Mining Novel Allergens from Coconut Pollen Employing Manual De

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Mining novel allergens from Coconut Pollen employing manual denovo sequencing and homology driven proteomics. Bodhisattwa Saha, Gaurab Sircar, Naren Pandey, and Swati Gupta Bhattacharya J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00657 • Publication Date (Web): 01 Oct 2015 Downloaded from http://pubs.acs.org on October 6, 2015

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Journal of Proteome Research

Mining novel allergens from Coconut Pollen employing manual denovo sequencing and homology driven proteomics. Bodhisattwa Saha1, Gaurab Sircar1, Naren Pandey2 and Swati Gupta Bhattacharya1* 1

Division of Plant Biology, Bose Institute, 93/1 Acharya Prafulla Chandra Road, Kolkata 700009, West Bengal, India

2

Department of Allergy and Asthma, Belle View Clinic, 9, Dr U.N. Brahmachari Street, Kolkata 700001, West Bengal, India

*

Corresponding Author: Swati Gupta Bhattacharya, Division of Plant Biology, Bose Institute, 93/1 Acharya Prafulla Chandra Road, Kolkata 700009, West Bengal, India. Tel No: +91 33 2303-1131/1129; Fax: +91 33 2350-6790; Email: [email protected]

Running title: Coconut pollen allergens

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ABSTRACT Coconut pollen, one of the major palm pollen grains is an important constituent amongst vectors of inhalant allergens in India and a major sensitizer for respiratory allergy in susceptible patients. To gain insight into its allergenic components, pollen proteins were analysed by two-dimensional electrophoresis, immunoblotted with coconut pollen sensitive patient sera followed by mass spectrometry of IgE reactive proteins. Coconut being largely unsequenced, a proteomic workflow has been devised that combines the conventional database dependent analysis of tandem mass spectral data and manual de novo sequencing followed by a homology-based search for identifying the allergenic proteins. N-terminal acetylation helped to distinguish ‘b’ ions from others, facilitating reliable sequencing. This led to the identification of 12 allergenic proteins. Cluster analysis with individual patient sera recognised vicilin-like protein as a major allergen which was purified to assess its in-vitro allergenicity and partially sequenced. Other IgE-sensitive spots showed significant homology with well known allergenic proteins such as 11S globulin, enolase, isoflavone reductase along with a few which are reported as novel allergens. The allergens identified can be used as potential candidates for hypoallergenic vaccine development, design specific immunotherapy trials and enrich the repertoire of existing IgE reactive proteins. Keywords: Pollen allergens, de novo sequencing, MS BLAST, Vicilin, MALDI, 2D gel electrophoresis


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INTRODUCTION Allergic diseases pose a burden to both patients and society as a whole affecting more than 25% of the world population, especially in industrialized countries1. It is associated with high levels of IgE antibodies to certain allergenic proteins that direct basophils and mast cells to release elevated levels of eosinophils, histamine, leukotrienes leading to elicitation of atopic syndromes2. Inhalant aeroallergens, mainly harboured by vectors such as pollen grains are responsible for rhinitis, asthma and other respiratory allergies in susceptible individuals3. Coconut is one of the most economically important palm trees widely cultivated mainly for its endosperm which is used as food and oil all over the world. India is also one of the prime producers of coconut. Amongst many, Cocos nucifera (Coconut) pollen grains have been documented as an important aeroallergen in India4. Approximately 47% of the atopic population in Kolkata, India suffering from asthma and rhinitis was found to be immunoreactive to this pollen 5. Most of the research on coconut pollen allergy is limited to its clinical manifestations and basic immunochemical studies. Protein purification through chromatographic techniques of coconut pollen extract showed a 158kD and 2.9 kD IgE reactive regions through immunoblot analysis6.Coconut pollen also demonstrated crossreactivity amongst three different palm pollen grains predicting presence of common antigenic epitopes7.Though there are reports on coconut as a food allergen sharing epitopes with other nut allergens8, evidence of pollen related allergies was rarely reported in Western countries. However, its prevalence in the Indian subcontinent gears up its importance to be investigated thoroughly. Proteomic investigations are being increasingly used in recent times to rapidly identify IgE sensitive proteins from various allergenic pollen grains9. Yet, a major drawback in mass spectrometry-based proteomics is that it depends heavily on the presence of complete protein databases and hence determining correct proteins from unsequenced species is challenging. 3 ACS Paragon Plus Environment


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De novo sequencing may overcome such problems by sequencing the mass spectral data without using any prior database knowledge and have been used efficiently in identification of several allergens10,11. To understand better and obtain more definitive information about allergens from coconut pollen we undertook an immunoproteomics approach: 1 and 2D gel electrophoresis of total pollen proteome, western blotting with patients’ sensitive sera, determination of IgE sensitive proteins, identification of the allergens by gel-based mass spectrometry. A proteomic workflow has been described which led to a homology-based identification of 12 IgE reactive proteins. In addition to a normal database search, spectral data were manually sequenced using preset rules followed by a similarity search. Further individual 2D immunoblot maps described personalised IgE sensitivity profiles with the identified allergens which were correlated with their specific IgE and histamine release. EXPERIMENTAL SECTION Isolation of pollen and preparation of antigenic extract Fresh pollen was harvested by passing the mature buds from inflorescence of coconut flowers through descending sized pores until pure pollen was obtained and checked under the microscope. The pollen grains were defatted using diethyl ether and incubated in 0.1 M phosphate buffer pH 7.2 in 1:15 ratio (gram/ml) overnight at 4⁰C with constant slow stirring. The slurry was thereafter centrifuged at 20000xg for 20 minutes. The supernatant was recentrifuged to remove any debris. The extract was passed through a 0.22µm filter and stored at -20 ⁰C for use as an antigenic extract as well as skin prick testing (SPT). Clinical study and patient selection The antigenic extract was diluted in phosphate buffer 1:50 (w/v) and applied for SPT by pricking on the skin with a sterile lancet. The wheal reaction was monitored after 20 minutes 4 ACS Paragon Plus Environment

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and graded from +1 to +3 as described earlier12. Histamine diphosphate (1mg/ml) and phosphate buffered saline (0.01M, pH 7.2) was used as positive and negative controls respectively. Patients visiting Mediland Diagnostics, Kolkata, who showed +2/+3 cutaneous response to coconut pollen extract with complaints of serious allergic rhinitis and bronchial asthma were initially screened. Finally ten patients amongst them with total IgE more than 100International Units, histamine release more than 100nM, high specific IgE and substantial history of coconut pollen allergy agreed to provide blood on a written consent for further immunological assays (Table S1, Supplementary Information). Serum samples from whole blood were collected after centrifugation and stored at -80⁰C until use. Smokers and patients suffering from immunodeficiency disorders and undergoing immunotherapy trials were excluded from the study. Sera from two non-atopic patients were taken as controls. The entire study was approved by the ethical committee of the concerned institute. Specific IgE ELISA: 100ng/µl of antigenic extract or purified allergen (50µl) was coated in the wells of ELISA plates (Maxisorp, Nunc, USA) and incubated overnight at 4⁰C. Wells were then washed with 100µl of 0.1 M Phosphate Buffer Saline (pH 7.2) containing 0.05% Tween 20 (PBST) followed by blocking in 50µl of 1% Bovine serum albumin (BSA) in PBST for 3hrs at 4⁰C. Wells were incubated with 50µl of patient sera diluted at 1:10 overnight in blocking solution (PBST-BSA) at 37⁰C. Bound antibodies were detected by incubation in anti-human IgE alkaline phosphatase tagged produced in mouse (Sigma) diluted at 1:1000 in blocking solution for 3h at 37 ⁰C followed by adding 50 µl of para-nitrophenyl phosphate substrate solution (Sigma) and kept at 37⁰C in dark for 30 minutes. The reaction was stopped by 3N



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NaOH and absorbance measured at 405nm in ELISA reader (Multiskan, Labsystems). Three replicates were used in each case, and the mean values are represented. Protein extraction for proteome analysis: 100mg pure pollen was crushed into fine powder in liquid nitrogen and mixed with 4ml chilled acetone containing 10% trichloroacetic acid (w/v), 1% Dithiothreitol (DTT) and 10µl/ml protease inhibitor cocktail(Sigma), vortexed for 30 minutes and incubated in -20 ⁰C overnight. After centrifuging for 20 minutes at 20000xg the pellet was washed thrice with chilled acetone containing 1% DTT, air dried, dissolved in rehydration solution (7M urea, 2M thiourea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)) and incubated overnight at -20⁰C. Final protein was extracted by centrifuging at 20000xg for 20 minutes at 4⁰C and concentrations were estimated by Bradford reagent method13. 1D, 2D Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Antigenic extract (100µg/well) was profiled in 12% SDS-PAGE for 1D electrophoresis (1DE) under reducing conditions. For 2D electrophoresis (2DE) 100µg (3-10 pI) and 150µg (4-7 pI) of protein in 125µl rehydration solution along with 40mM DTT (Himedia, India), 0.75% (v/v) IPG buffer (GE Lifesciences) and 0.002% (w/v) bromophenol blue was used for passive rehydration loading in 7cm Immobiline DryStrips (GE Lifesciences) on a re-swelling tray overnight at room temperature. Isoelectric focusing was done in Ettan IPG Phor system (GE Lifesciences) at 50µA/strip current up to a total of 8KVh. Thereafter the strips were equilibrated in equilibration buffer 1 (6M urea, 75 mM Tris-HCl, pH 8.8, 29.3 % (v/v) glycerol, 2% SDS and 1% (w/v) DTT) and Equilibration buffer 2 (same as equilibration buffer 1, replacing DTT with 2.5% (w/v) iodoacetamide) each for 15 minutes. For 2nd dimension strips were laid on 12% SDS-PAGE gels (100X105X1.5mm) run in MiniVE vertical gel apparatus (GE Lifesciences) at 40mA/gel using Lamelli buffer14 and stained in 6 ACS Paragon Plus Environment

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Coomassie brilliant blue R 250. 2D gels were performed in triplicates for increasing reproducibility and analysed in Imagemaster 2D Platinum (GE Lifesciences) for molecular weight and pI. 1D, 2D western blotting and image analysis: Total protein from 1DE and 2DE gels were electrotransferred to High Bond LFT Polyvinylidene Difluoride (PVDF) membrane (GE Lifesciences) by semi-dry transfer (GE Lifesciences) through a constant current of 1.2 mA/cm2 for 1 hour and blocked in Tris Buffered saline+0.05% tween 20 (TBST) containing 3% BSA for 3hrs at 4⁰C followed by washing in TBST for 15 minutes. Primary antibody incubation was done using patient specific sera diluted1:10 in blocking solution at 4⁰C. After washing with TBST thrice, the membrane was incubated in monoclonal anti-human IgE alkaline phosphatase tagged produced in mouse (Sigma) at 1:1000 dilution as secondary antibody in blocking solution for 3 hours at room temperature. IgE reactive bands/spots were observed in nitro-blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) in dark for 30 minutes, and the reaction stopped by 0.5M Ethylenediaminetetraacetic acid. Efficient transfer of bands/spots in PVDF membrane was ensured by Ponceau S staining. Gels for 2D blotting and staining were run in parallel under exactly same protein loads and experimental conditions to assume spots in blot exists in the same coordinates of the stained gel. Localization of spots was done by creating match sets between blot and gel followed by filtering artefacts in Imagemaster2D Platinum. Further confirmation was done by visually comparing stained gel, Ponceau S stained blot and immunoblot. 2D immunoblots from pooled sera were performed thrice to reduce false positive identifications. Another similar membrane was blotted with pooled sera from 2 non-atopic patients for control experiment.



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Glycoprotein staining and deglycosylation study: Glycoprotein staining was done with 2D gel using Glycoprotein staining kit (G Biosciences, USA) following manufacturer’s instructions. Deglycosylation was done according to protocol described previously15. Briefly, after blotting, the membrane was incubated in 10mM sodium acetate buffer (pH 5.0) containing 50 mM sodium metaperiodate for 3 hrs in the dark. Sorbitol at a concentration of 0.015mol/l along with a drop of ethylene was added to inactivate periodate. The membrane was then incubated with sodium borohydride (1 mg/ml) for 12 hrs at 4 °C ,acidified with 0.001% acetic acid followed by repeated washing in TBST and then blocked with 3% BSA. Detection of IgE reactive spots was done with pooled patient sera diluted 1:10 in blocking solution according to protocol mentioned earlier. In-gel digestion and MALDI-TOF MS/MS analysis Sample preparation for mass spectrometry was done according to the protocol by Shevchenko et al16. Single spots were excised from the coomassie stained gel using sterile scalpels and destained with 1:1 ratio of 50mM ammonium bicarbonate: ethanol and dehydrated by acetonitrile. Gel pieces were subsequently reduced in 10mM DTT and alkylated with 55mM iodoacetamide followed by alternate hydration and dehydration using 50mM ammonium bicarbonate and 100% acetonitrile. In-gel digestion was performed by adding 12.5 ng/µl of trypsin (Promega, Madison, USA) in 50mM ammonium bicarbonate and incubated overnight at 37⁰C. After trypsin removal, peptides were extracted using 3% Trifluoroacetic acid (TFA) and 30% acetonitrile by vigorous vortexing for 20 minutes. The extracted peptides were vacuum dried in a speed vac (Savant, Thermo Scientific, USA) and reconstituted in 5µl of 0.1% TFA and acetonitrile (1:1v/v). 2.5µl of peptide was mixed with equal volumes of 0.5mg/ml of α cyano 4 hydroxycinnamic acid matrix (Bruker Daltonics, Bremen, Germany) and 2.5µl of the mixture was spotted on an MTP 384 ground steel target plate (Bruker Daltonics) in duplicates. External calibration 8 ACS Paragon Plus Environment

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was done using peptide calibration standard II mixture (Bruker Daltonics). Acquisition of spectra was done using Autoflex II MALDI-TOF/TOF machine in linear mode equipped with a pulsed nitrogen laser (λ=337 nm, 50 Hz) at 54% power in the positive ion mode. Highintensity peaks having a good signal to noise ratio (>15) and were manually selected from each MS data and subjected to MS/MS by changing the mass spectrometer to the lift mode. The parent and the product ions were analysed using the SNAP algorithm in Flex analysis, version 3.4 (Bruker Daltonics). Baseline subtraction and smoothing of mass spectra were done in the flex analysis software. Database search and analysis The processed peaks were transferred to biotools 3.2 (Bruker Daltonics) which inputs data directly in MASCOT search engine version2.4.1. (http://www.matrixscience.com) for protein identification. The parameters set were: maximum of one missed cleavage, parent ion mass tolerance of 1.2Da and fragment ion mass tolerance of 0.5Da, variable modification: methionine oxidation, fixed modification: carbamidomethylation of cysteines,

taxonomy:

Viridiplantae. The search was done in NCBI nr database against 59642736 sequences and 21322359764 residues as on 31.12.2014. Protein identifications in MASCOT were considered reliable if the identification was beyond significance (p

Mining Novel Allergens from Coconut Pollen Employing Manual De

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