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PubMed2005

ADAMTS-1-knockout mice do not exhibit abnormalities in aggrecan turnover in vitro or in vivo.

OBJECTIVE: To determine the role of the proteinase ADAMTS-1 in normal and accelerated catabolism of aggrecan in articular and growth plate cartilage of mice. METHODS: Expression of ADAMTS-1 was determined using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of RNA isolated from microdissected chondrocytes from different zones of mouse growth plate and articular cartilage. Real-time RT-PCR for ADAMTS-4, ADAMTS-5, and ADAMTS-9 was performed on femoral head cartilage of wild-type (WT) and ADAMTS-1-knockout (KO) mice. Histologic and immunohistologic evaluation of growth plate and articular cartilage was performed in WT and KO mice from birth to 12 weeks of age. The effect of ADAMTS-1 ablation on cartilage proteoglycan loss was studied in antigen-induced arthritis (AIA). Aggrecan catabolism in WT and KO mice was studied in an in vitro model of cartilage degradation, by quantitation of glycosaminoglycan loss and histologic, immunohistologic, and Western immunoblot analyses. RESULTS: ADAMTS-1 messenger RNA (mRNA) was expressed in normal mouse articular and growth plate cartilage and was up-regulated in terminal hypertrophic differentiation of growth plate chondrocytes. There was no difference in mRNA levels in the cartilage of WT compared with KO mice for the other potential aggrecanases ADAMTS-4, ADAMTS-5, or ADAMTS-9. ADAMTS-1-KO mice were significantly smaller than their WT littermates; however, no morphologic differences between the genotypes were evident in growth plate or articular cartilage from birth to skeletal maturity (12-16 weeks). Similarly, no difference in cartilage aggrecan content or presence of aggrecan degradation products was detected between WT and KO mice. There was no difference between WT and KO mice in the degree of synovial inflammation or depletion of cartilage aggrecan in AIA. There was no difference between WT and KO cartilage in either basal or stimulated aggrecan loss in vitro; however, subtle changes in the aggrecanase-generated aggrecan catabolites were observed in interleukin-1-treated cartilage. CONCLUSION: Although ADAMTS-1 is expressed in articular and growth plate cartilage and is able to cleave aggrecan at physiologically relevant sites, our results indicate that it does not play a significant nonredundant role in normal cartilage and bone development and growth. Similarly, ablation of ADAMTS-1 offered no protection from accelerated aggrecanolysis in an inflammatory model of arthritis or in an in vitro model of early cartilage degradation. ADAMTS-1 does not appear to be a viable target for treatment of cartilage destruction in arthritis.

ADAM Proteins↗
PubMed1999

Down-regulation of chondrocyte aggrecan and type-II collagen gene expression correlates with increases in static compression magnitude and duration.

The goal of this study was to examine the simultaneous effects of mechanical compression of chondrocytes on mRNA expression and macromolecular synthesis of aggrecan and type-II collagen. Bovine cartilage explants were exposed to different magnitudes and durations of applied mechanical compression, and levels of aggrecan and type-IIa collagen mRNA normalized to glyceraldehyde-3-phosphate dehydrogenase were measured and quantified by Northern blot analysis. Synthesis of aggrecan and type-II collagen protein was measured by radiolabel incorporation of [35S]sulfate and [3H]proline into macromolecules. The results showed a dose-dependent decrease in mRNA levels for aggrecan and type-II collagen, with increasing compression relative to physiological cut thickness applied for 24 hours. Radiolabel incorporation into glycosaminoglycans and collagen also decreased with increasing compression in a dose-related manner similar to the changes seen in mRNA expression. The modulation of aggrecan and type-II collagen mRNA and protein synthesis were dependent on the duration of the compression. Aggrecan and type-II collagen mRNA expression increased during the initial 0.5 hours of static compression; however, 4-24 hours after compression was applied total mRNA levels had significantly decreased. The synthesis of aggrecan and collagen protein decreased more rapidly than did mRNA levels after the application of a step compression. Together, these results suggest that mechanical compression rapidly alters chondrocyte aggrecan and type-II collagen gene expression on application of load. However, our results indicate that the observed decreases in biosynthesis may not be related solely to changes in mRNA expression. The mechanisms by which mechanical forces affect different segments of the biosynthetic pathways remain to be determined.

Aggrecans↗
PubMed1992

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.

Aggrecans↗
PubMed2006

Autoantibodies against aggrecan in systemic rheumatic diseases.

OBJECTIVE: This study was undertaken to investigate the presence of autoantibodies against the main cartilage proteoglycan, aggrecan, in systemic rheumatic disease sera, and to identify substructure(s) responsible for the autoimmune response. METHODS: Sera were obtained from 86 patients with various systemic rheumatic diseases, 14 with osteoarthritis (OA), 18 with cancer and 40 healthy individuals. The presence of autoantibodies against aggrecan was examined by a solid phase assay and by Western blotting, using proteoglycan aggregates treated with proteolytic enzymes. The positive bands were subjected to nanohigh performance liquid chromatography (nanoHPLC)-MS, in order to identify the aggrecan substructures involved in the autoimmune response. RESULTS: Autoantibodies against aggrecan were identified in all systemic rheumatic disease sera at a high titre, almost three times that observed in healthy controls. OA and cancer sera produced a reaction equal to that of the healthy. Western blotting analysis of aggrecan proteolytic fragments revealed the presence of a triple band, reacting with the patients' sera, of about 37 kDa, which also reacted with a polyclonal antibody against hyaluronan-binding region. NanoHPLC-MS analysis suggested that this band belonged to the G2 domain of aggrecan. CONCLUSION: At least a part of the autoimmune reaction to aggrecan, displayed by the systemic disease sera, involves the G2 domain. The significant difference observed between these sera and those from other diseases, especially cancer, may suggest a possible discriminatory role of anti-aggrecan antibodies. This may help in the differential diagnosis in complicated clinical cases. However, for this to be confirmed, studies in larger cohorts of patients should be performed.

Adult↗
PubMed2006

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.

ADAM Proteins↗
PubMed2002

Highly sulfated glycosaminoglycans inhibit aggrecanase degradation of aggrecan by bovine articular cartilage explant cultures.

The catabolism of 35S-labeled aggrecan and loss of tissue glycosaminoglycans was investigated using bovine articular cartilage explant cultures maintained in medium containing 10(-6) M retinoic acid or 40 ng/ml recombinant human interleukin-1alpha (rHuIL-1alpha) and varying concentrations (1-1000 microg/ml) of sulfated glycosaminoglycans (heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and keratan sulfate) and calcium pentosan polysulfate (10 microg/ml). In addition, the effect of the sulfated glycosaminoglycans and calcium pentosan polysulfate on the degradation of aggrecan by soluble aggrecanase activity present in conditioned medium was investigated. The degradation of 35S-labeled aggrecan and reduction in tissue levels of aggrecan by articular cartilage explant cultures stimulated with retinoic acid or rHuIL-1alpha was inhibited by heparin and heparan sulfate in a dose-dependent manner and by calcium pentosan polysulfate. In contrast, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and keratan sulfate did not inhibit the degradation of 35S-labeled aggrecan nor suppress the reduction in tissue levels of aggrecan by explant cultures of articular cartilage. Heparin, heparan sulfate and calcium pentosan polysulfate did not adversely affect chondrocyte metabolism as measured by lactate production, incorporation of [35S]-sulfate or [3H]-serine into macromolecules by articular cartilage explant cultures. Furthermore, heparin, heparan sulfate and calcium pentosan polysulfate inhibited the proteolytic degradation of aggrecan by soluble aggrecanase activity. These results suggest that highly sulfated glycosaminoglycans have the potential to influence aggrecan catabolism in articular cartilage and this effect occurs in part through direct inhibition of aggrecanase activity.

Aggrecans↗
PubMed1996

Species-specific alternative splicing of the epidermal growth factor-like domain 1 of cartilage aggrecan.

Aggrecan transcripts contain two alternatively spliced exons that code for two epidermal growth factor-like domains (EGF1 and EGF2). Whereas the EGF2 sequence is expressed at a uniform level among different species, the EGF1 sequence has been detected only in human aggrecan transcripts. In this study we have used the nested primer reverse transcription-PCR (RT-PCR) method to compare the expression of the EGF1 exon in human, bovine and dog aggrecan transcripts. Our results indicate that this exon is expressed in a species-specific manner. In addition to its significant expression level in human transcripts, the EGF1 sequence can be detected in a small portion of bovine aggrecan transcripts as shown with nested primer RT-PCR. In contrast, the same module is not detectable in dog aggrecan transcripts, although an EGF1 sequence is present in the dog aggrecan gene. The expression level of the EGF1 exon in the aggrecan transcripts correlates with the strength of the polypyrimidine tract upstream of the exon. The EGF1 sequence also shows much less conservation between the species than the EGF2 sequence. The species-specific expression and high sequence variation of the EGF1 exon imply that this sequence is likely to code for an aggrecan domain having no cartilage-specific function.

Aggrecans↗
PubMed1995

Cell-mediated catabolism of aggrecan. Evidence that cleavage at the "aggrecanase" site (Glu373-Ala374) is a primary event in proteolysis of the interglobular domain.

A rat chondrosarcoma cell line and primary bovine chondrocytes have been used to study cell-mediated aggrecan catabolism. Addition of 1 microM retinoic acid to chondrosarcoma cultures resulted in aggrecan proteolysis with the release of greater than 90% of the cell layer aggrecan into the medium within 4 days. NH2-terminal sequencing of chondroitin sulfate-substituted catabolic products gave a single major NH2-terminal sequence of ARGNVILTXK, initiating at Ala374. This showed that the proteinase, commonly referred to as "aggrecanase," which cleaves the Glu373-Ala374 bond of the interglobular domain of aggrecan (Sandy, J. D., Neame, P. J., Boynton, R. E., and Flannery, C. R. (1990) J. Biol. Chem. 266, 8683-8685), is active in this cell system. Aggrecan G1 domain, generated by cleavage of the interglobular domain, was also liberated during catabolism and this was characterized with three antipeptide antisera. Anti-CDAGWL was used as a general probe for G1 domain. Anti-FVDIPEN was used to specifically detect G1 domain with COOH terminus of Asn341, the form which is readily generated by cleavage of aggrecan by a wide range of matrix metalloproteinases. Anti-NITEGE antiserum was used to specifically detect G1 domain with COOH terminus of Gln373, the form which is the expected product of "aggrecanase"-mediated cleavage of aggrecan. Western blot analysis indicated that a single form of G1 domain of about 60 kDa was formed. G1 domain of this size reacted with both anti-CDAGWL and anti-NITEGE but not with anti-FVDIPEN. Similar experiments with primary bovine chondrocyte cultures, treated with either retinoic acid or interleukin 1, showed that two forms of catabolic G1 domain, of about 62 and 66 kDa, were formed. Both of these forms reacted on Western blots with anti-CDAGWL and also with anti-NITEGE. It is suggested that cell-mediated catabolism of the aggrecan interglobular domain in these culture systems, whether promoted by retinoic acid or interleukin 1, primarily involves cleavage of the Glu373-Ala374 bond by aggrecanase. The accumulation of G1 domain with a COOH-terminal of Glu373 shows that such aggrecanase-mediated cleavage can occur independent of the cleavage of the Asn341-Phe342 bond by matrix metalloproteinases.

Aggrecans↗
PubMed1998

The interglobular domain of cartilage aggrecan is cleaved by hemorrhagic metalloproteinase HT-d (atrolysin C) at the matrix metalloproteinase and aggrecanase sites.

Two primary cleavage sites have been identified within the interglobular domain of the cartilage aggrecan core protein: one is between amino acid residues Asn 341 and Phe342, where many matrix metalloproteinases (MMP) have been shown to cleave; and the other is between amino acid residues Glu373 and Ala374. Although cleavage at the Glu373-Ala374 site is believed to play a critical role in cartilage aggrecan degradation in arthritic diseases, the enzyme responsible for cleavage at this site, "aggrecanase," has not been identified. Members of the ADAM (a disintegrin and metalloproteinase) family of proteins, which shows structural homology to the snake venom hemorrhagic metalloproteinases (reprolysins), have recently been demonstrated to be expressed in articular chondrocytes. Because many ADAM family members have a putative proteinase function, this raises the possibility that aggrecanase may be a member of this family of proteases. To examine whether reprolysins have the ability to cleave aggrecan at either the aggrecanase site or the MMP site, the snake venom hemorrhagic toxin metalloproteinase HT-d (atrolysin C) was tested for its ability to cleave bovine aggrecan monomer. Cleavage was monitored using the BC-3 antibody, which recognizes aggrecan fragments with the new NH2 terminus ARGSV generated by cleavage at the aggrecanase site, and with the AF-28 antibody, which recognizes aggrecan fragments with the new NH2 terminus FFGVG generated by cleavage at the MMP site. Cleavage at both the aggrecanase and MMP sites occurred in a concentration-dependent manner with 100 nM atrolysin C or greater. AF-28-reactive fragments were generated by 30 min of incubation, and levels were maximal by 8 h; BC-3-reactive fragments were detected at 2 h and continued to increase through 48 h, thus suggesting that atrolysin C can cleave at the MMP and aggrecanase sites. NH2-terminal aggrecan fragments generated by cleavage at the aggrecanase site were also detected using antisera recognizing the new COOH terminus, NITEGE, formed by cleavage at the Glu373-Ala374 bond, indicating that cleavage at this site does not require prior cleavage at the MMP site. These data provide the first demonstration that a reprolysin can cleave the core protein of aggrecan and the first example of a specific protease that can cleave at the aggrecanase site independent of cleavage at the MMP cleavage site.

Aggrecans↗
PubMed2000

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

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

Age Factors↗
PubMed2006

Lateral nanomechanics of cartilage aggrecan macromolecules.

To explore the role of the brush-like proteoglycan, aggrecan, in the shear behavior of cartilage tissue, we measured the lateral resistance to deformation of a monolayer of chemically end-attached cartilage aggrecan on a microcontact printed surface in aqueous NaCl solutions via lateral force microscopy. The effects of bath ionic strength (IS, 0.001-1.0 M) and lateral displacement rate (approximately 1-100 microm/s) were studied using probe tips functionalized with neutral hydroxyl-terminated self-assembled alkanethiol monolayers. Probe tips having two different end-radii (R approximately 50 nm and 2.5 microm) enabled access to different length-scales of interactions (nano and micro). The measured lateral force was observed to depend linearly on the applied normal force, and the lateral force to normal force proportionality constant, mu, was calculated. The value mu increased (from 0.03 +/- 0.01 to 0.11 +/- 0.01) with increasing bath IS (0.001-1.0 M) for experiments using the microsized tip due to the larger compressive strain of aggrecan that resulted from increased IS at constant compressive force. With the nanosized tip, mu also increased with IS but by a smaller amount due to the fewer number of aggrecan involved in shear deformation. The variations in lateral force as a function of applied compressive strain epsilon(n) and changes in bath IS suggested that both electrostatic and nonelectrostatic interactions contributed significantly to the shear deformational behavior of the aggrecan layers. While lateral force did not vary with lateral displacement rate at low IS, where elastic-like electrostatic interactions between aggrecan dominated, lateral force increased significantly with displacement rate at physiological and higher IS, suggestive of additional viscoelastic and/or poroelastic interactions within the aggrecan layer. These data provide insights into molecular-level deformation of aggrecan macromolecules that are important to the understanding of cartilage behavior.

Aggrecans↗
PubMed1991

1,25-Dihydroxyvitamin D3 down-regulates aggrecan proteoglycan expression in immortalized rat chondrocytes through a post-transcriptional mechanism.

We have examined the effects of various analogs of vitamin D on the expression of the aggrecan proteoglycan by an immortalized rat chondrocyte cell line. The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), produced a concentration-dependent reduction in the synthesis of aggrecan as monitored by histochemical staining of the matrix, incorporation of [35S]sulfate, and the level of aggrecan core protein. Other analogs of vitamin D were much less potent or had no activity whatsoever. The reduced expression of aggrecan was caused by a dramatic decrease in the steady-state level of the mRNA coding for the aggrecan core protein. A nuclear run-off analysis revealed that the rate of transcription of the aggrecan gene was not significantly altered by 1,25(OH)2D3 treatment, suggesting that the metabolite was acting through a post-transcriptional mechanism. Experiments using the transcriptional inhibitor actinomycin D also supported a nondirect effect of 1,25(OH)2D3 on the expression of the aggrecan gene. These results suggest that the vitamin D metabolite activates a new pattern of gene expression which results in a more rapid turnover of the aggrecan mRNA. This system should be useful for characterizing the regulation of chondrocyte gene expression by vitamin D.

Aggrecans↗
PubMed1994

Aggrecan synthesized by mature bovine chondrocytes suspended in alginate. Identification of two distinct metabolic matrix pools.

Proteoglycans synthesized by chondrocytes in alginate beads are found in two compartments: the cell-associated matrix and the further removed matrix (Häuselmann, H. J., Aydelotte M. B., Schumacher B. L., Kuettner K. E., Gitelis, S. H., and Thonar, E. J.-M. A. (1992) Matrix 12, 116-129). To study the metabolism of aggrecan in these two compartments, mature bovine articular chondrocytes in alginate beads were pulsed with [35S]sulfate for 30 min or 16 h on day 7 of culture and then chased in isotope-free medium for up to 21 days. At different times, the two matrix pools were separately isolated, and the 35S-proteoglycans quantified, purified, and characterized. Radiolabeled aggrecan molecules exhibited a very long average half-life in the beads (t1/2 = 95 days). In contrast, small non-aggregating proteoglycans, which made up approximately 4% of the 35S-proteoglycans synthesized, were rapidly lost from the beads (t1/2 = < 24 h). Approximately half the 35S-aggrecan subunits, representing mostly molecules which showed a delay in ability to form aggregates in the presence of exogenous hyaluronan and link protein, spent only a short time (t1/2 = 4 h) in the cell-associated matrix before moving into the further removed matrix. They exhibited a much longer average half-life in the beads than 35S-aggrecan molecules which became resident of the cell-associated matrix (t1/2 = > 95 days versus 15 days). Radiolabeled aggrecan subunits in the two matrix compartments had a similar average hydrodynamic size and polydispersity; importantly, the size of these molecules did not change during the chase period. Catabolism of 35S-aggrecan in the cell-associated matrix was the only significant contributor to the appearance in the medium of partially degraded 35S-aggrecan which had lost the ability to bind to hyaluronan. These results strongly suggest aggrecan molecules which reside in the pericellular and territorial matrix compartments in close proximity to the chondrocytes have a much faster rate of turnover than their counterpart in the interterritorial areas further removed from the cells.

Aggrecans↗
PubMed2003

G1 domain of aggrecan cointernalizes with hyaluronan via a CD44-mediated mechanism in bovine articular chondrocytes.

OBJECTIVE: To determine whether aggrecan fragments bound to hyaluronan (HA) can be retained and internalized by articular chondrocytes and whether these events are dependent on HA and its receptor, CD44. An additional objective was to determine whether partial degradation of aggrecan is a prerequisite for internalization. METHODS: Binding and internalization of a variety of fluorescein isothiocyanate (FITC)- or biotin-labeled HA/proteoglycan probes were investigated on normal bovine articular cartilage chondrocytes, bovine articular chondrocytes transfected with a dominant-negative construct of CD44, or COS-7 cells transfected with wild-type CD44. The probes were defined as being internalized by the presence of label associated with the cells following extensive trypsinization of the cell surface. RESULTS: Biotinylated aggrecan fragments bound to FITC-HA were cointernalized in bovine articular chondrocytes or COS-7 cells transfected with CD44. Intracellular vesicles containing FITC-HA colocalized with a fluorescent probe for lysosomes. The internalization of the aggrecan fragments was dependent on the presence of HA as well as the presence of functional CD44. Intact aggrecan/FITC-HA complexes bound to the cell surface but were not internalized. However, following brief trypsin digestion of the aggrecan/HA complex, the remaining proteoglycan fragments were bound and internalized. CONCLUSION: Partially degraded aggrecan fragments (e.g., aggrecan G1 domains bound to HA) can be internalized by articular chondrocytes via a mechanism involving HA/CD44-mediated endocytosis. Further, the presence of an intact aggrecan monomer bound to HA inhibits the internalization of HA as well as HA-bound fragments.

Aggrecans↗
PubMed1996

Compression of cartilage results in differential effects on biosynthetic pathways for aggrecan, link protein, and hyaluronan.

The differential effects of static compression and recovery from compression on biosynthesis and biosynthetic pathways of aggrecan, link protein, and hyaluronan were assessed. During compression, biosynthesis of aggrecan and link protein were inhibited to approximately 25 and approximately 40%, respectively, of free-swelling control levels. In marked contrast, hyaluronan synthesis was unaffected by static compression. After release from 12-h 50% static compression, aggrecan synthesis remained inhibited for up to 2.5 days; however, link protein synthesis completely recovered to free-swelling control levels within 8 h after release. Hyaluronan synthesis remained at control levels after release of compression. During compression, aggrecan core protein pool size was decreased, whereas the rate of processing into the proteoglycan form remained essentially the same as in free swelling control tissue. Four hours after release from compression, aggrecan core protein pool size remained small and the rate of intracellular processing of aggrecan had become slower than that of free swelling control tissue. Due to the altered core-protein processing kinetics, fewer but longer chondroitin sulfate chains were added to the core proteins. Sulfation was not markedly altered. The differential effects of static compression and release on the biosynthesis of aggrecan, link protein, and hyaluronan are similar to the changes in the biosynthetic pathways that are affected in response to IL-1 treatment, suggesting that the response to static compression is not a general inhibition of cellular activity, but appears to be part of a specific transduction mechanism.

Aggrecans↗
PubMed1995

The biochemically and immunologically distinct CSPG of notochord is a product of the aggrecan gene.

Using the monoclonal antibody S103L, which reacts specifically with an epitope in the chondroitin sulfate-rich domain of the chick cartilage chondroitin sulfate proteoglycan (CSPG) core protein, we have identified the predominant CSPG expressed by notochord. This large notochord CSPG is first detected immunohistochemically as early as stage 16, long before chondrogenesis occurs, and is expressed continuously during the time of active neural crest migration and through the onset of sclerotomal differentiation. Because of the cross-reactivity of both notochord and cartilage CSPGs with the S103L antibody, extensive molecular and biochemical analysis of the two CSPGs was carried out. Striking differences distinguish the notochord and cartilage (aggrecan) CSPGs at the level of posttranslational modification. Notably, cartilage aggrecan carries a significant content of keratan sulfate (KS) chains, while the notochord CSPG is devoid of KS. In contrast, cartilage aggrecan lacks the HNK-1 epitope, while the notochord CSPG has a high content of HNK-1. Three different approaches were used to establish the relationship of the two CSPGs at the molecular level. Northern blot analysis, using aggrecan probes, detected same-sized messages from notochord and cartilage RNA. Overlapping fragments, generated by RT-PCR using primers covering 98% of the entire coding sequence from the known cartilage structure, were of identical size in notochord and cartilage. Taking advantage of our recent studies, which demonstrated a single base change in the aggrecan gene resulting in conversion of Glu to a STOP codon in exon 12 of chick aggrecan as the molecular basis of the defect nanomelia, we demonstrated that the same mutation was present in notochord mRNA from nanomelic chicks. These results provide evidence that the chick aggrecan gene is expressed very early in development in notochord and confirm that the core proteins expressed in chick notochord and cartilage are derived from the same gene. These findings strongly support the hypothesis that the final structural characteristics of each proteoglycan are determined not only by the core protein but also by tissue-specific, developmentally regulated posttranslational mechanisms, functioning within the context of the requirement for specific extracellular matrices.

Aggrecans↗
PubMed1994

Molecular cloning and analysis of the protein modules of aggrecans.

The large aggregating chondroitin sulfate proteoglycan of cartilage, aggrecan, has served as a prototype of proteoglycan structure. Molecular cloning has elucidated its primary structure and revealed both known and unknown domains. To date the complete structures of chicken, rat and human aggrecans have been deduced, while partial sequences have been reported for bovine aggrecan. A related proteoglycan, human versican, has also been cloned and sequenced. Both aggrecan and versican have two lectin domains, one at the amino-terminus which binds hyaluronic acid and one at the carboxyl-terminus whose physiological ligand is unknown. Both lectins have homologous counterparts in other types of proteins. Within the aggrecans the keratan sulfate domain may be variably present and also has a prominent repeat in some species. The chondroitin sulfate domain has three distinct regions which vary in their prominence in different species. The complex molecular structure of aggrecans is consistent with the concept of exon shuffling and aggrecans serve as suitable prototypes for comprehending the evolution of multi-domain proteins.

Aggrecans↗
PubMed1993

Molecular cloning and analysis of the protein modules of aggrecans.

The large aggregating chondroitin sulfate proteoglycan of cartilage, aggrecan, has served as a prototype of proteoglycan structure. Molecular cloning has elucidated its primary structure and revealed both known and unknown domains. To date the complete structures of chicken, rat and human aggrecans have been deduced, while partial sequences have been reported for bovine aggrecan. A related proteoglycan, human versican, has also been cloned and sequenced. Both aggrecan and versican have two lectin domains, one at the amino-terminus which binds hyaluronic acid and one at the carboxyl-terminus whose physiological ligand is unknown. Both lectins have homologous counterparts in other types of proteins. Within the aggrecans the keratan sulfate domain may be variably present and also has a prominent repeat in some species. The chondroitin sulfate domain has three distinct regions which vary in their prominence in different species. The complex molecular structure of aggrecans is consistent with the concept of exon shuffling and aggrecans serve as suitable prototypes for comprehending the evolution of multi-domain proteins.

Aggrecans↗