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Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site engineering of sphingomyelin synthases.

ABSTRACT: SM is a fundamental component of mammalian cell membranes that contributes to mechanical stability, signaling, and sorting. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, a reaction catalyzed by SM synthase (SMS) 1 in the Golgi and SMS2 at the plasma membrane. Mammalian cells also synthesize trace amounts of the SM analog ceramide phosphoethanolamine (CPE), but the physiological relevance of CPE production is unclear. Previous work revealed that SMS2 is a bifunctional enzyme producing both SM and CPE, whereas a closely related enzyme, sphingomyelin synthase-related protein (SMSr)/SAMD8, acts as a monofunctional CPE synthase in the endoplasmatic reticulum. Using domain swapping and site-directed mutagenesis on enzymes expressed in defined lipid environments, we here identified structural determinants that mediate head group selectivity of SMS family members. Notably, a single residue adjacent to the catalytic histidine in the third exoplasmic loop profoundly influenced enzyme specificity, with glutamic acid permitting SMS-catalyzed CPE production and aspartic acid confining the enzyme to produce SM. An exchange of exoplasmic residues with SMSr proved sufficient to convert SMS1 into a bulk CPE synthase. This allowed us to establish mammalian cells that produce CPE rather than SM as the principal phosphosphingolipid and provide a model of the molecular interactions that impart catalytic specificity among SMS enzymes.

SUBMITTER: Kol M

PROVIDER: S-EPMC4918856 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site engineering of sphingomyelin synthases.

Kol Matthijs M Panatala Radhakrishnan R Nordmann Mirjana M Swart Leoni L van Suijlekom Leonie L Cabukusta Birol B Hilderink Angelika A Grabietz Tanja T Mina John G M JG Somerharju Pentti P Korneev Sergei S Tafesse Fikadu G FG Holthuis Joost C M JC

Journal of lipid research 20160510 7

SM is a fundamental component of mammalian cell membranes that contributes to mechanical stability, signaling, and sorting. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, a reaction catalyzed by SM synthase (SMS) 1 in the Golgi and SMS2 at the plasma membrane. Mammalian cells also synthesize trace amounts of the SM analog ceramide phosphoethanolamine (CPE), but the physiological relevance of CPE production is unclear. Previous work revealed that SM ...[more]

PMID: 27165857

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Project description:ObjectiveSphingomyelin synthase (SMS) catalyzes the conversion of ceramide to sphingomyelin and sits at the crossroads of sphingolipid biosynthesis. SMS has 2 isoforms: SMS1 and SMS2. Although they have the same SMS activity, they are different enzymes with distinguishable subcellular localizations and cell expression patterns. It is conceivable that these differences could yield different consequences, in terms of sphingolipid metabolism and its related atherogenesis.Methods and resultsWe created Sms1 gene knockout mice and found that Sms1 deficiency significantly decreased plasma, liver, and macrophage sphingomyelin (59%, 45%, and 54%, respectively), but only had a marginal effect on ceramide levels. Surprisingly, we found that Sms1 deficiency dramatically increased glucosylceramide and GM3 levels in plasma, liver, and macrophages (4- to 12-fold), whereas Sms2 deficiency had no such effect. We evaluated the total SMS activity in tissues and found that Sms1 deficiency causes 77% reduction in SMS activity in macrophages, indicating SMS1 is the major SMS in macrophages. Moreover, Sms1-deficient macrophages have a significantly higher glucosylceramide synthase activity. We also found that Sms1 deficiency significantly attenuated toll-like 4 receptor-mediated nuclear factor-κB and mitogen-activated protein kinase activation after lipopolysaccharide treatment. To evaluate atherogenicity, we transplanted Sms1 knockout mouse bone marrow into low-density lipoprotein receptor knockout mice (Sms1(-/-)→Ldlr(-/-)). After 3 months on a western diet, these animals showed a significant decrease of atherosclerotic lesions in the root and the entire aorta (35% and 44%, P<0.01, respectively) and macrophage content in lesions (51%, P<0.05), compared with wild-type→Ldlr(-/-) mice.ConclusionsSms1 deficiency decreases sphingomyelin, but dramatically increases the levels of glycosphingolipids. Atherosclerosis in Sms1(-/-)→Ldlr(-/-) mice is significantly decreased.

Dataset Information

Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site engineering of sphingomyelin synthases.

Publications

Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site engineering of sphingomyelin synthases.

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