ADAMTS‐4_v1 Is a Splice Variant of ADAMTS‐4 That Is Expressed as a Protein in Human Synovium and Cleaves Aggrecan at the Interglobular Domain. Issue 11 (28th October 2013)

• Record Type: Journal Article
• Authors: Wainwright, Shane D.; Bondeson, Jan; Caterson, Bruce; Hughes, Clare E.
• Is Part Of: Arthritis and rheumatism. Volume 65:Issue 11(2013:Nov.)
• Page Start: 2866
• Page End: 2875
• Publication Date: 2013-10-28
• Subjects: Arthritis -- Periodicals; Rheumatism -- Periodicals; Arthritis -- Periodicals; Rheumatic Diseases -- Periodicals; Rhumatisme -- Périodiques; Arthrite -- Périodiques; 616.72
• DOI: 10.1002/art.38102
• Languages: English
• ISSNs: 0004-3591

Abstract

<abstract abstract-type="main"> <title> <x xml:space="preserve">Abstract</x> </title> <sec id="art38102-sec-0001" sec-type="section"> <title>Objective</title> <p>We previously described a messenger RNA variant of <italic>ADAMTS4</italic> (<italic>ADAMTS4_v1</italic>) in human synovial cell cocultures obtained from patients with osteoarthritis (OA). This RNA message has been found only in OA synovium and, if translated, would result in a protein identical to ADAMTS‐4, except that the C‐terminal spacer domain would be different. The purpose of this study was to determine whether <italic>ADAMTS4_v1</italic> is translated into a protein, is expressed in vivo, and acts as a functional aggrecanase.</p> </sec> <sec id="art38102-sec-0002" sec-type="section"> <title>Methods</title> <p>Polyclonal antibodies were raised against unique C‐terminal sequences of ADAMTS‐4_v1. An immunohistochemical study of human OA synovium was performed. A mammalian expression vector coding for FLAG‐tagged human <italic>ADAMTS4</italic> was mutated to contain the different sequences of <italic>ADAMTS4_v1</italic>, and the resultant plasmid was used to transfect HEK 293 cells. ADAMTS‐4_v1 produced by these cells was purified via the FLAG epitope, and the ability of this recombinant protein to cleave aggrecan, biglycan, and decorin was investigated.</p> </sec> <sec id="art38102-sec-0003" sec-type="section"> <title>Results</title> <p>An antibody specific for ADAMTS‐4_v1 was found to bind to the synovial membrane surface on cryosections, and the protein was detected in cell lysates from synovium obtained from OA patients. The recombinant ADAMTS‐4_v1 demonstrated enzyme activity toward the target substrate in a commercial aggrecanase 1 enzyme‐linked immunosorbent assay and was also found to cleave aggrecan at the pathologically important Glu<sup>373↓374</sup>Ala aggrecanase site.</p> </sec> <sec id="art38102-sec-0004" sec-type="section"> <title>Conclusion</title> <p>ADAMTS‐4_v1 is expressed as a protein in vivo in human OA synovium, functions as an aggrecanase, and cleaves other proteoglycan substrates. This splice variant may be a major contributor to loss of aggrecan from the superficial zone of OA cartilage.</p> </sec> </abstract>