Chimeric hypoallergens of Der p 1 and Blo t 1 (QBD2/QBD4) reduce IgE reactivity and airway inflammation in mice. QBD4 promotes Th1/regulatory responses, suppressing mucus and eosinophilia more effectively than QBD2.

Many plant species have evolved surfaces that reduce insect attachment. Among such plants are deceptive trap flowers of Ceropegia. Their gliding zones consist of convex epidermal cells, each with a bristle-like central protuberance and a single small liquid droplet on its tip. So far, the molecular and physical mechanisms controlling the function of these droplets are unknown. We analyzed the droplets of Ceropegia sandersonii flowers by microscopic approaches, studied how they behave when getting in contact with the feet of fly pollinators, and analyzed their chemical composition. The droplets contaminate the insect feet, on which they solidify. As its main component, a negatively charged polysaccharide containing a β1,3-galactan backbone and Rha-α1,4-GlcA-β1,6-[Araf-α1,3-]Gal-β1,6 side chains or truncated versions of it was identified. The chemical structure represents a rudimentary version of an arabinogalactan, which is supported by its binding to β-d-glucosyl Yariv reagent. Candidates of arabinogalactan proteins were identified to which the polysaccharide might be connected. The high amount of GlcA in the polysaccharide helps to explain the unusual physical characteristics of the droplets, like viscoelasticity and hygroscopy. We add a new function to arabinogalactans and discuss why the identified polymer is well suited for catching and temporarily trapping pollinators.

Background Reverse transcription quantitative PCR (RT-qPCR) with intercalating dyes is one of the main techniques to assess gene expression levels used in basic and applied research as well as in diagnostics. However, primer design for RT-qPCR can be complex due to the high demands on primer quality. Primers are best placed on exon junctions, should avoid polymorphic regions, be specific to the target transcripts and also prevent genomic amplification accurately, among others. Current software tools manage to meet all the necessary criteria only insufficiently. Here, we present ExonSurfer, a novel, user-friendly web-tool for qPCR primer design.Results ExonSurfer combines the different steps of the primer design process, encompassing target selection, specificity and self-complementarity assessment, and the avoidance of issues arising from polymorphisms. Amplification of potentially contaminating genomic DNA is avoided by designing primers on exon-exon junctions, moreover, a genomic alignment is performed to filter the primers accordingly and inform the user of any predicted interaction. In order to test the whole performance of the application, we designed primer pairs for 26 targets and checked both primer efficiency, amplicon melting temperature and length and confirmed the targeted amplicon by Sanger sequencing. Most of the tested primers accurately and selectively amplified the corresponding targets.Conclusion ExonSurfer offers a comprehensive end-to-end primer design, guaranteeing transcript-specific amplification. The user interface is intuitive, providing essential specificity and amplicon details. The tool can also be used by command line and the source code is available. Overall, we expect ExonSurfer to facilitate RT-qPCR set-up for researchers in many fields.

The PDB database provides more than 150,000 entries for biological macromolecular structures [...]

Knowledge of MHC II binding peptides is highly desired in immunological research, particularly in the context of cancer, autoimmune diseases, or allergies. The most successful prediction methods are based on machine learning methods trained on sequences of experimentally characterized binding peptides. Here, we describe a complementary approach called MHCII3D, which is based on structural scaffolds of MHC II-peptide complexes and statistical scoring functions (SSFs). The MHC II alleles reported in the Immuno Polymorphism Database are processed in a dedicated 3D-modeling pipeline providing a set of scaffold complexes for each distinct allotype sequence. Antigen protein sequences are threaded through the scaffolds and evaluated by optimized SSFs. We compared the predictive power of MHCII3D with different sequence-based machine learning methods. The Pearson correlation to experimentally determine IC50 values for MHC II Automated Server Benchmarks data sets from IEDB (Immune Epitope Database) is 0.42, which is in the competitor methods range. We show that MHCII3D is quite robust in leaving one molecule out tests and is therefore not prone to overfitting. Finally, we provide evidence that MHCII3D can complement the current sequence-based methods and help to identify problematic entries in IEDB. Scaffolds and MHCII3D executables can be freely downloaded from our web pages.

Allergen‐specific immunotherapy (AIT) represents a curative approach for treating allergies. In the tropical and subtropical regions of the world, Blomia tropicalis (Blo t 5 and Blo t 21) is the likely dominant source of indoor allergens.

To analyze the impact of Ascaris lumbricoides infection on the pathogenesis and diagnosis of allergic diseases, new allergens should be identified. We report the identification of a new Ascaris lumbricoides allergen, Asc l 5. The aim of this study was to evaluate the physicochemical and immunological features of the Asc l 5 allergen. We constructed an A. lumbricoides cDNA library and Asc l 5 was identified by immunoscreening. After purification, rAsc l 5 was physicochemically characterized. Evaluation of its allergenic activity included determination of Immunoglobulin E (IgE) binding frequency (in two populations: 254 children and 298 all-age subjects), CD203c based-basophil activation tests (BAT) and a passive cutaneous anaphylaxis (PCA) mouse model. We found by amino acid sequence analysis that Asc l 5 belongs to the SXP/RAL-2 protein family of nematodes. rAsc l 5 is a monomeric protein with an alpha-helical folding. IgE sensitization to rAsc l 5 was around 52% in general population; positive BAT rate was 60%. rAsc l 5 induced specific IgE production in mice and a positive PCA reaction. These results show that Asc l 5 has structural and immunological characteristics to be considered as a new allergen from A. lumbricoides.

Introduction Protein fold stability has been proposed to represent an intrinsic feature contributing to immunogenicity and immune polarization by influencing the amount of peptide-MHC II complexes (pMHCII). Using in silico prediction, we introduced point mutations in proteins that either increase or decrease their fold-stability without altering immunodominant epitopes or changing the overall structure of the protein. Here, we investigated how modulation of the fold-stability of the grass pollen allergen Phl p 6 affects its ability to stimulate immune responses and T cell polarization. Methods Using the MAESTRO software tool, stabilizing or destabilizing mutations were selected and verified by molecular dynamics simulations. The mutants were expressed in E. coli, purified tag-free, and analyzed for thermal stability and resistance to endolysosomal proteases. The resulting peptides were analysed by degradome assay and mass spectrometry. The structure of the most stable mutant protein was obtained by X-ray crystallography. We evaluated the capacity of the mutants to stimulate T cell proliferation in vitro , as well as antibody responses and T cell polarization in vivo in an adjuvant-free BALB/c mouse model. Results Four stabilizing and two destabilizing mutations were identified by MAESTRO. Experimentally determined changes in thermal stability compared to the wild type protein ranged from -5 to +14 °C. Destabilization led to faster proteolytic processing in vitro , whereas highly stabilized mutants were degraded very slowly. However, the overall pattern of identified peptides remained very similar. This was confirmed in bone marrow derived dendritic cells that processed and presented the immune dominant epitope from a destabilized mutant more efficiently. In vivo , stabilization resulted in a shift in immune polarization as indicated by higher levels of IgG2a and increased secretion of TH1/TH17 cytokines. Conclusion MAESTRO was very efficient in detecting single point mutations that increase or reduce fold-stability. Thermal stability correlated well with susceptibility to protease resistance and presentation of pMHCII on the surface of dendritic cells in vitro . This change in processing kinetics significantly influenced the polarization of T cell responses in vivo . Modulating the fold-stability of proteins thus has the potential to optimize and polarize immune responses, which opens the door to more efficient design of molecular vaccines.

Identification of antibody binding epitopes is crucial to understand immunological mechanisms. It is of particular interest for allergenic proteins with high cross-reactivity as observed in the lipid transfer protein (LTP) syndrome that is characterized by severe allergic reactions. Art v 3, a pollen LTP from mugwort is frequently involved in this cross-reactivity, but no antibody binding epitopes have been determined so far.  To reveal human IgE binding regions of Art v 3, we produced three murine high-affinity monoclonal antibodies (mAbs), which showed 70-90% coverage of the allergenic epitopes from mugwort pollen allergic patients. As reliable methods to determine structural epitopes with tightly interacting intact antibodies under native conditions are lacking, we developed a straightforward NMR approach termed hydrogen/deuterium exchange memory (HDXMEM). It relies on the slow exchange between the invisible antigen-mAb complex and the free 15N-labeled antigen whose 1H-15N correlations are detected. Due to a memory effect, changes of NH protection during antibody binding are measured. Differences in H/D exchange rates and analyses of mAb reactivity to homologous LTPs revealed three structural epitopes: two partially cross-reactive regions around α-helices 2 and 4, as well as a novel Art v 3-specific epitope at the C-terminus. Protein variants with exchanged epitope residues confirmed the antibody-binding sites and revealed strongly reduced IgE reactivity. Using the novel HDXMEM for NMR epitope mapping allowed identification of the first structural epitopes of an allergenic pollen LTP. This knowledge enables improved cross-reactivity prediction for patients suffering from LTP-allergy and facilitates design of therapeutics.

Plant cell wall proteins play major roles during plant development and in response to environmental cues. A bioinformatic search for functional domains has allowed identifying the PAC domain (Proline-rich, Arabinogalactan proteins, conserved Cysteines) in several proteins (PDPs) identified in cell wall proteomes. This domain is assumed to interact with pectic polysaccharides and O-glycans and to contribute to non-covalent molecular scaffolds facilitating the remodeling of polysaccharidic networks during rapid cell expansion. In this work, the characteristics of the PAC domain are described in detail, including six conserved Cys residues, their spacing, and the predicted secondary structures. Modeling has been performed based on the crystal structure of a Plantago lanceolata PAC domain. The presence of β-sheets is assumed to ensure the correct folding of the PAC domain as a β-barrel with loop regions. We show that PDPs are present in early divergent organisms from the green lineage and in all land plants. PAC domains are associated with other types of domains: Histidine-rich, extensin, Proline-rich, or yet uncharacterized. The earliest divergent organisms having PDPs are Bryophytes. Like the complexity of the cell walls, the number and complexity of PDPs steadily increase during the evolution of the green lineage. The association of PAC domains with other domains suggests a neo-functionalization and different types of interactions with cell wall polymers

ScopeAllergies to lipid transfer proteins involve severe adverse reactions; thus, effective and sustainable therapies are desired. Previous attempts disrupting disulfide bonds failed to maintain immunogenicity; thus, the aim is to design novel hypoallergenic Pru p 3 variants and evaluate the applicability for treatment of peach allergy.Methods and resultsPru p 3 proline variant (PV) designed using in silico mutagenesis, cysteine variant (CV), and wild‐type Pru p 3 (WT) are purified from Escherichia coli. Variants display homogenous and stable protein conformations with an altered secondary structure in circular dichroism. PV shows enhanced long‐term storage capacities compared to CV similar to the highly stable WT. Using sera of 33 peach allergic patients, IgE‐binding activity is reduced by 97% (PV) and 71% (CV) compared to WT. Both molecules show strong hypoallergenicity in Pru p 3 ImmunoCAP cross‐inhibition and histamine release assays. Immunogenicity of PV is demonstrated with a phosphate‐based adjuvant formulation in a mouse model.ConclusionsAn in silico approach is used to generate a PV without targeting disulfide bonds, T cell epitopes, or previously reported IgE epitopes of Pru p 3. PV is strongly hypoallergenic while structurally stable and immunogenic, thus representing a promising candidate for peach allergen immunotherapy.

Purpose: Mutations in SZT2 have been previously reported in several cases of early onset epilepsy and intellectual disability. In this study we investigate potential causal mutations in two male siblings affected by early onset epilepsy, intellectual disability and macrocephaly. Methods: We use family-based whole-exome sequencing to identify candidate variants. Results: We report the identification of two potential causal SZT2 mutations in compound heterozygous state. We observe considerable differences in the clinical phenotype severity of the two affected individuals. The cerebral MRI revealed no abnormalities in the older affected brother, while in the youngest one it revealed a right frontal polymicrogiria. Moreover, while good seizure control was achieved in the older affected individual the younger brother is affected by pharmacoresistant epilepsy, progressive spastic paraplegia, cortical myoclonus and a more severe intellectual disability. We also analyzed the relative location of the reported pathogenic mutations in the SZT2 protein. Conclusion: Variable phenotypic expressivity is observed for this condition, while the location and type of mutations in SZT2 also has a potential impact on epilepsy severity. These findings extend our knowledge of epileptogenic conditions related to SZT2 and mTOR signaling.

Pollen tubes are the fastest growing cells in nature with elongation rates of >20 mu m min(-1). The pollen tubes grow as fast as possible through the stigma tissue towards the egg cells where they release two sperm cells for fertilization. In addition to speed, the growth process has to be precise and well balanced to find the targets and to avoid bursting of the tube, respectively. Transport of ions across the plasma membrane generates a typical polar pattern of endogenous currents that accompanies pollen tube growth, and which is thought to function as a pacemaker to synchronize cellular processes. The endogenous currents are generated by the activity of various ion transporters in the plasma membrane, which were investigated by biophysical and molecular biology approaches. Using an unbiased database search for pollen-expressed transporter genes in Arabidopsis and Lilium, a large number of putative transporters was identified that might contribute to the current pattern formation. Furthermore, we hypothesize that the distribution of the transporters to specific regions in the pollen tube is the basis for the current pattern and is achieved by a self-organization process based on the electrophoretic mobility of charged membrane proteins in an electrical field. Thus, the activity of an ion transporter causes a local change in the transmembrane potential, which in turn would attract similar transporters and repel others leading to a heterogeneous distribution of transporters in the plasma membrane and the observed current pattern.

Persistent activation of hedgehog (HH)/GLI signaling accounts for the development of basal cell carcinoma (BCC), a very frequent nonmelanoma skin cancer with rising incidence. Targeting HH/GLI signaling by approved pathway inhibitors can provide significant therapeutic benefit to BCC patients. However, limited response rates, development of drug resistance, and severe side effects of HH pathway inhibitors call for improved treatment strategies such as rational combination therapies simultaneously inhibiting HH/GLI and cooperative signals promoting the oncogenic activity of HH/GLI. In this study, we identified the interleukin‐6 (IL6) pathway as a novel synergistic signal promoting oncogenic HH/GLI via STAT3 activation. Mechanistically, we provide evidence that signal integration of IL6 and HH/GLI occurs at the level of cis‐regulatory sequences by co‐binding of GLI and STAT3 to common HH‐IL6 target gene promoters. Genetic inactivation of Il6 signaling in a mouse model of BCC significantly reduced in vivo tumor growth by interfering with HH/GLI‐driven BCC proliferation. Our genetic and pharmacologic data suggest that combinatorial HH‐IL6 pathway blockade is a promising approach to efficiently arrest cancer growth in BCC patients.

The Characeae are multicellular green algae with very close relationship to land plants. Their internodal cells have been the subject of numerous (electro-)physiological studies. When exposed to light, internodal cells display alternating bands of low and high pH along their surface in order to facilitate carbon uptake required for photosynthesis. Here we investigated for the first time the subcellular membrane protein composition of acidic and alkaline regions in internodal cells of Chara australis R. Br. using MS-proteomics. The identified peptides were annotated to Chara unigenes using a custom-made Chara database generated from a transcriptome analysis and to orthologous Arabidopsis genes using TAIR (The Arabidopsis Information Resource) database. Apart from providing the first public-available, functionally-annotated sequence database for Chara australis, the proteome study, which is supported by immunodetection, identified several membrane proteins associated with acidic regions that contain a high density of specific plasma membrane (PM) invaginations, the charasomes, which locally increase the membrane area to overcome diffusion limitation in membrane transport. An increased abundance of PM H+ ATPases at charasomes is consistent with their role in the acidification of the environment, but the characean PM H+ ATPase sequence suggests a different regulation compared to higher plant PM H+ ATPases. A higher abundance of H+ co-transporters in the charasome-rich, acidic regions possibly reflects enhanced uptake of ions and nutrients. The increase in mitochondrial proteins confirms earlier findings about the accumulation of cortical mitochondria in the acidic zones. The significant enrichment of clathrin heavy chains and clathrin adaptor proteins as well as other proteins involved in trafficking indicate a higher activity of membrane transport in the charasome-rich than in charasome-poor areas. New and unexpected data, for instance the upregulation and abundance of vacuolar transporters correlating with the charasome-rich, acidic cell regions account for new perspectives in the formation of charasomes.

Pollen is one of the most common causes of allergy worldwide, making the study of their molecular composition crucial for the advancement of allergy research. Despite substantial efforts in this field, it is not yet clear why some plant pollens strongly provoke allergies while others do not. However, proteases and protease inhibitors from allergen sources are known to play an important role in the development of pollen allergies. In this study, we aim to uncover differences in the transcriptional pattern of proteases and protease inhibitors in Betula verrucosa and Pinus sylvestris pollen as models for high and low allergenic potential, respectively. We applied RNA sequencing to Betula verrucosa and Pinus sylvestris pollen. After de-novo assembly we derived general functional profiles of the protein coding transcripts. By utilization of domain based functional annotation we identified potential proteases and protease inhibitors and compared their expression in the two types of pollen. Functional profiles are highly similar between Betula verrucosa and Pinus sylvestris pollen. Both pollen contain proteases and inhibitors from 53 and 7 Pfam families, respectively. Some of the members comprised within those families are implicated in facilitating allergen entry, while others are known allergens themselves. Our work revealed several candidate proteins which, with further investigation, represent exciting new leads in elucidating the process behind allergic sensitization.

Allergic diseases are considered a major problem for healthcare systems in both developed and developing countries. House dust mites are well-known triggers of allergic manifestations. While the Dermatophagoides genus is widely distributed globally, Blomia tropicalis is the most prominent mite species in the tropical and subtropical regions of the world. Over the last decades, an increase in sensitization rates to B. tropicalis has been reported, leading to increased research efforts on Blomia allergens. In fact, 8 new allergens have been identified and characterized to different degrees. Here, we provide an overview of recent developments concerning the identification and production of recombinant Blomia allergens, as well as their structural and immunological characterization. Although considerable progress has been achieved, detailed molecule-based studies are still needed to better define the clinical relevance of Blomia allergens. Thus, the establishment of a well-standardized and fully characterized panel of allergens remains a challenge for the development of better diagnosis and therapy of allergic diseases induced by B. tropicalis.

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.

Birch pollen allergy is highly prevalent, with up to 100 million reported cases worldwide. Proteases in such allergen sources have been suggested to contribute to primary sensitisation and exacerbation of allergic disorders. Until now the protease content of Betula verrucosa, a birch species endemic to the northern hemisphere has not been studied in detail. Hence, we aim to identify and characterise pollen and bacteria-derived proteases found within birch pollen. The pollen transcriptome was constructed via de novo transcriptome sequencing and analysis of the proteome was achieved via mass spectrometry; a cross-comparison of the two databases was then performed. A total of 42 individual proteases were identified at the proteomic level. Further clustering of proteases into their distinct catalytic classes revealed serine, cysteine, aspartic, threonine, and metallo-proteases. Further to this, protease activity of the pollen was quantified using a fluorescently-labelled casein substrate protease assay, as 0.61 ng/mg of pollen. A large number of bacterial strains were isolated from freshly collected birch pollen and zymographic gels with gelatinase and casein, enabled visualisation of proteolytic activity of the pollen and the collected bacterial strains. We report the successful discovery of pollen and bacteria-derived proteases of Betula verrucosa.