scholarly journals Turnover of proteoglycans in articular-cartilage cultures. Characterization of proteoglycans released into the medium

1989 ◽  
Vol 259 (1) ◽  
pp. 21-25 ◽  
Author(s):  
M A Campbell ◽  
C J Handley ◽  
S E D'Souza
Keyword(s):  
Articular Cartilage ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Keratan Sulphate ◽  
Bovine Articular Cartilage ◽  
Definitive Evidence ◽  
Core Proteins ◽  
The Matrix

By using an e.l.i.s.a. method it was demonstrated that the majority of proteoglycans released into the medium of both control and retinoic acid-treated explant cultures of bovine articular cartilage did not contain a hyaluronate-binding region. This supports our previous findings [Campbell & Handley (1987) Arch. Biochem. Biophys. 258, 143-155] that proteoglycans released into the medium of both cultures were of smaller hydrodynamic size, more polydisperse and unable to form aggregates with hyaluronate. Analysis of 35S-labelled core proteins associated with proteoglycans released into the medium of both cultures by using SDS/polyacrylamide-gel electrophoresis and fluorography indicated the presence of a series of core-protein bands (Mr approx. 300,000, 230,000, 215,000, 200,000, 180,000, 140,000, 135,000, 105,000, 85,000 and 60,000) compared with three core proteins derived from the proteoglycans remaining in the matrix (Mr 300,000, 230,000 and 215,000). Further analysis of the core proteins released into the medium indicated that the larger core proteins associated with medium proteoglycans contain both chondroitin sulphate and keratan sulphate glycosaminoglycans whereas the smaller core proteins contain only chondroitin sulphate chains. These experiments provide definitive evidence that the loss of proteoglycans from the matrix involves proteolytic cleavage at various sites along the proteoglycan core protein.

scholarly journals Separation and characterization of two populations of aggregating proteoglycans from cartilage

1985 ◽  
Vol 225 (1) ◽  
pp. 95-106 ◽  
Author(s):  
D Heinegård ◽  
J Wieslander ◽  
J Sheehan ◽  
M Paulsson ◽  
Y Sommarin
Keyword(s):  
Chondroitin Sulphate ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Rich Region ◽  
Keratan Sulphate ◽  
Core Proteins ◽  
Lower Mobility ◽  
Cartilage Proteoglycans

Intermediary gel immunoelectrophoresis was used to show that purified aggregating cartilage proteoglycans from 2-year-old steers contain two distinct populations of molecules and that only one of these is immunologically related to non-aggregating cartilage proteoglycans. The two types of aggregating proteoglycans were purified by density-gradient centrifugation in 3.5M-CsCl/4M-guanidinium chloride and separated by zonal rate centrifugation in sucrose gradients. The higher-buoyant-density faster-sedimenting proteoglycan represented 43% of the proteoglycans in the extract. It had a weight-average Mr of 3.5 × 10(6), did not contain a well-defined keratan sulphate-rich region, had a quantitatively dominant chondroitin sulphate-rich region and contained 5.9% protein and 23% hexosamine. The lower-buoyant-density, more slowly sedimenting, proteoglycan represented 15% of the proteoglycans in the extract. It had a weight-average Mr of 1.3 × 10(6), contained both the keratan sulphate-rich and the chondroitin sulphate-rich regions and contained 7.3% protein and 23% hexosamine. Each of the proteoglycan preparations showed only one band on agarose/polyacrylamide-gel electrophoresis. The larger proteoglycan had a lower mobility than the smaller. The distribution of chondroitin sulphate chains along the chondroitin sulphate-rich region was similar for the two types of proteoglycans. The somewhat larger chondroitin sulphate chains of the larger proteoglycan could not alone account for the larger size of the proteoglycan. Peptide patterns after trypsin digestion of the proteoglycans showed great similarities, although the presence of a few peptides not shared by both populations indicates that the core proteins are partially different.

scholarly journals Characterization of proteoglycans isolated from associative extracts of human articular cartilage

1993 ◽  
Vol 293 (1) ◽  
pp. 165-172 ◽  
Author(s):  
V Vilím ◽  
A J Fosang
Keyword(s):  
Articular Cartilage ◽  
Molecular Mass ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Gel Chromatography ◽  
Human Articular Cartilage ◽  
Keratan Sulphate ◽  
Core Proteins ◽  
Sds Page

Approx. 10% of the total proteoglycan content of normal young human articular cartilage was extracted under associative conditions with Dulbecco's PBS. Proteoglycans isolated from the extract by Q-Sepharose chromatography were separated by gel chromatography and characterized by gradient gel SDS/PAGE and immunoblotting. Three species of small proteoglycans, two main populations of aggrecan and a population of its smaller fragments were identified. The major populations of aggrecan contained chondroitin sulphate chains, all or part of the N-terminal G1 and G2 domains and, therefore, intact keratan sulphate domains. The larger population was estimated by gradient SDS/PAGE to have a molecular mass of approx. 600 kDa or greater. The second population had an apparent molecular mass of approx. 300-600 kDa. Core proteins derived from these populations of proteoglycans separated on SDS/PAGE into several clusters of bands in the range from 120 to approx. 360 kDa. The extract further contained smaller fragments which lacked chondroitin sulphate but reacted with antibodies against keratan sulphate, and against epitopes present in the G2 domain of aggrecan. The presence of the G2 domain in a broad range of populations of decreasing size indicated extensive cleavage of the aggrecan core protein within its chondroitin sulphate domain. These findings suggest that fragmentation of aggrecan probably occurs in vivo in normal articular cartilage of young individuals. Associative extracts also contained decorin, biglycan and fibromodulin. These were resolved from aggrecan by gel chromatography and identified by immunodetection.

scholarly journals A novel keratan sulphate domain preferentially expressed on the large aggregating proteoglycan from human articular cartilage is recognized by the monoclonal antibody 3D12/H7

1996 ◽  
Vol 318 (3) ◽  
pp. 1051-1056 ◽  
Author(s):  
Dagmar-Christiane FISCHER ◽  
Hans-Dieter HAUBECK ◽  
Kirsten EICH ◽  
Susanne KOLBE-BUSCH ◽  
Georg STÖCKER ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Human Articular Cartilage ◽  
Rich Region ◽  
Keratan Sulphate ◽  
The Core ◽  
Gel Shift Assay ◽  
Chemical Deglycosylation

Monoclonal antibodies (mAbs) were prepared against aggrecan which has been isolated from human articular cartilage and purified by several chromatographic steps. One of these mAbs, the aggrecan-specific mAb 3D12/H7, was selected for further characterization. The data presented indicate that this mAb recognizes a novel domain of keratan sulphate chains from aggrecan: (1) immunochemical staining of aggrecan is abolished by treatment with keratanase/keratanase II, but not with keratanase or chondroitin sulphate lyase AC/ABC; (2) after chemical deglycosylation of aggrecan no staining of the core-protein was observed; (3) different immunochemical reactivity was observed against keratan sulphates from articular cartilage, intervertebral disc and cornea for the mAbs 3D12/H7 and 5D4. For further characterization of the epitope, reduced and 3H-labelled keratan sulphate chains were prepared. In an IEF–gel-shift assay it was shown that the 3H-labelled oligosaccharides obtained after keratanase digestion of reduced and 3H-labelled keratan sulphate chains were recognized by the mAb 3D12/H7. Thus it can be concluded that the mAb 3D12/H7 recognizes an epitope in the linkage region present in, at least some, keratan sulphate chains of the large aggregating proteoglycan from human articular cartilage. Moreover, this domain seems to be expressed preferentially on those keratan sulphate chains which occur in the chondroitin sulphate-rich region of aggrecan, since the antibody does not recognize the keratan sulphate-rich region obtained after combined chondroitinase AC/ABC and trypsin digestion of aggrecan.

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

2004 ◽  
Vol 382 (1) ◽  
pp. 253-259 ◽  
Author(s):  
Hidefumi OSHITA ◽  
John D. SANDY ◽  
Kiichi SUZUKI ◽  
Atsushi AKAIKE ◽  
Yun BAI ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Articular Cartilage ◽  
Chondroitin Sulphate ◽  
Globular Domain ◽  
Cleavage Sites ◽  
Keratan Sulphate ◽  
Bovine Articular Cartilage ◽  
The Matrix ◽  
A Site

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

scholarly journals Dermatan sulphate proteoglycans of human articular cartilage. The properties of dermatan sulphate proteoglycans I and II

1989 ◽  
Vol 262 (3) ◽  
pp. 823-827 ◽  
Author(s):  
P J Roughley ◽  
R J White
Keyword(s):  
Articular Cartilage ◽  
Gel Electrophoresis ◽  
Core Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Human Articular Cartilage ◽  
Amino Acid Residues ◽  
Dermatan Sulphate ◽  
Sulphate Proteoglycan ◽  
Core Proteins

Dermatan sulphate proteoglycans were purified from juvenile human articular cartilage, with a yield of about 2 mg/g wet wt. of cartilage. Both dermatan sulphate proteoglycan I (DS-PGI) and dermatan sulphate proteoglycan II (DS-PGII) were identified and the former was present in greater abundance. The two proteoglycans could not be resolved by agarose/polyacrylamide-gel electrophoresis, but could be resolved by SDS/polyacrylamide-gel electrophoresis, which indicated average Mr values of 200,000 and 98,000 for DS-PGI and DS-PGII respectively. After digestion with chondroitin ABC lyase the Mr values of the core proteins were 44,000 for DS-PGI and 43,000 and 47,000 for DS-PGII, with the smaller core protein being predominant in DS-PGII. Sequence analysis of the N-terminal 20 amino acid residues reveals the presence of a single site for the potential substitution of dermatan sulphate at residue 4 of DS-PGII and two such sites at residues 5 and 10 for DS-PGI.

scholarly journals Articular cartilage cultured with interleukin 1. Increased release of link protein, hyaluronate-binding region and other proteoglycan fragments

1986 ◽  
Vol 238 (2) ◽  
pp. 571-580 ◽  
Author(s):  
A Ratcliffe ◽  
J A Tyler ◽  
T E Hardingham
Keyword(s):  
Articular Cartilage ◽  
Interleukin 1 ◽  
Binding Region ◽  
Rapid Loss ◽  
Keratan Sulphate ◽  
Link Protein ◽  
The Matrix ◽  
The Media ◽  
Average Size

Pig articular cartilage was maintained in culture for 3 days with and without porcine interleukin 1. The proteoglycans remaining in the cartilage and those released into the medium were analysed by using radioimmunoassays for the hyaluronate-binding region, link protein and keratan sulphate. In interleukin 1-treated cultures after 3 days there was 38% release of total glycosaminoglycans into the medium, 18% release of binding region, 14% release of link protein and 20% release of keratan sulphate epitope, whereas in control cultures the proportions released were much less (16, 9, 10 and 7% respectively). Characterization of the proteoglycans in the media after 1.5 days and 3 days of culture showed that interleukin 1 promoted the release of proteoglycan of large average size and also the release of link protein and of low-Mr binding region which was unattached to proteoglycan. Both the link protein and binding region released were able to bind to exogenously added hyaluronate, whereas the proteoglycan in the medium was not. The proteoglycans extracted from cultured cartilage were similar to those from fresh cartilage: they contained a high proportion of aggregating proteoglycans and some low-Mr binding region. The proportion of this binding region extracted from the interleukin 1-treated cartilage was increased. The presence of interleukin 1 in the cultures therefore appeared to increase the rate of proteolytic degradation of proteoglycan in the matrix and to lead to a more rapid loss of intact binding region, of link protein and of large proteoglycan fragments into the medium.

scholarly journals Structure and interactions of proteoglycans in the extracellular matrix produced by cultured human fibroblasts

1985 ◽  
Vol 232 (1) ◽  
pp. 161-168 ◽  
Author(s):  
S Johansson ◽  
K Hedman ◽  
L Kjellén ◽  
J Christner ◽  
A Vaheri ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Human Fibroblasts ◽  
Heparan Sulphate ◽  
Human Articular Cartilage ◽  
Sulphate Proteoglycan ◽  
Fibroblast Cultures ◽  
Core Proteins ◽  
The Matrix

Subconfluent cultures of human embryonic skin fibroblasts were labelled with [35S]sulphate for 3 days, after which cell-free extracellular matrix was isolated. A chondroitin sulphate proteoglycan (CSPG) and a heparan sulphate proteoglycan (HSPG) were purified from the matrix. Chromatography on Sepharose CL-2B gave peak Kav. values of 0.35 and 0.38 respectively for the CSPG and the HSPG. The polysaccharide chains released from the two PGs were of similar size (Kav. 0.50 on Sepharose CL-4B). Approx. 50% of the CSPG showed affinity for hyaluronic acid (HA). However, it differed immunologically from the HA-aggregating CSPG of human articular cartilage, and had a larger core protein (apparent molecular mass 290 kDa) than had the cartilage PG. Neither metabolically [35S]sulphate-labelled PGs, isolated from the medium of fibroblast cultures, nor chemically 3H-labelled polysaccharides (HA, CS, HS and heparin) were incorporated into the extracellular matrix when added to unlabelled cell cultures. These results indicate that the matrix PGs are not derived from the PGs present in the medium and that an interation between polysaccharide chains and matrix components is not sufficient for incorporation of PGs into the matrix. Incubation of cell-free 35S-labelled matrix with unlabelled polysaccharides did not lead to the release of any 35S-labelled material, supporting this conclusion. Furthermore, so-called ‘link proteins’ were not present in the fibroblast cultures, indicating that the CSPGs were anchored in the matrix in a manner different from the link-stabilized association of CSPG with HA in chondrocyte matrix. The identification of a proteinase, secreted by fibroblasts in culture, that after activation with heparin has the ability to release 35S-labelled PGs from the matrix may also indicate that the core proteins are important for the association of the PGs to the matrix.

scholarly journals Independent secretion of proteoglycans and collagens in chick chondrocyte cultures during acute ascorbic acid treatment

1990 ◽  
Vol 272 (1) ◽  
pp. 193-199 ◽  
Author(s):  
M Pacifici
Keyword(s):  
Acid Treatment ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Proteoglycan Synthesis ◽  
Collagen Types ◽  
Keratan Sulphate ◽  
Sulphate Proteoglycan ◽  
Chondrocyte Cultures ◽  
Chondroitin Sulphate Proteoglycan ◽  
Core Proteins

The mechanisms regulating the secretion of proteoglycans and collagens in chondrocytes, in particular those operating at the level of the rough endoplasmic reticulum (RER), are largely unknown. To examine these mechanisms, I studied the effects of acute ascorbate treatment on the secretion of two collagen types (types II and IX) and two proteoglycan types (PG-H and PG-Lb, the major keratan sulphate/chondroitin sulphate proteoglycan and the minor chondroitin sulphate proteoglycan respectively in cartilage) in scorbutic cultures of chick vertebral chondrocytes. I found that the scorbutic chondrocytes synthesized underhydroxylated precursors of types II and IX collagen that were secreted very slowly and accumulated in the RER. When the cultures were treated acutely with ascorbate, both macromolecules underwent hydroxylation within 1-1.5 h of treatment, and began to be secreted at normal high rates starting at about 2 h. Proteoglycan synthesis and secretion, however, remained largely unaffected by ascorbate treatment. Both the half-time of newly synthesized PG-H core protein in the RER and its conversion into completed proteoglycan were unchanged during treatment. Similarly, the overall rates of synthesis and secretion of both PG-H and PG-Lb remained at control levels during treatment. The data indicate that secretion of types II and IX collagen is regulated independently of secretion of PG-H and PG-Lb. This may be mediated by the ability of the RER of the chondrocyte to discriminate between procollagens and proteoglycan core proteins.

scholarly journals Characterization of a non-reducing terminal fragment from bovine articular cartilage keratan sulphates containing α(2-3)-linked sialic acid and α(1-3)-linked fucose. A sulphated variant of the VIM-2 epitope

1996 ◽  
Vol 319 (1) ◽  
pp. 137-141 ◽  
Author(s):  
Gavin M. BROWN ◽  
Thomas N. HUCKERBY ◽  
Beverley L. ABRAM ◽  
Ian A. NIEDUSZYNSKI
Keyword(s):  
Articular Cartilage ◽  
Sialic Acid ◽  
Spectroscopic Analysis ◽  
Anion Exchange Column ◽  
Keratan Sulphate ◽  
Bovine Articular Cartilage ◽  
Sialyl Lex ◽  
Strong Anion Exchange ◽  
Keratanase Ii

Alkaline-borohydride-reduced keratan sulphate chains were isolated from bovine articular cartilage (6–8-year-old animals) and digested with keratanase II, an endo-β-N-acetylglucosaminidase. The resulting oligosaccharides were borohydride-reduced and fractionated on a strong anion-exchange column. 1H-NMR spectroscopic analysis of the products revealed one containing both α(2-3)-linked sialic acid and α(1-3)-linked fucose which was shown to have the structure (I) shown. This structure is a sulphated variant of the VIM-2 epitope (CD65), a putative ligand of E-selectin. No oligosaccharide containing the sialyl-Lex structure [NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-] was identified in this study.

scholarly journals Proteoglycans isolated from dissociative extracts of differently aged human articular cartilage: characterization of naturally occurring hyaluronan-binding fragments of aggrecan

1994 ◽  
Vol 304 (3) ◽  
pp. 887-894 ◽  
Author(s):  
V Vilim ◽  
A J Fosang
Keyword(s):  
Articular Cartilage ◽  
Density Gradient ◽  
Chondroitin Sulphate ◽  
Gradient Centrifugation ◽  
Proteolytic Processing ◽  
Human Articular Cartilage ◽  
Globular Domain ◽  
Keratan Sulphate ◽  
Core Proteins

Proteoglycans extracted with 4 M guanidinium chloride from young (mean 20 years) or old (mean 79 years) macroscopically normal human articular cartilage were separated by density gradient centrifugation and Q-Sepharose chromatography and characterized by gradient gel SDS/PAGE and immunodetection before and after removal of glycosaminoglycan chains. The extracts contained two large populations of aggrecan, a population of small N-terminal aggrecan fragments, as well as decorin, biglycan and fibromodulin. The distribution of all these species in density gradient fractions has been determined. The large aggrecan populations comprised four different chondroitin sulphate-bearing core proteins while the population of smaller fragments comprised eight different components. The two smallest fragments (35 and 42 kDa), identified as the first globular domain of aggrecan (N-terminal) (G1) and containing no glycosaminoglycan, were detected only in extracts of old cartilage. A 55 and a 70 kDa fragment of G1 were present in both keratan sulphate-containing and non-keratan sulphate-containing forms. Four other fragments, each containing keratan sulphate epitopes, were identified and these contained either G1 epitopes (one 95 kDa species), or G1 and G2 epitopes (three species). These results have suggested that proteolytic processing at the N-terminus is more extensive than has previously been recognized and raises the possibility that more than one proteinase may be involved in aggrecan degradation in vivo. With the exception of the two smallest G1 fragments, the repertoire of proteoglycan fragments found in young and old human articular cartilage is essentially the same, although the relative abudnance of various species differed. The older tissue contains a larger proportion of C-terminally truncated aggrecan fragments and a significantly decreased content of decorin and biglycan.

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