Rapid cartilage degradation in the joints is observed in rheumatoid arthritis (RA). ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs-4) degrades aggrecan, the primary component of cartilage, therefore contributing to joint erosion in RA. The proteolytic activity of ADAMTS4 is inhibited by fibronectin (FN). FN is abundantly expressed in the synovia in RA and is modified by citrullination, the conversion of peptidylarginine to citrulline. This study aims to investigate the binding ability of citrullinated FN (cFN) to ADAMTS4 and the effect of cFN on aggrecanase activity.
The full-length recombinant ADAMTS4 was purified from HEK293 cells that were transiently transfected with a full-length cDNA coding for human ADAMTS4. A 40-kDa FN fragment exhibiting heparin binding was citrullinised with rabbit peptidylarginine deaminase. The binding activity of the full-length recombinant ADAMTS4 to cFN was investigated in an in vitro binding assay. The proteolytic activity of ADAMTS4 after incubation with cFN was determined using an aggrecanase activity kit, in which the ARGSVIL peptide is produced by digestion with aggrecanase.
cFN displayed significantly decreased binding activity with ADAMTS4 compared with FN. The full-length ADAMTS4 produced large amounts of the ARGSVIL peptide, but the amount was markedly decreased in the presence of FN. The production of this peptide approached the normal level when the full-length ADAMTS4 was incubated with cFN.
FN following citrullination is less effective in inhibiting the proteolytic activity of ADAMTS4. It is known that PADI4, an enzyme active in citrullination, is highly expressed in the synovial tissue in RA. Our results suggest that PADI4 in the RA synovium may contribute to cartilage destruction via the citrullination of FN.