J. Thomas
Hannich
ac,
Denia
Mellal‡
bc,
Suihan
Feng
ac,
Andreas
Zumbuehl§
*bc and
Howard
Riezman§
*ac
aDepartment of Biochemistry, University of Geneva, CH-1205 Geneva, Switzerland. E-mail: howard.riezman@unige.ch
bDepartment of Chemistry, University of Fribourg, CH-1700 Fribourg, Switzerland. E-mail: andreas.zumbuehl@unifr.ch
cNational Centre of Competence in Research (NCCR) “Chemical Biology”, Switzerland
First published on 6th March 2017
Sphingolipids are bio-active metabolites that show structural diversity among eukaryotes. They are essential for growth of all eukaryotic cells but when produced in an uncontrolled manner can lead to cell death and pathologies including auto-immune reactions, cancer, diabetes and neurodegeneration. Caenorhabditis elegans is an important genetic model organism both to find new drug-targets against parasitic nematodes and to study the conserved roles of sphingolipids in animals like their essential functions in very basic cellular processes ranging from maintenance of cell polarity and mitochondrial repair to growth and survival. C. elegans produces sphingoid bases which are structurally distinct from those of other animals as both iso- and anteiso-branched species have been reported. Using metabolic labeling we show that most worm sphingoid bases are iso-branched. We have synthesized the nematode-specific C17 iso-branched sphinganine and its 1-deoxy analogue and could show that both the iso-branch and the 1-hydroxyl group are essential to form functional nematode sphingolipids which are needed to maintain intestinal function. The organism specificity was examined by complementation experiments in Saccharomyces cerevisiae yeast cells lacking sphingoid base synthesis. We found that iso-branched sphingoid base did not support growth of mutant cells and was toxic to wild type yeast. 1-Deoxy sphingolipids have been linked to the hereditary disease HSAN1A and other metabolic disorders including diabetes. We found that in C. elegans the 1-deoxy analogue cannot rescue the intestinal phenotype caused by sphingoid base depletion. In fact, in wild-type animals with normal sphingoid base biosynthesis, exogenous 1-deoxy analogue had a disruptive effect on apical cytoskeletal organization of intestinal cells indicating that atypical bases can interfere with normal sphingolipid function.
Fig. 1 The nematode C. elegans has mostly C17 iso-branched sphingoid bases. (a) Metabolic labelling of nematodes with heavy isotope leucine, isoleucine, valine, and lysine represented as percentage heavy of total free amino acid (average of n = 4), error bars show standard deviation; (b) incorporation of heavy isotope label from amino acids into sphingoid bases after isotopic peak correction (average of n = 4), error bars show standard deviation; (c) structures of detected worm sphingoid bases with incorporated 13C label, see also Scheme 1 and ESI Fig. 1c† (d) major sphingoid bases from budding yeast: sphinganine (d18:0) and 4-hydroxysphinganine (t18:0, changes in cyan), from nematodes: C17 iso-branched sphinganine (id17:0) and C17 iso-branched sphingosine (id17:1 changes in cyan), from mammals: sphinganine (d18:0) and sphingosine (d18:1, changes in cyan); shorthand nomenclature for sphingoid bases “aXX:Y”: a-number of hydroxyl groups (m = 1, d = 2, t = 3), XX-number of carbon atoms, Y-number of double bonds, the prefix “i” indicates the iso-branch. |
Wild-type N2 (var Bristol) and bacterial strains were received from the Caenorhabditis Genetics Centre (University of Michigan, Michigan, USA) VJ402 (fgEx11[ERM-1::GFP]) were a kind gift of Dr Verena Göbel. A complete list of bacteria, yeast strains and nematodes used in this study can be found in Table 1. Unless stated otherwise bacteria were grown on LB medium, yeast on rich medium (YPUATD: 1% yeast extract, 2% bacto peptone, each 40 mg l−1 uracil, adenine, tryptophan, 2% glucose, 2% bacto agar) and worms on NGM medium seeded with OP50 bacteria.36
Name | Genotype | Source |
---|---|---|
E. coli | ||
OP50 | Uracil auxotrophic E. coli B (Berkley strain) | CGC |
OP50 GFP | OP50 expressing plasmid pFVP25.1 | CGC |
HT115 (DE3) | F-, mcrA, mcrB, IN(rrnD-rrnE)1, lambda-, rnc14::Tn10(DE3 lysogen:lacUV5 promoter-T7 polymerase) | Kamath et al. 2003 |
HT115 sptl-1 | HT115 expressing plasmid L4440 (pPD129.36) containing a fragment of the sptl-1 cDNA | Kamath et al. 2003 |
S. cerevisiae | ||
RH448 | MATa ura3 leu2 his4 lys2 can1 bar1 | Laboratory WT |
RH1201 | MATα/a ura3/ura3 leu2/leu2 his4/his4 lys2/lys2 can1/can1 bar1/bar1 | This study |
RH6995 | MATα/a lcb1::KanMX6/LCB1 ura3/ura3 leu2/leu2 his4/his4 lys2/lys2 can1/can1 bar1/bar1 | This study |
RH6998 | MATa lcb1::KanMX6 ura3 leu2 his4 lys2 can1 bar1 | This study |
C. elegans | ||
N2 (var. Bristol) | C. elegans wild isolate | Brenner S. 1974 |
VJ402 | fgEx11[ERM-1::GFP] | Zhang et al. 2011 |
For immunostaining with MH33 anitbody against IFB-2 ERM-1::GFP animals were grown for three days on media containing ethanol or sphingoid bases at a concentration of 50 μM, intestines were dissected in buffer containing 0.1% tricaine, and 0.001% levamisole. Fixation and immunostaining was performed according to standard procedures.26,32 For quantification of confocal images 20 μm line profiles were taken across the intestinal lumen. Average apical signal was divided by average cytosolic. Student's t-test was used to compare the different conditions.
Footnotes |
† Electronic supplementary information (ESI) available: Experimental procedures and supplementary figures. See DOI: 10.1039/c6sc04831e |
‡ Current address: MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK. |
§ Equal contribution. |
This journal is © The Royal Society of Chemistry 2017 |