We have examined the abundance and structure of intermediates in the chondrocyte-mediated degradation of
aggrecan by
aggrecanase(s). Degradation products were identified by Western-blot analysis with
antibodies to cleavage-site neoepitopes and to
peptides within the globular domains. Rat
chondrosarcoma tumour contained full-length
aggrecan and all of the individual
peptides expected from single independent cleavages at each of the four
aggrecanase sites in the
chondroitin sulphate (CS) domain. Kinetic analysis of the products present in rat
chondrosarcoma cell cultures treated with interleukin-1b showed that the first
aggrecanase-mediated cleavages occurred at the four sites within the CS attachment region to generate two stable intermediates, Val(1)-Glu(1459) and Val(1)-Glu(1274). These species were subsequently cleaved at the Glu(373) site in the interglobular domain to form the terminal products, Val(1)-Glu(373), Ala(374)-Glu(1274) and Ala(374)-Glu(1459). It therefore appears that the
aggrecanase-mediated processing of native
aggrecan by chondrocytes in situ is initiated within the CS-attachment region and completed by cleavage within the interglobular domain. Since it has been shown that digestion of
aggrecan monomer in
solution with recombinant ADAMTS-4 [Tortorella, Pratta, Liu, Austin, Ross, Abbaszade,
Burn and Arner (2000) Sites of
aggrecan cleavage by recombinant human
aggrecanase-1 (ADAMTS-4). J. Biol. Chem. 275, 18566-18573] exhibits similar kinetics, it appears that preferential
proteinase cleavage in the CS-rich region is determined by properties inherent in the
aggrecan monomer itself, such as preferred
peptide sequences for
enzyme binding or enhanced accessibility to the core
protein at these sites.