Differential Expression of Odorant-Binding Proteins in the Mandibular

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Differential Expression of Odorant-Binding Proteins in the Mandibular Glands of the Honey Bee According to Caste and Age Immacolata Iovinella,†,^ Francesca Romana Dani,‡,^ Alberto Niccolini,§ Simona Sagona,§ Elena Michelucci,‡ Angelo Gazzano,§ Stefano Turillazzi,|| Antonio Felicioli,§ and Paolo Pelosi*,† †

Department of Biology of Agricultural Plants, University of Pisa, Pisa, Italy CISM, University of Firenze, Firenze, Italy § Department of Physiological Sciences, University of Pisa, Pisa, Italy Department of Evolutionary Biology, University of Firenze, Firenze, Italy

)

‡

bS Supporting Information ABSTRACT: Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) mediate both perception and release of chemical stimuli in insects. The genome of the honey bee contains 21 genes encoding OBPs and 6 encoding CSPs. Using a proteomic approach, we have investigated the expression of OBPs and CSPs in the mandibular glands of adult honey bees in relation to caste and age. OBP13 is mostly expressed in young individuals and in virgin queens, while OBP21 is abundant in older bees and is prevalent in mated queens. OBP14, which had been found in larvae, is produced in hive workers’ glands. Quite unexpectedly, the mandibular glands of drones also contain OBPs, mainly OBP18 and OBP21. We have expressed three of the most represented OBPs and studied their binding properties. OBP13 binds with good specificity oleic acid and some structurally related compounds, OBP14 is better tuned to monoterpenoid structures, while OBP21 binds the main components of queen mandibular pheromone as well as farnesol, a compound used as a trail pheromone in the honey bee and other hymenopterans. The high expression of different OBPs in the mandibular glands suggests that such proteins could be involved in solubilization and release of semiochemicals. KEYWORDS: odorant-binding protein, chemosensory protein, mandibular glands, proteomics, Apis mellifera, ligand-binding, farnesol

’ INTRODUCTION Chemical communication makes use of soluble proteins, odorant-binding proteins (OBPs) in vertebrates,1,2 OBPs3 and chemo-sensory proteins (CSPs)4 in insects in the recognition of chemical stimuli, as well as in releasing the same chemical messengers.5,6 In mammals, the role of OBPs in broadcasting chemical information in the environment is well documented. Urinary proteins of rodents have been known for a long time,7,8 before the discovery of OBPs, but it was only after their structural similarity (in several cases identity) with OBPs had been revealed that their role as pheromone carriers was suggested.9,10 Other examples of pheromone-carrier proteins in mammals include salivary proteins in the male pig11,12 and in rodents,13 aphrodisin in the vaginal secretion of the hamster,14,15 and sweat proteins of horse16 and humans.17 Generally, these proteins are sex and/or age specific, are under hormonal control, and contain species specific semiochemicals as endogenous ligands.11,15,16,18 In insects, proteins assisting the release of pheromone in the environment have been studied more recently and comprise both classes of odorant-binding proteins of insects, OBPs and CSPs. r 2011 American Chemical Society

The first report describes the expression of CSPs in the pheromone gland of the lepidopteran Mamestra brassicae.19 Another CSP, named EjB, is expressed in the ejaculatory bulb of Drosophila melanogaster, the same organ producing the pheromone vaccenyl acetate.20 In Aedes aegypti, a member of the OBP family (OBP22) seems to be involved in semiochemical transfer from male to female during mating.21 Very recently, a number of genes encoding both OBPs and CSPs have been detected in the pheromone glands of the moth Heliothis virescens, although no evidence for the presence of their expression products is provided.22 Finally, using a proteomic approach, we were able to identify one OBP and several CSPs in the female pheromone glands of the silk moth Bombyx mori.23 Although these are the only experimental data available in insects, it is conceivable that the strategy of using OBPs and CSPs for delivering chemical stimuli is general and widespread among insect species, as it is in mammals. The honey bee, being a social insect, relies on a complex and rich olfactory language to regulate several aspects of the Received: January 26, 2011 Published: June 28, 2011 3439

dx.doi.org/10.1021/pr2000754 | J. Proteome Res. 2011, 10, 3439–3449

Journal of Proteome Research colony life, including colony demography, task partitioning, worker fertility, recruitment to food sources, larvaeadult interaction, as well as recognition of nestmates vs alien conspecifics.2429 Several pheromones have been identified in the honey bee and for most of them their role has been established. The queen mandibular pheromone is a complex mixture containing 9-keto2-decenoic acid, 9-hydroxy-2-decenoic acid, methyl p-hydroxybenzoate, 4-hydroxy-3-methoxyphenylethanol, and several other minor components. Its main function is to inhibit the fertility of workers,25 but other roles can be associated with some of these semiochemicals. The brood pheromone is a mixture of 10 esters of long-chain fatty acids26 produced by the larvae to stimulate the workers to take care of them. Alarm pheromones, such as 2-heptanone and amyl acetate, have also been described.27 The complex behaviors of honey bees certainly make use of a greater number of semiochemicals, such as the cuticular lipids used by guards to recognize individuals of their own family from those of other colonies,28 compounds used to recognize infected individuals,29 and other still unidentified compounds. Moreover, the different tasks assigned to workers during their lives are regulated by specific pheromones, yet to be identified. The genome of the honey bee31 contains 21 OBPs32 and 6 CSPs,33 both numbers being small enough to allow a complete study of the repertoire of soluble olfactory proteins in this species. In particular, 13 OBPs (113) are classic OBPs, containing the full set of six conserved cysteines, 7 (1521) belong to the subclass of C-minus OBPs, presenting only four cysteine residues and one (OBP14) contains five cysteines. In a previous paper,34 we have investigated the expression of these proteins in the antennae of honey bees and studied their binding characteristics. In this work we focus on the other complementary aspect of chemical communication, the release of pheromones and semiochemicals in the environment. Using the same proteomic approach, we have mapped the presence of OBPs and CSPs in the mandibular glands of different castes and age of the honey bee. Moreover, we have expressed three of the best represented OBPs and measured their ligand-binding properties.

’ MATERIALS AND METHODS Insects

Honey bees (Apis mellifera ligustica) were sampled in an experimental apiary of the Veterinary Faculty of the University of Pisa and stored at 80 C until use. In particular, 50 foragers, 50 hive-bees, 50 newly emerged bees, 50 drones, and 60 virgin queens were sampled in the experimental apiary of the Veterinary Faculty of Pisa University, while 200, 2 year old mated queens were collected from various beekeepers resulting from the usual practice of substituting 2 year old queens with new ones. Sampling Procedures

A total of 10 foragers were collected from each of 5 DadantBlatt hives after they gathered in front of the purposely closed entrance for a total amount of 50 foragers. To discriminate old foragers from guardians or scouts, only the bees with worn out wing endings were chosen. All the sampled foragers were then pooled and transported alive to the lab. Here they were stored at 80 C until use. A total of 10 hive-bees were collected from a peripheral frame of each of 5 hives for a total amount of 50 bees. Sampling from

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peripheral frames instead of frames with unsealed comb allowed us to discriminate hive-bees from nurse bees. All the sampled hive-bees were then pooled and transported alive to the lab. Here they were stored at 80 C until use. A total of 10 newly emerged bees were collected from a sealed comb frame of each of 5 hives, for a total of 50 bees as soon as they emerged. All the sampled newly emerged bees were then pooled and transported alive to the lab. Here they were stored at 80 C until use. A total of 10 drones were collected from each hive for a total of 50 drones. All the sampled drones were then pooled and transported alive to the lab. Here they were stored at 80 C until use. In total, 20 virgin queens have been reared from each of three orphanized colonies for a total output of 60 queens. The queen cells were placed inside an incubator until emergence occurred. To avoid killing each other after emergence, the queen cells were individually caged. After emergence, all the queens were individually frozen at 80 C and then pooled. The 200 mated alive queens provided by beekeepers were kept in their individual cages and frozen at 80 C. The mandibular glands were separated from the head by gently pulling the mandible with forceps until the whole gland was extracted, each gland was rapidly washed by a gentle spray of Milli-Q water. In total, 30 glands belonging to 15 bees have been used for each experiment. Reagents

All enzymes were from New England Biolabs. Oligonucleotides were custom synthesized at Eurofins MWG GmbH, Ebersberg, Germany. All other chemicals were purchased from SigmaAldrich and were of reagent grade, except selected compounds used in binding assays, that were prepared along with conventional synthetic routes. RNA Extraction and cDNA Synthesis

Total RNA was extracted from TRI Reagent (Sigma), following the manufacturer’s protocol. cDNA was prepared from total RNA by reverse transcription using 200 units of SuperScript III Reverse Transcriptase (Invitrogen) and 0.5 μg of an oligo-dT primer in a 50 μL total volume. The mixture also contained 0.5 mM of each dNTP (GE-Healthcare), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, and 0.1 mg/mL BSA in 50 mM Tris-HCl, pH 8.3. The reaction mixture was incubated at 50 C for 60 min, and the product was directly used for PCR amplification or stored at 20 C. Polymerase Chain Reaction

Aliquots of 1 μL of crude cDNA were amplified in a Bio-Rad Gene CyclerTM thermocycler, using 2.5 units of Thermus aquaticus DNA polymerase (GE-Healthcare), 1 mM of each dNTP (GE-Healthcare), 1 μM of each PCR primer, 50 mM KCl, 2.5 mM MgCl2, and 0.1 mg/mL BSA in 10 mM Tris-HCl, pH 8.3, containing 0.1% v/v Triton X-100. At the 50 end, we used specific primers corresponding to the sequence encoding the first six amino acids of the mature protein. The primers also contained an Nde I restriction site, for ligation into the expression vector and providing at the same time the ATG codon for an additional methionine in position 1. At the 30 end, specific primers were used, encoding the last six amino acids, followed by a stop codon and an EcoRI restriction site (a BamHI site in the case of AmelOBP14) for ligation into the expression vector. Therefore, 3440

dx.doi.org/10.1021/pr2000754 |J. Proteome Res. 2011, 10, 3439–3449

Journal of Proteome Research

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Figure 1. Two-dimensional gel electrophoretic separation of crude extracts from honey bee mandibular glands. Only low molecular weight regions of the gels are shown, where OBPs and CSPs are to be found. The figure reports for each sample a representative gel of three replicates. Classic OBPs are marked by triangles, C-minus OBPs by squares, and CSPs by circles. The full set of data for the identified proteins is reported in Table 2 for OBPs and CSPs and in Table S1 in the Supporting Information for other proteins.

we used the following primers for the each protein (enzyme restriction sites are underlined):

with the same enzymes. The resulting plasmids were sequenced and shown to encode the mature proteins. Preparation of the Proteins

After a first denaturation step at 95 C for 5 min, we performed 35 amplification cycles (1 min at 95 C, 30 s at 50 C, 1 min at 72 C) followed by a final step of 7 min at 72 C. In all experiments, we obtained amplification products of 300400 bp, in agreement with the expected sizes. Cloning and Sequencing

The crude PCR products were ligated into a pGEM (Promega) vector without further purification using a 1:5 (plasmidinsert) molar ratio and incubating the mixture overnight, at room temperature. After transformation of E. coli XL-1 Blue competent cells with the ligation products, positive colonies were selected by PCR using the plasmid’s primers SP6 and T7 and grown in LB/ampicillin medium. DNA was extracted using the Plasmid MiniPrep Kit (Euroclone) and custom sequenced at Eurofins MWG (Martinsried, Germany). Cloning in Expression Vectors

pGEM plasmids containing the appropriate sequences were digested with Nde I and EcoRI restriction enzymes for 2 h at 37 C, and the digestion products were separated on agarose gel. The obtained fragments were purified from gel using QIAEX II Extraction kit (Qiagen) and ligated into the expression vector pET5b (Novagen, Darmstadt, Germany), previously linearized

For expression of recombinant proteins, each pET-5b vector containing the appropriate OBP sequence was used to transform BL21(DE3)pLysS E. coli cells. Protein expression was induced by addition of IPTG to a final concentration of 0.4 mM when the culture had reached a value of OD600 = 0.8. Cells were grown for a further 2 h at 37 C, then harvested by centrifugation, and sonicated. After centrifugation, OBPs were present as inclusion bodies. To solubilize them, the pellet from 1 L of culture was dissolved in 10 mL of 8 M urea, 1 mM DTT in 50 mM Tris buffer, pH 7.4, then diluted to 100 mL with Tris buffer and dialyzed three times against Tris buffer. Purification of the proteins was accomplished by combinations of chromatographic steps anion-exchange resins, such as DE-52 (Whatman), QFF, or Mono-Q (GE-Healthcare), followed by gel filtration on Sephacryl-100 or Superose-12 (GEHealthcare) along with standard protocols previously adopted for other odorant-binding proteins.35,36 Preparation of Antisera

Antisera were obtained by injecting rabbits subcutaneously and intramuscularly with 300 μg of recombinant protein, followed by two additional injections of 150 μg after 15 and 30 days. The protein was emulsified with an equal volume of Freund’s complete adjuvant for the first injection and incomplete adjuvant for further injections. Animals were bled 10 days after the last injection, and the serum was used without further purification. Rabbits were individually housed in large cages, at constant temperature, and all operations were performed according to ethical guidelines to minimize pain and discomfort to animals. Western Blot Analysis

After electrophoretic separation under denaturing conditions (14% SDS-PAGE), duplicate gels were stained with 0.1% Coomassie blue R250 in 10% acetic acid, 20% ethanol, or electroblotted 3441



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Table 1. Relative Abundance of OBPs and CSPs in Mandibular Glands of Different Ages and Castes of Honey Bee, Compared with Previous Data on the Expression of the Same Proteins in Foragers’ Antennae and Larvae34 a mandibular glands protein

larvae

OBP1

**

OBP2

****

OBP4 OBP5

*** *

OBP11

virgin queens

newly emerged workers

hive workers

foragers

2 year old queens

drones

**

*

OBP13

**

OBP14

****

OBP15

*

****

****

** *

****

OBP17

*

*

OBP18 OBP19

*** **

**

OBP20

**

OBP21

***

CSP1

**

*

*

***

**

**

**

*

*

OBP16

CSP3 a

foragers antennae

* **

*

**

**

**

**

*

*

**

**

***

*

*

****

****

****

****

**

**

* ***

***

**

The levels of expression were visually judged from the relative intensity of the spots in the 2D gels.

on a Trans-Blot nitrocellulose membrane (Bio-Rad Lab) by the procedure of Kyhse-Andersen.37 After treatment with 2% powdered skimmed milk/0.05% Tween 20 in PBS overnight, the membrane was incubated with the crude antiserum against the protein at a dilution of 1:500 (2 h) and then with goat anti(rabbit IgG) horseradish peroxidase conjugate (dilution 1:1000; 1 h). Immunoreacting bands were detected by treatment with 4-chloro1-naphthol and hydrogen peroxide. Fluorescence Measurements

Emission fluorescence spectra were recorded on a Jasco FP750 instrument at 25 C in a right angle configuration, with a 1 cm light path quartz cuvette and 5 nm slits for both excitation and emission. The protein was dissolved in 50 mM Tris-HCl buffer, pH 7.4, while ligands were added as 1 mM methanol solutions. Fluorescence Binding Assays

To measure the affinity of the fluorescent ligand 1-NPN (Nphenyl-1-naphthylamine) to each protein, a 2 μM solution of the protein in 50 mM Tris-HCl, pH 7.4, was titrated with aliquots of 1 mM ligand in methanol to final concentrations of 216 μM. The probe was excited at 337 nm, and emission spectra were recorded between 380 and 450 nm. The affinity of other ligands was measured in competitive binding assays using 1-NPN as the fluorescent reporter at 2 μM concentration and 216 μM concentrations of each competitor. In order to save protein, we performed a preliminary screening of a number of ligands using a single concentration value for each competitor (10 mM). On the basis of these results, we have selected for each protein a subset of ligands that were tested over the entire concentration range. For determination of binding constants, the intensity values corresponding to the maximum of fluorescence emission were plotted against free ligand concentrations. Bound ligand was evaluated from the values of fluorescence intensity assuming that the protein was 100% active, with a stoichiometry of 1:1 proteinligand at saturation. The curves were linearized using

Scatchard plots. Dissociation constants of the competitors were calculated from the corresponding IC50 values (concentrations of ligands halving the initial fluorescence value of 1-NPN) using the equation: KD = [IC50]/1 + [1-NPN]/K1-NPN, [1-NPN] being the free concentration of 1-NPN and K1-NPN being the dissociation constant of the complex protein1-NPN. Two-Dimentional Electrophoresis of Proteins

Mandibles and mandibular glands were dissected from specimens by gently pulling the mandible with forceps. The sample (30 from 15 individuals) was then homogenized in 1.0 mL of 0.1% aqueous TFA by grinding in a mortar followed by sonication and centrifuged at 19 000g for 40 min at 4 C. Along with our previously described protocol,34 the obtained supernatants were concentrated to 50 μL and diluted to 250 μL with a buffer containing 7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 1% (v/v) IPG buffer (GE-Healthcare), and 60 mM DTT. The samples were loaded by rehydration for 11.5 h in IPG strips (pH 311, 7 cm). Isoelectrofocusing was performed with an Ettan IPG Phor III system (GE-Healthcare) using the following conditions: 50 V (2 h), 100 V (2 h), 500 V (2 h), 1000 V (2 h), 6000 V (1.5 h). Strips were then equilibrated for 15 min in a TrisHCl 1.5 M pH 8.8 solution containing glycerol 29.3%, urea 6 M, SDS 2% (w/v), DTT 1% and then for a further 15 min in a TrisHCl 1.5 M pH 8.8 solution, containing glycerol 29.3%, urea 6 M, SDS 2%, and iodoacetamide 2.5%. The second dimension electrophoresis was perfomed in 14% acrylamide gels using a SE 600 Ruby equipment (GE-Healthcare). Gels were stained with Brilliant Blue G-Colloidal Concentrate (Sigma). Three replicates were performed for each tissue, preparing new samples for each of the three gels. The gels relative to the second and third replicates are available as Supporting Information (Figures S2S7). Identification of Proteins from 2-D Gel Spots

Spots of interest were excised and processed as described in Dani et al.34 The peptide mixture was submitted to nano HPLCESI FT MS analysis on an Ultimate 3000 (Dionex, San Donato Milanese, 3442



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Table 2. Identification of OBPs and CSPs in the 2D Gels of Apis mellifera Mandibular Glandsa spot number and sample 2 VQ; D; F; HW 2 VQ; D; F 5 VQ; D 7 VQ; OQ,D; F; HW; NEW 8 VQ; F 9 VQ 10 VQ; OQ, F; HW; NEW 10A F; HW 10B F 11 VQ; OQ,D; F; HW; NEW 11 VQ

11 OQ; D; HW; F; NEW 13 VQ; F; HW; NEW 14 VQ 15 VQ; D; F; HW; NEW 19 VQ; OQ,D; F; HW; NEW

NCBI accessiona number NP_001035297 (DQ435334) NP_001071288 (GB17875) NP_001011583 (GB18819) NP_001011583 (GB18819) NP_001035314 (DQ435330) NP_001035317 DQ435335 NP_001035317 (DQ435335) NP_001035295 (DQ435333) NP_001011589 (AF393495) NP_001035317 (DQ435335) NP_001011583 (GB18819) NP_001035296 (DQ435338) NP_001035314 (DQ435330) NP_001035314 (DQ435330) NP_001035314 (DQ435330) NP_001035313 (DQ435331)

name

number of peptides

coverageb

P (pro) 7

consensus score

1.0 10

2

17; 5366; 103109; 112119 30.25% 4669 24.74%

3.0 104

20.1

5

4657; 96109 23.85%

1.4 106

50.21

4.7 1011

150.2

2

128; 4557; 72109 72.5% 110; 7181 18.26%

1.2 106

20.1

7

1898 68.64%

1.3 105

70.1

odorant binding protein 17 OBP17 chemosensory protein 1

5

chemosensory protein 3 antennal specific protein 3c chemosensory protein 3 antennal specific protein 3c odorant binding protein 13 OBP13 odorant binding protein 18 OBP18 odorant binding protein 18 OBP18 odorant binding protein 16 OBP16 odorant binding protein 4 OBP4 odorant binding protein 18 OBP18 chemosensory protein 3 antennal specific protein 3c odorant binding protein 21 OBP21 odorant binding protein 13 OBP13 odorant binding protein 13 OBP13 odorant binding protein 13 OBP13 odorant binding protein 14 OBP14

15

11

50.1

10

11101; 11118 83.90%

8.2 10

10

798; 102118 92.37%

1.2 1013

120.3

3

1119; 95103; 106117 25.64% 11101; 11118 83.90%

5.7 107

30.1

1.2 1010

130.2

13

4

100.2

2

4657; 96109; 23.85%

1.8 10

21

11101; 111; 118 85.59%

2.2 1015

210.0

7

125; 5397 60.87%

5.4 108

70.2

6

125; 5397 60.87%

5.6 107

60.1

9

125; 5270; 101115 51.30%

6.5 1011

90.3

7

1841; 3101 62.71%

3.1 10

14

20.0

70.3

a

Names of OBPs and CSPs and codes (in parentheses) are as in Forêt et al.32,33 b Amino acidic positions covered by considering the protein sequence after removal of the signal peptide as predicted by signalP. VQ, virgin queens; OQ, old queens; NEW, newly emerged workers; HW, hive workers; F, foragers; D, drones.

ilano, Italy) coupled to a LTQ Orbitrap mass spectrometer (Thermo Fisher, Bremen, Germany). Peptides were concentrated on a precolumn cartridge PepMap100 C18 (300 μm i.d. 5 mm, 5 μm, 100 Å, LC Packings Dionex) and then eluted on a C18 PepMap100 column (75 μm i.d. 15 cm, 5 μm, 100 Å, LC Packings Dionex) at 300 nL/min. The mobile phase compositions were H2O 0.1% formic acid/CH3CN 97/3 (phase A) and CH3CN 0.1% formic acid/H2O 97/3 (phase B). The gradient program was 0 min, 4% B; 10 min, 40% B; 30 min, 65% B; 35 min, 65% B; 36 min, 90% B; 40 min, 90% B; 41 min, 4% B; 60 min, 4% B. Mass spectra were acquired in positive ion mode, setting the spray voltage at 1.9 kV, the capillary voltage and temperature, respectively, at 40 V and 200 C, and the tube lens at 130 V. Data were acquired in data-dependent mode with dynamic exclusion enabled (repeat count 2); survey MS scans were recorded in the Orbitrap analyzer in the mass range 3002000 Th at a 15 000 nominal resolution, then up to the three most intense ions in each full MS scan were fragmented and analyzed in the Orbitrap analyzer at a 7 500 nominal resolution. Monocharged ions did not trigger MS/MS experiments. The acquired data were searched with Bioworks 3.2 (Thermo Fisher) using Sequest as the search algorithm against a database created by merging the sequences of the peptides predicted from Amel_pre_release2_OGS_pep.fa (downloaded from BeeBase; http://www.beebase.org) together with the entries reporting the “A. mellifera” “OBP” and “CPS” in the NCBI database. Searches and acceptance of the proteins identified followed the same criteria as

described.33 Each identified protein present in the gel at a position compatible with its calculated MW was submitted to a BLAST search in NCBI. MALDI Mass Spectrometry on Tissue Extracts

Proteins extracts obtained from mandibular glands were analyzed on a MALDI-TOF/TOF mass spectrometer Ultraflex III (Bruker Daltonics, Bremen, Germany) by using Flex Control 3.0 as data acquisition software. A 1 μL volume of the sample was mixed to 1 μL of the matrix (sinapinic acid 10 mg/mL in CH3CNH2O, 0.1% TFA, 70:40) on the target and allowed to dry. Spectra were acquired in linear mode over the m/z range 5 00020 000 for a total of 500 shots. The instrumental parameters were chosen by setting ion source 1 at 25 kV, ion source 2 at 23.45 kV, lens at 6.0 kV, and pulsed ion extraction at 80 ns. The instrument was externally calibrated prior to analysis using the Bruker Protein I calibrant standard kit (5 00017 000 Da).38 Molecular Modeling

Three-dimensional models of OBP13 and OBP21 were generated using the online program Swiss Model.3941 For OBP13, we used the structure of OBP1 of A. mellifera,42,43 accession no. 3d78A, as a template (identity between the two proteins, 22%), for OBP21 the template was the structure of Bombyx mori GOBP2,44 accession no. 2wcjA (identity between the two proteins, 20%). 3443



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Figure 2. Expression and purification of three selected OBPs. The proteins were expressed in a bacterial system and purified by combinations of ion-exchange chromatography and gel filtration. The figure reports for each protein the SDS-PAGE analysis relative to crude bacterial extracts before (Un) and after (In) induction with IPTG, the crude solubilized protein (C), and three fractions relative to the last step of purification. All three OBPs were synthesized as inclusion bodies and had to be denatured and renatured before purification. Molecular weight markers (M) are bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), trypsin inhibitor (20 kDa), and R-lactalbumin (14 kDa).

Models were displayed using the SwissPdb Viewer program “Deep-View”39 (http://www.expasy.org/spdbv/).

’ RESULTS AND DISCUSSION Proteomic Analysis

Using a classical proteomic approach, we separated mandibular extracts from different castes and ages of A. mellifera by twodimensional electrophoresis and analyzed spots migrating with apparent molecular weight lower than 20 kDa. The 2D gels are reported in Figure 1, where classic OBPs are marked by triangles, C-minus OBPs by squares, and CSPs by circles. The assignments of the spot are listed in Table 2. Although our focus was on OBPs and CSPs, other proteins were identified during this work and are listed in Table S1 in the Supporting Information but are however outside the scope of the present paper. We cannot exclude that other proteins, present in our samples, were not identified because their concentration was below the sensitivity of our method. However, we were interested in abundant proteins that could be of physiological significance in complexing the abundant semiochemicals produced in the glands. We can also exclude the possibility that other OBPs and CSPs could be present in the glands but were not efficiently extracted; on the basis of that, these proteins are known to be extremely soluble in water. For a better visualization of the results obtained in the different samples and a comparison with our previously published data,34 Table 1 summarizes the expression of OBPs and CSPs in a semiquantitative way. Along with this approach, we could identify seven OBPs and two CSPs, variously expressed in different castes and ages. In particular, OBP13 (spot no. 15) is most abundantly expressed in newly emerged workers and virgin queens. This protein is completely absent in mated queens, while low expression could be detected in foragers and only traces were found in hive workers and drones. It seems therefore that the expression of this protein is concentrated in the very first period of imaginal life of female honey bees. This OBP was also found in larvae.34 OBP21 (spot 11A), on the contrary, that was not found in the larvae appears to be characteristic of older bees, mated queens, foragers, and drones. OBP18 was also present in high concentration in the mandibular glands of drones but also, at a lower level, in all the samples examined. Among the four most

Figure 3. Expression of OBP13 in the mandibular glands of the honey bee evaluated by Western blot analysis. Proteins of crude extracts were separated by SDS-PAGE and blotted on nitrocellulose membrane using a semidry system. A polyclonal antiserum prepared against the recombinant protein was used, as reported in the Materials and Methods. The top panel reports the Coomassie stained gel and the lower panel the relative Western blot membrane. P, recombinant OBP13; dr, drones; vq, virgin queens; mq, mated queens; ne, newly emerged workers; hb, hive bees; fo, foragers. Molecular weight markers (M) are as in Figure 3. The expression of OBP13 was highest in virgin queens, moderate in newly emerged workers, low in hive workers, and completely absent in drones, mated queens, and foragers, in agreement with the data of the 2D gels.

abundant OBPs there is also OBP14 (spot no. 19), present with an intense spot in the glands of hive workers but also detected in all the other samples. Among the CSPs, we found only two, the same that were also reported in the antennae of foragers,34 CSP3 (spot no. 7) present at medium to high levels in all the samples, and CSP1 expressed in much lower concentrations. Bacterial Expression

On the basis of these findings, we expressed the three OBPs most highly and widely present in the mandibular glands of queens and workers, numbers 13, 14, and 21, to perform binding experiments and verify the hypothesis that these proteins could act as pheromone carriers. In the present study, we did not include OBP18 that was mainly expressed in drones, as there is little information on male pheromones in the honey bee. However, the abundant presence of OBPs in male maxillary glands poses interesting questions on the role of such glands in drones and their putative ligands. In order to minimize possible consequences of additional regions (such as His-tag) on the structural and functional characteristics of the proteins, our construct contained the DNA fragment encoding the amino acid sequence of the mature protein, with the only addition of an initial methionine. The bacterial expression of the three proteins followed well established protocols34 and was successful in all three cases with yields around 20 mg/L of culture. The purification also adopted common separation procedures (ion-exchange and gel filtration) and yielded the purified proteins after three chromatographic steps. Figure 2 reports the electrophoretic analysis of crude

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Figure 4. Binding of selected ligands to the three recombinant OBPs of A. mellifera. Top left panel: Binding curves of 1-NPN and Scatchard plots (inset). A 2 μM solution of the protein in Tris was titrated with a 1 mM solution of 1-NPN in methanol to final concentrations of 216 μM. Dissociation constants (average of three replicates) were OBP13, 6.0 μM (SD 1.7); OBP14, 92 nM (SD 6.2); OBP21, 1.9 μM (SD 0.07). The other panels report binding of the best ligands for each protein measured in competition assays. In each experiment, a mixture of the protein and 1-NPN in Tris, both at a concentration of 2 μM, was titrated with the competing ligand to final concentrations of 216 μM. Fluorescence intensities are reported as the percent of the values in the absence of a competitor. The calculated dissociation constants for all the ligands used are listed in Table 3.

bacterial pellet before and after induction as well as of samples of the purified proteins. Cysteine Pairing in OBP14

The amino acid sequences of the three expressed OBPs are aligned in Figure 5. Each of the three OBPs contains a different number of cysteines. OBP13 belongs to the classic group with all six conserved cysteines, while OBP14 presents only five cysteines and OBP21 only four. In classic OBPs, the pairing of the six cysteines is well conserved (13, 25, 46) and has been verified in several cases. The C-minus OBPs, to which OBP21 belongs, lack cysteines 2 and 5; therefore, it is reasonable to assume that the disulfide bridge between these two residues is missing. OBP14, instead, presents five cysteines. Cys no. 5 is missing as in C-minus OBPs, but Cys no. 2 seems to be misplaced by two positions and is separated from Cys no. 3 by a single residue. The recombinant OBP14 was analyzed through MALDI-TOF MS in order to verify its molecular weight and to investigate the cysteines oxidation state. The spectrum of the purified protein contains two peaks at MH+ 13 534.8 and 13 403.0, the first in good agreement with the mass of the expected protein (MH+

calculated by considering four oxidized cysteines 13 532.6), the second with the mass of the protein without the initial methionine (MH+calculated by considering four oxidized cysteines 13 401.4). The identity of the second species was confirmed through topdown MALDI ion source decay (ISD) experiments, which allows one to verify the amino acid sequence of intact proteins. 1,5-Diaminonaphthalene was used as the matrix.45 Most of the ions present in the ISD spectrum belong to the C series deriving from two species differing for the presence or the absence of the N-terminal methionine (Figure S1 in the Supporting Information). The recombinant proteins were then reacted with iodoacetamide in order to determine the number of free cysteines. The spectrum contains two peaks (m/z 13 589.87 and 13 456.57) corresponding to the two molecular species (with and without the initial methionine) derivatized with a single residue of acetamide (calculated masses 13 589.6 and 13 458.4 Da, respectively). Therefore, OBP14 contains a single free cysteine. The IAM reacted sample was then digested with trypsin and analyzed through MALDI MS. The spectrum of this sample presents, among others, two peaks at m/z 2310.00 and 3445



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Table 3. Values of IC50 (μM) and Dissociation Constants (KD, μM) of Complexes of A. mellifera Recombinant OBPs with Various Ligands, Measured in Competitive LigandBinding Assays a OBP13 ligand

OBP14

OBP21

IC50 KD IC50 KD IC50 KD

dodecanoic acid

20

16

tetradecanoic acid

3.5

2.8

hexadecanoic acid

6.5

5.2

oleic acid

1

0.8

linoleic acid

2.5

2

retinol

6.2

4.96

stearic acid methyl tetradecanoate

>50 / 2.5 2

methyl hexadecanoate

3.3

2.64

methyl octadecanoate

4.2

3.36

dodecanal

4.5

3.6

octadecanal

16

12.8

oleamide

>50 /

linalool

>50 /

>50 /

farnesol 3,7-dimethyl-1-octanol

>50 / 20 16

10 12

0.58 0.5 0.69

geraniol

>50 /

20

1.16 100 /

citronellal

>50 /

18.5 1.07 7.5

9-oxo-dec-2-enoic acid

Ligand-Binding Experiments

>50 /

0.28

4.19

10.5 5.86

methyl-p-hydroxybenzoate

10

0.58 10

5.59

citralva

2.5

0.14 5

2.79

retinal

>50 /

2-heptanone eugenol

3.8 0.5

4-hydroxy-3-methoxycinnamaldehyde

0.22 >50 / 0.03 >50 /

>50 /

>50 /

>50 /

4-hydroxy-3-methoxycinnamyl alcohol >50 /

>50 /

>50 /

homovanillic acid

>50 /

>50 /

isoamyl acetate

7

0.4

>50 /

geranyl acetate

5

0.3

7

R-methyl-cinnamaldehyde

1

0.06 >50 /

R-metoxy-cinnamaldehyde citral

10 8

0.58 >50 / 0.46 >50 /

1-hexadecanol

>50 /

parts of the body, we used polyclonal antibodies prepared against the recombinant protein in Western blot analysis. The results are reported in Figure 3. In the first experiment, we compared extracts from the mandibular glands of different castes and ages, the same utilized for 2D-gels. The expression of OBP13 was highest in virgin queens, moderate in newly emerged workers, low in hive workers, and completely absent in drones, mated queens, and foragers, in total agreement with our proteomic results. In a second experiment, we investigated the expression of the same protein in different parts of the body of newly emerged workers and virgin queens. In both cases, the protein seems to be ubiquitous, supporting the idea that the expression of OBP13 is related to age rather than organs or caste.

1

3.9

0.56

Mixtures of protein and 1-NPN, both at a concentration of 2 μM, were titrated with 1 mM solutions of each ligand to final concentrations of 216 μM. Dissociation constants were calculated from the values of ligands halving the 1-NPN fluorescence (IC50), as described in the Materials and Methods. a

2673.21); the first peak indicates the presence of peptide 1018 linked through a disulfide bridge to peptide 4352 which bears an alkylated cystein, while the second is relative to peptides 8399 joined to peptide 103109. This pairing corresponds to what was expected, assuming that the conserved cysteines could be paired as in classic OBPs but the data did not allow us to determine whether Cys46 or Cys48 is paired with Cys16 (the numbers refer to the mature protein without the initial methionine, Figure 5). Expression of OBP13

To validate the results obtained through our classical proteomic study and to investigate the expression of OBP13 in different

As the mandibular glands are the site of production of several known pheromones in the honey bee, we decided to investigate the affinity of our recombinant OBPs to pheromones and other potential ligands. First we measured the affinity of the proteins to the fluorescent probe N-phenyl-1-naphthylamine (1-NPN), a ligand that proved efficient for most OBPs and CSPs of insects. The emission spectrum of this compound, upon binding the protein, undergoes a major change, with a shift of its maximum from 480 nm to about 405410 nm and a strong increase in intensity, thus allowing reliable measurements of the bound ligand in the presence of free ligand.1-NPN and other fluorescent probes occupy the single binding pocket present in OBP following a saturable binding isotherm. Moreover, the presence of the fluorescent ligand 1-anilino-8-naphthalene sulfonate, a compound structurally similar to 1-NPN, in the binding pocket of the cockorach PBP has been visualized in the crystal structure of the complex.46 The binding curves of the three recombinant OBPs are reported in the top left panels of Figure 4. OBP13 and OBP21 exhibited moderate affinity to 1-NPN with dissociation constants of 6.0 μM (SD 1.7, three replicates) and 1.9 μM (SD 0.07), respectively, well in the range of affinities measured with most insect OBPs.6 The same fluorescent compound, however, proved to be a much stronger ligand for OBP14 with a dissociation constant of 92 nM (SD 6.2). With this protein we also observed that the binding curve reaches saturation when only half of the sites are occupied. This could indicate the presence of an endogenous ligand or a cooperative effect between the two subunits of the protein homodimer. Next we measured the affinity of several pure chemicals, including some pheromones of the honey bee, to the three recombinant OBPs. In these experiments, we titrated a mixture of protein and 1-NPN, both at a concentration of 2 μM, with increasing amounts of each potential ligand, measuring the percent of fluorescence displaced by the ligand. The three other panels of Figure 4 show typical results of these experiments, limited for each protein to its best ligands. The full set of data with the calculated dissociation constants is reported in Table 3. The three proteins show different spectra of binding. OBP13 seems to be tuned to oleic acid and other long chain molecules. The model of OBP13 (Figure 5), based on the three-dimensional structure of the honey bee OBP1,42,43 accession no. 3d78A, shows a single polar residue (S98) in the binding pocket and several hydrophobic groups, including an aromatic residue (F55). The molecule of oleic acid could fit into such cavity, establishing a hydrogen bond between the 3446



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Figure 5. Sequence alignment of the three OBPs expressed in bacteria and three-dimensional models of OBP13 and OBP21. The model of OBP13 is based on the structure of the A. mellifera OBP1,42,43 accession no. 3d78a, and shows a serine (S98) as the only hydrophilic residue in its binding pocket and an aromatic residue (F55) besides a number of branched amino acids. The model is compatible with the strong affinity found for this protein toward oleic acid. The model of OBP21 is based on the structure of GOBP2 of Bombyx mori,44 accession no. 2wcjA and presents two serines at the entrance of the binding pocket (S99 and S100) and several branched residues that could establish hydrophobic interactions with the branched backbone of farnesol, the best ligand for this protein, while its terminal hydroxyl group could sit between the two serines exposed to the outside. Models have been generated using the program Swiss Model.3941 Amino- and carboxy termini are indicated as Nt and Ct, respectively.

carboxylic group and S98 and a ππ interaction between its double bond and the benzene ring of F55. Oleic acid has been reported as one of the major components of mandibular secretion in young queens, its concentration decreasing with age.47 As OBP13 is also mostly expressed in young bees, it is tempting to speculate that the function of this protein could be related to solubilization and release of oleic acid. On the other hand, OBP21, which is expressed in old bees, binds with moderate affinity components of the queen mandibular pheromone, such as 9-oxo-2-decenoic acid and methyl p-hydroxybenzoate. This same protein also shows exceptionally high affinity to farnesol, a compound secreted by the Nasonov gland and used in the honey bee as a trail pheromone48 but whose presence in the mandibular glands has not been reported. The model of OBP21 (Figure 5), based on the structure of GOBP2 of Bombyx mori,44 accession no. 2wcjA, shows two serine residues at the entrance of the binding cavity (S99 and S100) and a number of branched chain amino acids, lining the hydrophobic pocket, with total absence of other types of residues. This arrangement matches the highly branched structure of farnesol, whose terminal hydroxyl group could establish hydrogen bonds with the two mentioned serines. OBP14, whose structure could not be reliably modeled on the basis of the present information, binds several terpenoid compounds, such as geranyl acetate and citralva, but also with lower affinity other molecules, such as isoamyl acetate and 2-heptanone used by the honey bee as alarm pheromones. The best ligand for this protein is however eugenol. The close structural similarity of this molecule with

4-hydroxy-3-methoxycinnamyl alcohol, another component of queen mandibular pheromone, prompted us to test such compounds as well as the corresponding aldehyde and homovanillic acid in binding experiments with the three proteins. Despite the apparent similarity of these structures with that of eugenol, none of the three compounds showed good affinity for any of the three OBPs. Apparently, the additional polar group present in these molecules as compared to eugenol plays a strongly negative role in the interaction with the hydrophobic pocket of the proteins. OBP14 was mainly found to be expressed in the mandibular glands of hive bees, besides being abundant in the larvae. Any attempt to correlate expression of OBPs and presence of pheromones in the glands would be too speculative and not reliable and also considering that probably other volatile components of mandibular glands might be still undiscovered, while the picture of OBPs expression during the life cycle of honey bees is far from being complete.

’ CONCLUSIONS In this work we have shown that several OBPs are produced in the mandibular glands of honey bee and that their expression is regulated with castes and age. This suggests that OBPs in pheromone glands might be involved in solubilizing hydrophobic semiochemicals and assisting their release in the environment or transfer them to the appropriate receiver. In particular, OBP13 and OBP21 seem to be complementary in relation to age, as the first is mainly expressed in virgin queens and newly emerged workers, 3447


Journal of Proteome Research while the second is synthesized in old individuals of the three castes. Particularly intriguing is the case of OBP14, which is most abundant in hive workers and present only at lower levels in younger as well as in older bees. This same protein was absent in the antennae of foragers but was found to be the most abundant in larvae. The small number of OBPs expressed in the mandibular glands, as compared to the larger number of semiochemicals secreted by the same glands, suggests that a combinatorial code might control the composition of the released blend of semiochemical as much as it occurs in the perception of the same olfactory messages. Under such a hypothesis, the relative concentration of each component in the final bouquet that is released will depend upon (i) the level of synthesis of the semiochemical, (ii) its volatility, (iii) its affinity to the different OBPs present in the organ, and (iv) the level of expression of the OBPs capable of binding that particular chemical. Therefore, regulation of the pheromonal message could be performed by regulating both the synthesis of the semiochemicals and the expression of their binding proteins. Finally, we would like to observe that the chemical communication system in the honey bee is extremely complex and far from having been fully understood. A recent paper49 reports that queens can control the fertility of workers even when deprived of their mandibular glands and in the total absence of its main component (E)-9-oxo-2-decenoic acid, considered so far to be the main chemical responsible for such effect. It is evident, therefore, that other semiochemicals are still waiting to be discovered and that the chemical language of honey bees could be even richer and more sophisticated.

’ ASSOCIATED CONTENT

bS

Supporting Information Table S1: Identification of proteins other than OBPs and CSPs (Figure 1). Table S2: amino acid sequences and accession numbers of the odorant-binding proteins and chemosensory proteins identified in the mandibular glands of the honey bee Apis mellifera. Figure S1: MALDI ion source decay (ISD) MS spectrum of recombinant OBP14. This topdown approach allowed one to confirm that the two proteins present in the samples (giving signals at MH+ 13 534.8 and 13 403.0) correspond, respectively, to the expected protein and to the same sequence lacking the N-terminal methionine. Figure S2: 2D gel replicates of extracts from mandibular glands of virgin queens. Figure S3: 2D gel replicates of extracts from mandibular glands of 2 year-old queens. Figure S4: 2D gel replicates of extracts from mandibular glands of drones. Figure S5: 2D gel replicates of extracts from mandibular glands of foragers. Figure S6: 2D gel replicates of extracts from mandibular glands of hive workers. Figure S7: 2D gel replicates of extracts from mandibular glands of newly emerged bees. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Address: Paolo Pelosi, Dipartimento di Biologia delle Piante Agrarie, University of Pisa, via S. Michele, 4, 56124 Pisa, Italy. Phone: +39 050 2216626. Fax: +39 050 2216630. E-mail: [email protected]. Author Contributions ^

These authors have equally contributed to the work.

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Differential Expression of Odorant-Binding Proteins in the Mandibular

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