Prosaposin Is Cleaved Into Saposins by Multiple Cathepsins in a Progranulin‐Regulated Fashion

Prosaposin (PSAP) is a lysosomal protein that plays a key role in sphingolipid metabolism. PSAP is cleaved into four bioactive disulfide-rich saposins (SapA, SapB, SapC, and SapD) that catalyze sphingolipidases to promote sphingolipid breakdown. Maintaining optimal levels of PSAP and saposins is crucial for proper lysosomal function and sphingolipid homeostasis, and PSAP dysfunction is associated with juvenile-onset lysosomal storage disorders and age-associated neurodegenerative disorders. Despite this, the mechanism by which saposins are released from PSAP, and thus available to modulate sphingolipidases, sphingolipid homeostasis, and downstream lysosomal function, is not well understood. Here, we performed a comprehensive study to identify lysosomal enzymes that regulated prosaposin cleavage into saposins. In vitro cleavage assays identified multiple enzymes that could process human prosaposin into multi- and single-saposin fragments. We confirmed the role of cathepsins D and B in PSAP processing and identified several additional lysosomal proteases (cathepsins E, K, L, S, V, G, and asparagine-specific endopeptidase) that were able to process PSAP in distinctive, pH-dependent manners. In addition, we found that PGRN and multi-granulin fragments (MGFs) directly regulated the cleavage of PSAP by cathepsin D. With this study, we have shown that multiple cathepsins, PGRN, and MGFs work in concert to produce saposins under different conditions, which could present novel opportunities to modulate saposin levels in disease.