Mutations in the interglobular domain of aggrecan alter matrix metalloproteinase and aggrecanase cleavage patterns - Evidence that matrix metalloproteinase cleavage interferes with aggrecanase activity

We have expressed G1-G2 mutants with amino acid changes at the DIPEN(341)down arrow (342)FFGVG and ITEGE(373)down arrow (374)-ARGSV cleavage sites, in order to investigate the relationship between matrix metalloproteinase (MMP) and aggrecanase activities in the interglobular domain (IGD) of aggrecan. The mutation DIPEN341 to DIGSA(341) partially blocked cleavage by MMP-13 and MMP-8 at the MMP site, while the mutation (342)FFGVG to (342)GTRVG; completely blocked cleavage at this site by MMP-1, -2, -3, -7, -8, -9, -13, -14. Each of the MMP cleavage site mutants, including a four-amino acid deletion mutant lacking residues ENFF343, were efficiently cleaved by aggrecanase, suggesting that the primary sequence at the MMP site had no effect on aggrecanase activity in the IGD. The mutation (374)ARGSV to (NVYSV)-N-374 completely blocked cleavage at the aggrecanase site by aggrecanase, MMP-8 and atrolysin C but had no effect on the ability of MMP-8 and MMP-13 to cleave at the Asn(341)down arrow Phe bond. Susceptibility to atrolysin C cleavage at the MMP site was conferred in the DIGSA(341) mutant but absent in the wildtype, (342)GTRVG, (NVYSV)-N-374, and deletion mutants. To further explore the relationship between MMP and aggrecanase activities, sequential digest experiments were done in which MMP degradation products were subsequently digested with aggrecanase and vice versa. Aggrecanase-derived G1 domains with ITEGE(373) C termini were viable substrates for MMPs; however, MMP-derived G2 fragments were resistant to cleavage by aggrecanase. A 10-mer peptide FVDIPENFFG, which is a substrate analogue for the MMP cleavage site, inhibited aggrecanase cleavage at the Glu(373)down arrow Ala bond. This study demonstrates that MMPs and aggrecanase have unique substrate recognition in the IGD of aggrecan and suggests that sequences at the C terminus of the DIPEN341 G1 domain may be important for regulating aggrecanase cleavage.