Glycosaminoglycans act as activators of peptidylarginine deiminase 4

Peptidylarginine deiminase 4 (PAD4) is a citrullinating enzyme that is gathering increasing attention due to its possible involvement in physiological processes as well as in the pathogenesis of diseases like rheumatoid arthritis or thrombosis. PAD4 is activated by calcium ions, but the details of this mechanism are elusive, because in the human body, Ca2+ concentrations are too low for full activity. Given that glycosaminoglycans (GAGs) are also implicated in the development and progression of rheumatoid arthritis, we investigated the activation of PAD4 by GAGs using heparin as a model. We employed activity assays, chromatography techniques, molecular interaction measurements (MST and SPR), FACS, and immunocytochemistry to demonstrate the activation of PAD4 by GAGs. Our data show that PAD4 binds heparin with high affinity and forms high molecular weight complexes with heparin, consistent with heparin-bound tetramer formation. Heparin activates PAD4 by increasing the enzymes Ca2+ affinity threefold. We also show that the effectiveness of activation with heparin depends on the length of GAG used and its negative charge. Direct measurement of heparin binding to PAD4 confirmed tight interaction with nanomolar affinity. Mutagenesis of regions likely responsible for heparin binding showed that dimerization of PAD4 is necessary for efficient activation, but the distinct binding site was not determined as interaction with heparin likely occurs over larger surface of PAD4. Furthermore, we show that other GAG family members, including heparan and chondroitin sulphates, are also able to activate PAD4. We also found that disturbed production of GAGs by CHO cells results in reduced PAD4 binding efficiency. Finally, heparin induces NETosis in hPMNs in concentration-dependent manner, as measured by the release of DNA and citrullination of histone H3. In summary, we identify the first natural coactivator of PAD4, which is present in all individuals, potentially explaining the regulation of PAD4 activity in physiological conditions, and providing new insight into the development of rheumatoid arthritis and other PAD4-related diseases.