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

Abstract

Objective We previously described a messenger RNA variant of ADAMTS4 (ADAMTS4-v1) 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 ADAMTS4-v1 is translated into a protein, is expressed in vivo, and acts as a functional aggrecanase. Methods 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 ADAMTS4 was mutated to contain the different sequences of ADAMTS4-v1, 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. Results 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 Glu373↓374Ala aggrecanase site. Conclusion 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. © The Authors. Arthritis & Rheumatism is published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology.