and severe disc degeneration was present in the participants with shorter variable numbers of tandem repeat length of the aggrecan gene. This suggests that subjects with shorter variable numbers of tandem repeat length of the aggrecan gene have a risk of having multilevel disc degeneration develop at an early age.

Adult↗
PubMed1997

Aggrecan degradation in human cartilage. Evidence for both matrix metalloproteinase and aggrecanase activity in normal, osteoarthritic, and rheumatoid joints.

To examine the activity of matrix metalloproteinases (MMPs) and aggrecanase in control and diseased human articular cartilage, metabolic fragments of aggrecan were detected with monospecific antipeptide antibodies. The distribution and quantity of MMP-generated aggrecan G1 fragments terminating in VDIPEN341 were compared with the distribution of aggrecanase-generated G1 fragments terminating in NITEGE373. Both types of G1 fragments were isolated from osteoarthritic cartilage. The sizes were consistent with a single enzymatic cleavage in the interglobular domain region, with no further proteolytic processing of these fragments. Both neoepitopes were also detected by immunohistochemistry in articular cartilage from patients undergoing joint replacement for osteoarthritis (OA), rheumatoid arthritis (RA), and in cartilage from adults with no known joint disease. In control specimens, the staining intensity for both G1 fragments increased with age, with little staining in cartilage from 22-wk-old fetal samples. There was also an increase with age in the extracted amount of MMP-generated neoepitope in relation to both aggrecan and collagen content, confirming the immunohistochemical results. After the age of 20-30 yr this relationship remained at a steady state. The staining for the MMP-generated epitope was most marked in control cartilage exhibiting histological signs of damage, whereas intense staining for the aggrecanase-generated fragment was often noted in adult cartilage lacking overt histological damage. Intense staining for both neoepitopes appeared in the more severely fibrillated, superficial region of the tissue. Intense immunostaining for both VDIPEN- and NITEGE- neoepitopes was also detected in joint cartilage from patients with OA or RA. Cartilage in these specimens was significantly more degraded and high levels of staining for both epitopes was always seen in areas with extensive cartilage damage. The levels of extracted VDIPEN neoepitope relative to collagen or aggrecan in both OA and RA samples were similar to those seen in age-matched control specimens. Immunostaining for both types of aggrecan fragments was seen surrounding the cells but also further removed in the interterritorial matrix. In some regions of the tissue, both neoepitopes were found while in others only one was detected. Thus, generation and/or turnover of these specific catabolic aggrecan fragments is not necessarily coordinated. Our results are consistent with the presence in both normal and arthritic joint cartilage of proteolytic activity against aggrecan based on both classical MMPs and "aggrecanase."

Adolescent↗