Age-related changes in aggrecan glycosylation affect cleavage by aggrecanase.
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.
Pratta
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MA
Inflammatory Diseases Research, The DuPont Pharmaceutical Company, Experimental Station Wilmington, Delaware 19880-0400, USA. Michael.A.Pratta@dupontpharma.com
Tortorella
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MD
Arner
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EC
D000367:Age Factors; D053674:Aggrecans; D000375:Aging; D000409:Alanine / Q000737:chemistry; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002809:Chondroitin Sulfates / Q000378:metabolism; D002810:Chondroitinases and Chondroitin Lyases / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D016326:Extracellular Matrix Proteins; D005615:Freezing; D005973:Glutamine / Q000737:chemistry; D006025:Glycosaminoglycans / Q000378:metabolism; D006026:Glycoside Hydrolases; D006031:Glycosylation; D007632:Keratan Sulfate / Q000378:metabolism; D037181:Lectins, C-Type; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry; D011509:Proteoglycans / Q000378:metabolism*; D012694:Serine / Q000737:chemistry; D013997:Time Factors; D001616:beta-Galactosidase / Q000494:pharmacology
D053674:Aggrecans; D016326:Extracellular Matrix Proteins; D006025:Glycosaminoglycans; D037181:Lectins, C-Type; D011509:Proteoglycans; D005973:Glutamine; D012694:Serine; D010649:Phenylalanine; D002809:Chondroitin Sulfates; D007632:Keratan Sulfate; D006026:Glycoside Hydrolases; C026093:keratan-sulfate endo-1,4-beta-galactosidase; D001616:beta-Galactosidase; D010450:Endopeptidases; D019278:Matrix Metalloproteinase 3; C083641:aggrecanase; D002810:Chondroitinases and Chondroitin Lyases; D000409:Alanine