Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Aggrecans”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 163 records · Page 9Linked to original sources
PubMed1996

Distribution of hyaluronan in articular cartilage as probed by a biotinylated binding region of aggrecan.

The proportion of total tissue hyaluronan involved in interactions with aggrecan and link protein was estimated from extracts of canine knee articular cartilages using a biotinylated hyaluronan binding region-link protein complex (bHABC) of proteoglycan aggregate as a probe in an ELISA-like assay. Microscopic sections were stained with bHABC to reveal free hyaluronan in various sites and zones of the cartilages. Articular cartilage, cut into 20 microns-thick sections, was extracted with 4 M guanidinium chloride (GuCl). Aliquots of the extract (after removing GuCl) were assayed for hyaluronan, before and after papain digestion. The GuCl extraction residues were analyzed after solubilization by papain. It was found that 47-51% of total hyaluronan remained in the GuCl extraction residue, in contrast to the 8-15% of total proteoglycans. Analysis of the extract revealed that 24-50% of its hyaluronan was directly detectable with the probe, while 50-76% became available only after protease digestion. The extracellular matrix in cartilage sections was stained with the bHABC probe only in the superficial zone and the periphery of the articular surfaces, both sites known to have a relatively low proteoglycan concentration. Trypsin pretreatment of the sections enhanced the staining of the intermediate and deep zones, presumably by removing the steric obstruction caused by the chondroitin sulfate binding region of aggrecans. Enhanced matrix staining in these zones was also obtained by a limited digestion with chondroitinase ABC. The results indicate that a part of cartilage hyaluronan is free from endogenous binding proteins, such as aggrecan and link protein, but that the chondroitin sulfate-rich region of aggrecan inhibits its probing in intact tissue sections. Therefore, hyaluronan staining was more intense in cartilage areas with lower aggrecan content. A large proportion of hyaluronan resists GuCl extraction, even from 20-micrograms-thick tissue sections.

Aggrecans↗
PubMed1996

Degradation of cartilage aggrecan by collagenase-3 (MMP-13).

Degradation of the large cartilage proteoglycan aggrecan in arthritis involves an unidentified enzyme aggrecanase, and at least one of the matrix metalloproteinases. Proteinase-sensitive cleavage sites in the aggrecan interglobular domain (IGD) have been identified for many of the humman MMPs, as well as for aggrecanase and other proteinases. The major MMP expressed by chondrocytes stimulated with retinoic acid to degrade their matrix is collagenase-3 or MMP-13. Because of its potential role in aggrecan degradation we examined the specificity of MMP-13 for an aggrecan substrate. The results show that MMP-13 cleaves aggrecan in the IGD at the same site (..PEN314-FFG..) identified for other members of the MMP family, and also at a novel site ..VKP384-VFE.. not previously observed for other proteinases.

Aggrecans↗
PubMed1994

Determination of kinetic changes of aggrecan-hyaluronan interactions in solution from its rheological properties.

The kinetics of interactions between aggregating cartilage proteoglycan (aggrecan) and hyaluronan was examined through their rheological flow behavior using a cone on plate viscometer. The mixing of the two types of molecules was carried out directly on the plate of the viscometer, and aggregation process was monitored through the changes of the sample's steady-shear viscosity and/or dynamic shear modulus as a function of time. The effect of flow conditions on the aggrecan-hyaluronan interaction rates was examined by subjecting samples to steady-shearing motions at specified shear rates, and to oscillatory shear motions of specified frequencies and amplitudes. The characteristics of the kinetics of interaction between aggrecan and hyaluronan molecules depended not only on the flow conditions under which proteoglycan aggregation took place, but also on the concentration of the components in the solution. At high shear rates (> 10 s-1), viscosity of the mixture solution increased monotonically, starting near the viscosity of the aggrecan solution, and reaching the viscosity of the aggregate solution in approximately 35 min. Surprisingly, under slow shearing motions (< 10 s-1), the viscosities of the mixture solutions exceeded those of control aggregate solutions at identical hyaluronan: aggrecan ratios and concentrations. In addition, the aggregation under oscillatory motions took place near physiologic frequency (10 rad s-1) although the rate of aggregation process was much slower than under steady-shearing motion (> 100 min). However, the high-frequency oscillatory shearing (62.8 rad s-1) tended to impede aggregation resulting in a reduction of dynamic modulus over time.(ABSTRACT TRUNCATED AT 250 WORDS)

Aggrecans↗
PubMed2006

Estimation of the identity of proteolytic aggrecan fragments using PAGE migration and Western immunoblot.

OBJECTIVE: To develop calculation models, using Western immunoblot, as a tool for the estimation of proteolytic human aggrecan fragment identity. METHOD: Seven human aggrecan fragments (calibrators), purified by CsCl gradient centrifugation and identified by Western immunoblot of N- and C-terminals, were used to develop calculation models. The models were used for identification of unknown aggrecan fragments each having one of their N- or C-terminals identified. RESULTS: The calibrator molecular weights (Mw) from sodium dodecyl sulfate (SDS)-gels (m), the Mw of amino acids (a) and the Mw of their carbohydrate substitution (g) were expressed as K = m/(a+g), or as K = 1.085m/(a+g) when compensation for the G1 domain was required. Using these models together with average K-values, 12 out of the 17 immuno-detected aggrecan fragments were calculated to a known protease cleavage site, while five were identified to domain levels. CONCLUSIONS: With six neoepitope antibodies together with antibodies against the G1- and G3-domain it was possible to predict the identity of several proteolytic fragments from different regions within the aggrecan monomer.

Aggrecans↗
PubMed1998

Aggrecan and link protein affect cell adhesion to culture plates and to type II collagen.

Cartilage is a hypocellular tissue in which a balance of matrix molecules, especially aggrecan and link protein, play a critical role in maintaining structural integrity. To study the role of aggrecan and link protein in mediating cell activities, we have stably expressed them in NIH/3T3 fibroblasts and observed the effect on cell-substratum interactions. Overexpression of either protein destabilized the cell-substratum interaction. However, when both were co-expressed, the interaction between cell and substratum was less impaired. Similar results were obtained on type II collagen-coated plates. The addition of exogenous gene products into fibroblast cell lines and chondrocyte culture had the same effect as expression of the genes. The addition of exogenous hyaluronan to the growth medium or treatment of cells with hyaluronidase also decreased cell adhesion, indicating that hyaluronan also plays a role in the cell-substratum adhesion. The presence of aggrecan seems to increase the amount of link protein on the cell surface. Chondrocytes expressing high concentrations of aggrecan and link protein were maintained within a matrix network and were able to survive in suspended culture. Imbalances in aggrecan or link protein concentrations, or degradation of hyaluronan, disrupted the network and caused the chondrocytes to aggregate or adhere to the plates.

3T3 Cells↗
PubMed2001

Nonlinear viscoelasticity of concentrated solutions of aggrecan aggregate.

Aggrecan, the major cartilage proteoglycan, is the macromolecular species primarily involved in the resiliency of cartilage tissue. Most aggrecan molecules occur in cartilage extracellular matrix as aggregates. Each aggregate has a supramolecular structure, with many highly anionic, brushlike aggrecan subunits noncovalently bound to a hyaluronan chain. To better examine the viscoelastic properties of aggrecan aggregate, contaminating subunits were removed by exclusion chromatography. At physiologic ionic strength, concentrated solutions of purified aggrecan aggregate exhibit predominantly elastic behavior at small shear strains. However, above a critical strain, gamma c, the shear moduli show a pronounced strain-softening transition, where the storage modulus decreases suddenly, and the loss modulus exhibits a maximum. At small stresses, the creep function is also highly elastic, exhibiting an equilibrium compliance and large recoverable compliance. When the stress is amplified, a discrete transition to viscous flow occurs at a yield stress sigma y. These nonlinear responses are similar to previous observations for close-packed colloidal suspensions of soft spheres, such as microgel or emulsion particles, for which a yield transition occurs when the stress and deformation are sufficient to move a particle past its neighbors.

Aggrecans↗
PubMed2006

Solution structure and dynamics of cartilage aggrecan.

We studied the structure and dynamics of porcine laryngeal aggrecan in solution using a range of noninvasive techniques: dynamic light scattering (DLS), small-angle neutron scattering (SANS), video particle tracking (VPT) microrheology, and diffusing wave spectroscopy (DWS). The data are analyzed within the framework of a combined static and dynamic scaling model, and evidence is found for reptation of the comb backbones with unentangled side-chain dynamics. Small-angle neutron scattering indicated standard polyelectrolyte scaling of the mesh size (xi) with concentration (c) in semidilute solutions for the whole aggrecan aggregate, xi = Ac(-0.47+/-0.04), with the prefactor (A) implying there is on average 60 nm between the aggrecan subunits along the backbone. VPT demonstrated large exponents for the power law dependence of the intrinsic viscosity (eta) on the polymer concentration in the semidilute concentration regime, eta approximately c(alpha); with alpha equal to 2.04 +/- 0.06 and 1.95 +/- 0.08 for the assembled and disassembled aggrecan aggregates, respectively. DWS at high frequencies (10(4)-10(5) Hz) gave evidence for internal Rouse modes of the aggrecan monomers, independent of the degree of self-assembly of the molecules.

Aggrecans↗
PubMed1994

Mouse cartilage matrix deficiency (cmd) caused by a 7 bp deletion in the aggrecan gene.

Mouse cartilage matrix deficiency (cmd) is an autosomal recessive mutation characterized by cleft palate, short limbs, tail and snout. Heterozygous mice show normal size and phenotype, while homozygous mice die just after birth due to respiratory failure. Biochemical and immunohistochemical characterization of cmd cartilage reveals normal levels of type II collagen and link protein, but an absence of the large cartilage proteoglycan, aggrecan. Here, we have mapped the aggrecan gene to a region of mouse chromosome 7 near the cmd locus. DNA sequencing of the aggrecan gene identified a 7 bp deletion in exon 5 resulting in a severely truncated molecule. The finding of an aggrecan mutation in the cmd mouse confirms the critical role of aggrecan in cartilage formation.

Aggrecans↗
PubMed2002

The mechanism of aggrecan release from cartilage differs with tissue origin and the agent used to stimulate catabolism.

The mechanisms of aggrecan degradation in adult human articular, adult bovine nasal and fetal bovine epiphyseal cartilage in response to either interleukin-1beta (IL-1beta) or retinoic acid were compared using an explant culture system. Bovine nasal cartilage cultured with either IL-1beta or retinoic acid exhibited significant release of glycosaminoglycan (GAG). For both factors, aggrecan proteolysis occurred predominantly at the 'aggrecanase' site, with no evidence for the action of matrix metalloproteinases, and resulted in the appearance of the corresponding G1 fragment in tissue extracts and in culture media. In human cartilage, little effect of IL-1beta was seen, but abundant release of GAG occurred in the presence of retinoic acid, with evidence of aggrecanase action. Treatment of fetal epiphyseal cartilage with retinoic acid resulted in significant GAG release, whereas treatment with IL-1beta did not. In the retinoic acid-treated tissue, however, no evidence for the cleavage of aggrecan in the interglobular region was apparent. Thus, in the fetal system, agents in addition to aggrecanase and matrix metalloproteinases appear to be active. Taken together, these data demonstrate that the pathways utilized for aggrecan catabolism may vary between different cartilages for a given stimulatory agent, and that, for a given tissue, different factors may elicit aggrecan release via different pathways.

Adult↗
PubMed1992

Modulation of aggrecan and link-protein synthesis in articular cartilage.

The addition of serum or insulin-like growth factor-I (IGF-I) to the medium of explant cultures of bovine articular cartilage is known to stimulate the synthesis of aggrecan in a dose-dependent manner. The half-life of the pool of proteoglycan core protein was measured in adult articular cartilage cultured for 6 days in the presence and absence of 20 ng of IGF-I/ml and shown to be 24 min under both sets of conditions. The half-life of the mRNA pool coding for aggrecan was also determined and shown to be approx. 4 h in cartilage maintained in culture with or without IGF-I. The pool size of mRNA coding for aggrecan core protein increased 5-6-fold in cartilage explants maintained in culture in medium containing 20% (v/v) fetal-calf serum; however, in tissue maintained with medium containing IGF-I there was no increase in the cellular levels of this mRNA. This suggests that aggrecan synthesis is stimulated by IGF-I at the level of translation of mRNA coding for the core protein of this proteoglycan and that other growth factors are present in serum that stimulate aggrecan synthesis at the level of transcription of the core-protein gene. Inclusion of serum or IGF-I in the medium of cartilage explant cultures induced increases in the amounts of mRNA coding for type II collagen and link protein, whereas only serum enhanced the amount of mRNA for the core protein of decorin.

Aggrecans↗
PubMed1995

Development of a cleavage-site-specific monoclonal antibody for detecting metalloproteinase-derived aggrecan fragments: detection of fragments in human synovial fluids.

We have developed a monoclonal antibody AF-28 that specifically recognizes a neo-epitope on polypeptides with N-terminal FFGVG ... sequences. This sequence is found at the N-terminus of aggrecan fragments that have been digested with matrix metalloproteinases (MMPs). By immunoblotting, monoclonal antibody AF-28 specifically detected G2 fragments derived from an aggrecan G1-G2 substrate digested with stromelysin, collagenase, gelatinase and matrilysin, but failed to detect G2 fragments obtained from elastase, trypsin or cathepsin B digests. Undigested G1-G2 was not detected. In addition, AF-28 antibody detected fragments derived from whole aggrecan and this detection did not require prior treatment with chondroitinase or keratanase. Competition experiments confirmed that peptides containing internal ... FFGVG ... sequences were not detected by the antibody, while native MMP-digested aggrecan fragments and a synthetic 32-mer peptide with FFGVG ... N-termini were equally competitive on a molar basis. An FFGVG 5-mer, and an FGVGGEEDI9-mer which lacked the N-terminal phenylalanine residue, were 50 times and 230 times respectively less competitive than the FFGVG ... 32-mer. Two fragments from the interglobular domain, F342-F373 and F342-D441, that are predicted products of G1-G2 digestion by neutrophil collagenase but have not previously been detected, could be detected with AF-28. The epitope recognized by AF-28 was also detected in human synovial fluids by Western blot analysis. A broad band of 100-200 kDa was detected in some patients and a dominant band of 40-60 kDa was found in two patients. The size of this small fragment corresponds with that seen for the porcine F342-E373 product and may represent the natural physiological product of aggrecan cleaved in vivo at both the MMP site (... DIPEN341 decreases F342FGVG ...) and the aggrecanase site (... ITEGE373 decreases A374RGSVI ...).

Aggrecans↗
PubMed1996

Quantification of aggrecan and link-protein mRNA in human articular cartilage of different ages by competitive reverse transcriptase-PCR.

A competitive reverse transcriptase-PCR (RT-PCR) assay has been developed for the quantification of particular mRNA species in human articular cartilage. Competitor RNA species were synthesized that differed from the amplified target sequence only by the central insertion of an EcoRI restriction site. By using known amounts of synthetic target and competitor RNA, it was shown that competitor RNA molecules designed in this way are reverse-transcribed and amplified with equal efficiency to the target of interest. Furthermore quantification could be performed during the plateau phase of the PCR, which was necessary when using ethidium bromide fluorescence as a detection system. The inhibition of aggrecan and link-protein mRNA expression by interleukin 1 or tumour necrosis factor in monolayers of human articular chondrocytes quantified by this competitive RT-PCR method compared favourably with Northern hybridization studies. The main advantage of this technique is that it can be used to quantify levels of mRNA with RNA extracted directly from 100 mg wet weight of human articular cartilage. Age-related changes in aggrecan and link-protein mRNA were therefore quantified in human articular cartilage directly after dissection from the joint. The concentration of link-protein mRNA was higher in immature cartilage than in mature cartilage when expressed relative to the amount of glyceraldehyde-3-phosphate dehydrogenase mRNA, but no age-related changes were observed in aggrecan mRNA expression. The ratio of aggrecan to link-protein mRNA was higher in mature cartilage than in immature tissue. These age-related differences in the molecular stoichiometry of aggrecan and link-protein mRNA might have implications with respect to the regulation of the formation and the stability of the proteoglycan aggregates in cartilage.

Aggrecans↗
PubMed2003

In situ hybridization and immunohistochemistry of versican, aggrecan and link protein, and histochemistry of hyaluronan in the developing mouse limb bud cartilage.

We investigated the expression pattern of versican, aggrecan, link protein and hyaluronan in the developing limb bud cartilage of the fetal mouse using in situ hybridization and/or immunohistochemistry. Versican mRNA and immunostaining were detected in the mesenchymal cell condensation of the future digital bone at E13. Versican mRNA expression rapidly disappeared from the tibial cartilage, as cartilage formation progressed during E13-15, but the immunostaining was gradually replaced by aggrecan immunostaining from the diaphysis. Immunostaining for both molecules thus had a 'nega-posi' pattern and consequently versican immunostaining was still detected at the epiphyseal end at E15. This result indicated that versican functions as a temporary framework in newly formed cartilage matrix. An aggrecan-positive region within the cartilage invariably had intense hyaluronan staining, whereas a versican-positive region also had affinity for hyaluronan within the cartilage, but not in the mesenchymal cell condensation. Therefore, the presence of versican aggregates was not confirmed in the developing limb bud cartilage. Furthermore, although link protein was more closely related with aggrecan than versican during limb bud cartilage formation, there was a discrepancy between the expression of aggrecan and link protein in tibial cartilage at E15. In particular, only a link protein-positive region was present in the marginal area of the metaphysis and the epiphysis at this stage. This finding may indicate a novel role for link protein.

Aggrecans↗
PubMed1996

Interaction of cartilage matrix protein with aggrecan. Increased covalent cross-linking with tissue maturation.

Cartilage matrix protein (CMP) is a trimeric protein present in many types of cartilage extracellular matrix. It has recently been purified under native conditions that allowed the proposal of a structural model (Hauser, N., and Paulsson, M. (1994) J. Biol. Chem. 269, 25747-25753). To examine the functional properties of CMP we studied its interaction with aggrecan within cartilage extracellular matrix. Aggrecan-enriched fractions were purified from bovine tracheal cartilage of different ages under nondenaturing and denaturing conditions, respectively, and characterized by a combination of biochemical methods and electron microscopy. The fractions contained a pool of CMP noncovalently associated with aggrecan as well as a pool of CMP that appears covalently cross-linked to the aggrecan core protein. Only about two thirds of the CMP subunits could be released even upon reduction under denaturing conditions. It appears that CMP is attached by a nonreducible covalent interaction of one of its subunits with the protein core. The amount of CMP strongly bound to aggrecan increases with age. Electron microscopy revealed interaction sites for CMP in the extended chondroitin-sulfate attachment domain E2. In old tissue five distinct binding sites for CMP were found while in young cartilage only three of these were occupied. The extent of decoration of E2 with CMP increases with age.

Aggrecans↗
PubMed2001

cis elements that control the expression of chick aggrecan.

Aggrecan is a large chondroitin sulfate proteoglycan whose expression is both cell-specific and developmentally regulated. Cloning and sequencing of the 1.8-kilobase genomic 5'-flanking sequence of the chick aggrecan gene revealed the presence of potential tissue-specific control elements including a consensus sequence found in the cartilage-associated silencers, CSIIS1 and CSIIS2, that were first characterized in the type II collagen promoter sequences, as well as numerous other cis elements. Transient transfections of chick sternal chondrocytes and fibroblasts with reporter plasmids bearing progressively deleted portions of the chick aggrecan promoter and enhancer region demonstrated cell type-specific promoter activity and identified a 420-base pair region in the genomic 5-flanking region responsible for negative regulation of the aggrecan gene. In this report, three complementary methods, DNase I footprinting assays, transient transfections, and electrophoretic mobility shift assays (EMSA), provided an integral approach to better understand the regulation of the aggrecan gene. DNase I footprinting revealed that six regions of this genomic sequence bind to nuclear proteins in a tissue-specific manner. Transient transfection of reporter constructs bearing ablations of these protected sequences showed that four of the six protected sequences, which contain the sequence TCCTCC or TCCCCT, had repressor activities in transfected chick chondrocytes. Cross-competition EMSA using nuclear protein extracted from chondrocytes or fibroblasts explored the contributions of the different sequence elements in formation of DNA-protein complexes specific to cell type. This is the first parallel examination of the EMSA patterns for six functionally defined cis elements with highly similar sequences, using protein from primary cultured cells.

5' Untranslated Regions↗
PubMed2002

Cleavage of the carboxyl tail from the G3 domain of aggrecan but not versican and identification of the amino acids involved in the degradation.

Aggrecan, a major structural proteoglycan in cartilage, contains three globular domains, G1, G2, and G3, as well as sequences for glycosaminoglycan modification. A large number of proteases are implicated in aggrecan cleavage in normal metabolism, aging, and arthritis. These proteases are known to cleave at the IGD, KS, and CS domains. Here we report for the first time evidence of cleavage at a novel site, the carboxyl tail of aggrecan. Results from deletion mutants of the tail indicated that the likely cleavage sites were two consensus sequences, RRLXK and RSPR, present in the aggrecan analogs of many species. This was confirmed by site-directed mutagenesis. A construct containing two G3 domains (G3G3) was also found to cleave between the G3 duplicates. When G3 tail was linked to a glycosaminoglycan-modifying sequence, it was protected from cleavage. Furin inhibitor also reduced the levels of tail cleavage. The carboxyl tails of chicken and human versican were not cleaved, despite the presence of the consensus sequence. Our studies indicate that the basic amino acids present in the tail play an important role in cleavage, and this mechanism is specific to aggrecan.

Aggrecans↗
PubMed2004

Link protein has greater affinity for versican than aggrecan.

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

Aggrecans↗
PubMed2006

Aggrecan turnover in human intervertebral disc as determined by the racemization of aspartic acid.

We have used the racemization of aspartic acid as a marker for the "molecular age" of aggrecan components of the human intervertebral disc matrix (aggregating and non-aggregating proteoglycans as well as the different buoyant density fractions of aggrecan). By measuring the D/L(Asp) ratio of the various aggrecan species as a function of age and using the values of the racemization constant, k(i), found earlier for aggrecan in articular cartilage, we were able to establish directly the relative residence time of these molecules in human intervertebral disc matrix. For A1 preparations taken from normal tissue, turnover rates of 0.059 +/- 0.01 and 0.063 +/- 0.01/year correspond to half-life values of 12 +/- 2.0 and 11.23 +/- 1.9 years for nucleus pulposus and annulus fibrosus, respectively; the turnover rates of 0.084 +/- 0.022 and 0.092 +/- 0.034/year for degenerate tissue correspond to half-life values of 8.77 +/- 2.2 and 8.41 +/- 2.8 years, suggesting increased rate of removal of small aggrecan fragments. For the large monomer, fraction A1D1, turnover is 0.13 +/- 0.04/year, corresponding to a half-life of 5.56 +/- 1.58 years, similar to 3.4 years in human articular cartilage. For the binding region (A1D6), turnover is 0.033 +/- 0.0012/year, corresponding to a half-life of 21.53 +/- 0.6 years, similar to 23.5 years in articular cartilage. A1 preparations from nucleus pulposus contain a lower proportion of aggregating proteoglycans as compared with annulus fibrosus, suggesting increased proteolytic modification in the nucleus pulposus. D/L(Asp) values in aggregating and non-aggregating proteoglycans of a 24-year-old individual show similar results, suggesting that the non-aggregating molecules are synthesized initially as aggregating proteoglycans, which thereafter undergo cleavage and detachment from hyaluronan.

Adolescent↗