Abstract
BACKGROUND: ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, 13), primarily synthesized in hepatic stellate and endothelial cells, plays a pivotal role in regulation of hemostasis by proteolytic cleavage of von Willebrand factor. Severe deficiency of plasma ADAMTS13 activity may result in thrombotic thrombocytopenic purpura, a potentially fatal blood disorder. ADAMTS13 undergoes posttranslational modifications including glycosylation, citrullination, oxidation. The present study determines the impact of arginine methylation by PRMT1 (protein arginine methyltransferase 1) on ADAMTS13 secretion and function. METHODS: Cell culture, recombinant protein, biochemical analysis, site-directed mutagenesis, and animal models were utilized. RESULTS: An inhibition of arginine methylation by a type I methyl transferase PRMT inhibitor (MS023) in HEK (human embryonic kidney) 293 cells expressing recombinant ADAMTS13 and in mice results in a significant reduction of ADAMTS13 secretion, but the secreted ADAMTS13 shows an increased specific activity; conversely, an overexpression of PRMT1 in HEK-293 cells and in transgenic mice results in an increase of ADAMTS13 secretion, but the secreted ADAMTS13 exhibits a significantly reduced specific activity. The altered ADAMTS13 activity appeared to be related to its conformational changes. LC-MS/MS (liquid chromatography with tandem mass spectrometry) identified greater than100 arginine methylation events on purified recombinant ADAMTS13. Site-directed mutagenesis performed on 5 highly conserved methylation sites (R193, R498, R692, R1123, and R1206) identifies the critical role of R1206 in ADAMTS13 function. The ADAMTS13 R1206K variant exhibits a 4- to 5-fold increase of specific activity, likely resulting from an alleviation of allosteric inhibition. CONCLUSIONS: These results demonstrate the crucial role of arginine methylation in ADAMTS13 secretion and function. Our findings may shed new light on the mechanism of allosteric regulation of ADAMTS13, which may have a therapeutic implication.