title: 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 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.
authors.affiliation: From the Department of Paediatrics, University of Melbourne, Parkville 3052, Australia, the Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville 3052, Australia.
authors.forename: Hansen J
authors.initials: HJ
authors.lastname: Kosasih
authors.affiliation: From the Department of Paediatrics, University of Melbourne, Parkville 3052, Australia, the Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville 3052, Australia.
authors.forename: Karena
authors.initials: K
authors.lastname: Last
authors.affiliation: From the Department of Paediatrics, University of Melbourne, Parkville 3052, Australia, the Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville 3052, Australia.
authors.forename: Fraser M
authors.initials: FM
authors.lastname: Rogerson
authors.affiliation: From the Department of Paediatrics, University of Melbourne, Parkville 3052, Australia, the Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville 3052, Australia.
authors.forename: Suzanne B
authors.initials: SB
authors.lastname: Golub
authors.affiliation: the Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville 3052, Australia.
authors.forename: Stephanie J
authors.initials: SJ
authors.lastname: Gauci
authors.affiliation: the Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville 3052, Australia.
authors.forename: Vincenzo C
authors.initials: VC
authors.lastname: Russo
authors.affiliation: From the Department of Paediatrics, University of Melbourne, Parkville 3052, Australia, the Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville 3052, Australia.
authors.forename: Heather
authors.initials: H
authors.lastname: Stanton
authors.affiliation: the University of Tasmania, Hobart 7000, Australia.
authors.forename: Richard
authors.initials: R
authors.lastname: Wilson
authors.affiliation: the Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville 3052, Australia.
authors.forename: Shireen R
authors.initials: SR
authors.lastname: Lamande
authors.affiliation: the Department of Orthopaedics & Rehabilitation, Oregon Health & Science University, Portland, Oregon 97239, and.
authors.forename: Paul
authors.initials: P
authors.lastname: Holden
authors.affiliation: From the Department of Paediatrics, University of Melbourne, Parkville 3052, Australia, the Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville 3052, Australia, amanda.fosang@mcri.edu.au.
authors.forename: Amanda J
authors.identifier: http://orcid.org/0000-0002-5523-5427
authors.initials: AJ
authors.lastname: Fosang
mesh_terms: D051722:ADAM Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000071116:ADAMTS5 Protein; D053674:Aggrecans / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001169:Arthritis, Experimental / Q000201:enzymology* / Q000276:immunology / Q000473:pathology; D020134:Catalytic Domain; D003432:Cross-Linking Reagents / Q000737:chemistry; D003433:Crosses, Genetic; D019281:Dimerization; D004789:Enzyme Activation; D017353:Gene Deletion; D057809:HEK293 Cells; D006801:Humans; D007719:Knee Joint / Q000201:enzymology* / Q000276:immunology / Q000473:pathology; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D008970:Molecular Weight; D050505:Mutant Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: C508320:Acan protein, mouse; D053674:Aggrecans; D003432:Cross-Linking Reagents; D050505:Mutant Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D051722:ADAM Proteins; D000071116:ADAMTS5 Protein; C121532:ADAMTS5 protein, human; C498489:Adamts5 protein, mouse
keywords: ADAMTS; ADAMTS5; aggrecan; cartilage; cartilage biology; metalloprotease; oligomer; oligomerization