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

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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 [Ca2+]i due to IP3R-mediated ER Ca2+ release followed by stimulation of the intrinsic apoptotic pathway (Blood, 133, 2222-2232). We also demonstrated that L-asparaginase triggers ER Ca2+ 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 S67-V68 residue downstream of the tethered ligand (TL) and removing it from PAR2, abrogates L-asparaginase-induced ER Ca2+ release, indicating that L-asparaginase targets the TL-containing PAR2 N-terminal extracellular domain to induce ER Ca2+ release. Inactive forms (T111V/K184T or D112T/K184T) of L-asparaginase do not induce ER Ca2+ 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 I26-G71 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 N30-R31 and R31-S32 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 Ca2+ homeostasis and aLL cell apoptosis.