Parietaria judaica pollen is a common
cause of airway allergic disease in the Mediterranean
area. Proteome analysis of mature Parietaria judaica
pollen by two-dimensional gel electrophoresis (2-DE)
and mass spectrometry has established the first
reference proteome map of this weed. Proteins
involved in a variety of cellular functions as well as
the occurrence of allergens were detected. By using
2-DE and immunoblotting with sera from Parietaria
judaica allergic patients we obtained a more detailed
characterization of Parietaria judaica allergen profil

ALESSANDRO, Riccardo

BARRANCA, Marilisa

Becchi, M

DE LEO, Giacomo

Duro, G

FONTANA, Simona

TAVERNA, Simona

Zanella Cleon, I

Archivio istituzionale della ricerca - Università di Palermo

ORIGINAL RESEARCH PAPERProteomic analysis of Parietaria judaica pollen and allergenprofiling by an immunoproteomic approachMarilisa Barranca • Simona Fontana • Simona Taverna •Giovanni Duro • Isabelle Zanella-Cleon • Michel Becchi •Giacomo De Leo • Riccardo AlessandroReceived: 12 November 2009 / Revised: 13 November 2009 / Accepted: 17 November 2009 / Published online: 2 December 2009 Springer Science+Business Media B.V. 2009Abstract Parietaria judaica pollen is a commoncause of airway allergic disease in the Mediterraneanarea. Proteome analysis of mature Parietaria judaicapollen by two-dimensional gel electrophoresis (2-DE)and mass spectrometry has established the firstreference proteome map of this weed. Proteinsinvolved in a variety of cellular functions as well asthe occurrence of allergens were detected. By using2-DE and immunoblotting with sera from Parietariajudaica allergic patients we obtained a more detailedcharacterization of Parietaria judaica allergen profileso to improve our comprehension of the pathogenesisof pollen-induced allergic reaction.Keywords Allergens  Parietaria judaica Immunoproteome  PollenIntroductionParietaria judaica belongs to the Urticaceae familyand is commonly found in urban and rural areas.Its pollen is one of the main causes of allergy in theMediterranean area (Masullo et al. 1996). Parietariajudaica pollen allergens have been identified andcharacterized by immunochemical and molecular-bio-logical techniques (Amoresano et al. 2003; Stumvollet al. 2003). Par j 1 and Par j 2 represent two majorallergens belonging to a family of glycoproteins knownas non-specific lipid transfer proteins (Salcedo et al.2004). Recently, the presence of minor allergens likeprofilin (Par j 3) and 2-EF-hand (Par j 4) proteins havebeen also described (Asturias et al. 2004; Bonura et al.2008). However, other proteins, specific isoforms orpost-translational modifications (PTM) of known aller-gens, can contribute to hypersensitivity reactions inallergic individuals. The accurate diagnosis of hyper-sensitive patients as well as the in-deep knowledge ofthe mixtures for a effective immunotherapy depend onthe molecular characterization of all allergenic compo-nents from a biological source.Marilisa Barranca and Simona Fontana contributed equally tothis work.Electronic supplementary material The online version ofthis article (doi:10.1007/s10529-009-0180-5) containssupplementary material, which is available to authorized users.M. Barranca  S. Fontana  S. Taverna G. De Leo  R. Alessandro (&)Sezione di Biologia e Genetica, Dipartimento diBiopatologia e Metodologie Biomediche, Universita` diPalermo, Via Divisi 83, 90133 Palermo, Italye-mail: ricale@unipa.itG. DuroIstituto di Biomedicina ed Immunologia Molecolare‘‘Alberto Monroy’’, Consiglio Nazionale delle Ricerche,Palermo, ItalyI. Zanella-Cleon  M. BecchiInstitut de Biologie et Chimie des Proteines, UMR 5086CNRS, IFR128 Lyon, France123Biotechnol Lett (2010) 32:565–570DOI 10.1007/s10529-009-0180-5Proteomic profiling of pollen extracts coupledwith classical immunological approaches have beenapplied to examine the presence of novel allergeniccomponents in maize, grass, and other species (Gon-za´lez-Buitrago et al. 2007; Petersen et al. 2006). Anunderstanding of P. judaica pollen proteome wouldprovide insight in the molecular nature of allergens aswell as of other proteins involved in the functionalspecialization of pollen. Using 2-DE followed byLC-MS/MS analyses, we generated the first referencemap of the P. judaica mature pollen proteome. In total,we identified 70 protein spots including isoforms, andby using an immunoproteomic approach with serumfrom P. judaica allergic patients, we have obtained amore detailed characterization of Parietaria judaicaallergen profile so to improve our comprehension ofthe molecular determinants of P. judaica-inducedallergic reaction. In particular, we have evidenced thatthe IgE from patients recognise Par j 1 and the Par j 2P2 allergens while they don’t recognise the polypep-tide Par j 2 P8. These results could provide importantindications to develop new and more efficient vaccinesfor allergic patients.Materials and methodsPollen collection and protein extractionPollen from Parietaria judaica was purchased fromAllergon (A¨ngelholm, Sweden) and extracted aspreviously described (Alessandro et al. 2009).Briefly, the pollen grains were defatted with diethylether, extracted in PBS at 4C, dialysed against waterand finally lyophilised and stored at -80C until use.2-DE, image acquisition and analysisAliquots of the lyophilised proteins, 60 lg foranalytical gels and 1 mg for preparative gels, weresolubilized in a buffer containing 8 M urea, 4% w/vCHAPS, 40 mM Tris and 65 mM DTE and a traceamount of Bromophenol Blue.The first dimension IEF was carried out usingimmobilized pH gradient (IPG) strips, pH 3–10 non-linear and pH 4–9 linear, 18 cm long. The first dimension,equilibration steps and the second dimension wereperformed as previously described (Fontana et al. 2007).Gels were stained with ammoniacal silver nitratefor analytical gels (Hochstrasser et al. 1988) orComassie staining for preparative gels. The gels werescanned by densitometer ImageScanner II (GEHealthcare Life Science, NJ, USA) and the Image-Master 2D Platinum 6.0 software (GE HealthcareLife Science, NJ, USA) was used to perform thequalitative and quantitative analysis of the 2-DEmaps. Human serum was used as internal standard forisoelectric point (pI) and Molecular Weight (MW)calibration of the 2-DE gel with pH 3–10 non lineargradient (Bjellqvist et al. 1993).In-gel protein digestionCoomassie Blue or silver-stained protein spots wereexcised from the preparative gels and cut into 1-mmpieces. In-gel digestion was performed as describedby Shevchenko et al. (1996) with minor modifica-tions. De-staining was obtained by successive washeswith 20 mM NH4HCO3 buffer and H2O/acetonitrile(50/50 v/v) mixture for Coomassie Blue. For prote-olytic digestion, the gel was treated by 5–15 lltrypsin (20 ng/ll in 50 mM NH4HCO3) for 5 h at37C. The resulting tryptic peptides were extractedfrom the gel by centrifugation and the supernatantfraction was recovered. A second extraction step wasperformed using 10–15 ll H2O/acetonitrile/formicacid (60/36/4; by vol.) for 30 min and, finally, allextracts were pooled. For LC-ESI/MS/MS experi-ments, extracts were dried under vacuum andre-suspended in 0.1% TFA solution.NanoLC/nanospray/tandem mass spectrometry(LC-ESI/MS/MS)Experiments were performed on a Q-STAR XLinstrument (Applied Biosystems, Courtaboeuf, France)equipped with a nanospray source using a distal coatedsilica-tip emitter (FS 150-20-10-D-20, New Objective,USA) set at 2300 V. Information Dependent Acquisi-tion (IDA) mode allowed ?2 to ?4 charged peptideions within a m/z 400–2000 survey scan mass range tobe analyzed for subsequent fragmentation. MS/MSspectra were acquired in the m/z 65–2000 range. Thecollision energy was automatically set by the software(Analyst 1.0) and was related to the charge of theprecursor ion. The MS and MS/MS data were recal-ibrated using internal reference ions from a trypsin566 Biotechnol Lett (2010) 32:565–570123autolysis peptide at m/z 842.510 [M ? H]? and m/z421.759 [M?2H]2?. Deconvolution of the TOF MSspectra were done with Bayesian Peptide Reconstructtool of Analyst. Multicharged ions in 700–1,800 m/zrange with 0.2 Da mass tolerance were used tocalculate glycopeptide masses from 2,000 to 4,000 Da.Tryptic peptides were separated using an Ultimate-nanoLC (Dionex, Voisins Le Bretonneux, France)with a C18 PepMap micro-precolumn (5 lm; 100 A˚´;300 lm 9 5 mm; Dionex) and a C18 PepMap nano-column (3 lm; 100 A˚´ ; 75 lm 9 150 mm; Dionex).After a 3 min wash with 0.05% TFA in H2O/acetonitrile (98/2 v/v) of the injected sample solutionon the micro-precolumn, the chromatographic sepa-ration was developed using a linear 60 min gradientfrom 0 to 50% B, where solvent A was 0.1% formicacid in H2O/acetonitrile (95/5 v/v) and solvent B was0.08% formic acid in H2O/acetonitrile (20/80 v/v) atapprox 200 nl/min.Protein identifications were performed using theParagon Algorithm thorough search mode in Protein-PilotTM software (Applied Biosystems, Courtaboeuf,France).Patient seraFor the identification of IgE-reactive proteins, serafrom patients (n = 10) sensitized to Parietariajudaica whose radio-allergosorbent test (RAST)exhibited scores over 3, were used. Control serawere from healthy, non-allergic subjects and fromsubjects sensitized to other allergic sources (dustmite, Olea and Cupressus). All sera were stored at-80C until use.ImmunoblottingFor immunoblot analysis, 2-DE gel was transferred tonitrocellulose membrane (Hybond-ECL, AmershamBioscience, Little Chalfont, UK) and the membranewas incubated in blocking solution (5% w/v non-fatdry milk, 20 mM Tris, 140 mM NaCl, 0.1% Tween20) (TBST) at room temperature. After severalwashings in TBST buffer, the filters were incubatedovernight at room temperature with sera from allergicpatients diluted 1:10 followed by horseradish perox-idase (HRP)-conjugated rabbit anti human IgE(1:2,000 dilution). After washings in TBST buffer,immunocomplexes were detected by the enhancedchemiluminescence detection system (Super Signal,Pierce, Rockford, IL, USA).Results and discussionProteome map of Parietaria judaica pollenParietaria judaica represents the main cause ofallergy in the Mediterranean area where up to 80%of pollinosis patients are sensitized against theproteins composing the P. judaica pollen. With theaim of obtaining a comprehensive and detailedanalysis of Parietaria judaica pollen content, weextracted total protein from 1.5 g mature pollen.Aliquots of the protein samples were subjected to2-DE analysis and representative silver-stained 2Dgel is shown in Fig. 1. Good reproducibility andresolution were achieved throughout the experimentalprocedures. On average 975 protein spots wereclearly resolved using IPG strips pH 3–10 non-linear.Protein identificationTo obtain a first reference proteomic map of thepollen, 100 protein spots were excised from Coo-massie Blue-stained gels and among these, 70 proteinspots (corresponding to 21 different proteins) wereidentified by LC-MS/MS technology (SupplementaryTable 1). Identified proteins ranged in calculatedmolecular mass from 92 to 7.5 kDa and in calculatedpI from 4.3 to 7.1. All proteins were designated witharbitrary spot numbers as shown in Fig. 1. While theidentification of proteins by proteomics methodologyhas found extensive application with organismswhich have a complete sequence of their genomesalready available, cross species identification hasbeen used to identify proteins from several plantspecies (Faulkner et al. 2005; Fernando 2005; Wang,et al. 2005). In fact, in our work, due to the poorprotein and DNA sequence database coverage inParietaria judaica (only 7 proteins are present inUni-Prot database for this plant), MS analysesallowed the recognition of 25 protein spots from P.jand 45 from other species (Supplementary Table 1).Multiple spots corresponding to the same proteinwere also identified, as described in other proteomicstudies, and reasonably corresponding to post-Biotechnol Lett (2010) 32:565–570 567123translational modifications, protein degradation, dif-ferent isoforms derived from different genes of amultigene family or products of alternatively splicedmRNAs.Figure 2 shows the distribution of P.j pollenproteins according to their functional classification.Briefly, the proteins were grouped in four distinctfunctional classes: proteins involved in carbon metab-olism and energy generation, cytoskeletal and calcium-binding proteins, stress-related proteins, pollenallergens and proteins involved in the methioninemetabolism. With respect to specific allergens, weidentified 11 spots (37–47) corresponding to Par j 2 P2protein, 13 spots (49–61) corresponding to Par j 2 P8protein, while 1 spot (36) corresponded to Par j 1 P1protein. A search of Par j 1 and 2 protein sequences,using specific softwares supported by ExPaSy Proteo-mics Server evidenced both O-glycosylation sites andphosphorylation sites (http://ogpet.utep.edu/OGPET,http://www.cbs.dtu.dk/services/NetPhos).In particular, Par j 1 P1 protein has one glycosyl-ation sites and five phosphorylation sites, Par j 2 P8protein has five phosphorylation sites and Par j 2 P2protein has three phosphorylation sites. These post-translational modifications do not change signifi-cantly the molecular weight of a protein but they mayFig. 1 Representativeproteomic map ofParietaria judaica pollen,proteins (60 lg) wereloaded on IPG strips(18 cm, 3.5–10 non-linearpH). The second dimensionwas performed on a verticallinear-gradient slab gel(9–16%). Gel was silverstainedFig. 2 Pie-chart representing the percentage distribution ofidentified pollen proteins within functional classes568 Biotechnol Lett (2010) 32:565–570123be responsible of the shifts in isoelectric points. Atthe same time, post-translational modifications mighthave an influence on the Ig binding so that theknowledge of the structure of allergens is importantto improve diagnosis and therapy of allergy (Ferreiraet al. 2006).Identification of IgE reactive spots by 2-DEand pollen allergensAlthough molecular biology and immunologicalmethodologies have allowed the identification ofPar j 1 and Par j 2 as the major pollen componentsresponsible of allergic reaction and profilin as aminor allergen (Asturias et al. 2007), the study of P.jpollen allergens have never been completed by usingan immunoproteomic approach. Since Par j 1 andPar j 2 are basic proteins, to improve the separationof their several isoforms, the pollen protein sampleswere run on IPG strips 6–9 linear, which allows abetter separation of polypeptides with a pI higherthan 7. Figure 3a shows a representative Coomassie-stained 2 D gels and corresponding immunoblotobtained by using pooled sera of allergic patients(Fig. 3b). In order to better characterize the biochem-ical identities of immunoreactive spots (Fig. 3a,numbers 1–7) they were excised from Coomassie-stained 2-DE gel and subjected to LC-ESI MS/MS.Table 2 in Supplementary Material shows the resultsof protein spot identifications. Protein spots numbers1 and 2 are isoforms of Par j 1 allergen, protein spotsnumbers 3–5 are isoforms of Par j 2 P8 while proteinspots 6 and 7 are isoforms of Par j 2 P2 allergen.Figure 3b, shows a representative western blot usingthe pooled sera from patients. In particular, the spotindicated with arrows corresponds to Par j 1 and spotincluded in the square corresponds to Par j 2 P2.Control experiments, carried out with pooled serafrom 10 nonsensitized subject, indicated the speci-ficity of antibody detection (Fig. 3c). Interestingly,no reaction was ever observed against Par j 2 P8suggesting that Par j 2 P2, together with Par j 1, arethe main allergenic components present in Parietariajudaica pollen.The data presented in our study show for the firsttime the identification of Parietaria judaica pollenallergens through bidimensional western blot thusrepresenting satisfactory tools for immunologicalcharacterization and classification of the proteinsfrom a raw plant extract.Acknowledgments M.B. was a fellow of Regional Agency forEnvironmental Prevention, ARPA Sicilia. This work was supportedby ARPA Sicilia; Universita` di Palermo (International Coop-eration) to R.A.; ex 60% MURST to R.A. and to G.D.L.ReferencesAlessandro R, Gallo A, Barranca M, Principe S, Taverna S,Duro G, Cassata G, Becchi M, Fontana S, De Leo G(2009) Production of an egg yolk antibody againstParietaria judaica 2 allergen. Poult Sci 88(8):1773–1778Amoresano A, Pucci P, Duro G, Colombo P, Costa M, Izzo V,Lamba D, Geraci D (2003) Assignment of disulphidebridges in Par j 2.0101, a major allergen of Parietariajudaica pollen. Biol Chem 384(8):1165–1172Asturias J, Ibarrola I, Eseverri J, Arilla M, Gonza´lez-Rioja R,Martı´nez A (2004) PCR-based cloning and immunologi-cal characterization of Parietaria judaica pollen profilin.J Investig Allergol Clin Immunol 14(1):43–48Fig. 3 2-DE analysis of Parietaria judaica pollen extractusing a non linear pH 6–9 gradient in the first dimensionfollowed by a 9–16% SDS-PAGE gradient in the seconddimension. 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Proteomic analysis of Parietaria judaica pollen and allergen profiling by an immunoproteomic approach

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Proteomic analysis of Parietaria judaica pollen and allergen profiling by an immunoproteomic approach

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