Sphingomyelin synthase‐related protein generates diacylglycerol via hydrolysis of phosphatidic acid, phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine without ceramide

Abstract

Introduction Sphingomyelin synthase-related protein (SMSr) produces diacylglycerol (DG) and ceramide phosphoethanolamine (CPE) by the transfer of phosphoethanolamine from phosphatidylethanolamine (PE) to ceramide. We previously reported that SMSr functionally interacts with DG kinase (DGK) δ via steraile α motif domains (SAMD), protein-protein interaction module [Murakami et al. J. Biol. Chem. 295, 2932–47, 2020]. Moreover, SMSr provided DG to DGKδ. Puzzlingly, mammalian SMSr produces only trace amounts of CPE and DG. However, we previously reported that DG levels, particularly saturated fatty acid (SFA)- and/or monounsaturated fatty acid (MUFA)-containing DG molecular species, were significantly increased in COS-7 cells overexpressing human SMSr. To clarify this discrepancy, we purified human SMSr and determined whether purified SMSr showed other DG-generating activity via hydrolyis of phospholipids without ceramide in the present study. Results and Conclusion Human SMSr was expressed using the baculovirus-insect cell expression system and highly purified. Interestingly, purified SMSr showed DG-generating activity by hydrolysis of PE, phosphatidylcholine (PC), phosphatidylinositol (PI) and phosphatidic acid (PA) in the absence of ceramide. PA phosphatase (PAP) activity of SMSr was about 300-fold stronger than CPE synthase activity. Moreover, SMSr showed substrate selectivity for SFA- and/or MUFA-containing PA molecular species, but not arachidonic acid-containing PA, which is exclusively generated in the PI(4,5)P2 cycle. We confirmed that SMSr expressed in COS-7 cells showed PAP and PI-phospholipase C (PLC) activities. Collectively, our study indicated that SMSr possesses previously unrecognized enzyme activities (multi-glycerophospholipid PLC hydrolase) and constitutes a novel DG signaling pathway together with DGKd independent of the PI(4,5)P2 cycle.