Functional characterization of enzymes catalyzing ceramide phosphoethanolamine biosynthesis in mice

Bickert, Andreas; Ginkel, Christina; Kol, Matthijs; Dorp, Katharina vom; Jastrow, Holger; Degen, Joachim; Jacobs, René L.; Vance, Dennis E.; Winterhager, Elke; Dörmann, Peter; Somerharju, Pentti; Holthuis, Joost C. M.; Willecke, Klaus

doi:10.1194/jlr.m055269

Cited by 40 publications

(46 citation statements)

References 47 publications

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“…Swapping these resi dues with SMSr allowed conversion of the Golgi-resident SMS, SMS1, into a bulk producer of CPE and the establish ment of a mammalian cell line that synthesizes CPE rather than SM as the dominant phosphosphingolipid. CPE nor mally represents only a minor fraction of the mammalian sphingolipid pool, with steady state levels 300 to 1,500fold below those of SM (20). In contrast, CPE is the dominant phosphosphingolipid in most insects, including Drosophila (22,43).…”

Section: Discussionmentioning

confidence: 99%

“…After neu tralization with 0.3 M HCl, the lipids were extracted as above and dissolved in 40 l of LC/MS quality methanol. CPE and SM spe cies were separated on Acquity Ultra Performance LC system equipped with an Acquity BEHC18 1.0 × 150 mm column (Wa ters Inc.) as in Bickert et al (20). The column eluent was infused to a Quattro Premier mass spectrometer (Waters) operated in the positive ion mode, and the CPE and SM molecular species were detected using selective reaction monitoring.…”

Section: Lipid Msmentioning

confidence: 99%

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

Kol¹,

Panatala²,

Nordmann³

et al. 2016

Journal of Lipid Research

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Section: Discussionmentioning

confidence: 99%

Section: Lipid Msmentioning

confidence: 99%

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

Kol¹,

Panatala²,

Nordmann³

et al. 2016

Journal of Lipid Research

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“…These phenotypes are suppressed by blocking de novo ce ramide synthesis, stimulating ER export of ceramides or targeting SMS1 to the ER, supporting a role of SMSr as critical regulator of ER ceramide levels. However, ubiqui tous inactivation of SMSr catalytic activity in mice primarily disrupted CPE biosynthesis in the brain without any obvious impact on steadystate ceramide levels, cell integrity, or sur vival (20,21). These contradictory outcomes could be due to compensatory mechanisms that overcome a deregulation of ER ceramides over time.…”

Section: Synthesis Of Clickable Ceramide Analogmentioning

confidence: 99%

“…Besides bulk amounts of SM, mammals also produce small quantities of ceramide phos phoethanolamine (CPE), a widespread but poorly studied SM analog. We and others previously showed that SMS2 is a bifunctional enzyme that produces both SM and CPE on the cell surface, whereas a closely related enzyme, sphingo myelin synthaserelated protein (SMSr) SAMD8, acts as a monofunctional CPE synthase in the lumen of the ER (18)(19)(20)(21). Interestingly, SMSr is by far the bestconserved SMS family member, with homologs in insects and various marine organisms that lack SM (18,22).…”

Section: Synthesis Of Clickable Ceramide Analogmentioning

confidence: 99%

“…SMS1null cells display only residual SMS ac tivity (15%) compared with control KBM7 cells (38). This residual activity is likely due to SMS2, which is barely expressed in lymphoid cells (13,20). SMS1null cells stably expressing HAtagged SMS1 or SMS1 CPE were created by retroviral transduction and expression of the enzymes was verified by immunoblotting (Fig.…”

Section: Switching Head Group Selectivity In Mammalian Sphingolipid Bmentioning

confidence: 99%

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Erratum

2017

Journal of Lipid Research

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821Regarding "Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site-engineering of sphingomyelin synthases" ( J. Lipid Res. 57: 1273-1285 : This article has been withdrawn by the authors. The left panel of Fig. 1E contained data from their previously published work (Bickert et al., without proper attribution in the figure legend. Additionally, one lane in this panel represented the results of different experimental conditions due to an inadvertent error in cropping of the original TLC image. As the authors state that these errors were unintentional and do not affect the conclusions of the work, the article will be resubmitted by them to JLR upon correction of Fig. 1E. SM is a major structural component of mammalian cell membranes and one of the end points in sphingolipid bio synthesis. The bulk of SM is produced in the Golgi lumen and delivered by vesicular transport to the plasma mem brane, where it accumulates in the exoplasmic leaflet (1). Owing to its unique ability to form extensive hydrogen bonds with other membrane molecules, SM participates in a multitude of cellular processes. SM is the preferred inter action partner of cholesterol, and this interaction has important physiological consequences. Cell surface SM degradation causes cholesterol to redistribute to the endo plasmatic reticulum (ER) (2), leading to downregulation of HMG-CoA reductase, the rate-limiting step in choles terol biosynthesis (3). Besides directly influencing cellular cholesterol homeostasis, SM (along with cholesterol) likely contributes to the high packing density and thickening of the lipid bilayer of the trans Golgi and plasma membrane, which may influence protein sorting through hydrophobic mismatching of membrane spans (4, 5). Specific inter actions between SM and membrane spans have been re ported to influence the activity of various integral membrane 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 p...

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Lipid composition of membrane microdomains isolated detergent‐free from PUFA supplemented RAW264.7 macrophages

Hellwing

Tigistu-Sahle

Fuhrmann

et al. 2017

Journal Cellular Physiology

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Profound alterations in the lipid profile of raft and non-raft plasma membrane microdomains were found when RAW264.7 macrophages were supplemented with polyunsaturated fatty acids (PUFAs) in physiologically relevant concentrations. For the first time lipids in the detergent-free isolated membrane domains of phagocytic immune cells were characterized by mass spectrometry. The extent of remodeling of the membrane lipids differed with different n3 and n6 PUFA supplements. The mildest effects were detected for α-linolenic acid (LNA) and linoleic acid (LA), the C18 precursors of the n3 and n6 families, respectively. When the effects of highly unsaturated PUFAs were compared, eicosapentaenoic acid (EPA) caused more extensive restructuring of membrane lipids than docosahexaenoic acid (DHA) or arachidonic acid (AA). The supplements altered the lipid species composition of both the raft and non-raft membrane fractions. The rafts containing elevated proportions of highly unsaturated lipid species may relocate sterically incompatible lipids and proteins originally belonging to this microdomain. Such effect was evident for sphingomyelin, which favored non-rafts instead of rafts after EPA supplementation. The current work suggests that the different functional consequences found previously when supplementing macrophages with either EPA or DHA have their origin in the different effects of these PUFAs on membrane architecture.

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Functional characterization of enzymes catalyzing ceramide phosphoethanolamine biosynthesis in mice

Cited by 40 publications

References 47 publications

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

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

Erratum

Lipid composition of membrane microdomains isolated detergent‐free from PUFA supplemented RAW264.7 macrophages

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