OC 49.1 - Arginine Methylation by Protein arginine methyltransferase 1 (PRMT1) on ADAMTS13 Affects Its Secretion and Proteolytic Activity

Background: ADAMTS13 (A Disintegrin And Metalloproteinase with ThromboSpondin type 1 repeats, 13) is a critical plasma metalloprotease, synthesized primarily in hepatic stellate cells and endothelial cells. It plays a pivotal role in regulating adhesion and aggregation of platelets through proteolytic cleavage of von Willebrand factor (VWF). Severe deficiency of plasma ADAMTS13 activity results in a life-threatening blood disorder thrombotic thrombocytopenic purpura (TTP). ADAMTS13 undergoes posttranslational modifications including glycosylation, citrullination, and oxidation.

Aims: The present study determines the impact of arginine methylation, catalyzed by protein arginine methyltransferase 1 (PRMT1), on ADAMTS13 secretion and function.

Methods: Biochemical, cell culture, recombinant expression, and mass spectrometric analysis, as well as an animal model will be employed.

Results: We showed that inhibition of arginine methylation by a PRMT1 inhibitor led to significant reduction of recombinant ADAMTS13 secretion from HEK293 cells stably expressing recombinant ADAMTS13. However, the secreted recombinant ADAMTS13 exhibited an increased proteolytic activity towards FRETS-VWF73 substrate. Conversely, an overexpression of PRMT1 in the stably transfected HEK293 significantly increased recombinant ADAMTS13 secretion but the secreted recombinant ADAMTS13 exhibited a reduced proteolytic activity (Fig.1). Transgenic mice overexpressing PRMT1 in prmt1-/- background showed an increased plasma ADAMTS13 antigen and reduced ADAMTS13 proteolytic activity (Fig. 2). LC-MS/MS mass spectrometry identified more than 100 arginine methylation events on purified recombinant ADAMTS13. Site-directed mutagenesis on several conserved methylation sites among zebrafish, mouse, and human including R193K, R498K, R692K, R1123K, and R1206K revealed the critical role of arginine 1206 in ADAMTS13 secretion and proteolytic activity. Recombinant ADAMTS13 R1206K variant exhibited 4-5 fold increase in its proteolytic activity compared with the wild type control.

Conclusion(s): Our results demonstrate for the first time the importance of arginine methylation on ADAMTS13 in regulation of its secretion and proteolytic activity. Our findings may help resign novel therapeutics for TTP and other inflammatory and thrombotic disorders.