scholarly journals Quantification of aggrecan and link-protein mRNA in human articular cartilage of different ages by competitive reverse transcriptase-PCR

1996 ◽  
Vol 319 (2) ◽  
pp. 489-498 ◽  
Author(s):  
Mark C BOLTON ◽  
Jayesh DUDHIA ◽  
Michael T BAYLISS
Keyword(s):  
Articular Cartilage ◽  
Reverse Transcriptase ◽  
Mrna Expression ◽  
Northern Hybridization ◽  
Human Articular Cartilage ◽  
Rt Pcr ◽  
Link Protein ◽  
Reverse Transcriptase Pcr ◽  
Age Related ◽  
Age Related Changes

A competitive reverse transcriptase–PCR (RT-PCR) assay has been developed for the quantification of particular mRNA species in human articular cartilage. Competitor RNA species were synthesized that differed from the amplified target sequence only by the central insertion of an EcoRI restriction site. By using known amounts of synthetic target and competitor RNA, it was shown that competitor RNA molecules designed in this way are reverse-transcribed and amplified with equal efficiency to the target of interest. Furthermore quantification could be performed during the plateau phase of the PCR, which was necessary when using ethidium bromide fluorescence as a detection system. The inhibition of aggrecan and link-protein mRNA expression by interleukin 1 or tumour necrosis factor in monolayers of human articular chondrocytes quantified by this competitive RT-PCR method compared favourably with Northern hybridization studies. The main advantage of this technique is that it can be used to quantify levels of mRNA with RNA extracted directly from 100 mg wet weight of human articular cartilage. Age-related changes in aggrecan and link-protein mRNA were therefore quantified in human articular cartilage directly after dissection from the joint. The concentration of link-protein mRNA was higher in immature cartilage than in mature cartilage when expressed relative to the amount of glyceraldehyde-3-phosphate dehydrogenase mRNA, but no age-related changes were observed in aggrecan mRNA expression. The ratio of aggrecan to link-protein mRNA was higher in mature cartilage than in immature tissue. These age-related differences in the molecular stoichiometry of aggrecan and link-protein mRNA might have implications with respect to the regulation of the formation and the stability of the proteoglycan aggregates in cartilage.

Age-related Changes in the Antigenicity of Human Articular Cartilage Proteoglycans

1982 ◽  
Vol 2 (1) ◽  
pp. 45-60 ◽  
Author(s):  
Brian R. Champion ◽  
Agnes Reiner ◽  
Peter J. Roughley ◽  
A. Robin Poole
Keyword(s):  
Articular Cartilage ◽  
Human Articular Cartilage ◽  
Age Related ◽  
Cartilage Proteoglycans ◽  
Age Related Changes

scholarly journals Age-related changes in the sulphation of the chondroitin sulphate linkage region from human articular cartilage aggrecan

2001 ◽  
Vol 358 (2) ◽  
pp. 523 ◽  
Author(s):  
Robert M. LAUDER ◽  
Thomas N. HUCKERBY ◽  
Gavin M. BROWN ◽  
Michael T. BAYLISS ◽  
Ian A. NIEDUSZYNSKI
Keyword(s):  
Articular Cartilage ◽  
Chondroitin Sulphate ◽  
Human Articular Cartilage ◽  
Linkage Region ◽  
Age Related ◽  
Age Related Changes

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.

Age-related changes in the sulphation of the chondroitin sulphate linkage region from human articular cartilage aggrecan

2001 ◽  
Vol 358 (2) ◽  
pp. 523-528 ◽  
Author(s):  
Robert M. LAUDER ◽  
Thomas N. HUCKERBY ◽  
Gavin M. BROWN ◽  
Michael T. BAYLISS ◽  
Ian A. NIEDUSZYNSKI
Keyword(s):  
Articular Cartilage ◽  
Chondroitin Sulphate ◽  
Basic Structure ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Human Articular Cartilage ◽  
Linkage Region ◽  
Age Related ◽  
Age Related Changes

The chondroitin sulphate (CS) linkage regions have been isolated from human articular cartilage aggrecan (from 10- to 72-year-olds) by chondroitin ABC endolyase digestion and size-exclusion chromatography. Linkage region hexasaccharides have been characterized and their abundance estimated by high-pH anion-exchange chromatography. The basic structure for the CS linkage region oligosaccharides identified from human aggrecan is as follows: ΔUA(β1–3)GalNAc[0S/4S/6S](β1–4)GlcA(β1–3)Gal[0S/6S](β1–3)Gal(β1–4)Xyl, where ΔUA represents 4,5-unsaturated hexuronic acid, 4S and 6S represent an O-ester sulphate group on C-4 and C-6 respectively, and 0S represents zero sulphation. There are significant age-related changes in the abundance of the various N-acetylgalactosamine (GalNAc) sulphation forms identified, occurring up to approx. 20 years old. During the period from 10 to 20 years old the level of GalNAc 6-sulphation at the linkage region increases from approx. 43% to approx. 75%, while there is a corresponding reduction in unsulphated (approx. 30% to approx. 20%) and 4-sulphated (approx. 25% to approx. 6%) GalNAc residues. There is also an increase in the incidence of linkage region galactose 6-sulphation (approx. 2% to approx. 10%) which was only observed in linkage regions with GalNAc 6-sulphation. Beyond 20 years old there are few changes in the relative abundance of these GalNAc sulphation variants; however, there is a slight increase in the abundance of 6-sulphation between approx. 20 years old and approx. 40 years old and a slight decrease in its abundance beyond approx. 40 years old. Our data show that in the majority of chains from tissues of all ages the GalNAc residue closest to the linkage region is 6-sulphated, but the level of GalNAc 6-sulphation within the linkage region is lower than the average level observed within the repeat region.

scholarly journals Age-related changes in the content of the C-terminal region of aggrecan in human articular cartilage

1996 ◽  
Vol 313 (3) ◽  
pp. 933-940 ◽  
Author(s):  
Jayesh DUDHIA ◽  
Catherine M. DAVIDSON ◽  
Terri M. WELLS ◽  
Demis H. VYNIOS ◽  
Timothy E. HARDINGHAM ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Terminal Region ◽  
Human Articular Cartilage ◽  
Amino Acid Residues ◽  
Turnover Rates ◽  
Human Cartilage ◽  
Age Related ◽  
Sds Page ◽  
High Concentrations ◽  
Age Related Changes

The content of the C-terminal region of aggrecan was investigated in samples of articular cartilage from individuals ranging in age from newborn to 65 years. This region contains the globular G3 domain which is known to be removed from aggrecan in mature cartilage, probably by proteolytic cleavage, but the age-related changes in its abundance in human cartilage have not been described previously. The analysis was performed by immunosorbant assay using an antiserum (JD5) against recombinant protein expressed from a cDNA clone encoding the terminal 598 amino acid residues of human aggrecan, on crude extracts of cartilage without further purification of aggrecan. The results showed that the content of the C-terminal region decreased with age relative to the G1 domain content (correlation coefficient = 0.463). This represented a 92% fall in the content of this region of the molecule from newborn to 65 years of age. Furthermore, when the G1 content of the cartilage extracts was corrected to only include the G1 attached to aggrecan and to exclude the G1 fragments which accumulate as a by-product of normal aggrecan turnover (free G1), the age-related decrease in the C-terminal region remained very pronounced. Analysis by composite agarose/PAGE showed that the number of subpopulations of aggrecan resolved increased from one in newborn to three in adult cartilage. All of these reacted with an antiserum to the human G1 domain, but only the slowest migrating species reacted with the C-terminal region antiserum (JD5). Similar analysis by SDS/PAGE confirmed the presence of high-molecular-mass (200 kDa) proteins reactive with JD5, but no reactive fragments of lower electrophoretic mobility were detected. In contrast, when probed with the antiserum to the human G1 domain, the immunoblots showed protein species corresponding to the free G1 and G1-G2 fragments, which were present at high concentrations in adult cartilage. The results suggest that the loss of the C-terminal region is not directly part of the process of aggrecan turnover, but it is a slow independent matrix process that occurs more extensively with aging as turnover rates become slower. Young cartilage with the fastest turnover contains least molecules lacking the C-terminal region, whereas in old tissue with slow turnover few molecules retain this region. An increase in the cleavage of this region with age may also contribute to this change. The content of the C-terminal region may thus give a measure of the abundance of newly synthesized aggrecan.

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