2D NMR-based metabolomics uncovers interactions between conserved biochemical pathways in the model organism Caenorhabditis elegans

Abstract

Ascarosides are small-molecule signals that play a central role in C. elegans biology, including dauer formation, aging, and social behaviors, but many aspects of their biosynthesis remain unknown. Using automated 2D NMR-based comparative metabolomics, we identified ascaroside ethanolamides as shunt metabolites in C. elegans mutants of daf-22, a gene with homology to mammalian 3-ketoacyl-CoA thiolases predicted to function in conserved peroxisomal lipid β-oxidation. Two groups of ethanolamides feature β-keto functionalization confirming the predicted role of daf-22 in ascaroside biosynthesis, whereas α-methyl substitution points to unexpected inclusion of methylmalonate at a late stage in the biosynthesis of long-chain fatty acids in C. elegans. We show that ascaroside ethanolamide formation in response to defects in daf-22 and other peroxisomal genes is associated with severe depletion of endocannabinoid pools. These results indicate unexpected interaction between peroxisomal lipid β-oxidation and the biosynthesis of endocannabinoids, which are major regulators of lifespan in C. elegans. Our study demonstrates the utility of unbiased comparative metabolomics for investigating biochemical networks in metazoans.

Figure 1

Ascarosides are signaling molecules in C. elegans. (a) Example structures and biological functions of ascaroside-based signaling molecules in C. elegans. (b) Model for peroxisomal biosynthesis of ascaroside side chains and roles of peroxisomal β-oxidation enzymes ACOX-1, MAOC-1, DHS-28, and the putative 3-ketoacyl-CoA thiolase DAF-22.

Figure 2

mvaDANS reveals structural motifs upregulated in daf-22 mutants. (a) Schematic overview of mvaDANS for identification of up- and downregulated metabolites in daf-22 mutants. In the Principal Component Analysis (PCA), PC 1 separates the four wild-type data sets (blue dots) from both daf-22 mutant data sets (red and brown dots), whereas PC2 separates the two daf-22 alleles. In the back projection (lower left), the coloring of cross speaks corresponds to the PC 1 loading coefficients; blue peaks represent signals downregulated and red peaks signals upregulated in the two daf-22 mutant strains. See Figure S4 for actual spectra. (b) Partial structures inferred from manual analysis of daf-22-specific crosspeaks suggesting α-methyl branched fatty acids, methyl ketones, and β-keto fatty acid derivatives.

Figure 3

Long-chain ascarosides identified in the daf-22-mutant metabolome and interaction with NEA biosynthesis. (a) HPLC-MS analysis confirms long-chain ascarosides and ethanolamides in the daf-22 metabolome that are absent in wild-type. (b) daf-22 mutant-specific VLCAs identified via mvaDANS and subsequent HPLC-MS analysis. (c) Quantitative distribution of daf-22-upregulated ascarosyl methylketones. (d) Mutation of maoc-1, dhs-28, and daf-22 greatly reduces EPEA production and addition of ascarosides (2.5 μM ascr#3 and ascr#9, see reference (4) for structures) to daf-22(m130) cultures does not rescue EPEA production.* P<0.05, ** P<0.01, *** P<0.001. (e) Interactions of peroxisomal β-oxidation and endocannabinoid biosynthesis. Mutation of daf-22 abolishes processing of long-chain ascaroside CoA esters, whose conversion into ethanolamides is associated with reduced endocannabinoid production (13), likely due to depletion of phosphatidylethanolamine pools.