scholarly journals Age-related changes in protein-related epitopes of human articular-cartilage proteoglycans

1986 ◽  
Vol 236 (1) ◽  
pp. 71-75 ◽  
Author(s):  
T T Glant ◽  
K Mikecz ◽  
P J Roughley ◽  
E Buzás ◽  
A R Poole
Keyword(s):  
Core Protein ◽  
Buoyant Density ◽  
Human Articular Cartilage ◽  
Human Cartilage ◽  
Keratan Sulphate ◽  
Cartilage Proteoglycan ◽  
Age Related ◽  
Age Dependent ◽  
Old Adult ◽  
Cartilage Proteoglycans

Monoclonal antibodies were prepared that recognize different age-related epitopes on proteoglycan subunits of high buoyant density isolated from human epiphysial and articular cartilages. Antibody EFG-4 (IgG1) recognizes a proteinase-sensitive segment associated with the core protein. Antibody BCD-4 (IgG1) reacts with keratan sulphate bound to core protein. Both epitopes are minimally expressed in foetal cartilage and increase with age after birth to become maximally expressed in adult cartilage by about 30 years of age. In contrast, monoclonal antibody alpha HFPG-846 (IgM) recognizes a core-protein-related epitope that is maximally expressed in young foetal cartilage, declines up to birth and thereafter and is almost absent after about 30 years of age. Antibody alpha HFPG-846 was used to isolate by immuno-affinity chromatography two subpopulations of proteoglycan subunits from a 16-year-old-human cartilage proteoglycan subunit preparation. Only the antibody-unbound population showed a significant reaction with antibodies EGF-4 and BCD-4. The amino acid and carbohydrate compositions of these proteoglycan fractions were different, and one (antibody-bound) resembled those of foetal and the other (antibody-unbound) resembled those of adult proteoglycans isolated from 24-27-week-old-foetal and 52-56-year-old-adult cartilage respectively. These observations demonstrate that human cartilages contain at least two chemically and immunochemically distinct populations of proteoglycans, the proportions and content of which are age-dependent. It is likely that these populations represent the products of different genes, though their heterogeneity may be compounded by the result of different post-translation modifications.

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 Treatment of cartilage proteoglycan aggregate with hydrogen peroxide. Relationship between observed degradation products and those that occur naturally during aging

1987 ◽  
Vol 247 (2) ◽  
pp. 349-357 ◽  
Author(s):  
C R Roberts ◽  
J S Mort ◽  
P J Roughley
Keyword(s):  
Transition Metal ◽  
Metal Ion ◽  
Core Protein ◽  
Degradation Products ◽  
Transition Metal Ions ◽  
Human Articular Cartilage ◽  
Human Aging ◽  
Human Cartilage ◽  
Cartilage Proteoglycan ◽  
Age Related

The effects of treatment of purified neonatal human articular-cartilage proteoglycan aggregate with H2O2 were studied. (1) Exposure of proteoglycan aggregate to H2O2 resulted in depolymerization of the aggregate and modification of the core protein of both the proteoglycan subunits and the link proteins. (2) Treatment of the proteoglycan aggregate with H2O2 rendered the proteoglycan subunits unable to interact with hyaluronic acid, with minimal change in their hydrodynamic size. (3) Specific cleavages of the neonatal link proteins occurred. The order in which the major products were generated and their electrophoretic mobilities resembled the pattern observed during human aging. (4) The proteolytic changes in the link proteins were inhibited in the presence of transition-metal-ion chelators, thiourea or tetramethylurea, suggesting that generation of hydroxyl radicals from H2O2 by trace transition-metal ions via a site-specific Fenton reaction may be responsible for the selective cleavages observed. (5) Cleavage of the link proteins in proteoglycan aggregates by H2O2 was shown to have a limited effect on the susceptibility of these proteins to cleavage by trypsin. (6) The relationship between these changes and those observed in cartilage during human aging suggests that some of the age-related changes in the structure of human cartilage proteoglycan aggregate may be the result of radical-mediated damage.

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.

scholarly journals Monoclonal antibodies to different protein-related epitopes of human articular cartilage proteoglycans

1986 ◽  
Vol 234 (1) ◽  
pp. 31-41 ◽  
Author(s):  
T T Glant ◽  
K Mikecz ◽  
A R Poole
Keyword(s):  
Monoclonal Antibodies ◽  
Articular Cartilage ◽  
Alkali Treatment ◽  
Buoyant Density ◽  
Limb Bud ◽  
Human Articular Cartilage ◽  
Human Cartilage ◽  
Keratan Sulphate ◽  
Human Adult ◽  
Cartilage Proteoglycans

Monoclonal antibodies produced against chondroitinase-treated human adult cartilage proteoglycans were selected for their ability to recognize epitopes on native proteoglycans. Binding analyses revealed that four of these monoclonal antibodies (BCD-4, BCD-7, EFG-4 and KPC-190) each recognized a different epitope on the same proteoglycan molecule which represents a subpopulation of a high buoyant density (D1) fraction of human articular cartilage proteoglycans (10, 30, 50 and 60% in fetal-newborn, 1.5 years old, 15 years old and 52-56 years old cartilages, respectively). Analysis of epitope specificities revealed that BCD-7 and EFG-4 monoclonal antibodies recognized epitopes on proteoglycan monomer which are associated with the protein structure in that they are sensitive to cleavage by Pronase, papain and alkali treatment and do not include keratan sulphate, chondroitin sulphate or oligosaccharides. The BCD-4 and KPC-190 epitopes also proved to be sensitive to Pronase or papain digestion or to alkali treatment, but keratanase or endo-beta-galactosidase also reduced the immunoreactivity of these epitopes. These observations indicate that the BCD-4 and KPC-190 epitopes represent peptides substituted with keratan sulphate or keratan sulphate-like structures. The BCD-4 epitope is, however, absent from a keratan sulphate-rich fragment of human adult proteoglycan, while the other three epitopes were detected in this fragment. None of these four epitopes were detected in the link proteins of human cartilage, in the hyaluronic acid-binding region of human newborn cartilage proteoglycan, in Swarm rat chondrosarcoma proteoglycan, in chicken limb bud proteoglycan monomer and in the small dermatan sulphate-proteoglycan of bovine costal cartilage. EFG-4 and KPC-190 epitopes were not detected in human fetal cartilage proteoglycans, although fetal molecules contained trace amounts of epitopes reactive with BCD-4 and BCD-7 antibodies.

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

1998 ◽  
Vol 337 (1) ◽  
pp. 77-82 ◽  
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.

scholarly journals Extraction and characterization of proteoglycan from human meniscus

1980 ◽  
Vol 185 (3) ◽  
pp. 705-713 ◽  
Author(s):  
D McNicol ◽  
P J Roughley
Keyword(s):  
Chondroitin Sulphate ◽  
Specific Interaction ◽  
Specific Area ◽  
Human Articular Cartilage ◽  
Tissue Concentrations ◽  
Keratan Sulphate ◽  
Cartilage Proteoglycan ◽  
Age Related ◽  
Core Proteins

This study consists of (1) the extraction of proteoglycan from the human meniscus under dissociative conditions, (2) an investigation of the changes that occur in the abundance and structure of this proteoglycan with age and (3) a comparison of these findings with those for human articular-cartilage proteoglycan. Adult meniscus was found to possess proteoglycan molecules of similar size and glycosaminoglycan content to those present in cartilage, although tissue concentrations were considerably lower. In addition, age-related changes, with respect to the occurrence of keratan sulphate and the sulphation of chondroitin sulphate chains, were common to both tissues. The presence of aggregated proteoglycan was demonstrated, although specific interaction with hyaluronic acid was not conclusively shown biochemically. Differences were, however, noted in the structure of the proteoglycan between the two tissues: dermatan sulphate was found in the meniscus proteoglycan preparation and the core proteins exhibited some dissimilarities. A proteoglycan structure of this type would be compatible with its participation in meniscus elasticity, especially as the material is localized in a specific area.

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 Demonstration of link protein in proteoglycan aggregates from human intervertebral disc

1984 ◽  
Vol 222 (1) ◽  
pp. 85-92 ◽  
Author(s):  
A Tengblad ◽  
R H Pearce ◽  
B J Grimmer
Keyword(s):  
Intervertebral Disc ◽  
Density Gradient ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Intervertebral Discs ◽  
Human Articular Cartilage ◽  
Link Protein ◽  
Nasal Cartilage ◽  
Proteoglycan Aggregates ◽  
Cartilage Proteoglycans

Proteoglycan aggregates free of non-aggregating proteoglycan have been prepared from the annuli fibrosi and nuclei pulposi of intervertebral discs of three human lumbar spines by extraction with 4M-guanidinium chloride, associative density gradient centrifugation, and chromatography on Sepharose CL-2B. The aggregate (A1-2B.V0) was subjected to dissociative density-gradient ultracentrifugation. Three proteins of Mr 38 900, 44 200 and 50 100 found in the fraction of low buoyant density (A1-2B.V0-D4) reacted with antibodies to link protein from newborn human articular cartilage. After reduction with mercaptoethanol, two proteins of Mr 43 000 and two of Mr 20 000 and 14 000 were seen. The A1-2B.V0-D4 fraction, labelled with 125I, coeluted with both hyaluronate and a hyaluronate oligosaccharide (HA14) on a Sepharose CL-2B column. HA10 and HA14 reduced the viscosity of A1 fractions; HA4, HA6 and HA8 did not. HA14 decreased the viscosity of disc proteoglycans less than it did that of bovine cartilage proteoglycans. Thus, although a link protein was present in human intervertebral disc, it stabilized proteoglycan aggregates less well than did the link protein from bovine nasal cartilage.

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