家蚕触角蛋白质组学分析

Proteomic Analysis of Silkworm Antennae

Yunpo Zhao1&Haichao Li1&Xuexia Miao1

Received:3June2015/Revised:7September2015/Accepted:8October2015/Published online:29October2015 #Springer Science+Business Media New York2015

Abstract The silkworm Bombyx mori is an oligophagous in-sect that feeds mainly on mulberry leaves.The olfactory sys-tem of silkworm is a good model to study olfaction in Lepidoptera.Here,we carried out shotgun proteomic analysis and MS sequencing of the silkmoth antennae.A total of 364proteins were detected,77were female specific,143 were male specific,and144were expressed in both male and female antennae.Five odorant-binding proteins,two chemosensory proteins,and one olfactory receptor were identified.They may play a major role in the perception of odorants.An esterase and an aldehyde dehydrogenase were found only in male antennae.Glutathione S-transferases (GSTs)and cytochrome P450s,also found in silkworm anten-nae,may be involved in the degradation of xenobiotics. Additionally,antioxidation proteins and immunity proteins were identified.Juvenile hormone binding proteins(JHBP), juvenile hormone resistance protein II,and juvenile hormone episode hydrolase(JHEH)were found in the proteomic anal-ysis,which suggests that the antennae are a target for juvenile hormone in the silkworm.Our results provide insight into the expression of proteins in the antennae of silkworm and will facilitate the future functional analysis of silkworm antennae. Keywords Proteomics.Chemosensory.Odorant-binding proteins.Olfaction.Bombyx mori

Introduction

Insects use their antennae to sense odorant signals that guide their interactions with hosts and mating partners.They are able to locate hosts or edible plants by discriminating emitted volatiles.Odorants are sensed by olfactory receptor neurons (ORNs)housed in the sensilla on the antennae.ORNs express specific odorant receptors(ORs)and project axons to the brain (Leal2013).The silkworm,Bombyx mori is an oligophagous insect that feeds mainly on mulberry leaves(Horie1980). Silkworm is a well-established model for studying insect olfaction.

Female silkmoths produce two pheromones,bombykol and bombykal(Sakurai et al.2014).V olatile pheromones are hydro-phobic,thus,they need to bind to pheromone binding proteins (PBPs)to pass the aqueous sensillum lymph.Silkworm PBP was isolated from male antennae,but also found to be expressed in female antennae(Maida et al.1993).The PBP family is a subfamily of the odorant binding protein family in insects.The silkworm genome encodes forty-four candidate BmorOBP genes,with some expressed specifically in olfactory tissues,and others expressed more broadly in non-olfactory tissues(Gong et al.2009).

RNA-seq has been used widely to analyze the antennal transcriptome of various insects,including Drosophila melanoganster(Menuz et al.2014;Younus et al.2014), Anopheles gambiae(Rinker et al.2013),Spodoptera frugiperda(Legeai et al.2014),Helicoverpa armigera

Electronic supplementary material The online version of this article (doi:10.1007/s10886-015-0643-1)contains supplementary material, which is available to authorized users.

*Xuexia Miao

xxm@https://www.360docs.net/doc/6316580154.html,

Yunpo Zhao

ypzhao@https://www.360docs.net/doc/6316580154.html,

Haichao Li

lihaichao@https://www.360docs.net/doc/6316580154.html,

1Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences,

Shanghai200032,People’s Republic of China

J Chem Ecol(2015)41:1037–1042 DOI10.1007/s10886-015-0643-1

(Liu et al.2012),Manduca sexta(Grosse-Wilde et al.2011). At the protein level,Anholt and Williams(2010)analyzed the soluble proteome of Drosophila antenna using a shotgun pro-teomic approach.The antennal proteomes of honeybees have been studied(Fang et al.2012;Feng et al.2011).Bombyx mori is an important model in chemosensory science,however,no antennal transcriptome or proteome of Bombyx mori is avail-able.Two draft genome sequence maps for Bombyx mori were published in2004(Mita et al.2004;Xia et al.2004),which propelled genomics and functional genomics research on Bombyx mori(Xia et al.2014).In this study,SDS-PAGE and shotgun LC-MS/MS were employed to characterize the proteome of the silkworm antennae.

Methods and Materials

Sample Preparation and Gel Electrophoresis Silkworm strain Dazao was obtained from the Sericulture Research Institute,Chinese Academy of Agriculture Sciences(SRI-CAAS),and reared on fresh mulberry leaves at26±1°C. The antennae of adult male and female silkmoths(1–3d-old) were cut and collected separately.The samples were snap-frozen in liquid nitrogen and then stored at?80°C.The an-tennae were ground to a powder in liquid nitrogen with a mortar and pestle.The resulting deep-frozen powder was transferred into400μl of lysis buffer(containing2.5% SDS,10%glycerin,5%β-mercaptoethanol,and50mM Tris-HCl pH8.8).The homogenate was kept for10min at room temperature,and then subjected to continued sonication treatment in an ice-bath four times,each time for15s with a 15s interval.The samples then were boiled for2min,and 60μg of protein were loaded into two lanes of an SDS-PAGE gel(8%stacking gel,12.5%resolving gel).The gels were stained with Coomassie Brilliant Blue R250(Sigma).

In-gel Digestion and Mass Spectrometry Each Coomassie-stained gel lane was manually cut into10slices(Fig.1).Each slice was cut into1×1mm pieces,and then subjected to in-gel tryptic digestion.The gel pieces were rinsed twice with Milli-Q water(Millipore)and destained thrice with25mM NH4HCO3in50%acetonitrile(ACN,Amersham).The sam-ples were evaporated to dryness in a vacuum centrifuge (Eppendorf)and then incubated with10mM dithiothreitol (BIO-RAD)in25mM NH4HCO3at56°C for1h with suf-ficient solution to cover the gel pieces.Upon cooling to room temperature,the same amount of55mM iodoacetamide in 25mM NH4HCO3was added,and the resulting solution was incubated at room temperature in the dark for45min. One hundred microliters of25mM NH4HCO3in50% ACN were added and incubated at room temperature with rotation for10min.This step was repeated twice to dehydrate the gel pieces.The samples were evaporated to dryness in the vacuum centrifuge.This was followed by the addition of tryp-sin(20ng/μl trypsin(sequence grade,Promega)in25mM NH4HCO3).The gel pieces were rehydrated at4°C for 30min.The extra trypsin solution was removed.The diges-tion was carried out at37°C overnight.The tryptic peptides mixture was extracted from the gel pieces with100μl Milli-Q water,followed by two extractions with5%trifluoroacetic acid(TFA,Fluka)in50%ACN solution.The pooled extracts were evaporated in a vacuum centrifuge to a volume of about 50μl.Liquid chromatography tandem mass spectrometry (LC–MS/MS)analysis was carried out as previously described.

Data Analysis A database was constructed with the protein sequences downloaded from the NCBInr database containing Bombyx and Drosophila,and the sequences from silkDB (https://www.360docs.net/doc/6316580154.html,/silkdb/doc/download.html).The 20raw datasets from the LC-MS/MS were searched against the database with the algorithm SEQUEST(Thermo Finnigan) on a local server.The mass tolerances of precursor and fragmentation ions were set to2and0.8,respectively.Only b and y ions were taken into account.The trypsin-cleavage was at both ends,and two missing cleavage sites were allowed. Static modification on cysteine(carbamidomethyl),and dynamic modification on methionine(oxidation)were set. Results

The Silkworm Antennal Proteome The proteomic profiles of the male and female silkworm antennae were obtained using a shotgun proteomics strategy and searched against an in-house compiled database.Tandem mass spectrometric analysis of eluted chromatographic peaks allowed for the identification of proteins through SEQUEST database search algorithms under stringent criteria.We identified265and

338 Fig.1SDS-PAGE analysis of the silkworm antenna proteome.The numbers indicate the ten slices used for sample fractionation prior to in-gel digestion

proteins from female and male silkworm antennae,respectively. Since the in-house database was built with protein sequences downloaded from the NCBI nr database and silkDB,there are identical protein sequences within this database.The redundant proteins were manually removed,and a total number of364 unique proteins were identified.Among them,77proteins are female specific,143are male specific,and144are expressed in both male and female antennae(Table1).Due to the limitations of the silkworm protein database,not all proteins had complete functional annotations.Some of those specific proteins identi-fied from the proteome of silkworm antennae are listed in Table2.

Odorant Binding Proteins,Chemosensory Proteins,and Olfactory Receptors Zhou et al.(2009)showed that the tran-scripts of BmorPBP1,BmorGOBP1,BmorGOBP2,and BmorABPx are highly enriched in the antennae of silkworm. In our proteomic analysis,we detected4of5OBPs (BmorPBP1,BmorGOBP2,BmorABPX,and antennal bind-ing protein precursor)both in male and female antennae (Table2).General odorant binding protein1(BmorGOBP1) was found only in female antennae.Chemosensory proteins (CSPs)are widely expressed in different tissues.The silk-worm genome encodes about20candidate CSPs(Gong et al.2007).In this study,two CSPs(CSP4and CSP13)were detected in the antennae(Table2).CSP4is transcribed pre-dominantly in the antennae,while CSP13is more widely expressed(Gong et al.2007).The olfactory receptor,OR45 (gi:162,461,258)was male-specific.

Esterases,Aldehyde Dehydrogenases,Glutathione S-Transferases(GSTs),Cytochrome P450s(CYPs),and Im-munity Related Proteins We identified an esterase and an aldehyde dehydrogenase in male antennae(Table2, Supplementary Table3).Glutathione S-transferases(GSTs) are a superfamily of multifunctional enzymes involved in the cellular detoxification of both xenobiotic and endobiotic com-pounds(Salinas and Wong1999).Twenty three GSTs have been identified in silkworm genome(Yu et al.2008).We detected8GSTs in silkworm antennae(Table2).Three GSTs(GST epsilon1,epsilon4,and omega1)were found specifically in female antennae,the remaining five(GST2, delta1,delta2,sigma1,and sigma2)were found in both male and female antennae.Antioxidant proteins(peroxiredoxin, thiol peroxiredoxin,thioredoxin peroxidase,thioredoxin-like protein,Mn superoxide dismutase,and glutathione peroxi-dase)also were detected in silkworm antennae(Table2). The cytochrome P450(CYP)superfamily is a large and di-verse group of enzymes that catalyze the oxidation of lipids, steroidal hormones,and xenobiotics(Guengerich2008).We identified7CYPs that showed a mutual exclusive pattern in male(4CYPs)and female(3CYPs)antennae(Table2).We also identified immune related proteins.Phenoloxidase is a key component of the insect immune system(Gonzalez-Santoyo and Cordoba-Aguilar2012).Phenoloxidase exists in circulating hemocytes and the cuticle in silkworm(Asano and Ashida2001;Ling et al.2005).GlcAT-S encodes a gly-cosyl transferase and its expression is induced by immune challenge(Kim et al.2005).

Juvenile Hormone-Related Proteins and Heat Shock-Related Proteins Juvenile hormone binding proteins(JHBPs), juvenile hormone resistance protein II,and juvenile hormone episode hydrolase(JHEH)were detected in the proteomic analysis,as were8heat shock-related proteins(Table2). Discussion

BmorPBP1,BmorPBP2,and BmorABPx bind the pheromone component bombykal equally well,whereas BmorGOBP2 binds bombykol more than it does to bombykal(Zhou et al. 2009).The silkworm genome encodes44odorant binding protein(OBP)genes(Gong et al.2009),however only a small number of OBPs were found in our proteomic analysis.These four OBPs are highly expressed(Zhou et al.2009).We spec-ulate the remaining OBPs are not expressed or at least are expressed at low levels in the antennae.The high concentra-tions of PBP1,ABPX,GOBP1,GOBP2,CSP4,and CSP13in silkworm antennae facilitate the perception of pheromones,as well as odorants(Supplementary Table1-3).The odorant binding protein OBP49a was able to attenuate nerve firings in sugar-activated taste sensory neurons when bitter com-pounds were combined with sucrose in Drosophila(Jeong et al.2013).It is surprising that an OBPs regulates taste per-ception.Silkworm feeds mainly on mulberry leaves.It will be interesting to determine whether these OBPs play a role in taste,as well as their primary role in olfaction.

The olfactory receptor,OR45was found only in male an-tennae.Wanner et al.(2007)showed that OR45is expressed in a female-biased manner,with7×higher levels of transcript found in female compare to male antennae by quantitative real-time PCR.There is a discrepancy between the proteomic data and this OR45transcript data.One possibility is that OR45transcript levels may not correlate well with its protein levels.In addition,we cannot rule out the possibility that female moth antennae also express the OR45protein but this was not detected in our study.Tanaka et al.(2009)also

Table1Numbers of proteins identified from female and male antennae

Female specific Male specific Common Total

SEQUEST84157181422 Redundancy removed77143144364

Table2The specific proteins identified from the proteome of Bombyx mori antennae

Accession number Annotation

Odorant binding proteins,chemosensory proteins,and olfactory receptors

gi|112,984,442*pheromone binding protein[Bombyx mori]

gi|112,984,426*general odorant-binding protein1precursor[Bombyx mori]

gi|112,984,436general odorant binding protein2[Bombyx mori]

BGIBMGA002626-PA antennal binding protein[Bombyx mori]

gi|164,448,664antennal binding protein precursor[Bombyx mori]

gi|112,983,094chemosensory protein4precursor[Bombyx mori]

gi|112,984,474chemosensory protein13precursor[Bombyx mori]

gi|162,461,258*olfactory receptor45[Bombyx mori]

Oxidative enzymes and defense mechanisms

gi|114,053,31126S protease regulatory subunit6B[Bombyx mori]

gi|112,983,471antichymotrypsin-1precursor[Bombyx mori]

gi|112,983,920chitinase-like protein EN03precursor[Bombyx mori]

BGIBMGA009106-PA glutathione S-transferase2[Bombyx mori]

gi|112,984,484glutathione S-transferase delta1[Bombyx mori]

gi|112,983,444glutathione S-transferase delta2[Bombyx mori]

gi|112,983,028glutathione S-transferase sigma1[Bombyx mori]

gi|160,333,678glutathione S-transferase sigma2[Bombyx mori]

gi|112,984,522glutathione S-transferase epsilon1[Bombyx mori]

gi|169,234,678glutathione S-transferase epsilon4[Bombyx mori]

gi|114,052,210glutathione S-transferase omega1[Bombyx mori]

BGIBMGA007286-PA ommochrome-binding protein[Bombyx mori]

gi|114,052,472peptidylprolyl isomerase B precursor[Bombyx mori]

gi|114,051,191peroxiredoxin[Bombyx mori]

gi|112,983,667phenoloxidase subunit1precursor[Bombyx mori]

gi|112,983,448phenoloxidase subunit2precursor[Bombyx mori]

gi|24,641,095stress-sensitive B,isoform B[Drosophila melanogaster]

gi|112,982,996thiol peroxiredoxin[Bombyx mori]

BGIBMGA013098-PA immune-related Hdd13[Hyphantria cunea]

gi|114,052,941thioredoxin peroxidase[Bombyx mori]

gi|148,298,796thioredoxin-like protein[Bombyx mori]

BGIBMGA003073-P A translationally controlled tumor protein[Bombyx mori]

gi|112,982,683catalase[Bombyx mori]

gi|19,921,004GlcAT-S,isoform B[Drosophila melanogaster]

gi|112,983,348glutathione peroxidase[Bombyx mori]

gi|190,341,026alpha-esterase25[Bombyx mori]

gi114052408mitochondrial aldehyde dehydrogenase[Bombyx mori]

gi|160,358,387cytochrome P450CYP6AE9[Bombyx mori]

gi|168,823,415cytochrome P450CYP366A1precursor[Bombyx mori]

gi|119,226,184cytochrome P4506AB4[Bombyx mori]

gi|162,462,656cytochrome P450,family307,subfamily a,polypeptide1precursor[Bombyx mori]

BGIBMGA002272-PA cytochrome P450[Bombyx mori]

gi|160,333,255CYP6AE family cytochrome P450CYP6AE21[Bombyx mori]

gi|163,838,680cytochrome P450CYP4G25[Bombyx mori]

BGIBMGA011085-PA putative toll[Danaus plexippus]

BGIBMGA006920-P A PREDICTED:similar to leucine-rich repeats and immunoglobulin-like domains3[Tribolium castaneum] Juvenile hormone related proteins

BGIBMGA004613-PA takeout/JHBP like protein[Papilio xuthus]

gi|112,983,194juvenile hormone binding protein an-0921precursor[Bombyx mori]

analyzed OR45transcription in male and female moth anten-nae.Although they did not quantify this,they did not see female biased transcription of OR45(Tanaka et al.2009).

Pheromones are thought to diffuse through100?-wide pore tubules in the cuticular wall of sensilla to enter the recep-tor lymph(Steinbrecht1980;V ogt and Riddiford1981).Thus, the receptor lymph is also exposed to oxidative stress and xenobiotic challenge.Rapid clearing of xenobiotics,as well as residual pheromone,from the sensilla is very important to the animal.Esterases are involved in pheromone degradation in insects.An esterase from female antennae of another moth was found to bind and degrade pheromone(Supplementary Table2;V ogt and Riddiford1981),and a male-specific ester-ase can also degrade pheromone(Supplementary Table3; Ishida and Leal2005;V ogt and Riddiford1981).Since the silkworm sex pheromone comprises an alcohol(bombykol) and an aldehyde(bombykal),sex pheromone components are unlikely to be a substrate for alpha-esterase25.An alde-hyde dehydrogenase was identified,however,in male anten-nae.Rybczynski et al.(1990)reported that a130kDa alde-hyde oxidase enriched in male antennae was able to metabo-lize bombykal.GSTs catalyze xenobiotic and endobiotic com-pound detoxification by conjugation with reduced glutathi-one,GSH(Salinas and Wong1999).They also are involved in regulating cell signaling pathways(Cho et al.2001).An antennal-specific GST can modify trans-2-hexenal,a plant derived green leaf aldehyde(Rogers et al.1999).Eight GSTs were identified in our proteomic analysis,three of them were female specific.Tan et al.(2014)described BmGSTD4, which was highly expressed and specific to male silkmoth antenna.However,BmGSTD4was not identified in our re-search.This suggests a limitation of the proteomics approach. Nevertheless,both studies revealed that some GSTs are highly expressed in silkmoth antennae.Anti-microbial peptide (AMP)genes are constitutively expressed in Spodoptera frugiperda antennae and palps at high levels(Legeai et al. 2014).Immunity-related proteins also were found in our study.This suggests that immunity pathways are activated in insect antennae.What is the importance of activating immu-nity pathways in the olfactory organs?Do they play a role in the odorants perception?It will be interesting to determine whether AMPs also are activated in silkmoth antennae and which transcription factor(s)regulate their expression.GSTs, oxidative enzymes,CYPs,and immunity proteins likely work together to maintain a favorable environment in the antennal hemolymph,which enables the insects to detect pheromones and plant volatiles in a very sensitive way.

JHBPs may protect JHs from nonspecific degradation and nonspecific adsorption,and transport JHs to the appropriate cellular compartment,while Juvenile hormone resistance pro-tein II and JHEH regulate JH titer(Suzuki et al.2011;Touhara and Prestwich1993).Expression of such proteins in silkworm antenna suggest that antennae are a target organ for JH,and that JH may play a role in the silkworm olfactory system.In honeybee,JH has a profound effect on short term olfactory memory(Maleszka and Helliwell2001).JH also induces GST activity(Wu and Lu2008).

Acknowledgments This work is funded by the National Basic Re-search Program of China(2015CB755703),grants from NSFC (31172152and31402012)and CAS(KSZD-EW-Z-021-2-1).

Author contributions X.M.designed the experiment and wrote the manuscript.Y.Z.and H.L.performed experiments and analyzed the data. Compliance with Ethical Standards

Conflict Interests The authors declare no conflict interests.

Table2(continued)

Accession number Annotation

gi|169,234,671juvenile hormone resistance protein II[Bombyx mori]

gi|112,984,538juvenile hormone epoxide hydrolase precursor[Bombyx mori]

gi|112,983,082cytosolic juvenile hormone binding protein36kDa subunit[Bombyx mori]

Heat shock proteins

BGIBMGA007950-P A heat shock protein70–3[Bombyx mori]

gi|28,571,719heat shock protein cognate4,isoform E[Drosophila melanogaster]

BGIBMGA004540-P A heat shock protein hsp19.9[Bombyx mori]

gi|112,983,414heat shock protein hsp21.4[Bombyx mori]

gi|112,982,828heat shock cognate protein[Bombyx mori]

gi|112,983,55690-kDa heat shock protein[Bombyx mori]

gi|112,983,152heat shock protein20.4[Bombyx mori]

gi|148,298,772heat shock protein70B[Bombyx mori]

*Bold font entries designate proteins found in both female and male antennal extracts;*underlined entries indicate proteins found in female antennae only;*italic font indicates proteins found in male antennal extracts only

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