Clustering of olive pollens into model cultivars on the basis of their allergenic content

Chapter 2 © 2012 Morales et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Clustering of Olive Pollens into Model Cultivars on the Basis of Their Allergenic Content Sonia Morales, Antonio Jesús Castro, Carmen Salmerón, Francisco Manuel Marco, María Isabel Rodríguez-García and Juan de Dios Alché Additional information is available at the end of the chapter http://dx.doi.org/10.5772/49964 1. Introduction Olive pollen allergy is a leading cause of seasonal allergic disease in the Mediterranean countries, where olive trees are intensively cultivated and pollen grain count reaches very high levels during the pollination season (Wheeler, 1992, Liccardi et al., 1996). The level of sensitization to olive pollen among the general population is directly related to the abundance of trees as this determines allergen exposure. Nevertheless, apart from tree abundance, other factors such as genetic background may influence the incidence of sensitization to olive pollen even in areas of very high exposition (Geller-Bernstein et al. 1996).Olive trees have been cultivated in the Mediterranean basin for several millennia and this has led to the selection of a wide variety of cultivars with agronomic importance. Olive germplasm is exceptionally wide, with more than 250 cultivars only in Spain (Barranco and Rallo, 2005), probably as a direct consequence of intensive cultivation. Material commonly used for clinical and biological analysis corresponds in most cases to commercially available pollen, obtained from uncertain varietal sources. Previous studies have determined that most allergens isolated and characterized up to date are highly polymorphic (Villalba et al. 1993, 1994; Lombardero et al. 1994; Asturias et al. 1997; Alché et al. 1998; Tejera et al. 1999; Huecas et al. 2001; Martínez et al. 2002; Jiménez-López et al. 2012). Besides polymorphism, olive cultivars display broad differences in the expression levels for many allergens (Carnés et al. 2002; Conde Hernandez et al. 2002; Castro et al. 2003; Morales 2012) as well as in the number and molecular characteristics of the expressed allergen isoforms (Hamman-Khalifa et al. 2003, 2008; Hamman-Khalifa 2005; Castro et al. 2010; Jiménez-López et al. 2012). These differences are in a certain degree Current Insights in Pollen Allergens 28 maintained over the years, and have been demonstrated to be associated to the genetic background of the different olive cultivars (Fernandez Caldas et al. 2007; Morales 2012; Morales et al. this volume). Differences in the allergen composition of the extracts, particularly as regard to the olive pollen major allergen Ole e 1, are responsible of large differences in the biological potency of the extracts. Thus, Castro et al. (2003) analysed the allergenicity and Ole e 1 content in pollen samples of 10 cultivars of olive trees, and compared it to a commercial extract with no indication on varietal origin, probably representing a mixture of several cultivars. The authors found that there are important differences in the content of this major allergen and that Ole e 1 abundance correlated with total allergenicity when extracts were tested by skin prick test (SPT) on allergic patients. Interestingly some patients (about 10%) did not react with a commercial extract and only reacted to extracts coming from specific cultivars. These findings may have important implications in both diagnosis and therapy of olive pollen allergy, and in the efficacy and safety of the preparations used for specific immunotherapy (SIT) (Castro et al., 2003; Alché et al. 2007; Hamman-Khalifa et al. 2008; Jiménez-López 2008; Morales 2012). The basis for personalized SIT, based in the individual usage of olive cultivar extracts, have been described and are protected by several Spanish patents (Alché et al. 2005, 2006). However, and as handling and characterization of a large number of cultivar extracts is impracticable under industrial and clinical standards, the present work intends to define a limited number of model cultivar, characterized by distinctive pollen allergen profiles. For this purpose, a number of olive pollen extracts have been analysed in their content for several relevant allergens. After appropriate quantitation, several model cultivars have been defined to group the cultivars analysed. This model can be used as the basis for a future classification and inclusion of the numerous olive cultivars available. 2. Materials and methods 2.1. Pollen samples Olea europaea L. pollen samples were obtained during May and June of 2005-2010 from cultivated trees of the following cultivars: ΄Picual΄, ΄Manzanilla΄, ΄Arbequina΄, ΄Blanqueta΄, ΄Cornicabra΄, ΄Verdial΄, ΄Lechín΄, ΄Hojiblanca΄, ΄Lucio΄and ΄Loaime΄. Pollen samples were collected from numerous branches of at least two trees of each cultivar by shaking flowering shoots inside paper bags. Prior to its storage in liquid nitrogen, the harvested pollen was sieved through a 150 µm mesh in order to eliminate fallen corollas, anthers and other rests. After light microscopy observation, foreign-species pollen was estimated to be <0.1% and other plant parts <0.5% for all the cultivars used. 2.2. Preparation of crude protein extracts and SDS-PAGE Crude protein extracts were obtained by stirring 1 g of pollen for each cultivar in 10 ml extraction buffer (0.01 M ammonium bicarbonate, pH 8.0, and 2 mM phenylmethylsulfonyl Clustering of Olive Pollens Into Model Cultivars on the Basis of Their Allergenic Content 29 fluoride) for 8 h at 4°C. After centrifugation (2 x 30 minutes at 14,000 rpm at 4°C), the supernatants were filtered through a 0.2 µm filter, and stored in aliquots at –20°C. Protein concentration in the different samples was measured using the Bio-Rad reagent (Bio-Rad, Hercules, CA, USA) and bovine serum albumin (BSA) as standard. Proteins (30 µg per lane) and Mw1 (New England BioLabs, Ipswich, MA, USA) and Mw2 standards (MBI Fermentas, Vilnius, Lithuania) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 15% gels in a MiniProtean II system (Bio-Rad). The resulting gels were stained with Coomassie blue. The same procedure described here was applied to a commercially available extract used for olive pollen allergy diagnosis. 2.3. Immunoblotting Gels obtained as described above were transferred onto BioTrace® polyvinylidene difluoride (PVDF) membranes (Pall BioSupport, Port Washington, NY, USA) at 100 V for 1.5 hours using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad). Immunoblots were performed independently in the case of Ole e 1 (Figure 2) and Ole e 2 (Figure 3). Ole e 5 and Ole e 9 were simultaneously detected in the same membrane (Figure 4). Prior to the treatment with antibodies, the membranes were blocked with TBST buffer (Tris buffered saline: TBS + 0.3% v/v Tween 20) + 10% w/v dried skimmed milk. The membranes were probed with antibodies to the following allergenic proteins: Ole e 1, (olive pollen major allergen), Ole e 2 (profilin), Ole e 5 (Cu,Zn superoxide dismutase) and Ole e 9 (1,3-β-glucanase). The anti-Ole e 1 mAb was kindly provided by Dr. Carlos Lahoz (Fundación Jiménez Díaz, Madrid, Spain) (Lauzurica et al. 1988). The anti-Ole e 2 polyclonal antibody (PoAb) was produced by immunization of rabbits with a keyhole limpet hemocyanin (KLH)-linked synthetic peptide (AQSATFPQFKPEEM) designed from the predicted amino acid sequence of an olive profilin (Ole e 2). Specificity of the antibody was already reported by Western blotting experiments and immunolocalization of the allergen (Morales et al. 2008). The anti-Ole e 9 polyclonal Ab was produced as described above using a synthetic peptide (YPYFAYKNQPTPDT) from the Ole e 9 amino acid sequence (Huecas et al. 2001; Duffort et al. 2006). Finally, we also purchased a commercially available PoAb that recognizes a chloroplastidic isoform of Cu/Zn-superoxide dismutase (SOD) from Arabidopsis thaliana (Agrisera, city, Sweden, Product No AS06 170), with probed cross-reactivity to Ole e 5 (Zafra 2007). Primary Abs were diluted in blocking solution and incubated for 2 h at room temperature, whereas secondary Abs were diluted in TBST buffer and incubated for 1 h at room temperature in the dark. After Ab incubation, membranes were rinsed in TBST buffer four times for 5 min each. The different Abs used in this work and their corresponding dilutions are summarized in Table 1. Each experiment described below was repeated in triplicate. Negative controls included preimmune serum in the cases of Ole e 2 and Ole e 9. Current Insights in Pollen Allergens 30 Target Primary antibody Dilution Secondary antibody Dilution Ole e 1 Mouse anti-olive Ole e 1 mAb (Lauzurica et al. 1988) 1:20,000 Goat anti-mouse IgG Ab, Alexa fluor 488-conjugated (Molecular Probes) 1:10,000 Ole e 2 Rabbit anti-olive Ole e 2 PoAb (Morales et al. 2008) 1:20,000 Donkey anti-rabbit IgG (Fab fragment) Ab, Cy3conjugated (Jackson ImmunoResearch) 1:10,000 Ole e 5 Rabbit anti-Cu/Zn SOD PoAb (Agrisera Prod. No. AS06 170) 1:250 Goat anti-rabbit IgG Ab, Alexa fluor 633-conjugated (Molecular Probes) 1:10,000 Ole e 9 (Ndomain) Rabbit anti-olive Ole e 9 PoAb 1:10,000 Goat anti-rabbit IgG Ab, Alexa fluor 633-conjugated (Molecular Probes) 1:10,000 Table 1. Antibodies and dilutions used for immunoblotting experiments. mAb: monoclonal antibody; PoAb: polyclonal antibody. Imaging was carried out with a Pharos FX Plus Molecular Imager (Bio-Rad) using the Quantity One v4.6.2 software (Bio-Rad). 2.4. Absolute and relative quantitation of allergens The intensity of each fluorescent band was calculated using the quantitation tools of the Quantity One v4.6.2 software. In order to increase sensitivity of measurements and to avoid disturbing factors like the intensity of the background, the presence of individual nonspecific spots, etc., two different methods for quantitation were used:  For each allergen studied, reactive bands were identified, their optical density individually measured and then their absolute values added for each cultivar. Relative percentages of each allergen were then calculated for each cultivar, taking the cultivar with the highest optical density as the reference, which was assigned 100%.  Simultaneous measurement of the optical density corresponding to all reactive bands from a given allergen in each cultivar was also performed. As before, relative percentages were also calculated, referred to the cultivar with the highest optical density, which was assigned 100%. Finally, average of the percentages calculated by both methods was worked out, and the resulting percentages were newly made relative to the cultivar with the highest percentage, which was re-assigned 100%. Clustering of Olive Pollens Into Model Cultivars on the Basis of Their Allergenic Content 31 3. Results 3.1. SDS-PAGE protein profiles Figure 1 shows the protein profiles of the extracts analysed after SDS-PAGE and Coomassie staining. The patterns observed for the major protein species were somewhat similar for all the cultivars tested. However, clear quantitative differences were distinguished, from which the most conspicuous were those in the protein range of 17-20 kDa. Proteins within this range were relatively abundant in the extracts corresponding to the cvs. ΄Picual΄, ΄Manzanilla΄, ΄Cornicabra΄, ΄Hojiblanca΄, ΄Loaime΄, ΄Blanqueta΄ and ΄Lucio΄. When the commercial pollen extract was assayed by SDS-PAGE, a protein profile similar to the profile corresponding to the individual cultivars was observed, although several bands were absent or poorly resolved. Proteins in the range 17-20 kDa represented a low proportion of the total protein for this extract. Figure 1. Coomassie stained SDS-PAGE gel of the univarietal pollen extracts and the commercial extract (Olea europaea) after using denaturing, reducing conditions. Gels contained 30 µg total protein per lane. 3.2. Immunoblot detection and quantitation of Ole e 1 Immunoblots probed with the monoclonal antibody to Ole e 1 resulted in the presence of two major immunoreactive bands of 18 and 20 kDa, corresponding to the monomeric nonglycosylated and mono-glycosylated forms (Figure 2). Other immunoreactive bands with low quantitative relevance were observed (36, 40 y 44 kDa) in several lanes. Bands corresponding to the Mw of 18 and 20 kDa were quantitated according to the methods described above. Absolute measurements of the intensity of each individual band and both bands simultanously are displayed in Table 2, as well as the relative percentages calculated as described above. Current Insights in Pollen Allergens 32 Figure 2. Immunoblot probed with the anti-Ole e 1 monoclonal antibody. Two major bands were observed (yellow arrows), corresponding to apparent molecular weights of 18 and 20 kDa. O. europaea Picual Manzanilla Arbequina Cornicabra Verdial Lechín Hojiblanca Loaime Blanqueta Lucio 18 kDa 3672 14385 16746 11894 20033 12174 17668 30706 41345 40388 17436 20 kDa 2348 10666 11233 7983 11716 3204 11804 22780 21143 32768 13087 Σ 18 and 20 kDa 6021 25051 27979 19877 31749 15378 29472 52784 62489 73156 30523 Relative % 8.23 34.24 38.25 27.1 7 43.40 21.02 40.28 72.15 85.41 100 41.72 18 and 20 kDa 6659 26741 29626 22044 34260 20950 32493 54095 60906 73900 30345 Relative % 9.01 36.18 40.09 29.83 46.36 28.35 43.9 7 73.21 82.42 100 41.06 Average relative % 8.62 35.21 39.17 28.5 41.88 24.68 42.125 72.68 83.915 100 41.39 Table 2. Quantitation of the two major bands cross-reactive to the anti Ole e 1 antibody. Absolute data in volume units (INT*mm2). 3.3. Immunoblot detection and quantitation of Ole e 2 Immunoblots probed with the polyclonal antiserum to Ole e 2 resulted in the presence of up to five major immunoreactive bands of c.a. 14, 13.7, 14.2, 14.9 and 15.7 kDa (Figure 3). Bands corresponding to the five Mws were quantitated according to the methods described above. Absolute measurements of the intensity of each individual band and all five bands simultanously are displayed in Table 3, as well as the relative percentages calculated as described above. Clustering of Olive Pollens Into Model Cultivars on the Basis of Their Allergenic Content 33 Figure 3. Immunoblot probed with the anti-Ole e 2 polyclonal antiserum. Five major bands were observed (orange arrows), corresponding to apparent molecular weights of 14, 13.7, 14.2, 14.9 and 15.7 kDa. O. europaea Picual Manzanilla Arbequina Cornicabra Verdial Lechín Hojiblanca Loaime Blanqueta Lucio 13.0 kDa 357152 277747 248153 405554 165942 394519 537187 356024 910640 476889 350778 13.7 kDa 398025 305569 261364 312528 198300* 250846 484703 463371 1004748 454397 486318 14.2 kDa 264210 198300* 198300* 223593 147305 198300* 198300* 198300* 987772 410790 198300* 14.9 kDa 198300* 198300* 198300* 198300* 290636 198300* 198300* 198300* 198300* 198300* 198300* 15.7 kDa 198300* 198300* 198300* 217632 198300* 198300* 198300* 198300* 198300* 198300* 198300* Σ bands above 1415987 1178216 1104417 1357607 1000483 1240265 1616790 1414295 3299760 1738676 1431996 Relative % 42.91 35.70 33.47 41.14 30.31 37.59 49 42.86 100 52.70 43.39 All bands 2281004 2385713 2464789 2550651 2499993 2472138 2437099 2312637 2913894 2092950 1973168 Relative % 78.3 81.87 84.59 87.53 85.80 84.84 83.64 79.37 100 71.83 67.71 Average relative % 60.605 58.78 59.03 64.335 58.055 61.215 66.32 61.115 100 62.265 55.55 Table 3. Quantitation of the five major bands cross-reactive to the anti Ole e 2 antibody. Absolute data in volume units (INT*mm2). *: band not present. The indicated value corresponds to the average of 5 measurements made in the background. Current Insights in Pollen Allergens 34 3.4. Immunoblot detection and quantitation of Ole e 5 and Ole e 9 Immunoblots probed with the commercial antibody to Cu,Zn SOD (Ole e 5) and the polyclonal antiserum to Ole e 9 resulted in the presence of up to five major immunoreactive bands of c.a. 16, 16.5, 22, 26 and 50 kDa for Ole e 5, and two immunoreactive bands of c.a. 36 and 46.5 kDa for Ole e 9 (Figure 4). Figure 4. Immunoblot probed with the antiCu,Zn-SOD (Ole e 5) commercial antibody and the polyclonal antiserum to Ole e 9. Five major bands were observed (blue arrows), corresponding to apparent molecular weights of 16, 16.5, 22, 26 and 50 kDa for Ole e 5, and two immunoreactive bands of c.a. 36 and 46.5 kDa for Ole e 9 (red arrows). Bands corresponding to the five Mws of Ole e 5 and two of Ole e 9 were quantitated according to the methods described above. Absolute measurements of the intensity of each individual band and all five bands simultanously are displayed in Tables 4 and 5, as well as the relative percentages calculated as described above. 4. Clustering of cultivars according to their relative allergenic content Table 6 summarizes the final relative averages of reactivity calculated for each cultivar and allergen. Relative values present a wide range in the case of allergens Ole e 1 and Ole e 9, whereas Ole e 2 and Ole e 5 allergens maintain values relatively constant, higher than 50% for all cultivars, with a single exception (Ole e 5 in the cultivar ΄Lucio΄). Therefore, the following thresholds have been defined in order to divide cultivars into cultivars with high/average/low allergenic content for the allergens Ole e 1 and Ole e 9. In the case of Ole e 1, we have considered that percentages of 30% and 35% may represent reasonable limits, taking into account the extremely high content of some cultivars in this allergen, which may represents up to 23% of the total protein content for these cultivars (Castro et al. 2003). For Ole e 9, the percentages of 40% and 60% were selected as the thresholds. Clustering of Olive Pollens Into Model Cultivars on the Basis of Their Allergenic Content 35 O. europaea Picual Manzanilla Arbequina Cornicabra Verdial Lechín Hojiblanca Loaime Blanqueta Lucio 16.0 kDa 12522 10472 13810 16174 8515 16875 20320 19682 1054 793 714 16.5 kDa 33503 21491 23191 27710 33630 28904 36422 39682 27218 23811 4304 22.0 kDa 9451 7946 9623 14385 12453 9703 13812 17211 6380 5201 5041 26.0 kDa 6221 3389 3688 7134 7617 4447 7775 11424 12698 9191 7098 50.0 kDa 5611 7474 9643 4818 3941 5794 8238 19507 12979 5625 4200 Σ bands above 67308 50772 59955 70221 66156 65723 86567 107506 47350 44621 21357 Relative % 62.61 47.23 55.77 65.32 61.54 61.13 80.52 100 44.04 41.51 19.87 All bands 122909 95529 107694 136847 128879 118503 157103 155113 136354 107779 79135 Relative % 78.23 60.81 68.55 87.11 82.03 75.43 100 98.73 86.79 68.60 50.37 Average relative % 70.42 54.02 62.16 76.215 71.785 68.28 90.26 99.365 65.415 55.055 35.12 Average relative to 100 % 70.87 54.36 62.55 76.70 72.24 68.72 90.83 100 65.83 55.41 35.34 Table 4. Quantitation of the five major bands cross-reactive to the anti Cu,Zn SOD (Ole e 5) antibody. Absolute data in volume units (INT*mm2). In this case, the average relative percentage was again referred to 100%, as the maximun relative percentages previously calculated corresponded to two different cultivars (΄Hojiblanca΄ and ΄Lechín΄). O. europaea Picual Manzanilla Arbequina Cornicabra Verdial Lechín Hojiblanca Loaime Blanqueta Lucio 36 kDa 20241 18902 25868 18912 15583 17605 24746 44273 29766 28377 20736 46.5 kDa 39567 23419 17751 16764 13584 13916 20517 53338 22784 13160 12730 Σ 36 and 46.5 kDa 59808 42322 43619 35677 29167 31521 45264 97612 52550 41538 33467 Relative % 61.27 43.36 44.68 36.55 29.88 32.29 46.37 100 53.83 42.55 34.28 36 and 46.5 kDa 72676 51950 56389 47169 39589 45075 59672 102195 74531 52213 39717 Relative % 71.11 50.83 55.17 46.15 38.73 44.10 58.39 100 72.93 51.09 38.86 Average relative % 66.19 47.095 49.925 41.35 34.305 38.195 52.38 100 63.38 46.82 36.57 Table 5. Quantitation of the two major bands cross-reactive to the anti Ole e 9 antibody. Absolute data in volume units (INT*mm2).