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

J Biol Chem. 2016 Feb 12;291(7):3197-208. doi: 10.1074/jbc.M115.704817. Epub 2015 Dec 14.

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.

Keywords: ADAMTS; ADAMTS5; aggrecan; cartilage; cartilage biology; metalloprotease; oligomer; oligomerization.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADAM Proteins / chemistry
  • ADAM Proteins / genetics
  • ADAM Proteins / isolation & purification
  • ADAM Proteins / metabolism*
  • ADAMTS5 Protein
  • Aggrecans / isolation & purification
  • Aggrecans / metabolism*
  • Animals
  • Arthritis, Experimental / enzymology*
  • Arthritis, Experimental / immunology
  • Arthritis, Experimental / pathology
  • Catalytic Domain
  • Cross-Linking Reagents / chemistry
  • Crosses, Genetic
  • Dimerization
  • Enzyme Activation
  • Gene Deletion
  • HEK293 Cells
  • Humans
  • Knee Joint / enzymology*
  • Knee Joint / immunology
  • Knee Joint / pathology
  • Mice, Inbred C57BL
  • Mice, Mutant Strains
  • Molecular Weight
  • Mutant Proteins
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / isolation & purification
  • Peptide Fragments / metabolism
  • Protein Interaction Domains and Motifs
  • Proteolysis
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism

Substances

  • Acan protein, mouse
  • Aggrecans
  • Cross-Linking Reagents
  • Mutant Proteins
  • Peptide Fragments
  • Recombinant Fusion Proteins
  • ADAM Proteins
  • ADAMTS5 Protein
  • ADAMTS5 protein, human
  • Adamts5 protein, mouse