Succinylated octopamine ascarosides and a new pathway of biogenic amine metabolism in Caenorhabditis elegans

Abstract

The ascarosides, small-molecule signals derived from combinatorial assembly of primary metabolism-derived building blocks, play a central role in Caenorhabditis elegans biology and regulate many aspects of development and behavior in this model organism as well as in other nematodes. Using HPLC-MS/MS-based targeted metabolomics, we identified novel ascarosides incorporating a side chain derived from succinylation of the neurotransmitter octopamine. These compounds, named osas#2, osas#9, and osas#10, are produced predominantly by L1 larvae, where they serve as part of a dispersal signal, whereas these ascarosides are largely absent from the metabolomes of other life stages. Investigating the biogenesis of these octopamine-derived ascarosides, we found that succinylation represents a previously unrecognized pathway of biogenic amine metabolism. At physiological concentrations, the neurotransmitters serotonin, dopamine, and octopamine are converted to a large extent into the corresponding succinates, in addition to the previously described acetates. Chemically, bimodal deactivation of biogenic amines via acetylation and succinylation parallels posttranslational modification of proteins via acetylation and succinylation of L-lysine. Our results reveal a small-molecule connection between neurotransmitter signaling and interorganismal regulation of behavior and suggest that ascaroside biosynthesis is based in part on co-option of degradative biochemical pathways.

Keywords: Biosynthesis; Dauer; Dopamine; Neurotransmitters; Pheromone; Serotonin; Small-molecule signaling.

FIGURE 1.

Ascarosides in C. elegans and detection of ascarosides specific to starved L1 larvae. A, structures and functions of ascarosides produced by different developmental stages of C. elegans. In this study, we investigated ascarosides specifically produced by arrested L1 larvae. B, HPLC-MS analysis of exo-metabolome samples obtained from starved L1 worms using negative-ion electrospray ionization reveals a novel ascaroside derivative at m/z 482 (osas#9). Its mass spectrometric fragmentation pattern suggests that it is derived from the known ascr#9. C, proposed structures of osas#9 and two related compounds, osas#2 and osas#10.

FIGURE 2.

Synthesis, biosynthesis, and biological properties of osas#9. A, synthesis of osas#9 and tsas#9. (a) succinic anhydride, N,N-diisopropylethylamine, 4-dimethylaminopyridine, dimethylformamide; (b), 4-dimethylaminopyridine, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, dichloromethane; (c), H₂(g), Pd/C, MeOH; (d), LiOH, tetrahydrofuran/dioxane/H₂O. B, comparison of relative abundances of major ascarosides in starved L1, fed L1, and mixed stage exo-metabolomes of C. elegans (N2). C, model for the biosynthesis of octopamine, tyramine, and derived ascarosides tsas#9 and osas#9 in C. elegans. D, osas#9 induces avoidance behavior in the absence of bacterial food (*, no avoidance response). E, medium from starved tdc-1 L1 larvae is less deterrent to young adult worms than medium from wild-type L1 larvae. This experiment was conducted in the absence of bacterial food. F, ratio of N-succinyl to N-acetyl derivatives of serotonin in N2 L1 medium as a function of exogenously added serotonin as determined by positive-ion electrospray ionization MS. G, concentrations of osas ascarosides strongly decrease in response to exogenously added serotonin (5-hydroxy tryptamine (5-HT)), whereas levels of all other ascarosides are only marginally affected.