To develop and characterize a polyclonal antiserum (RAM 3.2), which recognizes the neo-C terminal cleavage product generated by the action of aggrecanase (ADAMts 4/5) on the G1-domain of human aggrecan. We also intend to use this antiserum to investigate normal, age-related changes in human articular cartilage.
The antiserum was raised in rabbits and its localization in cryosections of normal articular cartilage was investigated by immunohistochemistry. The concentration of the aggrecanase neo-epitope was also investigated in extracts of the tissue using SDS-PAGE and electrophoresis in large pore/agarose gels.
The product of aggrecanase action appears to accumulate in the extracellular matrix during normal aging of the tissue. Furthermore, the concentration of the fragment depended on the topographical site on the femoral condyle from which the sample was selected. Electrophoretic and immunohistochemical analysis of the fragment in normal cartilage showed that in immature cartilage it was deposited mainly in the surface layers, whereas in mature samples it was distributed throughout the depth of the tissue. In contrast, immunoreactivity of osteoarthritic cartilage was always less and the distribution was more variable than in normal cartilage of the same age.
(1) The proteolytic cleavage of aggrecan by aggrecanase is a normal homeostatic event and much of the neo-C terminal fragment produced by the enzyme is retained in the tissue. (2) The presence of this immunoreactive product in normal cartilage can be used as an indication of aggrecan turnover. (3) That in osteoarthritic cartilage there is a reduction in the concentration of the G1-fragments.