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Degradation of the cartilage proteoglycan aggrecan is an early event in the development of osteoarthritis, and a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4) and ADAMTS-5 are considered to be the major aggrecan-degrading enzymes. We have recently found that ADAMTS-5 is rapidly endocytosed via low density lipoprotein receptor-related protein 1 (LRP1) and degraded by chondrocytes. Here we report that this regulatory mechanism also applies to ADAMTS-4, although its rate of endocytosis is slower than that of ADAMTS-5. Domain deletion mutagenesis of ADAMTS-4 identified that the cysteine-rich and spacer domains are responsible for binding to LRP1, whereas the thrombospondin 1 and spacer domains are responsible in ADAMTS-5. The estimated t½ value of ADAMTS-4 endocytosis was about 220 min, whereas that of ADAMTS-5 was 100 min. The difference in half-lives between the two enzymes is explained by the 13-fold lower affinity of ADAMTS-4 for LRP1 compared with that of ADAMTS-5. Studies using soluble ligand binding clusters of LRP1 showed that ADAMTS-4 binds to clusters II and IV with similar KD,app values of 98 and 73 nm, respectively, whereas ADAMTS-5 binds to cluster II, III, and IV with KD,app values of 3.5, 41, and 9 nm, respectively. Thus, ADAMTS-5 competitively inhibits ADAMTS-4 endocytosis but not vice versa. This study highlights that the affinity between a ligand and LRP1 dictates the rate of internalization and suggests that LRP1 is a major traffic controller of the two aggrecanases, especially under inflammatory conditions, where the protein levels of ADAMTS-4 increase, but those of ADAMTS-5 do not.

Original publication

DOI

10.1074/jbc.m113.545376

Type

Journal article

Journal

The Journal of biological chemistry

Publication Date

03/2014

Volume

289

Pages

6462 - 6474

Addresses

From the Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Roosevelt Drive, Headington, Oxford OX3 7FY, United Kingdom.

Keywords

Cartilage, Articular, Cells, Cultured, Animals, Swine, Humans, Osteoarthritis, Procollagen N-Endopeptidase, Endocytosis, Sequence Deletion, Catalytic Domain, Protein Binding, Half-Life, ADAM Proteins, Low Density Lipoprotein Receptor-Related Protein-1, ADAMTS4 Protein, ADAMTS5 Protein