Abstract
Loss of articular cartilage because of extracellular matrix breakdown is the hallmark of arthritis. Degradative fragments of cartilage oligomeric matrix protein (COMP), a prominent noncollagenous matrix component in articular cartilage, have been observed in the cartilage, synovial fluid, and serum of arthritis patients. The molecular mechanism of COMP degradation and the enzyme(s) responsible for it, however, remain largely unknown. ADAMTS-12 (a disintegrin and metalloprotease with thrombospondin motifs) was shown to associate with COMP both in vitro and in vivo. ADAMTS-12 selectively binds to only the epidermal growth factor-like repeat domain of COMP of the four functional domains tested. The four C-terminal TSP-1-like repeats of ADAMTS-12 are shown to be necessary and sufficient for its interaction with COMP. Recombinant ADAMTS-12 is capable of digesting COMP in vitro. The COMP-degrading activity of ADAMTS-12 requires the presence of Zn2+ and appropriate pH (7.5-9.5), and the level of ADAMTS-12 in the cartilage and synovium of patients with both osteoarthritis and rheumatoid arthritis is significantly higher than in normal cartilage and synovium. Together, these findings indicate that ADAMTS-12 is a new COMP-interacting and -degrading enzyme and thus may play an important role in the COMP degradation in the initiation and progression of arthritis.
Highlights
More than 15% of the world population older than 18 years are affected by arthritic disorders, including osteoarthritis (OA)3 and rheumatoid arthritis (RA) [1]
ADAMTS-12 Associates with Cartilage oligomeric matrix protein (COMP) in Yeast—Our unpublished observation that the EGF-like domain of COMP binds to the C-terminal TSP1like repeats of ADAMTS-7, whose domain organization and structure are similar to those of ADAMTS-12, prompted us to investigate whether ADAMTS-12 interacts with COMP
The plasmid encoding the EGF-like domain of COMP linked to Gal4DBD and the plasmid encoding the four C-terminal TSP1-like repeats of ADAMTS-12 fused to VP16AD were used to cotransform the yeast strain MAV203
Summary
More than 15% of the world population older than 18 years are affected by arthritic disorders, including osteoarthritis (OA)3 and rheumatoid arthritis (RA) [1]. In Vitro GST Pulldown Assay—To determine whether COMP binds to ADAMTS-12 in vitro, glutathione-Sepharose beads (50 l) preincubated with either purified GST (0.5 g; serving as control) or GSTCOMP-EGF (0.5 g) were incubated with 500 g of cell lysates prepared from COS-7 cells transfected with an expression plasmid either wild-type ADAMTS-12 (pcDNA3-ADAMTS12-HA) or ADAMTS-12 with mutant catalytic domain (pcDNA3-ADAMTS12-MUT, provided by Drs Cal and Lopez-Otin) [39] in 150 l of buffer AM
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