Phosphorylated glycosphingolipids essential for cholesterol mobilization in C. elegans

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1 SUPPLEMENTARY INFORMATION Phosphorylated glycosphingolipids essential for cholesterol mobilization in C. elegans Sebastian Boland, Ulrike Schmidt, Vyacheslav Zagoriy, Julio L. Sampaio, Raphael Fritsche, Regina Czerwonka, Tilo Lübken, Jakob Reimann, Sider Penkov, Hans-Joachim Knölker & Teymuras V. Kurzchalia 1

2 Supplementary Results Supplementary Figure 1 HPLC-MS separation of fraction 4 into more confined fractions, which were tested for their capability to abolish L2* arrest caused by sterol depletion. (a) Base-peak chromatogram of fraction 4 after column chromatography using rp-c18 LC-MS. The red labeled parts of the chromatogram represent the more confined fractions 6.1 to 6.4 and 10.1 to 10.4 which were (b) tested for their activity to rescue the L2* arrest. The results from experiments with biological duplicates in technical triplicates were pooled together and expressed as a percent value. Error bar, SD. 2

3 Supplementary Figure 2 High-resolution mass spectrometry analysis of the bioactive fraction 6. (a) Fourier transform MS (FTMS) analysis in a LTQ Orbitrap XL of the combined bioactive fraction 6 displays mmpegc-c22 in the protonated (m/z = ) and sodiated (m/z = ) forms. (b) Higher-energy collisional dissociation (HCD) MS2 analysis of the precursor mass displays the fragments of the ceramide containing the C22-FA (m/z = ) and the polar head (m/z = ). (c) Ion trap MS3 (ITMS3) analysis of the precursor mass followed by the polar head (300.1) fragment displays the mono-methyl phosphoethanolamine fragment. Water losses are marked with *. 3

4 Supplementary Figure 3 High-resolution mass spectrometry analysis of the bioactive fraction 10. (a) FTMS analysis in a LTQ Orbitrap XL of the combined bioactive fraction 10 displays mmpegc-c24 in the protonated (m/z = ) and sodiated (m/z = ) forms. (b) HCD MS2 analysis of the precursor mass displays the fragments of the ceramide containing the C24-FA (m/z = ) and the polar head (m/z = ). (c) ITMS3 analysis of the precursor mass followed by the polar head (300.1) fragment displays the monomethyl phosphoethanolamine fragment. Water losses are marked with *. 4

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6 Supplementary Figure 4 Structural elucidation of the different PEGC species. FTMS2 experiments in positive ion mode of the precursors with HCD fragmentation: (a) m/z = 927.7, (b) m/z = 941.7, (c) m/z = 955.7, and (d) m/z = Fragments corresponding to different ceramide backbones (642.6, 656.6, and 670.6) and their respective water losses (*). The inlets correspond to a zoom in on the low m/z where the polar head fragments are present. Two different polar heads, depending on the species, (methylated head group) and (nonmethylated head group) are present. (e-h) ITMS2 experiments in negative ion mode of the precursors with CID fragmentation: (e) m/z = 925.7, (f) m/z = 939.7, (g) m/z = 953.7, and (h) m/z = In these spectra, the ceramide structure of the different species was resolved by measuring the intense fragments corresponding to the amide-linked α-hydroxylated fatty acids (C22), (C23), and (C24). 6

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8 Supplementary Figure 5 Comparison of isolated mmpegc-c22 and synthetic mmpegc- C22 (1) by LC-MS and MS2. (a,b) Extracted ion chromatograms of (a) isolated mmpegc-c22 and (b) synthetic mmpegc-c22 (1) displayed no difference in the retention time. (c,d) Comparison of the TOFMS and TOFMS2 spectra acquired on a Xevo G2-S revealed the same fragmentation pattern in the case of (c) the isolated mmpegc-c22 and (d) the synthetic mmpegc-c22 (1). (c,d) TOFMS2 data were acquired using a collision energy ramp from 20 ev to 50 ev. 8

9 Supplementary Figure 6 Expansion of an overlay of the COSY spectra of isolated and synthetic mmpegc-c22 (1). The COSY spectra (600 MHz, CD 3 OD) of isolated mmpegc-c22 in red and synthetic mmpegc-c22 (1) in green are superimposed. Overlapping cross peaks appear yellow. Due to the small amount of the isolated sample, some cross peaks could not be detected. 9

10 Supplementary Figure 7 Synthetic mmpegc-c22 (1) exhibits sterol-starvation rescue activity similar to the isolated mmpegc-c22. (a) The rescue capability of PEGC was investigated by the rescue ability of daf-12 mutant worms from sterol-starvation induced larval arrest (empty arrowheads). When either isolated mmpegcc22 or synthetic mmpegc-c22 (1) is fed to worms deprived of sterols the worms do not interrupt development but become fertile adults (filled arrowheads). The scale bars represent 0.5 mm. (b) Comparison of the activities of isolated mmpegc-c22 versus synthetic mmpegc-c22 (1) over a range of concentrations that were determined by LC-MS analysis. The results from experiments with biological duplicates in technical triplicates were pooled together and expressed as a percent value with concentrations ranging from 35 μm to 330 μm. The development of worms was scored after 4.5 d grown at 20 C. Error bar, SD. 10

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12 Supplementary Figure 8 bre-3 but not cgt-3;cgt-1 extract is capable to reverse sterolstarvation induced larval arrest (L2*) due to different amounts of PEGC. (a) Simplified biosynthetic pathway of sphingolipids and complex glycolipids in C. elegans, including the catalytic enzymes (blue) and the corresponding genes (green). (b) Second generation of daf-12 mutants on sterol-free agarose plates arrest as L2* larvae (empty arrowheads) whereas daf-12 worms on 13 μm cholesterol develop to adults (filled arrowhead, offspring marked with asterisk). Feeding a cgt-3;cgt-1 extract/bacterial suspension does not rescue the L2* arrest (empty arrowheads) whereas a bre-3 extract/bacterial suspension leads to fertile adults (filled arrowheads, offspring marked with asterisk). Pictures were taken 4.5 d after bleaching and the scale bars represent 1 mm. (c) Quantification of PEGC content (the sum of individual PEGC species) in extracts derived from different genetic backgrounds (wild-type, bre-3, cgt-3;cgt-1 and ncr-2;ncr-1 mutant worms) by LC-MS. For quantification, two independent biological samples for each strains have been analyzed. Wild-type extract was regarded as the reference. 12

13 Supplementary Figure 9 PEGC does not facilitate worm development by dissolution of residual sterols from the medium. (a+b) Common detergents and substances similar to PEGC (e.g., d17iso-glccer (11) and sulfatides) were tested for their ability to rescue daf-12 worms from the sterol depletion-induced growth arrest. None of these substances promoted development to adulthood. (b) Quantification of the adult formation after 5 days. The experiments were performed in biological duplicates in technical triplicates pooled together and expressed as a percent value. Error bar, SD. 13

14 Supplementary Figure 10 Quantification of PEGC content in different generations. Histogram displays the sum of individual PEGC species in extracts derived from L2/L2* wild-type worms grown on sterol supplemented (P0) or on sterol-depleted conditions (F1 and F2). 14

15 Supplementary Figure 11 Full COSY spectrum of isolated mmpegc-c22. COSY spectra (600 MHz, CD 3 OD) of isolated mmpegc-c22. 15