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. 2017 May;58(5):962-973.
doi: 10.1194/jlr.M076133. Epub 2017 Mar 23.

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

Matthijs Kol  1   2 , Radhakrishnan Panatala  3   2 , Mirjana Nordmann  3 , Leoni Swart  2 , Leonie van Suijlekom  2 , Birol Cabukusta  3 , Angelika Hilderink  3 , Tanja Grabietz  3 , John G M Mina  3 , Pentti Somerharju  4 , Sergei Korneev  3 , Fikadu G Tafesse  5 , Joost C M Holthuis  1   2
Affiliations

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

Matthijs Kol et al. J Lipid Res. 2017 May.

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, SMS-related protein (SMSr)/SAMD8, acts as a monofunctional CPE synthase in the endoplasmic reticulum. Using domain swapping and site-directed mutagenesis on enzymes expressed in defined lipid environments, we here identified structural determinants that mediate the 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 Glu permitting SMS-catalyzed CPE production and Asp confining the enzyme to produce SM. An exchange of exoplasmic residues with SMSr proved sufficient to convert SMS1 into a bulk CPE synthase. This allowed us to establish mammalian cells that produce CPE rather than SM as the principal phosphosphingolipid and provide a model of the molecular interactions that impart catalytic specificity among SMS enzymes.

Keywords: Golgi apparatus; cell-free expression; ceramide phosphoethanolamine; click chemistry; enzyme mechanisms; lipid biochemistry; lipidomics; model membranes; protein engineering.

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Figures

Fig. 1.
Fig. 1.
The exoplasmic loops of SMSr and SMS1 harbor structural determinants of substrate selectivity. A: Predicted membrane topology of SMSr and SMS1. Active site residues are marked in red. The positions of two N-linked glycosylation sites introduced by site-directed mutagenesis in human SMSr are indicated. SAM, sterile α motif. B: Anti-V5 immunoprecipitates prepared from HeLa cells transfected with V5-tagged human SMSr, SMSrM180N, or SMSrG284N constructs were mock-treated or treated with EndoH and then subjected to immunoblot analysis with anti-V5 antibody. SMSr-G marks the migration of an N-glycosylated EndoH-sensitive form of SMSr that is produced exclusively in SMSrM180N- and SMSrG284N-expressing cells. C: Sequence alignment of loop c in SMS family members from vertebrates and fruit fly. The position of a residue critical for discriminating the phospholipid head group donors, PC and PE (Asp or Glu), is marked by an arrow. Database accession numbers are: human SMS1, BAD16809.1; chicken SMS1, ADY69193.1; frog SMS1, NP_001008197.1; zebrafish SMS1, NP_001071082.1; human SMS2, NP_689834.1; chicken SMS2, XP_420492.1; frog SMS2, AAH88568.1; zebrafish SMS2, zgc:100911; human SMSr, Q96LT4; chicken SMSr, XP_426501.3; frog SMSr, Q28CJ3; zebrafish SMSr, zgc:162183; fruit fly SMSr, CG32380. D: TLC analysis of reaction products formed when lysates of yeast strains expressing V5-tagged human SMS2 or SMS2E271D were incubated with C6-NBD-ceramide (NBD-Cer). EV denotes yeast lysate from strain transfected with empty vector. SMS expression was verified by immunoblotting with anti-V5 antibody (bottom). Note that residue substitution E271D in loop c converts SMS2 into a monofunctional SMS. E: TLC analysis of reaction products formed when lysates of yeast strains expressing V5-tagged human SMSr, SMSrE343D, SMS1, SMS1D327E, or SMS1/r chimera in which loop b was swapped (SMSrLb1, SMS1Lbr) were incubated with NBD-Cer. SMS expression was verified by immunoblotting with anti-V5 antibody (bottom). Note that residue substitution D327E combined with swapping loop b against that of SMSr converts SMS1 into a monofunctional CPE synthase (SMS1D327E-Lbr or SMS1CPE). Data shown in (D) and (E) are representative of two independent experiments.
Fig. 2.
Fig. 2.
Cell-free expression and functional analysis of human SMS family members. A: Schematic outline of the wheat germ-based dialysis mode for cell-free translation of SMS-V5 mRNA. Unless indicated otherwise, translation reactions were supplemented with liposomes containing equal amounts of phospholipid head group donors, PC and PE. B: Translation reactions with or without SMS-V5 mRNA were subjected to immunoblot analysis using anti-V5 antibody. Known amounts of a 75 kDa V5-tagged reference protein, pRef-V5, were included to allow quantification of cell-free-produced SMS-V5 protein. Note that translation reactions with SMS-V5 mRNA in each case yielded an immunoreactive protein of the expected size. C: SMS2-V5 mRNA was translated in the absence or presence of 2 mM liposomes. Translation reactions were subjected to density gradient fractionation and immunoblotting using anti-V5 antibody. D: SMS2-V5 mRNA was translated in wheat germ extract in the presence of the indicated amounts of liposomes. Cell-free produced SMS2-V5 was incubated with NBD-Cer and reaction products were analyzed by TLC. Expression of SMS2-V5 was verified by immunoblotting using anti-V5 antibody (bottom). E: TLC analysis of reaction products formed when SMS2-V5 produced in the presence of 2 mM liposomes was incubated with NBD-Cer for the indicated period of time. Sensitivity of detection was increased 10-fold compared with (D) to visualize SMS2-mediated production of NBD-CPE. Migration of an unidentified fluorescent lipid that was also present in reactions lacking SMS2 is marked by an asterisk. F: Quantification of reaction products formed by cell-free-produced SMS2-V5 when incubated with NBD-Cer for the indicated period of time. G: Functional analysis of cell-free-produced SMS1, SMS1CPE, and SMS2. TLC analysis of reaction products formed when the indicated SMS enzymes produced in the presence of 2 mM liposomes were incubated with NBD-Cer. SMS expression was verified by immunoblotting using anti-V5 antibody (bottom). Data shown are representative of three independent experiments.
Fig. 3.
Fig. 3.
Tracing catalytic activity of cell-free-produced SMS enzymes by click chemistry. A: A clickable ceramide analog, clickCer (1), was synthesized by condensation of D-erythro-sphingosine and a C15-fatty acid carrying a terminal alkyne group (7). The latter was synthesized in five steps, as detailed in the Materials and Methods. Reagents and conditions: a, Mg, Et2O, C11H19O3Cl, room temperature; b, NaBH4, methanol-water), room temperature; c, TsCl, Et3N, DMAP, room temperature; d, NaBH4, DMSO, 75°C; e, KOH, methanol, 50°C; f, sphingosine, EDCI, HOBT, room temperature. B: Schematic outline of the click-chemistry-based SMS assay. ClickCer was incorporated as SMS substrate in liposomes present during the cell-free translation of SMS-V5 mRNA. After lipid extraction the alkyne moiety was click-reacted with the fluorogenic dye, 3-azido-7-hydroxycoumarin, to yield fluorescently labeled lipids. The scheme was adapted from (29). C: V5-tagged SMS1, SMS1CPE, SMS2, and SMSr were produced cell-free in the presence of liposomes containing 2 mol% clickCer. After lipid extraction, SMS reaction products were click-reacted with 3-azido-7-hydroxycoumarin, separated by TLC, and analyzed by fluorescence detection. SMS expression was verified by immunoblotting using anti-V5 antibody (bottom). Data shown are representative of two independent experiments. Boldface numbers correspond to intermediates of clickCer synthesis described in Materials and Methods.
Fig. 4.
Fig. 4.
Switching head group selectivity in mammalian sphingolipid biosynthesis. A: HeLa cells were transfected with empty vector (EV), SMS1-HA, or SMS1CPE-HA, fixed, and then double-labeled with antibodies against the HA-tag (green) and Golgi marker, GM130 (red). Note that both SMS enzymes localize to the Golgi. Scale bar, 10 μm. B: Immunoblots of KBM7-derived WT and SMS1-null cells (SMS1KO) transfected with empty vector (EV), SMS1-HA, or SMS1CPE-HA were stained with antibodies against the HA-tag and β-actin. C: KBM7-derived WT and SMS1KO cells transfected with EV, SMS1-HA, or SMS1CPE-HA were metabolically labeled with [14C]ethanolamine for 24 h and then subjected to lipid extraction, TLC analysis, and autoradiography. In some extracts, glycerolipids were deacylated by mild alkaline hydrolysis (hydr. +) prior to TLC analysis. Note that only SMS1CPE-expressing cells produce a radiolabeled lipid resistant to alkaline hydrolysis and with an Rf of CPE. Metabolic labeling of insect Sf21 cells, which produce bulk amounts of CPE, served as control. D: SM and CPE levels in lipid extracts of KBM7-derived WT and SMS1KO cells expressing SMS1-HA or SMS1CPE-HA were determined by LC-MS/MS and expressed as mole percent of total phospholipid analyzed. E: Levels of SM and CPE species in KBM7-SMS1KO cells expressing SMS1-HA or SMS1CPE-HA were determined as in (D). Data shown in (D) and (E) are representative of two independent experiments.
Fig. 5.
Fig. 5.
Structural model of SMS head group selectivity. A: Membrane topology and structural elements that contribute to substrate specificity of SMS enzymes. The invariant His and Asp residues that form the catalytic triad are marked in red. Head group selectivity of SMS enzymes is determined by a single residue adjacent to the catalytic His in exoplasmic loop c, i.e., Asp in SMS1 (marked in green) or Glu in SMS2 and SMSr (marked in yellow), along with structural information in loop b (marked in blue). SAM, sterile α motif. B: Model explaining how structural elements in loops b and c of SMS enzymes cooperate to determine head group selectivity. See text for details.
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