Sphingolipidomic Analysis of C. elegans reveals Development- and Environment-dependent Metabolic Features

Abstract

Sphingolipids (SLs) serve as structural and signaling molecules in regulating various cellular events and growth. Given that SLs contain various bioactive species possessing distinct roles, quantitative analysis of sphingolipidome is essential for elucidating their differential requirement during development. Herein we developed a comprehensive sphingolipidomic profiling approach using liquid chromatography-mass spectrometry coupled with multiple reaction monitoring mode (LC-MS-MRM). SL profiling of C. elegans revealed organism-specific, development-dependent and environment-driven metabolic features. We showed for the first time the presence of a series of sphingoid bases in C. elegans sphingolipid profiles, although only C17-sphingoid base is used for generating complex SLs. Moreover, we successfully resolved growth-, temperature- and nutrition-dependent SL profiles at both individual metabolite-level and network-level. Sphingolipidomic analysis uncovered significant SL composition changes throughout development, with SMs/GluCers ratios dramatically increasing from larva to adult stage whereas total sphingolipid levels exhibiting opposing trends. We also identified a temperature-dependent alteration in SMs/GluCers ratios, suggesting an organism-specific strategy for environmental adaptation. Finally, we found serine-biased GluCer increases between serine- versus alanine-supplemented worms. Our study builds a “reference” resource for future SL analysis in the worm, provides insights into natural variability and plasticity of eukaryotic multicellular sphingolipid composition and is highly valuable for investigating their functional significance.

iso‐Fatty Acid Metabolism in Caenorhabditis elegans’ Ceramide Biosynthesis

“…Inspection of ESI-(À )-HR-MS e spectra also confirmed that the [ 13 C 5 ]-labels are exclusively located in the homologous N-iso-acyl building blocks. Taken together, these results demonstrate that the biogenesis of N-iso-acyl substituted ceramides 18 (R' = CH 3 ) in wildtype C. elegans incorporates two L-leucine (12) units in the d17 : 1iso sphingosine (17) and N-iso-acyl moieties, respectively, and further indicates that these two iso-acyl building blocks are derived from two distinct biosynthetic pathways, the daf-22 dependent de novo biogenesis of d17 : 1iso sphingosine ( 17) and the daf-22 independent lipogenesis of very long chain iso-fatty acids (Scheme 2).…”

“…[16,29,30,49] Isomeric structures were assigned based on the incorporation of branchedchain amino acids (BCAA). A total of 48 ceramides 18 were identified, which constitute a homologous series characterized by a conserved d17 : 1iso sphingosine (17) backbone that is N-acylated with straight and isobranched acyl and 2-hydroxy-acyl moieties ranging from 16 to 27 carbons (Figure 1,a -1,c, Tables S1 and S2). C. elegans' ceramides are dominated by Cer d17 : 1iso/22:0(2OH) (18, n = 3, R' = H, X=OH) with a N-(2-hydroxy)docosanoyl moiety.…”

The lipidomes of C. elegans with mutations in asm-3/acid sphingomyelinase and hyl-2/ceramide synthase show distinct lipid profiles during aging

“…We note that our protocol called for thawing frozen samples prior to lipidomics sample preparation. This freeze and thaw have been previously done on C. elegans for lipidomics [ 36 , 85 ], and our quality control does not suggest degradation of any particular class of lipids. However, others have prepped from frozen C. elegans extracts directly [ 31 ].…”

Genetic and lipidomic analyses suggest that Nostoc punctiforme, a plant-symbiotic cyanobacterium, does not produce sphingolipids

“…However, although they appear to be much less common than unbranched LCBs, branched sphingolipids can still be found across kingdoms. For example, C17 methyl-branched LCBs are produced by Sphingobacterium species [31] and Caenorhabditis elegans [83, 84], whereas a C19 methyl-branched LCB is produced by a marine strain of the fungus Aspergillus niger [85]. Iso- and anteiso-branched fatty acids do occur in Nostoc and Anabaena species [86].…”