Localization of the Expression of Type I, II, III Collagen, and Aggrecan Core Protein Genes in Developing Human Articular Cartilage

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.

Download Full-text

Characterization of proteoglycans isolated from associative extracts of human articular cartilage

Biochemical Journal ◽

10.1042/bj2930165 ◽

1993 ◽

Vol 293 (1) ◽

pp. 165-172 ◽

Cited By ~ 8

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.

Download Full-text

Development of Enzyme Immunoassays Specific for Keratan Sulphate- and Core-Protein-Epitopes of the Large Aggregating Proteoglycan from Human Articular Cartilage

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm.1994.32.4.285 ◽

1994 ◽

Vol 32 (4) ◽

Author(s):

Dagmar-Christiane Fischer ◽

Susanne Kolbe-Busch ◽

G. Stöcker ◽

A. Hoffmann ◽

H.-D. Haubeck

Keyword(s):

Articular Cartilage ◽

Core Protein ◽

Human Articular Cartilage ◽

Keratan Sulphate ◽

Enzyme Immunoassays

Download Full-text

Hyaluronate degradation as an alternative mechanism for proteoglycan release from cartilage during interleukin-1β-stimulated catabolism

Biochemical Journal ◽

10.1042/bj3620473 ◽

2002 ◽

Vol 362 (2) ◽

pp. 473-479 ◽

Cited By ~ 25

Author(s):

Robert SZTROLOVICS ◽

Anneliese D. RECKLIES ◽

Peter J. ROUGHLEY ◽

John S. MORT

Keyword(s):

Core Protein ◽

Cartilage Matrix ◽

Interleukin 1Β ◽

Alternative Mechanism ◽

Human Articular Cartilage ◽

Link Protein ◽

Nasal Cartilage ◽

Fetal Bovine ◽

Proteoglycan Release ◽

Aggrecan Core Protein

Data presented previously suggest that release of components of the cartilage matrix, in response to catabolic agents, cannot be accounted for by proteolytic mechanisms alone. In the present study, the release of glycosaminoglycan-containing components from bovine nasal cartilage cultured in the presence of interleukin-1β, and from bovine nasal, fetal bovine epiphyseal and adult human articular cartilage cultured in the presence of retinoic acid, was accompanied by the loss of link protein and hyaluronate into the culture medium. Chromatographic analysis of the released hyaluronate showed it to be markedly reduced in size relative to that extracted from the corresponding tissue. It is proposed that, under stimulation by catabolic agents, two independent, but concurrent, mechanisms act to promote the release of aggrecan from the cartilage matrix. First, proteolytic cleavage of the aggrecan core protein results in the production of glycosaminoglycan-containing fragments that are free to diffuse from the tissue. Secondly, cleavage of hyaluronate renders portions of the proteoglycan aggregate small enough so that complexes of aggrecan (or fragments containing its G1 domain) and link protein are released from the tissue. It is likely that both mechanisms contribute to cartilage metabolism in normal physiology and pathology.

Download Full-text

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

Biochemical Journal ◽

10.1042/bj3181051 ◽

1996 ◽

Vol 318 (3) ◽

pp. 1051-1056 ◽

Cited By ~ 10

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.

Download Full-text

Non-proteoglycan forms of biglycan increase with age in human articular cartilage

Biochemical Journal ◽

10.1042/bj2950421 ◽

1993 ◽

Vol 295 (2) ◽

pp. 421-426 ◽

Cited By ~ 96

Author(s):

P J Roughley ◽

R J White ◽

M C Magny ◽

J Liu ◽

R H Pearce ◽

...

Keyword(s):

Articular Cartilage ◽

Intervertebral Disc ◽

Core Protein ◽

Proteolytic Processing ◽

Human Articular Cartilage ◽

Minor Constituent ◽

Structural Variations ◽

Core Proteins ◽

A Minor ◽

Peptide Antibodies

Polyclonal anti-peptide antibodies were raised to the C-terminal regions of human biglycan and decorin. These antibodies were used in immunoblotting to study structural variations with age in the proteoglycan core proteins present in extracts of human articular cartilage and intervertebral disc. Three forms of the biglycan core protein were identified. The largest form was detected only after chondroitinase treatment and represents the proteoglycan form of the molecule from which the glycosaminoglycan chains have been removed. However, chondroitinase treatment did not alter the electrophoretic mobility of the two smaller proteins, which appear to represent non-proteoglycan forms of the molecule, resulting either from a failure to substitute the intact proteoglycan core protein with glycosaminoglycan chains during its synthesis or from proteolytic processing of the intact proteoglycan causing removal of the N-terminal region bearing the glycosaminoglycan chains. The non-proteoglycan forms constituted a minor proportion of biglycan in the newborn, but were the major components in the adult. A similar trend was seen in both articular cartilage and intervertebral disc. In comparison, decorin appears to exist predominantly as a proteoglycan at all ages, with two core protein sizes being present after chondroitinase treatment. Non-proteoglycan forms were detected in the adult, but they were always a minor constituent.

Download Full-text

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

Biochemical Journal ◽

10.1042/bj3370077 ◽

1998 ◽

Vol 337 (1) ◽

pp. 77-82 ◽

Cited By ~ 24

Author(s):

Mark C. BOLTON ◽

Jayesh DUDHIA ◽

Michael T. BAYLISS

Keyword(s):

Articular Cartilage ◽

De Novo ◽

Core Protein ◽

Buoyant Density ◽

Aggregate Stability ◽

Exchange Chromatography ◽

Human Articular Cartilage ◽

Post Translational Modification ◽

Link Protein ◽

Age Related

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-trancriptional 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.

Download Full-text

Progenitor Cells in Healthy and Osteoarthritic Human Cartilage Have Extensive Culture Expansion Capacity while Retaining Chondrogenic Properties

Cartilage ◽

10.1177/19476035211059600 ◽

2021 ◽

pp. 194760352110596

Author(s):

M. Rikkers ◽

J.V. Korpershoek ◽

R. Levato ◽

J. Malda ◽

L.A. Vonk

Keyword(s):

Articular Cartilage ◽

Progenitor Cells ◽

Cartilage Regeneration ◽

Surface Marker ◽

Type I ◽

Human Articular Cartilage ◽

Human Cartilage ◽

Promising Alternative ◽

Surface Marker Expression ◽

Marker Expression

Objective Articular cartilage-derived progenitor cells (ACPCs) are a potential new cell source for cartilage repair. This study aims to characterize endogenous ACPCs from healthy and osteoarthritic (OA) cartilage, evaluate their potential for cartilage regeneration, and compare this to cartilage formation by chondrocytes. Design ACPCs were isolated from full-thickness healthy and OA human cartilage and separated from the total cell population by clonal growth after differential adhesion to fibronectin. ACPCs were characterized by growth kinetics, multilineage differentiation, and surface marker expression. Chondrogenic redifferentiation of ACPCs was compared with chondrocytes in pellet cultures. Pellets were assessed for cartilage-like matrix production by (immuno)histochemistry, quantitative analyses for glycosaminoglycans and DNA content, and expression of chondrogenic and hypertrophic genes. Results Healthy and OA ACPCs were successfully differentiated toward the adipogenic and chondrogenic lineage, but failed to produce calcified matrix when exposed to osteogenic induction media. Both ACPC populations met the criteria for cell surface marker expression of mesenchymal stromal cells (MSCs). Healthy ACPCs cultured in pellets deposited extracellular matrix containing proteoglycans and type II collagen, devoid of type I collagen. Gene expression of hypertrophic marker type X collagen was lower in healthy ACPC pellets compared with OA pellets. Conclusions This study provides further insight into the ACPC population in healthy and OA human articular cartilage. ACPCs show similarities to MSCs, yet do not produce calcified matrix under well-established osteogenic culture conditions. Due to extensive proliferative potential and chondrogenic capacity, ACPCs show potential for cartilage regeneration and possibly for clinical application, as a promising alternative to MSCs or chondrocytes.

Download Full-text