CELL-MEDIATED CATABOLISM OF AGGRECAN EVIDENCE THAT CLEAVAGE AT THE AGGRECANASE SITE (GLU(373)-ALA(37) PROTEOLYSIS OF THE INTERGLOBULAR DOMAIN

A rat chondrosarcoma cell line and primary bovine chondrocytes have be en used to study cell-mediated aggrecan catabolism. Addition of 1 mu M retinoic acid to chondrosarcoma cultures resulted in aggrecan proteol ysis with the release of greater than 90% of the cell layer aggrecan i nto the medium within 4 days. NH2-terminal sequencing of chondroitin s ulfate-substituted catabolic products gave a single major NH2-terminal sequence of ARGNVILTXK, initiating at Ala(374). This showed that the proteinase, commonly referred to as ''aggrecanase,'' which cleaves the Glu(373)-Ala(374) bond of the interglobular domain of aggrecan (Sandy , J. D., Neame, P. J., Boynton, R. E., and Flannery, C. R. (1990) J. B iol. Chem. 266, 8683-8685), is active in this cell system. Aggrecan G1 domain, generated by cleavage of the interglobular domain, was also l iberated during catabolism and this was characterized with three antip eptide antisera. Anti-CDAGWL was used as a general probe for G1 domain . Anti-FVDIPEN was used to specifically detect G1 domain with COOH ter minus of Asn(341), the form which is readily generated by cleavage of aggrecan by a wide range of matrix metalloproteinases. Anti-NITEGE ant iserum was used to specifically detect G1 domain with COOH terminus of Glu(373), the form which is the expected product of ''aggrecanase''-m ediated cleavage of aggrecan. Western blot analysis indicated that a s ingle form of G1 domain of about 60 kDa was formed. G1 domain of this size reacted with both anti-CDAGWL and anti-NITEGE but not with anti-F VDIPEN. Similar experiments with primary bovine chondrocyte cultures, treated with either retinoic acid or interleukin 1, showed that two fo rms of catabolic G1 domain, of about 62 and 66 kDa, were formed. Both of these forms reacted on Western blots with anti-CDAGWL and also with anti-NITEGE. It is suggested that cell-mediated catabolism of the agg recan interglobular domain in these culture systems, whether promoted by retinoic acid or interleukin 1, primarily involves cleavage of the Glu(373)-Ala(374) bond by aggrecanase. The accumulation of G1 domain w ith a COOH-terminal of Glu(373) shows that such aggrecanase-mediated c leavage can occur independent of the cleavage of the Asn(341)-phe(342) bond by matrix metalloproteinases.