The intermediates of aggrecanase-dependent cleavage of aggrecan in rat chondrosarcoma cells treated with interleukin-1

We have examined the abundance and structure of intermediates in the chondr ocyte-mediated degradation of aggrecan by aggrecanase(s). Degradation produ cts were identified by Western-blot analysis with antibodies to cleavage-si te neoepitopes and to peptides within the globular domains. Rat chondrosarc oma tumour contained full-length aggrecan and all of the individual peptide s expected from single independent cleavages at each of the four aggrecanas e sites in the chondroitin sulphate (CS) domain. Kinetic analysis of the pr oducts present in rat chondrosarcoma cell cultures treated with interleukin -lb showed that the first aggrecanase-mediated cleavages occurred at the fo ur sites within the CS attachment region to generate two stable intermediat es, 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 termin al products, Val(1)-Glu(373), Ala(374)-Glu(1274) and Ala(374)-Glu(1459). It therefore appears that the aggrecanase-mediated processing of native aggre can by chondrocytes in situ is initiated within the CS-attachment region an d completed by cleavage within the interglobular domain. Since it has been shown that digestion of aggrecan monomer in solution with recombinant ADAMT S-4 [Tortorella, Pratta, Liu, Austin, Ross, Abbaszade, Burn and Amer (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 pr operties inherent in the aggrecan monomer itself, such as preferred peptide sequences for enzyme binding or enhanced accessibility to the core protein at these sites.