Comparative metabolomics reveals biogenesis of ascarosides, a modular library of small-molecule signals in C. elegans

Article abstract of DOI:10.1021/ja210202y

In the model organism Caenorhabditis elegans, a family of endogenous small molecules, the ascarosides function as key regulators of developmental timing and behavior that act upstream of conserved signaling pathways. The ascarosides are based on the dideoxysugar ascarylose, which is linked to fatty-acid-like side chains of varying lengths derived from peroxisomal β-oxidation. Despite the importance of ascarosides for many aspects of C. elegans biology, knowledge of their structures, biosynthesis, and homeostasis remains incomplete. We used an MS/MS-based screen to profile ascarosides in C. elegans wild-type and mutant metabolomes, which revealed a much greater structural diversity of ascaroside derivatives than previously reported. Comparison of the metabolomes from wild-type and a series of peroxisomal β-oxidation mutants showed that the enoyl CoA-hydratase MAOC-1 serves an important role in ascaroside biosynthesis and clarified the functions of two other enzymes, ACOX-1 and DHS-28. We show that, following peroxisomal β-oxidation, the ascarosides are selectively derivatized with moieties of varied biogenetic origin and that such modifications can dramatically affect biological activity, producing signaling molecules active at low femtomolar concentrations. Based on these results, the ascarosides appear as a modular library of small-molecule signals, integrating building blocks from three major metabolic pathways: carbohydrate metabolism, peroxisomal β-oxidation of fatty acids, and amino acid catabolism. Our screen further demonstrates that ascaroside biosynthesis is directly affected by nutritional status and that excretion of the final products is highly selective.

Full text of DOI:10.1021/ja210202y

Article
pubs.acs.org/JACS
Comparative Metabolomics Reveals Biogenesis of Ascarosides, a
Modular Library of Small-Molecule Signals in C. elegans
Stephan H. von Reuss,† Neelanjan Bose,† Jagan Srinivasan,‡ Joshua J. Yim,† Joshua C. Judkins,†
Paul W. Sternberg,‡ and Frank C. Schroeder*,†
†Boyce Thompson Institute and Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853,
United States
‡Howard Hughes Medical Institute and Division of Biology, California Institute of Technology, Pasadena, California 91125, United
States
S
* Supporting Information
ABSTRACT: In the model organism Caenorhabditis elegans, a
family of endogenous small molecules, the ascarosides function
as key regulators of developmental timing and behavior that
act upstream of conserved signaling pathways. The ascarosides
are based on the dideoxysugar ascarylose, which is linked to
fatty-acid-like side chains of varying lengths derived from
peroxisomal β-oxidation. Despite the importance of ascaro-
sides for many aspects of C. elegans biology, knowledge of their
structures, biosynthesis, and homeostasis remains incomplete.
We used an MS/MS-based screen to profile ascarosides in C.
elegans wild-type and mutant metabolomes, which revealed a
much greater structural diversity of ascaroside derivatives than previously reported. Comparison of the metabolomes from wild-
type and a series of peroxisomal β-oxidation mutants showed that the enoyl CoA-hydratase MAOC-1 serves an important role in
ascaroside biosynthesis and clarified the functions of two other enzymes, ACOX-1 and DHS-28. We show that, following
peroxisomal β-oxidation, the ascarosides are selectively derivatized with moieties of varied biogenetic origin and that such
modifications can dramatically affect biological activity, producing signaling molecules active at low femtomolar concentrations.
Based on these results, the ascarosides appear as a modular library of small-molecule signals, integrating building blocks from
three major metabolic pathways: carbohydrate metabolism, peroxisomal β-oxidation of fatty acids, and amino acid catabolism.
Our screen further demonstrates that ascaroside biosynthesis is directly affected by nutritional status and that excretion of the
final products is highly selective.
of both the dauer pheromone and male-attracting signals.3,9,10
daf-22 encodes a protein with strong homology to human sterol
carrier protein SCPx and in C. elegans functions in peroxisomal
Caenorhabditis elegans are regulated by ascarosides, glycosides
1. INTRODUCTION
Several different aspects of the life history of the nematode
of the dideoxysugar ascarylose (Figure 1).1−5 The ascarosides
ascr#1−3 were originally identified as major components of the
dauer pheromone, a population-density signal that promotes
entry into an alternate larval stage, the nonfeeding and highly
persistent dauer diapause.1,2,4−6 Entry into the dauer stage is
mediated by several highly conserved signaling pathways,
including insulin/IGF-1 signaling and transforming growth
factor beta (TGF-β) signaling,7 which contributed to interest in
the ascarosides’ structures, their biosynthesis, and their mode of
action. More recent work showed that specific mixtures of
ascarosides including the 4-aminobenzoic acid derivative ascr#8
act as strong male-specific attractants,3,5 whereas ascarosides
including a tryptophan-derived indole-3-carboxy moiety
function as aggregation signals at femtomolar concentrations.8
The biosynthetic pathways that control specific assembly of
the ascarylose, lipid side chain, and peripheral building blocks
are largely unknown. Earlier work showed that worms carrying
a mutation in the gene daf-22 are defective in the biosynthesis
β-oxidation of long-chain fatty acids, producing the 3−9-carbon
side chains of the ascarosides.6 Two other components of
peroxisomal β-oxidation were shown to participate in ascaro-
side biosynthesis: the acyl-CoA oxidase ACOX-111 and the β-
hydroxyacyl-CoA dehydrogenase DHS-28,6,12 a partial homo-
logue of human multifunctional enzyme type 2 (MFE-2).
However, the exact roles of these enzymes in ascaroside
biosynthesis remained unclear, because the effects of acox-1 and
dhs-28 mutations on ascaroside production were only partially
characterized.
The recent discovery of new structural variants with
important divergent functions, e.g., icas#3 and icas#9,8,13
suggested that knowledge of ascaroside structures and functions
in C. elegans remains incomplete. Results from biological studies
further indicate that even small structural differences between
Received: October 30, 2011
Published: January 5, 2012
© 2012 American Chemical Society
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Figure 1. Ascarosides that regulate development and behavior in C.
elegans: ascr#1−4, ascr#5, and icas#9 were identified via activity-guided
fractionation;1−4,13 ascr#7 and ascr#8 were identified using differential
analysis of 2D NMR spectra;5 and ascr#9 and #10 were detected using
mass spectrometric techniques.8
ascarosides can be associated with significant functional
differences; for example, ascr#3 is a potent male attractant,
whereas the structurally similar ascr#7 is nearly inactive in this
assay.5,14 Correspondingly, several previous studies indicate that
ascaroside biosynthesis is tightly controlled by environmental
factors such as temperature, nutrient availability, and
population density.6,11,15 Therefore, detailed investigation of
ascaroside structures and their biosynthetic pathways is
essential for many aspects of the biology of this model
organism.
In this study, we introduce HPLC-MS/MS-based metab-
olomics as a tool for ascaroside profiling in C. elegans.
Application of this method to wild-type C. elegans revealed
that the previously described ascarosides are part of a much
larger, structurally diverse library of compounds derived from
modular combination of building blocks from three different
metabolic pathways. Subsequently we used HPLC-MS/MS of
mutant metabolomes to interrogate ascaroside biosynthesis and
homeostasis.
Figure 2. (a) LC-MS total ion current chromatogram of wild-type C.
elegans excretome (ESI−). (b) MS/MS fragmentation of ascarosides.
(c) LC-MS/MS screen (precursors of m/z 73) of wild-type excretome
reveals known ascarosides (black), new homologues (blue), new ω-
oxygenated isomers (red), and new 4′-acylated derivatives (green) (*
indicates signals from non-ascarosides). The highly polar ascr#5 elutes
at 6.5 min, outside of the retention time range shown.
unit; however, we found that ESI+ MS/MS detection of
ascarosides is generally less sensitive than ESI− MS/MS
detection, as ascarosides fragment less predictably under ESI+
conditions. Next, we tested whether a screen for precursor ions
of m/z 73 could be used to detect known as well as yet-
unidentified ascarosides in the C. elegans wild-type metabolome.
For this purpose we used liquid culture metabolite extracts,
which contain accumulated excreted metabolites from large
numbers of worms (the worm “excretome”). The resulting
HPLC-MS/MS chromatograms showed a large number of well-
resolved peaks, most of which we found to represent
ascarosides, including several families of previously undetected
compounds.
We first confirmed the identities of the known ascarosides
using synthetic standards. In addition we found that the known
saturated ascarosides ascr#1, ascr#9, and ascr#10 are accom-
panied by substantial quantities of homologues with 6−16-
carbon side chains (Figures 2c and 3a). Identification of this
homologous series was based on high-resolution MS/MS, LC
retention times, and synthesis of representative members (see
Supporting Information section 1.7 and Figure S2). The LC-
MS/MS screen further revealed that ascarosides with side
chains of 5−11 carbons are accompanied by smaller quantities
of slightly less polar isomers. These ascaroside isomers are
produced more abundantly by acox-1 mutant worms (vide
infra).
2. RESULTS
2.1. LC-MS/MS Reveals New Ascarosides in Wild-Type
C. elegans. We aimed at developing a method that would (1)
facilitate sensitive detection and quantitation of the known
ascarosides in the metabolomes of different C. elegans strains
and mutants and (2) aid with the discovery of new ascaroside
derivatives. Because of the vast complexity of the C. elegans
metabolome, HPLC-MS analysis of metabolite extracts results
in extremely crowded chromatograms that are difficult to
interpret (Figure 2a). However, it seemed likely that
structurally related ascarosides would exhibit characteristic
MS/MS fragmentation patterns, providing a much more
selective and sensitive means for their detection. Therefore
we investigated ESI MS/MS fragmentation of a series of
synthetic ascarosides. We found that with negative-ion
electrospray ionization (ESI−), ascarosides give rise to an
intense and highly diagnostic product ion at m/z 73.02939
[C3H5O2] which originates from the ascarylose unit (Figures 2b
and S1). This detection method proved suitable for all known
ascarosides, except for ascr#2 and ascr#4 which do not ionize
well under ESI− conditions. For detection of ascarosides that
ionize only under ESI+ conditions, we monitored neutral loss of
130.0663 amu [C6H10O3] due to cleavage of the ascarylose
Several additional peaks in the wild-type MS/MS chromato-
grams could not be assigned to any of the known ascaroside
classes. For two of these compounds, MS/MS product ions at
m/z 301.1651 [C15H25O6] suggested that they represent ascr#3
derivatives. The putative ascr#3 derivatives were isolated by
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functions appear to be separated in C. elegans such that the
hydratase and dehydrogenase are distinct proteins (Figure
S6).18 Previous work showed that C. elegans DHS-28, a protein
with homology to the (R)-selective β-hydroxyacyl-CoA
dehydrogenase domain of human MFE-2, likely participates
in converting β-hydroxyacyl-CoA-derivatives into the corre-
sponding β-ketoacyl-CoA intermediates, which are subse-
quently cleaved by the β-ketoacyl-CoA thiolase DAF-22.6,12
However, it remained unclear which enzyme serves as the
enoyl-CoA hydratase that catalyzes the essential second step of
the β-oxidation cascade. Using our MS/MS-based ascaroside
screen, we reinvestigated the ascaroside profiles of acox-
1(ok2257), dhs-28(hj8), and daf-22(ok693) mutant worms.
Additionally, we analyzed the excretomes of several other
peroxisomal mutants, including maoc-1(hj13) worms, because a
recent study indicated that maoc-1 encodes a peroxisomal 2-
enoyl-CoA hydratase,19 which we hypothesized could partic-
ipate in the hydration step of ascaroside β-oxidation.
2.3. Side-Chain Functionalization Precedes β-Oxida-
tion. LC-MS/MS analysis of the excretome of acox-1(ok2257)
mutant worms revealed that the abundance of the α,β-
unsaturated ascr#3, the dominating component of wild-type
media, was greatly reduced (Figure 4c). This decrease in ascr#3
and other α,β-unsaturated ascarosides does not appear to be the
result of overall down-regulation in ascaroside production, but
instead is accompanied by accumulation of a series of saturated
ascarosides. For example, ascr#10, the dihydro derivative and
direct precursor of ascr#3, is 13.6 times more abundant in acox-
1(ok2257) than in the wild-type excretome. The corresponding
homologues with 11- and 13-carbon side chains, ascr#18 and
ascr#22, are 29 times and 66 times more abundant in acox-1
than in the wild-type excretome, respectively. The buildup of
longer chained saturated ascarosides in the acox-1(ok2257)
excretome confirms the importance of ACOX-1 in α,β-
dehydrogenation of the ascaroside side chain (Figure 4a).
Because ascaroside biosynthesis is not abolished in acox-
1(ok2257) worms, it seems likely that other, yet-unidentified
ACOX-enzymes contribute to peroxisomal β-oxidation of long-
chain ascaroside precursors. However, LC-MS/MS analysis of
excretome of several other peroxisomal acox mutants (see
Supporting Information, Figure S7) revealed largely wild-type-
like ascaroside profiles.
Further analysis of the acox-1(ok2257) excretome revealed
the complete absence of ascr#5, one of the major dauer-
inducing ascarosides produced abundantly in wild-type (Figure
4d). Ascr#5 differs from all other previously identified
ascarosides in that its side chain is attached to the ascarylose
sugar via the terminal carbon (“ω linkage”) and not the
penultimate carbon (“ω−1 linkage”) (Figure 3b). Instead of
ascr#5, we detected large quantities of a new homologous series
of saturated ascarosides in acox-1(ok2257), smaller amounts of
which were also present in the wild-type excretome (Figure
4d). The most abundant component of this series of isomers
was isolated via preparative HPLC and identified by NMR
spectroscopy as an ω-linked ascaroside with a C5 side chain
(Figures 3b and S9). This suggested that the additional series of
compounds observed in acox-1 represents ω-linked saturated
ascarosides (Table S2), which we confirmed by synthesis of C5
and C9 ω-linked ascarosides (see Supporting Information). In
order to differentiate the ω-linked ascarosides from their
previously described (ω−1)-linked isomers, we name the newly
found ω-linked compounds with the SMID “oscr”, e.g., the
Figure 3. Ascarosides identified in wild-type, acox-1, maoc-1, dhs-28,
and daf-22 worms via LC-MS/MS: (a) (ω−1)-oxygenated ascarosides,
(b) ω-oxygenated ascarosides, and (c) examples for 4′-acylated
derivatives. The stereochemistry of compounds that were not
synthesized (see Supporting Information for syntheses and a complete
list of the 146 characterized ascarosides) was proposed as shown on
the basis of analogy and HPLC-MS retention times (Figure S2).
preparative HPLC and analyzed using 2D NMR spectroscopy
(dqfCOSY, see Figures S3 and S4), which confirmed that these
compounds are ascr#3-based and further indicated the presence
of a 4-hydroxybenzoyl or (E)-2-methyl-2-butenoyl (tigloyl)
moiety attached to the 4-position of the ascarylose (Figure 3c).
We corroborated these structural assignments via total
synthesis of authentic samples (see Supporting Information).
In analogy to the recently reported indole-3-carboxy derivative
of ascr#3 (“icas#3”),8 we named the 4-hydroxybenzoyl and (E)-
2-methyl-2-butenoyl derivatives of ascr#3 with the small-
molecule identifiers (SMIDs)16 “hbas#3” and “mbas#3”,
respectively. hbas#3 and mbas#3 are the first ascarosides to
incorporate 4-hydroxybenzoyl and (E)-2-methyl-2-butenoyl
moieties, which in analogy to the indole-3-carboxy moiety in
icas#38 could be derived from amino acid precursors. Because
of structural similarity to the aggregation-inducing indole
ascarosides,8 we tested hbas#3 for its effect on worm behavior.
We found that hbas#3 strongly attracts C. elegans at
concentrations as low as 10 fM (see Figure S5), which exceeds
the potency of any previously known C. elegans small-molecule
signal.3,8 Low femtomolar activity is unusual for small-molecule
signals in animals but matched by some classes of peptide
hormones.17
2.2. Peroxisomal β-Oxidation in Ascaroside Biosyn-
thesis. Next we used comparative LC-MS/MS to investigate
ascaroside biogenesis. Previous studies suggested that the side
chains of the ascarosides are derived from peroxisomal β-
oxidation of longer-chained precursors and that the acyl-CoA
oxidase ACOX-1 participates in the first step of ascaroside side-
chain β-oxidation, introducing α,β-unsaturation (Figure 4a,b).11
In vertebrates as well as in Drosophila, the next two steps in
peroxisomal β-oxidation, hydration of the double bond and
subsequent dehydrogenation to the β-ketoacyl-CoA ester, are
catalyzed by one protein, e.g., MFE-2. These two enzymatic
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Figure 4. (a) Proposed roles of peroxisomal β-oxidation enzymes ACOX-1, MAOC-1, DHS-28, and DAF-22 in ascaroside biosynthesis. (b) Gene
structures of acox-1, maoc-1, dhs-28, and daf-22 mutant alleles, showing point mutations (vertical bars) and deletions (horizontal bars). (c) (ω−1)-
Oxygenated ascarosides in wild-type and β-oxidation mutants (acox-1, maoc-1, dhs-28, and daf-22) with saturated (blue), α,β-unsaturated (red), and
β-hydroxylated (green) side chains. (d) ω-Oxygenated ascarosides in wild-type and acox-1 mutants (see also Figure S8).
synthesized ω-linked isomers of ascr#9 and ascr#10 were
named oscr#9 and oscr#10 (Figure 3).
however, the possibility that β-oxidation occurs prior to
ascarylose attachment cannot be excluded.
2.4. MAOC-1 and DHS-28 Are Functional Homologues
of Human MFE-2. In contrast to wild-type and acox-
1(ok2257) worms, short-chain (<C9) ascarosides were not
detected in maoc-1(hj13) and dhs-28(hj8) worms, which
instead accumulate several series of (ω−1)- and ω-oxygenated
medium- and long-chain ascarosides (≥C9). The ascaroside
profile of the maoc-1(hj13) excretome was dominated by α,β-
unsaturated ascarosides such as ascr#21 (C13) and ascr#25
(C15) (Figure 4c), supporting our hypothesis that MAOC-1
functions as an enoyl-CoA hydratase in the ascaroside
biosynthetic pathway (Figure 4a). In addition, the maoc-
1(hj13) excretome contained smaller amounts of the
corresponding saturated ascarosides, along with a third
homologous series of compounds, whose HRMS and MS/MS
fragmentation suggested that they represent a series of long-
chain β-hydroxylated ascarosides (Figures 4c and S1, Tables S3
and S4). These structural assignments were confirmed via
synthesis of representative members of this series (see
Thus production of ω-linked ascr#5 is abolished in acox-
1(ok2257) worms, whereas production of longer chain
homologues with 5−13-carbon side chains, e.g., oscr#9, is
starkly upregulated (Figure 4d). These results indicate that β-
oxidation in acox-1(ok2257) worms is strongly dependent on
whether the side chain is (ω−1)- or ω-functionalized. Chain
shortening of (ω−1)-oxygenated substrates appears to stall at a
chain length of 9 carbons as in ascr#10, whereas ω-oxygenated
substrates are processed for two additional rounds of β-
oxidation to afford large quantities of ω-oxygenated oscr#9
featuring a 5-carbon side chain. This suggests that side-chain
oxygenation precedes peroxisomal β-oxidation. In contrast, the
time point of ascarylose attachment seems less certain. The
absence of any (ω−1)- or ω-hydroxylated fatty acids in the
investigated C. elegans mutant metabolome samples suggests a
biosynthetic model in which very long-chain ascarosides
(VLCAs) serve as substrates for peroxisomal β-oxidation;
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Supporting Information), the C9 and C13 β-hydroxylated (ω−
1)-ascarosides, bhas#10 and bhas#22, as well as the C15 β-
hydroxylated ω-ascaroside bhos#26 [SMIDs for (ω−1)- and ω-
oxygenated β-hydroxylated ascarosides are bhas and bhos].
Since β-hydroxylated ascarosides are putative products of
enoyl-CoA hydratases such as MAOC-1, their presence in the
excretomes of both studied maoc-1 mutant strains, maoc-
1(hj13), which carries a point mutation in the active site
(D186N), and the 2110 base pair deletion mutant maoc-
1(ok2645) (Figure 4b), suggests that additional enoyl-CoA
hydratases may participate in ascaroside biosynthesis.
ascaroside biosynthesis the indole-3-carbonyl moiety is
attached. Comparison of ascaroside and indole ascaroside
profiles revealed that indole ascaroside biosynthesis is tightly
controlled. For example, we found that in acox-1 mutants the
ω-ascaroside oscr#9 is over 100 times more abundant than the
(ω−1)-ascaroside ascr#9, whereas (ω−1)-indole ascaroside
icas#9 is much more prominent than ω-indole ascaroside
icos#9 (Figure 5a). Similarly, icas#3 and icas#9, the dominating
Similar to our results for maoc-1(hj13), we found that the
dhs-28(hj8) ascaroside profile is dominated by compounds with
side chains ranging from C9−C21 (Figure 4c). However, in
contrast to maoc-1(hj13), saturated and α,β-unsaturated
ascarosides constitute a relatively smaller portion of total
ascarosides in dhs-28(hj8). Instead, we found (ω−1)- and ω-
oxygenated β-hydroxyascarosides (bhas and bhos) with odd-
numbered side chains from C13−C21 as major components
(Figures 4c and S8), which is consistent with the proposed
biosynthetic role of DHS-28 as a β-hydroxyacyl-CoA
dehydrogenase.6,12 Analysis of the daf-22(ok693) excretome
revealed the absence of all ascarosides with side chains shorter
than 12 carbons, as reported earlier (Figures 4c and S8).5,6,12
We found that daf-22(ok693) contains small amounts of
homologous series of (ω−1)- and ω-oxygenated long-chain
ascarosides featuring saturated (ascr and oscr) and β-
hydroxylated side chains (bhas and bhos). In addition, the
daf-22(ok693) excretome contains VLCAs (>C22) with side-
chain lengths of up to 33 carbons, as reported earlier.5,20
2.5. Identification of New Indole Ascarosides. Indole-3-
carbonylated ascarosides are much less abundant than the
corresponding unfunctionalized ascarosides and have recently
been shown to function as highly potent aggregation signals.8
Our LC-MS/MS screen revealed several new types of indole
ascarosides (Figure 3c, Tables S5−S8). Using synthetic samples
of icas#3, icas#9, and icas#1,8 we showed that indole
ascarosides exhibit a characteristic fragmentation pattern that
includes neutral loss of 143 amu [C9H5NO] due to cleavage of
the indole-3-carbonyl unit, as well as the ascaroside-diagnostic
product ion at m/z 73 (see Figure S1). LC-MS/MS screening
for components with this fragmentation pattern revealed that
known (ω−1)-oxygenated isomers icas#1, icas#9, and icas#10
in acox-1(ok2257) are accompanied by a series of ω-oxygenated
indole ascarosides (SMIDs icos#1, icos#9, and icos#10), which
was confirmed by chemical synthesis of icos#10 as a
representative example (see Supporting Information). Perox-
isomal β-oxidation mutants that do not produce short-chain
ascarosides (<9 carbon side chains), for example, maoc-1 and
dhs-28 worms, also do not produce any of the corresponding
indole ascarosides. Instead, we found that the maoc-1 and dhs-
28 excretomes contain significant amounts of (ω−1)- and ω-
oxygenated long-chain indole ascarosides (SMIDs icas and
icos) and indole β-hydroxyascarosides (SMIDs ibha and ibho)
with side chains from 9−17 carbons (see Tables S5−S8).
2.6. Indole Ascaroside Biogenesis. Using application
experiments with deuterium-labeled tryptophan and axenic in
vitro cultures, we have previously shown that the indole-3-
carbonyl moiety of indole ascarosides originates from L-
tryptophan.8 A similar L-tyrosine or L-phenylalanine origin
seems likely for the 4-hydroxybenzoyl moiety of hbas#3,
whereas the tigloyl group of mbas#3 could be derived from L-
isoleucine.21 However, it remained unclear at what stage in
Figure 5. Indole ascaroside biosynthesis. (a) Relative abundance of
(ω−1) and ω-oxygenated C5-ascarosides ascr#9 and oscr#9 and their
corresponding indole ascarosides icas#9 and icos#9 in acox-1 indicates
that indole-3-carbonyl attachment is highly specific. (b) LC-MS ion
traces of daf-22(m130) excretome following incubation with a 1:1
mixture of ascr#10 and oscr#10, showing a preference for indole-3-
carbonyl attachment to the (ω−1)-oxygenated ascr#10.
indole ascarosides in the wild-type excretome, are produced in
roughly equal amounts, whereas ascr#3 is about 20 times more
abundant than ascr#9 (Figure S10). This strong dependence of
indole ascaroside biosynthesis on side-chain length and (ω−1)-
vs ω-oxygenation suggested that these compounds originate
from specific attachment of an indole-3-carbonyl unit to the
corresponding non-indole ascarosides.
To test whether non-indole ascarosides serve as precursors
for indole ascarosides, we incubated daf-22(m130) worms
(which are devoid of all short-chain indole and non-indole
ascarosides) with a 1:1 mixture of ascr#10 and oscr#10 for 5
days. Subsequent analysis by LC-MS showed partial conversion
into icas#10 and icos#10 (Figure 5b), indicating that non-
indole ascarosides serve as specific precursors to their
corresponding indole derivatives. Moreover, conversion of
(ω−1)-ascaroside ascr#10 was preferred over conversion of ω-
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ascaroside oscr#10, reflecting the ratios of these compounds in
wild-type and acox-1 mutants. Similarly, we found that daf-
22(m130) worms convert added ascr#3 into icas#3. Further
conversion of indole or non-indole ascarosides into shorter
chain derivatives (e.g., ascr#1 or icas#1) was not observed.
These results indicate that attachment of an L-tryptophan-
derived indole-3-carbonyl unit represents the final step in
indole ascaroside biosynthesis.
2.7. Ascaroside Excretion Is Selective. Despite detailed
investigations of ascaroside function, little is known about how
ascarosides are released and transported from their site of
biosynthesis. We compared the ascaroside profiles of the wild-
type excretomes (liquid culture supernatant extracts) and worm
body metabolomes (worm pellet extracts) to identify possible
nonexcreted ascaroside derivatives and to determine quantita-
tive differences. We found that ascaroside profiles of worm
pellet extracts differ significantly from those excreted into the
media, indicating that ascarosides are differentially released by
C. elegans (Figure 6a). In the worm pellets, the most abundant
transport or storage forms of the ultimately excreted signaling
molecules.
In addition, we found that saturated ascarosides are retained
in the worm bodies to a much greater extent than their α,β-
unsaturated derivatives (Figure 6a). Differential release was also
observed for ω-oxygenated components (Figure S13). There-
fore, it appears that C. elegans exhibits remarkable control over
the release of ascaroside signals.
2.8. Nutritional State Affects Ascaroside Biosynthesis.
Ascaroside biosynthesis has been reported to depend on
various environmental factors including food availability,6
developmental stage,15 and temperature.11 We used LC-MS
to investigate the effect of nutritional state on ascaroside
biosynthesis by comparing the excretomes of well-fed and
starved cultures of wild-type and mutant worms. We found that
the ratio of (ω−1)- to ω-linked ascarosides strongly depends
on nutritional state. Production of long-chain (ω−1)-oxy-
genated ascarosides in starved cultures of dhs-28 was about 5
times higher than those of well-fed cultures (Figures 6b and
S14). Similarly, starved wild-type worms excrete significantly
larger amounts of the (ω−1)-linked ascr#3 than well-fed
worms, relative to the amounts of ω-linked ascr#5 (Figure
S15). Both ascr#5 and ascr#3 are major components of the
dauer pheromone; however, they affect worm behavior
differently.1,3,4,22
Therefore, it appears that ascaroside signaling is actively
regulated in response to changes in nutrient availability via
modulation of (ω−1) and ω-functionalization of very long-
chain fatty acids (VLCFAs) upstream of peroxisomal β-
oxidation. Together with the recent finding that ω- and (ω−
1)-functionalized ascarosides are sensed by different families of
G-protein-coupled receptors,23 these results suggest that ω- and
(ω−1)-functionalized ascarosides target separate downstream
signaling pathways.
3. DISCUSSION
Ascarosides play important roles for several different aspects of
C. elegans biology. This functional diversity is paralleled by
corresponding structural diversity and a complex biosynthetic
pathway. Our MS/MS-based study revealed 146 ascarosides,
including 124 previously unreported analogues, which show
several unexpected features including ω-oxygenation of the
fatty-acid-derived side chains, 4-hydroxybenzoylation or (E)-2-
methyl-2-butenoylation of the ascarylose unit, and glucosyl
esters. Most ascarosides occur as members of homologous
series of compounds with (ω−1)- or ω-linked saturated, α,β-
unsaturated, or β-hydroxylated side chains ranging from 3 to 21
(occasionally more) carbons. Importantly, only a few members
of each series are produced abundantly in wild-type, and
incorporation of specific structural features such as modification
in position 4 of the ascarylose (e.g., as in indole ascarosides) or
incorporation of α,β-unsaturation appears to be tightly
controlled. Given their assembly from carbohydrate, lipid, and
amino-acid-derived building blocks, the ascarosides appear as a
modular library of small-molecule signals that integrate inputs
from three basic metabolic pathways (Figure 7a). The
ascarosides then transduce input from these pathways via
their diverse signaling functions in C. elegans’s behavior and
development, including dauer formation, mate attraction,
hermaphrodite repulsion, and aggregation.8,14 Their specific
biosyntheses suggest that many of the newly identified
ascarosides also contribute to known or as yet undetermined
functions in C. elegans. As an example, we show that hbas#3 acts
Figure 6. (a) Analysis of worm body ascaroside profiles reveals
ascaroside glucosides (e.g., glas#10) and indicates preferential
excretion of unsaturated ascarosides. (b) Ratio of (ω−1)- to ω-linked
saturated ascarosides in dhs-28 mutant excretome shows strong
dependence on nutritional conditions (for β-hydroxyascarosides see
Figure S14), reflecting starvation dependence of ascr#3/ascr#5 ratio in
wild-type C. elegans (see Figure S15) (Welch’s t test; * indicates P <
0.05; ** indicates P < 0.001).
ascarosides, for example ascr#3 in wild-type and ascr#10 in
acox-1 worms, are accompanied by significantly more-polar
derivatives, which are absent from the media extracts (Figure
S11). MS/MS analysis suggested that these components
represent ascaroside O-glycoside esters. Isolation of the putative
ascr#10 glycoside from acox-1(ok2257) worm pellet extracts
and subsequent NMR spectroscopic analysis (Figure S12)
indicated esterification of ascr#10 with the anomeric hydroxy
group of β-glucose, which was subsequently confirmed via
synthesis (SMID for 1-β-D-glucosyl ascr#10 is glas#10, Figure
6a). The fact that large quantities of highly polar glas#10 and
other ascaroside glucosides (see Table S9) are retained in the
worm bodies but not excreted suggests that they represent
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Article
though the presence of a promiscuous ascarosyl transferase
cannot be excluded (Figure 7b).
Chain shortening of VLCA then progresses via repetitive
cycles of peroxisomal β-oxidation. The results from our LC-
MS/MS screen allow us to propose precise roles for enzymes
participating in each of the four steps of the β-oxidation cycle:
acyl-CoA oxidase ACOX-1, enoyl-CoA hydratase MAOC-1, β-
hydroxyacyl-CoA dehydrogenase DHS-28, and β-ketoacyl-CoA
thiolase DAF-22. We show that mutations in acox-1, maoc-1,
and dhs-28 result in specific changes of the corresponding
ascaroside profiles, in agreement with their proposed functions.
The acyl-CoA oxidase ACOX-1 has been the subject of a
previous study which suggested that mutations in acox-1
primarily affect the biosynthesis of ascr#2 and ascr#3, but not
of ascr#1.11 However, our results indicate that acox-1(ok2257)
mutants have a reduced ability to process C9 (ω−1)-
functionalized ascarosides, resulting in diminished production
of all shorter-chained ascarosides and buildup of C9 and longer-
chained saturated ascarosides. Mutations of maoc-1 (as well as
dhs-28 and daf-22) have been shown to result in expansion of
intestinal lipid droplets and cause an increase in fasting- and
lipolysis-resistant triglycerides.19 Our results show that MAOC-
1 participates in ascaroside biosynthesis, acting as the previously
unidentified enoyl-CoA hydratase. These findings further
demonstrate that hydration of enoyl-CoAs and dehydrogen-
ation of β-hydroxyacyl-CoAs in C. elegans are catalyzed by two
distinct enzymes, MAOC-1 and DHS-28, as had been suggested
on the basis of their homology to separate functional domains
of human MFE-2.19
We further show that attachment of the tryptophan-derived
indole-3-carbonyl unit in indole ascarosides likely represents
the last step in their biosynthesis, and that this step is highly
specific. As attachment of an indole-3-carbonyl group to
ascarosides can dramatically alter their biological function, such
tight regulation makes sense. For example, indole-3-carbonyl
addition to the dauer-inducing and strongly repulsive signal
ascr#3 results in the potent hermaphrodite attractant icas#3.8
Therefore, identification of the enzymes that attach indole-3-
carbonyl and other functional groups to the ascarosides will be
of great interest.
The biosynthesis of ascarylose in C. elegans has not been
investigated, but detection of ascarosides in axenic C. elegans
cultures demonstrated that C. elegans produce ascarylose
endogenously.8 Ascarylose biosynthesis in bacteria is well
understood, and the C. elegans genome includes several
homologues of bacterial genes in this pathway, for example,
ascE from Yersinia pseudotuberculosis (Figure S16),26 providing
potential entry points for the study of ascarylose biosynthesis
and its regulation in nematodes. In addition, the oxidases
catalyzing (ω−1)- or ω-functionalization of VLCFA precursors
remain to be identified. Our finding that the ratio of (ω−1)/ω-
oxygenated ascarosides in wild-type and peroxisomal β-
oxidation mutants is strongly affected by starvation suggests
that this step may encode information about nutritional state.
Finally, we demonstrate that ascaroside excretion is surprisingly
specific. Given the high sensitivity and selectivity of LC-MS/
MS, we believe that ascaroside profiling using this method will
aid identification of additional genes and environmental factors
that participate in ascaroside biosynthesis and homeostasis.
Figure 7. (a) Modular assembly of ascarosides from amino acid
(green), fatty acid (blue), and carbohydrate (red) building blocks. (b)
Model for ascaroside biogenesis. Chain elongation of fatty acids (by
putative elongase homologues elo-1−925) is followed by (ω−1)- or ω-
oxygenation of VLCFAs and ascarylose attachment. The resulting
VLCAs enter peroxisomal β-oxidation via ACOX-1, MAOC-1, DHS-
28, and DAF-22, producing short-chain ascarosides, which are linked
to amino-acid-derived moieties and other building blocks.
as an attraction signal whose potency exceeds that of all
previously known small molecules in this model organism.
More comprehensive biological evaluation will require
consideration of synergistic activities (i.e., testing of compound
mixtures), which, given the very large number of compounds,
may necessitate high-throughput approaches, for example based
on microfluidic devices.24
Our results allow us to propose a working model for
ascaroside biogenesis (Figure 7b). The finding that ascarosides
are members of several homologous series with side chains up
to 21 (and more) carbons suggests their origin from
peroxisomal β-oxidation of very long-chain precursors. Previous
studies reported the presence of VLCAs with C29 and C31 side
chains in wild-type and daf-22 mutants, which could represent
precursors or intermediates in ascaroside biosynthesis.5,20
Alternatively, VLCFAs could undergo peroxisomal β-oxidation
prior to (ω−1)- or ω-functionalization and subsequent
attachment of the ascarylose. Our observation that the acox-1
mutation affects (ω−1)- and ω-oxygenated ascarosides differ-
ently suggests that (ω−1)- and ω-functionalization of VLCFA
precursors occurs upstream of their breakdown by peroxisomal
β-oxidation. Given the large range in side-chain lengths, it
seems likely that resulting (ω−1)- and ω-hydroxy VLCFAs are
linked to ascarylose prior to entering the β-oxidation pathway,
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this database is to introduce searchable, gene-style identifiers
“SMIDs”for all small molecules newly identified from C. elegans and
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Matsuoka, Y.; van Dyck, C. H.; Thal, L. CNS Drug Rev. 2005, 11, 353−
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ASSOCIATED CONTENT
* Supporting Information
Detailed experimental procedures including the synthesis of
reference compounds, a full list of identified ascarosides, and
their LC-MS and NMR spectroscopic data. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
(18) Haataja, T. J. K.; Koski, M. K.; Hiltunen, J. K.; Glumoff, T.
Biochem. J. 2011, 435, 771−781.
AUTHOR INFORMATION
Corresponding Author
schroeder@cornell.edu
■
(19) Zhang, S. O.; Box, A. C.; Xu, N.; Le Men, J.; Yu, J.; Guo, F.;
Trimble, R.; Mak, H. Y. Proc. Natl. Acad. Sci. U.S.A. 2010, 10, 4640−
4645.
ACKNOWLEDGMENTS
(20) Zagoriy, V.; Matyash, V.; Kurzchalia, T. Chem. Biodivers. 2010, 7,
■
2016−2022.
We thank Arthur Edison (University of Florida, Gainesville) for
helpful suggestions, the Caenorhabditis Genetics Center, Ho Yi
Mak (Stowers Institute), and Shohei Mitani (Tokyo Women’s
Medical University) for providing C. elegans mutant strains, and
Maciej Kukula (BTI Mass Spectrometry Facility) and Wei
Chen (Proteomics and Mass Spectrometry Core Facility,
Cornell University) for assistance with HR-MS. This work
was supported in part by the National Institutes of Health
(GM088290, GM085285, and T32GM008500) and the
Cornell/Rockefeller/Sloan-Kettering Training Program in
Chemical Biology.
(21) Attygalle, A. B.; Wu, X.; Will, K. W. J. Chem. Ecol. 2007, 33,
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(22) Macosko, E. Z.; Pokala, N; Feinberg, E. H.; Chalasani, S. H.;
Butcher, R. A.; Clardy, J.; Bargmann, C. I. Nature 2009, 458, 1171−
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Ragains, J. R.; Clardy, J.; Touhara, K.; Sengupta, P. Science 2009, 326,
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