Identification of a family of animal sphingomyelin synthases - PubMed (original) (raw)
Comparative Study
Identification of a family of animal sphingomyelin synthases
Klazien Huitema et al. EMBO J. 2004.
Abstract
Sphingomyelin (SM) is a major component of animal plasma membranes. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, yielding diacylglycerol as a side product. This reaction is catalysed by SM synthase, an enzyme whose biological potential can be judged from the roles of diacylglycerol and ceramide as anti- and proapoptotic stimuli, respectively. SM synthesis occurs in the lumen of the Golgi as well as on the cell surface. As no gene for SM synthase has been cloned so far, it is unclear whether different enzymes are present at these locations. Using a functional cloning strategy in yeast, we identified a novel family of integral membrane proteins exhibiting all enzymatic features previously attributed to animal SM synthase. Strikingly, human, mouse and Caenorhabditis elegans genomes each contain at least two different SM synthase (SMS) genes. Whereas human SMS1 is localised to the Golgi, SMS2 resides primarily at the plasma membrane. Collectively, these findings open up important new avenues for studying sphingolipid function in animals.
Figures
Figure 1
Selection and phylogenetic analysis of CSSs. (A) Animal entries in SwissProt/TrEMBL were searched for the presence of a sequence motif shared by LPPs and Aur1p proteins and then further selected on the basis of three additional criteria, as indicated. (B) Phylogenetic tree of human CSSs and previously characterised members of the human LPP superfamily. (C) Phylogenetic tree of CSS proteins from human (Hs), mouse (Mm), C. elegans (Ce) and D. melanogaster (Dm), and of Aur1p proteins from S. cerevisiae (Sc), Schizosaccharomyces pombe (Sp) and Candida albicans (Ca). Asterisks denote CSS proteins expressed in S. cerevisiae and tested for SM synthase activity. SM synthases (SMS) are marked in red. SwissProt/TrEMBL accession numbers of CSS proteins are: (1) Q9TYV2; (2) Q20696; (3) Q9VS60/Q9VS61; (4) Q96LT4; (5) Q9DA37; (6) Q86VZ5; (7) Q8VCQ6; (8) Q8NHU3; (9) Q9D4B1; (10) Q9XTV2; (11) Q20735; (12) Q965Q4; (13) Q9D606; (14) Q9NXE2; (15) Q8VCY8; (16) Q96GM1; (17) Q96MP0; (18) AAP57768; (19) Q9BQF9; (20) AAP57767; (21) Q22250; (22) Q9TXU1; (23) Q9VNT9; (24) Q9VNU1; (25) Q10022; (26) Q9D4F2; (27) Q8IY26; (28) Q91WB2; (29) Q96SS7; (30) Q8T8T9; (31) Q22461. Note that Q96LT4 contains a partial protein sequence and that the complete ORF was deduced from a corresponding EST clone (Table I).
Figure 2
A subset of CSS3 family members displays SM synthase activity upon expression in yeast. (A) Immunoblots of cells expressing various CSS proteins were stained with antibodies recognising the V5 epitope-tagged carboxy termini of CSS proteins. Control denotes cells transformed with empty vector. (B) TLC separation of reaction products generated when NBD-ceramide (NBD-Cer) was incubated with lysates of control or CSS-expressing cells in the presence (+) or absence (−) of the IPC synthase inhibitor aureobasidin A (Aba). (C) Metabolic labelling of cells expressing human CSS3α1/SMS1 or CSS3α2/SMS2 with [14C]-choline and NBD-ceramide. The lipids were extracted, separated by two-dimensional TLC and analysed for fluorescence and radioactivity. Note that SMS1- and SMS2-expressing cells, but not control cells, synthesised NBD-SM, and that this NBD-SM was labelled with [14C]-choline. (D) Metabolic labelling of cells expressing human CSS3α2/SMS2 with [14C]-choline. The lipids were extracted, deacylated by mild alkaline hydrolysis (+NaOH) or control incubated (−NaOH) and separated by two-dimensional TLC before autoradiography. Note that SMS2-expressing cells synthesised alkaline-resistant species of [14C]-choline-labelled lipids (phytoSM) that were absent in control cells.
Figure 3
Detergent extracts of human CSS3α2/SMS2-expressing yeast cells support SM formation from bovine brain ceramides. Lines A and B show the reconstituted ion chromatograms of m/z 731.6, corresponding to the m/z ratio of protonated 18:0 SM, during the separation of molecular species of choline-containing phospholipids after solid-phase extraction of membrane extracts that had been incubated with bovine brain ceramides and egg PC. The elution time of an authentic standard of 18:0 SM is indicated in the chromatogram by an arrow (42.3 min). Line A was derived from detergent extracts of human CSS3α2/SMS2-expressing cells and line B from detergent extracts of control cells. Mass spectra recorded at the elution time of 18:0 SM confirmed that formation of 18:0 SM occurred in CSS3α2/SMS2-containing extracts (top right panel) but not in control extracts (bottom right panel).
Figure 4
Human SMS1 and SMS2 function as PC:ceramide cholinephosphotransferases. (A) Detergent extracts of yeast cells expressing human SMS1, SMS2 or transformed with empty vector (control) were incubated with NBD-ceramide (18 μM) in the presence or absence of different potential head group donors (220 μM), as indicated. Formation of NBD-SM or NBD-IPC was monitored by one-dimensional TLC and quantified as described in Materials and methods. Note that addition of PI stimulated formation of NBD-IPC in all three extracts (asterisks). PC, phosphatidylcholine; SM, sphingomyelin; Ch, choline; Ch-P, phosphorylcholine; CDP-Ch, cytidine-5′ diphosphocholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PA, phosphatidic acid; PG, phosphatidylglycerol. (B) Detergent extracts of SMS2-expressing and control cells were incubated with NBD-ceramide and [3H]-choline-labelled PC. The lipids were extracted, separated by two-dimensional TLC and analysed for fluorescence and radioactivity. Note that only SMS2-expressing cells synthesised NBD-SM, and that this NBD-SM was labelled with [3H]-choline.
Figure 5
Human SMS1 and SMS2 exhibit reverse activity. Detergent extracts of yeast cells expressing human SMS1, SMS2 or transformed with empty vector (control) were incubated with NBD-diacylglycerol (NBD-DAG; 18 μM) in the presence or absence of different head group donors (220 μM), as indicated. Formation of NBD-PC was monitored by one-dimensional TLC and quantified as described in Materials and methods.
Figure 6
Conserved sequence motifs in SMS1, SMS2 and SMSr proteins. (A) Alignment of human SMS1, SMS2 and SMSr amino-acid sequences. Identical residues are highlighted in black and conservative amino-acid substitutions in grey. Conserved residues within four homology motifs, designated D1–D4, are highlighted in blue and conservative amino-acid substitutions in the D2 and D4 motifs of SMSr proteins in green. Note that motifs D3 and D4 display similarity to the C2 and C3 domains in LPPs, with identical residues highlighted in red. Regions predicted to form transmembrane domains, TM1–TM6, are marked by a black line. (B) Alignment of the four homology motifs in SMS and SMSr proteins from humans (Hs), C. elegans (Ce), P. falciparum (Pf) and D. melanogaster (Dm). PlasmoDB accession numbers of _Pf_SMS1 and _Pf_SMS2 are MAL6P1.178 and MAL6P1.177, respectively. Putative active site residues in consensus sequences are underlined. Symbols used are: n, small neutral amino acid; Φ, aromatic amino acid; b, branched amino acid; x, any amino acid.
Figure 7
Human SMS1 and SMS2 are encoded by ubiquitously expressed genes. Northern blot analysis of SMS1 and SMS2 transcripts (arrows) in various human tissues. Random prime-labelled human SMS1 or SMS2 cDNA was hybridised to a human poly(A)+ RNA blot (Origene, Rockville, MD). As a control for loading, the RNA blot was stripped and rehybridised with a human β-actin cDNA probe. Mobilities of RNA size markers are indicated.
Figure 8
Human SMS1 and SMS2 localise to different cellular organelles. (A) HeLa cells co-transfected with V5-tagged SMS1 and _myc_-tagged sialyltransferase were incubated in the presence or absence of 10 μM nocodazole for 60 min at 37°C, fixed and then co-stained with rabbit anti-V5 and mouse anti-myc antibodies. Counterstaining was with FITC-conjugated goat anti-rabbit and Texas red-conjugated goat anti-mouse antibodies. (B) HeLa cells transfected with V5-tagged SMS2 were biotinylated on ice, fixed and then co-stained with mouse anti-V5 and rabbit anti-biotin antibodies. Counterstaining was with Texas red-conjugated goat-anti mouse and FITC-conjugated goat anti-rabbit antibodies. Bar, 10 μm.
Figure 9
Membrane topology of human SMS1 and SMS2 proteins. (A) Schematic view of the predicted membrane topologies of V5-tagged SMS1 and SMS2, and the Golgi-associated type I membrane protein, p24. (B) Immunoblot of intact or lysed HeLa cells expressing V5-tagged SMS1 or SMS2 and pretreated with 8 mM trypsin for 30 min at 30°C in the presence or absence of 0.4% Triton X-100, as indicated. Immunoblots were stained with antibodies against the V5 epitope (α-V5) or against p24 (α-p24).
Similar articles
- Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site engineering of sphingomyelin synthases.
Kol M, Panatala R, Nordmann M, Swart L, van Suijlekom L, Cabukusta B, Hilderink A, Grabietz T, Mina JG, Somerharju P, Korneev S, Tafesse FG, Holthuis JC. Kol M, et al. J Lipid Res. 2016 Jul;57(7):1273-85. doi: 10.1194/jlr.M068692. Epub 2016 May 10. J Lipid Res. 2016. PMID: 27165857 Free PMC article. Retracted. - Inhibition of sphingomyelin synthase (SMS) affects intracellular sphingomyelin accumulation and plasma membrane lipid organization.
Li Z, Hailemariam TK, Zhou H, Li Y, Duckworth DC, Peake DA, Zhang Y, Kuo MS, Cao G, Jiang XC. Li Z, et al. Biochim Biophys Acta. 2007 Sep;1771(9):1186-94. doi: 10.1016/j.bbalip.2007.05.007. Epub 2007 Jun 6. Biochim Biophys Acta. 2007. PMID: 17616479 Free PMC article. - Both sphingomyelin synthases SMS1 and SMS2 are required for sphingomyelin homeostasis and growth in human HeLa cells.
Tafesse FG, Huitema K, Hermansson M, van der Poel S, van den Dikkenberg J, Uphoff A, Somerharju P, Holthuis JC. Tafesse FG, et al. J Biol Chem. 2007 Jun 15;282(24):17537-47. doi: 10.1074/jbc.M702423200. Epub 2007 Apr 22. J Biol Chem. 2007. PMID: 17449912 - Role of ceramide/sphingomyelin (SM) balance regulated through "SM cycle" in cancer.
Taniguchi M, Okazaki T. Taniguchi M, et al. Cell Signal. 2021 Nov;87:110119. doi: 10.1016/j.cellsig.2021.110119. Epub 2021 Aug 19. Cell Signal. 2021. PMID: 34418535 Review. - Phosphatidylinositol synthase from yeast.
Nikawa J, Yamashita S. Nikawa J, et al. Biochim Biophys Acta. 1997 Sep 4;1348(1-2):173-8. doi: 10.1016/s0005-2760(97)00103-3. Biochim Biophys Acta. 1997. PMID: 9370330 Review.
Cited by
- Complex sphingolipid profiling and identification of an inositol-phosphorylceramide synthase in Dictyostelium discoideum.
Listian SA, Mazur AC, Kol M, Ufelmann E, Eising S, Fröhlich F, Walter S, Holthuis JCM, Barisch C. Listian SA, et al. iScience. 2024 Jul 30;27(9):110609. doi: 10.1016/j.isci.2024.110609. eCollection 2024 Sep 20. iScience. 2024. PMID: 39286488 Free PMC article. - Elongation of Very Long-Chain Fatty Acids (ELOVL) in Atopic Dermatitis and the Cutaneous Adverse Effect AGEP of Drugs.
Blaess M, Csuk R, Schätzl T, Deigner HP. Blaess M, et al. Int J Mol Sci. 2024 Aug 28;25(17):9344. doi: 10.3390/ijms25179344. Int J Mol Sci. 2024. PMID: 39273293 Free PMC article. Review. - Emerging role of sphingolipids and extracellular vesicles in development and therapeutics of cardiovascular diseases.
Bhat OM, Mir RA, Nehvi IB, Wani NA, Dar AH, Zargar MA. Bhat OM, et al. Int J Cardiol Heart Vasc. 2024 Jul 23;53:101469. doi: 10.1016/j.ijcha.2024.101469. eCollection 2024 Aug. Int J Cardiol Heart Vasc. 2024. PMID: 39139609 Free PMC article. Review. - Integrating transcriptomics and metabolomics to reveal the protective effect and mechanism of Bushen Kangshuai Granules on the elderly people.
Hu J, Yang F, Yang G, Pan J, Tan Y, Tang Y, Liu Y, Zhang H, Wang J. Hu J, et al. Front Pharmacol. 2024 Jul 29;15:1361284. doi: 10.3389/fphar.2024.1361284. eCollection 2024. Front Pharmacol. 2024. PMID: 39135783 Free PMC article. - Regulation of cellular and systemic sphingolipid homeostasis.
Kuo A, Hla T. Kuo A, et al. Nat Rev Mol Cell Biol. 2024 Oct;25(10):802-821. doi: 10.1038/s41580-024-00742-y. Epub 2024 Jun 18. Nat Rev Mol Cell Biol. 2024. PMID: 38890457 Review.
References
- Andrieu-Abadie N, Levade T (2002) Sphingomyelin hydrolysis during apoptosis. Biochim Biophys Acta 1585: 126–134 - PubMed
- Bankaitis VA (2002) Cell biology Slick recruitment to the Golgi. Science 295: 290–291 - PubMed
- Bourteele S, Hausser A, Doppler H, Horn-Muller J, Ropke C, Schwarzmann G, Pfizenmaier K, Muller G (1998) Tumor necrosis factor induces ceramide oscillations and negatively controls sphingolipid synthases by caspases in apoptotic Kym-1 cells. J Biol Chem 273: 31245–31251 - PubMed
- Brauer AU, Savaskan NE, Kuhn H, Prehn S, Ninnemann O, Nitsch R (2003) A new phospholipid phosphatase, PRG-1, is involved in axon growth and regenerative sprouting. Nat Neurosci 6: 572–578 - PubMed
- Brose N, Rosenmund C (2002) Move over protein kinase C, you've got company. J Cell Sci 115: 4399–4411 - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Molecular Biology Databases
Research Materials
Miscellaneous