Structural Similarities, in Relation with the Cross-Reactivity, of Hymenoptera Allergenic Dipeptidyl Peptidases IV-An Overall Comparison Including a New Dipeptidyl Peptidase IV Sequence from Vespa velutina.

(1) Background: Dipeptidyl Peptidases IV (DPPIVs), present in many organisms, are minor components in the venoms of Hymenoptera, where they have been identified as cross-reactive allergenic molecules. Considering that the structure of homologous DPPIVs is well characterized, we aimed to explain which regions have higher similarity among these proteins and present a comparison among them, including a new Vespa velutina DPPIV sequence. Moreover, two cases of sensitization to DPPIVs in wasp- and honeybee-sensitized patients are presented. (2) Methods: Proteomic analyses have been performed on the venom of the Asian hornet Vespa velutina to demonstrate the sequence of its DPPIV (allergen named Vesp v 3, with sequence accession number P0DRB8, and with the proteomic data available via ProteomeXchange with the identifier PXD046030). A comparison performed through their alignments and analysis of the three-dimensional structure showed a region with higher similarity among Hymenoptera DPPIVs. Additionally, ImmunoCAP™ determinations (including specific inhibition experiments), as well as IgE immunoblotting, are performed to demonstrate the allergenicity of Api m 5 and Ves v 3. (3) Results and Conclusions: The data presented demonstrate that the similarities among Hymenoptera DPPIVs are most likely localized at the C-terminal region of these enzymes. In addition, a higher similarity of the Vespa/Vespula DPPIVs is shown. The clinical cases analyzed demonstrated the allergenicity of Api m 5 and Ves v 3 in the sera of the allergic patients, as well as the presence of this minor component in the preparations used in venom immunotherapy.

Purification and molecular characterization of phospholipase, antigen 5 and hyaluronidases from the venom of the Asian hornet (Vespa velutina).

The aim of this study was to purify potential allergenic components of Vespa velutina venom, the yellow legged Asian Hornet, and perform a preliminary characterization of the purified proteins. Starting from the whole venom of V.velutina, several chromatographic steps allowed to purify the phospholipase (named Vesp v 1), as well as the antigen 5 (Vesp v 5, the only allergenic component described as such so far). The two hyaluronidase isoforms found (Vesp v 2A and Vesp v 2B) cannot be separated from each other, but they are partially purified and characterized. Purity of the isolated proteins in shown by SDSPAGE, as well as by the results of the N-terminal sequencing. This characterization and nLC-MS/MS data provide most of the sequence for Vesp v 1 and Vesp v 5 (72 and 84% coverage, respectively), confirming that the whole sequences of the isolated natural components match with the data available in public transcriptomic databases. It is of particular interest that Vesp v 1 is a glycosylated phospholipase, a fact that had only described so far for the corresponding allergen components of Dolichovespula maculata and Solenopsis invicta. The availability of the complete sequences of Vespa velutina components permits comparison with homologous sequences from other Hymenoptera. These data demonstrate the higher similarity among the species of the genera Vespa and Vespula, in comparison to Polistes species, as it is especially observed with the hyaluronidases isoforms: the isoform Vesp v 2A only exists in the former genera, and not in Polistes; in addition, the most abundant isoform (Vesp v 2B) exhibits 93% sequence identity with the Ves v 2 isoform of Vespula vulgaris. Finally, the isolated components might be useful for improving the diagnosis of patients that could be allergic to stings of this invasive Asian hornet, as it has been the case of an improved diagnosis and treatment of other Hymenoptera-sensitized patients.

A recombinant Sal k 1 isoform as an alternative to the polymorphic allergen from Salsola kali pollen for allergy diagnosis.

BACKGROUND: The incidence of Amaranthaceae pollen allergy has increased due to the desertification occurring in many countries. In some regions of Spain, Salsola kali is the main cause of pollinosis, at almost the same level as olive and grass pollen. Sal k 1 - the sensitization marker of S. kali pollinosis - is used in clinical diagnosis, but is purified at a low yield from pollen. We aimed to produce a recombinant (r)Sal k 1 able to span the structural and immunological properties of the natural isoforms from pollen, and validate its potential use for diagnosis. METHODS: Specific cDNA was amplified by PCR, cloned into the pET41b vector and used to transform BL21 (DE3) Escherichia coli cells. Immunoblotting, ELISA, basophil activation and skin-prick tests were used to validate the recombinant protein against Sal k 1 isolated from pollen. Sera and blood cells from S. kali pollen-sensitized patients and specific monoclonal and polyclonal antisera were used. RESULTS: rSal k 1 was produced in bacteria with a yield of 7.5 mg/l of cell culture. The protein was purified to homogeneity and structural and immunologically validated against the natural form. rSal k 1 exhibited a higher IgE cross-reactivity with plant-derived food extracts such as peanut, almond or tomato than with pollen sources such as Platanus acerifolia and Oleaceae members. CONCLUSIONS: rSal k 1 expressed in bacteria retains intact structural and immunological properties in comparison to the pollen-derived allergen. It spans the immunological properties of most of the isoforms found in pollen, and it might substitute natural Sal k 1 in clinical diagnosis.

Molecular aerobiology - Plantago allergen Pla l 1 in the atmosphere.

INTRODUCTION: Exposure to airborne pollen from certain plants can cause allergic disease, but allergens can also be found in non-pollen-bearing fractions of ambient air. This may explain why the allergic response in susceptible patients does not always coincide with the presence and magnitude of airborne pollen counts. Plantago pollen is an important cause of pollinosis in northern Mediterranean countries, but it is difficult to determine its incidence in allergies because Plantago pollen appears in the atmosphere at the same time as grass pollen. OBJECTIVE: The study aimed to investigate the relationship between the atmospheric concentration of Pla l 1 aeroallergen and Plantago pollen, and its incidence in a population group. MATERIALS AND METHOD: Pollen was sampled using a Hirst-type volumetric trap (Burkard) and Burkard Cyclone sampler (Burkard) for Pla l 1 allergen. Allergen was determined with a Pla l 1-specific ELISA. Serum-specific IgE levels to several plant allergens were measured with the EAST system. RESULTS: The aerobiological dynamics of Plantago pollen grains and Pla l 1 did not follow the same trend, whereas the sum of Plantago with some other pollen types showed a more similar behaviour. Of the 118 subjects tested, sera from 52 contained IgE to Plantago pollen, but only 5 were monosensitized. CONCLUSIONS: The presence of Pla l 1 in the atmosphere depends not only on Plantago pollen but also on the pollen of other species from the Oleaceae family. Knowledge of the behaviour of allergen Pla l 1 in the atmosphere can help understand better asthma exacerbations associated with aeroallergens.

Cross-reactivity between Anisakis spp. and wasp venom allergens.

BACKGROUND: Anisakiasis is caused by the consumption of raw or undercooked fish or cephalopods parasitized by live L3 larvae of nematode Anisakis spp. Larvae anchor to stomach mucosa releasing excretion/secretion products which contain the main allergens. It has been described that nematode larvae release venom allergen-like proteins among their excretion/secretion products. We investigated potential cross-reactivity between Anisakis and wasp venom allergens. METHODS: Two groups of 25 patients each were studied: wasp venom- and Anisakis-allergic patients. Sera from patients were tested by ImmunoCAP, dot-blotting with recombinant Anisakis allergens and ADVIA-Centaur system with Hymenoptera allergens. Cross-reactivity was assessed by IgE immunoblotting inhibition assays. Role of cross-reactive carbohydrate determinants (CCDs) was studied by inhibition with bromelain and periodate treatment. RESULTS: A total of 40% of wasp venom-allergic patients had specific IgE to Anisakis simplex and 20% detected at least one of the Anisakis recombinant allergens tested. Likewise, 44% of Anisakis-allergic patients had specific IgE to Vespula spp. venom and 16% detected at least one of the Hymenoptera allergens tested. Wasp venom-allergic patients detected CCDs in Anisakis extract and peptide epitopes on Anisakis allergens rAni s 1 and rAni s 9, whereas Anisakis-allergic patients only detected CCDs on nVes v 1 allergen from Vespula spp. venom. The only Anisakis allergen inhibited by Vespula venom was rAni s 9. CONCLUSIONS: This is the first time that cross-sensitization between wasp venom and Anisakis is described. CCDs are involved in both cases; however, peptide epitopes are only recognized by wasp venom-allergic patients.

Structural and immunological characterization of the N-glycans from the major yellow jacket allergen Ves v 2: the N-glycan structures are needed for the human antibody recognition.

Yellow jacket (Vespula vulgaris) hyaluronidase (Ves v 2) is a glycoprotein and a mixture of two isoallergens, Ves v 2.01 and Ves v 2.02. Wasp and bee sensitized individuals frequently show IgE antibodies that in vitro recognize common carbohydrate structures across the hymenoptera species. The aim of the study was to characterize the glycosylation patterns in Ves v 2 isoallergens and to assess their immunological properties regarding antibody binding and T cell activation. The glycosylation sites and the carbohydrate structures were verified by use of tandem mass spectrometry (MS/MS). The immunological characterization of the N-glycan structures was assessed by antibody binding, T cell proliferation and T cell epitope assays comparing native (n) and non-glycosylated recombinant (r) Ves v 2. Analyses of the Ves v 2 glycopeptides revealed that glycan attachments were found for residues 79, 99 and 127 of Ves v 2.01, and residues 66 and 81 of Ves v 2.02. Structural analysis of the glycopeptides showed that the majority of the N-glycans contained at least one alpha1,3-fucose and/or alpha1,6-fucose residues in a structure. Interestingly, serum IgE antibodies from vespid allergic patients recognized nVes v 2 but not rVes v 2. Non-glycosylated rVes v 2, however, induced T cell and cytokine responses comparable to glycosylated nVes v 2. The present study shows that N-glycan structures are needed for the antibody recognition but not for the T cell reactivity of Ves v 2 in vitro. The occurrences of carbohydrate-specific antibodies against nVes v 2, however, suggest that non-mammalian glycan structures as in nVes v 2 may provide a link between T cells and other effector cells in allergic responses.

Structure of recombinant Ves v 2 at 2.0 Angstrom resolution: structural analysis of an allergenic hyaluronidase from wasp venom.

Wasp venom from Vespula vulgaris contains three major allergens: Ves v 1, Ves v 2 and Ves v 5. Here, the cloning, expression, biochemical characterization and crystal structure determination of the hyaluronidase Ves v 2 from family 56 of the glycoside hydrolases are reported. The allergen was expressed in Escherichia coli as an insoluble protein and refolded and purified to obtain full enzymatic activity. Three N-glycosylation sites at Asn79, Asn99 and Asn127 were identified in Ves v 2 from a natural source by enzymatic digestions combined with MALDI-TOF mass spectrometry. The crystal structure of recombinant Ves v 2 was determined at 2.0 A resolution and reveals a central (beta/alpha)(7) core that is further stabilized by two disulfide bonds (Cys19-Cys308 and Cys185-Cys197). Based on sequence alignments and structural comparison with the honeybee allergen Api m 2, it is proposed that a conserved cavity near the active site is involved in binding of the substrate. Surface epitopes and putative glycosylation sites have been compared with those of two other major group 2 allergens from Apis mellifera (honeybee) and Dolichovespula maculata (white-faced hornet). The analysis suggests that the harboured allergic IgE-mediated cross-reactivity between Ves v 2 and the allergen from D. maculata is much higher than that between Ves v 2 and the allergen from A. mellifera.

Recombinant pronapin precursor produced in Pichia pastoris displays structural and immunologic equivalent properties to its mature product isolated from rapeseed.

2S albumin storage proteins from rapeseed (Brassica napus), called napins, consist of two different polypeptide chains linked by disulphide bridges, which are derived by proteolytic cleavage from a single precursor. The precursor form of the napin BnIb (proBnIb) has been cloned using a PCR strategy and sequenced. The amino-acid sequence deduced from the clone includes 31 residues of the small chain and 75 of the large chain, which are connected by the peptide Ser-Glu-Asn. Expression of the cDNA encoding proBnIb has been carried out in the methylotrophic yeast Pichia pastoris. The induced protein was secreted to the extracellular medium at a yield of 80 mg.L(-1) of culture and was purified by means of size-exclusion chromatography and reverse phase-HPLC. Recombinant proBnIb appeared properly folded as its molecular and spectroscopic properties were equivalent to those of the mature heterodimeric protein. As 2S albumin storage proteins from Brassicaceae have been shown to be type I allergy inducers, the immunological activity of the recombinant proBnIb was analysed as a measure of its structural integrity. The immunological properties of the recombinant precursor and the natural napin were indistinguishable by immunoblotting and ELISA inhibition using polyclonal antisera and sera of patients allergic to mustard and rapeseed. In conclusion, the recombinant expression of napin precursors in P. pastoris has been shown to be a successful method for high yield production of homogeneous and properly folded proteins whose polymorphism and complex maturation process limited hitherto their availability.