Characterization of an ADAMTS-5-mediated cleavage site in aggrecan in OSM-stimulated bovine cartilage.
OBJECTIVE
In a previous study, we identified a 50-kDa G3-containing aggrecan degradation product in bovine cartilage, released from the tissue after interleukin-1 (IL-1) stimulation in the presence of oncostatin M (OSM). Our objective was to purify, determine the N-terminal sequence of this fragment and verify whether this cleavage could be attributed to a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 action in vitro.


METHODS
Collected media from bovine cartilage explant cultures stimulated with IL-1+OSM were subjected to anion-exchange chromatography. The N-terminal sequence of the fragment of interest in the purified fractions was determined by automated Edman sequencing. Fetal bovine aggrecan was digested with full-length recombinant ADAMTS-4 and ADAMTS-5 and resulting degradation products were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) and immunoblotting using an anti-G3 antiserum and an anti-neoepitope antibody that had been generated to the new N-terminus of the G3 fragment.


RESULTS
Characterization of the 50-kDa fragment showed that it possesses chondroitin sulfate (CS) and is the result of a cleavage within the C-terminal portion of the CS-2 domain, adjacent to the G3 region. Sequence analysis identified the cleavage region as TQRPAE(2047)-(2048)ARLEIE, suggesting an aggrecanase-derived product. Using an anti-neoepitope antibody specific for the additional cleavage site, it was shown that the product is generated in vitro upon digestion of aggrecan by ADAMTS-5 and, to a much lesser extent, by ADAMTS-4.


CONCLUSIONS
The abundance and rapid rate of release of this degradation product in organ cultures in the presence of OSM suggest that it could result from a unique aggrecan proteolysis mediated by aggrecanases.
Durigova
M
M
Shriners Hospital for Children, 1529 Cedar Avenue, Montreal, Quebec H3G 1A6, Canada.
Soucy
P
P
Fushimi
K
K
Nagase
H
H
Mort
J S
JS
Roughley
P J
PJ
D051722:ADAM Proteins / Q000737:chemistry / Q000378:metabolism*; D053674:Aggrecans / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002358:Cartilage, Articular / Q000378:metabolism; D002417:Cattle; D019902:Chondrocytes / Q000378:metabolism; D002809:Chondroitin Sulfates / Q000737:chemistry; D007375:Interleukin-1; D053683:Oncostatin M; D011509:Proteoglycans / Q000737:chemistry / Q000378:metabolism*
D053674:Aggrecans; D007375:Interleukin-1; D011509:Proteoglycans; D053683:Oncostatin M; D002809:Chondroitin Sulfates; D051722:ADAM Proteins