The high molecular weight dipeptidyl peptidase IV Pol d 3 is a major allergen of Polistes dominula venom

Abstract

Hymenoptera venom allergy can cause severe anaphylaxis in untreated patients. Polistes dominula is an important elicitor of venom allergy in Southern Europe as well as in the United States. Due to its increased spreading to more moderate climate zones, Polistes venom allergy is likely to gain importance also in these areas. So far, only few allergens of Polistes dominula venom were identified as basis for component-resolved diagnostics. Therefore, this study aimed to broaden the available panel of important Polistes venom allergens. The 100 kDa allergen Pol d 3 was identified by mass spectrometry and found to be a dipeptidyl peptidase IV. Recombinantly produced Pol d 3 exhibited sIgE-reactivity with approximately 66% of Polistes venom-sensitized patients. Moreover, its clinical relevance was supported by the potent activation of basophils from allergic patients. Cross-reactivity with the dipeptidyl peptidases IV from honeybee and yellow jacket venom suggests the presence of exclusive as well as conserved IgE epitopes. The obtained data suggest a pivotal role of Pol d 3 as sensitizing component of Polistes venom, thus supporting its status as a major allergen of clinical relevance. Therefore, Pol d 3 might become a key element for proper diagnosis of Polistes venom allergy.

Conflict of interest statement

MS has received travel support from ALK-Abelló and Bencard. BE has received research funding from Bühlmann Laboratories. UD has been speaker, investigator and /or been a member of advisory boards for Allergopharma, ALK-Abelló, Bencard, GSK, Hermal, MEDA, Novartis Pharma, Stallergenes, Stiefel. TB has received research funding, speaker’s honorarium and consultancy fees from Thermo Fisher Scientific, has received research support from DFG, Novartis and Thermo Fisher Scientific, has received lecture fees from MSD, Novartis, HIPP GmbH & Co, ALK-Abelló, MedComms Ltd and Astellas Pharma GmbH. CBS-W has received grants from Allergopharma, Leti, PLS-Design and Regeneron; is member of the scientific advisory board of Leti and Bencard; has received consultancy fees from Leti, GLG Consultancy, Allergopharma and Bencard. MO has received consultancy fees from Siemens Healthcare, Hitachi Chemical Diagnostics and Bencard; has received lecture fees from Thermo Fisher Scientific, Bencard and Siemens Healthcare; is co-founder of PLS-Design GmbH. SB has received speaker’s honorarium and/or travel support from ALK-Abelló, Bencard and Thermo Fisher Scientific; has received consultancy fees as an advisory board member and research support from Bencard and Allergy Therapeutics. The other authors declare that they have no conflict of interest.

Figures

Figure 1

Detection of Pol d 3 in Polistes dominula venom. SDS-PAGE and protein staining (SyproRuby staining) of PDV (left) and sIgE-immunoreactivity of pooled sera from PDV-allergic patients with PDV in Western blot (right). The arrow indicates the 100 kDa band that was subjected to tandem mass spectrometry and MALDI-TOF analyses. Asterisks indicate the band that was identified as Pol d 5 and black bars the area of the gel/blot in which Pol 1 and Pol d 4 were identified. Shown are parts of the gel. Blot and full-length gels are shown in Supplemental Fig. S3. The kDa values correspond to the protein marker (not shown) which can be found in Fig. S3.

Figure 2

Alignment of Pol d 3 with Ves v 3 and Api m 5. Mature amino acid sequences of Pol d 3 (Polistes dominula), Ves v 3 (Vespula vulgaris) and Api m 5 (Apis mellifera) are shown. Black shaded amino acids are identical between all three proteins, gray shaded amino acids are shared by two proteins and amino acids not shaded are unique to the individual protein. Peptides identified by tandem mass spectrometry are underlined in black and potential N-glycosylation sites in gray. The residues involved in the conserved active center of the enzymes are represented boxed. Overall amino acid identity between the different proteins is stated in percent.

Figure 3

Recombinant expression and characterisation of Pol d 3. SDS-PAGE and Western blot analyses of Pol d 3 recombinantly produced in Sf9 insect cells in comparison with the HBV and YJV homologues Api m 5 and Ves v 3 either by Coomassie blue staining or anti-V5 epitope antibody, GNA (Galanthus nivalis agglutinin), anti-HRP antiserum, anti-Api m 5 antiserum and pooled sera of PDV-allergic patients. Shown are parts of one or more gels and blots and full-length gels and blots are given in Supplemental Fig. S3.

Figure 4

Basophil activation tests of (a) PDV- and/or YJV-allergic patients from Spain (area of Barcelona) and (b) HBV- and/or YJV-allergic patients from Germany with recombinant DPP IV allergens Pol d 3, Ves v 3 or Api m 5. Basophils were exposed to different concentrations of the DPP IV allergens. Additionally, stimulation with anti-FcεRI antibody (positive control) and plain stimulation buffer (negative control) is shown. Activation is shown as percentage of CD63+ out of total basophilic cells. The cut-off of the assay (10%) is represented as dotted line.

Figure 5

sIgE reactivity of individual hymenoptera venom-allergic patients with recombinant DPP IV allergens in ELISA. (a) sIgE immunoreactivity of PDV- (n = 30), HBV- (n = 28) and YJV-allergic patients (n = 20) with Pol d 3. (b) Comparative sIgE immunoreactivity of Pol d 3-positive HBV-allergic patients with Pol d 3 and Api m 5. (c) Comparative sIgE immunoreactivity of Pol d 3-positive YJV-allergic patients with Pol d 3 and Ves v 3. The lower end cut-off of the ELISAs is represented by dotted lines.