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 peptides, saposins A, B, C, and D, that catalyze sphin-golipidases to promote sphingolipid breakdown. Considering the key role of PSAP and saposins in sphingolipid metabolism and the existence of genetic mutations in PSAP associated with juve-nile-onset lysosomal storage and adult-onset neurodegenerative diseases, maintaining optimal levels of PSAP and saposins is crucial for proper lysosomal function and sphingolipid homeosta-sis. 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 which regulate prosaposin cleavage into saposins. In vitro cleavage assays identified multiple enzymes that can process human prosaposin into multi- and single-saposin fragments in a pH-dependent manner. We confirmed the role of cathepsins D and B in PSAP processing and showed that these and several previously unidentified lysosomal proteases (cathepsins E, K, L, S, V, G and AEP/LGMN) are able to process PSAP in distinctive, pH-dependent manners. In addition, we have demonstrated a novel role for progranulin (PGRN) in the regulation of PSAP cleavage. We found that PGRN and multi-granulin fragments (MGFs) directly regulate the cleavage of PSAP by cathepsin D. With this study, we have identified 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.