Lgmn targets two distinct GPCRs, PAR2 and µ-OR1, and induces cell death in acute lymphoblastic leukemia through an intracellular Ca²⁺ imbalance triggered by ER Ca²⁺ release.

Legumain (Lgmn) is a virulence factor found in the protozoan parasites Blastocystis and Trichomonas, which affect both humans and animals. However, its specific targets and cytotoxic mechanisms on host cells are not well understood. Recent findings show that Lgmn cleaves PAR2 at the N(30)-R(31) residue, a site also targeted by L-asparaginase, a vital treatment for acute lymphoblastic leukemia (ALL), a severe hematologic cancer that poses high risks to children. This emphasizes the urgent need for more effective therapeutic strategies. Here, we demonstrate that Lgmn induces ER Ca(2+) release via the µ-OR1-G(αi) and PAR2-G(αq) pathways. In PAR2-knockdown ALL cells, the stimulation of adenylate cyclase (AC) with forskolin or treatment with 8-CPT-cAMP effectively inhibits Lgmn-induced µ-OR1-mediated ER Ca(2+) release, indicating that Lgmn's stimulation of µ-OR1 results in the downregulation of AC and a subsequent decrease in cAMP levels. Additionally, the PKA-specific inhibitor 14-22 amide alone triggers ER Ca(2+) release, and subsequent treatment with Lgmn does not enhance this effect, suggesting that PKA inhibition plays a role and that the Lgmn-µ-OR1-AC-cAMP axis can be bypassed in µ-OR1-mediated ER Ca(2+) release. Furthermore, we observed a corresponding reduction in the phosphorylation of PLCβ3 at Ser1105 and BAD at Ser118, both of which are regulated by PKA. The Lgmn-induced ER Ca(2+) release ultimately leads to apoptosis in ALL cells, which can be reversed by blocking ER Ca(2+) release. Our results thus provide novel insights into the specific targets of Lgmn secreted from the protozoa and demonstrate how this virulence factor induces cytotoxic effects on host cells, paving the way for innovative therapeutic strategies for patients with ALL.