Sphingosine-1-Phosphate Lyase Deficient Cells as a Tool to Study Protein Lipid Interactions

Abstract

Cell membranes contain hundreds to thousands of individual lipid species that are of structural importance but also specifically interact with proteins. Due to their highly controlled synthesis and role in signaling events sphingolipids are an intensely studied class of lipids. In order to investigate their metabolism and to study proteins interacting with sphingolipids, metabolic labeling based on photoactivatable sphingoid bases is the most straightforward approach. In order to monitor protein-lipid-crosslink products, sphingosine derivatives containing a reporter moiety, such as a radiolabel or a clickable group, are used. In normal cells, degradation of sphingoid bases via action of the checkpoint enzyme sphingosine-1-phosphate lyase occurs at position C2-C3 of the sphingoid base and channels the resulting hexadecenal into the glycerolipid biosynthesis pathway. In case the functionalized sphingosine looses the reporter moiety during its degradation, specificity towards sphingolipid labeling is maintained. In case degradation of a sphingosine derivative does not remove either the photoactivatable or reporter group from the resulting hexadecenal, specificity towards sphingolipid labeling can be achieved by blocking sphingosine-1-phosphate lyase activity and thus preventing sphingosine derivatives to be channeled into the sphingolipid-to-glycerolipid metabolic pathway. Here we report an approach using clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated nuclease Cas9 to create a sphingosine-1-phosphate lyase (SGPL1) HeLa knockout cell line to disrupt the sphingolipid-to-glycerolipid metabolic pathway. We found that the lipid and protein compositions as well as sphingolipid metabolism of SGPL1 knock-out HeLa cells only show little adaptations, which validates these cells as model systems to study transient protein-sphingolipid interactions.

Downregulation of S1P Lyase Improves Barrier Function in Human Cerebral Microvascular Endothelial Cells Following an Inflammatory Challenge

“…In agreement with this, other sphingolipid species may mediate the response towards inflammatory stimuli, as we have seen from the LC-MS/MS data that S1P and dihydro-S1P, which are both modestly increased by TNFα in control cells, synergistically increase by TNFα in SPL-kd ( Figure 8). Ceramides of various chain lengths were decreased in HCMEC/D3 SPL-kd (Table S1), which is in accordance to a previous study in SPL-deficient Hela cells [81]. According to Linder et al [84], ceramides have the potential to alter endothelial cell permeability.…”

“…In this regard, PKC-α, PKC-β, -θ, and −ζ activities were attributed to increased endothelial permeability [74][75][76][77], whereas PKC-δ and PKC-ε can mediate barrier protection [78,79]. It is also worth noting that in the livers of SPL-deficient mice [80] and in MEFs isolated from SPL-deficient mice [81], diacylglycerols, which are well known direct PKC activators, were reported to be significantly increased, thus further supporting the conclusion that SPL-kd couples to enhanced PKC activity.…”

Monitoring Sphingolipid Trafficking in Cells using Fluorescence Microscopy

“…SGPL1 knockout (SGPL1-KO) cells (for gene editing strategy, see Gerl et al, 2016) DMEM FBS, charcoal stripped (delipidated FBS; e.g., Thermo Fisher, A3382101) PhotoClick Sphingosine (pacSphingosine; e.g., Avanti, 900600) FBS PBS Electron microscopy-grade paraformaldehyde (e.g., Electron Microscopy Sciences) Saponin Click-iT TM Cell Reaction Buffer Kit (e.g., Thermo Fisher, C10269) Alexa Fluor TM 647 azide (e.g., Thermo Fisher, A10277) Glass-bottom culture dishes (e.g., MatTeK, P35GC-1.5-14-C) Tissue culture hood 37°C, 5% CO 2 incubator Water bath sonicator Fluorescence microscope…”

“…The use of photoactivatable and clickable Sphingosine (pacSphingosine) to visualize sphingolipids requires cells that are deficient in sphingosine 1–phosphate lyase activity. Pioneering studies used SGPL1‐/‐ mouse cells generated by a genetic knockout strategy (Colie et al., ; Haberkant et al., ) and more recently in cultured human cells using CRISPR/Cas9 gene editing approaches (Gerl et al., ). In our laboratory, we have used the CRISPR/Cas9‐based method to generate Sgpl1 null HeLa cells.…”

Activation of Sphingolipid Pathway in the Livers of Lipodystrophic Agpat2−/− Mice

“…We have shown that conversion of diacylglycerol to PA by diacylglycerol kinase can occur in the livers of Agpat2 −/− mice [ 9 ]. It is also possible that the downstream product of ceramide, sphingosine 1-phosphate, might provide an additional substrate for synthesis of phospholipids as a first step in converting sphingolipids to phospholipids [ 35 ]. The enzymes for these conversions have recently been identified in yeast [ 36 ] and in Drosophila [ 37 ] (reviewed in [ 38 ]) (see Fig.…”