Abstract
Besides bulk amounts of SM, mammalian cells produce small quantities of the SM analog ceramide phosphoethanolamine (CPE). Little is known about the biological role of CPE or enzymes responsible for CPE production. Heterologous expression studies revealed that SM synthase (SMS)2 is a bifunctional enzyme producing both SM and CPE, whereas SMS-related protein (SMSr) serves as monofunctional CPE synthase. Acute disruption of SMSr catalytic activity in cultured cells causes a rise in endoplasmic reticulum (ER) ceramides, fragmentation of ER exit sites, and induction of mitochondrial apoptosis. To address the relevance of CPE biosynthesis in vivo, we analyzed the tissue-specific distribution of CPE in mice and generated mouse lines lacking SMSr and SMS2 catalytic activity. We found that CPE levels were >300-fold lower than SM in all tissues examined. Unexpectedly, combined inactivation of SMSr and SMS2 significantly reduced, but did not eliminate, tissue-specific CPE pools and had no obvious impact on mouse development or fertility. While SMSr is widely expressed and serves as the principal CPE synthase in the brain, blocking its catalytic activity did not affect ceramide levels or secretory pathway integrity in the brain or any other tissue. Our data provide a first inventory of CPE species and CPE-biosynthetic enzymes in mammals.
Highlights
Besides bulk amounts of SM, mammalian cells produce small quantities of the SM analog ceramide phosphoethanolamine (CPE)
Studies in cultured cells previously revealed a critical role of SMS-related protein (SMSr) in ceramide homeostasis and as suppressor of ceramide-induced mitochondrial apoptosis
We show that ubiquitous catalytic inactivation of SMSr in mice primarily disrupts CPE biosynthesis in the brain without having any obvious impact on steady state ceramide levels, cell integrity, or survival
Summary
Besides bulk amounts of SM, mammalian cells produce small quantities of the SM analog ceramide phosphoethanolamine (CPE). While SMSr is widely expressed and serves as the principal CPE synthase in the brain, blocking its catalytic activity did not affect ceramide levels or secretory pathway integrity in the brain or any other tissue. Mammalian cells contain two SMS isoforms, namely, Abbreviations: Ad.GFP, adenovirus expressing only green fluorescent protein; Ad.PEMT, adenovirus expressing both phosphatidylethanolamine N-methyltransferase and green fluorescent protein; CPE, ceramide phosphoethanolamine; DTA, diphtheria toxin A; eGFP, (enhanced) green fluorescent protein; ER, endoplasmic reticulum; ES, embryonic stem; Flp, flippase; frt, Flp recognition target; GFP, green fluorescent protein; HR, homologous region; lacZ, -galactosidase gene; loxP, locus of X-over P1; mSMSr, mouse sphingomyelin synthase-related protein; NBD, nitrobenzoxadiazole; NBD-Cer, nitrobenzoxadiazole-ceramide; NBDGlcCer, nitrobenzoxadiazole glucosylceramide; PC, phosphatidylcholine, PE, phosphatidylethanolamine; PEMT; phosphatidylethanolamine N-methyltransferase; SAM, sterile ␣ motif; SMS, sphingomyelin synthase; SMSr, sphingomyelin synthase-related protein; UTR, untranslated region. Together with a closely related enzyme, SMS-related protein (SMSr), they form the SMS protein family [9]
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