Animal Venom Studies

23 Pages·2019·2.672 MB·English

by  Roy Waidler

(cid:9)
Toaicon, Vo1.20, No.l, pp.317-321, 1982 0041-0101/82/010317-0502.00/0
Printed in Great Britain. Pergamon Press Ltd.
ADOLAPIN-ANEWLYISOLATED ANALGETICANDANTI-INFLAMMATORY
POLYPEPTIDEFROMBEEVENOM
Stefan ShkenderovandKrasimiraKoburova
Institutefas StateControlofDrugs, Bul.Vl.Zaimov26,Sofia,Bulgaria
ABSI'RACT
Adolapinwas isolatedbyatwo-stepprocedure:gelfiltrationand chromatographyonCMcellulose. The molecularmam
of the polvpepddeas determinedby SDSelectrophoresis andaminoacidwmposition provedtobe11500and 11092
respectively.Adolapiaerdubitedapotentanalgetic effectdemonstratedbythe "writhing"test(ED50-0,016mq/kg)and
by the RandaIlSellito'a teat (ED50-0,013 mg/!<g).The anti-inflammatory activity of adolapin was most marked with
regard to carragsenin, pro~aglandin and adjuvant rat hindpawedemas andadjuvantpolyrarthritis.Theadolapin effects
are presumably due to its capadty toiahrbit the prostaglandin synthetase system, followingabiphasic doss-response
relationship.It islikely thatcentralmechanicsarealso involvedintheanalgeticaction ofadokpin.
KEYWORDS
Beevenompolypeptide:analgetic polypeptide:anti-inflammatorypolypeptide;cyclooayvenaeeinhibitorypolypeptide:
non~terordalanti-inflammatorydrugs, biphasic dose-responserelationship.
IN'T'RODUCTION
Habermann and Reis(1%5)inaremarkable separation of beevenomcomponents by gel9tration,desi~atedasmall
fraction appearing in the beginning ofthemelittin peakas Oa.Fraction Oaprovedtobenon-tonic. Itwasconsidered
biologicalyinactive.Forthesereasonsitwasignoredbytheinvestigators.
In a screening studyofbeevenomcomponents we demonstratedthatfraction Oapossessed potent anti-infJamrnatay
properties (Shkenderov, 1976). Inasubsequentinvestigation werevealed the analgetic and cydoozygenaee~nhibitory
activity of the fraction (Shkenderovand Koburova,1979).Thesefindingspromptedthe presentinvestigationofitsbio-
chemicaland phnrrnacolodcalcharacteristics.
MATERIALSANDMETHODS
BeevenomwasaproductofNectarooop -Sofia, obtained by electrical"mi>kirg" ofbem. SephadeaG50waspur-
chased from Pharmade (Sweden), CM cellulose C-52 from Whatman (England). Ampholine plates were from I~KB.
Prostaglandin Erwas a giftfrom Upjohn (USA), Nalosbae was kindly supplied byWinthrop (Is'nglaad). Hi~+ami*~.
hydrochloride was obtained from Merck(FRG), serotonin,oreatine sulphate andbradykinin-triacetate-tnhydrate were
from Koch-Light (England), and carrageenin from Sigma (USA). Complete Freund adjuvant contained kr~ed
Mycobacteriatuberculoals Ra,6ma/ml.
All chromatographic procedures wereperformed usingL~KBequipment,inanLI{&Combl-ColdRackat4oC.Themole-
cularmawwasdeterminedby SDSelectrophoresis usingtheCombithek teatkitofBoehringer (FRG).The aminoadd
wmpoeition was studiedin aLKB 4400 Aminoaddanalyserafter48 hourshydrolysisinGNHCI.Tryptophenwaaas-
317
(cid:9)
31 8 STEFAN SHIO;NDEBOV and ICRASIMIRA KOBUROVA
savedaedescribedbyScoffoneandFontana(1975).TheN-ta~minalgroupwa~determinedbythemethodofIvanovand
Mancheva (1973). Microsomesfrom cat spleen were prepared accordingto Dembinaka-Kieb, Zaùuda andKrupinsira
(1977) andratbrainmiaosomes- as deecnbed by Nakamaru (1968). Cydooxygenaseactivitywasdeterminedbythe
method of Taieauahi, Kohono andSih (1971). Theanalgeticactivityvrasstudiedbythemethod ofRandallandSelitto
(1957) with an Uqo 19asa1eanalgesimeterandbythe"writhing"teat,using1mlof0,3percentaceticadduaperitoneal
irritant,by themethodofSiegmund,CadmusandLu (1957) and0,3micros.prostaglandinEiesan irritant,udescribed
by CollierandSchneider(1972). Rathindpawedemaswere measured bymeansofadüferentialvolumeterUvoliasile.
InaIItheeaperimentthepolvpeptideswereinfectedaubcutaneously.
RESULTS
FractbnOa wasobtained by ~~tration of beevenomoaSephadexG50(Fig.la).Furtherseparationofthecompo-
nentsoffractionOa,wasachievedbychromatographyonCMceIIuloseWhatmanCM-50applyingaconcentrationaada
pHgradient (FIg. lb). Five polypeptidepeakswere eluted,thefirstappearingbeforethestartofthegradient.Thedrat
twopeaks(1and2) were identicalandappeared tobehomogeneousinSDSeleca~opharetis.Inisoeh+ctrofocuainqthey
showed isoelsctricpouts8,9;9,2reap.Theeepeaksdidnothemolyseerythrocytesanddidnotliberatehistaminefrom
mastceps.Therernaininvpeakswweheterogeneous.
iM
'~~'pH7.5
;a,M
DHh,6
50 bY~~
Fig. la.Gelfiltrationof8q beevenomonSephadexGSOf9necohunn (300/55cm,0.1 M
amm^niA - formate buffer, pH 4b,fbwrate 3,6ml/cm2/h, 18 ml fractions).The frac-
tbnsseparatedbyverticallisescontainthecoreapondinqenaimesandpeptides.
Fig. lb. Chromatography of 190mg fraction Oa (dottedfractionsinFig. la )on CM -
oeAuk~se cohmm (40/?,5cm), equilibratedwith 0,1 Mamonia-formatsbufferpH4,6.Elu-
tionrate -32ml/h,8mlfiacdonswere collected.Thegradient(j) was2Mammonia-for-
mate buffer pH 7,5.Thehatchedandnumberedpeakswarspooled.Penl~1and2 -ado-
lapin 1andadolapfn 2.
Peaks 1 and 2have identicalaminoacidcomposition.It ischaracterisedby thelackofcystein,methioninandtrypto-
phatt.TheN-terminal aminoaddis glydne.Theminimalmolecuarmass,derivedfromtheamino addcomposition, is
11092Daltons,containing 108aminoaddreerlduea.
Of allthecomponents of Fraction Oa,peaks1and2exhibitthestrongestcyclooxygenase inhibitoryeffect,expressed
toacomparabledegreeinthetwopeaks.(Fig.2)
Catspleen microeomal cYclooxYgenase wasinhibitedby polypeptides 1and2toameterextentthantheratbrainen-
syms.DiPfarsrnphasesoftheinhibitoryactions,dependingonthedone,should benoted.Spleencydooaysenasewasin-
hibitedbylassthen 50 percentbythepolypeptideeat adoes of30 mia~ograaw,/ral, whereas1,5and3mia~ograma/ml
inhibitedtheensymeactivitybym~ethan85percent.(Fig.2)
Peptides 1and2possessapotentanalgsticeffect,asshownbybothtestsused. Theanalgeticeffect ofthetwodosesap-
plied-50mia+og./kgand20mlcroaJkq,wasalmostidentical.IntheRandaIISetftto'steata~ifkxntdifferenceinthe
effectsexertedby dffferem doses wasnotedonly atthefourthhour.ED50 oftheanalgeticeffectprovedtobe0,016
mq/kg(bythe"writtdna"feet)and0,013mg/kq(bytheRandall~elitto'stest).
Ths peptidesposedapowerful anti-inf]emrnatary effect demonstrated by cairageeninrathind pawedema(65per
(cid:9)(cid:9)
Adolapin from Bee Venom 319
H
m
Z
F
Z
W
Ci
2
W
d
Fiq.2.Effectsofthepolypeptides from peak 1 (dottedcolumas),peak 2 (empty aoiumas)
and indometha~a (i) on cydooxygenase acüvity of catspleen (a) andratbrain (b)miao-
somss. The microeomes hadbeen flreincubated wtth thepolypeptides for 15 minutes.C-
cycloxygenassactivityoftheuntreatedmians~mes.
cent inhibitionproduced by20mic:oa./kg)andproetaglsndin Ei edema (70parcent inhibitioncausedby20miaogJ
kg). ]3radykinin, eerotonin and histamiae hind paw edemas were suppre®ed to a lower degree,is. 20 to 30 per
cent by20microc./kg.Theanti-inflammatoryeffect waadose-independent atdosesexceeding50microgJka.Theanti-
inflammatayaction ofthepolypeptideauponthemodeledemaswasactinfluencedbyadrenalectoaly.
~~1~~-'~ !~1Î~1!~Î~1
~gkg 20 20
~oo~y,u, .coi, wKOmn,
Fig.3a.Analgetfceffect of adolapin 1 (peak 1),adolapin 2 (peak2) oathep~itoneel con-
tractionsin iatact ar pretreatedwith aaloxon(1 mg/log)miceinducedbyiatraperitaaeslin-
jectionof 0.1 mlSpercoatofacetic add(.Ac)ar0.3inicrog.ot ardlnEi(Ei)/0.1 ml
saline. Thepolypeptides hadbeen igjected foe30minbefore theiniectbnsoftheirrltaata.
Fig.3b. Analgetic effect of adolapin 1 (peak 1) onrats meeaured byRanda>l~elitto's feet.
Ratshadbeeniulecradwith 20microgJka(dottedlice)a50miorog./kq (solidline)adolapin
1,30minbsfaro themeama~satsweremade.
Durlng iavestiastfonoftheeffect of thepeptides onadjuvant rathindpawedema,uelnaadailydoseof20microgJloa,
maztmaleupaees"loaof theedema, ti3nercent,wasachieved onday9aftertheinoculation.Aweakerinh~bfdonwas
elidtedbyadoseof100microchgrenchiaqama>~nu<nof30percentoaday14.(!FIg.4h)
(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)
320 STEFAN SHICENDEROV and KRAS]MIRA KOBUROVA
s
12 16 20 Isys 14 18 22 26 30 34 38 dqt
Fig.4.Effectofdailyinjectionsof20 microg./kg (-._.-), 100microg./kq(-),ofadola-
pin 1 ar saline ("-""-"~juntilday14 fromtheinitiation ofadjuvant hind-paw edema (a)
andadjuvantnolyarthritia(b)in rats.
Ac(juvantpolyartluitisdevelopedafterday14 oftheexperiment.Thepeptidetreatmentwasdiscontinuedatthesame
time.Polyarthritiswasinhibitedby about70 percent inthegroupwhichhadreceived adose of20microgJkgofthe
peptides.Theeffectof 100microg./kgdose wanweaker (Fig.4b),
DISCUSSION
Thechemical characteristicsandthepharnmcologicalProperties ofthepolypeptidefrompeaks1and2,isolated by us,
demonstratethat representanewbiologically active componentof beevenom. Theseparationofthis componentinto
twoveekson ionßxchangechromntoarauhy, similarto the other oeptidee from been venom (melittin, apemineand
MCD peptide), is probably dus to the amidatbn or acylation of some of the fimctional groups ofthepolypeptide
chains.
Weproposetoname thenewlyidentlflednolvpeptideadolapin,whichsuggestsbothitsanalgeticactivityandrtevenom
The inlribitory effect on microsomal cydooxyaenase (proamglandin-symthetase) is acharacteristic andweII expressed
property ofadolapia.Onamolarbaskadolapin isabout70timesmare potent inhibitorofbraincyclooxycenasethat
theweIIknownanti-rheumatic preparationindomethacin.Thebiphaaicpatternoftheinhibitory effectofadolapin38a
particularcaseofenayme-inhibitor interactionanditseluddatbndeservesfurtherinvestigations.
Adolapin is arepresentative of anewclassofpharmacologioaIIyactive subataaoes -naturalinhibitcasoftheprostaglan-
din-synthesishavingpeptidestructure.
It may be suggestedthatthecyclooaygenase-inhibitory activity ofadole~pinis thebasicmechanimnofitsantt~nnflam-
matory andanalgeticeffects.This suugestionisbaseduponthewaIIestablishedrelationshipbetweentheinhibitationof
proateglandin"synthetase and theanti-inflammatory None, 1971) andanaloetic (Moaoada, FettersandVane, 1978)
action of non~teroidal anti-inflammatorydrugs. Thetwo effectsare manifested atsimilarchess. Thereis similarity
betweenthe biphasicinhibitoryeffect gadthenon-linear doseteeponse dependence oftheanti-inflammatoryandthe
(cid:9)
Adolapin from Bee Venom 321
analgesicactivity. It is likely,however,that centralmechanismsarealso involvedintheanalgesicactivity.This issucges-
ted by the fact that naloxon, aMocker of theopiatereceptorsin thebrain, eliminates partly the analgesiceffect of
Adoapininmice.
Thepharmaookxdcalactivityof Adoapiniedemonstrated at ahightherapeuticindex(LD50/ED~isabout5000).This
indexezceedshundredfoldthetherapeuticindezofthenon~steroidalanti-inflammatoryPreparations.Adolapinisalsoa
pharmacologicalantagonistofprostaglandinEunlikethemostnon~teroidalanti-inflammatorydrugs.
REFERENCES
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morphine.NatureNewBiol,23~141-143.
Dembinska-Kieç,A., A.Zrniuda, andI.Krupinska(1977).Inhibitionofprostaglandinsynthetaaeby aspirin-like
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andThromboxarteResearch,Vol.1, RavenPress,New-York,pp.99-112.
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denedesaentralwirkeamenPeptideApamin.Biochem. Z., 341, 451-466.
Ivanov,Ch.P.,andI.N.Mancheva (1973).Thin-layerchromatographicseparationofdiphenylindolyhubstitutedthiohy-
daatoinderivativesofaminoadds.J. Chromatogr,75.129-132.
Monkada, S.,S.H.Ferreira,and J.R.Vane(1978).Painandinflammatorymediators.InJ.R.VaneandS.H.Ferreira
(Eds.)Handbookofexperimentalpharmacology,SOlI.Inflammation,Springer Verlaq,Berlin-New-York.pp.588-
616.
Nakaaaru,T.(1968).Magneaiumadenashietriphosphataeeactivatedbyalowconcentration ofcaldumbrainmica+oeomes.
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Randall, L.O., andJ.J.Selitto(1957).Amethodformeasurementofanalgesicactivity on inflamedtissue.Arch. Int.
Pharmacodyn, 128,409-419.
Scoffone,E., andA.Fontana(1975).Identificationofspecificaminoacidresidues.InS.Needelman(Ed.),Proteinse~
quencedetermination, 2nded.,SpringerV~lag, Berlin-Heidelberg-New-York.pp.162-203.
Shkenderov,S.(1976).Newpharmocobiochemicaldataontheanti-inflAR+R+~toryeffect ofbeevenom.InA.Ohaaka,
K.HayashiandSawai(Eds.),Animal,plantandmicrobialtoxins,Vol.2, PlenumPubl.Corp.,NewYork,pp.
319-336.
Shkenderov,S.andK.Koburova(1979).AnalgesiceffectsofbeevenomanditspeptidefractionsOsandOP.Toxtcon,
suppl., No 1, 17. 171.
Siegmund,E.A.,R.A.Cadmus,andG.Lu (1957).Amethod forevaluatingboth non-narwticandnarcoticanalgeafa.
Proc.Soc. exp.BioL (N.Y.) 95,729-731.
Takeguchi,C.,E.Kohono,andC.J.Sih(1971).Mechanismofprostaglandinbiosynthesis.I.Characterisationaadassayof
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Vane,J.R.(1971)Inhibitionofprostaglandinsynthesisasamechanismofaction fanaspirin-like drugs.NatureNewBio-
logy,231, 232-235.
Proteomics2007,7,1615–1623 DOI10.1002/pmic.200600800 1615
R A
ESEARCH RTICLE
A proteomic study of the major allergens from yellow
jacket venoms
DanielKolarich1*,AndreasLoos1*,RenaudLéonard1,LukasMach2,GorjiMarzban3,
WolfgangHemmer4andFriedrichAltmann1
1BiochemistryDivision,DepartmentofChemistry,UniversityofNaturalResourcesandApplied
LifeSciences(BOKU),Vienna,Austria
2InstituteofAppliedGeneticsandCellBiology,UniversityofNaturalResourcesandAppliedLife
Sciences(BOKU),Vienna,Austria
3PlantBiotechnologyUnit,DepartmentofBiotechnology,UniversityofNaturalResourcesand
AppliedLifeSciences(BOKU),Vienna,Austria
4FloridsdorfAllergyCentre(FAZ),Vienna,Austria
The venomsofstinging insectsbelongtothe mostdangerousallergen sourcesandcancause Received:October19,2006
fatal anaphylactic reactions. Reliable prediction of a patient’s risk to anaphylactic reactions is Revised:December12,2006
vital, and diagnosis requires the knowledge of the relevant allergens. Recently, a new hyalu- Accepted:January10,2007
ronidase-likeglycoproteinfromVespulavulgaris(Vesv2b)wasidentified.Thisledustoinvesti-
gatehyaluronidasesandalsoothermajorallergensfromV.germanicaandfouradditionalVespula
species.ByMALDI-Q-TOF-MS,thenewhyaluronidase-likeproteinwasshowntobethemajor
componentofthe43-kDabandinallVespulaspeciesstudied.LC-ESI-Q-TOF-MS/MSsequencing
ofVesg2aandVesg2bfacilitatedthecloningoftheircDNA.Vesv2bandVesg2bturnedoutto
beessentiallyidenticalonproteinlevel.Whereasthelessabundant“a”formdisplayedenzymatic
activity,thenew“b”homologuedidnot.Thisisprobablycausedbyaminoacidexchangesinthe
activesite,anditraisesquestionsaboutthephysiologicalroleofthisprotein.Sequencecompar-
isonsbyMS/MSofantigen5andphospholipasesfromV.vulgaris,germanica,maculifrons,pen-
sylvanica,flavopilosaandsquamosarevealedthelatterasataxonomicoutlierandledtothedis-
coveryofseveralnotpreviouslyreportedaminoaciddifferences.
Keywords:
Allergens/Denovosequencing/Hyaluronidase/Vespula
1 Introduction pollen-allergic patients, who suffer seasonally from diverse
allergicsymptoms,insect-allergicindividualsareaffectedby
Yellow jackets (Vespula) represent a latent risk for people insect stings only and may therefore not be aware of their
allergicagainstthevenomoftheseinsects[1].Incontrastto dispositionuntilstungforasecondtime.Thismakesinsect
allergylesspredictablethanpollenallergyandmoredanger-
ous, because without immediate treatment a single insect
Correspondence: Dr. Daniel Kolarich, Division of Biochemistry,
stingcanleadtothedeathofthepatientduetoanaphylactic
DepartmentofChemistry,UniversityofNaturalResourcesand
shock.
Applied Life Sciences, Vienna, Muthgasse 18, A-1190 Vienna,
Austria Venoms of the yellow jacket species considered so far
E-mail:[email protected] contain three major allergens: antigen 5 (Ag5 or Ves x 5),
Fax:[phone] phospholipase(PLAorVesx1)andhyaluronidase(Hyaseor
Vesx2),thelatterbeingaglycoprotein[2–6].Otherproteins
Abbreviations:Ag5,antigen5;CCD,cross-reactivecarbohydrate
suchasVmac1andVmac3haveadditionallybeendescribed
determinants;cDNA,complementaryDNA;GlcNAc,N-acetylglu-
cosamine; GlcUA, glucuronic acid; Hyase, hyaluronidase; PLA,
phospholipase * Bothauthorscontributedequallytothiswork.
© 2007WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim www.proteomics-journal.com
1616 D.Kolarichetal. Proteomics2007,7,1615–1623
asminorallergensfromV.maculifrons[7].Thephysiological 2.3 cDNAcloningofV.germanicaallergens
roleofAg5hasnotyetbeendiscovered,however,theenzy-
matic activity of the two other major proteins has been ThecDNAcloninghasbeenperformedfromvenomsacsof
revealedbyHoffmanandKingabout25yearsago[2,8–10]. wasps as previously published [5]. The primers used to
Theirpioneeringworkledtothesequencingofseveralaller- amplify cDNA for V.germanica venom proteins were
gensfromselectedyellowjackets,butsofar,Ag5remained TCCGAGAGACCGAAAAGAGTC and TTAGTTGACGG-
the only allergen where protein sequences have been CTTCTGTCACG for Ves 2a, GTGATTACAATCTGGCC-
obtainedforalltheusuallyconsideredyellowjacketspecies TAAG and CTAAAAGTTTAACGGTGTG for Ves 2b, GAA-
[2]. In the case of PLA, the protein sequence is known for GAGTTAAGAAGAAACCTTCG and AAAGGTCATTTGG-
V.vulgarisandV.maculifrons(andduringthepreparationof TAATCTTT for Ag5 and CATGGTGATCCGTTATCTTACG
thispapertheV.germanicaPLAnucleotidesequencebecame andTTAAATTATCTTCCCCTTGTTATTGforPLA.
available online), while for Hyase, only the V. vulgaris en-
zymehashithertobeenclonedandsequenced[11].
The structures of the Asn-linked oligosaccharides of 2.4 ExpressionofVesv2variantsininsectcells
HyasesfromV.vulgarishaverecentlybeendeterminedand
shown to be cross-reactive carbohydrate determinants Vesv2aandVesv2bconstructsencodingaminoacids1–331
(CCDs)duetothecorea1,3-linkedfucose[12,13].CCDsare and 1–340, respectively, were generated by PCR from the
a frequent cause of cross-reactions of patients’ sera with correspondingcDNA[5],usingthefollowingprimercombi-
allergens from insects and other sources [3, 14, 15] but nations: 5’-AAAACTGCAGCCATCCGAGAGACCGAAAA-
appear to be of low clinical significance [13, 16, 17]. The GAGTCTT-3’ and 5’-GGAATTCCTTAGTTGACGGCTTCT-
relative contributions of the protein and the carbohydrate GTCACGTT-3’ (Ves v 2a), and 5’-AAAACTGCAGCCAGA-
moietyofinsectvenomHyasestoinvitroIgEbindingandto CAGAACAATTTGGCCTAAGAAG-3’and5’-GGAATTCCC-
elicitingallergicsymptomsinvivohasnotyetbeendefined. TAAAAGTTTAACGGTGTGTTTTCTTTG-3’(Vesv2b).The
The most surprising finding of this glycoproteomic PCR products were cleaved with PstI and EcoRI restriction
analysis was that the major component of the Hyase band enzymesattheunderlinedsitesandligatedintopVTBacHis-
was a protein dissimilar from but 76% homologous to the 1baculovirustransfervector[20].Inthisconstruct,theVesv
knownallergenVesv2[5].Noteworthy,thetwocatalytically 2proteinsareplaceddownstreamofthemelittinsignalpep-
importantresiduesinVesv2a,Asp107andGlu109[6,18]), tide,a66His-tagandanenterokinasecleavagesite.
are both exchanged to His in Ves v 2b [5], raising doubts A pVTBacHis-1 construct encoding untagged Ves v 2b
abouttheenzymaticactivityofthishomolog. was produced as above, using a different forward primer
Intheframeworkofthisstudy,wehavecharacterizedthe (5’-CGGGATCCCGACAGAACAATTTGGCCTAAGAAG-3’),
major allergens from six Vespula species by MS. The com- the same reverseprimer (5’-GGAATTCCCTAAAAGTTTAA
plementary DNA (cDNA) encoding the major allergens of CGGTGTGTTTTCTTTG-3’), and the restriction enzymes
V.germanicahavebeenclonedandtheenzymaticactivityof BamHI and EcoRI. This procedure places the Ves v 2b
thetwoHyasehomologuesfromV.vulgariswasexamined. sequencedirectlybehindthemelittinsignalpeptide,thereby
removingthe66His-tagandtheenterokinasecleavagesite.
Expression in Spodoptera frugiperda Sf21 cells was per-
2 Materialsandmethods formed exactly as described previously [21]. Briefly, the
recombinant transfer vector (1mg) was co-transfected with
2.1 Materials 200ng ofBaculoGoldviralDNA (BD Biosciences,Erembo-
degem,Belgium)intoSf9cellsusingLipofectin(Invitrogen)
Vespulavenomsintheformofvenomsacextractswerepur- asrecommendedbythemanufacturer.After5daysat277C,
chased from Sweden Diagnostics (Uppsala, Sweden). Se- supernatants containing recombinant virus were used for
quencing-grade trypsin was obtained from Roche (Basel, infection of Sf21 cells. Cells and culture media were har-
Switzerland).SpecimensofV.vulgarisandV.germanicawere vestedafter4daysat277CandsubjectedtoSDS-PAGEand
collectedinandaroundViennaandstoredat–807Cuntiluse. immunoblottinganalysis.
For immunoblotting analysis, baculovirus-infected and
2.2 Samplepreparation,WesternblottingandMS non-infected Sf21 cells were lysed in SDS-PAGE sample
andMS/MSanalysis buffer.CelllysatesandculturesupernatantsofinfectedSf21
cells were subjected to 12.5% SDS-PAGE under reducing
Sample preparation of wasp venom, SDS-PAGE, tryptic conditions. Fractionated proteins were electrophoretically
digest, MALDI and ESI-Q-TOF-MS and MS/MS for the transferredontoNCmembranes(ProtranR,Schleicherand
identificationandsequencingoftrypticpeptidesofthemajor Schuell Bioscience) and subsequently incubated with a
allergenproteinswasperformedasdescribedindetailelse- mouse mAb recognizing the His-Tag (Sigma). Detection of
where [5, 19]. Western blots with anti-HRP serum (Sigma) bound antibodies was achieved with goat anti-mouse IgG
wereperformedasdescribedpreviously[14]. antibodies conjugated to alkaline phosphatase (Jackson
© 2007WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim www.proteomics-journal.com
Proteomics2007,7,1615–1623 AnimalProteomics 1617
ImmunoResearch,Soham,UK)usingtheNBT/BCIPdevel- featureoftheparticularvenombatchorarespecificforthe
opmentkit(Sigma). species has not been further investigated. The protein
bands were excised as indicated in Fig.1 and subjected to
2.5 Purificationandactivityassaysofrecombinant MS characterization.
VespulaHyases
3.2 VespulavenomAg5
RecombinantHis-taggedVesv2aand2bwerepurifiedusing
Ni-NTA agarose (Qiagen, Vienna) as described previously The majority of the MS/MS results obtained agreed with
[22]. Besides the purified enzymes, diluted supernatants the published sequences. However, a number of single-
frominsectcellcultureswerealsotestedforHyaseactivity. amino acid differences were found. In V.maculifrons Ag5
Total bee venom, purified bee venom Hyase [23] as well as (P35760) the predicted tryptic peptide 107–128 was detec-
bovinetestesHyase(Sigma)wereusedaspositivecontrols. ted, however, MALDI-Q-TOF MS showed an additional
Hyaluronic acid (Sigma) degradation was measured using signal with a mass increment of 2. Using MS/MS this
the BSA-test and the Morgan-Elson assay as describedpre- signal could be shown to be the same peptide, however,
viously[24–26].Thelatterassaywasalsoperformedwiththe with Thr instead of Val in position 122. As deduced from
substrates chondroitin sulfate A and chondroitin sulfate C signal intensities of the two peptides, approx. 80% of the
(Sigma). Ag5 in the analyzed venombatch contained Thr instead of
Weak anion-exchange chromatography of 2-amino- Val in the particular position. For V.flavopilosa Ag5
benzamide-labeled glycosaminoglycans obtained after deg- (P35783) an amino acid substitution in position 38 was
radationwithpurifiedVesv2awasperformedasdescribed identified, where a Glu replaced the Lys, resulting in a
previously[27]. different tryptic cleavage pattern.
An interesting accumulation of changes in a particular
region was detected in Ag5 from V.squamosa. While the
3 Results majority of the sequence corresponded to the in silico pre-
dictedpeptides(P35786), the tryptic peptide 108–132could
3.1 Generalremarks atfirstnotbedetectedbyeitherMALDI-Q-TOF-MSorESI-
Q-TOF-LC-MSand MS/MS. However, peptides of [MH]1=
SDS-PAGE separation of the six yellow jacket venoms 1019.59and1676.90Da,respectively,asdetectedbyMALDI-
yielded strong bands for two major allergens, Ag5 and Q-TOFcouldnotbeexplainedbytheinsilicoprediction.Se-
PLA, and more or less closely migrating double bands of quencing by ESI-MS/MS identified them as the missing
about 45kDa for Hyase (Fig.1). Whether the observeddif- sequencebutwitharemarkableaccumulationofsixamino
ferenceinconcentrationofAg5andPLApresentespecially acidsubstitutionswithoneevenintroducingatrypticcleav-
in the venom of V. flavopilosa and V. germanica are just a agesite(Fig.2).
Figure1. SDS-PAGE(right)andanti-HRPantibodyWesternblot(left)ofsixyellowjacketvenoms.Thethreemajorallergenproteinswere
detectedinallvenoms:Ag5(,25kDa),PLA(,33kDa)andHyase(,44kDa).Thehyaluronidasebandsanalyzedaremarkedwithbrackets.
MSoftheHyasedoublebandsdidnotshowanydetectabledifferencesbetweentheupperandthelowerbands.Blotstainingusingan
antibodyspecificforcorea1,3-linkedfucosehighlightstheHyasebands,however,especiallyintheV.squamosavenomalsoPLA-con-
tainingbandsshowastaining,thatisnotseenintheotherfivevenoms.Abbreviations:P:V.pensylvanica;S:V.squamosa;F:V.flavopilosa;
G:V.germanica;V:V.vulgarisandM:V.maculifrons.pos:positivecontrolusingHRP;neg:negativecontrolusinghumantransferrin.
© 2007WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim www.proteomics-journal.com
1618 D.Kolarichetal. Proteomics2007,7,1615–1623
Figure 2. MALDI-Q-TOF MS
spectrum of V. squamosa Ag5.
The sequence of peptide 108–
132isgivenasrevealedbyLC-
ESI-Q-TOF-MS/MS with the six
aminoacidsdifferentfromdata-
base entry P35786 written in
largerfont.Becauseoftheaddi-
tional Lys, the peptide gave
three signals. Other peptides
were in accordance with the
Swiss-Prot entry as confirmed
by MS/MS. Leucine could
equally be isoleucine, as they
are not discriminated by their
mass.
3.3 VespulavenomPLA cies analyzed in this study, V.squamosa PLA contains two
N-glycosylation consensus sequences (N125 and N161,
PLAsequencesarejustreportedforV.vulgaris(P49369)and aminoacidnomenclatureadjustedtotheV.vulgarisPLA,see
V.maculifrons (P51528) in the Swiss-Prot database and just Fig.3).AlthoughnoglycopeptidescouldbedetectedbyMS
recently (during the course of this work) the nucleotide andpeptide156–168(LVTDYNVSMADIR)wasidentifiedin
sequenceforV.germanicaPLAbecameavailable(NCBIEntry its unglycosylated form by MS/MS, results from Western
AM083318).Inafirstsurvey,thesixPLAsweresubjectedtoa blotanalysesusinganantibodyknowntobespecificfora1,3
standard tryptic peptide mapping with MALDI-Q-TOF-MS. boundfucose(andb1,2xylose)indicatethatV.squamosaPLA
These preliminary results confirmed the database informa- might at least be partially glycosylated and contains CCD-
tion for V. vulgaris and V.germanica PLA, whereas several reactiveN-glycans(Fig.1).
peaksdidnotcorrespondtotheinsilicodigestofV.maculi-
frons PLA. ESI-Q-TOF-LC-MS/MS indicated four yet 3.4 VespulavenomHyases
unknown differences in the V.maculifrons sequence (96:
K?T, 133: A?L, 238: I?M and 296: R?S; amino acid The identification of V. vulgaris Hyase (Ves v 2a) and the
numbering see Fig.3). Since the predicted peptides corre- detection of the major component of the Hyase band as
sponding to these stretches of the previously reported Hyase-likeprotein(Vesv2b)hasalreadybeendescribedina
sequencewerenotfound,weconcludethatthefourexchan- recentpublication,whichalsodealtwiththeglycosylationof
ges occurred on one polypeptide and not on two or more HyasefromvariousVespulaspecies[5].TheMALDI-Q-TOF-
isoformsasdescribedaboveforV.maculifronsAg5. MSanalysisoftrypticpeptidesfromtheHyasebandsofyel-
Theclosetaxonomicrelationofthedifferentyellowjack- lowjacketvenomsrevealedastrikingsimilaritybetweenall
ets set the basis for the MS investigation of the hitherto Vespulaspecies(Fig.4). Only V. squamosagave a rather dif-
unknown PLA from V.pensylvanica, V.flavopilosa and the ferent picture (data not shown). Instead of continuing the
partially described PLA from V.squamosa, respectively [28]. MSsequencingofthehighlyconservedHyases,wedecided
The peptide sequences identified for V.pensylvanica and to determine the nucleotide sequences of the Hyases from
V.flavopilosa displayed very high homologies at the protein V.germanica, the yellow jacket species endemic in Central
level to the already known PLA sequences from V.vulgaris, Europeandtoinvestigatethebiologicalfunctionofthetwo
V.maculifronsandV.germanica(Fig.3).However,thepeptide homologues.
sequences for V.squamosa PLA differed significantly from
those of the other five species. By MALDI-Q-TOF-MS and 3.5 cDNAcloningofV.germanicaallergens
LC-ESI-MS/MSthe V.squamosaAg5 sequencedescribedby
Hoffman et al. [28] previously could be confirmed (Fig.3) cDNAcorrespondingtoV.germanicaVesv2bandPLAcould
andonesingle-aminoaciddifferenceinposition136(V?S) beamplifiedbyPCRusingprimersdesignedonthebasisof
couldbedetected(seealsoSupportingInformation).Incon- thehighsequencehomologiestotheV.vulgarishomologues
trasttothePLAsequencesfromtheotheryellowjacketspe- as revealed by the proteomic study. At first, an mRNA
© 2007WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim www.proteomics-journal.com