cerpe d18  (Avanti Polar)

Journal: EMBO Reports

Article Title: Functional profiling and visualization of the sphingolipid metabolic network in vivo

doi: 10.1038/s44319-025-00632-0

Figure Lengend Snippet: ( A ) OlyA w expression and SMases RNAi knockdowns in neurons. UAS-OlyA w expression and SMases RNAi were driven by nSyb -GAL4 ( nSyb -GAL4> OlyA w + UAS-RNAi ). Confocal images were captured from the posterior view of the Calyx in the brain of young adult flies (1 week old). (Right) Quantitative analysis of OlyA w intensity in the cortical region. P values [dSMPD4-IR (BDSC 51682), P < 0.001; dSMPD4-IR (VDRC 110163), P = 0.03; nSMase-IR (BDSC 36759), P = 0.09; nSMase-IR (VDRC 107062), P = 0.25; aSMase-IR (VDRC 12227), P = 0.46] were calculated using one-way ANOVA with Dunnett’s multiple comparisons. Data are represented as mean ± SEM ( n ≥ 4, biological repeats). Data are representative of at least two independent experiments. ( B ) OlyA w expression in neurons and SMases RNAi knockdowns in glia. LexAop-OlyA w expression is driven by nSyb -LexA, while SMases RNAi were driven by repo -GAL4 ( nSyb -LexA> LexAop-OlyA w ; repo -GAL4 > UAS-RNAi ). Confocal images were captured from the posterior view of the Calyx in the adult brain of young adult flies (1 week old). (Right) Quantifications of OlyA w intensity in the cortical region. P values [dSMPD4-IR (BDSC 51682), P = 0.10; dSMPD4-IR (VDRC 110163), P = 0.49; nSMase-IR (BDSC 36759), P = 0.90; nSMase-IR (VDRC 107062), P = 0.41; aSMase-IR (VDRC 12227), P < 0.001] were calculated using one-way ANOVA with Tukey’s multiple comparisons. Data are represented as mean ± SEM ( n ≥ 4, biological repeats). Data are representative of at least two independent experiments. ( C ) Quantification of the fold change in total lipid levels of dSMPD4 KO and nSMase KO compared to w 1118 brains (day 3 females). P values ( nSMase KO vs. w 1118 : Total lipids, P = 0.973; PS, P = 0.955; PE, P = 0.995; PC, P = 0.294; CerPE, P < 0.001; Cer, P < 0.001. dSMPD4 KO vs. w 1118 : Total lipids, P = 0.946; PS, P = 0.988; PE, P = 0.938; PC, P = 0.779; CerPE, P = 0.101; Cer, P = 0.768.) were calculated using one-way ANOVA with Dunnett’s multiple comparisons. Data are represented as mean ± SEM ( n = 4, biological repeats). ( D ) Quantification of the fold change in CerPE of dSMPD4 -knockout compared to w 1118 brains (day 3 females). P values (d16:2/22:0, P = 0.345; d16:2/20:0, P = 0.832; d16:2/18:0, P = 0.428; d16:1/22:0, P = 0.842; d16:0/22:0, P = 0.122; d14:2/24:0, P = 0.932; d14:2/22:0, P = 0.227; d14:2/20:0, P = 0.096; d14:2/18:1, P = 0.005; d14:2/18:0, P = 0.027; d14:1/24:1, P = 0.171; d14:1/24:0, P = 0.766; d14:1/22:0, P = 0.379; d14:1/20:0, P = 0.024; d14:1/18:0, P = 0.001; d14:0/22:1, P = 0.152; d14:0/20:1, P = 0.061) were calculated using two-tailed unpaired Student’s t test. Data are represented as mean ± SEM ( n = 4, biological repeats). ( E ) Quantification of the fold change in CerPE of nSMase -knockout compared to w 1118 brains (day 3 females). P values (d16:2/22:0, P = 0.081; d16:2/20:0, P < 0.001; d16:2/18:0, P < 0.001; d16:1/22:0, P < 0.001; d16:1/20:0, P < 0.001; d16:1/18:0, P < 0.001; d14:2/24:0, P = 0.610; d14:2/22:0, P = 0.663; d14:2/20:0, P < 0.001; d14:2/18:1, P < 0.001; d14:2/18:0, P < 0.001; d14:1/24:0, P = 0.203; d14:1/22:0, P = 0.016; d14:1/20:0, P < 0.001; d14:1/18:0, P < 0.001; d14:1/16:0, P < 0.001; d14:0/22:1, P = 0.001; d14:0/20:1, P < 0.001; d14:0/18:1, P < 0.001) were calculated using two-tailed unpaired Student’s t-test. Data are represented as mean ± SEM ( n = 4, biological repeats). ( F ) Anti-HA and anti-Lamin co-staining of the adult brain from dSMPD4 -HG line. Lamin is used as a marker of the nuclear envelope (NE). ( G ) Quantification of dSMPD4-3XHA subcellular localization. Box plot shows the Pearson’s coefficient of anti-HA and organellar markers, including anti-Lamin (nuclear envelope), anti-ATP5A (mitochondria), anti-Na + -K + -ATPase (Plasma membrane), and anti-Cathepsin L (Lysosome). Box plots illustrate the data distribution. The center line within the box represents the median, which is the 50th percentile. The lower and upper bounds of the box correspond to the 25th and 75th percentiles, respectively. The ends of the whiskers indicate the minima and maxima. Data points represent biological repeats. P values (Nuclear envelope vs. Mitochondria, P < 0.001; Nuclear envelope vs. Plasma membrane, P < 0.001; Nuclear envelope vs. Lysosome, P < 0.001) were calculated using one-way ANOVA with Tukey’s multiple comparisons. Representative images of anti-HA and organellar markers co-stainings are presented in Appendix Fig. . ( H ) Morphology of the nuclear membrane (anti-Lamin, magenta) in adult brains (1 week old) of indicated genotypes. ( I ) Quantification of the percentage of nuclei with blebs. Data are represented as mean ± SEM ( n = 3, biological repeats). P values ( w 1118 vs. dSMPD4 KO , P < 0.001; w 1118 vs. dSMPD4 KO ; actin > dSMPD4-myc , P = 0.999; w 1118 vs. nSMase KO , P = 0.547) were calculated using one-way ANOVA with Dunnett’s multiple comparisons. Data are representative of at least two independent experiments. ( J ) Olfactory associative memory assay in dSMPD4 -knockout, nSMase -knockout, and w 1118 control flies (1-week-old). Data are represented as mean ± SEM. P values ( w 1118 vs. dSMPD4 KO , P = 0.018; w 1118 vs. nSMase KO , P = 0.915) were calculated using one-way ANOVA with Dunnett’s multiple comparisons. Data are representative of four independent experiments of biological repeats. .

Article Snippet: Following ISTD were added: Glucosyl-beta-Ceramide d18:1/8:0 (Avanti #860540), CerPE d18:1/24:0 (Avanti #860067) and EquisplashTM LIPIDOMIX® Quantitative Mass Spec Internal Standard (Avanti #330731).

Techniques: Expressing, Knock-Out, Two Tailed Test, Staining, Marker, Clinical Proteomics, Membrane, Control

Journal: EMBO Reports

Article Title: Functional profiling and visualization of the sphingolipid metabolic network in vivo

doi: 10.1038/s44319-025-00632-0

Figure Lengend Snippet: ( A ) OlyA w expression and the aSMases RNAi knockdown in glia. UAS-OlyA w expression and aSMases RNAi were driven by repo -GAL4 ( repo -GAL4 > UAS-OlyA w +aSMase-RNAi ). Confocal images were captured from the posterior view of the Calyx in the adult brain of young adult flies (1 week old). (Right) Quantifications of OlyA w intensity in the cortical region. The P value ( P = 0.003) was calculated using two-tailed unpaired Student’s t test. Data are represented as mean ± SEM ( n ≥ 7). Data are representative of 4 independent experiments. ( B ) Quantification of the total CerPE levels in control ( repo -GAL4/+) and aSMase knockdown ( repo -GAL4/UAS-aSMase-RNAi) flies (day 10 female). The P value ( P = 0.01) were calculated using two-tailed unpaired Student’s t test. Data are represented as mean ± SEM (n > 3). Data are representative of 3 independent experiments. ( C ) Ref(2)P/p62 immunostainings in the optic lobe of adult brains from control or aSMase conditional knockout flies (1-week-old). Cell type-specific aSMase knockout was achieved by using small guide RNA targeting aSMase and Cas9 driven by neuronal ( nSyb -GAL4) or glial ( repo -GAL4) drivers. Control: UAS-Cas9 /+; UAS-aSMase-sgRNA /+; neuronal cKO: nSyb > Cas9+aSMase-sgRNA; glial cKO: repo > Cas9+aSMase-sgRNA . ( D ) Quantification of Ref(2)P/p62 puncta counts. Data are represented as mean ± SEM ( n ≥ 6). P values [Control vs. aSMase cKO (neuron), P = 0.910; Control vs. aSMase cKO (glia), P = 0.012] were calculated using one-way ANOVA with Tukey’s multiple comparisons. Data are representative of >5 independent experiments. ( E ) Climbing assay of control or aSMase conditional knockout flies (1-week-old). Cell type-specific aSMase knockout was achieved by using small guide RNA targeting aSMase and Cas9 driven by neuronal ( nSyb -GAL4) or glial ( repo -GAL4) drivers. Data are represented as mean ± SEM ( n = 5). P values [Control vs. aSMase cKO (neuron), P = 0.109; Control vs. aSMase cKO (glia), P < 0.001] were calculated using one-way ANOVA with Tukey’s multiple comparisons. Data are representative of 5 independent experiments. Control: UAS-Cas9 /+; UAS-aSMase-sgRNA /+; neuronal cKO: nSyb > Cas9+aSMase-sgRNA; glial cKO: repo > Cas9+aSMase-sgRNA .

Article Snippet: Following ISTD were added: Glucosyl-beta-Ceramide d18:1/8:0 (Avanti #860540), CerPE d18:1/24:0 (Avanti #860067) and EquisplashTM LIPIDOMIX® Quantitative Mass Spec Internal Standard (Avanti #330731).

Techniques: Expressing, Knockdown, Two Tailed Test, Control, Knock-Out, Climbing Assay

Journal: The Journal of Biological Chemistry

Article Title: Multiple activities of sphingomyelin synthase 2 generate saturated fatty acid– and/or monounsaturated fatty acid–containing diacylglycerol

doi: 10.1016/j.jbc.2024.107960

Figure Lengend Snippet: Purification of SMS2 and PLC/SMS/CPES activity assay in the presence of detergents. A and B , C-terminal Twin-Strep–tagged human SMS2 (SMS2-TS) was expressed in HEK293 cells and purified by affinity chromatography using Strep-Tactin XT beads. Purified SMS-TS was analyzed using SDS-PAGE (12% gel), followed by Coomassie brilliant blue staining ( A ) or immunoblotting using an anti-Strep tag II antibody (1:1000 dilution in Can Get Signal Solution 1 (Toyobo)). BlueStar Prestained Protein Ladder (#NE-MWP03, Nippon Genetics) was used for molecular mass markers. C–E , DG-, SM-, and CPE-producing activities of SMS2-TS, which was purified in the presence of detergents, were measured using LC–MS/MS. d18:1/18:0-Cer and/or 16:0/18:1-phospholipids (PC, PE, PA, PI, PG, or PS) in the detergent mixed micelles were used as substrates. Purified SMS2-TS (10 μl (approximately 500 ng protein) of the sample) was used for the enzyme assays. The peak area ratio of 16:0/18:1-DG ( C ), d18:1/18:0-SM ( D ), and d18:1/18:0-CPE ( E ) in the samples were calculated using an internal standard (I.S.) (0.2 ng/μl of 15:0/18:1-DG, d18:0/12:0-SM, and d18:1/24:0-CPE). Purified SMS2-TS contained detectable 16:0/18:1-DG, d18:1/18:0-SM that probably came from HEK293 cell membranes. We calculated DG-producing activity of SMS2-TS after subtracting these backgrounds. The absolute quantities of 16:0/18:1-DG and d18:1/18:0-SM in the samples were determined from calibration curves generated using commercially available purified lipids (16:0/18:1-DG and d18:1/18:0-SM) ( Fig. S2 ). The micelles contained detectable 16:0/18:1-DG, which was likely contaminated by the phospholipid samples. Therefore, we calculated DG-generating activity after subtracting these backgrounds (the purified SMS2 mixed with the micelle without incubation at 37 °C for 2 h). The values are presented as the mean ± SD (n = 3, technical replicates). ND, not detectable. ns, not significant, ∗∗∗, p < 0.005 ( versus PC/Cer); ††† , p < 0.005 ( versus PC without Cer); ‡‡‡ , p < 0.005 ( versus PE/Cer). One-way ANOVA with Tukey–Kramer’s post hoc test was used. F , the DG-generating activity of SMS2-TS in the presence of Cer and phospholipids was measured using LC–MS/MS. The micelles containing d18:1/18:0-Cer and 16:0/18:1-glycerophospholipid (50 μM each) were incubated with purified SMS2. The values are presented as the mean ± SD (n = 3, technical replicates). ∗∗∗, p < 0.005 ( versus PC/Cer); ††† , p < 0.005 ( versus PE/Cer). One-way ANOVA with Tukey’s post hoc test was used. G , SMS activity of SMS2-TS in the presence of Cer/PC or Cer/phosphocholine ( PCho ) was measured using LC–MS/MS. The micelles containing d18:1/18:0-Cer and 16:0/18:1-PC (50 μM each) or PCho (50 μM each) was incubated with purified SMS2 (approximately 500 ng). The SMS activity in the samples was determined by the quantitation of d18:1/18:0-SM using LC–MS/MS. Values are presented as percentages of the SMS activity in the presence of PC and Cer (set to 100%). Values are presented as the mean ± SD (n = 3 technical replicates). ND, not detectable. ∗∗∗, p < 0.005 (PCho/Cer versus PC/Cer). Student’s t test was used. Cer, ceramide; CPE, ceramide phosphoethanolamine; CPES, ceramide phosphoethanolamine synthase; DG, diacylglycerol; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PLC, phospholipase C; PS, phosphatidylserine; SM, sphingomyelin; SMS, sphingomyelin synthase.

Article Snippet: Lipids: 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphate (16:0/18:1-PA, cat. no. 840857), 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoinositol (16:0/18:1-PI, Cat. No. 850142), 1-palmitoyl-2-oleoyl- sn -glycero-3-phospho-L-serine (16:0/18:1-PS, Cat. no. 840034), and 1-pentadecanoyl-2-oleoyl- sn -glycerol (15:0/18:1-DG, Cat. no. 330722), 1-palmitoyl-2-oleoyl- sn -glycero-3-PCho (16:0/18:1-PC, Cat. no. 850457); 1-palmitoyl-2-oleoyl- sn -glycero- sn -glycerol (16:0/18:1-DG, Cat. no. 800815); 1,2-dioleoyl- sn -glycero-3-PCho (18:1/18:1-PC; Cat. no. 850375), 1-stearoyl-2-arachidonoyl- sn -glycero-3-PCho (18:0/20:4-PC, Cat. no. 850469), 1-palmitoyl-2-oleoyl- sn -3-phosphoglycerol (16:0/18:1-PG, Cat. No. 840457), and N-lignoceroyl-D-erythro-sphingosylphosphoethanolamine (d18:1/24:0-CPE, Cat. no. 860067), and N-lauroyl-D-erythro-sphingosylphosphorylcholine (d18:1/12:0-SM, Cat. no. 860583) were purchased from Avanti Polar Lipids (Alabaster).

Techniques: Purification, Activity Assay, Affinity Chromatography, SDS Page, Staining, Western Blot, Strep-tag, Liquid Chromatography with Mass Spectroscopy, Generated, Incubation, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Multiple activities of sphingomyelin synthase 2 generate saturated fatty acid– and/or monounsaturated fatty acid–containing diacylglycerol

doi: 10.1016/j.jbc.2024.107960

Figure Lengend Snippet: Effects of Cer and PC concentrations on SMS/PC-PLC activities of SMS2. A and B , 16:0/18:1-DG ( A ) and d18:1/18:0-SM ( B ) generated by SMS2-TS, which was purified in the presence of detergents, were analyzed in the presence of 50 μM of 16:0/18:1-PC and various concentrations of d18:1/18:0-Cer in detergent mixed micelles as indicated. The detected intensity of the analyte in the sample was quantified using an internal standard (I.S.) (0.2 ng/μl of 15:0/18:1-DG, d18:0/12:0-SM, and d18:1/24:0-SM). The absolute quantities of 16:0/18:1-DG and d18:1/18:0-SM were determined from calibration curves generated using commercially available lipids. Values are presented as the mean ± SD (n = 3, technical replicates). C , 16:0/18:1-DG–generating activity (PC-PLC activity + SMS activity) ( A ), d18:1/18:0-SM–generating activity (SMS activity) ( B ), and PC-PLC activity (subtracted values (16:0/18:1-DG – d18:1/18:0-SM)) of SMS2-TS were plotted. D , enzyme kinetic analysis of purified SMS2 (PC-PLC) with 16:0/18:1-PC mixed micelles. DG-generating activity (PC-PLC activity) was plotted as a function of the 16:0/18:1-PC concentration in the mixed micelles (mol%). Values represent averages of triplicate measurements. Data are represented as the mean ± SD. Cer, ceramide; DG, diacylglycerol; PC, phosphatidylcholine; PLC, phospholipase C; SM, sphingomyelin; SMS, sphingomyelin synthase; TS, Twin-Strep.

Article Snippet: Lipids: 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphate (16:0/18:1-PA, cat. no. 840857), 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoinositol (16:0/18:1-PI, Cat. No. 850142), 1-palmitoyl-2-oleoyl- sn -glycero-3-phospho-L-serine (16:0/18:1-PS, Cat. no. 840034), and 1-pentadecanoyl-2-oleoyl- sn -glycerol (15:0/18:1-DG, Cat. no. 330722), 1-palmitoyl-2-oleoyl- sn -glycero-3-PCho (16:0/18:1-PC, Cat. no. 850457); 1-palmitoyl-2-oleoyl- sn -glycero- sn -glycerol (16:0/18:1-DG, Cat. no. 800815); 1,2-dioleoyl- sn -glycero-3-PCho (18:1/18:1-PC; Cat. no. 850375), 1-stearoyl-2-arachidonoyl- sn -glycero-3-PCho (18:0/20:4-PC, Cat. no. 850469), 1-palmitoyl-2-oleoyl- sn -3-phosphoglycerol (16:0/18:1-PG, Cat. No. 840457), and N-lignoceroyl-D-erythro-sphingosylphosphoethanolamine (d18:1/24:0-CPE, Cat. no. 860067), and N-lauroyl-D-erythro-sphingosylphosphorylcholine (d18:1/12:0-SM, Cat. no. 860583) were purchased from Avanti Polar Lipids (Alabaster).

Techniques: Generated, Purification, Activity Assay, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Multiple activities of sphingomyelin synthase 2 generate saturated fatty acid– and/or monounsaturated fatty acid–containing diacylglycerol

doi: 10.1016/j.jbc.2024.107960

Figure Lengend Snippet: Effects of Cer and PE concentrations on CPES/PE-PLC activities of SMS2. A and B , 16:0/18:1-DG ( A ) and d18:1/18:0-CPE ( B ) generated by SMS2-TS, which was purified in the presence of detergents, were analyzed in the presence of 50 μM of 16:0/18:1-PE and various concentrations of d18:1/18:0-Cer in detergent mixed micelles as indicated. The detected intensity of the analyte in the sample was quantified using an internal standard (I.S.) (0.2 ng/μl of 15:0/18:1-DG, d18:0/12:0-SM, and d18:1/24:0-CPE). The absolute quantities of 16:0/18:1-DG were determined based on calibration curves generated using commercially available lipids. The values are presented as the mean ± SD (n = 3, technical replicates). C , relative 16:0/18:1-DG- and d18:1/18:0-CPE-generating activities of SMS2-TS (A and B). The subtracted values (relative 16:0/18:1-DG – relative d18:1/18:0-CPE) were also plotted as the relative PE-PLC activity of SMS2-TS. D , enzyme kinetic analysis of purified SMS2 (PE-PLC) with 16:0/18:1-PE. DG-generating activity (PE-PLC activity) was plotted as a function of the 16:0/18:1-PE concentration in the mixed micelles (mol%). Values represent averages of triplicate measurements. Data are presented as the mean ± SD (n = 3, technical replicates). Cer, ceramide; CPE synthase; CPE, ceramide phosphoethanolamine; CPES, DG, diacylglycerol; PE, phosphatidylethanolamine; PLC, phospholipase C; SM, sphingomyelin; SMS, sphingomyelin synthase; TS, Twin-Strep.

Article Snippet: Lipids: 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphate (16:0/18:1-PA, cat. no. 840857), 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoinositol (16:0/18:1-PI, Cat. No. 850142), 1-palmitoyl-2-oleoyl- sn -glycero-3-phospho-L-serine (16:0/18:1-PS, Cat. no. 840034), and 1-pentadecanoyl-2-oleoyl- sn -glycerol (15:0/18:1-DG, Cat. no. 330722), 1-palmitoyl-2-oleoyl- sn -glycero-3-PCho (16:0/18:1-PC, Cat. no. 850457); 1-palmitoyl-2-oleoyl- sn -glycero- sn -glycerol (16:0/18:1-DG, Cat. no. 800815); 1,2-dioleoyl- sn -glycero-3-PCho (18:1/18:1-PC; Cat. no. 850375), 1-stearoyl-2-arachidonoyl- sn -glycero-3-PCho (18:0/20:4-PC, Cat. no. 850469), 1-palmitoyl-2-oleoyl- sn -3-phosphoglycerol (16:0/18:1-PG, Cat. No. 840457), and N-lignoceroyl-D-erythro-sphingosylphosphoethanolamine (d18:1/24:0-CPE, Cat. no. 860067), and N-lauroyl-D-erythro-sphingosylphosphorylcholine (d18:1/12:0-SM, Cat. no. 860583) were purchased from Avanti Polar Lipids (Alabaster).

Techniques: Generated, Purification, Activity Assay, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Multiple activities of sphingomyelin synthase 2 generate saturated fatty acid– and/or monounsaturated fatty acid–containing diacylglycerol

doi: 10.1016/j.jbc.2024.107960

Figure Lengend Snippet: Effects of PC and PE concentrations on PLC/SMS/CPES activities of SMS2. A and B , the effect of PC concentrations on the enzyme activities of SMS2, which was purified in the presence of detergents, was determined. Relative d18:1/18:0-CPE- and d18:1/18:0-SM–producing activities ( A ) and relative DG-producing activities ( B ) of SMS2-TS were analyzed in the presence of 50 μM each of 16:0/18:1-PE and d18:1/18:0-Cer, and various concentrations of 16:0/18:1-PC in mixed micelles. C and D , the effect of PE concentrations on the enzyme activities of SMS2, which was purified in the presence of detergents, was determined. Relative d18:1/18:0-CPE- and d18:1/18:0-SM–producing activities ( C ) and relative DG-producing activities ( D ) of SMS2-TS were analyzed in the presence of 50 μM each of 16:0/18:1-PC, d18:1/18:0-Cer, and various concentrations of 16:0/18:1-PE in detergent mixed micelles. The DG, SM, and CPE produced by SMS2 were quantified using LC–MS/MS. Data are represented as the mean ± SD (n = 3, technical replicates). Cer, ceramide; CPE, ceramide phosphoethanolamine; CPES, CPE synthase; DG, diacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PLC, phospholipase C; SM, sphingomyelin; SMS, sphingomyelin synthase; TS, Twin-Strep.

Article Snippet: Lipids: 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphate (16:0/18:1-PA, cat. no. 840857), 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoinositol (16:0/18:1-PI, Cat. No. 850142), 1-palmitoyl-2-oleoyl- sn -glycero-3-phospho-L-serine (16:0/18:1-PS, Cat. no. 840034), and 1-pentadecanoyl-2-oleoyl- sn -glycerol (15:0/18:1-DG, Cat. no. 330722), 1-palmitoyl-2-oleoyl- sn -glycero-3-PCho (16:0/18:1-PC, Cat. no. 850457); 1-palmitoyl-2-oleoyl- sn -glycero- sn -glycerol (16:0/18:1-DG, Cat. no. 800815); 1,2-dioleoyl- sn -glycero-3-PCho (18:1/18:1-PC; Cat. no. 850375), 1-stearoyl-2-arachidonoyl- sn -glycero-3-PCho (18:0/20:4-PC, Cat. no. 850469), 1-palmitoyl-2-oleoyl- sn -3-phosphoglycerol (16:0/18:1-PG, Cat. No. 840457), and N-lignoceroyl-D-erythro-sphingosylphosphoethanolamine (d18:1/24:0-CPE, Cat. no. 860067), and N-lauroyl-D-erythro-sphingosylphosphorylcholine (d18:1/12:0-SM, Cat. no. 860583) were purchased from Avanti Polar Lipids (Alabaster).

Techniques: Purification, Produced, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Multiple activities of sphingomyelin synthase 2 generate saturated fatty acid– and/or monounsaturated fatty acid–containing diacylglycerol

doi: 10.1016/j.jbc.2024.107960

Figure Lengend Snippet: Effects of the mutations of the conserved catalytic triad of SMS2 on PLC/SMS/CPES activities. A , WT human SMS2-TS (WT), SMS2 point-mutated in catalytic triad (H229A, H272A, or D276A) (50), and SMS2 point-mutated in loop b (E271D), which is crucial for CPES activity of SMS2 (53), were expressed in HEK293 cells and purified in the presence of DDM/CHS detergents. Purified proteins were detected using SDS-PAGE followed by Coomassie brilliant blue staining. BlueStar Prestained Protein Ladder was used for molecular weight markers. B – G , the effects of the point-mutations on SMS activities (analyte: DG ( B ) and SM ( C )), PC-PLC activity ( D ), CPES activity (analyte: DG ( E ) and CPE ( F )), and PE-PLC activity ( G ) of SMS2. Purified proteins (approximately 500 ng) were incubated with 16:0/18:1-PC, 16:0/18:1-PE, and/or d18:1/18:0-Cer in detergent mixed micelles for 2 h at 37 °C. The DG-, SM-, and CPE-producing activities were determined using LC–MS/MS and normalized based on the protein concentration of samples. The relative activity was standardized through comparison to the WT SMS2 (set at 100%). The data are represented as the mean ± SD (n = 3, technical replicates). ∗∗, p < 0.01 and ∗∗∗, p < 0.005. One-way ANOVA with Dunnett’s post hoc test was used. Cer, ceramide; CHS, cholesteryl hemisuccinate; CPE, ceramide phosphoethanolamine; CPES, CPE synthase; DDM, n-dodecyl-β-D-maltoside; DG, diacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PLC, phospholipase C; SM, sphingomyelin; SMS, sphingomyelin synthase; TS, Twin-Strep.

Article Snippet: Lipids: 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphate (16:0/18:1-PA, cat. no. 840857), 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoinositol (16:0/18:1-PI, Cat. No. 850142), 1-palmitoyl-2-oleoyl- sn -glycero-3-phospho-L-serine (16:0/18:1-PS, Cat. no. 840034), and 1-pentadecanoyl-2-oleoyl- sn -glycerol (15:0/18:1-DG, Cat. no. 330722), 1-palmitoyl-2-oleoyl- sn -glycero-3-PCho (16:0/18:1-PC, Cat. no. 850457); 1-palmitoyl-2-oleoyl- sn -glycero- sn -glycerol (16:0/18:1-DG, Cat. no. 800815); 1,2-dioleoyl- sn -glycero-3-PCho (18:1/18:1-PC; Cat. no. 850375), 1-stearoyl-2-arachidonoyl- sn -glycero-3-PCho (18:0/20:4-PC, Cat. no. 850469), 1-palmitoyl-2-oleoyl- sn -3-phosphoglycerol (16:0/18:1-PG, Cat. No. 840457), and N-lignoceroyl-D-erythro-sphingosylphosphoethanolamine (d18:1/24:0-CPE, Cat. no. 860067), and N-lauroyl-D-erythro-sphingosylphosphorylcholine (d18:1/12:0-SM, Cat. no. 860583) were purchased from Avanti Polar Lipids (Alabaster).

Techniques: Activity Assay, Purification, SDS Page, Staining, Molecular Weight, Incubation, Liquid Chromatography with Mass Spectroscopy, Protein Concentration, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Multiple activities of sphingomyelin synthase 2 generate saturated fatty acid– and/or monounsaturated fatty acid–containing diacylglycerol

doi: 10.1016/j.jbc.2024.107960

Figure Lengend Snippet: Effects of D609, a PC-PLC/SMS inhibitor, on PC-PLC, PE-PLC, SMS, and CPES activities of SMS2. A , DG-generating activities of SMS2, which was purified in the presence of detergents, were analyzed in the presence of various concentrations of D609 (0, 0.25, 0.5, and 1 mM). Purified WT SMS2-TS (approximately 500 ng) was incubated in the presence of 16:0/18:1-PC alone, 16:0/18:1-PC and d18:1/18:0-Cer, 16:0/18:1-PE alone, or 16:0/18:1-PE and d18:1/18:0-Cer–containing detergent mixed micelles for 2 h at 37 °C. Produced 16:0/18:1-DG was quantified using LC–MS/MS. Values are presented as percentages of the activity of SMS2 in the absence of D609 (set at 100%). Values are presented as the mean ± SD (n = 6, technical replicates). ∗∗∗, p < 0.005 ( versus control (without D609)). One-way ANOVA with Dunnett’s post hoc test was used. B , SM-producing activities of SMS2, which was purified in the presence of detergents, in the presence of 16:0/18:1-PC and d18:1/18:0-Cer–containing detergent mixed micelles were analyzed in the presence of various concentrations of D609 (0, 0.25, 0.5, and 1 mM). Values are presented as percentages of the SMS activity of SMS2 in the absence of D609 (set to 100%). The values are presented as the mean ± SD (n = 4, technical replicates). ∗, p < 0.05; ∗∗, p < 0.01 ( versus control (without D609)). One-way ANOVA with Dunnett’s post hoc test was used. C , CPE-producing activities of SMS2, which was purified in the presence of detergents, in the presence of 16:0/18:1-PE and d18:1/18:0-Cer–containing detergent mixed micelles were analyzed in the presence of various concentrations of D609 (0, 0.25, 0.5, and 1 mM). Values are presented as percentages of the CPES activity of SMS2 in the absence of D609 (set at 100%). Values are presented as the mean ± SD (n = 6, technical replicates). ∗∗, p < 0.01; ∗∗∗, p < 0.005 ( versus control (without D609)). One-way ANOVA with Dunnett’s post hoc test was used. Cer, ceramide; CPE, ceramide phosphoethanolamine; CPES, CPE synthase; DG, diacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PLC, phospholipase C; SM, sphingomyelin; SMS, sphingomyelin synthase; TS, Twin-Strep.

Article Snippet: Lipids: 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphate (16:0/18:1-PA, cat. no. 840857), 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoinositol (16:0/18:1-PI, Cat. No. 850142), 1-palmitoyl-2-oleoyl- sn -glycero-3-phospho-L-serine (16:0/18:1-PS, Cat. no. 840034), and 1-pentadecanoyl-2-oleoyl- sn -glycerol (15:0/18:1-DG, Cat. no. 330722), 1-palmitoyl-2-oleoyl- sn -glycero-3-PCho (16:0/18:1-PC, Cat. no. 850457); 1-palmitoyl-2-oleoyl- sn -glycero- sn -glycerol (16:0/18:1-DG, Cat. no. 800815); 1,2-dioleoyl- sn -glycero-3-PCho (18:1/18:1-PC; Cat. no. 850375), 1-stearoyl-2-arachidonoyl- sn -glycero-3-PCho (18:0/20:4-PC, Cat. no. 850469), 1-palmitoyl-2-oleoyl- sn -3-phosphoglycerol (16:0/18:1-PG, Cat. No. 840457), and N-lignoceroyl-D-erythro-sphingosylphosphoethanolamine (d18:1/24:0-CPE, Cat. no. 860067), and N-lauroyl-D-erythro-sphingosylphosphorylcholine (d18:1/12:0-SM, Cat. no. 860583) were purchased from Avanti Polar Lipids (Alabaster).

Techniques: Purification, Incubation, Produced, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: Multiple activities of sphingomyelin synthase 2 generate saturated fatty acid– and/or monounsaturated fatty acid–containing diacylglycerol

doi: 10.1016/j.jbc.2024.107960

Figure Lengend Snippet: Effects of metal ions on the PLC/SMS/CPES activities of SMS2. A–F the effects of several metal ions (500 μM), Mg 2+ , Ca 2+ , Mn 2+ or Zn 2+ , or EDTA (500 μM) on SMS (analyte: 16:0/18:1-DG ( A ) and d18:1/18:0-SM ( B )), PC-PLC ( C ), CPES (analyte: 16:0/18:1-DG ( D ) and d18:1/18:0-CPE (E)), and PE-PLC ( F ) activities of SMS2, which was purified in the presence of detergents, in the presence of mixed micelles (50 μM lipids) were determined using LC–MS/MS. The absolute quantities of 16:0/18:1-DG and d18:1/18:0-SM in the samples were determined based on the calibration curves. Values are presented as the mean ± SD (n = 3, technical replicates). ∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001 ( versus control (without metal ions)). One-way ANOVA with Dunnett’s post hoc test was used. Cer, ceramide; CPE, ceramide phosphoethanolamine; CPES, CPE synthase; DG, diacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PLC, phospholipase C; SM, sphingomyelin; SMS, sphingomyelin synthase; TS, Twin-Strep.

Article Snippet: Lipids: 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphate (16:0/18:1-PA, cat. no. 840857), 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoinositol (16:0/18:1-PI, Cat. No. 850142), 1-palmitoyl-2-oleoyl- sn -glycero-3-phospho-L-serine (16:0/18:1-PS, Cat. no. 840034), and 1-pentadecanoyl-2-oleoyl- sn -glycerol (15:0/18:1-DG, Cat. no. 330722), 1-palmitoyl-2-oleoyl- sn -glycero-3-PCho (16:0/18:1-PC, Cat. no. 850457); 1-palmitoyl-2-oleoyl- sn -glycero- sn -glycerol (16:0/18:1-DG, Cat. no. 800815); 1,2-dioleoyl- sn -glycero-3-PCho (18:1/18:1-PC; Cat. no. 850375), 1-stearoyl-2-arachidonoyl- sn -glycero-3-PCho (18:0/20:4-PC, Cat. no. 850469), 1-palmitoyl-2-oleoyl- sn -3-phosphoglycerol (16:0/18:1-PG, Cat. No. 840457), and N-lignoceroyl-D-erythro-sphingosylphosphoethanolamine (d18:1/24:0-CPE, Cat. no. 860067), and N-lauroyl-D-erythro-sphingosylphosphorylcholine (d18:1/12:0-SM, Cat. no. 860583) were purchased from Avanti Polar Lipids (Alabaster).

Techniques: Purification, Liquid Chromatography with Mass Spectroscopy, Control

Journal: The Journal of Biological Chemistry

Article Title: Multiple activities of sphingomyelin synthase 2 generate saturated fatty acid– and/or monounsaturated fatty acid–containing diacylglycerol

doi: 10.1016/j.jbc.2024.107960

Figure Lengend Snippet: Effects of 18:1/18:1-DG on PC-PLC and SMS activities of SMS2. SMS2 was purified in the presence of detergents. SMS activity (substrates: 16:0/18:1-PC and d18:0/18:1-Cer detergent mixed micelles) ( A ) and PC-PLC activity (substrate: 16:0/18:1-PC detergent mixed micelles) ( B ) were determined in the presence or absence of 18:0/18:1-DG (16 mol%). The detected intensity of the analyte in the sample was determined using an internal standard (I.S.) 0.2 ng/μl of 15:0/18:1-DG, d18:0/12:0-SM, and 15:0/18:1-PC. Values are presented as the mean ± SD (n = 3, technical replicates). ∗∗, p < 0.01 ( vs . control (without 18:1/18:1-DG)). Student’s t test was used. Cer, ceramide; CPE, ceramide phosphoethanolamine; CPES, CPE synthase; DG, diacylglycerol; PC, phosphatidylcholine; PLC, phospholipase C; SM, sphingomyelin; SMS, sphingomyelin synthase; TS, Twin-Strep.

Article Snippet: Lipids: 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphate (16:0/18:1-PA, cat. no. 840857), 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoinositol (16:0/18:1-PI, Cat. No. 850142), 1-palmitoyl-2-oleoyl- sn -glycero-3-phospho-L-serine (16:0/18:1-PS, Cat. no. 840034), and 1-pentadecanoyl-2-oleoyl- sn -glycerol (15:0/18:1-DG, Cat. no. 330722), 1-palmitoyl-2-oleoyl- sn -glycero-3-PCho (16:0/18:1-PC, Cat. no. 850457); 1-palmitoyl-2-oleoyl- sn -glycero- sn -glycerol (16:0/18:1-DG, Cat. no. 800815); 1,2-dioleoyl- sn -glycero-3-PCho (18:1/18:1-PC; Cat. no. 850375), 1-stearoyl-2-arachidonoyl- sn -glycero-3-PCho (18:0/20:4-PC, Cat. no. 850469), 1-palmitoyl-2-oleoyl- sn -3-phosphoglycerol (16:0/18:1-PG, Cat. No. 840457), and N-lignoceroyl-D-erythro-sphingosylphosphoethanolamine (d18:1/24:0-CPE, Cat. no. 860067), and N-lauroyl-D-erythro-sphingosylphosphorylcholine (d18:1/12:0-SM, Cat. no. 860583) were purchased from Avanti Polar Lipids (Alabaster).

Techniques: Purification, Activity Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: Multiple activities of sphingomyelin synthase 2 generate saturated fatty acid– and/or monounsaturated fatty acid–containing diacylglycerol

doi: 10.1016/j.jbc.2024.107960

Figure Lengend Snippet: SMS/PC-PLC activities of SMS2-TS in proteoliposomes in the absence of detergents. A and B , SMS2-TS was expressed in HEK293 cells and purified using affinity chromatography with Strep-Tactin XT beads in the presence of detergents. Purified SMS-TS was reconstituted into the liposomes (18:1/18:1-PC). The SMS2 containing liposomes (detergent-free) were subjected to SDS-PAGE (12% gel), followed by Coomassie brilliant blue staining ( A ) and immunoblotting ( B ). C–E , quantification of 16:0/18:1-DG ( C ), d18:1/18:0-SM ( D ) and d18:1/18:0-CPE ( E ) generated by SMS2-TS containing liposomes (detergent-free) when incubated with 16:0/18:1-PC or PE and/or d18:1/18:0-Cer for 2 h at 37 °C. The detected intensity of the analyte in the sample was quantified using an internal standard (I.S.) (0.2 ng/μl of 15:0/18:1-DG, d18:0/12:0-SM, or d18:1/24:0-CPE). Values are presented as the mean ± SD (n = 3, technical replicates). ND, not detectable. ns, not significant, ∗∗∗, p < 0.005 ( versus PC/Cer); ††† , p < 0.005 ( versus PC without Cer). One-way ANOVA with Tukey’s post hoc test was used. Cer, ceramide; CPE, ceramide phosphoethanolamine; DG, diacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PLC, phospholipase C; SM, sphingomyelin; SMS, sphingomyelin synthase; TS, Twin-Strep.

Article Snippet: Lipids: 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphate (16:0/18:1-PA, cat. no. 840857), 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoinositol (16:0/18:1-PI, Cat. No. 850142), 1-palmitoyl-2-oleoyl- sn -glycero-3-phospho-L-serine (16:0/18:1-PS, Cat. no. 840034), and 1-pentadecanoyl-2-oleoyl- sn -glycerol (15:0/18:1-DG, Cat. no. 330722), 1-palmitoyl-2-oleoyl- sn -glycero-3-PCho (16:0/18:1-PC, Cat. no. 850457); 1-palmitoyl-2-oleoyl- sn -glycero- sn -glycerol (16:0/18:1-DG, Cat. no. 800815); 1,2-dioleoyl- sn -glycero-3-PCho (18:1/18:1-PC; Cat. no. 850375), 1-stearoyl-2-arachidonoyl- sn -glycero-3-PCho (18:0/20:4-PC, Cat. no. 850469), 1-palmitoyl-2-oleoyl- sn -3-phosphoglycerol (16:0/18:1-PG, Cat. No. 840457), and N-lignoceroyl-D-erythro-sphingosylphosphoethanolamine (d18:1/24:0-CPE, Cat. no. 860067), and N-lauroyl-D-erythro-sphingosylphosphorylcholine (d18:1/12:0-SM, Cat. no. 860583) were purchased from Avanti Polar Lipids (Alabaster).

Techniques: Purification, Affinity Chromatography, Liposomes, SDS Page, Staining, Western Blot, Generated, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Multiple activities of sphingomyelin synthase 2 generate saturated fatty acid– and/or monounsaturated fatty acid–containing diacylglycerol

doi: 10.1016/j.jbc.2024.107960

Figure Lengend Snippet: Cell-free expression of SMS2-TS in the presence of proteoliposomes and its PLC/SMS/CPES activities in the absence of detergents. A , SMS2-TS was expressed using the human cell-free protein expression system (detergent-free) and reconstituted in the presence of liposomes (18:1/18:1-PC, detergent-free). After translation reactions with or without SMS2-TS–containing plasmid, the SMS2-TS protein in the sample was detected using immunoblotting with anti-Strep tag II antibody. BlueStar Prestained Protein Ladder was used for molecular weight markers. An empty vector (vector alone) was used for the expression system as a control. B–D , quantification of 16:0/18:1-DG ( B ), d18:1/18:0-SM ( C ), and d18:1/18:0-CPE ( D ) generated through SMS2-TS expressed by the cell-free expression system. After expression, the sample containing SMS2-TS or negative control (vector alone) was incubated with 16:0/18:1-glycerophospholipids and/or d18:0/18:1-Cer for 16 h at 37 °C. The detected intensity of the analyte in the sample was quantified using an internal standard (I.S.) (0.2 ng/μl of 15:0/18:1-DG, d18:0/12:0-SM, or d18:1/24:0-CPE). The vector alone samples contained detectable 16:0/18:1-DG, d18:1/18:0-SM, and d18:1/18:0-CPE that probably came from cell-free expression mixtures or lipid solutions. To determine whether SMS2-TS exhibits the activities, we compared the DG, SM, and CPE levels of SMS2-TS expressing samples to that of vector alone. Values are presented as the mean ± SD (n = 3, technical replicates). ∗∗, p < 0.01; ∗∗∗, p < 0.005; ns, not significant. Student’s t test was used. Cer, ceramide; CPE, ceramide phosphoethanolamine; CPES, CPE synthase; DG, diacylglycerol; PC, phosphatidylcholine; PLC, phospholipase C; SM, sphingomyelin; SMS, sphingomyelin synthase; TS, Twin-Strep.

Article Snippet: Lipids: 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphate (16:0/18:1-PA, cat. no. 840857), 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoinositol (16:0/18:1-PI, Cat. No. 850142), 1-palmitoyl-2-oleoyl- sn -glycero-3-phospho-L-serine (16:0/18:1-PS, Cat. no. 840034), and 1-pentadecanoyl-2-oleoyl- sn -glycerol (15:0/18:1-DG, Cat. no. 330722), 1-palmitoyl-2-oleoyl- sn -glycero-3-PCho (16:0/18:1-PC, Cat. no. 850457); 1-palmitoyl-2-oleoyl- sn -glycero- sn -glycerol (16:0/18:1-DG, Cat. no. 800815); 1,2-dioleoyl- sn -glycero-3-PCho (18:1/18:1-PC; Cat. no. 850375), 1-stearoyl-2-arachidonoyl- sn -glycero-3-PCho (18:0/20:4-PC, Cat. no. 850469), 1-palmitoyl-2-oleoyl- sn -3-phosphoglycerol (16:0/18:1-PG, Cat. No. 840457), and N-lignoceroyl-D-erythro-sphingosylphosphoethanolamine (d18:1/24:0-CPE, Cat. no. 860067), and N-lauroyl-D-erythro-sphingosylphosphorylcholine (d18:1/12:0-SM, Cat. no. 860583) were purchased from Avanti Polar Lipids (Alabaster).

Techniques: Expressing, Liposomes, Plasmid Preparation, Western Blot, Strep-tag, Molecular Weight, Control, Generated, Negative Control, Incubation