AGGRECANASE ACTIVITY IS IMPLICATED IN TUMOR-NECROSIS-FACTOR-ALPHA MEDIATED CARTILAGE AGGRECAN BREAKDOWN BUT IS NOT DETECTED BY AN IN-VITRO ASSAY

Aims-To develop an in vitro assay for the putative glutamyl endopeptid ase, ''aggrecanase'', which is thought to degrade cartilage aggrecan, and to examine the role of the enzyme in tumour necrosis factor stimul ated aggrecan cleavage. Methods-Aggrecan fragments released by bovine nasal cartilage explants, with and without exposure to tumour necrosis factor alpha, were purified and analysed by western blotting and N-te rminal sequencing. Intact bovine aggrecan was incubated with extracts of cartilage, lysed chondrocytes, or cartilage explant conditioned cul ture medium under a variety of conditions. Deglycosylated aggrecan was incubated with nasal cartilage explants. Proteoglycan breakdown was a ssessed by metachromatic assay of fragments in culture media, and clea vage of the substrate at the aggrecanase cleavage site was detected an d measured using the antibody BC3, which recognises a neoepitope produ ced by aggrecanase cleavage of aggrecan. Results-Aggrecan fragments ge nerated from explants treated with tumour necrosis factor had N-termin al sequences consistent with cleavage of aggrecan at a restricted numb er of glutamyl bonds. Aggrecanase generated fragments were found in ca rtilage explant culture medium and chondrocyte monolayers, However, no aggrecanase activity could be detected in extracts of cartilage, or c hondrocytes from which endogenous aggrecan fragments had been removed, under a variety of assay conditions. Deglycosylated aggrecan, added t o explant cultures, efficiently inhibited endogenous aggrecan breakdow n. Conclusions-Aggrecanase is active in cartilage and in chondrocyte m onolayers, and its action is stimulated by tumour necrosis factor a. H owever, activity due to this enzyme could not be detected in vitro und er our assay conditions, although a deglycosylated version of the subs trate inhibited aggrecan breakdown in explant cultures.