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At least 19 recordsLinked to original sources
PubMed1992

High pressure gel-permeation assay for the proteolysis of human aggrecan by human stromelysin-1: kinetic constants for aggrecan hydrolysis.

The adaptation of an analytical procedure for aggrecan based upon gel-permeation chromatography to an FPLC-based protocol has significantly sped up the analysis. The faster assay has permitted determination of the kinetic constants for digestion of human aggrecan by human stromelysin-1. Monomeric aggrecan appeared to be hydrolyzed by stromelysin-1 to multiple forms with lower molecular weight. The disappearance of high-molecular-weight aggrecan was first-order, showing Km much larger than 2 microM and kc/Km = 4000 M-1 s-1 at pH 7.5. The disappearance of high-molecular-weight aggrecan upon hydrolysis by stromelysin-1 at pH 5.5 was also first-order, with kc/Km = 10,700 M-1 s-1. The disappearance of high-molecular-weight aggrecan at pH 7.5 was first-order for digestion by human leukocyte elastase with kc/Km = 230,000 M-1 s-1, by human cathepsin G with kc/Km = 4200 M-1 S-1, and by human plasma plasmin with kc/Km = 2800 M-1 s-1, all with Km much larger than 2 microM.

Aggrecans↗
PubMed1992

Molecular cloning of chicken aggrecan. Structural analyses.

The large, aggregating chondroitin sulphate proteoglycan of cartilage, aggrecan, has served as a generic model of proteoglycan structure. Molecular cloning of aggrecans has further defined their amino acid sequences and domain structures. In this study, we have obtained the complete coding sequence of chicken sternal cartilage aggrecan by a combination of cDNA and genomic DNA sequencing. The composite sequence is 6117 bp in length, encoding 1951 amino acids. Comparison of chicken aggrecan protein primary structure with rat, human and bovine aggrecans has disclosed both similarities and differences. The domains which are most highly conserved at 70-80% identity are the N-terminal domains G1 and G2 and the C-terminal domain G3. The chondroitin sulphate domain of chicken aggrecan is smaller than that of rat and human aggrecans and has very distinctive repeat sequences. It has two separate sections, one comprising 12 consecutive Ser-Gly-Glu repeats of 20 amino acids each, adjacent to the other which has 23 discontinuous Ser-Gly-Glu repeats of 10 amino acids each; this latter region, N-terminal to the former one, appears to be unique to chicken aggrecan. The two regions contain a total of 94 potential chondroitin sulphate attachment sites. Genomic comparison shows that, although chicken exons 11-14 are identical in size to the rat and human exons, chicken exon 10 is the smallest of the three species. This is also reflected in the size of its chondroitin sulphate coding region and in the total number of Ser-Gly pairs. The putative keratan sulphate domain shows 31-45% identity with the other species and lacks the repetitive sequences seen in the others. In summary, while the linear arrangement of specific domains of chicken aggrecan is identical to that in the aggrecans of other species, and while there is considerable identity of three separate domains, chicken aggrecan demonstrates unique features, notably in its chondroitin sulphate domain and its keratan sulphate domain. Thus different variants of chondroitin sulphate and keratan sulphate domains may have evolved separately to fulfil specific biochemical and physiological functions.

Aggrecans↗
PubMed1992

Aggrecan core protein is expressed in membranous bone of the chick embryo. Molecular and biomechanical studies of normal and nanomelia embryos.

The recessive mutation nanomelia blocks the synthesis of a large aggregating proteoglycan (aggrecan) by avian embryo chondrocytes. Lack of aggrecan is associated with short stature, multiple morphological defects in cartilage, and embryo lethality. Bony defects have also been described, but were assumed to be a secondary consequence of the cartilage defect. However, two lines of evidence presented in this paper indicate that the aggrecan deficiency directly affects intramembranous bone. First, the morphology (i.e. projected area and shape) of certain membranous bones of nanomelia embryos was abnormal. Second, membranous bone from nanomelia embryos proved to be significantly stiffer in biomechanical tests that measured functional properties of the extracellular matrix. These findings were unexpected because intramembranous bones normally develop from mesenchyme and not from a cartilage intermediate, and they prompted a search for evidence of aggrecan expression in the bone of normal chick embryos. We report that: 1) aggrecan mRNA was identified by PCR analysis of total RNA isolated from day-13 chick embryo calvarium, 2) the PCR method successfully amplified aggrecan mRNA from primary chick embryo osteoblasts in culture, 3) in situ hybridization of membranous bone tissue sections demonstrated aggrecan expression by chick embryo osteoblasts in vivo, and 4) the aggrecan message was identified in Northern blots of calvarial mRNA probed at high stringency. The results of the molecular and biomechanical studies provide evidence that aggrecan is indeed expressed in membranous bone as well as cartilage. Altogether, these results suggest that aggrecan may contribute to the functional properties and the normal growth and development of avian membranous bone.

Aggrecans↗
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↗
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↗
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↗
PubMed1992

Monoclonal antibodies directed against epitopes within the core protein structure of the large aggregating proteoglycan (aggrecan) from the swarm rat chondrosarcoma.

The core protein of the large hyaline cartilage proteoglycan, aggrecan, is composed of six distinct domains: globular 1 (G1), interglobular, globular 2 (G2), keratan sulfate attachment, chondroitin sulfate (CS) attachment, and globular 3 (G3). Monoclonal antibodies that recognize epitopes in these domains were raised against Swarm rat chondrosarcoma aggrecan that was either denatured through reduction and alkylation or partially deglycosylated through chondroitinase ABC digestion or alkali elimination, the latter with or without sulfite addition. Monoclonal antibodies were further characterized for reactivity to purified aggrecan substructures including rat chondrosarcoma G1 and CS attachment domains, a recombinant rat chondrosarcoma G3 domain fusion protein, bovine articular cartilage G2 domain, and rat chondrosarcoma link protein (LP). Biochemical characterization of the specificities of these monoclonal antibodies indicated that one (1C6) recognized an epitope shared by both the G1 and the G2 domains; one (5C4) recognized an epitope shared by both LP and the G1 domain; one (7D1) recognized an epitope shared by both the G1 and the CS attachment domains; two (14A1 and 15B2) recognized epitopes in the CS attachment domain; one (14B4) recognized an epitope in the G3 domain; and one (13D1) recognized a ubiquitous epitope shared by the G1, G2, G3, and CS attachment domains of aggrecan and also LP. Collectively the specificities of these antibodies confirm the occurrence of multiple repeated epitopes (both carbohydrate and protein in nature) throughout the different domain structures of aggrecan. These antibodies have been proven to be useful for identifying aggrecan-like molecules in several connective tissues other than cartilage.

Aggrecans↗
PubMed1992

Variability in the G3 domain content of bovine aggrecan from cartilage extracts and chondrocyte cultures.

The content of the globular domains G1, G2 and G3 on the core protein of high-density (A1D1) aggrecan isolated from newborn and mature bovine cartilage and from cultures of bovine chondrocytes was examined. Quantitation based on the 220 nm absorbance of tryptic marker peptides from each domain isolated by reversed-phase HPLC showed that while the content of G1 and G2 was essentially the same for all samples, the content of G3 varied markedly. The molar yield of G3 and G1 marker peptides indicated that approximately 55% of the G1-bearing aggrecan from immature cartilage carried the G3 domain, while for mature cartilage this figure was markedly reduced, at about 35%. Aggrecan prepared from the cell layer matrix of calf chondrocyte cultures had an apparent G3 content similar to newborn cartilage (55%), whereas aggrecan prepared from the medium of these cultures had a markedly higher G3 content, at about 80%. The high content of G3 in cell medium samples compared to cartilage extracts was supported by electron microscopic analysis of A1D1 preparations. The G3 content of the two subpopulations of aggrecan present in mature cartilage and separable by flat bed agarose gel electrophoresis was also determined at about 45% (Band I) and 20% (Band II) respectively. These results are discussed in terms of the likely origin of the marked variability in the G3 domain content of aggrecan.

Aggrecans↗
PubMed1992

Localization of the expression of type I, II, III collagen, and aggrecan core protein genes in developing human articular cartilage.

The expression of mRNAs for collagen types I, II, III and for aggrecan core protein was studied in developing human femoral cartilage by in situ hybridization, with special attention given to the cartilage covered by the perichondrium and to the articular surface. In parallel, the synthesis of the related proteins was monitored by immunohistochemistry. The cells metabolically active for type I and type III collagen expression were identified by hybridization using [32P]-labeled cDNA clones coding for human alpha 1(I) and alpha 1(III), respectively. Type II collagen and core protein mRNAs were detected by hybridization with specific [32P]-labeled oligonucleotide probes. In the femoral heads of one 22-week old fetus and of one newborn, our in situ hybridization and immunohistochemical analysis revealed that chondrocytes located immediately subjacent to the perichondrium produced collagen types I, II, III as well as aggrecan; whereas only type II collagen and aggrecan gene expression was detected deeper in the cartilage covered by the perichondrium. This observation supports the hypothesis that the inner cell layers of perichondrium are chondrogenic, with a transient state where cells express all the markers studied here. At the articular surface different patterns of expression were observed at the two developmental stages. After 22 weeks of fetal development only collagen types I and III were expressed by the surface zone cells while in the newborn cartilage, these cells expressed all the molecules studied (collagen types I, II, III and cartilage proteoglycan). At both ages the underlying cartilage cells expressed only the cartilage-specific molecules (type II collagen and aggrecan). Thus a progressive transformation of cartilaginous matrix occurs with time from the deep cartilage up to the surface by addition of new components, i.e. aggrecan and type II collagen. These results supplemented by an immunofluorescence analysis on 20-, 26- and 38-week old fetal femoral heads suggest that expression of collagen and aggrecan in the cartilage covered by the perichondrium and in the cartilage at the articular surface are subject to different regulatory mechanisms during development. Furthermore, the appearance of hybridizable core protein and type II collagen mRNAs at the articular surface, closely followed by the appearance of the proteins for which they code, indicates that core protein and type II collagen expression is regulated primarily at the transcriptional level in this region. Finally, the similar topography observed for the expression of these two proteins suggests that the genes for these two major constituents of cartilage matrix are coordinately regulated during growth of articular cartilage.

Aggrecans↗
PubMed1991

Distribution of cartilage proteoglycan (aggrecan) core protein and link protein gene expression during human skeletal development.

The distribution of cartilage proteoglycan core protein (aggrecan) and cartilage proteoglycan link protein was investigated by in situ hybridization during different stages of human skeletal development. Aggrecan and link protein expression were confined to chondrocytes of the developing skeleton and other cartilaginous structures. Distribution and intensity of the signal was identical with aggrecan as compared to link protein probes. Parallel to the calcification of cartilaginous matrix, chondrocytes of this area lost the expression of aggrecan and link protein specific mRNA and stayed negative throughout the following stages of skeletal development. Highest expression was found in the lower proliferative and upper hypertrophic zone whereas the resting zone showed less expression. Aggrecan gene expression was additionally investigated in iliac crest biopsies of 3 patients with pseudoachondroplasia and compared to age-matched controls. Distribution and intensity of staining revealed no abnormalities. Thus, the phenotypic changes during chondrocyte maturation are accompanied by distinct changes in aggrecan and link protein gene expression. This pattern was maintained in the growth plate of patients with pseudoachondroplasia.

Aggrecans↗
PubMed1992

The structure of aggrecan fragments in human synovial fluid. Evidence for the involvement in osteoarthritis of a novel proteinase which cleaves the Glu 373-Ala 374 bond of the interglobular domain.

Synovial fluid was collected from patients with recent knee injury and from patients with early or late stage osteoarthritis. Chondroitin sulfate-substituted aggrecan fragments present in these fluids, and in normal bovine synovial fluid, were purified by cesium chloride gradient centrifugation, enzymically deglycosylated and fractionated by gel filtration on Superose-12. Each sample contained two major aggrecan core protein populations with apparent molecular masses of approximately 90 kD and 150 kD. For all samples, NH2-terminal analysis of both populations gave a single major sequence beginning ARGSV. This NH2 terminus results from cleavage of the human aggrecan core protein at the Glu 373-Ala 374 bond within the interglobular domain between the G1 and G2 domains. Cleavage at this site also occurs during control and interleukin-1 stimulated aggrecan catabolism in bovine cartilage explant cultures (Sandy, J., P. Neame, R. Boynton, and C. Flannery. 1991. J. Biol. Chem. 266:8683-8685). These results indicate that the major aggrecan fragments present in both osteoarthritic human synovial fluid and in normal bovine synovial fluid are large, being composed of a short NH2-terminal stretch of the interglobular domain, the G2 domain, the keratan sulfate domain, and variable lengths of the chondroitin sulfate domain(s). We conclude that the release of aggrecan fragments from articular cartilage into the synovial fluid seen at all stages of human osteoarthritis (Lohmander, L. S. 1991. Acta Orthop. Scand. 62:623-632) is promoted by the action of a normal cartilage proteinase which cleaves the Glu 373-Ala 374 bond of the interglobular domain.

Aggrecans↗
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↗
PubMed1992

The high molecular weight Cat-301 chondroitin sulfate proteoglycan from brain is related to the large aggregating proteoglycan from cartilage, aggrecan.

Monoclonal antibodies Cat-301 and Cat-304 recognize a neuronal cell surface-associated chondroitin sulfate proteoglycan (CSPG), which is expressed during critical periods of postnatal development in the mammalian central nervous system (CNS). In the present study we show that the CNS CSPG identified by Cat-301/304 is similar to aggrecan, the high molecular weight CSPG from cartilage. By Western blot analysis, cartilaginous tissues, which are rich sources of aggrecan, have a high concentration of a high molecular weight CSPG which is immunoreactive with Cat-301 and 304. The Cat-301 and 304 epitopes, however, are partially masked by chondroitin sulfate glycosamino-glycan and are unmasked by digestion of the antigen with chondroitinase ABC. Although the antigen from both cartilage and CNS can be purified by CsCl buoyant density gradient centrifugation, a standard technique for purifying aggrecan, most of the antigen from the CNS has a lower buoyant density than that of cartilage. This may be due, in part, to the paucity of keratan sulfate substitution on the CNS antigen compared with that of the cartilage antigen. Both the CNS and cartilage antigens bind to hyaluronic acid, a feature characteristic of aggrecan. The physiochemical, biochemical, and functional properties of the Cat-301/304 antigen from cartilage are identical to aggrecan. The CNS antigen is similar, but not identical, to the cartilage antigen, and may thus represent another member of the family of high molecular weight CSPGs which bind to and aggregate with hyaluronic acid.

Aggrecans↗
PubMed1991

Complete coding sequence and deduced primary structure of the human cartilage large aggregating proteoglycan, aggrecan. Human-specific repeats, and additional alternatively spliced forms.

We have obtained the complete coding sequence of the large aggregating chondroitin sulfate proteoglycan of human cartilage (aggrecan) from a combination of cDNA and genomic exon sequencing. We screened a human costal chondrocyte cDNA library, using rat aggrecan cDNA probes, and obtained three nonoverlapping clones totaling 6.2 kilobases in length. These clones were sequenced, and the sequence of the gaps between clones was obtained from genomic exon fragments and polymerase chain reaction-amplified cDNA. The composite sequence is 7137 nucleotides long, encoding 2316 amino acids. The human and rat aggrecan amino acid sequences are about 75% identical, with domains ranging from 100% to about 60% of conserved amino acids. The human sequence contains two regions of highly conserved repeats not found in rat aggrecan: 11 repeats of a hexameric sequence in the keratan sulfate attachment domain, E-E-P-(S,F)-P-S; and a 19-amino acid sequence reiterated 19 times, in the CS-1 portion of the serine-glycine-containing region. There are at least three forms of aggrecan transcripts, generated by alternative exon usage, and the form reported here is the shortest and also the most prevalent, lacking both the epidermal growth factor-like domain, and the complement regulatory protein-like sequence.

Aggrecans↗
PubMed1992

The interglobular domain of cartilage aggrecan is cleaved by PUMP, gelatinases, and cathepsin B.

The action of three matrix metalloproteinases (MMPs), 72- and 95-kDa gelatinases (MMP-2 and MMP-9) and PUMP (MMP-7), and a cysteine proteinase, cathepsin B, were investigated on aggrecan the major proteoglycan of cartilage. All the enzymes cleaved aggrecan although the activity of the 95-kDa gelatinase was very low. Specific cleavage sites were investigated following incubation with a purified aggrecan G1-G2 domain fragment (150 kDa). Both gelatinases produced 110-kDa G2 and 56-kDa G1 products by a single cleavage at an Asn-Phe bond within the interglobular domain close to the G1 domain. This was similar to the action of stromelysin (MMP-3) (Fosang, A. J., Neame, P. J., Hardingham, T. E., Murphy, G., and Hamilton, J. A. (1991) J. Biol. Chem. 266, 15579-15582). Cathepsin B also produced two fragments from a single cleavage at a Gly-Val bond only three amino acids C-terminal to the metalloproteinase cleavage site. PUMP cleaved at the metalloproteinase Asn-Phe site, but in addition produced a low yield of a smaller G2 fragment (56 kDa) corresponding to cleavage between Asp441 and Leu442 (human sequence), within the interglobular domain, close to the G2 domain. The apparent difference in size between the two G2 fragments released by PUMP (110 and 56 kDa) was much greater than predicted from the peptide length between the cleavage sites (100 amino acids). However, keratanase digestion greatly reduced the size of the 110-kDa G2 fragment, while producing only a small reduction in size of the 56-kDa product, showing that there was approximately 30-40 kDa of keratan sulfate attached to the interglobular domain between the PUMP cleavage sites. This new structural information on aggrecan may account for the previously observed stiffness of the interglobular domains when viewed by rotary shadowing electron microscopy (Paulsson, M., Morgelin, M., Wiedemann, H., Beardmore-Gray, M., Dunham, D. G., Hardingham, T. E., Heinegard, D., Timpl, R., and Engel, J. (1987) Biochem. J. 245, 763-772). These results show that in spite of a high keratan sulfate content the interglobular domain provides important sites for cleavage by different proteinases, including several members of the matrix metalloproteinase family.

Aggrecans↗
PubMed1992

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

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

Aged↗
PubMed1991

Catabolism of aggrecan in cartilage explants. Identification of a major cleavage site within the interglobular domain.

The catabolism of aggrecan has been studied in calf articular cartilage explant cultures. The chondroitin sulfate-rich, high buoyant density products that accumulate in culture medium have been purified, and NH2-terminal sequence data have been obtained. Aggrecan released from the tissue in the presence or absence of interleukin-1 alpha, whether analyzed before or after reduction and alkylation, exhibited only one major and one minor NH2-terminal sequence. The major sequence, ARGXVILXAKPDF, shows very high similarity to a region of the interglobular domain (between the G1 and G2 domains) of both human and rat aggrecan. The minor sequence, VEVS, was that previously described for the NH2 terminus of the intact core protein. These results indicate that catabolism of aggrecan in cartilage explants involves proteolytic cleavage within a conserved region of the interglobular domain and that this results in the separation of the G1 domain from the remainder of the molecule. A major product of this process is a large nonaggregating species that consists of an NH2-terminal sequence beginning with ARG (and composed of about 100 residues of the interglobular domain) that is attached to an intact G2 domain followed by an extended section of the chondroitin sulfate-bearing domain toward the COOH terminus.

Aggrecans↗
PubMed1991

Increased aggrecan (cartilage proteoglycan) production in the sclera of myopic chicks.

A previously characterized chick model of myopia was used to evaluate biochemical changes in the sclera which are associated with ocular enlargement and myopia. Chicks were monocularly occluded for 10 days and the DNA, hydroxyproline, and glycosaminoglycan contents of the sclera were compared between the normal and the myopic eyes. No significant differences could be detected in total DNA or hydroxyproline content. There was, however, a 34% increase in glycosaminoglycans and a 20.7% decrease in cell density within the posterior sclera of myopic eyes. The biosynthesis of scleral proteoglycans was determined by measuring 35SO4 incorporation in the sclera of chicks visually occluded for 5, 10, and 15 days. No differences could be detected in 35SO4 incorporation into the cornea or the anterior sclera. However, 35SO4 incorporation was significantly increased in the posterior sclera of myopic eyes by 64% at Day 5, 39% at Day 10, and 49% at Day 15. When fractionated on Sepharose CL-4B, scleral proteoglycans were resolved into two peaks which were identified by Western blot analysis as aggrecan (cartilage proteoglycan) and decorin. Furthermore, Western blot and dot blot analyses indicated that significantly more aggrecan core protein was present in the sclera of myopic eyes compared with equivalent amounts of sclera from control eyes. These results indicate that increased synthesis and accumulation of aggrecan, which increases the volume of extracellular matrix in the posterior sclera, are responsible for the ocular enlargement observed in this model of myopia.

Aggrecans↗