Major Pollen Allergen Research Articles

The Influence of Recombinant Production on the Immunologic Behavior of Birch Pollen Isoallergens

BackgroundAllergic reactions towards the birch major pollen allergen Bet v 1 are among the most common causes of spring pollinosis in the temperate climate zone of the Northern hemisphere. Natural Bet v 1 is composed of a complex mixture of different isoforms. Detailed analysis of recombinant Bet v 1 isoforms revealed striking differences in immunologic as well as allergenic properties of the molecules, leading to a classification of Bet v 1 isoforms into high, medium, and low IgE binding proteins. Especially low IgE binding Bet v 1 isoforms have been described as ideal candidates for desensitizing allergic patients with allergen specific immunotherapy (SIT). Since diagnosis and therapy of allergic diseases are highly dependent on recombinant proteins, continuous improvement of protein production is an absolute necessity.MethodologyTherefore, two different methods for recombinant production of a low IgE binding Bet v 1 isoform were applied; one based on published protocols, the other by implementing latest innovations in protein production. Both batches of Bet v 1.0401 were extensively characterized by an array of physicochemical as well as immunological methods to compare protein primary structure, purity, quantity, folding, aggregation state, thermal stability, and antibody binding capacity.ConclusionThe experiments demonstrated that IgE antibody binding properties of recombinant isoallergens can be significantly influenced by the production method directly affecting possible clinical applications of the molecules.

Targeting the cysteine-stabilized fold of Art v 1 for immunotherapy of Artemisia pollen allergy

Plants of the genus Artemisia domestic in Northern and Central Europe, USA and parts of Asia are a major cause of allergic symptoms from late summer to autumn. Art v 1, the major mugwort pollen allergen appears as two-domain glycoprotein, consisting of an N-terminal defensin-like and a proline/hydroxyproline-rich C-terminal part. Patients sensitized to Art v 1 commonly display IgE antibodies against the cysteine-stabilized defensin fold. Site-directed mutagenesis of eight cysteines was used to disrupt disulfide bonds to generate molecules with altered IgE-binding capacity. Engineered constructs were expressed in E. coli and recombinant proteins were tested for their allergenic and T cell reactivity as well as for their physicochemical characteristics. Three cysteine variants (C22S, C47S, and C49S) exhibited extremely low IgE-binding activity in immunoblot and ELISA using sera from Art v 1-allergic patients. Mediator release assays using rat basophil leukemia cells showed that these variants displayed a 1×105-fold reduced allergenic potency as compared to wild-type protein. All variants were able to activate allergen-specific T cells in PBMC, as well as Art v 1-specific T cell lines and clones. Variant C49S displayed an increased hydrophobic surface potential which correlated with an advanced activation of allergen-specific T cells. The low allergenicity and high immunogenic activity of Art v 1 variant C49S renders the molecule an attractive candidate for hypoallergen-based immunotherapy of Artemisia pollen allergy.

Graph-theoretical comparison of protein surfaces reveals potential determinants of cross-reactivity and the molecular mimicry

Different proteins, even without sequence similarity, still can contain similar surface regions involved in protein–protein interactions with common target. These regions can serve as structural determinants of cross-reactivity and molecular mimicry. Molecular mimicry, defined as the process in which structural properties of one molecule are simulated by the dissimilar molecules, is implicated in several biologically important processes, including autoimmune and allergic reactions, binding of some ligands to common receptor, and interactions in cell signaling. The problem of identification of the determinants of molecular mimicry is not completely solved at this time. We hypothesize that identification of structurally and chemically similar surface regions of two protein molecules capable of binding to the same target will allow us to identify sites involved in cross-reactivity including determinants of the molecular mimicry. We used a graph-theoretical approach in order to determine highly similar surface regions of two proteins with known three-dimensional structures. This approach uses a variation of Maximal Common Subgraph (MCS) isomorphism, where an association graph is constructed based on the surface-exposed residues of the two molecules and the matching regions are found based on the maximum cliques in the association graph. Testing the proposed method on the targets of autoantibody involved in antiphopholipid syndrome (APS) – β2-GPI, PC, thrombin, factor IX, factor X, and plasmin allowed identifying potential epitopes for antibody that can inhibit coagulation proteases. Application of this method to the Activated Protein C and factor VII Gla-domains revealed surface regions involved in EPCR and plasma membrane binding, consistent with known experimental results. Analysis of major pollen allergen that can cause food allergies through cross-reactivity found known epitopes involved in cross-reactivity and also revealed additional surface regions that can complement the list of epitopes. Taken together, our results suggest that the proposed graph-theoretical approach can identify determinants of cross-reactivity and molecular mimicry.

Timothy grass pollen major allergen Phl p 1 activates respiratory epithelial cells by a non‐protease mechanism

Group 1 allergens from grass pollen (e.g. Phl p 1, the major allergen of timothy grass Phleum pratense) cause IgE reactivity in about 95% of allergic subjects and exist in all grass species. The respiratory epithelium represents a first line of contact of the immune system with airborne allergens, functions as physical barrier and is an important immunological regulation system. The aim of this study was to investigate the interaction of Phl p 1 with human respiratory epithelium to elucidate the contribution of epithelial cells to the development of allergic reactions. Purified Phl p 1 was used to stimulate A549 cells and transient transfected HEK293 cells. mRNA level of different mediators were investigated by real-time PCR, release of the mediators was determined by ELISA. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and an ex vivo model of the murine trachea were used to investigate a potential proteolytic activity of Phl p 1. Phl p 1 activates respiratory epithelial cells as measured by induction of IL-6, IL-8 and TGF-beta mRNA and release. Phl p 1, in contrast to Der p 1 from the house dust mite, does not exert proteolytic activity, as investigated by microscopic observation and MTT test. In an ex vivo model of the murine trachea we were able to show that Der p 1, in contrast to Phl p 1, enhances the transportation velocity of particles by the trachea, presumably by ATP released from the injured epithelium. We conclude that under physiological conditions Phl p 1 affects tracheal epithelial cells through a non-proteolytic activity. Enhancement of TGF-beta expression induced by Phl p 1 together with the increased release of IL-6 and IL-8 might provide an indirect mechanism through which the allergen may cross the epithelial barrier and attracts immunocompetent cells.

Modulation of allergen-specific T-lymphocyte function by virus-like particles decorated with HLA class II molecules

T(H)2 lymphocytes play an important role in the induction and maintenance phase of type I allergy. Modulation of the responses of T(H)2 lymphocytes by novel forms of antigen-presenting platforms may help shape the immune response to allergen and palliate allergic diseases. To present HLA class II/allergen-peptide complexes on virus-like particles (VLPs) and to evaluate their potential to modulate allergen-specific T-cell responses. Virus-like particles that express the immunodominant T-cell epitope Art v 1(25-34) of the major mugwort pollen allergen in the context of HLA-DR1 and costimulatory molecules were produced by transfection of 293 cells. The effect of VLPs on IL-2 promoter activity, proliferation, and cytokine production of allergen-specific T cells derived from donors with and without mugwort pollen allergy was determined. Flow-cytometric analyses showed that HLA class II molecules, invariant chain::Art v 1 fusion proteins, and costimulatory molecules were expressed on 293 cells. Biochemical analyses confirmed that these molecules were efficiently targeted to VLPs. The engineered VLPs activated Art v 1-specific T cells in a costimulation-dependent manner. VLPs lacking costimulators induced T-cell unresponsiveness, which was overcome by addition of exogenous IL-2. Costimulation could be provided by CD80, CD86, or CD58 and induced distinct cytokine profiles in allergen-specific T cells. Unlike the other costimulatory molecules, CD58 induced IL-10/IFN-gamma-secreting T cells. Virus-like particles represent a novel, modular, acellular antigen-presenting system able to modulate the responses of allergen-specific T cells in a costimulator-dependent fashion. Allergen-specific VLPs show promise as tools for specific immunotherapy of allergic diseases.

Reduction of Antigenicity of Cry j 1, a Major Allergen of Japanese Cedar Pollen, by Thermal Denaturation

The soluble aggregates of Cry j 1, a major allergen of Japanese cedar pollen, were formed without any coagulates during heat treatment at acidic pH 5, as shown in HPLC and SDS-PAGE patterns. A remarkable change in the CD spectrum was observed between native and heat-denatured Cry j 1 at a linear rate of 1 degrees C/min from 40 to 90 degrees C. The negative peak of native Cry j 1 at 222 nm was moved to 218 nm, suggesting the transition of an alpha-helix to beta-structure during heat denaturation. The increase in beta-structure was also observed during heat denaturation by monitoring the fluorescence with Thioflavin T. These results suggest that Cry j 1 forms intermolecular cross-beta-structure between denatured proteins during heating at 90 degrees C. The antigenicity of Cry j 1 detected by dot-blotting was greatly diminished during heating at a linear rate of 1 degrees C/min from 40 to 90 degrees C without any coagulates. These results suggest that IgE epitopes exposed on the molecular surface of Cry j 1 was buried inside soluble aggregates through intermolecular beta-structure formed by heating.

Role of the polypeptide backbone and post-translational modifications in cross-reactivity of Art v 1, the major mugwort pollen allergen

Artemisia vulgaris (mugwort) is one of the main causes of late summer pollinosis in Europe, with >95% of patients sensitized to the glycoallergen Art v 1. Despite the importance of this allergen, little is known about its cross-reactive behavior. Here we investigated the occurrence of conserved Art v 1 antigenic determinants in sources known to display clinically relevant cross-reactivity with mugwort pollen. For this purpose, monoclonal antibodies specific for a cysteine-stabilized epitope of the Art v 1 defensin domain and for carbohydrates attached to the proline domain were produced by hybridoma and phage display technologies. Using polyclonal Art v 1-specific rabbit sera and antibodies against both the Art v 1 carbohydrate and polypeptide moieties, we could identify cross-reactive structures in pollen from botanically related Asteraceae weeds (Artemisia absinthium, Helianthus annuus and Ambrosia sp.). Homologous allergens were also recognized by IgE from mugwort-sensitized patients and the reactivity could be decreased by serum pre-incubation with natural and recombinant Art v 1. As no cross-reactive structures could be found in foods associated with mugwort pollinosis, we conclude that Art v 1 is poorly involved in mugwort cross-reactivity to food allergens.

3-Layer-based analysis of peptide–MHC interaction: In silico prediction, peptide binding affinity and T cell activation in a relevant allergen-specific model

CD4+ T cells recognize peptides bound to major histocompatibility complex (MHC) class II molecules on the surface of antigen presenting cells by their T cell receptor (TCR). Using a well-characterized allergen-specific model we studied peptide/MHC (pMHC) interactions by combining computational methods with experimental analyses. A 12-mer and an 18-mer peptide, both containing the human leukocyte antigen (HLA)-DR1-restricted, immunodominant T cell epitope of Art v 1, the major mugwort pollen allergen, were compared. A Molecular Dynamics simulation for a real time of 20 ns using GROMACS was performed. To this aim, the peptides were modelled into the binding groove of HLA-DRB1*0101 using different amino acid substitution tools. Binding of synthetic peptides to purified HLA-DRB1*0101 molecules was analysed in competition assays. The potency of the peptides to activate Art v 1-specific T cells was assessed using oligo- and monoclonal Art v 1-specific T cell cultures expanded from mugwort allergic individuals. All approaches revealed that the 18-mer peptide possessed higher HLA DR affinity as compared to the 12-mer. Computer modelling indicated that a loop-like structure within the additional N-terminal peptide flanking region of the 18-mer contributed to the pMHC interaction. Our approach, to combine computational methods validated by experimental results, demonstrates that Molecular Dynamics simulation may be a useful tool for the prediction of pMHC interactions in the future with possible applications in T cell-based immunotherapy e.g. in Type I allergy.

Allergenicity and immunogenicity of the major mugwort pollen allergen Art v 1 chemically modified by acetylation

Treating allergies with modified allergens is an approach to make the treatment safer and more efficient. Art v 1 is the most prominent allergen of mugwort pollen and a significant cause of hayfever around Europe. The aim of this study was to reduce the allergenicity of Art v 1 by acetylation, and to investigate the capacity of the modified protein to generate blocking antibodies. The reduction of allergenicity of Art v 1 following acetylation was monitored by immunoblot, ELISA inhibition using a pool of sera from mugwort pollen allergic patients, basophil activation assay and by skin prick testing of mugwort-allergic patients. Rabbits were immunized against Art v 1 and acetylated Art v 1 (acArt v 1) and the rabbit antisera were tested for their capacity to block human IgE binding in ELISA. Human T cell proliferation against Art v 1 and acArt v 1 was examined in peripheral blood mononuclear cells (PBMCs) of mugwort pollen allergic patients and cytokine release in PBMC cultures was monitored. Acetylation of Art v 1 gave a derivative of reduced allergenicity in the in vitro and ex vivo tests applied. The skin test reactivity to acArt v 1 was significantly reduced in 19 patients when compared with the reactivity to Art v 1. Rabbit antibodies to acArt v 1 and Art v 1 showed similar capacity to block human IgE binding to Art v 1 in inhibition ELISA. Both proteins were able to induce proliferation of PBMCs and CD3/CD4(+) cells of mugwort-allergic patients. Release of IL-5 was significantly reduced in cultures stimulated with acArt v 1. Art v 1 modified by acetylation had a significantly reduced allergenicity in vitro and in vivo, while its immunogenicity was retained. Modification of allergens by acetylation could be a new strategy for allergen-specific immunotherapy.

Chemical modification of Art v 1, a major mugwort pollen allergen, by cis-aconitylation and citraconylation

Art v 1 is the major allergen of mugwort (Artemisia vulgaris) pollen, a significant cause of hay fever all over Europe. Specific immunotherapy is the only treatment modality for allergic disease. Application of modified allergens makes the treatment safer and more efficient. In this work, two out of three (citraconic anhydride, cis-aconitic anhydride, 2,3-dimethylmaleic anhydride) tested anhydrides were proven to be suitable for chemical modifications of allergens. Art v 1 was modified by cis-aconitylation and citraconylation in order to obtain derivatives of Art v 1 that may be suitable for further immunological testing. Acylation of Art v 1 gave derivatives (caaArt v 1 and citArt v 1) with about 80 % modified amino groups. The derivatives were in the monomeric form and had dramatically reduced pI values. Both derivatives were relatively stable at neutral pH values, while the acyl groups undergo hydrolysis under acidic conditions. Modification of allergens by cis-aconitylation and citraconylation could be a new tool for obtaining allergoids.

Immune recognition of novel isoforms and domains of the mugwort pollen major allergen Art v 1

Allergen isoforms can differ in their IgE and T cell recognition patterns, and thus might have an impact on the selection of candidates for molecule-based diagnostic and therapeutic approaches. The present study aimed at the identification and characterization of isoforms of Art v 1, the mugwort pollen major allergen. In addition, single Art v 1 domains were physicochemically and immunologically characterized. For this purpose, the Art v 1 cDNA was radiolabeled and used to screen a mugwort pollen cDNA library. Positive clones were sequenced and used for the production of recombinant proteins in Escherichia coli using the pHIS-Parallel2 vector. Protein purification was performed by affinity- and ion exchange chromatography. Antibody binding to the recombinant proteins was determined by immunoblot, ELISA, cross-inhibition experiments, and mediator release assays. We could identify 7 Art v 1 isoforms differing in 1–6 amino acid residues. Interestingly, all amino acid variations were restricted to the proline domain carrying the molecule's post-translational modifications. No significant difference in IgG or IgE reactivity could be observed between Art v 1 isoforms and the defensin domain produced in E. coli. When expressed in E. coli, the proline domain was not recognized by Art v 1-specific antibodies. Our results demonstrated that the relevant IgE epitopes of Art v 1 are located on the defensin domain and suggest the involvement of carbohydrates in the allergenicity of natural Art v 1. Plant-based expression systems could help to reveal possibly different glycosylation patterns and IgE binding properties of Art v 1 isoforms. These findings have direct implications on the development of novel tools for mugwort pollen allergy diagnosis and therapy.

Alternative statistical methods for interpreting airborne Alder (Alnus glutimosa (L.) Gaertner) pollen concentrations

This paper reports on the behaviour of Alnus glutinosa (alder) pollen grains in the atmosphere of Ponferrada (León, NW Spain) from 1995 to 2006. The study, which sought to determine the effects of various weather-related parameters on Alnus pollen counts, was performed using a volumetric method. The main pollination period for this taxon is January-February. Alder pollen is one of the eight major airborne pollen allergens found in the study area. An analysis was made of the correlation between pollen counts and major weather-related parameters over each period. In general, the strongest positive correlation was with temperature, particularly maximum temperature. During each period, peak pollen counts occurred when the maximum temperature fell within the range 9 degrees C-14 degrees C. Finally, multivariate analysis showed that the parameter exerting the greatest influence was temperature, a finding confirmed by Spearman correlation tests. Principal components analysis suggested that periods with high pollen counts were characterised by high maximum temperature, low rainfall and an absolute humidity of around 6 g m(-3). Use of this type of analysis in conjunction with other methods is essential for obtaining an accurate record of pollen-count variations over a given period.

Molecular cloning, expression and immunological characterisation of Pas n 1, the major allergen of Bahia grass Paspalum notatum pollen

Bahia grass, Paspalum notatum, is a clinically important subtropical grass with a prolonged pollination season from spring to autumn. We aimed to clone and characterise the major Bahia grass pollen allergen, Pas n 1. Grass pollen-allergic patients presenting to a tertiary hospital allergy clinic were tested for IgE reactivity with Bahia grass pollen extract by skin prick testing, ImmunoCAP, ELISA and immunoblotting. Using primers deduced from the N-terminal peptide sequence of a group 1 allergen of Bahia grass pollen extract separated by two-dimensional gel electrophoresis, the complete Pas n 1 cDNA was obtained by rapid amplification of cDNA ends and cloned. Biological relevance of recombinant Pas n 1 expressed in Escherichia coli was assessed by serum IgE reactivity and basophil activation. Twenty-nine of 34 (85%) consecutive patients presenting with grass pollen allergy were skin prick test positive to Bahia grass pollen. The Pas n 1 cDNA has sequence homology with the β-expansin 1 glycoprotein family and is more closely related to the maize pollen group 1 allergen (85% identity) than to ryegrass Lol p 1 or Timothy grass Phl p 1 (64 and 66% identity, respectively). rPas n 1 reacted with serum IgE in 47 of 55 (85%) Bahia grass pollen-allergic patients, activated basophils and inhibited serum IgE reactivity with the 29 kDa band of Bahia grass pollen extract. In conclusion the cDNA for the major group 1 allergen of the subtropical Bahia grass pollen, Pas n 1, was identified and cloned. rPas n 1 is immunologically active and is a valuable reagent for diagnosis and specific immunotherapy of grass pollen allergy.

Production and characterization of monoclonal antibody against Saffron pollen profilin, Cro s 2.

Allergy to Saffron (Crocus sativus) pollen has been described in people involved in processing of saffron flower stamens. Profilins have been identified as a pan-allergen in different plant pollens and foods. This molecule is an actin-binding protein with a molecular weight of 12-16 kDa found in eukaryotic species. The aim of this study was to generate monoclonal antibody against Cro s 2 in order to characterize this major allergen of saffron pollen. BALB/c mice were immunized to obtain adequate humoral response. Splenocytes were prepared from the immunized animals, mixed with the P3-X63-Ag8.653 myeloma cells and fused by means of PEG 1500. After two weeks of culturing in HAT-containing media, the supernatant from those wells growing hybridomas were screened by ELISA using plates coated with Cro s 2. Cells from positive wells were cloned at least 3 times by limiting dilution. Specificity and cross-reactivity of the mAbs were determined by Western blot analysis and sandwich ELISA. Two stable hybridoma clones secreting mAbs against Cro s 2 were obtained and expanded. The anti-Cro s 2 mAbs were also found to cross-react with other plant profilins. Isotype of this mAb was identified as micro heavy chain and k light chain. The anti-Cro s 2 mAb could be a useful tool for characterization and standardization of many pollen and fruit-derived profilins.

Characterization of HLA Class II/Peptide-TCR Interactions of the Immunodominant T Cell Epitope in Art v 1, the Major Mugwort Pollen Allergen

More than 95% of mugwort pollen-allergic individuals are sensitized to Art v 1, the major allergen in mugwort pollen. Interestingly, the CD4 T cell response to Art v 1 involves only one single immunodominant peptide, Art v 1(25-36) (KCIEWEKAQHGA), and is highly associated with the expression of HLA-DR1. Therefore, we investigated the molecular basis of this unusual immunodominance among allergens. Using artificial APC expressing exclusively HLA-DRB1*0101 and HLA-DRA*0101, we formally showed that DR1 acts as restriction element for Art v 1(25-36)-specific T cell responses. Further assessment of binding of Art v 1(25-36) to artificial HLA-DR molecules revealed that its affinity was high for HLA-DR1. Amino acid I27 was identified as anchor residue interacting with DR molecules in pocket P1. Additionally, Art v 1(25-36) bound with high affinity to HLA-DRB1*0301 and *0401, moderately to HLA-DRB1*1301 and HLA-DRB5*0101, and weakly to HLA-DRB1*1101 and *1501. T cell activation was also inducible by Art v 1(25-36)-loaded, APC-expressing HLA molecules other than DR1, indicating degeneracy of peptide binding and promiscuity of TCR recognition. Specific binding of HLA-DRB1*0101 tetramers containing Art v 1(19-36) allowed the identification of Art v 1(25-36)-specific T cells by flow cytometry. In summary, the immunodominance of Art v 1(25-36) relies on its affinity to DR1, but is not dictated by it. Future investigations at the molecular HLA/peptide/TCR and cellular level using mugwort pollen allergy as a disease model may allow new insights into tolerance and pathomechanisms operative in type I allergy, which may instigate new, T cell-directed strategies in specific immunotherapy.

Sojaallergie durch Kreuzreaktionen gegen Birkenpollen-Majorallergen Bet v 1

The most prevalent soy allergy in Central Europe is based on birch pollen-related cross reactions. Due to structure similarities of birch pollen major allergen Bet v 1 and soy allergen Gly m 4, 75% of birch pollen-allergic subjects show serological IgE-mediated cross reactions to the soy protein Gly m 4 from the family of Bet v 1 -homologue, pathogenesis-related (PR)-10 proteins. Interestingly, only 10% of birch pollen-allergic patients develop clinical symptoms after ingestion of non- or slighty processed soy products (soy flakes, soy milk, tofu, diatery powders with soy isolate). Predominantly local oropharyngeal symptoms can lead to massive and severe swellings of throat or facial areas with dramatic severity. In addition, systemic reactions with urticarial, gastrointestinal and cardiovascular symptoms have been described. A typical case history of reactions with quick onset after ingestion of mildly processed soy products together with a strong allergic sensitization to birch pollen represent highly suggestive diagnostic clues. Native soy products are more useful for skin testing than commercial soy extracts due to labile Gly m 4 and its low abundance of 0.01 - 0.1% in total soy protein. For the same reason crude soy extracts are not sensitive enough for allergen-specific IgE detection in case of suspected Gly m 4-induced soy allergy. Recently introduced recombinant Gly m 4 is superior in demonstrating specific IgE. In case of established birch pollen-associated, clinically relevant soy allergy non- or hardly processed soy products should be avoided. Subsequent declaration of soy products about potential risks for birch pollen-allergic subjects might be helpful for future avoidance and instant diagnosis of the usually unexpected reactions. Heated and strongly processed products like soy sauce, textured soy proteins, roasted soy beans or soy oil are unlikely to represent inherent risks due to their low Gly m 4 content. Soy allergy on the basis of birch pollen-related crossreactions should be publicized more frequently.

Vegetable Proteomics: The Detection of Ole e 1 Isoallergens by Peptide Matching of MALDI MS/MS Spectra of Underivatized and Dansylated Glycopeptides

Ole e 1 (NCBI entry gi|14424429) is the major allergen of Oleaceae family. Multiple isoforms and variants are present in varying degrees of distribution. In this report, we present a new approach to the resolution of multiple forms of Ole e 1 from whole antigen extracts, based on a preliminary chemical fractionation procedure followed by MALDI MS and MS/MS measurements. The characterization of Ole e 1 isoallergens was accomplished through the identification of the amino acid sequence including the glycosylation site and the structure of the glycan moieties. The structure feature of the identified Ole e 1.0102 (gi|2465127), main olive allergen [Olea europaea] (gi|13195753), Major pollen allergen Ole e 1 (gi|33329740) and Ole e 1c (gi|1362131) is represented by the point mutation K(106) --> I and by the presence of a glycan moiety. Two other variants Major pollen allergen (Allergen Ole e1) (Ole e I) (gi|14424429) and Ole e 1.0103 protein [Olea europea] (gi|2465129) were identified as nonglycosylated species. These results, partially in disagreement with Swiss-Prot annotation, were validated by matching the MALDI MS/MS spectra of the natural tryptic mixture with those obtained after deglycosylation.

Grass pollen allergens

SummaryTo date, eleven groups of grass pollen allergens eliciting a specific IgE response in atopic individuals have been identified. Groups 1 and 5 allergens are the most critical (major) pollen allergens leading to the sensitization of 90% and 65–85% allergic patients, respectively. Other allergens frequently involved in the IgE response include groups 2/3, 4, 6, 7, 10–13 allergens. Allergens found in various Pooideae exhibit high homology in terms of their amino acid sequence composition, which translates into significant cross‐reactivity in terms of antibody (IgE and IgG) as well as T cell responses. Nevertheless, for a given allergen group, there is evidence of both interspecies (i.e. differences in amino acid sequences) and intraspecies (multigenes, post‐translational modification, mRNA splicing or editing) molecular variability.

Purification and Characterization of the Major Oak Pollen Allergen Que a 1 for Component-Resolved Diagnostics Using ImmunoCAP®

Background: The aim of this study was to purify the major oak pollen allergen, Que a 1, to perform biochemical and immunological characterization of the allergen and to develop an experimental native (n) Que a 1 ImmunoCAP®. Methods: Que a 1 was purified from oak pollen extract using affinity chromatography and characterized by SDS-PAGE, two-dimensional (2D) PAGE, mass spectrometry (MS), N-terminal sequencing and specific IgE inhibition on ImmunoCAP. Samples from 16 subjects sensitized to oak pollen were analyzed by ImmunoCAP for IgE reactivity to nQue a 1, and recombinant (r)Bet v 1 and 2 (profilin). They were also studied in IgE immunoblotting. Results: The purity of nQue a 1 was >95%, since a single band was observed on silver-stained SDS-PAGE. The identity was verified by MS analysis, and 2D-PAGE revealed several isoforms. The obtained N-terminal sequence of 50-amino-acid residues from nQue a 1 showed a 58–74% sequence identity with other pathogenesis-related class 10 allergens. Specific IgE inhibition verified a preserved immunoreactivity (70–92% inhibition). All subjects were sensitized to Que a 1 and Bet v 1, and two to profilin. The IgE antibody levels to nQue a 1 were generally lower than to rBet v 1. The obtained results correlated well with IgE immunoblotting. Conclusions: We present a highly purified and extensively characterized preparation of nQue a 1. Que a 1 seems to be an allergen of equal importance in oak pollen as Bet v 1 in birch pollen. An nQue a 1 ImmunoCAP will be useful in component-resolved diagnostics.

Solution structure of the C‐terminal domain of Ole e 9, a major allergen of olive pollen

Ole e 9 is an olive pollen allergen belonging to group 2 of pathogenesis-related proteins. The protein is composed of two immunological independent domains: an N-terminal domain (NtD) with 1,3-beta-glucanase activity, and a C-terminal domain (CtD) that binds 1,3-beta-glucans. We have determined the three-dimensional structure of CtD-Ole e 9 (101 amino acids), which consists of two parallel alpha-helices forming an angle of approximately 55 degrees , a small antiparallel beta-sheet with two short strands, and a 3-10 helix turn, all connected by long coil segments, resembling a novel type of folding among allergens. Two regions surrounded by aromatic residues (F49, Y60, F96, Y91 and Y31, H68, Y65, F78) have been localized on the protein surface, and a role for sugar binding is suggested. The epitope mapping of CtD-Ole e 9 shows that B-cell epitopes are mainly located on loops, although some of them are contained in secondary structural elements. Interestingly, the IgG and IgE epitopes are contiguous or overlapped, rather than coincident. The three-dimensional structure of CtD-Ole e 9 might help to understand the underlying mechanism of its biochemical function and to determine possible structure-allergenicity relationships.