Cell-mediated catabolism of aggrecan. Evidence that cleavage at the "aggrecanase" site (Glu373-Ala374) is a primary event in proteolysis of the interglobular domain.
A rat chondrosarcoma cell line and primary bovine chondrocytes have been used to study cell-mediated aggrecan catabolism. Addition of 1 microM retinoic acid to chondrosarcoma cultures resulted in aggrecan proteolysis with the release of greater than 90% of the cell layer aggrecan into the medium within 4 days. NH2-terminal sequencing of chondroitin sulfate-substituted catabolic products gave a single major NH2-terminal sequence of ARGNVILTXK, initiating at Ala374. This showed that the proteinase, commonly referred to as "aggrecanase," which cleaves the Glu373-Ala374 bond of the interglobular domain of aggrecan (Sandy, J. D., Neame, P. J., Boynton, R. E., and Flannery, C. R. (1990) J. Biol. Chem. 266, 8683-8685), is active in this cell system. Aggrecan G1 domain, generated by cleavage of the interglobular domain, was also liberated during catabolism and this was characterized with three antipeptide antisera. Anti-CDAGWL was used as a general probe for G1 domain. Anti-FVDIPEN was used to specifically detect G1 domain with COOH terminus of Asn341, the form which is readily generated by cleavage of aggrecan by a wide range of matrix metalloproteinases. Anti-NITEGE antiserum was used to specifically detect G1 domain with COOH terminus of Gln373, the form which is the expected product of "aggrecanase"-mediated cleavage of aggrecan. Western blot analysis indicated that a single 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-FVDIPEN. Similar experiments with primary bovine chondrocyte cultures, treated with either retinoic acid or interleukin 1, showed that two forms 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 aggrecan interglobular domain in these culture systems, whether promoted by retinoic acid or interleukin 1, primarily involves cleavage of the Glu373-Ala374 bond by aggrecanase. The accumulation of G1 domain with a COOH-terminal of Glu373 shows that such aggrecanase-mediated cleavage can occur independent of the cleavage of the Asn341-Phe342 bond by matrix metalloproteinases.
Department of Immunology, Merck Research Laboratories, Rahway, New Jersey 07065.
M W
MW
Lark
J T
JT
Gordy
J R
JR
Weidner
J
J
Ayala
J H
JH
Kimura
H R
HR
Williams
R A
RA
Mumford
C R
CR
Flannery
S S
SS
Carlson
M
M
Iwata
D053674:Aggrecans; D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002813:Chondrosarcoma / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D016326:Extracellular Matrix Proteins; D018698:Glutamic Acid / Q000378:metabolism; D006868:Hydrolysis; D007106:Immune Sera; D037181:Lectins, C-Type; D008969:Molecular Sequence Data; D011509:Proteoglycans / Q000378:metabolism*; D051381:Rats; D014407:Tumor Cells, Cultured
C508321:Acan protein, rat; D053674:Aggrecans; D016326:Extracellular Matrix Proteins; D007106:Immune Sera; D037181:Lectins, C-Type; D011509:Proteoglycans; D018698:Glutamic Acid; D010450:Endopeptidases; C083641:aggrecanase; D000409:Alanine