A Disintegrin and Metalloproteinase with Thrombospondin Motifs-5 (ADAMTS-5) Forms Catalytically Active Oligomers

Abstract

The metalloproteinase ADAMTS-5 (A disintegrin and metalloproteinase with thrombospondin motifs) degrades aggrecan, a proteoglycan essential for cartilage structure and function. ADAMTS-5 is the major aggrecanase in mouse cartilage, and is also likely to be the major aggrecanase in humans. ADAMTS-5 is a multidomain enzyme, but the function of the C-terminal ancillary domains is poorly understood. We show that mutant ADAMTS-5 lacking the catalytic domain, but with a full suite of ancillary domains inhibits wild type ADAMTS activity, in vitro and in vivo, in a dominant-negative manner. The data suggest that mutant ADAMTS-5 binds to wild type ADAMTS-5; thus we tested the hypothesis that ADAMTS-5 associates to form oligomers. Co-elution, competition, and in situ PLA experiments using full-length and truncated recombinant ADAMTS-5 confirmed that ADAMTS-5 molecules interact, and showed that the catalytic and disintegrin-like domains support these intermolecular interactions. Cross-linking experiments revealed that recombinantADAMTS-5formedlarge,reduction-sensitiveoligomers with a nominal molecular mass of ϳ400 kDa. The oligomers were unimolecular and proteolytically active. ADAMTS-5 truncates comprising the disintegrin and/or catalytic domains were able to competitively block full-length ADAMTS-5-mediated aggrecan cleavage, measured by production of the G1-EGE 373 neoepitope. These results show that ADAMTS-5 oligomerization is required for full aggrecanase activity, and they provide evidence that blocking oligomerization inhibits ADAMTS-5 activity. The data identify the surface provided by the catalytic and disintegrin-like domains of ADAMTS-5 as a legitimate target for the design of aggrecanase inhibitors.ADAMTS-5 (TS5) is one member of a family of secreted, multidomain, zinc-dependent proteinases known as A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS).2 TS5 is a major aggrecan-degrading enzyme (aggrecanase) (1) mediating proteolysis and loss of aggrecan from articular cartilage in arthritic diseases. TS5 inhibitors are currently in development for arthritis therapies (2, 3), although ADAMTS-4 (TS4) might also have a role in human joint disease (4 -6). LGHG (7, 8). Fragments arising from proteolysis at these sites have been detected in cartilage and synovial fluids from patients with arthritic diseases including rheumatoid arthritis, osteoarthritis, and juvenile idiopathic arthritis (9 -12).TS5 is a multidomain enzyme comprising an N-terminal pro (Pro)-domain, a catalytic (Cat) domain, a disintegrin (Dis)-like domain, two thrombospondin (TS)-type 1 motifs, a cysteinerich (CysR) region, and a spacer (Sp) domain as reviewed previously (13-15). It is expressed as a zymogen that requires cleavage by pro-protein convertases (16, 17) at a polyarginine consensus sequence to remove the pro-domain. Further processing at the C terminus by autocatalysis gives rise to multiple forms of TS5, and recombinant C-terminal truncates have altered enzyme activity and substrate specificity (18,19). The active and ...

ADAMTS-10 and -6 differentially regulate cell-cell junctions and focal adhesions

“…Secreted ADAMTS6 was also found to form SDS resistant multimers, which are possibly trimeric and tetrameric in nature. It has been recently reported that ADAMTS5 also forms oligomers via the catalytic and disintegrin-like domains, with a nominal molecular weight of ~400 kDa31. Oligomerisation of ADAMTS5 was required for aggrecanase activity, which maybe also the case for the enzymic activity of ADAMTS6.…”

Interaction with macrophages attenuates equine fibroblast‐like synoviocyte ADAMTS5 (aggrecanase‐2) gene expression following inflammatory stimulation

“…We observed a variety of immunopositive bands present in the cell layers of EFLS but only one major band in the media fraction, which migrated at 50 kDa. This could be analogous to a similarly sized form missing the C‐terminal cysteine rich and spacer domains but still retaining the pro‐domain which was observed by Kosasih et al This would suggest that the most abundant isoform of ADAMTS5 secreted into the media by EFLS is not active. We should not currently rule out the possibility of other isoforms being present in the EFLS culture media however, as we used a precipitation method to concentrate the secreted protein which may have led to non‐uniform isoform enrichment.…”

“…We detected an approximately 50 kDa band, in western blot analysis of EFLS media (Fig. B), which potentially corresponds to ADAMTS5 which has undergone auto‐catalytic removal of the C‐terminal domains . After 6 h, levels of the 50 kDa ADAMTS5 were 2.9 times higher than controls.…”

“…Protein (40 µg) was extracted from each culture media sample using StrataClean™ Resin according to manufacturer protocol and eluted by boiling in 15 µl 1× SDS sample buffer. Cell lysate and culture media protein samples were run on SDS‐PAGE gels and blotted onto nitrocellulose membranes, which were then probed using a sheep anti‐mouse ADAMTS5 antibody (a kind gift from Amanda Fosang, Melbourne Australia) at a dilution of 1:500 followed by an anti‐goat/sheep IgG‐peroxidase secondary antibody at a dilution of 1:1000 (A9452; Sigma–Aldrich). Antibody localization was visualized using Western Lightening‐Plus chemiluminescence reagent (Perkin Elmer, Beaconsfield, UK), and imaged on a UVP ChemiDoc‐it imaging system.…”

Treatment of Dystrophic mdx Mice with an ADAMTS-5 Specific Monoclonal Antibody Increases the Ex Vivo Strength of Isolated Fast Twitch Hindlimb Muscles

“…This ADAMTS-5 mAb reduces aggrecan degradation via an allosteric lock mechanism. By binding to the catalytic and disintegrin-like domains of ADAMTS-5, the enzyme's ability to engage and cleave substrate is inhibited [33], as the catalytic and disintegrin-like domains are necessary for full proteolytic activity [34]. Intraperitoneal injection (IP) of the ADAMTS-5 mAb leads to distribution within the musculoskeletal system.…”