Aggrecanase cleavage at the Glu(373)-Ala(374) site in the interglobular domain of the cartilage
proteoglycan aggrecan is a key event in arthritic diseases. The observation that substrates representing only the
aggrecanase cleavage site are not catabolized efficiently by
aggrecanase prompted us to investigate the requirement of
aggrecanase for additional structural elements of its substrate other than the actual cleavage site. Based on the recombinant substrate rAgg1mut we constructed deletion mutants with successively truncated N- or C-termini of the interglobular domain. Catabolism by
aggrecanase 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 rAgg1mut. 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 the Glu(373)-Ala(374) site,
aggrecanase cleavage was completely inhibited. In contrast, C-terminal deletions did not negatively affect
aggrecanase cleavage up to the reduction to 13
amino acids C-terminal to the cleavage site. Unlike
aggrecanase(s), membrane type 1-matrix
metalloprotease (MT1-MMP), able to cleave rAgg1mut both at the
aggrecanase and the
MMP site, was insensitive 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 rAgg1mut, containing the
MMP site, plays an important role for efficient cleavage by
aggrecanase(s), possibly by serving as a further site of interaction between the
enzyme and its substrate.