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

Hansen J Kosasih,Karena Last,Fraser M Rogerson,Suzanne B Golub,Stephanie J Gauci,Vincenzo C Russo,Heather Stanton,Richard Wilson,Shireen R Lamande,Paul Holden,Amanda J Fosang

doi:10.1074/jbc.m115.704817

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 recombinant ADAMTS-5 formed large, reduction-sensitive oligomers with a nominal molecular mass of ∼ 400 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.

Highlights

(aggrecanase) [1] mediating proteolysis and loss of aggrecan from articular cartilage in arthritic diseases
When the level of induced aggrecan loss was compared between genotypes, the results showed that there was no significant difference between aggrecan loss from wild type and het TS5 null explants (p ϭ 0.0856), aggrecan loss from the het TS5⌬cat explants was significantly less than from wild type (p ϭ 0.0041) and significantly less than from the het TS5 null mice (p ϭ 0.0347) (Fig. 2A)
When increasing amounts of purified TS5–5-V5 competitor (Fig. 4E, lanes 3–5) or TS5– 6-V5 competitor (Fig. 4E, lanes 8 –11) were incubated with TS5-FLAG, levels of the EGE373 neoepitope were markedly reduced. These results suggest that TS5 oligomerization is required for aggrecanase activity, and that oligomerization is mediated via the Dis and/or Cat domains

Summary

Introduction

(aggrecanase) [1] mediating proteolysis and loss of aggrecan from articular cartilage in arthritic diseases. This unexpected result suggested the possibility of an interaction between wild type and mutant TS5 molecules in TS5ϩ/ ⌬cat cartilage, in which binding of the mutant TS5⌬cat protein, with its full set of ancillary domains, to wild type TS5 inhibits enzyme activity in a dominant-negative manner.

Results

Conclusion