Ceramide phosphoethanolamine biosynthesis in Drosophila is mediated by a unique ethanolamine phosphotransferase in the Golgi lumen

Abstract

Sphingomyelin (SM) is a vital component of mammalian membranes, providing mechanical stability and a structural framework for plasma membrane organization. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, a reaction catalyzed by SM synthase in the Golgi lumen. Drosophila lacks SM and instead synthesizes the SM analogue ceramide phosphoethanolamine (CPE) as the principal membrane sphingolipid. The corresponding CPE synthase shares mechanistic features with enzymes mediating phospholipid biosynthesis via the Kennedy pathway. Using a functional cloning strategy, we here identified a CDP-ethanolamine:ceramide ethanolamine phosphotransferase as the enzyme responsible for CPE production in Drosophila. CPE synthase constitutes a new branch within the CDP-alcohol phosphotransferase superfamily with homologues in Arthropoda (insects, spiders, mites, scorpions), Cnidaria (Hydra, sea anemones), and Mollusca (oysters) but not in most other animal phyla. The enzyme resides in the Golgi complex with its active site facing the lumen, contrary to the membrane topology of other CDP-alcohol phosphotransferases. Our findings open up an important new avenue to address the biological role of CPE, an enigmatic membrane constituent of a wide variety of invertebrate and marine organisms.

FIGURE 1.

Drosophila S2 cells contain a CDP-Eth-dependent CPE synthase. A, TLC analysis of reaction products from lysates of Drosophila S2 cells (left) or human HeLa cells (right) incubated with NBD-Cer in the presence or absence of CDP-Eth and MnCl₂ (Mn²⁺). B, TLC separation of reaction products from lysates of dSMSr-depleted (dsSMSr) or mock-depleted (dsGFP) Drosophila S2 cells incubated with NBD-Cer and MnCl₂ in the absence (left) or presence (middle) of CDP-Eth. Right, immunoblots (WB) of dSMSr- or mock-depleted Drosophila S2 cells stained with anti-dSMSr and anti-dGolgi245 antibodies.

FIGURE 2.

Selection and phylogenetic analysis of candidate CPE synthases. A, reaction chemistry of PE and CPE biosynthesis in mammals and insects. See “Results and Discussion” for further details. DAG, diacylglycerol. B, phylogenetic tree of proteins in insects, Caenorhabditis elegans, human, and yeast containing a CAPT motif. The four candidate CPE synthases in Drosophila are marked dCCS1–4. The protein sequences were aligned with DIALIGN 2 (33) with manual editing to ensure correct alignment of the conserved pattern, D(X)₂DG(X)₂(A/Y)R(X)_8–16G(X)₃D(X)₃D. Alignment columns with a quality score of 4 or better were used to draw a phylogenetic tree with Protdist and Fitch from the PHYLIP package. The tree was displayed and edited with TreeIllustrator (34). NCBI GI numbers are: 1) 110750730 (translation of bases 327125–328027); 2) 158033532 (translation of bases 2339192–2338023); 3) O77475; 4) 6320059; 5) 23172318; 6) 10092647; 7) 71984834; 8) 6325370; 9) 17537129; 10) 5453906; 11) 24642243; 12) 6321915; 13) 42742307; 14) 50083289; 15) 28574275; 16) 28574587; 17) 71986977; 18) 193209951; 19) 115534720; 20) 50726996; 21) 24653393; 22) 5174415.

FIGURE 3.

Subcellular distribution of candidate CPE synthases. A, confocal sections of Drosophila S2 cells double-transfected with V5-tagged dCCS1, dCCS2, dCCS3, or dCCS4 and ER marker PE-methyltransferase-GFP. Cells were stained with anti-V5 antibody (top) or co-stained against the V5 epitope and Golgi marker dGMAP (bottom). Arrows indicate nuclear envelope staining, whereas arrowheads mark immunopositive Golgi structures. B, confocal sections of human HeLa cells transfected with V5-tagged dCCS1, dCCS2, dCCS3, or dCCS4 and stained with anti-V5 and anti-calnexin (ER) or anti-GM30 (Golgi) antibodies. Scale bars: 5 μm.

FIGURE 4.

dCCS4 is required for CDP-Eth-dependent CPE production in Drosophila S2 cells. A, immunoblots of Drosophila S2 cells transfected with V5-tagged dCCS1, dCCS2, dCCS3, or dCCS4 and then treated with dsRNA targeting individual dCCS proteins (ds dCCS1–4). Cells treated with dsRNA targeting GFP (ds GFP) served as control. Blots were stained with anti-V5 and anti-dGolgin245 antibodies. B, lysates of Drosophila S2 cells treated with dsRNA as in A were incubated with NBD-Cer in the presence of CDP-Eth, MnCl₂, and UDP-glucose. The amount of NBD-CPE formed was determined by TLC analysis, normalized against NBD-GlcCer levels, and then expressed as the percentage of control (ds GFP-treated cells). Error bars: S.D., n = 3. C, lysates of Drosophila S2 cells depleted for dCCS4 (ds dCCS4) or mock-depleted for GFP (ds GFP) were incubated with CDP-[¹⁴C]ethanolamine and then subjected to lipid extraction, TLC analysis, and autoradiography. Levels of [¹⁴C]CPE were normalized against [¹⁴C]PE levels and expressed as the percentage of control (ds GFP-treated cells). Error bars: range, n = 2. D, Drosophila S2 cells depleted for dCCS4 (ds dCCS4) or mock-depleted for GFP (ds GFP) were metabolically labeled for 2 h at 27 °C with [¹⁴C]ethanolamine and then subjected to lipid extraction, TLC analysis, and autoradiography. Levels of ¹⁴C-labeled CPE were normalized against [¹⁴C]PE levels and expressed as percentage of control (ds GFP-treated) cells. Error bars: S.D., n = 3.

FIGURE 5.

dCCS4 shows CDP-Eth:ceramide ethanolamine phosphotransferase activity. A, lysates of human HeLa cells transfected with V5-tagged dCCS4 or empty vector (EV) were incubated with NBD-Cer in the absence or presence of CDP-Eth and MnCl₂ and then subjected to lipid extraction and TLC analysis. B, lysates of HeLa cells transfected with V5-tagged dCCS4 or empty vector (EV) were incubated with NBD-Cer in the presence of CDP-[¹⁴C]Eth and MnCl₂. Lipids were extracted, separated by TLC, and then analyzed for fluorescence (left) and radioactivity (right). Note that only dCCS4p-expressing cells synthesized NBD-CPE that was labeled with ¹⁴C.

FIGURE 6.

Structure and membrane topology of CPES and related enzymes. A, alignment showing the conserved amino acid motif D(X)₂DG(X)₂(A/Y)R(X)_8–16G(X)₃D(X)₃D in the CAPT domain of various members of the CDP-alcohol phosphotransferase superfamily. Note the change in spacing between the two halves of the motif in CLS and CPES. The conserved Ala residue in the CAPT motif of CDP-alcohol phosphotransferases is substituted by a Tyr residue in CPES from Hydra and sea anemone (asterisk), which might have to do with the use of a phosphonate analogue of CDP-Eth as substrate in Cnidaria. Protein sequences were aligned with T-Coffee (35) for each family separately (CEPT, PIS, CLS, CPES). The alignment was trimmed manually and displayed with prettyplot from the EMBOSS package (36). NCBI GI numbers or UniProtKB identifiers are 24653393, 5174415, 193209951, 6321915, and 15222885 for CEPT; 24642243, 5453906, 6325370, 17537129, and 15220618 for PIS; 23172318, 10092647, 71984834, 6320059, and 18412722 for CLS; and O77475, 110750730 (translation of bases 327125–328027), B5M6F6, 204837661 (translation of bases 20637–21278), and A7S4T3 for CPES. B, hydrophobicity plots of CPES homologues from D. melanogaster (fly), Aedes aegypti (mosquito), and Bombyx mori (moth) created according to Kyte and Doolittle (37). Positions of putative transmembrane domains (TM1–6) and the CAPT motif are indicated. C, confocal sections of HeLa cells transfected with V5-tagged dCCS4 or empty vector (EV) and stained with anti-V5 antibodies before (intact) or after saponin-mediated membrane permeabilization (perm.). Scale bar: 15 μm. D, HeLa cells transfected with V5-tagged dCCS4 or empty vector (EV) were treated with trypsin in the presence or absence of Triton X-100 and then subjected to immunoblot analysis using anti-V5 and anti-β-actin antibodies. E, HeLa cells transfected with V5-tagged dCCS4 or empty vector were incubated at 15 °C with NBD-Cer in the presence or absence of CDP-Eth, MgCl₂, and streptolysin. NBD-labeled lipids formed on the cell surface were extracted by BSA, separated by TLC, and analyzed for fluorescence. F, schematic view of the predicted membrane topologies of CEPT (27) and CPES.

FIGURE 7.

Phylogenetic distribution of SM and CPE synthases in the animal kingdom. The phylogenetic distribution of SMS and CPES homologues in sequenced genomes correlates with the occurrence of their products SM, CPE, and CAEP (see under “Results and Discussion” for details). The phylogenetic tree was drawn with TreeViewX according to the classification in the NCBI/GenBank^TM Taxonomy Browser. The presence of SMS and CPES genes was checked by a BLAST search (blastp or tblastn) in the NCBI/GenBank genomic BLAST databases, the NCBI/GenBank nonredundant protein database, UniProtKB, and the Silkworm Genome Database: SilkDB (B. mori).