Age-related changes in aggrecan glycosylation affect cleavage by aggrecanase

Aggrecan degradation involves proteolytic cleavage of the core protein with in the interglobular domain. Because aggrecan is highly glycosylated with c hondroitin sulfate (CS) and keratan sulfate (KS), we investigated whether g lycosylation affects digestion by aggrecanase at the Glu(373)-Ala(374) bond . Treatment of bovine aggrecan monomers to remove CS and BS resulted in los s of cleavage at this site, suggesting that glycosaminoglycans (GAGs) play a role in cleavage at the Glu(373)-Ala(374) bond. In contrast, MMP3 cleavag e at the Ser(341)-Phe(342) bond was not affected by glycosidase treatment o f aggrecan. Removal of KS, but not CS, prevented cleavage at the Glu(373)-A la(374) bond. Thus, KS residues may be important for recognition of this cl eavage site by aggrecanase. KS glycosylation has been observed at sites adj acent to the Glu(373)-Ala(374) bond in steer aggrecan, but not in calf aggr ecan (Barry, F. P., Rosenberg, L. C., Gaw, J. U., Gaw, J. U., Koob, T. J., and Neame, P. J. (1995) J. Biol. Chem. 270, 20516-20524). Interestingly, al though we found that aggrecanase degraded both calf and steer cartilage agg recan, the proportion of fragments generated by cleavage at the Glu(373)-Al a(374) bond was higher in steer than in calf, consistent with our observati ons using aggrecan treated to remove BS. me conclude that the GAG content o f aggrecan influences the specificity of aggrecanase for cleavage at the Gl u(373)-Ala(374) bond and suggest that age may be a factor in aggrecanase de gradation of cartilage.