L-asparaginase is a PAR2 N-terminal protease that unmasks the PAR2 tethered ligand.

L-asparaginase is an indispensable chemotherapeutic drug for patients with acute lymphoblastic leukemia (aLL), a life-threatening lymphoid neoplasm and the prime cause of cancer death among children. Previously, we reported that L-asparaginase kills aLL cells via an excessive rise in [Ca(2+)](i) due to IP3R-mediated ER Ca(2+) release followed by stimulation of the intrinsic apoptotic pathway (Blood, 133, 2222-2232). We also demonstrated that L-asparaginase triggers ER Ca(2+) release by targeting the G-protein-coupled receptor (GPCR), protease-activated receptor 2 (PAR2) (Cell Death & Discovery, 10:366). However, how L-asparaginase stimulates PAR2 remains unknown. Here, we show that elastase, which can disarm trypsin-mediated PAR2 activation by cleaving a S(67)-V(68) residue downstream of the tethered ligand (TL) and removing it from PAR2, abrogates L-asparaginase-induced ER Ca(2+) release, indicating that L-asparaginase targets the TL-containing PAR2 N-terminal extracellular domain to induce ER Ca(2+) release. Inactive forms (T(111)V/K(184)T or D(112)T/K(184)T) of L-asparaginase do not induce ER Ca(2+) release in μ-opioid receptor 1 (µ-OR1)-knockdown aLL cells, suggesting that L-asparaginase action on PAR2 requires its enzymatic activity. Time-lapse confocal microscopy of cells expressing mRFP-hPAR2-eYFP and nanoluciferase (Nluc) reporter release assays of cells expressing Nluc-hPAR2-eYFP showed that L-asparaginase cleaves PAR2 at the N-terminal extracellular I(26)-G(71) domain. Cleavage assay of a PAR2 N-terminal peptide by L-asparaginase and subsequent LC-MS/MS analysis show that L-asparaginase is a PAR2 protease that cleaves N(30)-R(31) and R(31)-S(32) residues, unmasking the PAR2 TL. Thus, our findings reveal for the first time the molecular mechanism through which L-asparaginase activates PAR2, leading to perturbation of intracellular Ca(2+) homeostasis and aLL cell apoptosis.