Multiple IgE recognition on the major allergen of the Parietaria pollen Par j 2

Molecular Immunology 63 (2015) 412–419

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Multiple IgE recognition on the major allergen of the Parietaria pollen Par j 2 Valeria Longo a , Maria Assunta Costa b , Fabio Cibella a , Giuseppina Cuttitta a , Stefania La Grutta a , Paolo Colombo a,∗ a b

Istituto di Biomedicina ed Immunologia Molecolare “Alberto Monroy” del Consiglio Nazionale delle Ricerche, Via Ugo La Malfa 153, Palermo, Italy Istituto di Biofisica del Consiglio Nazionale delle Ricerche (UOS Palermo), Via Ugo La Malfa 153, Palermo, Italy

a r t i c l e

i n f o

Article history: Received 5 August 2014 Received in revised form 11 September 2014 Accepted 18 September 2014 Available online 3 October 2014 Keywords: Parietaria IgE Epitope mapping Molecular biology

a b s t r a c t The interaction between IgE antibodies and allergens is a key event in triggering an allergic reaction. The characterization of this region provides information of paramount importance for diagnosis and therapy. Par j 2 Lipid Transfer Protein is one of the most important allergens in southern Europe and a wellestablished marker of sensitization in Parietaria pollen allergy. The main aim of this study was to map the IgE binding regions of this allergen and to study the pattern of reactivity of individual Parietaria-allergic patients. By means of gene fragmentation, six overlapping peptides were expressed in Escherichia coli, and their IgE binding activity was evaluated by immunoblotting in a cohort of 79 Parietaria-allergic patients. Our results showed that Pj-allergic patients display a heterogeneous pattern of IgE binding to the different recombinant fragments, and that patients reacted simultaneously against several protein domains spread all the over the molecule, even in fragments which do not contain structural features resembling the native allergen. Our results reveal the presence of a large number of linear and conformational epitopes on the Par j 2 sequence, which probably explains the high allergenic activity of this allergen. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction An allergic reaction is initiated by a series of cellular events which are primarily mediated by the interaction between IgE antibodies and allergens. The major event is the cross-linking of IgE on mast cells and basophils, leading to the release of the inflammatory mediators responsible for the immediate reaction, but other immunological mechanisms involving IgE antibodies have also been described (Acharya et al., 2010; van der Heijden et al., 1993; Ying et al., 2001). However, despite the pivotal role established for IgE in allergic reactions, little is known about the interaction between allergens and antibodies, mainly due to the low concentration of this class of immunoglobulin in the sera of patients and to the fact that IgE-producing B cells have been poorly characterized [reviewed by (Gadermaier et al., 2014)]. Most of the data related to this aspect are derived from in vitro assays using mouse monoclonal antibodies and/or recombinant chimeric human IgE derived from mouse antibodies (Tai et al., 2013; Tiwari et al., 2012). In this

∗ Corresponding author at: Istituto di Biomedicina ed Immunologia Molecolare “Alberto Monroy”, Via Ugo La Malfa 153, 90146 Palermo, Italy. Tel.: +39 91 6809535; fax: +39 91 6809548. E-mail address: [email protected] (P. Colombo). http://dx.doi.org/10.1016/j.molimm.2014.09.012 0161-5890/© 2014 Elsevier Ltd. All rights reserved.

respect, it has been demonstrated that human allergen-specific IgE may interact in a different way from mouse antibodies (Aalberse and Crameri, 2011); therefore, these strategies may not be able to fully elucidate the overall spectrum of antibody recognition. In addition, starting from the observation that the degree of effector cell degranulation is determined by IgE concentration and the number of recognized epitopes (Gieras et al., 2007), this information can be relevant for any strategy targeting IgE/allergen recognition. In this context, little is known about the Lipid Transfer Protein (LTP) family of allergens. LTPs are one of the more relevant classes of allergenic proteins in pollen and plant-derived food (Campana et al., 2011). These allergens are characterized by heat resistance and stability at acidic pH as a consequence of their compact, highly conserved three-dimensional structure, characterized by the presence of a conserved pattern of cysteine residues that forms four disulphide bonds compacting four alpha-helices. This overall fold has been detected in all the available structures of allergens belonging to this family (Pfam database PF00234). These features make this family of allergens of particular clinical interest since recent studies have shown that, in food-allergic patients, the majority of anaphylactic episodes occur in patients sensitized to LTPs (Asero et al., 2009). Parietaria judaica (Pj) pollen is one of the major outdoor allergenic sources in the Mediterranean area, with two major allergens

V. Longo et al. / Molecular Immunology 63 (2015) 412–419

413

HELIX 2

A HELIX 1 HELIX 4

HELIX 3

B

1

57

EEAC GKVVQDIMPCLHFV KGEEKEPSK ECCSGTKKLSEEV KTTEQKRE ACKCIVRATK helix 1 loop 1 helix 2 loop 2 helix 3 58

102

GISGIKNE LVAEVP KKCDIKTTLPPITA DFDCSKIQSTIFRGYY loop 3 helix 4 loop 4 beta strand Fig. 1. 3D model by homology. Panel A: Ribbon representation of the Par j 2 allergen determined by using the Swiss-Model Protein Modelling Server using the PDB entry 1FK1A as a template. Panel B: Mapping of the predicted ␣-helices and loops on the Par j 2 sequence.

which both belong to the LTP family (Par j 1 and Par j 2) (Colombo et al., 2003). In particular, the Par j 2 allergen has been recognized as the specie-specific allergen marker of Pj sensitization (Stumvoll et al., 2003) whose 3D model and disulphide bond assignment has been determined (Amoresano et al., 2003; Colombo et al., 1998). Some preliminary studies have been performed attempting to define B cell epitopes on both the Par j 1 and Par j 2 allergens (Asturias et al., 2003; Colombo et al., 1998; Costa et al., 2000). In this study, we analyzed the IgE binding activity of six overlapping regions of the Par j 2 allergen by using a set of 79 sera from Parietaria-allergic patients. This study allowed us to define a large set of IgE binding epitopes on a relatively small allergenic protein such as the Par j 2, which can explain the high allergenic potency of this protein and open the way to a rational approach for the design of hypoallergenic derivatives for the members of this family.

2. Materials and methods 2.1. Study population Between November 2005 and May 2006, 2150 children (10–17 years old) living in Palermo, in the Mediterranean area of southern Italy, completed a questionnaire based on SIDRIA and ISAAC surveys and underwent skin prick tests (SPT) at school (Cibella et al., 2011). Skin prick tests were performed according to EAACI recommendations, with a standard panel including Dermatophagoides mix, grass mix, P. judaica, olive, dog and cat dander, alternaria, and Blattella germanica, plus a positive (histamine 1%) and a negative (saline) control (Stallergènes Italia S.r.l., Milan, Italy). The reading was performed after 15 min: reactions were considered positive if the mean wheal diameter (computed as the maximum diameter plus its orthogonal divided by 2) was 3 mm or greater, after having subtracted the wheal diameter of the reaction to the negative control. Allergic sensitization was defined as the presence of at least one positive skin prick test. For the purpose of the present study, all

the 311 children showing allergic sensitization for P. judaica were recalled for further investigation. The study was approved by the Institutional Ethics Committee. All parents of invited adolescents signed a written informed consent form. According to Italian law, the respect of individual privacy was guaranteed. Of these, 79 children (mean age 15.7 years ± 0.9; 50 males) gave their consent and, on the day of the study, each subject underwent a new skin prick test and blood sampling. Sera were tested for the presence of Par j 2 specific antibodies by means of Western blot. A non-allergic subject was enrolled as a negative control. 2.2. In silico analysis 3D modelling was performed using the SWISS MODEL Workspace (http://swissmodel.expasy.org/workspace) and the Predict Protein software (https://www.predictprotein.org). Putative antigenic determinants were analyzed by the ElliPro Prediction software (http://tools.immuneepitope.org/tools/ElliPro/ iedb input). Based on the 3D structure of a protein antigen, ElliPro predicts linear and discontinuous antibody epitopes by homology modelling. ElliPro associates each predicted epitope with a score, defined as a PI (Protrusion Index) value, averaged over epitope residues. For each residue, a PI value is defined as percentage of the protein atoms enclosed in the ellipsoid, which approximates the protein surface, at which the residue first comes to lie outside the ellipsoid; for example, all residues that are outside the 90% ellipsoid will have PI = 9 (or 0.9 in ElliPro). Prediction was performed using the 1FK1A PDB entry. Peptide similarity was studied using the Structural Database of Allergenic Proteins (SDAP) software (https://fermi.utmb.edu/). 2.3. Gene fragmentation and recombinant protein expression Different clones were obtained by PCR amplification of the Par j2.0101 template (accession number X95865). The

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Fig. 2. In silico epitope prediction. Panel A: Predicted linear and discontinuous epitopes of the Par j 2 sequence. Numbers indicate the position of the amino acids involved in the predicted antibody binding. Score is the Protrusion Index of the predicted epitope. Panel B: Schematic representation of the predicted epitopes on the 3D model of the Par j 2 allergen.

oligonucleotides were flanked by the restriction enzyme EcoRI and XbaI recognition sites, plus 3 nucleotides for best recognition of the restriction sites. The oligos used for cloning were: Parj2/for 5 cgcGAATTCatgccgtgcctgcatttc 3 ; Parj/2rev 5 cgcTCTAGActaatagtaacctctgaa 3 (forward primer wild type Pa r j 2); Parj2/30 5 cgcTCTAGAtgcagcactccttcgacgg 3 (reverse primer fragment A); Parj2/54 5 cgcTCTAGAgcactatgcacttgcaggcctc 3 (reverse primer fragment B); Parj2/37 5 cgcGAATTCgcggcacgaagaagctgagc 3 (forward primer fragment C); Parj2/48 5 cgcGAATTCagacgacggagcagaagagg 3 (forward primer fragment D); Parj2/60 5 cgcGAATTCacgaagggcatctccg 3 (forward primer fragment E); Parj2/76 5 cgcTCTAGActtaatatcgcacttcttgg 3 (reverse primer fragments C–D). (Upper case indicates the restriction enzyme sites introduced for cloning in the expression vector; italics indicate nucleotides introduced for improving restriction enzyme cutting efficiency; lower case indicates the Par j 2 coding regions.) The fragments generated by PCR for subcloning mapped from amino acid 1 to 30 (fragment A); from amino acid 1 to 54 (fragment B); from amino acid 37 to 76 (fragment C); from amino acid 48 to 76 (fragment D); from amino acid 60 to 76 (fragment E) and from amino acid 60 to 102 (fragment F). Fragment A expresses a peptide containing the loop 1 region (from aa 20 to 26); Fragment B loops 1 and 2 (from aa 20 to 26 and from 42 to 46, respectively), Fragment C loops 2 and 3 (from aa 41 to 48 and from 58 to 65, respectively);

Fragment D loop 3 (from aa 58 to 65); Fragment E a portion of loop 3 (from aa 60 to 65); Fragment F a portion of loop 3 and all of loop 4 (from aa 60 to 65 and from 73 to 87, respectively). PCR products were purified, digested with EcoRI and XbaI restriction enzymes, and cloned in frame in the EcoRI-XbaI sites of the pMALC2 vector (BioLabs, UK). All the clones were sequenced to check the open reading frame. The recombinant clones were grown at 37 ◦ C to a density of 0.5 ± 0.6 OD600 in LB broth with the appropriate antibiotic and induced for 2 h with 0.3 mM isopropylthio-␤-d-galactoside (IPTG). The cells were harvested by centrifugation at 4000 × g for 20 min, and the pellet was then dissolved in 1× PBS (10 mM sodium phosphate, pH 7.2, 200 mM NaCl, 1 mM EDTA, and 1 mM NaN3) and lysed by sonication with the Heat System Ultrasonic, W-385. Cell debris was removed by centrifugation at 9000 × g for 30 min. Supernatants were collected and analyzed by SDS-PAGE and Coomassie blue staining. A similar procedure was performed with a nonrecombinant empty pMAL-C2 vector to yield Escherichia coli total extract for negative controls (pMAL lysate control). 2.4. Immunoblot analysis Twenty ␮g of total E. coli protein cell lysate from recombinant clones were fractionated on 16% SDS-PAGE and electroblotted

V. Longo et al. / Molecular Immunology 63 (2015) 412–419

1

4

rParj 2

102

14----s-s---- 29 --------------------------s-s ---------------52 L1

L3

L2

----s-s----39 42

20

1

415

46

26 27

L4

91 50------------------------s-s---------------

14 ----s-s----29 30

A

L1

20

1

4

B

26

----s-s----29 ------- 14 -------------------s-s --------------52 L1

20

54

L2

26 27

----s-s----39 42

46

37

C

76 L2

41

L3

48

58

65

48

76

D

L3

58

65

60

E

76

L3

60

F

102 L3

L4

73

87

Fig. 3. Schematic representation of w.t. Par j 2 and engineered peptides. Numbers indicate the size of the peptides and the position of the cysteine residues. Dotted lines show the putative disulphide bonds in the w.t. sequence (rPar j 2) and in the six recombinant derivatives. Empty white boxes indicate the predicted loop regions (loops 1–4).

onto PVDF membranes (Immobilon P Millipore, USA). After blotting, membranes were incubated for 1 h with blocking buffer (PBS supplemented with 3% BSA, 0.5% Tween-20, and 0.02% NaN3) and washed three times with PBS containing 0.1% Tween-20. The filters were then incubated overnight with the single sera (1:5 dilution). A non-allergic serum was used as negative control. After washing, the filters were incubated for 1 h with horseradish peroxidase HRP-conjugated rabbit antihuman IgE (1:5000 dilution) (Biosource International, USA). IgE reactive bands were visualized by using SuperSignal West Pico Maximum Sensitivity Substrate (Pierce Biotechnology Inc, Rockford, USA) and subsequent exposure to Kodak X-OMAT X-ray film (Kodak, New York, NY). The intensity of the Par j 2 signals was evaluated using a reference serum (++++ ≥60 kU/L; +++ ≥35 kU/L; ++ ≥10 kU/L and +≤10 kU/L) and densitometric analysis (Quantity ONE Software, Biorad, USA). 2.5. Statistical analysis Kendall Rank Correlation was used for evaluating the correlation between not normally distributed variables.

regions between amino acids 1–5, 19–28, 58–66 and 85–102 (Fig. 2, panel A). In addition, using the SDAP web server, these regions did not match with any other LTPs in the database, confirming the absence of cross-reactivity between Par j 2 and other LPTs in the database. Furthermore, using the same algorithm, three discontinuous epitopes were predicted (Fig. 2, panel A). The first one was mapped within the loop 1 region lying between helix 1 and helix 2. The remaining epitopes were mapped within the loop 3 and the loop 4 regions covering a large number of amino acids (Fig. 2, panel A). A 3D schematic representation of the predicted epitopes on the target surface is shown in panel B of Fig. 2. 3.2. Cloning and expression of recombinant Par j 2 fragments Six overlapping fragments covering the entirety of the Par j 2 sequence were generated by PCR, as schematically shown in Fig. 3. The recombinant fragments were expressed as fusion proteins with the E. coli Maltose-Binding Protein (MBP) to get a comparable and efficient expression of the small peptides (in particular peptides D and F). Protein expression was evaluated by SDS-PAGE, followed by Coomassie brilliant blue staining showing a high level of expression for all the fragments (Fig. 4).

3. Results 3.3. Immunoblot analysis 3.1. In silico epitope prediction In silico structural analysis of Par j 2 performed using the 1FK1A PDB structure as a model showed a canonical ␣–␣–␣–␣–␤ LTP structure (Fig. 1, panel A). The predicted position of ␣-helices and loops are indicated in panel B of Fig. 1. For the prediction of putative IgE binding regions within the Par j 2 allergen, the full-length sequence was scanned using the Ellipro tool. The software identified four predicted regions of putative linear epitopes mapping the

Seventy-nine Parietaria-allergic patients were enrolled in this study, and their individual IgE reactivity towards the sixengineered fragments was evaluated by Western blot analysis. Fig. 5 shows a representative analysis performed on 5 allergic patients and one non-allergic subject (negative control). The negative control did not show any Ab binding with the whole set of fragments (Fig. 5, panel NA). Non-recombinant MBP protein did not show any IgE binding in the whole set of sera studied (data not

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20

Number of patients

18 16 14 12 10 8 6 4 2 0 1 Fig. 4. SDS-PAGE. (A) Coomassie staining of purified recombinant MBP (UN) and the six recombinant Par j 2 derivatives (fragments A–F). M line indicates a molecular mass marker, and numbers display the relative molecular weight.

shown). All the subjects reacted against the full-length rPar j 2 (data not shown). Table 1 summarizes the pattern of reactivity for the Parietaria-allergic population, showing that all the fragments were recognized by the IgE Abs with a heterogeneous pattern of reactivity between individuals. In particular, fragments A and B, covering the regions from amino acid 1 to 30 and from amino acid 1 to 54, were recognized by the majority of the sera, with values of 80% and 76%, respectively. These two fragments are the only two engineered peptides capable of forming disulphide bonds resembling the native configuration. Indeed, a very high prevalence of recognition was observed for fragment C as well. Interestingly, this peptide does not contain any features of the native structure (i.e. formation of disulphide bonds) suggesting the presence of dominant linear epitope/s. Finally, patients reacted to a lower extent to fragments D–F, with percentages ranging from about 60% to 40%. Taken all together, these data demonstrate that IgE reactivity was higher in the NH2 terminal region of the protein, but antibody binding

2

3

4

5

6

Number of fragments Fig. 6. Frequency distribution of the number of fragments recognized by each subject’s serum.

was detected all over the molecule, independently of the threedimensional structure of the protein. These data are consistent with the in silico analysis, in which both linear and discontinuous epitopes were predicted. Furthermore, we observed that the majority of the sera recognized a large number of fragments. Fig. 6 shows a histogram indicating that more than 80% of the patients presented simultaneous IgE reactivity towards a number of fragments ≥3, supporting the hypothesis that patients produce IgE versus multiple sites along the molecule. This seems to be particularly relevant for the two non-overlapping fragments, A and C (containing loops 1–3), which were both recognized by 80% of all the tested sera. Although a positive trend between the intensity of the IgE binding to Par j 2 and the number of recognized fragments has been observed, no statistical correlation has been detected (Fig. 7 p = 0.13, Kendall Rank Correlation).

Fig. 5. Western blot analysis of purified recombinant MBP (UN) and the six recombinant Par j 2 derivatives (fragments A to F). The Figure shows a representative binding with 5 Parietaria-allergic patient sera (P01-P16-P26-P28-P43) and one non-allergic control serum (NA).

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417

Table 1 (Continued)

N

Par j 2

Fr. A

Fr. B

Fr. C

Fr. D

Fr. E

Fr. F

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76

+++ +++ +++ ++++ + ++ ++++ ++ +++ ++ +++ +++ ++++ ++++ +++ ++++ ++++ ++ ++ ++++ ++ +++ ++++ ++ ++ ++ +++ ++ ++++ ++ ++ +++ ++++ ++ ++++ ++++ ++ +++ +++ ++ ++ +++ ++ +++ ++ ++ +++ ++++ ++ ++ ++++ +++ ++ + + +++ ++++ + ++ ++++ ++++ ++ ++++ + ++++ + + + + +++ + + ++ ++++ ++ +

− − + + − − + + + − + + + + + − −− + + + + + − − + + + + + + + + + + + + + + − + + + + − − − + + + + + + + + + + + + + + + + + + + + + + − + + + + + + +

− + + + − − + + + − + + + + + + + + − + + − + − + + + − + − + + + + + + + + − + + + + − − − + + + + + + + − − + + + + + + + + − + + + + − − + + + + + +

+ − + + + + + + + + + + + + − + − − + + + + + + + + + + + + + + − + + + + + + − + + + + − + + + − + + + − + − + + + − + + + + + + + + + − + + + − + + +

− − + − + + + − + + − + − + − − − + − + + − + + + − − + + + + − − − − + − + + − + + − + + + + + + − + − − − + + − + + + + − + − + + + + − + + − − + + −

− − − − − + + + + + − + − + − − − + − + + + + − + − − + + + + − − − − + + + − − + + − + + + + − + − + − − + + + + + + + + + + + + + + + + + − − − + + −

− − − − − − + − + − − + − − − − − − − − − + − − − − − − − − − − − + − − + + + − + + − + − + + − + + − − − + + + + − + − + − − + + + + + + + − − + + + +

N

Par j 2

Fr. A

Fr. B

Fr. C

Fr. D

Fr. E

Fr. F

77 78 79 Percentage of recognition

++ ++ +

− + − 80%

− + + 76%

− + − 80%

− + + 60%

+ − + 60%

− − − 40%

5

Tot no. of recognized fragments

Table 1 Individual Par j 2 and its derivatives IgE recognition.

4

3

2

1

0 1

2

3

4

Intensity of the Par j 2 signal Fig. 7. Relationship between total number of recognized fragments and Par j 2 intensity. Horizontal bars indicate group medians. p = 0.13, Kendall Rank Correlation.

4. Discussion Allergic reactions to LTPs represent a relevant clinical problem since it has been demonstrated that this widespread class of proteins can act as life-threading allergens (Asero and Pravettoni, 2013). The experimentally determined 3D structure of several LTPs demonstrated the presence of four ␣-helixes separated by short turns, and a flexible and non-structured C-terminal coil (Fig. 1) packed with four disulphide bridges, forming a large internal hydrophobic tunnel-like cavity capable of harbouring lipids (Marion et al., 2007). However, despite such a conserved structure and homology at the amino acid level, studies of allergen crossreactivity have shown that not all the components of this family share common IgE epitopes (Lombardero et al., 2004; Tordesillas et al., 2011). In particular, Par j 2 LTP is the most important allergen involved in Parietaria allergy, with a very high prevalence of sensitization in southern Europe (Scala et al., 2010) and no demonstrated cross-reactivity with other LTPs (Tordesillas et al., 2011). The investigation of the IgE/allergen interactions of the Par j 2 LTP may be of paramount importance for the development of vaccine design, disease diagnosis and allergy research. Many algorithms have been developed to predict B cell epitopes, which are based on the characteristics of the amino acid properties together with the structural context in the molecules, such as hydrophilicity/hydrophobicity profiles, flexibility and accessibility (Pomes, 2010). However, in the case of allergens, this surface undergoes unknown modifications during the crossing of the epithelial barrier, which make this prediction more complex (Greenbaum et al., 2007). Furthermore, as shown for food allergens, processing or digestion can modify the accessibility of amino acid residues and increase the IgE binding properties of the allergen (Vissers et al., 2011). Using a PCR-based strategy to address this question, we designed six overlapping regions which were expressed in E. coli as recombinant MBP-fusion protein to allow the high and comparable expression of peptides of different sizes. Furthermore, considering the heterogeneity in the immunological response and

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the polyclonality of the IgE repertoire (Gadermaier et al., 2014), we decided to perform a study in a large set of patients (n = 79) to attempt to identify the domains of the protein most often involved in IgE recognition. The humoral response against the Par j 2 fragments confirmed the immunological relevance of the NH2 terminal region (Colombo et al., 1998) and further highlighted the identification of additional epitopes in the C-terminal domain of the allergen. In particular, our in vitro data showed that fragments A and B (containing the protein domain surrounding the loop 1 and loop 1 + loop 2 regions, respectively) were recognized by the majority of the patients (about 80%), suggesting that these protein domains contain highly immunogenic regions. It is noteworthy that these two derivatives are the only two engineered peptides capable of forming cysteine pairing like that contained in the full-length wild type Par j 2. Our previously published data demonstrated that the disruption of cysteine residues C4 , C29 and C30 dramatically reduced the IgE binding and allergenicity of both Par j 1 and Par j 2 (Bonura et al., 2007). Taken together, these data support the hypothesis that the N-terminal region of the allergen is highly immunogenic and that its IgE binding activity is dependent by the secondary structure of the protein. On the other hand, the correlation between natural folding and IgE reactivity does not seem to be essential for the C-terminal region of Par j 2. In fact, fragments C to F do not have any cysteine residue capable of forming native disulphide bonds yet still retain a strong capability of binding IgE antibodies as well. This seems to be particularly important for fragment C, which displays a high prevalence of reactivity in our population (about 80% of the patients). It is important to notice that fragments A and C express non-overlapping regions and contain 3 out of 4 of the highly protruding surface exposed loops (Bonura et al., 2012; Colombo et al., 1998) (loops 1 to 3) identified by the 3D modelling, revealing that both fragments contain immunodominant IgE epitopes. This first set of data can be summarized by showing that (1) all subjects reacted against the full-length Par j 2, showing that our population is a genuinely Parietaria-allergic population (Stumvoll et al., 2003); (2) Pj allergic patients displayed a heterogeneous pattern of IgE binding to the different recombinant fragments; (3) there was no correlation between the intensity of the signal to rPar j 2 and the number of fragments recognized by the same serum; (4) patients reacted simultaneously against several protein domains spread all the over the molecule, even in fragments which do not contain structural features resembling the native allergen (Table 1 and Fig. 3). A second relevant point coming out of our study is that the majority of the patients reacted against a large set of fragments. Our data show that more than 80% of the patients recognized more than three peptides (Fig. 6). Furthermore, it could be speculated that the disruption of the native structure of an allergen by gene fragmentation will probably lead to the underestimation of the number of epitopes in vivo, since this strategy identifies the most relevant epitopes and/or IgE antibodies with more affinity, thus missing epitopes which have been disrupted by the fragmentation itself, suggesting that a larger number of the Par j 2 regions can form an epitope. This observation raised the question of whether, despite the low molecular weight of the Par j 2 LTP (about 10 kDa), this allergen can be bound by several antibodies simultaneously and thus be extremely harmful in terms of allergenic potential. In fact, the number of IgE epitopes on an allergenic molecule (as well as the concentration and affinity of the allergen specific antibodies) not only determines the extent of degranulation (Gieras et al., 2007), but also influences the facilitated antigen presentation-mediated T cell activation (Holm et al., 2011) defining the potency of an allergen. The high density IgE recognition of Par j 2 described in our study is also in agreement with similar findings reported for other pollen (Bet v 1 (Gieras et al., 2011) and Der f 2 (Lollier et al., 2014))

and food major allergens (Bos d 5, Hev b 6 and Ara h 2) (Lollier et al., 2011) (see (Lollier et al., 2014) for a meta-analysis), implying that allergens can accommodate several different IgE antibodies on small areas. 5. Conclusions Our study provides new evidence of the presence of a large number of IgE epitopes distributed along the protein sequence of the major Parietaria Par j 2 allergen showing a highly immunogenic N-terminal region of the allergen and an unconstrained Ab recognition in the C-terminal region of the molecule. Individual IgE binding showed a heterogeneous pattern of recognition with a large number of epitopic regions detected by serum antibodies. These observations could be important for the development of new therapeutic options for LTP-induced mediated hypersensitivity reaction. References Aalberse, R.C., Crameri, R., 2011. IgE-binding epitopes: a reappraisal. Allergy 66, 1261–1274. 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