Abstract
Aggrecan degradation involves proteolytic cleavage of the core protein within the interglobular domain. Because aggrecan is highly glycosylated with chondroitin sulfate (CS) and keratan sulfate (KS), we investigated whether glycosylation affects digestion by aggrecanase at the Glu(373)-Ala(374) bond. Treatment of bovine aggrecan monomers to remove CS and KS resulted in loss of cleavage at this site, suggesting that glycosaminoglycans (GAGs) play a role in cleavage at the Glu(373)-Ala(374) bond. In contrast, MMP-3 cleavage at the Ser(341)-Phe(342) bond was not affected by glycosidase treatment of aggrecan. Removal of KS, but not CS, prevented cleavage at the Glu(373)-Ala(374) bond. Thus, KS residues may be important for recognition of this cleavage site by aggrecanase. KS glycosylation has been observed at sites adjacent to the Glu(373)-Ala(374) bond in steer aggrecan, but not in calf aggrecan (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, although we found that aggrecanase degraded both calf and steer cartilage aggrecan, the proportion of fragments generated by cleavage at the Glu(373)-Ala(374) bond was higher in steer than in calf, consistent with our observations using aggrecan treated to remove KS. We conclude that the GAG content of aggrecan influences the specificity of aggrecanase for cleavage at the Glu(373)-Ala(374) bond and suggest that age may be a factor in aggrecanase degradation of cartilage.
Highlights
The articular cartilage matrix consists primarily of collagen, which provides strength and support, and proteoglycan, which contributes qualities of compressibility and elasticity
Because aggrecan is highly glycosylated with chondroitin sulfate (CS) and keratan sulfate (KS), we investigated whether glycosylation affects digestion by aggrecanase at the Glu373–Ala374 bond
The interglobular domain (IGD) of the aggrecan core protein, which is located between G1 and G2, contains proteolytic cleavage sites that are believed to be critical to the overall loss of aggrecan function
Summary
The articular cartilage matrix consists primarily of collagen, which provides strength and support, and proteoglycan, which contributes qualities of compressibility and elasticity. Because the aggrecan core protein is heavily glycosylated, it is possible that the glycosaminoglycans on the aggrecan molecule may affect the ability of either MMPs or aggrecanase to cleave the core protein This notion is especially intriguing based on the finding by Barry et al [17] that glycosylation of aggrecan in articular cartilage is altered with age and could potentially play a role in regulating the susceptibility of aggrecan to degradation during aging. These authors reported that aggrecan isolated from steer (age of 1.5–2 years) articular cartilage is post-translationally modified with a keratan sulfate within the sequence 368NITEGEAR375 whereas aggrecan from calf (age of 1 week) articular cartilage lacks glycosylation in this region Because this sequence contains the aggrecanase cleavage site, the presence of glycosylation may play a role in regulating aggrecanase-mediated degradation. Studies by Bayliss and co-workers [18] detected aggrecan G1 fragments with the C terminus (ITEGE), indicating that they had been cleaved
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