scholarly journals Structure of proteoglycans from different layers of human articular cartilage

1983 ◽  
Vol 209 (2) ◽  
pp. 387-400 ◽  
Author(s):  
M T Bayliss ◽  
M Venn ◽  
A Maroudas ◽  
S Y Ali
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Chondroitin Sulphate ◽  
Articular Surface ◽  
Current Model ◽  
Gel Chromatography ◽  
Slice Thickness ◽  
Human Articular Cartilage ◽  
Guanidinium Chloride ◽  
Keratan Sulphate

Full-depth plugs of adult human articular cartilage were cut into serial slices from the articular surface and analysed for their glycosaminoglycan content. The amount of chondroitin sulphate was highest in the mid-zone, whereas keratan sulphate increased progressively through the depth. Proteoglycans were isolated from each layer by extraction with 4M-guanidinium chloride followed by centrifugation in 0.4M-guanidinium chloride/CsCl at a starting density of 1.5 g/ml. The efficiency with which proteoglycans were extracted depended on slice thickness, and extraction was complete only when cartilage from each zone was sectioned at 20 microns or less. When thick sections (250 microns) were extracted, hyaluronic acid was retained in the tissue. Most of the proteoglycans, extracted from each layer under optimum conditions, could interact with hyaluronic acid to form aggregates, although the extent of aggregation was less in the deeper layers. Two pools of proteoglycan were identified in all layers by gel chromatography (Kav. 0.33 and 0.58). The smaller of these was rich in keratan sulphate and protein, and gradually increased in proportion through the cartilage depth. Chondroitin sulphate chain size was constant in all regions. The changes in composition and structure observed were consistent with the current model for hyaline-cartilage proteoglycans and were similar to those observed with increasing age in human articular cartilage.

scholarly journals Biosynthesis of proteoglycan in vitro by cartilage from human osteochondrophytic spurs

1982 ◽  
Vol 206 (2) ◽  
pp. 329-341 ◽  
Author(s):  
Charles J. Malemud ◽  
Victor M. Goldberg ◽  
Roland W. Moskowitz ◽  
Lee L. Getzy ◽  
Robert S. Papay ◽  
...  
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Deae Cellulose ◽  
Culture Conditions ◽  
Human Articular Cartilage ◽  
Guanidinium Chloride ◽  
Tissue Slices ◽  
Keratan Sulphate ◽  
Defined Culture

Proteoglycan biosynthesis by human osteochondrophytic spurs (osteophytes) obtained from osteoarthritic femoral heads at the time of surgical joint replacement was studied under defined culture conditions in vitro. Osteophytes were primarily present in two anatomic locations, marginal and epi-articular. Minced tissue slices were incubated in the presence of [35S]sulphate or [14C]glucosamine. Osteophytes incorporated both labelled precursors into proteoglycan, which was subsequently characterized by CsCl-isopycnic-density-gradient ultracentrifugation and chromatography on Sepharose CL-2B. The material extracted with 0.5m-guanidinium chloride showed 78.1% of [35S]sulphate in the A1 fraction after centrifugation. Only 23.0% of the [35S]sulphate in this A1 fraction was eluted in the void volume of Sepharose CL-2B under associative conditions. About 60–80% of the [35S]sulphate in the tissue 4m-guanidinium chloride extract was associated with monomeric proteoglycan (fraction D1). The average partition coefficient (Kav.) of the proteoglycan monomer on Sepharose CL-2B was 0.28–0.33. Approx. 12.4% of this monomer formed stable aggregates with high-molecular-weight hyaluronic acid in vitro. Sepharose CL-2B chromatography of fractions with lower buoyant densities (fractions D2–D4) demonstrated elution profiles on Sepharose CL-2B substantially different than that of fraction D1, indicative of the polydisperse nature of the newly synthesized proteoglycan. Analysis of the composition and chain size of the glycosaminoglycans showed the following: (1) preferential elution of both [35S]sulphate and [14C]glucosamine in the 0.5m-LiCl fraction on DEAE-cellulose; (2) the predominant sulphated glycosaminoglycan was chondroitin 6-sulphate (60–70%), with 9–11% keratan sulphate in the monomer proteoglycan; (3) Kav. values of 0.38 on Sephadex G-200 and 0.48 on Sepharose CL-6B were obtained with papain-digested and NaBH4-treated D1 monomer respectively. A comparison of the synthetic with endogenous glycosaminoglycans indicated similar types. These studies indicated that human osteophytes synthesized in vitro sulphated proteoglycans with some characteristics similar to those of mature human articular cartilage, notably in the size of their proteoglycan monomer and predominance of chondroitin 6-sulphate. They differed from articular cartilage primarily in the lack of substantial quantities of keratan sulphate and aggregation properties associated with monomer interaction with hyaluronic acid.

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 Variations in the composition of bovine hip articular cartilage with distance from the articular surface

1981 ◽  
Vol 195 (3) ◽  
pp. 535-543 ◽  
Author(s):  
A Franzén ◽  
S Inerot ◽  
S O Hejderup ◽  
D Heinegård
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Chondroitin Sulphate ◽  
Articular Surface ◽  
Full Thickness ◽  
Rich Region ◽  
Keratan Sulphate ◽  
Average Size ◽  
Almost All ◽  
Cartilage Proteoglycans

Punch biopsies of bovine hip articular cartilage was sectioned according to depth and the proteoglycans were isolated. The mid-sections of the cartilage contained more proteoglycans than did either the superficial or the deepest portions of the cartilage proteoglycans than did either the superficial or the deepest portions of the cartilage. The most superficial 40 micrometer of the cartilage contained relatively more glucosaminoglycans compared with the remainder of the cartilage. The proteoglycans recovered from the surface 200 micrometer layer contained less chondroitin sulphate, were smaller and almost all of these molecules were able to interact with hyaluronic acid to form aggregates. From about 200 micrometer and down to 1040 micrometer from the surface, the proteoglycans became gradually somewhat smaller, probably owing to decreasing size of the chondroitin sulphate-rich region. The proportion of molecules that were able to interact with the hyaluronic acid was about 90% and remained constant with depth. The proteoglycans from the deepest layer near the cartilage-bone junction contained a large proportion of non-aggregating molecules, and the average size of the proteoglycans was somewhat larger. The alterations of proteoglycan structure observed with increasing depth of the articular cartilage beneath the surface layer (to 200 micrometer) are of the same nature as those observed with increasing age in full-thickness articular cartilage. The articular-cartilage proteoglycans were smaller and had much higher keratan sulphate and protein contents that did molecules isolated from bovine nasal or tracheal cartilage.

scholarly journals Age-related changes in the composition and structure of human articular-cartilage proteoglycans

1978 ◽  
Vol 176 (3) ◽  
pp. 683-693 ◽  
Author(s):  
M T Bayliss ◽  
S Y Ali
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Age Groups ◽  
Human Articular Cartilage ◽  
Human Cartilage ◽  
Keratan Sulphate ◽  
Age Related ◽  
Composition And Structure ◽  
Normal Human ◽  
Cartilage Proteoglycans

1. Analysis of the purified proteoglycans extracted from normal human articular cartilage with 4M-guanidinium chloride showed that there was an age-related increase in their content of protein and keratan sulphate. 2. The hydrodynamic size of the dissociated proteoglycans also decreased with advancing age, but there was little change in the proportion that could aggregate. 3. Results suggested that some extracts of aged-human cartilage had an increased content of hyaluronic acid compared with specimens from younger patients. 4. Dissociated proteoglycans, from cartilage of all age groups, bind to hyaluronic acid and form aggregates in direct proportion to the hyaluronic acid concentration. 5. Electrophoretic heterogeneity of the dissociated proteoglycans was demonstrated on polyacrylamide/agarose gels. The number of proteoglycan species observed was also dependent on the age of the patient.

scholarly journals Hyaluronic acid in cartilage and proteoglycan aggregation

1974 ◽  
Vol 139 (3) ◽  
pp. 565-581 ◽  
Author(s):  
Timothy E. Hardingham ◽  
Helen Muir
Keyword(s):  
Hyaluronic Acid ◽  
Buoyant Density ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Gel Chromatography ◽  
Guanidinium Chloride ◽  
Keratan Sulphate ◽  
Sulphate Content ◽  
Lower Protein ◽  
Proteoglycan Aggregates

1. Dissociation of purified proteoglycan aggregates was shown to release an interacting component of buoyant density higher than that of the glycoprotein-link fraction of Hascall & Sajdera (1969). 2. This component, which produced an increase in hydrodynamic size of proteoglycans on gel chromatography, was isolated by ECTEOLA-cellulose ion-exchange chromatography and identified as hyaluronic acid. 3. The effect of pH of extraction showed that the proportion of proteoglycan aggregates isolated from cartilage was greatest at pH4.5. 4. The proportion of proteoglycans able to interact with hyaluronic acid decreased when extracted above or below pH4.5, whereas the amount of hyaluronic acid extracted appeared constant from pH3.0 to 8.5. 5. Sequential extraction of cartilage with 0.15m-NaCl at neutral pH followed by 4m-guanidinium chloride at pH4.5 was shown to yield predominantly non-aggregated and aggregated proteoglycans respectively. 6. Most of the hyaluronic acid in cartilage, representing about 0.7% of the total uronic acid, was associated with proteoglycan aggregates. 7. The non-aggregated proteoglycans were unable to interact with hyaluronic acid and were of smaller size, lower protein content and lower keratan sulphate content than the disaggregated proteoglycans. Together with differences in amino acid composition this suggested that each type of proteoglycan contained different protein cores.

scholarly journals The synthesis of dermatan sulphate proteoglycans by fetal and adult human articular cartilage

1989 ◽  
Vol 261 (2) ◽  
pp. 501-508 ◽  
Author(s):  
L I Melching ◽  
P J Roughley
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Uronic Acid ◽  
Degradation Products ◽  
Chondroitin Sulphate ◽  
Human Articular Cartilage ◽  
Dermatan Sulphate ◽  
Sulphate Proteoglycan ◽  
Adult Human

Non-aggregating dermatan sulphate proteoglycans can be extracted from both fetal and adult human articular cartilage. The dermatan sulphate proteoglycans appear to be smaller in the adult, this presumably being due to shorter glycosaminoglycan chains, and these chains contain a greater proportion of their uronic acid residues as iduronate. Both the adult and fetal dermatan sulphate proteoglycans contain a greater amount of 4-sulphation than 6-sulphation of the N-acetylgalactosamine residues, in contrast with the aggregating proteoglycans, which always show more 6-sulphation on their chondroitin sulphate chains. In the fetus the major dermatan sulphate proteoglycan to be synthesized is DS-PGI, though DS-PGII is synthesized in reasonable amounts. In the adult, however, DS-PGI synthesis is barely detectable relative to DS-PGII, which is still synthesized in substantial amounts. Purification of the dermatan sulphate proteoglycans from adult cartilage is hampered by the presence of degradation products derived from the large aggregating proteoglycans, which possess similar charge, size and density properties, but which can be distinguished by their ability to interact with hyaluronic acid.

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 Differences in the rates of aggregation of proteoglycans from human articular cartilage and chondrosarcoma

1983 ◽  
Vol 215 (3) ◽  
pp. 705-708 ◽  
Author(s):  
M T Bayliss ◽  
G D Ridgway ◽  
S Y Ali
Keyword(s):  
Articular Cartilage ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Equilibrium Density ◽  
Gel Chromatography ◽  
Human Articular Cartilage ◽  
Guanidinium Chloride ◽  
Adult Human ◽  
Comparison Of The Results

Pieces of adult human articular cartilage and chondrosarcoma were incubated in the presence of [35S]sulphate. After continuous or pulse-change incorporation of radioactivity, proteoglycans were extracted with 4.0 M-guanidinium chloride, purified by equilibrium density-gradient centrifugation and fractionated by gel chromatography. A comparison of the results suggests that the formation of stable aggregates occurs at a lower rate in articular cartilage than in chondrosarcoma.

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.

scholarly journals The identification and characterization of two populations of aggregating proteoglycans of high buoyant density isolated from post-natal human articular cartilages of different ages

1987 ◽  
Vol 248 (3) ◽  
pp. 735-740 ◽  
Author(s):  
C Webber ◽  
T T Glant ◽  
P J Roughley ◽  
A R Poole
Keyword(s):  
Hyaluronic Acid ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Buoyant Density ◽  
Human Articular Cartilage ◽  
Keratan Sulphate ◽  
Different Ages ◽  
Cartilage Proteoglycans ◽  
Two Populations

After chromatography on Sepharose CL-2B under associative conditions, high-buoyant-density human articular-cartilage proteoglycans were analysed biochemically and by radioimmunoassay with monoclonal antibodies to a core-protein-related epitope and to keratan sulphate. An examination of proteoglycans from individuals of different ages revealed the presence at 1 year of mainly a single polydisperse population containing chondroitin sulphate (uronic acid) and keratan sulphate. From 4 years onwards a smaller keratan sulphate-rich and chondroitin sulphate-deficient population appears in increasing amounts until 15 years. At the same time the larger population shows a progressive decrease in size from 1 year onward. By 23 years and after the proportion of keratan sulphate in the larger chondroitin sulphate-rich proteoglycan increases. Both adult proteoglycan populations are shown immunologically to aggregate with hyaluronic acid, with the smaller showing a greater degree of interaction. The larger population is richer in serine and glycine, and the smaller population contains more glutamic acid/glutamine, alanine, phenylalanine, lysine and arginine; its protein content is also higher. Whether the larger post-natal population represents a different gene product from the single polydisperse population found in the human fetus, which has a different amino acid composition, remains to be established. The smaller population, which represents approximately one-third the mass of the larger population in the adult, may represent a degradation product of the larger population, in which the hyaluronic acid-binding region and keratan sulphate-rich region are conserved.

Sign in / Sign up

Export Citation Format

Share Document