Truncation of the amino-terminus of the recombinant aggrecan rAgg1(mut) leads to reduced cleavage at the aggrecanase site. Efficient aggrecanase catabolism may depend on multiple substrate interactions

Aggrecanase cleavage at the Glu(373)-Ala(374) Site in the interglobular dom ain of the cartilage proteoglycan aggrecan is a key event in arthritic dise ases. The observation that substrates representing only the aggrecanase cle avage site are not catabolized efficiently by aggrecanase prompted us to in vestigate the requirement of aggrecanase for additional structural elements of its substrate other than the actual cleavage site. Based on the recombi nant substrate rAgg1(mut) we constructed deletion mutants with successively truncated N- or C-termini of the interglobular domain. Catabolism by aggre canase activities induced in rat chondrosarcoma cells, porcine chondrocytes , and by human recombinant ADAMTS4 showed a gradually decreasing catabolism of progressively shortened, N-terminal deletion mutants of the substrate r Agg1(mut). A reduction to 32 amino acids N-terminal to the aggrecanase site resulted in a decrease of at least 42% of aggrecanase cleavage products as compared with the wild-type substrate. When only 16 amino acids preceded t he Glu(373)-Ala(374) site, aggrecanase cleavage was completely inhibited. I n contrast, C-terminal deletions did not negatively affect aggrecanase clea vage up to the reduction to 13 amino acids C-terminal to the cleavage sire. Unlike aggrecanase(s), membrane type 1-matrix metalloprotease (MT1-MMP), a ble to cleave rAgg1(mut), both at the aggrecanase and the MMP site, was ins ensitive to N-terminal deletions regarding aggrecanase cleavage, indicating that the importance of the N-terminus is characteristic for aggrecanase(s) . Taken together, the results demonstrate that the amino-terminus of rAgg1( mut) containing the MMP site, plays an important role for efficient cleavag e by aggrecanase(s), possibly by serving as a further site of interaction b etween the enzyme and its substrate. (C) 2000 Elsevier Science B.V./Interna tional Society of Matrix Biology. All rights reserved.