Novel IgE Recognized Components of Lolium perenne Pollen

Groups 1 and 5 include major allergens, recognized by IgE of about 95% and up ... Lyophilized extract was suspended in 50 mM Tris/HCl, pH 8.0, 5 M Ure...
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Novel IgE Recognized Components of Lolium perenne Pollen Extract: Comparative Proteomics Evaluation of Allergic Patients Sensitization Profiles Michele De Canio,†,# Simona D’Aguanno,‡,# Cristiano Sacchetti,† Francesca Petrucci,§ Giovanni Cavagni,| Marzia Nuccetelli,⊥ Giorgio Federici,†,| Andrea Urbani,*,†,‡,O and Sergio Bernardini†,|,O Department of Internal Medicine, University of Rome Tor Vergata, Rome, Italy, Laboratory of Proteomics and Metabonomics, S. Lucia Foundation-IRCCS, Rome, Italy, Department of Biomedical Science, University “G. D’Annunzio”, Chieti, Italy, Children’s Hospital “Bambino Gesu ` ”-IRCCS, Rome, Italy, and Department of Laboratory Medicine, Policlinico Tor Vergata Foundation, Rome, Italy Received April 6, 2009

Abstract: In the last years, proteomic investigation provided a powerful tool in molecular characterization of complex allergen sources with relevant implications in both diagnosis and immunotherapic treatment of allergies. We followed a proteomic approach to characterize ryegrass (Lolium perenne) pollen, a common cause of seasonal allergic diseases affecting an increasing part of world population. Peptide shotgun experiments performed on nanoLiquid Ultra Pressure Chromatography coupled with fast Q-TOF MS-MS/MS acquisition protocols (MSE) and 2-DE immunoblot combined with MALDI-TOFTOF analysis allowed the detection of all previously identified ryegrass allergens. Comparative analysis of immunoblot highlighted a class of patients characterized by a more complex 2-DE pattern associated with increased levels of IgE antibodies and by higher susceptibility to multiple sensitization toward different allergen sources. Cluster analysis revealed that all these patients recognized profilin, considered the main cross-reactive allergen in grass pollen. Furthermore, mass spectrometry analysis revealed the presence of other IgE reactive components in ryegrass pollen that might be involved in polysensitization, such as cyclophilin, fructosyltransferase and legumin-like protein. Keywords: IgE binding protein • pollen allergens • mass spectrometry • LC-MS/MS * To whom correspondence should be addressed. Prof. Andrea Urbani, University of Rome “Tor Vergata”, Dept. of Internal Medicine, Via Montpellier 1, 00133 Rome, Italy. E-mail: [email protected]. † University of Rome Tor Vergata. ‡ S. Lucia Foundation-IRCCS. # These authors made equal contributions and should be considered as “first authors”. § University “G. D’Annunzio”. | Children’s Hospital “Bambino Gesu ` ”-IRCCS. ⊥ Policlinico Tor Vergata Foundation. O Both authors acted as senior investigators and should be considered equal “last authors”. 10.1021/pr900315a CCC: $40.75

 2009 American Chemical Society

1. Introduction Allergic diseases are Immunoglobulin E (IgE)-mediated type I hypersensitivity reactions against common environmental substances. Atopic individuals respond to allergens exposure by production of IgE antibodies, leading basophils and tissue mast cells to secrete immuno and inflammatory response mediators, such as histamine, cytokines, leukotrienes. The final effect is the elicitation of allergic symptoms, ranging from dermatitis or rhinoconjunctivitis to bronchial asthma.1 Pollens are the most frequent cause of seasonal allergic rhinitis (SAR) affecting about 15% of European population.2 In literature, allergic reactions have been described against pollens from a variety of plant species. Only the Poaceae family (grasses) contains about 25% of allergenic pollen species and about 45% of sequenced allergens.3 Grass pollen allergens may have elevated sequence homology and exhibit similar biochemical and immunological properties.4 According to these features, the International Union of Immunological Societies (IUIS)-Allergen Nomenclature Sub-Committee (www.allergen.org) classified grass pollen allergens into 11 groups. However, molecular variability due to allelic polymorphism, post-translational modifications and alternative mRNA splicing can be also observed among allergens from the same group. Groups 1 and 5 include major allergens, recognized by IgE of about 95% and up to 85% of grass pollensensitized patients, respectively. Group 2/3 is recognized with a frequency of approximately 60%.5 In Lolium perenne (ryegrass), six allergen groups are reported. Lol p 1 (group 1) is a glycoprotein of 31-35 kDa, belonging to the β-expansins family of papain-related cysteine proteinases, involved in the loosening of plant cell walls during the extension.6 Lol p 2 and Lol p 3 are 11-12 kDa related proteins, showing sequence similarity to the C-terminal domains of group 1 allergens.7 Lol p 4, a basic 50-67 kDa protein, containing a FAD binding domain,8,9 and Lol p 11, a soybean trypsin inhibitor-related protein,10 are glycosilated allergens. Lol p 5 isoallergens consist of two main isoforms, A and B, showing 66% sequence identity11 with an apparent molecular mass of 27-33 kDa. Finally, cytochrome C was characterized as allergen and named Lol p 10,12 but further investigations have not supported its relevancy.4 Journal of Proteome Research 2009, 8, 4383–4391 4383 Published on Web 07/08/2009

technical notes In the last years, proteomic investigations in biochemical and immunological characterization of allergens allowed a more comprehensive knowledge of these allergy-eliciting molecules. In particular, 2-DE was successfully employed to resolve complex allergen sources, separating different allergen isoforms and studying post-translation modifications;13 at the same time, mass spectrometry analysis provided a useful tool to identify novel allergens.14 In this work, we pursued a proteomic investigation to perform a comparative evaluation of allergic patients sensitization profiles. Nanoliquid Ultra Pressure chromatography-mass spectrometry (nUPLC-MSE) and MALDITOF-TOF analysis were employed to examine the components recognized by IgE detecting all the previously identified allergens and identifying three new putative ones. Multivariate statistical analysis of 2-DE immunoblot maps revealed that about half of the examined patients showed complex pattern of sensitization in association with increased level of total serum IgE and sensitization to multiple allergen sources.

2. Materials and Methods 2.1. Patients. Serum samples, collected at the Department of Laboratory Medicine, “Policlinico Tor Vergata Foundation”, Rome, were obtained by centrifugation from whole blood collected in standard glass tubes for serological analysis containing clot activator and gel for serum separation and stored at -80 °C until use. Patient cohort was represented by 19 individuals sensitized to ryegrass pollen, while control cohort included 10 nonsensitized subjects (Table 1S, Supporting Information). All patients suffered from seasonal allergic rhinitis (SAR) for at least 2 consecutive years. Patients included in this study have total serum IgE levels above 100 International Unit/ mL (IU/mL) for adults, and 20 IU/mL for children. Ryegrass pollen sensitization was assessed measuring ryegrass-specific IgE and only patients with values above 5 IU/mL were selected. Total and specific IgE were measured by Capture Assay Radim Liquid Allergens (CARLA) system (RADIM Diagnostics, Pomezia, Italy). All patients declared that they had never received grass pollen immunotherapy. Allergic and control subjects were informed of this study and gave their written consent. An addictional cohort of 22 sera from patients sensitized to ryegrass pollen was provided by Pediatric Allergy Research Unit of Children’s Hospital “Bambino Gesu `” of Rome and employed to validate preliminary results. Sensitization to ryegrass pollen was assessed by skin prick test and by measurement of ryegrass specific IgE and/or total IgE. A control cohort of 10 non sensitized subjects was also included. 2.2. Ryegrass Pollen Extracts. Ryegrass pollen extract, used as starting material in preparation of reagents for allergy diagnosis, was kindly provided by RADIM Diagnostics. Lyophilized extract was suspended in 50 mM Tris/HCl, pH 8.0, 5 M Urea, 2 M Thiourea, 4% (w/v) CHAPS, and 80 mM DTT, and stored at -20 °C in aliquots until use. 2.3. Protein Identification by nUPLC-MSE. A total of 100 µg of pollen extract was precipitated in acetone and dissolved in 50 mM ammonium bicarbonate pH 8.5 containing 1.2 M Urea and 1% (w/v) CHAPS. Reduction and alkylation of proteins were obtained adding 10 mM DTT (1 h at 36 °C) and 50 mM Iodoacetamide (1 h at room temperature (RT)). Protein sample at final concentration of 1.6 µg/µL was digested with 1:20 (w/ w) sequence grade trypsin (Promega, Madison, WI) at 36 °C overnight. Reaction was stopped by the addition of 5 µL of 1% (v/v) trifluoroacetic acid (TFA). Digestion mixture was diluted 1:3 with 0.1% (v/v) formic acid and 2 µL was loaded onto a 4384

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De Canio et al. column for peptide separation. Peptides were trapped on a Symmetry C18 5 µm, 180 µm × 20 mm precolumn (Waters Corp., Milford, MA) and separated using a NanoEase BEH C18 1.7 µm, 75 µm × 25 cm nanoscale LC column (Waters Corp.). Sample was initially transferred with an aqueous 0.1% formic acid solution to the precolumn with a flow rate of 15 µL/min for 5 min. Mobile phase A was water with 0.1% formic acid, and mobile phase B was 0.1% formic acid in acetonitrile. Peptides were separated with a gradient of 3-40% mobile phase B over 120 min at flow rate of 250 nL/min, followed by a gradient of 40-90% mobile phase B over 5 min and a 15 min rinse with 90% mobile phase B. The column was re-equilibrated at initial conditions for 30 min. The column temperature was maintained at 35 °C. The lock mass ([Glu1]-Fibrinopeptide B, 250 fmol/µL) was delivered from the auxiliary pump of the nanoACQUITY UPLC System (Waters Corp.) with a constant flow rate of 200 nL/min. The Q-Tof Premier mass spectrometer (Waters Corp., Manchester, U.K.) was operating in “High-Low mode”: it was programmed to step between low (4 eV) and high (15-40 eV) collision energies on the gas cell, using a scan time of 1.5 s over 50-1990 m/z.15 Data were also collected in “Data dependent Mode” (DDA): the instrument alternatively acquired a full scan (m/z acquisition range from 400 to 1700 Da/e) and a tandem mass spectrum (m/z acquisition range from 70 to 1700 Da/e). The 8 most intense peaks in any full scan were selected as precursor and fragmented by collision energy. Continuum LC-MS data were processed and searched using ProteinLynx GlobalServer v2.3 (PLGS, Waters Corp.). Protein identifications were obtained with the embedded ion accounting algorithm of the software and searching in both Uniprot and Swiss-Prot Databases restricted to Poales taxonomy, 166 928 and 4207 entries, respectively. The search parameters for “High-Low” data were typically 15 ppm of tolerance for precursor ions, 20 ppm of tolerance for product ions, minimun 3 fragment ions matched per peptide, minimun 7 fragment ions matched per protein, minimun 2 peptides matched per protein, 1 missed cleavage, cysteine carbamidomethylation and methionine oxidation as modifications. The false positive rate of the ion accounting identification algorithm is typically 3-4% with a randomized database 5 times the size of the original utilized database. For DDA data, the mass accuracy was within 50 ppm for the peptide mass tolerance and within 0.20 Da for fragment mass tolerance, at the most one miscleavage for tryptic peptides was allowed, and the modifications accepted were carbamidomethylation of cysteines and oxidation of methionines. 2.4. 2-D Electrophoresis. A total of 100 µg of pollen extract was precipitated in acetone and resuspended in a buffer containing 5 M Urea, 2 M Thiourea, 4% (w/v) CHAPS, 80 mM DTT, 1.6% (v/v) IPG-buffer (pH 3-10NL, Amersham Biosciences, Buckinghamshire, U.K.), and 1% (w/v) bromophenol blue. Isoelectric focusing was performed in IPGphor system (Amersham Biosciences) using Immobiline Dry strips 7 cm, pH interval 3-10 nonlinear. After 1 h of passive and 8 h of active rehydratation at 30 V, proteins were focused by ramping to 300 V over 1 h, holding at 300 V for 1 h, ramping to 3500 V over 3 h, holding at 3500 V for 1 h, successively ramping to 5000 V over 1.5 h and plateau at 5000 V until 25 000 V/h. After IEF, the IPG strips were equilibrated in a reducing buffer containing 50 mM Tris/HCl, pH 8.8, 6 M Urea, 2% (w/v) SDS, 30% (v/v) glycerol, 2% (w/v) DTT, and alkylated in the same buffer replacing DTT with 2.5% (w/v) iodoacetamide. Second-dimension separation was performed by polyacrylamide SDS-PAGE

Novel IgE Recognized Components of L. perenne Pollen Extract 16

gel (13.5% T, 3.3% C) with the buffer system of Laemmli. After electrophoresis, proteins were either stained with Colloidal Coomassie or transferred onto a PVDF membrane (Amersham Biosciences) for the subsequent glycoprotein staining with Glycoprotein Detection Kit (Sigma-Aldrich, Poole, Dorset, U.K.). 2.5. Immunoblots and Image Analysis. Proteins resolved by 2-DE were electrotransferred onto nitrocellulose membranes and incubated 1 h with patient sera (1:10 or 1:20 working dilution depending on the level of ryegrass-specific IgE). Immunoreactivity was revealed by incubation with a goat antihuman IgE antibody conjugated with alkaline phosphatase, a gentle gift by RADIM Diagnostics, followed by detection with Immobilon Western AP Chemiluminescent Substrate (Millipore, Billerica, MA) and film exposure. Images were acquired using Image scanner UMAX (Amersham Biosciences) and analyzed by ImageMaster analysis software (Amersham Biosciences). To correct for variability due to spot detection or protein loading reproducibility, the individual spot value of each immunoblot was normalized automatically by the software. The raw data of each spot in a member gel was expressed as Intensity percentage. Spot with Intensity percentage values below 2 were not considered. 2.6. Statistical Analysis. Differential analysis of allergic patients patterns based on single allergens was performed adding intensity values of spots concerning different forms of the same allergen. The correlations between allergen intensity and among allergens and clinical variables were calculated using Spearman rank correlations. Results were showed by a correlation map visualizing the correlation values and the levels of significance marked as *p < 0.05, **p < 0.01, ***p < 0.001. A qualitative matrix designed on presence/absence of each allergen in all immunoblots was submitted to Hierarchical Clustering Analysis, processed with PermutMatrix according to the Euclidean distance and McQuitti’s aggregation method.17 Clinical differences between classes, investigated applying Mann-Whitney U-test, were considered significant for p < 0.05. 2.7. MALDI-TOF MS Analysis. Single spots were excised from Coomassie stained 2-DE gel and gradually dehydrated with 50 mM ammonium bicarbonate, 50 mM ammonium bicarbonate/acetonitrile (1:1) and acetonitrile. Proteins were reduced using a solution of 10 mM DTT and alkylated with 55 mM iodoacetamide. Gel plugs were alternatively hydrated and dehydrated with 50 mM ammonium bicarbonate and acetonitrile, respectively, and completely dried. A solution of trypsin (Promega) was added and proteins were digested at 37 °C overnight. The reaction was stopped with 1% (v/v) TFA in H2O. Peptides were desalted by C18 ZipTips (Millipore) and cocrystallized with a solution of 0.5 mg/mL R-cyano-4-hydroxycinnamic acid dissolved in acetonitrile/0.1% (v/v) TFA in H2O (1:1) on a Ground Steel plate (Bruker-Daltonics, Bremen, Germany) prespotted with a thin layer of 10 mg/mL R-cyano4-hydroxycinnamic acid dissolved in ethanol/acetonitrile/0.1% (v/v) TFA in H2O (49.5:49.5:10). Mass spectra were acquired with a Ultraflex III MALDI TOF/TOF spectrometer (BrukerDaltonics). External calibration was performed using the standard peptide mixture from Bruker-Daltonics (m/z 1046.54, 1296.68, 1347.73, 1619.82, 1758.93, 2093.08, 2465.19, 2932.59, 3494.65). Monoisotopic peptide masses were selected using FlexAnalysis software v3.0 (Bruker Daltonics). Internal calibration was performed using autolysis peaks from porcine trypsin (m/z 842.509 and 2211.104). After excluding contaminant ions (known matrix and human keratin peaks), database search was performed

technical notes

using MASCOT 2.2.03 algorithm (www.matrixscience.com) against NCBInr_20080606 database rescricted to Viridiplantae taxonomy (856 685 entries) with carbamidomethyl of cysteines as fixed modification, oxidation of methionines as variable modification, one missed cleavage site allowed for trypsin and 50 ppm as maximal tolerance. 2.8. MALDI-TOF MS/MS Analysis. After MS spectra acquisition, the instrument was switched in LIFT mode and precursor ions for fragmentation were selected manually. MS/MS spectra were acquired with a minimum of 4000 and a maximum of 8000 laser shots using the instrument calibration file. The precursor mass window was set automatically after the precursor ion selection. Spectra baseline subtraction, smoothing (Savitsky-Golay) and centroiding was performed by FlexAnalysis software v3.0 (Bruker Daltonics). Database search was performed setting the following criteria: maximum of one missed cleavage was allowed, the mass tolerance of precursor ions and fragments were set to 50 ppm and 0.4 Da, respectively; allowed variable modifications were carbamidomethylation on cysteine and methionine oxidation, and the taxonomy was restricted to Viridiplantae (856 685 entries).

3. Results 3.1. Identification of Ryegrass IgE-Binding Proteins. Ryegrass pollen extract quality was evaluated by shotgun proteomic analysis. Protein identification was obtained searching in Uniprot and Swiss-Prot databases restricted to Poales Taxonomy. Table 2S (Supporting Information) listed the 14 distinct proteins identified with significant PLGS score. The 6 ryegrass allergens annotated in the Official List of Allergens of IUIS were recognized: Lol p 1, Lol p 2, Lol p 3, Lol p 4, Lol p 5 (A and B isoforms) and Lol p 11. Further 7 items were recognized because they shared identical sequence peptides with homologous proteins of related grass species: alpha glucan phosphorylase H (Triticum aestivum); 14 3 3-like protein, fructose bisphosphate aldolase, actin and DEADbox ATP dependent RNA helicase 1 (Oryza sativa); glyceraldehyde 3 phosphate dehydrogenase (Hordeum vulgare) and polcalcin Phl p 7 (Phleum pratense). The number of identified peptides for each protein ranged from 3 to 28, with a protein sequence coverage from 19% to 87%. This preliminary experiment showed the integrity of starting material demonstrating the presence of all expected allergens in the extract. A more detailed investigation of pollen protein components was performed by 2-DE. Coomassie stained gel showed 120 ( 11 spots with pI from 3 to 10 and molecular mass from 10 to 100 kDa (Figure 1A). Most proteins had molecular masses lower than 50 kDa and neutral or acidic pI. To visualize IgE-binding proteins, immunoblot was performed incubating transferred nitrocellulose membrane with pooled sera obtained from 19 pollen-allergic patients (Figure 1B). Control experiment, carried out with pooled sera from 10 nonsensitized subjects, indicated the complete specificity of immunoblot detection (Figure 1C). Immunoreactive spots were excised from corresponding Coomassie-stained gel and analyzed by peptide mass fingerprinting (PMF) and MS/MS. Starting from a total of 70 detected spots, only those with intensity percentage values above 2 were selected and further analyzed by mass spectrometry analysis. These resulted in the identification of 27 protein spots (Table 3S, Supporting Information). Spots 7-11, showing differences in pI, were assigned to the major pollen allergen Lol p 1. Five spots corresponding to the major pollen allergen Lol p 5A were Journal of Proteome Research • Vol. 8, No. 9, 2009 4385

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De Canio et al.

Figure 1. 2-DE analysis of ryegrass pollen extract and corresponding immunoblot using pooled sera from allergic patients. A total of 100 µg of pollen extract was resolved by 2-DE using a nonlinear pH 3-10 gradient in the first dimension, followed by 13.5% SDS-PAGE in the second dimension. The separated proteins were stained by Coomassie (A) or transferred onto a PVDF membrane for the subsequent glycoprotein staining (D). IgE binding proteins were detected with pooled sera from pollen-allergic patients (B) and control subjects (C). Arrows indicate spot analyzed by MALDI-TOF-TOF.

determined (spots 14-18), whereas spots 5, 12, and 13 were identified as Lol p 5B. Reactive fragments belonging to Lol p 1 and Lol p 5A were also picked out in spots 24 and 25, respectively. In the basic high mass region of the gel, three different isoforms of Lol p 4 (spots 2, 3 and 4) were identified. At low molecular mass, two basic spots (26 and 27) were assigned to Lol p 3. Lol p 2 (spot 22), Lol p 11 (spot 20) and profilin Phl p 12 from P. pratense (spots 21 and 23) were identified in the acidic low mass region of the gel. Spot 1, corresponding to an acidic protein with apparent molecular mass of 50 kDa, was assigned to fructosyltransferase. Finally, two proteins were identified only by MS/MS sequencing. Spot 19 was assigned to a polypeptide homologous to cyclophilin (T. aestivum) by two identical sequence peptides. Spot 6 was assigned to a protein structurally related to legumin-like protein (Zea mays) and the Os05g0116000/11-S plant seed storage protein family protein (O. sativa). Since most of ryegrass pollen allergens are glycoproteins and carbohydrate moieties are involved in IgE recognition, 2-DE gel was transferred to a PVDF membrane for glycoprotein detection. A total of 62 ( 15 magenta positive spots were visualized (Figure 1D), 12 of them matched to IgE binding 4386

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proteins identified by MALDI-TOF analysis (Table 3S, Supporting Information). 3.2. Comparative Analysis of Patient Immunological Profiles. To define IgE reactivity profile of each patient, allergic sera were individually assayed in a series of immunoblot experiments with membranes obtained by 2-DE separation of pollen components. Spot immunoreactivities were evaluated by image analysis and expressed as intensity percentage. Differential analysis of allergic patients patterns based on single allergens was performed adding intensity values of spots identified as different 2-DE forms of the same protein. Lol p 5A and Lol p 5B were considered distinct allergens because of their structural and immunological differences. Grass pollen allergens Lol p 1 and Lol p 5A show higher prevalence, 95 and 100%, respectively. In a single case (patient V), we observed sensitization to a unique component on 2-DE immunoblot, identified as Lol p 5A. Allergen Lol p 5B was detected in 79% of patients, while Lol p 2 and Lol p 3 exhibited same frequency, 74%, and similar distribution among patients. Although Lol p 4 and Lol p 11 are considered major allergens, in these experimental conditions, Lol p 4 was detected only in a unique case (patient XVII) and Lol p 11 in two cases (patients II and XVIII). Profilin and cyclophilin were found in 42% of patient

Novel IgE Recognized Components of L. perenne Pollen Extract

technical notes

Figure 2. Distribution of component reactivities among patients. Histogram reports component frequencies (A); dot plot shows component percentage intensities (B). Lol p 1 (1), Lol p 5A (5A), Lol p 5B (5B), Lol p 2 (2), Lol p 3 (3), profilin (Pr), cyclophilin (Cy), legumin (Le), fructosyltransferase (Fr), Lol p 11 (11) and Lol p 4 (4).

Figure 3. Correlation map among single component intensities and clinical measurements of total and ryegrass-specific IgE. Values of component intensities and clinical results were analyzed by Spearman rank correlation analysis. The lower part of the map shows the distributions of variables and the regression lines. The upper part shows the correlation values. Positive values indicate direct relationships between variables, negative values inverse relationships. The significance levels are indicated as *p < 0.05, **p < 0.01, ***p < 0.001.

immunoblots, while legumin-like protein and fructosyltransferase were detected with frequency of 21 and 16%, respectively (Figure 2A). Comparative analysis of patient sensitization patterns was performed considering only the most relevant components, then excluding the low prevalence ones, Lol p 4, Lol p 11, legumin-like protein and fructosyltransferase. Moreover, patients V and XVII were not included in this analysis because of their unique serum profile (Figure 2B). Single allergen intensities were compared to results from total and specific IgE measurements (Figure 3). Two opposite trends were discriminated: Lol p 1 and Lol p 5A intensities were inversely related to total IgE and ryegrass specific IgE values, whereas the other allergens were directly related to them. Therefore, Lol p 1 and Lol p 5A intensity values were inversely related to the other allergens. Further analysis was performed using a matrix of binary data based on presence/absence of reactive components in each

immunoblot (Figure 4). Columns clustering showed three homogeneous classes according to frequency of components in examined cohort: cluster R, composed of Lol p 1 and Lol p 5A; cluster β composed of Lol p 5B, Lol p 2 and Lol p 3; cluster γ composed of profilin and cyclophilin. Because of their continue presence in the data set, Lol p 1 and Lol p 5A did not contribute to discriminate patients; consequently, cluster R was excluded to perform cluster algorithm of the rows. The algorithm identified three major row clusters, designated as Class A, B, C. Class A was composed of three patients whose sensitization profiles showed a prominent reactivity toward cluster R allergens, Lol p 1 and Lol p 5A, markers of genuine sensitization to grass pollen (Figure 4A). Class B pattern, found in six cases, was characterized by a more extensive sensitization to grass pollen components, comprising Lol p 2, Lol p 3, Lol p 5B and cyclophilin (Figure 4B). Last class C, composed of eight patients, had the most complex patterns of reactivity. All patients belonging to Class C showed sensitization toward Journal of Proteome Research • Vol. 8, No. 9, 2009 4387

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Figure 4. Hierarchical clustering analysis of single reactive components and patients. A qualitative summary matrix based on presence/ absence of each component in 2D immunoblots was processed with PermutMatrix according to the Euclidean distance and McQuitti’s aggregation method. Column clustering divided components into 3 groups: R, containing Lol p 1 (1) and Lol p 5A (5A); β, containing Lol p 5B (5B), Lol p 3 (3) and Lol p 2 (2); and γ, containing profilin (Pr) and cyclophilin (Cy). Row clustering divided patients into 3 classes indicated as A, B and C. Patients are indicated by roman numeral. On the right part of the panel, representative 2D immunoblots are shown: patient XIII for class A (A), patient XV for class B (B), and patient XII for class C (C).

profilin, marker of cross-reactivity among different plant pollens, while cyclophilin reactivity was detected in five of them (Figure 4C). For patient’s dendrogram, the amount of clustering structure of the data set was also estimated by determining the agglomerative coefficient. It was found to be near to optimal value 1. This component-based classification of patients was further combined with other acquired information. Class C patients were found to have more elevated Total IgE levels in comparison with the other two classes (Figure 5A), and a less relevant increment of specific IgE (Figure 5B). Moreover, we investigated the relationship between patients profiling complexity and multiple co-sensitization. A sensitization score, ranging from 1 to 5, was assigned to each patient on the basis of the number of positive tests to specific IgE (Table 1S, Supporting Information). Mann-Whitney test revealed that Class C patients showed sensitization toward a larger variety of allergen sources compared to Class A and B patients (Figure 5C). The same criterion of classification described above was applied to a different cohort of 22 patients sensitized to ryegrass pollen, whose sera were investigated by 2-DE immunoblot. Three cases were excluded because they showed a reactivity toward a unique component (as already observed for patient 4388

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V in the training group). The remaining 19 sera were divided into classes A, B, C according to the model proposed. Even in this case, Class C patients, characterized by profilin recognition, can be significantly discriminated from the other classes in having higher IgE values (Tables 1 and 2). When the MannWhitney test was applied on the whole of patients analyzed in this study, the resulting p-values increased, thus, demonstrating the effectiveness of the classification scheme.

4. Discussion The aim of the present work is to examine ryegrass pollen extract in order to evaluate the presence of putative novel allergens and to perform a comparative analysis of patient immunological profiles. Crude allergen extracts are widely employed in allergic diseases diagnosis and immunotherapy in spite of their limitations due to heterogeneity, presence of nonallergic components or degradation of allergens.14 Proteomic analysis can be a useful tool for quality assessment of allergen extracts and for standard methods development, allowing a direct comparison of these complex mixtures.18 nUPLC-MSE analysis of ryegrass pollen extract revealed the presence of all known allergens from L. perenne and further seven components were identified by sequencing peptides common to homologous proteins of other

technical notes

Novel IgE Recognized Components of L. perenne Pollen Extract

Table 2. Comparison of Ryegrass Specific IgE* of Class A + B and C in Training and Validation Groups class

§

Figure 5. Differential analysis of patient classes. Box and whisker plots show total IgE, specific IgE and sensitization trends in patient classes. Statistical analysis was performed applying Mann-Whitney test. Class C patients have elevated total (A, p < 0.05) and ryegrass specific IgE (B, p ) 0.1) and display sensitization against more allergen sources (C, p < 0.05) compared to the other two classes. Table 1. Comparison of Total IgE* of Class A + B and C in Training and Validation Groups class

§

group

A+B

C

Training (N ) 17) Validation (N ) 19) Total (N ) 36)

357 ( 251 (N ) 9) 429 ( 331 (N ) 12) 398 ( 294 (N ) 21)

692 ( 350 (N ) 8) 770 ( 313 (N ) 7) 728 ( 324 (N ) 15)

P-value§

0.03 0.04 0.004

* Total IgE are expressed in IU/mL and reported as Mean ( SD. P-value calculated by Mann-Whitney test.

grass species. Among them, Polcalcin,19 Fructose bisphosphate aldolase20 and glyceraldehyde 3 phosphate dehydrogenase21

group

A+B

C

P-value§

Training (N ) 17) Validation (N ) 14) Total (N ) 31)

81 ( 67 (N ) 9) 48 ( 56 (N ) 8) 65 ( 62 (N ) 17)

125 ( 72 (N ) 8) 114 ( 78 (N ) 6) 120 ( 72 (N ) 14)

0.19 0.11 0.03

* Specific IgE are expressed in IU/mL and reported as Mean ( SD. P-value calculated by Mann-Whitney test.

were already reported as IgE-binding proteins in ryegrass related species. Immunoreactivity of these proteins was not pointed out in the experimental data set presented, probably because the relatively restricted number of allergic sera assayed might limit the detection of less relevant or abundant allergens. Nevertheless, our methodological approach based on IgE recognition of single components allowed us to discriminate a group of patients, Class C, with a more severe clinical panel. These patients are characterized by a more complex 2-DE immunoblot pattern associated with increased levels of IgE antibodies. These observations could be explained considering the peculiar features of allergy diseases. Repeated exposures to one allergen elicit the immunologic response in atopic subjects, inducing them to develop IgE toward a growing number of allergens.22 At the same time, the polyclonality of immune response increases producing a more diverse repertoire of IgE antibodies. Consequently, cross-reactivity reactions might occur with higher probability.22 In fact, Class C patients are more susceptible to multiple sensitization toward different allergen sources. In this regard, it is important to underlie that all Class C patients recognize profilin, considered the main cross-reactive allergen in grass pollen.23,24 This allergen was discovered a long time ago in ryegrass pollen,23 although its amino acid sequence is still missing in the protein database. We recognized profilin by identity matching with peptides common to the homologous protein from P. pratense. The homology level was verified by sequencing a profilin transcript obtained from ryegrass pollen total RNA (Figure 1S, Supporting Information). The deduced amino acid sequence was compared to those included in the AllergenOnline database v.9.0 (1386 sequence entries; www.allergenonline.com) by Food Allergy Research and Resource Program (FARRP). The result indicates that ryegrass profilin has an elevated sequence identity with three entries of profilin from P. pratense CAA70610.1 (97.7%), P35079.1 (96.9%), CAA70609.1 (95.4%). The high identity scores of the alignments are a strong evidence of a potential for allergenic cross-reactions. This finding was somewhat expected considering that the two species are closely related. Moreover, mass spectrometry analysis revealed the presence of other IgE reactive components in ryegrass pollen that might be involved in polysensitization. Among them, a relevant role could be played by cyclophilin, that is recognized by most Class C patients. Cyclophilin, also known as peptidyl-prolyl cis-trans isomerase, is an ubiquitous enzyme involved in protein folding,25 recently associated with pollen tube growth during germination in O. sativa.26 It is supposed to be a new member of an emerging allergen family that seems to play an important role in polysensitization toward a variety of allergen sources.27 Journal of Proteome Research • Vol. 8, No. 9, 2009 4389

technical notes

De Canio et al. 28,29

This family also includes pollen allergen Bet v 7 from Betula verrucosa and fungal allergens, such as Mal s 6 from Malassezia sympodialis,30 Asp f 11 from Aspergillus fumigatus31 and Psi c 2 from Psilocybe cubensis.32 Moreover, an IgE-binding peptide of cyclophilin was identified in a recent proteomic analysis of wheat protein extract.21 Fructosyltransferase, better defined as Sucrose:sucrose 1-Fructosyltransferase (1-SST), is an enzyme with invertase activity, involved in metabolism of fructans, sucrose-derived fructose polymers, a main storage of carbohydrate in temperate grasses.33,34 Fucsin staining indicated that fructosyltransferase is a glycoprotein and multiple putative N-glycosilation sites have been predicted by sequence analysis.34 Our homology search revealed 76% similarity and 55% identity to the glycosilated allergen of tomato Lyc e 2.35 The ability of this allergen to be recognized by IgE and to trigger the histamine release from basophils in tomato sensitized patients is related to the presence of cross-reactive carbohydrate determinants (CCD) linked to polypeptide chain.35 CCD were found in many allergic sources such as pollen, insect venom and plant food; therefore, they were considered to have remarkable implication in crossreactivity phenomena among distantly related organisms.36 The clinical relevance of anti-CCD IgE is still under discussion. Because of its small size, a single CCD structure is thought to be unable to cross-link IgE bound to effector cells receptors. However, proteins with multiple glycosilation sites may overcome this structural limitation thus inducing allergic symptoms, as it occurs in Lyc e 2.35,37 Legumin-like protein/11-S seed storage protein belongs to Cupin superfamily, composed of functionally heterogeneous proteins but evolutionally conserved from archea to eucaryotes,38 sharing a 6-stranded β-barrel structural motif (cupin domain).39 Legumins, hexameric proteins containing two cupin domains for each monomer, comprise the major legume, tree nut and seed allergens,40 such as Cor a 9 from Corylus avellana,41 Ara h 3 from Arachis hypogaea42 and glycinin from Glycine max.39 Any members of Cupin superfamily were not reported as pollen allergen previously.3 Sensitization to 11-S seed storage protein, Cor a 9, was associated with severe systemic symptoms caused by hazelnut ingestion in allergic subjects.41 Mild symptoms restricted to the oral cavity, reported for individuals co-sensitized to birch pollen, could be explained by IgE cross-reactivity induced by shared epitopes of homologous allergen pairs Bet v 1/Cor a 1 and profilin Bet v 2/Cor a 2.43 Whether other pollen allergens may also be cross-reactive with hazelnut has not been welldocumented. In conclusion our proteomic analysis revealed that patients with a more complex 2-DE immunoblot pattern present higher total IgE levels and higher disposition to co-sensitization. This behavior is mainly associated to profilin IgE recognition but other minor components of grass pollen, such as IgE-binding proteins identified in this work, may be involved in crossreactivity phenomena in virtue of homology with allergens from other sources. However, it should be considered that the presence in patient sera of IgE antibodies toward specific pollen components does not necessarily indicate that the patients are clinically allergic to these proteins. Further investigation is needed to assess the actual allergenicity of these IgE reactive proteins. Moreover, comparative analysis of sensitization patterns demonstrated that each allergic patient develops an individual response to the single component from the allergen source. 4390

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Consequently, a correct evaluation of allergic disease should take in account these differences, particularly in the perspective of allergen-specific immunotherapy treatment. For this purpose, allergens might be purified from natural sources or expressed as recombinant proteins and employed in allergenspecific diagnostic tests. To achieve this goal, a more detailed characterization of allergen sources is necessary. By this point of view, proteomic platform is a promising approach in discovering novel allergens suitable for both diagnosis settings and immunotherapy vaccines.

Acknowledgment. The authors are grateful to Dr. Olindo Forini for providing sera from allergic patients and to Dr. Rosa Linda Cuffari, RADIM Spa, Pomezia, Rome, Italy, for providing L. perenne pollen extract and anti-human IgE antibody conjugated with alkaline phosphatase. This work was supported by Scientific Reserach Fund of Regione Lazio granted by Finanziaria Laziale di Sviluppo (FI.LA.S. Spa). Supporting Information Available: Supplementary Figure 1S and experimental procedures “Profilin cDNA sequencing”; Table 1S, patients serological features; Table 2S, protein identification by nUPLC-MSE analysis; Table 3S, protein identification by MALDI-TOF-TOF analysis. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Kay, A. B. N. Engl. J. Med. 2001, 344, 30–37. (2) Behrendt, H.; Becker, W. M. Curr. Opin. Immunol. 2001, 13, 709– 715. (3) Radauer, C.; Breiteneder, H. J. Allergy Clin. Immunol. 2006, 117, 141–147. (4) Andersson, K.; Lidholm, J. Int. Arch. Allergy Immunol. 2003, 130, 87–107. (5) Weber, R. W. J. Allergy Clin. Immunol. 2003, 112, 229–239. (6) Grobe, K.; Becker, W. M.; Schlaak, M.; Petersen, A. Eur. J. Biochem. 1999, 263, 33–40. (7) van Ree, R.; van Leeuwen, W. A.; van den Berg, M.; Weller, H. H.; Aalberse, R. C. Allergy 1994, 49, 254–261. (8) Jaggi, K. S.; Ekramoddoullah, A. K.; Kisil, F. T. Int. Arch. Allergy Appl. Immunol. 1989, 89, 342–348. (9) Nandy, A.; Petersen, A.; Wald, M.; Suck, R.; et al. Biochem. Biophys. Res. Commun. 2005, 337, 563–570. (10) van Ree, R.; Hoffman, D. R.; van Dijk, W.; Brodard, V.; et al. J. Allergy Clin. Immunol. 1995, 95, 970–978. (11) Ong, E. K.; Griffith, I. J.; Knox, R.; Singh, M. B. Gene 1993, 134, 235–240. (12) Ekramoddoullah, A. K.; Kisil, F. T.; Sehon, A. H. Mol. Immunol. 1982, 19, 1527–1534. (13) Petersen, A. Proteomics. 2003, 3, 1206–1214. (14) Gonzalez-Buitrago, J. M.; Ferreira, M.; Isidoro-Garcia, M.; Sanz, C.; et al. Clin. Chim. Acta 2007, 385, 21–27. (15) Vissers, J. P.; Langridge, J. I.; Aerts, J. M. Mol. Cell. Proteomics 2007, 6, 755–766. (16) Laemmli, U. K. Nature 1970, 227, 680–685. (17) Caraux, G.; Pinloche, S. Bioinformatics 2005, 21, 1280–1281. (18) Iraneta, S. G.; Acosta, D. M.; Duran, R.; Apicella, C.; et al. Clin. Exp. Allergy 2008, 38, 1391–1399. (19) Verdino, P.; Westritschnig, K.; Valenta, R.; Keller, W. EMBO J. 2002, 21, 5007–5016. (20) Kao, S. H.; Su, S. N.; Huang, S. W.; Tsai, J. J.; Chow, L. P. Proteomics 2005, 5, 3805–3813. (21) Sotkovsky, P.; Hubalek, M.; Hernychova, L.; Novak, P.; et al. Proteomics 2008, 8, 1677–1691. (22) Aalberse, R. C.; Akkerdaas, J. H.; van Ree, R. Allergy 2001, 56, 478– 490. (23) van Ree, R.; Voitenko, V.; van Leeuwen, W. A.; Aalberse, R. C. Int. Arch. Allergy Immunol. 1992, 98, 97–104. (24) Valenta, R.; Duchene, M.; Ebner, C.; Valent, P.; et al. J. Exp. Med. 1992, 175, 377–385. (25) Trandinh, C. C.; Pao, G. M.; Saier, M. H., Jr. FASEB J. 1992, 6, 3410– 3420. (26) Dai, S.; Li, L.; Chen, T.; Chong, K.; et al. Proteomics 2006, 6, 2504– 2529.

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(36) Foetisch, K.; Westphal, S.; Lauer, I.; Retzek, M.; et al. J. Allergy Clin. Immunol. 2003, 111, 889–896. (37) Vieths, S.; Scheurer, S.; Ballmer-Weber, B. Ann. N.Y. Acad. Sci. 2002, 964, 47–68. (38) Radauer, C.; Breiteneder, H. J. Allergy Clin. Immunol. 2007, 120, 518–525. (39) Adachi, M.; Kanamori, J.; Masuda, T.; Yagasaki, K.; et al. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 7395–7400. (40) Breiteneder, H.; Radauer, C. J. Allergy Clin. Immunol. 2004, 113, 821–830. (41) Beyer, K.; Grishina, G.; Bardina, L.; Grishin, A.; Sampson, H. A. J. Allergy Clin. Immunol. 2002, 110, 517–523. (42) Rabjohn, P.; Helm, E. M.; Stanley, J. S.; West, C. M.; et al. J. Clin. Invest. 1999, 103, 535–542. (43) Flinterman, A. E.; Akkerdaas, J. H.; Knulst, A. C.; van Ree, R.; Pasmans, S. G. Curr. Opin. Allergy Clin. Immunol. 2008, 8, 261–265.

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