Succinylated Octopamine Ascarosides and a New Pathway of Biogenic Amine Metabolism in ...

37 Pages·2015·3.2 MB·English

REPORT
THEJOURNALOFBIOLOGICALCHEMISTRYVOL.288,NO.26,pp.18778–18783,June28,2013
©2013byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PublishedintheU.S.A.
Succinylated Octopamine thepreviouslydescribedacetates.Chemically,bimodaldeacti-
vationofbiogenicaminesviaacetylationandsuccinylationpar-
Ascarosides and a New Pathway
allelsposttranslationalmodificationofproteinsviaacetylation
of Biogenic Amine Metabolism andsuccinylationofL-lysine.Ourresultsrevealasmall-mole-
cule connection between neurotransmitter signaling and
in Caenorhabditis elegans*□S
interorganismalregulationofbehaviorandsuggestthatascaro-
side biosynthesis is based in part on co-option of degradative
Receivedforpublication,April12,2013,andinrevisedform,May13,2013
Published,JBCPapersinPress,May20,2013,DOI10.1074/jbc.C113.477000 biochemicalpathways.
AlexanderB.Artyukhin‡§,JoshuaJ.Yim‡,JaganSrinivasan¶1,
YevgeniyIzrayelit‡,NeelanjanBose‡,StephanH.vonReuss‡2,
YearaJo¶,JamesM.Jordan(cid:1),L.RyanBaugh(cid:1),MicheongCheong§,
PaulW.Sternberg¶3,LeonAvery§,andFrankC.Schroeder‡4 Small-molecule signals that incorporate glycosides of the
Fromthe‡BoyceThompsonInstituteandDepartmentofChemistryand dideoxysugar ascarylose (“ascarosides”) play a central role in
ChemicalBiology,CornellUniversity,Ithaca,NewYork14853,the thebiologyofthemodelorganismCaenorhabditiselegans,reg-
§DepartmentofPhysiologyandBiophysics,VirginiaCommonwealth ulating several aspects of development and behavior. Ascaro-
University,Richmond,Virginia23298,the¶HowardHughesMedical
side-basedsignalingmoleculeswereinitiallyidentifiedascom-
InstituteandDivisionofBiology,CaliforniaInstituteofTechnology,
Pasadena,California91125,andthe(cid:1)DepartmentofBiology,DukeCenter ponents of the dauer pheromone, a population density signal Do
forSystemsBiology,DukeUniversity,Durham,NorthCarolina27708 thatinducesentryintothedauerstage,ahighlystress-resistant wn
alternatelarvalstagethatcansurviveformonthswithoutfood loa
d
Background: Ascarosides play central roles regulating intakebeforeresumingnormaldevelopment(1,2).Subsequent ed
C.elegansbehavioranddevelopment. studiesshowedthatascarosidesalsoregulateC.eleganslifespan fro
m
Results:L1larvaeproducestarvation-dependentascaro- (3),aswellasseveraldifferentbehaviors,includingsex-specific h
sCidoenscbluassieodno:nSusuccccininyylalatitoendoisctaonpaimmpinoert.ant pathway for aFtitgr.a1cAti)o.nThaevositdruanctcuer,edsiospfethrseala,scaanrdosaidggerseagraetihoingh(l4y–m1o0d)u(lsaere, ttp://ww
mSiegtnaibfioclaisnmceo:fbOiocgtoepnaicmaimneineasscinarCo.siedleegsancso.nnect neu- ipnrtiemgararytimngetbaubioldliicnpgabthlowcakyssf(r1o1m).Aamscianroosaidcieds,iglinpaidli,nagnisdhoigthhelyr w.jbc.o
rotransmitterandascarosidesignalinginC.elegans. conserved among nematodes, including both parasitic and rg
a/
non-parasiticspecies(12–14). t D
Theascarosides,small-moleculesignalsderivedfromcombi- The biosynthesis of nematode ascarosides is only partially uk
e
natorial assembly of primary metabolism-derived building understood. Although the lipid-like side chains have been U
n
blocks,playacentralroleinCaenorhabditiselegansbiologyand shown to derive from peroxisomal (cid:1)-oxidation of longer ive
regulate many aspects of development and behavior in this chainedprecursors(11,15–17),site(s)andenzymesinvolvedin rsity
model organism as well as in other nematodes. Using HPLC- attachingsubstituentstothe2-and4-positionsoftheascary- o
n
MS/MS-basedtargetedmetabolomics,weidentifiednovelasca- lose or the side chain have not been characterized. Here we A
u
g
rosidesincorporatingasidechainderivedfromsuccinylationof describetheidentificationofanewseriesofmodularascaro- u
s
the neurotransmitter octopamine. These compounds, named sides,inwhichthe4-positionoftheascaryloseisconnectedtoa t 26
osas#2,osas#9,andosas#10,areproducedpredominantlybyL1 sidechainderivedfromsuccinylationoftheneurotransmitter , 2
0
1
larvae, where they serve as part of a dispersal signal, whereas octopamine. These octopamine ascarosides are specifically 5
these ascarosides are largely absent from the metabolomes of producedbyL1larvae,whichhadnotpreviouslybeenstudied
other life stages. Investigating the biogenesis of these octop- in detail. The incorporation of N-succinylated octopamine in
amine-derivedascarosides,wefoundthatsuccinylationrepre- theseascarosidesthenledustoinvestigatewhethersuccinyla-
sents a previously unrecognized pathway of biogenic amine tion plays a general role in biogenic amine metabolism in
metabolism. At physiological concentrations, the neurotrans- C.elegans.
mittersserotonin,dopamine,andoctopamineareconvertedtoa
EXPERIMENTALPROCEDURES
large extent into the corresponding succinates, in addition to
C.elegansStrainsandGeneralCultureMethods—Wild-type
*ThisworkwassupportedinpartbyNationalInstitutesofHealthGrants C.elegans(N2,Bristol),MT13113tdc-1,MT9455tbh-1,RB859
GM088290 (to F.C.S.), GM008500 (to Y.I.), DK83593 (to L.A.), and daf-22,andVS8dhs-28mutantstrainswereobtainedfromthe
GM085285(toF.C.S.andP.W.S.),andNationalScienceFoundationGrant Caenorhabditis Genetics Center (CGC). Strains were main-
IOS1120206(toL.R.B.),aswellastheVassarCollegeMaryLandonSague
tainedat20°ConNGM5plateswithbacteria(Escherichiacoli
Fellowship(toY.I.).
□S ThisarticlecontainssupplementalFigs.S1–S11. OP50orHB101)asfood.
1Presentaddress:Dept.ofBiologyandBiotechnology,WorcesterPolytechnic Preparation of L1 Exo-metabolome Samples—Worms were
Institute,Worcester,MA01609.
grown at 20–22°C either on NGM plates seeded with E.coli
2Presentaddress:Dept.ofBioorganicChemistry,MaxPlanckInstitutefor
ChemicalEcology,07745Jena,Germany.
3AnInvestigatoroftheHowardHughesMedicalInstitute.
4Towhomcorrespondenceshouldbeaddressed.Tel.:[phone];Fax: 5Theabbreviationsusedare:NGM,nematodegrowthmedium;MS/MS,tan-
607-254-2958;E-mail:[email protected]. demmassspectrometry.
18778 JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER26•JUNE28,2013
REPORT: BiogenicAmineMetabolismviaSuccinylation
HB101orinliquidculture(S-completemediumsupplemented anterioramphidsensoryneurons.Avoidanceofosas#9isindi-
with3%HB101).CultureswerestartedwithsynchronizedL1 cated by a backward motion away from the drop. Such a
larvae. Gravid worms were collected and treated with 20% response was scored as a positive response. The avoidance
bleachand0.3MNaOHfor6mintoyieldacleaneggsuspen- index was calculated by dividing the number of positive
sion.TheeggswerewashedtwicewithM9buffer,resuspended responsestothetotalnumberoftrials(18).Thewormswere
infreshM9atadensityof30–500eggs/(cid:2)l,andincubatedwith allowed to freely move on the plate for about 20–30 min
shakingat25°Cfor24h.HatchedL1larvaewerespundown, beforetesting.After30min,wormsweretestedforresponseto
and the supernatant (starved L1 medium) was either used distilledwater.Forallthestagesofwormstested,weobserved
directlyforHPLC-MSanalysisinsingleionrecordingmodeor an avoidance index of less than 0.1 for water. 50 individual
frozen,lyophilized,extractedwithmethanol,andthenanalyzed animals per life stage were tested on each day using freshly
byHPLC-MSorMS/MS.Forpreparationofexo-metabolome prepared osas#9 concentrations. Shown data represent the
samples from fed L1 larvae, eggs were prepared similarly but meanfromassaysconductedon3ormoredays.
resuspendedinM9withHB101.Sampleforascarosideprofil- Dauer Assay—Dauer assays were performed in liquid as
ingwastaken14haftereggpreparationtoassurethatworms described previously with modifications (10). Eggs were sus-
arestillinL1stage. pended in 3 ml of S-complete medium in 20-ml scintillation
HPLC-MS Analysis—HPLC-MS and HPLC-MS/MS were vials at a concentration of 1 egg/(cid:2)l, and E.coli (HB101) was
performedusinganAgilent1100seriesHPLCsystemequipped addedforafinalconcentrationof1.3mg/ml.Aliquotsofasca-
with an Agilent Eclipse XDB-C18 column (9.4 (cid:1) 250 mm, rosidesolutionswereaddedtoachieve200nMascr#2,200nM
5-(cid:2)mparticlediameter)connectedtoaQuattroIIspectrome- ascr#5, and 1 (cid:2)M osas#9, and the worms were grown on a Do
w
ter (Micromass/Waters) using a 10:1 split. For HPLC, a 0.1% 180-rpm shaker at 20°C for 6 days. Dauer formation was n
lo
aceticacid-acetonitrilesolventgradientwasusedataflowrate assessedbysoakingthewormsin1%SDSfor50minandcount- ad
e
of3.6ml/min,startingwithanacetonitrilecontentof5%for5 ingsurvivingL4wormsafter24hofrecoveryonHB101plates. d fro
min, which was increased to 100% over a period of 40 min. L1SurvivalAssay—Eggsweresuspendedin6mlofM9buffer m
Exo-metabolome samples were analyzed by HPLC-electros- orosas#9solutioninM9in40-mlglassvialsataconcentration http
prayionization-MSinnegativeionmodeusingacapillaryvolt- of0.8egg/(cid:2)landincubatedinashakerat210rpmand25°C. ://w
age of 3.5 kV and a cone voltage of (cid:2)40 V. HPLC-MS/MS Triplicatealiquotsfromeachsampleweretakeneverydayor w
w
screeningforprecursorionsofm/z(cid:3)73.0wasperformedusing everyotherdayandseededonHB101platesforrecovery.Star- .jb
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argon as collision gas at 2.1 mtorr and 40 eV. Analysis and vationsurvivalwasassessedbycountingwormsthatresumed .o
rg
characterizationofosasascarosidesandN-acylatedderivatives growthafterrecoveryat20°Cfor2–3days. a/
of serotonin, dopamine, and octopamine were performed in Statistical Analysis—Statistical analyses of avoidance used t D
u
bothnegativeandpositiveionizationmodeswithconevoltages unpaired Student’s t tests with Welch correction using the ke
of(cid:2)40Vand(cid:2)20Vfornegativeand(cid:4)30Vforpositiveelec- InStatstatisticssoftware.pvaluesaredenotedasfollows:**,p(cid:5) Un
trosprayionizationmode.HighresolutionHPLC-MSwasper- 0.001;***,p(cid:5)0.0001. ive
rs
formedusingaWatersnanoACQUITYUPLCsystemequipped ity
with Waters ACQUITY UPLC HSS C-18 column (2.1 (cid:1) 100 RESULTSANDDISCUSSION on
A
mm,1.8-(cid:2)mparticlediameter)connectedtoaXevoG2QTof L1LarvaeProduceOctopamineAscarosides—Westartedour u
g
u
massspectrometer. investigationswithananalysisofchemicalsignalsproducedby s
t 2
tsas#9 Identification—High resolution HPLC-MS analysis starvedL1larvae,whichenteradistinctstateofdevelopmental 6
, 2
demonstrated a molecular formula of putative tsas#9 in the arrest that confers enhanced stress resistance and extended 0
1
5
naturalsample(calculatedmassofmolecularion466.2077and starvationsurvival(19–23).Toscreenforascarosidesspecifi-
ascr#9 fragment 247.1182; measured masses 466.2082 and cally produced by starved L1 worms, we used comparative
247.1184, respectively). To confirm the identity of tsas#9 in HPLC-MSanalysiscombinedwithanMS/MSscreenforpre-
tbh-1 L1 medium, we compared its retention time with the cursorionsoftheascaroside-characteristicfragmentionatm/z
retentiontimeofsynthetictsas#9.Bothhavethesamereten- 73,atechniquethatgreatlysimplifiesrecognitionofascaroside
tiontime(17.0minundertheconditionsdescribedabove)and derivativesincomplexmetabolomesamples(11).Comparison
uponco-injectioneluteasonepeak. of ascaroside profiles in the exo-metabolomes of starved L1
Avoidance Assays—A previously described avoidance assay larvae,adultworms,ormixedstageculturesshowedremarka-
was used to test the behavior of animals to osas#9 and octo- bledifferences.ascr#3,oneofthemostabundantascarosidesin
pamine (18). For these experiments, osas#9 was diluted into mixed stage cultures, is produced in L1 stage only in trace
doublydistilledwater,andwaterwasusedasacontrol.Assays amounts. Instead, HPLC-MS/MS analysis of the L1 exo-
wereconductedonunseededNGMplatesusinganimalsofdif- metabolome revealed a strong peak with MS/MS precursor
ferentstages.Totestanimalsforavoidancetoosas#9,worms ionsatm/z482andm/z247,whichdidnotmatchthemolecular
werewashedfrommixedstageplatesinM9buffertoremove ionsofanyknownascarosideandthereforesuggestedthepres-
bacteria.Afteracoupleofwashes,thewormsweresuspended enceofayetundescribedcompound(Fig.1B).Highresolution
inasmallvolumeofbufferanddispensedontounseededNGM MSanalysesindicatedmolecularformulaeofC H NO for
23 32 10
plates. For avoidance measurements, a drop of assay solution the molecular ion ([M-H](cid:2), calculated 482.2026; observed
containing osas#9 or control was delivered near the tail of a 482.2030) and C H O (calculated 247.1182; observed
11 19 6
movinganimal.Thedropsurroundstheanimalandreachesthe 247.1186),suggestingthatthiscompoundmaybederivedfrom
JUNE28,2013•VOLUME288•NUMBER26 JOURNALOFBIOLOGICALCHEMISTRY 18779
REPORT: BiogenicAmineMetabolismviaSuccinylation
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FIGURE1.AscarosidesinC.elegansanddetectionofascarosidesspecifictostarvedL1larvae.A,structuresandfunctionsofascarosidesproducedby h
differentdevelopmentalstagesofC.elegans.Inthisstudy,weinvestigatedascarosidesspecificallyproducedbyarrestedL1larvae.B,HPLC-MSanalysisof ttp
exo-metabolomesamplesobtainedfromstarvedL1wormsusingnegative-ionelectrosprayionizationrevealsanovelascarosidederivativeatm/z482 ://w
(osas#9).Itsmassspectrometricfragmentationpatternsuggeststhatitisderivedfromtheknownascr#9.C,proposedstructuresofosas#9andtworelated w
compounds,osas#2andosas#10. w
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sthimepaldedaitsicoanroosfidaeCfe1a2tHu1r3inNgOa4smatouireattyedtofitvhee-ckanrboownnsaidsecrc#h9ai,na aonfadsOu-cscuinbystligturoteudpCwHer-eCoHb2semrvoeiedt(ys,uapspwleelmlaesnstiaglnFailgs.sSu2g)g.Besatsievde at D/
u
withaterminalcarboxylgroupthathasbeenidentifiedinexo- onthechemicalshiftsofprotonsinthesetentativefragments ke
U
metabolomesamplesfrommixedstagecultures(11).Analysis andaputativemolecularformulaofC H NO ,weproposed n
23 33 10 iv
ofexo-metabolomesamplesfromfedL1larvaeandfedmixed astructureinwhichascr#9isconnectedviaasuccinylmoiety e
rs
stage cultures revealed only trace amounts of the compound tothenitrogenofoctopamine,abiogenicaminethatservesasa ity
o
with m/z 482, whereas it is consistently produced in large neurotransmitter in C.elegans (25) (Fig. 1C). We named this n
A
amounts by starved L1 larvae. Detailed examination of structure osas#9 (octopamine succinyl ascaroside, see Small u
g
u
HPLC-MS data from fed and starved L1 larvae revealed two Molecule Identifier Database (SMID DB) for nomenclature), s
t 2
additionalcompoundsatm/z538and480,whoseMSfragmen- named its putative longer chained homolog osas#10, and 6
, 2
tationpatternsweresimilartothefragmentationofthecom- namedtheascr#2-derivedmemberofthisseriesosas#2(Fig. 0
1
5
poundwithm/z482(supplementalFig.S1),suggestingthatthey 1C).
arechemicallyrelated.ThemolecularformulaeofC H NO Forconfirmationoftheproposedstructureofosas#9andto
27 40 10
([M-H](cid:2), calculated 538.2652; observed 538.2658) and determineitsstereochemistry,wedevelopedashortsynthesis
C H NO ([M-H](cid:2), calculated 480.2234; observed 480.2243) asshowninFig.2A(seesupplementaldataformethods).Bis-
24 34 9
obtainedfortheseionssuggestedthattheyrepresentascarosides succinylationofthebenzylester1ofascr#9providedinterme-
derivedfromtheadditionofaC H NO moietytoascr#10(fea- diate 2, which was reacted with (R)-octopamine 3 (26), the
12 13 4
turing a 9-carbon side chain) and the keto ascaroside ascr#2, naturally occurring enantiomer (27). The resulting bis-octo-
respectively. paminederivative5wasthenhydrogenatedtoremovetheben-
Toobtainlargerquantitiesofthedetectedcompounds,we zyl group and subsequently treated with lithium hydroxide,
fractionated large scale exo-metabolome samples by prepara- producingamixtureofosas#9andits2-O-substitutedisomer,
tiveHPLC(11).Fractionscontainingtheunknowncompounds fromwhichpuresyntheticosas#9wasisolatedviapreparative
were analyzed by two-dimensional NMR spectroscopy, using HPLC. Comparison of HPLC retention times (supplemental
highresolutiondoublequantumfilteredcorrelationspectros- Fig.S3),massspectra,andNMRspectroscopicdataconfirmed
copyspectraasdescribedpreviouslyfortheanalysisofcomplex thestructureofthenaturalcompoundwithm/z482asthatof
metabolite mixtures (10, 24). NMR spectroscopic analysis osas#9(supplementalFig.S4).Inparallel,werepeatedthesyn-
revealed cross-peaks representing the ((cid:3)-1)-oxygenated side thesisshowninFig.2Ausingracemicoctopamine.Themixture
chain of an ascaroside derivative. Furthermore, cross-peaks ofdiastereomersofosas#9obtainedfromracemicoctopamine
indicativeofa4-O-acylatedascarylose,aswellasspinsystems showeddistinctNMRspectroscopicsignalsforthemethylene
suggesting a p-hydroxy substituted phenyl group, a likely N- groupoftheoctopamine,whichenabledustoconfirmR-con-
18780 JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER26•JUNE28,2013
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FIGURE2.Synthesis,biosynthesis,andbiologicalpropertiesofosas#9.A,synthesisofosas#9andtsas#9.(a)succinicanhydride,N,N-diisopropylethyl- w
w
eaHxm2o(ig-nm)e,eP,4tda-/dbCio,mlMoemethOeyHsla;om(df)iCn,.LoeipOleyHgria,dtneinstre(aN,hd2yi)md.rCeo,tfhmuyrolafdoner/mdlifoaomxratindheee/;Hb(bi2o)O,s4.y-Bnd,tichmoemestisphayolrfaismoocnitnooopfparyemrliadintiinev,ee,tay1br-aeumtnhdiynale-n3,c-a(e3ns-ddofidmmeeraitvjhoeyrdlaaasmsccainaroorospsirdiodepesysiln)tcssaatrasbr#vo9eddaiimnLd1id,ofees,dadsLic#1h9,laoinnrodCmm.eeilxtehegadansnets;a.(gcDe),, .jbc.org
osas#9inducesavoidancebehaviorintheabsenceofbacterialfood(*,noavoidanceresponse).E,mediumfromstarvedtdc-1L1larvaeislessdeterrentto a/
youngadultwormsthanmediumfromwild-typeL1larvae.Thisexperimentwasconductedintheabsenceofbacterialfood.F,ratioofN-succinyltoN-acetyl t D
derivativesofserotonininN2L1mediumasafunctionofexogenouslyaddedserotoninasdeterminedbypositive-ionelectrosprayionizationMS.G,concen- u
trationsofosasascarosidesstronglydecreaseinresponsetoexogenouslyaddedserotonin(5-hydroxytryptamine(5-HT)),whereaslevelsofallotherascaro- ke
sidesareonlymarginallyaffected. U
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figurationoftheoctopaminesidechaininnaturalosas#9via mutantworms,whichproducetyramine,butnotoctopamine. ity
o
comparison of double quantum filtered correlation spectros- Wefoundthatbothtdc-1andtbh-1mutantwormsdonotpro- n
A
copyspectra(supplementalFig.S4). duce osas#9, whereas production of other ascarosides, e.g. u
g
u
ComparisonofstarvedandfedL1exo-metabolomesrevealed ascr#9andicas#9,isnotabolishedinthesemutants(supple- s
t 2
thatosas#9ismostabundantlyproducedbystarvedL1larvae, mentalFig.S6).Inaddition,wefoundthat,insteadofosas#9, 6
, 2
whereasosas#10isdominantinfedL1cultures(Fig.2B)and tbh-1mutantwormsproducelargeamountsofacorresponding 0
1
5
osas#2 is observed in smaller, highly variable amounts. We tyraminederivative,tsas#9(supplementalFig.S6,seesupple-
noticed a general shift from ascarosides with 9-carbon side mentaldataforstructureelucidation).Weonlydetectedtrace
chains (ascr#10, osas#10) in the presence of food to ascaro- amounts (1–2% of osas#9 ) oftsas#9 in starved wild-type L1
sideswithashorter5-carbonsidechaininstarvedconditions medium, suggesting that the great abundance of this com-
(ascr#9,osas#9,icas#9)(Fig.2B). pound in tbh-1 medium is a result of the roughly 20-fold
Biosynthesis of Octopamine Ascarosides—We then consid- increased tyramine levels in this mutant (28). These findings
ered possible biogenetic origins of osas#9. Previous work demonstrated that osas ascarosides are synthesized from
demonstrated that the fatty acid-derived side chains of the worm-producedoctopamine.
ascarosides are derived from peroxisomal (cid:1)-oxidation (11). osas#9 Induces Avoidance Behavior—In most metazoans,
Correspondingly,wefoundthatosas#9isnotproducedbynull monoamine neurotransmitters such as octopamine and sero-
mutantsofdaf-22anddhs-28,encodingtheperoxisomal3-ke- tonininteractwithneuropeptidestomodulatebehavioralstate
to-acyl-CoA thiolase and 3-hydroxyacyl-CoA dehydrogenase, (29). In C.elegans, octopamine down-regulates pharyngeal
respectively(11)(supplementalFig.S5).Octopamineisderived pumping(andtherebyfoodintake)aswellasegglaying(25,28,
fromdecarboxylationoftyrosine(catalyzedbytyrosinedecar- 30),opposingtheeffectsofserotonin.Inaddition,octopamine
boxylase(TDC-1))andhydroxylationoftheresultingtyramine signalingisrequiredforavoidancebehaviorinresponsetospe-
bytyramine(cid:1)-hydroxylase(TBH-1)(28)(Fig.2C).Toconfirm cific chemical stimuli (29), although octopamine itself is not
that osas#9 is derived from worm-produced octopamine, we knowntobedeterrent.Becausewefoundthatosas#9isspecif-
examinedtheexo-metabolomesoftdc-1mutantworms,which icallyassociatedwithstarvedL1larvae,weaskedwhetherthis
do not produce tyramine or octopamine, as well as of tbh-1 compound promoted dauer entry (a starvation response) or
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REPORT: BiogenicAmineMetabolismviaSuccinylation
otherwise affected larval survival. However, synthetic osas#9 deactivation mechanism or, as in the case of octopamine in
didnotinducedaueroraffectlifespanofstarvedL1larvae(sup- C.elegans, lead to the creation of new signaling molecules.
plementalFig.S7).Next,wetestedbehavioralresponsesoflar- Notably, competing succinylation and acetylation of biogenic
val and adult worms to synthetic osas#9 and found that all aminesappeartoparallelposttranslationalmodificationofpro-
larvalstagesarestronglydeterredbythiscompound(Fig.2D). teins via acetylation and succinylation of L-lysine (35). Use of
Notably,theadditionofE.coliasfoodoverridesosas#9deter- primarymetabolite-derivedbuildingblocksinthebiosynthesis
rence.Octopamineitselfdidnotelicitavoidancebehavior(sup- of more complex signaling molecules provides a direct link
plemental Fig. S8). We then tested whether the presence of betweenmetabolicstatusoftheorganismandsmall-molecule
osas#9contributestoavoidanceresponseselicitedbystarved signaling. Competition for shared building blocks (e.g. succi-
L1 medium. For this purpose, we compared avoidance nate)bydifferentbiochemicalreactionsmayresultininterde-
responses to medium from starved L1 larvae with those to pendenceofdifferentpathways,creatingthepotentialforaddi-
medium from starved tdc-1 mutant L1 larvae, which do not tionallayersofregulation.
produceosas#9ortsas#9.WefoundthatalthoughstarvedL1 The octopamine ascarosides thus connect intraorganismal
mediumfromwild-typewormsisstronglydeterrent,thedeter- neurotransmitter-basedsignalingpathwayswithinterorganis-
rence of medium from tdc-1 mutant worms is significantly malcommunicationbycreatingadeterrentpheromonesignal
weaker, especially at higher dilutions (Fig. 2E). The residual (osas#9)fromsuccinylatedoctopamineandtheperoxisomally
deterrenceofthetdc-1mediumlikelyresultsfromthepresence producedascarosideascr#9.Inconjunctionwiththeoriginof
ofotherascarosides,whichareproducedbywild-typeandtdc-1 ascarosides as end products of peroxisomal (cid:1)-oxidation (11),
D
mutantlarvaeinroughlyequalamounts(datanotshown)and theimplicationofoctopaminesuccinylationaspartofageneral o
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havepreviouslybeenshowntobedeterrentathighconcentra- pathwayforbiogenicaminedeactivationfurthersuggeststhat n
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tions(6,7).Takentogether,theseresultssuggestthatosas#9is ascaroside biosynthesis may have originated as a pathway for ad
e
partofadispersalsignalthatindicatesunfavorableconditions, waste product elimination that has been co-opted for addi- d fro
specificallytheabsenceoffood. tionalsignalingfunctions. m
C.elegansSuccinylatesBiogenicAmines—Acetylationofthe http
neurotransmitters octopamine, dopamine, and serotonin is Acknowledgment—WethankMaciejKukulaforhelpwithhighreso- ://w
generally thought of as a deactivation or degradation mecha- lutionMSanalysis. w
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nismandhasbeendescribedforinsects,nematodes,andother .jb
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invertebrates (31, 32), and several N-acetyl transferases with .o
octopamine-acetylating activity have been described (33, 34). REFERENCES arg/
Ouridentificationofsuccinylatedoctopamine(osas)andtyra- 1. Jeong,P.Y.,Jung,M.,Yim,Y.H.,Kim,H.,Park,M.,Hong,E.,Lee,W.,Kim, t D
u
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JUNE28,2013•VOLUME288•NUMBER26 JOURNALOFBIOLOGICALCHEMISTRY 18783
Reports:
Succinylated Octopamine Ascarosides and
a New Pathway of Biogenic Amine
Metabolism in Caenorhabditis elegans

Alexander B. Artyukhin, Joshua J. Yim, Jagan
Srinivasan, Yevgeniy Izrayelit, Neelanjan
Bose, Stephan H. von Reuss, Yeara Jo, James
M. Jordan, L. Ryan Baugh, Micheong
Cheong, Paul W. Sternberg, Leon Avery and
D
Frank C. Schroeder o
w
J. Biol. Chem. 2013, 288:18778-18783. n
lo
doi: 10.1074/jbc.C113.477000 originally published online May 20, 2013 ad
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Supporting Information

Succinylated octopamine ascarosides and a new pathway of biogenic amine
metabolism in C. elegans*
Alexander B. Artyukhin1,2, Joshua J. Yim1, Jagan Srinivasan3, Yevgeniy Izrayelit1,
Neelanjan Bose1, Stephan H. von Reuss1, Yeara Jo3, James M. Jordan4, L. Ryan Baugh4,
Micheong Cheong2, Paul W. Sternberg3, Leon Avery2, and Frank C. Schroeder1
1Boyce Thompson Institute and Department of Chemistry and Chemical Biology
Cornell University, Ithaca, New York 14853, USA
2Department of Physiology and Biophysics,
Virginia Commonwealth University, Richmond, VA 23298, USA
3Howard Hughes Medical Institute and Division of Biology,
California Institute of Technology, Pasadena, California 91125, USA
4Department of Biology, Duke Center for Systems Biology,
Duke University, Durham, NC 27708, USA

*Corresponding Author: email: [email protected] phone: [phone] fax: 607-254-
2958

S1
Table of Contents
1. Supporting Figures S1-S11 S3-S12
2. Supporting Methods S14
3. Synthesis of osas#9 and tsas#9 S15-S17
4. Synthesis of N-acetyl and N-succinyl derivatives of dopamine, octopamine, and serotonin S18-S19
5. NMR Spectra of intermediates, osas#9, and tsas#9 S20-S29
6. References S30
S2
1. Supporting Figures

Figure S1. Electron spray ionization MS spectra of osas#9, osas#2, and osas#10 in both positive
and negative ion modes. Fragmentation in ES+ yields the same ions for all three osas ascarosides
(m/z 136, 218, 236, 348, 366) resulting from fragmentation of octopamine succinyl ascarylose
moiety as well as [M+H-H O]+ and [M+Na]+. Fragmentation of osas#9 and osas#10 in ES-
2
produces ascr#9 and ascr#10 ions, respectively, but this fragmentation pathway is absent in
osas#2. All three osas ascarosides also produce ions at m/z 216 and 234 in ES- resulting from
octopamine succinyl moiety, in addition to molecular ions [M-H]-.
S3