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Journal

ADAMTS4_v1 is a splice variant of ADAMTS4 that is expressed as a protein in human synovium and cleaves aggrecan at the interglobular domain

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Published:25th Mar 2020

Objective:

We have previously reported a mRNA variant of ADAMTS4 (ADAMTS4_v1) in human synovial cell co-cultures obtained from osteoarthritic patients. This RNA message has only been found in OA synovium and if translated would result in a protein identical to ADAMTS4 except that the C-terminal spacer domain would be different. In this study, we set out to determine if ADAMTS4_v1 is translated into a protein, expressed in vivo and acts as a functional aggrecanase.

Methods:

Polyclonal antibodies were raised against unique C-terminus sequences of ADAMTS4_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 HEK293 cells. ADAMTS4_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 to ADAMTS4_v1 was found to bind to the synovial membrane surface on cryosections and the protein was detected in synovial cell lysates from human OA patient synovium. The recombinant ADAMTS4_v1 demonstrated enzyme activity towards the target substrate in a commercial aggrecanase-1 ELISA assay and was also found to cleave aggrecan at the pathologically important Glu373?374Ala aggrecanase site.

Conclusion:

ADAMTS4_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 superficial zone loss of aggrecan in OA cartilage.

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