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 Glu(373↓374) Ala 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.