Claudia Asam

Crystallographically Mapped Ligand Binding Differs in High and Low IgE Binding Isoforms of Birch Pollen Allergen Bet v 1

The ability of pathogenesis-related proteins of family 10 to bind a broad spectrum of ligands is considered to play a key role for their physiological and pathological functions. In particular, Bet v 1, an archetypical allergen from birch pollen, is described as a highly promiscuous ligand acceptor. However, the detailed recognition mechanisms, including specificity factors discriminating binding properties of naturally occurring Bet v 1 variants, are poorly understood. Here, we report crystal structures of Bet v 1 variants in complex with an array of ligands at a resolution of up to 1.2 Å. Residue 30 within the hydrophobic pocket not only discriminates in high and low IgE binding Bet v 1 isoforms but also induces a drastic change in the binding mode of the model ligand deoxycholate. Ternary crystal structure complexes of Bet v 1 with several ligands together with the fluorogenic reporter 1-anilino-8-naphthalene sulfonate explain anomalous fluorescence binding curves obtained from 1-anilino-8-naphthalene sulfonate displacement assays. The structures reveal key interaction residues such as Tyr83 and rationalize both the binding specificity and promiscuity of the so-called hydrophobic pocket in Bet v 1. The intermolecular interactions of Bet v 1 reveal an unexpected complexity that will be indispensable to fully understand its roles within the physiological and allergenic context.

Proteomic profiling of birch (Betula verrucosa) pollen extracts from different origins

Pollen of the European white birch is a major source of spring pollinosis in Europe. Pollen‐allergy diagnosis and treatment by specific immunotherapy commonly rely on extracts of natural origin. To gain insight into the protein content and its variability, we evaluated the profile of allergenic and non‐allergenic proteins in extracts of pollen from different origins by MS‐based proteomics. Aqueous extracts prepared from commercially available Swedish birch pollen, pollen collected from Austrian trees and a commercial skin prick extract were analyzed by 1‐DE, 2‐DE, immunoblotting and mass spectrometry, resulting in a complete inventory of extractable, disease‐relevant pollen proteins. A main focus of this study was on the isoform distribution of Bet v 1, the major allergen of birch pollen. Using a combination of intact mass determination and peptide sequencing, five isoforms (a, b, d, f and j) were unequivocally identified in Swedish and Austrian birch pollen extracts, while the skin prick extract contained only isoforms a, b and d. Using the same methods as for Bet v 1, divergencies in the sequence of birch profilin (Bet v 2), a plant panallergen, were solved. The molecular characterization of pollen extracts is relevant for standardization and development of new reagents for specific immunotherapy.

Reshaping the Bet v 1 fold modulates TH polarization

BACKGROUND Several alternative mechanisms have been proposed to explain why some proteins are able to induce a T(H)2-biased and IgE-mediated immune response. These include specific interactions with receptors of the innate immune system, proteolytic activities, allergen-associated carbohydrate structures, and intrinsic structural determinants. OBJECTIVES Available data suggest that a fold-dependent allergy-promoting mechanism could be a driving force for the T(H)2-polarization activity of Bet v 1, the major birch pollen allergen. METHODS Computer-aided sequence and fold analysis of the Bet v 1 family identified a short stretch susceptible for mutations inducing an altered fold of the entire molecule. With this knowledge, 7 consecutive amino acids of Bet v 1 were replaced with the homologous Mal d 1 sequence, creating the derivative BM4. RESULTS The minimal changes of the sequence led to a loss of the Bet v 1-like fold and influenced the immunologic behavior. Compared to wild-type Bet v 1, BM4 induced elevated T-cell proliferation of human PBMCs. In the mouse model, immunization with Bet v 1 absorbed to aluminum hydroxide triggered strong T(H)2 polarization, whereas BM4 immunization additionally recruited T(H)1 cells. Furthermore, the fold variant BM4 showed enhanced uptake by dendritic cells and a decreased susceptibility to endo-/lysosomal proteolysis. CONCLUSION Modifications in the 3-dimensional structure of Bet v 1.0101 resulted in a change of its immunologic properties. We observed that the fold alteration led to a modified crosstalk with dendritic cells and a shift of the immune response polarization toward a mixed T(H)1/T(H)2 cytokine production.

Tree pollen allergens—an update from a molecular perspective

It is estimated that pollen allergies affect approximately 40% of allergic individuals. In general, tree pollen allergies are mainly elicited by allergenic trees belonging to the orders Fagales, Lamiales, Proteales, and Pinales. Over 25 years ago, the gene encoding the major birch pollen allergen Bet v 1 was the first such gene to be cloned and its product characterized. Since that time, 53 tree pollen allergens have been identified and acknowledged by the WHO/IUIS allergen nomenclature subcommittee. Molecule‐based profiling of allergic sensitization has helped to elucidate the immunological connections of allergen cross‐reactivity, whereas advances in biochemistry have revealed structural and functional aspects of allergenic proteins. In this review, we provide a comprehensive overview of the present knowledge of the molecular aspects of tree pollen allergens. We analyze the geographic distribution of allergenic trees, discuss factors pivotal for allergic sensitization, and describe the role of tree pollen panallergens. Novel allergenic tree species as well as tree pollen allergens are continually being identified, making research in this field highly competitive and instrumental for clinical applications.

The impact of nitration on the structure and immunogenicity of the major birch pollen allergen Bet v 1.0101.

Allergy prevalence has increased in industrialized countries. One contributing factor could be pollution, which can cause nitration of allergens exogenously (in the air) or endogenously (in inflamed lung tissue). We investigated the impact of nitration on both the structural and immunological behavior of the major birch pollen allergen Bet v 1.0101 to determine whether nitration might be a factor in the increased incidence of allergy. Bet v 1.0101 was nitrated with tetranitromethane. Immune effects were assessed by measuring the proliferation of specific T-cell lines (TCLs) upon stimulation with different concentrations of nitrated and unmodified allergen, and by measurement of cytokine release of monocyte-derived dendritic cells (moDCs) and primary DCs (primDCs) stimulated with nitrated versus unmodified allergen. HPLC-MS, crystallography, gel electrophoresis, amino acid analysis, size exclusion chromatography and molecular dynamics simulation were performed to characterize structural changes after nitration of the allergen. The proliferation of specific TCLs was higher upon stimulation with the nitrated allergen in comparison to the unmodified allergen. An important structural consequence of nitration was oligomerization. Moreover, analysis of the crystal structure of nitrated Bet v 1.0101 showed that amino acid residue Y83, located in the hydrophobic cavity, was nitrated to 100%. Both moDCs and primDCs showed decreased production of TH1-priming cytokines, thus favoring a TH2 response. These results implicate that nitration of Bet v 1.0101 might be a contributing factor to the observed increase in birch pollen allergy, and emphasize the importance of protein modifications in understanding the molecular basis of allergenicity.

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

Background Allergic 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. Methodology Therefore, 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. Conclusion The 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.

Stabilization of the Dimeric Birch Pollen Allergen Bet v 1 Impacts Its Immunological Properties

Background: Frequently reported dimerization of allergens may contribute to their allergenicity. Results: Polysulfide-bridged allergen dimers exhibit different allergenic properties compared with the monomer. Conclusion: The N-terminal region has a distinct susceptibility for modifications and impacts its protein-protein interaction characteristics. Significance: The crystal structures well mimic transient dimerization of the allergens in solution, providing a rational for effective IgE cross-linking on effector cells. Many allergens share several biophysical characteristics, including the capability to undergo oligomerization. The dimerization mechanism in Bet v 1 and its allergenic properties are so far poorly understood. Here, we report crystal structures of dimeric Bet v 1, revealing a noncanonical incorporation of cysteine at position 5 instead of genetically encoded tyrosine. Cysteine polysulfide bridging stabilized different dimeric assemblies, depending on the polysulfide linker length. These dimers represent quaternary arrangements that are frequently observed in related proteins, reflecting their prevalence in unmodified Bet v 1. These conclusions were corroborated by characteristic immunologic properties of monomeric and dimeric allergen variants. Hereby, residue 5 could be identified as an allergenic hot spot in Bet v 1. The presented results refine fundamental principles in protein chemistry and emphasize the importance of protein modifications in understanding the molecular basis of allergenicity.

How relevant is panallergen sensitization in the development of allergies

Panallergens comprise various protein families of plant as well as animal origin and are responsible for wide IgE cross‐reactivity between related and unrelated allergenic sources. Such cross‐reactivities include reactions between various pollen sources, pollen and plant‐derived foods as well as invertebrate‐derived inhalants and foodstuff. Here, we provide an overview on the most clinically relevant panallergens from plants (profilins, polcalcins, non‐specific lipid transfer proteins, pathogenesis‐related protein family 10 members) and on the prominent animal‐derived panallergen family, tropomyosins. In addition, we explore the role of panallergens in the sensitization process and progress of the allergic disease. Emphasis is given on epidemiological aspects of panallergen sensitization and clinical manifestations. Finally, the issues related to diagnosis and therapy of patients sensitized to panallergens are outlined, and the use of panallergens as predictors for cross‐reactive allergy and as biomarkers for disease severity is discussed.

Ligand Binding Modulates the Structural Dynamics and Compactness of the Major Birch Pollen Allergen

Pathogenesis-related plant proteins of class-10 (PR-10) are essential for storage and transport of small molecules. A prominent member of the PR-10 family, the major birch pollen allergen Bet v 1, is the main cause of spring pollinosis in the temperate climate zone of the northern hemisphere. Bet v 1 binds various ligand molecules to its internal cavity, and immunologic effects of the presence of ligand have been discussed. However, the mechanism of binding has remained elusive. In this study, we show that in solution Bet v 1.0101 is conformationally heterogeneous and cannot be represented by a single structure. NMR relaxation data suggest that structural dynamics are fundamental for ligand access to the protein interior. Complex formation then leads to significant rigidification of the protein along with a compaction of its 3D structure. The data presented herein provide a structural basis for understanding the immunogenic and allergenic potential of ligand binding to Bet v 1 allergens.

Tackling Bet v 1 and associated food allergies with a single hybrid protein

Background Allergy vaccines should be easily applicable, safe, and efficacious. For Bet v 1–mediated birch pollen and associated food allergies, a single wild‐type allergen does not provide a complete solution. Objective We aimed to combine immunologically relevant epitopes of Bet v 1 and the 2 clinically most important related food allergens from apple and hazelnut to a single hybrid protein, termed MBC4. Methods After identification of T cell epitope–containing parts on each of the 3 parental allergens, the hybrid molecule was designed to cover relevant epitopes and evaluated in silico. Thereby a mutation was introduced into the hybrid sequence, which should alter the secondary structure without compromising the immunogenic properties of the molecule. Results MBC4 and the parental allergens were purified to homogeneity. Analyses of secondary structure elements revealed substantial changes rendering the hybrid de facto nonreactive with patients’ serum IgE. Nevertheless, the protein was monomeric in solution. MBC4 was able to activate T‐cell lines from donors with birch pollen allergy and from mice immunized with the parental allergens. Moreover, on immunization of mice and rabbits, MBC4 induced cross‐reactive IgG antibodies, which were able to block the binding of human serum IgE. Conclusion Directed epitope rearrangements combined with a knowledge‐based structural modification resulted in a protein unable to bind IgE from allergic patients. Still, properties to activate specific T cells or induce blocking antibodies were conserved. This suggests that MBC4 is a suitable vaccine candidate for the simultaneous treatment of Bet v 1 and associated food allergies. Graphical abstract Figure. No Caption available.