The development of allergic disease involves the production of IgE antibodies upon allergen exposure in a process called sensitization. IgE binds to receptors on the surface of mast cells and ...basophils, and subsequent allergen exposure leads to cross-linking of IgE antibodies and release of cell mediators that cause allergy symptoms. Although this process is quite well-understood, very little is known about the epitopes on the allergen recognized by IgE, despite the importance of the allergen-antibody interaction for the allergic response to occur. This review discusses efforts to analyze allergen-antibody interactions, from the original epitope mapping studies using linear peptides or recombinant allergen fragments, to more sophisticated technologies, such as X-ray crystallography and nuclear magnetic resonance. These state-of-the-art approaches, combined with site-directed mutagenesis, have led to the identification of conformational IgE epitopes. The first structures of an allergen (egg lysozyme) in complex with Fab fragments from IgG antibodies were determined in the 1980s. Since then, IgG has been used as surrogate for IgE, due to the difficulty of obtaining monoclonal IgE antibodies. Technical developments including phage display libraries have contributed to progress in epitope mapping thanks to the isolation of IgE antibody constructs from combinatorial libraries made from peripheral blood mononuclear cells of allergic donors. Most recently, single B cell antibody sequencing and human hybridomas are new breakthrough technologies for finally obtaining human IgE monoclonal antibodies, ideal for epitope mapping. The information on antigenic determinants will facilitate the design of hypoallergens for immunotherapy and the investigation of the fundamental mechanisms of the IgE response.
Topoisomerase 2 (TOP2) DNA transactions proceed via formation of the TOP2 cleavage complex (TOP2cc), a covalent enzyme-DNA reaction intermediate that is vulnerable to trapping by potent anticancer ...TOP2 drugs. How genotoxic TOP2 DNA-protein cross-links are resolved is unclear. We found that the SUMO (small ubiquitin-related modifier) ligase ZATT (ZNF451) is a multifunctional DNA repair factor that controls cellular responses to TOP2 damage. ZATT binding to TOP2cc facilitates a proteasome-independent tyrosyl-DNA phosphodiesterase 2 (TDP2) hydrolase activity on stalled TOP2cc. The ZATT SUMO ligase activity further promotes TDP2 interactions with SUMOylated TOP2, regulating efficient TDP2 recruitment through a “split-SIM” SUMO2 engagement platform. These findings uncover a ZATT-TDP2–catalyzed and SUMO2-modulated pathway for direct resolution of TOP2cc.
Allergy is defined as an inappropriate immune response to something normally considered harmless. The symptomatic immune response is driven by IgE antibodies directed against allergens. The study of ...allergens has contributed significantly to our understanding of allergic disease in 3 main areas. First, identifying allergens as the cause of symptoms and developing allergen standards has led to many advances in exposure assessment and patient diagnostics. Second, a biochemical understanding of allergens has suggested a number of hypotheses related to the mechanisms of allergic sensitization. And finally, studies of allergen-antibody interactions have contributed to understanding the cross-reactivity of allergens, mapping patient epitopes, and the development of hypoallergens. In this review, a few select cases are highlighted where structural biology, in particular, has contributed significantly to allergen research and provided new avenues for investigation.
Background
Over 100 million people worldwide suffer from birch pollen allergy. Bet v 1 has been identified as the major birch pollen allergen. However, the molecular mechanisms of birch allergic ...sensitization, including the roles of Bet v 1 and other components of the birch pollen extract, remain incompletely understood. Here, we examined how known birch pollen–derived molecules influence the endolysosomal processing of Bet v 1, thereby shaping its allergenicity.
Methods
We analyzed the biochemical and immunological interaction of ligands with Bet v 1. We then investigated the proteolytic processing of Bet v 1 by endosomal extracts in the presence and absence of ligands, followed by a detailed kinetic analysis of Bet v 1 processing by individual endolysosomal proteases as well as the T‐cell epitope presentation in BMDCs.
Results
We identified E1 phytoprostanes as novel Bet v 1 ligands. Pollen‐derived ligands enhanced the proteolytic resistance of Bet v 1, affecting degradation kinetics and preferential cleavage sites of the endolysosomal proteases cathepsin S and legumain. E1 phytoprostanes exhibited a dual role by stabilizing Bet v 1 and inhibiting cathepsin protease activity.
Conclusion
Bet v 1 can serve as a transporter of pollen‐derived, bioactive compounds. When carried to the endolysosome, such compounds can modulate the proteolytic activity, including its processing by cysteine cathepsins. We unveil a paradigm shift from an allergen‐centered view to a more systemic view that includes the host endolysosomal enzymes.
The newly identified birch pollen‐derived ligands, Phytoprostane E1 (PPE1), interacts with Bet v 1 affecting its stability and proteolytic processing. Ubiquitous plant phytoprostanes, PPE1, covalently inhibit lysosomal cysteine cathepsins, with multiple consequences including effects on antigen processing. PPE1 affected the presentation of Bet v 1 T‐cell epitope in BMDC to T‐cell.
Background Sensitization to cockroach allergens is a major risk factor for asthma. The cockroach allergen Bla g 1 has multiple repeats of approximately 100 amino acids, but the fold of the protein ...and its biological function are unknown. Objective We sought to determine the structure of Bla g 1, investigate the implications for allergic disease, and standardize cockroach exposure assays. Methods nBla g 1 and recombinant constructs were compared by using ELISA with specific murine IgG and human IgE. The structure of Bla g 1 was determined by x-ray crystallography. Mass spectrometry and nuclear magnetic resonance spectroscopy were used to examine the ligand-binding properties of the allergen. Results The structure of an rBla g 1 construct with comparable IgE and IgG reactivity to the natural allergen was solved by x-ray crystallography. The Bla g 1 repeat forms a novel fold with 6 helices. Two repeats encapsulate a large and nearly spherical hydrophobic cavity, defining the basic structural unit. Lipids in the cavity varied depending on the allergen origin. Palmitic, oleic, and stearic acids were associated with nBla g 1 from cockroach frass. One unit of Bla g 1 was equivalent to 104 ng of allergen. Conclusions Bla g 1 has a novel fold with a capacity to bind various lipids, which suggests a digestive function associated with nonspecific transport of lipid molecules in cockroaches. Defining the basic structural unit of Bla g 1 facilitates the standardization of assays in absolute units for the assessment of environmental allergen exposure.
Abstract
The Drosophila melanogaster protein Glorund (Glo) represses nanos (nos) translation and uses its quasi-RNA recognition motifs (qRRMs) to recognize both G-tract and structured UA-rich motifs ...within the nos translational control element (TCE). We showed previously that each of the three qRRMs is multifunctional, capable of binding to G-tract and UA-rich motifs, yet if and how the qRRMs combine to recognize the nos TCE remained unclear. Here we determined solution structures of a nos TCEI_III RNA containing the G-tract and UA-rich motifs. The RNA structure demonstrated that a single qRRM is physically incapable of recognizing both RNA elements simultaneously. In vivo experiments further indicated that any two qRRMs are sufficient to repress nos translation. We probed interactions of Glo qRRMs with TCEI_III RNA using NMR paramagnetic relaxation experiments. Our in vitro and in vivo data support a model whereby tandem Glo qRRMs are indeed multifunctional and interchangeable for recognition of TCE G-tract or UA-rich motifs. This study illustrates how multiple RNA recognition modules within an RNA-binding protein may combine to diversify the RNAs that are recognized and regulated.
Graphical Abstract
Graphical Abstract
In IgE-mediated food allergies, exposure to the allergen activates systemic allergic responses. Oral immunotherapy (OIT) treats food allergies through incremental increases in oral allergen exposure. ...However, OIT only induces sustained clinical tolerance and decreased basophil sensitivity in a subset of individuals despite increases in circulating allergen-specific IgG in all treated individuals. Therefore, we examined the allergen-specific antibodies from 2 OIT cohorts of patients with sustained and transient responses. Here, we compared antibodies from individuals with sustained or transient responses and discovered specific tolerance-associated conformational epitopes of the immunodominant allergen Ara h 2 recognized by neutralizing antibodies. First, we identified what we believe to be previously unknown conformational, intrahelical epitopes using x-ray crystallography with recombinant antibodies. We then identified epitopes only recognized in sustained tolerance. Finally, antibodies recognizing tolerance-associated epitopes effectively neutralized allergen to suppress IgE-mediated effector cell activation. Our results demonstrate the molecular basis of antibody-mediated protection in IgE-mediated food allergy, by defining how these antibodies disrupt IgE-allergen interactions to prevent allergic reactions. Our approach to studying the structural and functional basis for neutralizing antibodies demonstrates the clinical relevance of specific antibody clones in antibody-mediated tolerance. We anticipate that our findings will form the foundation for treatments of peanut allergy using neutralizing antibodies and hypoallergens.
There have been many attempts to identify common biophysical properties which differentiate allergens from their non-immunogenic counterparts. This review will focus on recent studies which examine ...two such factors: abundance and stability. Anecdotal accounts have speculated that the elevated abundance of potential allergens would increase the likelihood of human exposure and thus the probability of sensitization. Similarly, the stability of potential allergens dictates its ability to remain a viable immunogen during the transfer from the source to humans. This stability could also increase the resilience of potential allergens to both gastric and endosomal degradation, further skewing the immune system toward allergy. Statistical analyses confirm both abundance and stability as common properties of allergens, while epidemiological surveys show a correlation between exposure levels (abundance) and allergic disease. Additional studies show that changes in protein stability can predictably alter gastric/endosomal processing and immunogenicity, providing a mechanistic link between stability and allergenicity. However, notable exceptions exist to both hypotheses which highlight the multifaceted nature of immunological sensitization, and further inform our understanding of some of these other factors and their contribution to allergic disease.
•Beginning and evolution of the official Allergen Nomenclature system 1980–2018.•Allergen Names abbreviated genus, species and number.•Expected data including characterization of protein amino acid ...sequence, cDNA, human serum donors and experimental data.•Challenges of identifying allergens including exposure and complex human exposure and immunity.•Complexity of new methods including “omics”.
A systematic nomenclature for allergens originated in the early 1980s, when few protein allergens had been described. A group of scientists led by Dr. David G. Marsh developed a nomenclature based on the Linnaean taxonomy, and further established the World Health Organization/International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-Committee in 1986. Its stated aim was to standardize the names given to the antigens (allergens) that caused IgE-mediated allergies in humans. The Sub-Committee first published a revised list of allergen names in 1986, which continued to grow with rare publications until 1994. Between 1994 and 2007 the database was a text table online, then converted to a more readily updated website. The allergen list became the Allergen Nomenclature database (www.allergen.org), which currently includes approximately 880 proteins from a wide variety of sources. The Sub-Committee includes experts on clinical and molecular allergology. They review submissions of allergen candidates, using evidence-based criteria developed by the Sub-Committee. The review process assesses the biochemical analysis and the proof of allergenicity submitted, and aims to assign allergen names prior to publication. The Sub-Committee maintains and revises the database, and addresses continuous challenges as new “omics” technologies provide increasing data about potential new allergens. Most journals publishing information on new allergens require an official allergen name, which involves submission of confidential data to the WHO/IUIS Allergen Nomenclature Sub-Committee, sufficient to demonstrate binding of IgE from allergic subjects to the purified protein.
IgE Abs drive the symptoms of allergic disease upon cross-linking allergens on mast cells or basophils. If the IgE binding sites on the allergens could be identified, it may be useful for creating ...new forms of immunotherapy. However, direct knowledge of the human IgE (hIgE) epitopes is limited because of the very low frequency of IgE-producing B cells in blood. A new hybridoma technology using human B cells from house dust mite-allergic patients was used to identify four Der p 2-specific hIgE mAbs. Their relative binding sites were assessed and compared by immunoassays with three previously studied murine IgG mAbs. Immunoassays showed that the recognition of Der p 2 by the first three hIgE was inhibited by a single murine IgG, but the fourth hIgE recognized a different epitope from all the other mAbs. The functional ability of the hIgE that bind different epitopes to cross-link Der p 2 was demonstrated in a mouse model of passive systemic anaphylaxis. Nuclear magnetic resonance analyses of Der p 2 in complex with IgG and IgE Abs were used to identify specific residues in the epitopes. To our knowledge, the combination of immunoassays to distinguish overlapping epitopes and nuclear magnetic resonance analyses to identify specific residues involved in Ab binding provided the first epitope mapping of hIgE mAbs to an allergen. The technologies developed in this study will be useful in high-resolution mapping of human epitopes on other Ags and the design of improved therapeutics.
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