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PubMed2006

The involvement of aggrecan polymorphism in degeneration of human intervertebral disc and articular cartilage.

The functions of the intervertebral disc and of articular cartilage are intimately related to their aggrecan content. Aggrecan is a proteoglycan that interacts with hyaluronan to form large aggregates, which are responsible for the ability of the tissues to resist compressive loads. This function is related to the structure of aggrecan, and in particular to the large number of chondroitin sulphate chains present on its core protein. The chondroitin sulphate chains are present in two adjacent regions of the aggrecan core protein, termed the CS1 and CS2 domains. In the human, the region of the aggrecan gene encoding the CS1 domain exhibits size polymorphism, which can result in variation in the degree of chondroitin sulphate substitution of aggrecan in different individuals. This raises the possibility that the functional properties of aggrecan may vary between individuals, and that those individuals with an inferior aggrecan structure may be more susceptible to premature intervertebral disc or articular cartilage degeneration. Several studies have been performed to demonstrate such an association, but the results have been ambiguous. This review explains the relationship between aggrecan structure and function, describes the technique used to assess aggrecan polymorphism and the conclusions and limitations of the data obtained to date, and discusses the implications for tissue degeneration and clinical practice.

Aggrecans↗
PubMed1999

Age-related changes in the synthesis of link protein and aggrecan in human articular cartilage: implications for aggregate stability.

The rates of incorporation of radiolabelled leucine into aggrecan and link protein have been measured in human articular cartilage of different ages. Aggrecan and link protein were purified in the A1 fraction of CsCl gradients as a result of their ability to form high-buoyant-density proteoglycan aggregates with hyaluronic acid. Separation of the aggrecan from the link protein was achieved by Mono Q anion-exchange chromatography. The rates of synthesis of both aggrecan and link protein decreased with age. The age-related decrease in synthesis of aggrecan was paralleled by a decrease in the rate of sulphate incorporation into glycosaminoglycan chains. The synthesis of link protein decreased with age to a greater extent than that of aggrecan such that the ratio of the rates of link protein to aggrecan synthesis decreased from 1 in immature cartilage to 0.2 in mature cartilage. The age-related decrease in link protein synthesis is controlled at least in part by transcriptional or post-transcriptional mechanisms, as shown by the accompanying age-related decrease in link-protein mRNA. The absence of any age-related decrease in aggrecan mRNA suggests that the decrease in synthesis of aggrecan core protein is controlled by a translational mechanism. Measurement of the total tissue content of aggrecan and link protein by radioimmunoassay revealed an age-related increase in the accumulation of these matrix proteins, even though their de novo synthesis was decreasing. This illustrates the importance that the regulation of extracellular post-translational modification also has in controlling the overall turnover of the cartilage matrix.

Adolescent↗
PubMed1999

Changes in joint cartilage aggrecan after knee injury and in osteoarthritis.

OBJECTIVE: To determine the concentrations of aggrecan fragments in synovial fluid from patients with knee joint injury, osteoarthritis (OA), or acute pyrophosphate arthritis (PPA; pseudogout), and to test their relative reactivity with the 846 epitope, a putative marker of cartilage aggrecan synthesis. METHODS: Samples of knee joint fluid from 385 patients and 9 healthy-knee volunteers were obtained in a cross-sectional study. Study groups were acute PPA/ pseudogout (n = 60), anterior cruciate ligament (ACL) rupture (n = 159), meniscus lesion (n = 129), and primary knee OA (n = 37). The 846 epitope on aggrecan was assayed by competitive solution-phase radioimmunoassay. Aggrecan fragments were assayed by enzyme-linked immunosorbent assay using a monoclonal antibody (1-F21). Cartilage oligomeric matrix protein (COMP), C-propeptide of type II collagen (CPII), bone sialoprotein, matrix metalloproteinases 1 and 3, and tissue inhibitor of metalloproteinases 1 were previously quantified by immunoassays. RESULTS: Reactivity of the 846 epitope was increased in all study groups compared with the reference group, and was highest in patients with primary OA. The median levels (in microg fetal aggrecan equivalents/ml) of the epitope were 0.28 (range 0.24-0.47) in the reference group, 0.48 (range 0.26-1.32) in PPA/pseudogout, 0.61 (range 0.12-2.87) in ACL rupture, 0.53 (range 0.22-3.02) in meniscus lesion, and 0.68 (range 0.31-4.31) in primary OA. The 846 epitope reactivity per microg aggrecan fragments in the joint fluid was higher in late-stage OA than in early-stage OA. Epitope 846 reactivity correlated positively with several markers of matrix turnover, particularly with COMP (r(s) = 0.421) and CPII (r(s) = 0.307). CONCLUSION: The observed differences in 846 epitope reactivity in synovial fluid, and its concentration in relation to aggrecan and other markers of matrix turnover, were consistent with marked ongoing changes in aggrecan turnover after joint injury and in the development of OA. OA is thus a disease characterized by dynamic changes in tissue macromolecule turnover, which is reflected by measurable changes in aggrecan epitopes in the synovial fluid.

Aggrecans↗
PubMed2002

Human intervertebral disc aggrecan inhibits nerve growth in vitro.

OBJECTIVE: To assess the effects of human intervertebral disc aggrecan on nerve growth and guidance, using in vitro techniques. METHODS: Aggrecan extracted from human lumbar intervertebral discs was incorporated into tissue culture substrata for the culture of the human neuronal cell line, SH-SY5Y, or explants of chick dorsal root ganglia. The effects on nerve growth of different concentrations of aggrecan extracted from the anulus fibrosus and nucleus pulposus, and of these aggrecan preparations following enzymic deglycosylation, were compared. RESULTS: Disc aggrecan inhibited the growth of neurites from SH-SY5Y cells and induced growth cone turning of chick sensory neurites in a concentration-dependent manner. Aggrecan isolated from the anulus fibrosus was more inhibitory than that isolated from the nucleus pulposus, but enzymic pretreatments to reduce the glycosylation of both types of disc aggrecan partially abrogated their inhibitory effects. CONCLUSION: Nerve growth into degenerate intervertebral discs has been linked with the development of low back pain, but little is known about factors affecting disc innervation. The finding that disc aggrecan inhibits nerve growth in vitro, and that this inhibitory activity depends on aggrecan glycosylation, has important implications for our understanding of mechanisms that may regulate disc innervation in health and disease.

Aggrecans↗
PubMed2003

Localization of aggrecan and versican in the developing rat central nervous system.

The localization of aggrecan and mRNA splice variants of versican in the developing rat central nervous system has been examined by using specific polyclonal antibodies to the nonhomologous glycosaminoglycan attachment regions of these hyaluronan-binding chondroitin sulfate proteoglycans. At embryonic day 16 (E16), aggrecan and versican splice variants containing either or both the alpha-and beta-domains are present in the marginal zone and subplate of the cerebral cortex and in the amygdala, internal capsule, and the optic and lateral olfactory tracts. There is strong staining of versican but not of aggrecan in the hippocampus and dentate gyrus by E19, whereas both aggrecan and alpha-versican are present in the fimbria. At E19, aggrecan is seen throughout the cerebral cortex, whereas the distribution of versican is considerably more limited, being confined essentially to the marginal zone and subplate. At 1 week postnatal, both aggrecan and versican are present in the prospective white matter and in the molecular and granule cell layers of the cerebellum, but neither proteoglycan is seen in the external granule cell layer. alpha- but not beta-versican staining is seen in Purkinje cells, and aggrecan staining of Purkinje cells is also rather minimal. In the spinal cord at E13, aggrecan is present in the dorsal root entry zone, ventral funiculus, mantle layer, and floor plate, as well as in the dorsal root ganglia and ventral roots. However, alpha-versican is confined to the dorsal root entry zone and the ependyma surrounding the spinal canal, and beta-versican is not present in spinal cord parenchyma at this developmental stage, being limited to the surrounding connective tissue. By E19, there are significant amounts of all three proteoglycans in the spinal cord. Aggrecan staining is most intense in the lateral funiculus and the fasciculi gracilis and cuneatus, where alpha-versican staining is also strong. In contrast, beta-versican is seen predominantly in the motor columns. Differences in the localization and temporal expression patterns of these chondroitin sulfate proteoglycans suggest that, like neurocan and phosphacan, they have partially complementary roles during central nervous system development.

Aggrecans↗
PubMed2003

Aggrecan regulates telencephalic neuronal aggregation in culture.

Proteoglycans have been suggested to play roles in pattern formation in the developing central nervous system. In the chick embryo, aggrecan, a chondroitin sulfate proteoglycan, has a regionally-specific and developmentally-regulated expression profile. Telencephalic neuronal cultures, when aggregated, exhibit aggrecan expression patterns comparable to those observed in vivo. The chicken mutation nanomelia produces a truncated aggrecan species that cannot be processed further and is not secreted. Neurons from normal and nanomelic chick embryo telencephalon were scored for aggregate formation and analyzed for distribution of aggrecan protein and expression of aggrecan mRNA. Distinctly different pattern formation, with respect to aggregate size (smaller) and number (fewer) were observed in poly-L-lysine plated neuronal cultures derived from nanomelic embryos when compared to those derived from normal embryos. Significantly, the nanomelic phenotype was subsequently rescued upon addition of the brain-specific form of aggrecan. Modulation of neuronal aggregate formation was mimicked by treatment with chondroitinase ABC but not other glycanases, and was rescued by addition of chondroitin 6-sulfate to the culture media. Lastly, although broad and diffuse distribution of aggrecan among the cell aggregates in the culture paradigm was observed by immunocytochemistry, mRNA in situ hybridization revealed that only a small population of cells in the center of the aggregates was responsible for the production of the secreted aggrecan found associated with neuronal aggregates. These studies suggest a function for aggrecan as a diffusible signal in CNS histomorphogenesis.

Aggrecans↗
PubMed2003

Variations in aggrecan structure modulate its susceptibility to aggrecanases.

Proteoglycan aggregates and purified aggrecan from adult and fetal bovine cartilage and adult and neonatal human cartilage were subjected to in vitro degradation by recombinant aggrecanase-1 and aggrecanase-2. The ability of the aggrecanases to cleave within the aggrecan IGD (interglobular domain) and CS2 domain (chondroitin sulphate-rich domain 2) was monitored by SDS/PAGE and immunoblotting. Aggrecanase-2 showed a similar ability to cleave within the IGD of adult and immature aggrecan, whereas aggrecanase-1 was less efficient in cleavage in the IGD of immature aggrecan, for both the bovine and the human substrates. Both aggrecanases showed a similar ability to cleave within the CS2 domain of bovine aggrecan irrespective of age, but showed a much lower ability to cleave within the CS2 domain of human aggrecan. Equivalent results were obtained whether aggrecan was present in isolation or as part of proteoglycan aggregates. When proteoglycan aggregates were used, neither aggrecanase was able to cleave link protein. Thus, for aggrecan cleavage by aggrecanases, variations in cleavage efficiency exist with respect to the species and age of the animal from which the aggrecan is derived and the type of aggrecanase being used.

ADAM Proteins↗
PubMed1996

Age-related changes in the content of the C-terminal region of aggrecan in human articular cartilage.

The content of the C-terminal region of aggrecan was investigated in samples of articular cartilage from individuals ranging in age from newborn to 65 years. This region contains the globular G3 domain which is known to be removed from aggrecan in mature cartilage, probably by proteolytic cleavage, but the age-related changes in its abundance in human cartilage have not been described previously. The analysis was performed by immunosorbant assay using an antiserum (JD5) against recombinant amino acid residues of human aggrecan, on crude extracts of cartilage without further purification of aggrecan. The results showed that the content of the C-terminal region decreased with age relative to the G1 domain content (correlation coefficient = 0.463). This represented a 92% fall in the content of this region of the molecule from newborn to 65 years of age. furthermore, when the G1 content of the cartilage extracts was corrected to only include the G1 attached to aggrecan and to exclude the G1 fragments which accumulate as a by-product of normal aggrecan turnover (free G1), the age-related decrease in the C-terminal region remained very pronounced. Analysis by composite agarose/PAGE showed that the number of subpopulations of aggrecan resolved increased from one in newborn to three in adult cartilage. All of these reacted with an antiserum to the human G1 domain, but only the slowest migrating species reacted with the C-terminal region antiserum (JD5). Similar analysis by SDS/PAGE confirmed the presence of high-molecular-mass (200 kDa) proteins reactive with JD5, but no reactive fragments of lower electrophoretic mobility were detected. In contrast, when probed with the antiserum to the human G1 domain, the immunoblots showed protein species corresponding to the free G1 and G1-G2 fragments, which were present at high concentrations in adult cartilage. The results suggest that the loss of the C-terminal region is not directly part of the process of aggrecan turnover, but it is a slow independent matrix process that occurs more extensively with aging as turnover rates become slower. Young cartilage with the fastest turnover contains least molecules lacking the C-terminal region, whereas in old tissue with slow turnover few molecules retain this region. An increase in the cleavage of this region with age may also contribute to this change. The content of the C-terminal region may thus give a measure of the abundance of newly synthesized aggrecan.

Adolescent↗
PubMed2000

Age-related changes in aggrecan glycosylation affect cleavage by aggrecanase.

Aggrecan degradation involves proteolytic cleavage of the core protein within the interglobular domain. Because aggrecan is highly glycosylated with chondroitin sulfate (CS) and keratan sulfate (KS), we investigated whether glycosylation affects digestion by aggrecanase at the Glu(373)-Ala(374) bond. Treatment of bovine aggrecan monomers to remove CS and KS resulted in loss of cleavage at this site, suggesting that glycosaminoglycans (GAGs) play a role in cleavage at the Glu(373)-Ala(374) bond. In contrast, MMP-3 cleavage at the Ser(341)-Phe(342) bond was not affected by glycosidase treatment of aggrecan. Removal of KS, but not CS, prevented cleavage at the Glu(373)-Ala(374) bond. Thus, KS residues may be important for recognition of this cleavage site by aggrecanase. KS glycosylation has been observed at sites adjacent to the Glu(373)-Ala(374) bond in steer aggrecan, but not in calf aggrecan (Barry, F. P., Rosenberg, L. C., Gaw, J. U., Gaw, J. U., Koob, T. J., and Neame, P. J. (1995) J. Biol. Chem. 270, 20516-20524). Interestingly, although we found that aggrecanase degraded both calf and steer cartilage aggrecan, the proportion of fragments generated by cleavage at the Glu(373)-Ala(374) bond was higher in steer than in calf, consistent with our observations using aggrecan treated to remove KS. We conclude that the GAG content of aggrecan influences the specificity of aggrecanase for cleavage at the Glu(373)-Ala(374) bond and suggest that age may be a factor in aggrecanase degradation of cartilage.

Age Factors↗
PubMed2005

APBP-1, a DNA/RNA-binding protein, interacts with the chick aggrecan regulatory region.

Expression of the extracellular proteoglycan aggrecan is both cell-specific and developmentally regulated. Previous studies identified six functionally defined cis elements in the aggrecan promoter region which were shown to repress aggrecan gene expression (1). Using competition electrophoretic mobility shift assays (EMSAs) we have now identified in nuclear extracts a functional repressor cis element, (T/C)TCCCCT(A/C)RRC, which occurs at multiple locations within the chick aggrecan regulatory region. We purified the factor that binds to this cis element and established that it, APBP-1 (aggrecan promoter-binding protein-1), is a 19-kDa protein that has significant homology to CIRP (cold inducible RNA-binding protein). Recombinantly expressed APBP-1 mimics the native cis element-trans factor interaction in EMSAs. In situ hybridization demonstrates that aggrecan and APBP-1 RNA expression are restricted to complementary tissues in the developing limb, and Northern blot analysis of chick limb bud mRNA shows that APBP-1 mRNA expression is inversely correlated with aggrecan mRNA expression. Functional analyses by transient transfections and Northern blot analyses suggest APBP-1 has the capacity to repress aggrecan expression, indicating that this factor may be important regulator of aggrecan gene expression.

Aggrecans↗
PubMed2001

CD44 binds a chondroitin sulfate proteoglycan, aggrecan.

Here we report that CD44 binds a chondroitin sulfate (CS) proteoglycan, aggrecan, a major component of cartilage. Soluble CD44-IgG and CD44(+) cells bound to aggrecan from rat chondrosarcoma and bovine cartilage, immobilized on microtiter plates. In both cases, binding was blocked by a neutralizing anti-CD44 mAb or by the pretreatment of aggrecan with chondroitinase, but not hyaluronidase or keratanase, indicating that CD44 binds aggrecan in a manner dependent on CS side chains of aggrecan and that hyaluronic acid is not involved in the binding. Structural analysis showed that glycosaminoglycans of aggrecan from rat chondrosarcoma and bovine articular cartilage consist of mainly CS A and a mixture of CS A and C respectively. When immobilized on microtiter plates, both CS A and C bound CD44-IgG, and the reaction was specifically inhibited by an anti-CD44 mAb. In addition, aggrecan augmented apoptosis in cells expressing CD44-Fas chimeric molecules in synergy with a non-blocking anti-CD44 mAb IRAWB14.4, suggesting that CD44-aggrecan interaction can induce oligomerization of the chimeric molecules. These results suggest that aggrecan interacts with CD44 to mediate cell adhesion and to trigger oligomerization of CD44 molecules, which may lead to intracellular signaling.

Aggrecans↗
PubMed2005

DNA methylation is not likely to be responsible for aggrecan down regulation in aged or osteoarthritic cartilage.

BACKGROUND: Expression of aggrecan is reduced during aging and osteoarthritic cartilage degeneration. CpG methylation may have a role in the down regulation of aggrecan transcriptions. OBJECTIVE: To investigate whether a correlation between gene methylation and expression of aggrecan in chondrocytes exists. METHODS: The human aggrecan promoter region was analysed computationally for CpG-rich regions. These were investigated for the methylation of C residues in normal (aged) and osteoarthritic chondrocytes by the bisulphite method for modifying DNA as well as sequence analysis using DNA directly extracted from normal and osteoarthritic cartilage tissue. Additionally, chondrocytic cell lines were investigated for methylation within the aggrecan promoter region. RESULTS: The CpG-rich promoter region of the human aggrecan gene contains a 0.6 kb region that meets the criteria of a CpG island as defined by prediction programmes. A significant correlation of aggrecan mRNA expression levels and methylation status in normal (aged) and osteoarthritic chondrocytes as well as in different chondrocytic cell lines was not found. CONCLUSIONS: Expression of aggrecan in normal cartilage and diseased states is not modulated by gross changes of CpG methylation of its promoter region. CpG methylation does not have a central role in the switch off of aggrecan promoter activity in human adult articular chondrocytes.

Aged↗
PubMed1996

Changes in cartilage proteoglycan aggrecan after intra-articular injection of interleukin-1 in rabbits: studies of synovial fluid and articular cartilage.

OBJECTIVE: To determine how acute but transient inflammation affects the cartilage proteoglycan aggrecan and the value of analyses of synovial fluid to study this. METHODS: For 96 hours after a single intra-articular injection of rabbit knees with human interleukin-1 alpha (IL-1 alpha) or vehicle, articular cartilage and synovial fluid were examined using a putative indicator of aggrecan synthesis (aggrecan chondroitin sulphate epitope 846), immunoreactive keratan sulphate, and total glycosaminoglycan (GAG) content. Aggrecan extractability (with 0.5 M NaCl) followed by 4 M guanidine hydrochloride extraction permitted analyses of cartilage damage, total content and aggrecan heterogeneity. Aggrecan epitopes as well as GAG were assayed in synovial fluid. Changes were related to total joint leucocyte content in synovial fluid. RESULTS: At 10 ng, IL-1 alpha produced a transient increase in synovial fluid leucocytes at six hours and 24 hours. This accompanied a reduction in content and increased extractability of GAG, which was greatest in the tibial medial compartment of the knee. Further studies of this compartment showed no change in keratan sulphate epitope content, but a transient increase in extractability in 0.5 M NaCl. Epitope 846 content and extractability were unchanged. Total contents and extractability for GAG were inversely correlated in both controls and joints injected with IL-1 alpha. These changes were accompanied by transient increases in GAG, keratan sulphate epitope, and 846 content in synovial fluid. CONCLUSION: According to the aggrecan component measured, damage to the matrix of articular cartilage was sometimes reflected by a transient increased extractability and a net loss of aggrecan. There was always an increased release of GAG, and keratan sulphate, and 846 epitopes into synovial fluid. These studies show that changes in aggrecan epitopes and GAG in synovial fluid reflect changes in cartilage metabolism induced by acute transient inflammation.

Aggrecans↗
PubMed1996

Effects of prostaglandins on deoxyribonucleic acid and aggrecan synthesis in the RCJ 3.1C5.18 chondrocyte cell line: role of second messengers.

PGs play an important role in regulating articular chondrocyte function in both normal and pathological states. However, the mechanisms of the effects of PG on chondrocyte function remain undefined. We, therefore, examined the effects of PGE1, PGE2, and PGE2 alpha on second messenger generation in relation to DNA and aggrecan synthesis in the nontransformed rat RCJ 3.1C5.18 (RCJ) chondrocyte cell line. RCJ cells were grown under minimal attachment conditions on a composite collagen-agarose (0.15%/0.8%) gel to maintain a differentiated phenotype. PGE1 and PGE2 (0.001-100 microM) produced a similar dose-related increase in cAMP accumulation, with a maximal 8-fold increase over basal values, whereas PGF2 alpha produced a minimal 1.3-fold increase in cAMP levels only at 100 microM. On the other hand, both PGE2 and PGE2 alpha raised the intracellular free calcium ([Ca2+]i) concentration, derived primarily from extracellular sources, whereas PGE1 was without effect on [Ca2+]i. These three PGs also had divergent effects on DNA synthesis, as measured by [3H]thymidine ([3H]TdR) incorporation. PGF2 alpha (0.001-5 microM) produced a dose-related increase in [3H]TdR incorporation, with a maximal 1.6-fold increase over baseline values at 5 microM and a slight decline to below maximal levels at 10 microM. PGE2 exhibited a contrasting inverse biphasic response, with an initial small suppressive effect that was maximal at 0.1 microM and a secondary stimulatory phase producing a small increase over control values at 5 microM. PGE1 had a uniformly suppressive effect, producing a 30% decrease at 10 microM. Despite the divergent effects of PGE1, PGE2, and PGE2 alpha on second messenger generation and DNA synthesis, all three PGs produced a dose-related stimulation of aggrecan synthesis. PGF2 alpha was the most potent, producing significant stimulation at 0.001 microM and a maximal 104% increase at 5 microM. PGE1 and PGE2 were approximately equipotent and approximately 60% as effective as PGF2 alpha in stimulating aggrecan synthesis. Northern analysis demonstrated that the effects of PG on aggrecan synthesis were not accompanied by changes in aggrecan core protein steady state messenger RNA levels. Thus, the effects of PG on aggrecan production in RCJ cells appear to be regulated at the posttranscriptional level. Forskolin and (Bu)2cAMP mimicked the suppressive effects of PGE1 on [3H]TdR incorporation, as well as the stimulatory effect of PGE1 on aggrecan synthesis. In addition, the phorbol ester 12-O-tetradecanoyl phorbol acetate mimicked PGF2 alpha stimulation of [3H]TdR incorporation and aggrecan synthesis, and the effects of PGE2 alpha on these processes were blocked by protein kinase C inhibitors. Therefore, it appears that in mammalian chondrocytes, PGE1 primarily activates the cAMP-protein kinase A second messenger system, PGE2 alpha affects primarily the Ca2(+)-protein kinase C system, and PGE2 activates both pathways. Moreover, PG posttranscriptional regulation of aggrecan synthesis in chondrocytes involves both the cAMP-protein kinase A and Ca2(+)-protein kinase C second messenger systems.

Aggrecans↗
PubMed1999

Influence of polysulfated polysaccharides on aggrecans synthesized by differentiated human articular chondrocytes.

OBJECTIVE: Synthesis rates of aggrecans by phenotypically stable human articular chondrocytes and the immobilization of these aggrecans in large aggregates have been used as variables reflecting the capability of these cells to restore the extracellular matrix of articular cartilage in vitro. The effects of heparin, chondroitin polysulfate, and xylosan polysulfate on these variables were investigated. METHODS: Human articular chondrocytes were maintained in culture in gelled agarose. After 2 weeks of culture, the media were supplemented for one week with 10 microg/ml each of heparin, chondroitin polysulfate, and xylosan polysulfate. Synthesis of aggrecans was investigated using Na2(35SO4) as a radioactive precursor during the last 24 h of exposure to polysaccharides. Size exclusion chromatography was used to assay the proportions of aggrecans immobilized in aggregates. Native aggrecan aggregates accumulated during culture were liberated in associating conditions by agarase digestion of the artificial agarose matrix and studied by electron microscopy. Hyaluronan synthesis was studied in monolayer cultured chondrocyte derived fibroblasts. The cells were exposed to 3H glucosamine for 24 h after the nutrient media were supplemented with 3 polysaccharides for one week. Size exclusion chromatography was used to assess the length of the hyaluronan filaments. RESULTS: Xylosan polysulfate and chondroitin polysulfate but not heparin significantly increased total 35S incorporation rates in aggrecan. Electron microscopy study of aggrecan aggregates showed that xylosan and chondroitin polysulfate but not heparin significantly increased aggrecan aggregate sizes. The same polysulfated polysaccharides increased the synthesis of high molecular weight hyaluronan by chondrocyte derived fibroblast-like cells. CONCLUSION: Polysulfated polysaccharides significantly increased the synthesis rates and the accumulation of aggrecan in aggregates in the extracellular environment in this in vitro system.

Adult↗
PubMed1991

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.

Aggrecans↗
PubMed1994

Effects of brefeldin A on aggrecan core protein synthesis and maturation in rat chondrosarcoma cells.

In this paper, the effects of the fungal metabolite, brefeldin A, on the synthesis and maturation of aggrecan core protein precursor were studied in rat chondrosarcoma chondrocytes. The aggrecan core protein precursor was partially identified in total protein pools isolated from cell extracts based on its selective cleavage at a single site by the restriction protease factor Xa. During a 2-h labeling period with [3H]serine as precursor, brefeldin A inhibited the synthesis of mature aggrecan from its aggrecan core protein precursor consistent with an inhibition of chondroitin sulfate chain elongation and sulfation as described in the accompanying paper (Calabro, A., and Hascall, V. C. (1994) J. Biol Chem. 269, 22764-22770). This inhibition is presumably the result of the disruption of vesicular transport by brefeldin A, which isolates the aggrecan core protein precursor at the level of the trans-Golgi cisternae from the enzymes for chondroitin sulfate chain elongation and sulfation located in the trans-Golgi network. Brefeldin A also inhibited the exocytosis of all radiolabeled secretory proteins from the cell layer into the medium compartment, which is also consistent with the disruption of vesicular transport attributed to this metabolite. Although total protein synthesis was inhibited by 12% in the presence of brefeldin A, the aggrecan core protein precursor accumulated within the cell layer indicating that the inhibition of chondroitin sulfate synthesis by brefeldin A was not the result of a lack of aggrecan core protein precursor. When the brefeldin A block was removed and cultures chased in the presence of cycloheximide to prevent new protein synthesis, vesicular transport through the cell was re-established and chondroitin sulfate chains were added to a large proportion of the aggrecan core protein precursor that had accumulated during the brefeldin A block. These results suggest that the machinery for chondroitin sulfate synthesis and for protein exocytosis, that were disrupted by brefeldin A treatment, recover after removal of the brefeldin A, even in the presence of cycloheximide, and that the structures involved in these processes reassemble from previously existing proteins. Interestingly, two other proteins with the same relative abundance as the aggrecan core protein precursor were observed. An approximately 210-kDa protein with the characteristics of the fibronectin subunit, and an unidentified approximately 150-kDa protein which was efficiently cleaved by the protease Xa enzyme.

Aggrecans↗
PubMed1996

Brain aggrecan.

During development, the extracellular matrix (ECM) is a complex dynamic structure whose components and organization help to establish the requisite position and state of differentiation. Until recently, the large chondroitin sulfate proteoglycan, aggrecan, has been localized predominantly to skeletal tissue and considered a hallmark of cartilage differentiation. We have identified the presence of aggrecan in two other highly differentiated systems, brain and notochord, with clearly distinct expression patterns. In chick cartilage, aggrecan starts to be expressed at embryonic day 5 in limb rudiments, continues through the entire period of chondrocyte development, and remains a biochemical marker of the cartilage phenotype thereafter. In brain, aggrecan has a very low level of expression beginning at day 7, increases up to day 13, markedly decreases after day 16, and is not expressed posthatching. This pattern coincides with migration and establishment of neuronal nuclei in the chick telencephalon and has been proposed to be a component of the migration arrest mechanism. In very primitive embryos, aggrecan is detected as early as stage 16 in the notochord, long before chondrogenesis occurs, is then expressed up to day 5 and decreases thereafter. The expression of aggrecan occurs during the time of active neural crest migration and through the onset of sclerotomal differentiation, and correlates with the notochords' ability to inhibit neural crest cell migration. Animal models defective in aggrecan biosynthesis have been invaluable in delineating these functions. In addition we have characterized these proteoglycans by chemical, biosynthetic, and molecular analyses. Although significant post-translation differences distinguish the cell-specific aggrecan species, their core proteins are the products of a single gene. Our findings of the expression of the same gene (aggrecan) in multiple ontogenously unrelated differentiating tissue systems and at different times over the developmental life of an organism provide an elegant model system to study the regulation and interplay in expression of that gene, as well as the effect of alterations in that single gene simultaneously in several developing programs.

Aggrecans↗