AGE-RELATED-CHANGES IN THE CONTENT OF THE C-TERMINAL REGION OF AGGRECAN IN HUMAN ARTICULAR-CARTILAGE

The content of the C-terminal region of aggrecan was investigated in s amples of articular cartilage from individuals ranging in age from new born to 65 years. This region contains the globular G3 domain which is known to be removed from aggrecan in mature cartilage, probably by pr oteolytic cleavage, but the age-related changes in its abundance in hu man cartilage have not been described previously. The analysis was per formed by immunosorbant assay using an antiserum (JD5) against recombi nant protein expressed from a cDNA clone encoding the terminal 598 ami no acid residues of human aggrecan, on crude extracts of cartilage wit hout further purification of aggrecan. The results showed that the con tent of the C-terminal region decreased with age relative to the G1 do main 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 extra cts was corrected to only include the G1 attached to aggrecan and to e xclude the G1 fragments which accumulate as a by-product of normal agg recan turnover (free G1), the age-related decrease in the C-terminal r egion remained very pronounced. Analysis by composite agarose/PAGE sho wed that the number of subpopulations of aggrecan resolved increased f rom one in newborn to three in adult cartilage. All of these reacted w ith an antiserum to the human G1 domain, but only the slowest migratin g species reacted with the C-terminal region antiserum (JD5). Similar analysis by SDS/PAGE confirmed the presence of high-molecular-mass (20 0 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 s pecies corresponding to the free G1 and G1-G2 fragments, which were pr esent at high concentrations in adult cartilage. The results suggest t hat the loss of the C-terminal region is not directly part of the proc ess of aggrecan turnover, but it is a slow independent matrix process that occurs more extensively with aging as turnover rates become slowe r. Young cartilage with the fastest turnover contains least molecules lacking the C-terminal region, whereas in old tissue with slow turnove r few molecules retain this region. An increase in the cleavage of thi s region with age may also contribute to this change. The content of t he C-terminal region may thus give a measure of the abundance of newly synthesized aggrecan.