Calcium-binding proteins, particularly those in the EF-hand family, are found ubiquitously in nature, primarily for calcium transport and storage in the body. In this review, we discuss allergens in the parvalbumin, polcalcin, sarcoplasmic calcium-binding protein, and troponin C families, as well as additional allergens. Allergens from these protein families display a wide range of IgE reactivity and cross-reactivity. They are implicated in both inhaled and food allergies, and, due to their common presence, they are difficult to avoid.

In the environment, aromatic compounds are commonly introduced artificially or as natural plant secondary metabolites. A major agricultural pest, Tetranychus urticae, has acquired genes encoding for intradiol ring-cleavage dioxygenases (IDRCDs) that can detoxify such compounds. Studies of one such IDRCD, TuIDRCD930 (encoded by gene tetur07g05930), revealed a dimerization not seen before in enzymes of this class in T. urticae, including a different oligomerization mechanism observed in fungal and bacterial homologs. Through SEC-SAXS and gel filtration experiments, TuIDRCD930 was found to dimerize by utilizing two different interactions in the N-termini: changes in protonation states and the formation of a disulfide bond. Additionally, in vitro studies demonstrate catalytic efficiency of TuIDRCD930 towards polycyclic aromatic compounds, which represents an evolutionary advantage for T. urticae. It was shown that the dimerization process does not obscure access of relatively large substrates to the enzyme active site. We further displayed that the recombinant version of TuIDRCD930 is active in both monomeric and dimeric states. Overall, this work determines oligomerization and activity not previously described in IDRCDs from T. urticae and provides further evidence of how evolution has provided this pest with novel tools to overcome plant defenses.

Tetranychus urticae (T. urticae), commonly known as two spotted spider mites (TSSM), is a major agricultural pest worldwide that feeds on all major crops and has developed resistance to most chemical compounds used for its control. Genome sequence analysis of T. urticae revealed an expansion in gene families that play a role in digestion, detoxification, and transport of xenobiotics. This large detoxifying machinery, when paired with high transcriptional plasticity, has been linked to the unprecedented xenobiotic responsiveness of this pest. To better understand how T. urticae has evolved the extensive enzymes for xenobiotic detoxification, two closely related T. urticae Mu-class GSTs, TuGSTm06 (Tetur05g05220) and TuGSTm12 (Tetur05g05300), were structurally and functionally characterized. Enzymatic characterization of these two recombinant enzymes demonstrated different activity towards model substrates 1-chloro-2,4,-dinitrobenzene (CDNB) and isothiocyanates (ITCs). Some ITCs that we used in the studies are generated by plants and serve as defense compounds. We determined the crystal structures of TuGSTm06 and TuGSTm12 which revealed that the active sites of these enzymes differed only in three residues in the H-site. Single amino acid substitution suggested that these differences in the catalytic pocket may contribute to the specific catalytic attributes of each enzyme. Additionally, complementary molecular dynamics simulations predicted differences in the overall dynamic behavior of TuGSTm06 and TuGSTm12 and a correlation between the active site residues and protein dynamics in distant residues was determined. Our work highlights the complexity of the molecular basis underlying the activity of Mu-class TuGSTs and suggests that they may play a role in overcoming plant defenses using ITCs. Elucidating these molecular details is an essential step towards finding effective ways to manage this pest.

Growing ownership and presence of pets in households have contributed to increasing cases of pet-related allergies globally. In this study, we aim to analyze molecular sensitization patterns towards pet animals among a group of individuals (n = 84) who were experiencing respiratory symptoms upon exposure to furry pets. Specific IgEs (sIgEs) towards furry animal allergens from 84 individuals were tested in vitro using the ALEX2 (Allergy Explorer) macroarray. The sequence and structural homology of common pet allergens were analyzed to evaluate the molecular basis of sensitization. Tested individuals showed allergic responses towards 20 allergens belonging to secretoglobin, lipocalin, and serum albumin protein families from cat, dog, horse, rabbit, mouse, guinea pig, hamster, and cow. Sensitization towards cats and dogs was found to be dominant, and cat allergen Fel d 1 was identified as the major sensitizer for the tested population, with 84.5% of patients having IgE against the protein. Major dog allergens were shown to be Can f 1 (32.1% of patients reacting) and Can f 6 (26.1%). The majority of the population (61.9%) had IgE to multiple allergens, largely within the lipocalin and serum albumin allergen families. This study reports higher sensitization towards cat and dog allergens for the individuals incorporated in the study. Understanding patterns of IgE sensitization to pets may help in diagnosis and development of better allergy treatments.

We previously determined crystal structures of peanut allergen Ara h 8.0101 in the apo form as well as in complex with model ligands. These structures illustrated the varied ligand binding capabilities of PR-10s and Ara h 8’s structural similarity to the major birch allergen Bet v 1. Here, we expanded on those structural studies with structures of Ara h 8.0101 and Ara h 8.0201 in complex with 8-anilino-1-naphthalene sulfonate (ANS), as well as the apo form of Ara h 8.0201. Structural studies revealed that both proteins may bind more than one ANS molecule. We also examined the impact of ANS on the ligand binding cavities of Ara h 8.0101 and Ara h 8.0201 with fluorescence assays and compared the results to prototypic PR-10 Bet v 1.0101. Moreover, as ANS is often used in fluorescence-based ligand binding assays, we analyzed structures from the PDB and provided a summary on experimentally determined ANS binding sites. These analyses show that ANS is useful for investigation of ligand binding sites, but it may also participate in non-specific reactions on nonpolar surfaces of proteins.

Molecular analysis of interactions between IgE antibody and allergen allows the structural basis of IgE recognition to be defined. Human IgE (hIgE) epitopes of respiratory lipocalin allergens, including Can f 1, remain elusive due to a lack of IgE-allergen complexes. This study aims to map the structure of allergenic epitopes on Can f 1. The fragment antigen-binding (Fab) regions of Can f 1 specific human IgE monoclonal antibodies (hIgE mAb) were used to determine the structures of IgE epitopes. Epitope mutants were designed to target Can f 1 epitopes. Immunoassays and a human FcεRIα transgenic mouse model of passive anaphylaxis in vivo were used to assess the functional activity of epitope mutants. Crystal structures of natural or recombinant Can f 1 complexed with two hIgE mAb 1J11 and 12F3 Fabs, respectively, were determined. The hIgE mAb bound to two partially overlapping epitopes and recognized two different Can f 1 conformations. The hIgE mAb 12F3 showed an unusual mode of binding by protruding its heavy chain CDR3 inside the Can f 1 calyx. Epitope mutants generated based on the structural analyses displayed a 64%-89% reduction in IgE antibody binding and failed to induce passive anaphylaxis in a human FcεRIα transgenic mouse model. In summary, the structures of Can f 1-hIgE Fab complexes revealed two unique and partially overlapping epitopes on Can f 1. The modification of the identified IgE epitopes provides a pathway for the design of hypoallergens to treat dog allergies.

Background:Advancements in hybridoma technology have enabled the production of human IgE monoclonal antibodies (hIgE mAb) for successful IgE epitope mapping of major allergens. Here, we assessed the hypoallergenicity of three IgE-epitope mutants (single 4C8 or 2F10, and double 4C8 + 2F10 epitope mutants) of house dust mite allergen (HDM) Der p 2. Methods:Humanized rat basophilic leukemia (huRBL) cells, passively sensitized overnight with either pairs of Der p 2 specific hIgE mAb (2F10, 4C8 or 2G1) or HDM-allergic serum (n=8), were stimulated with either wildtype (WT) Der p 2 or an epitope mutant and mediator release was measured. Results:No degranulation was induced upon stimulation with all mutants, when cells were sensitized with pairs of hIgE mAb specific for at least one mutated epitope. HIgE mAb specific for non-mutated epitopes led to mediator release comparable to WT Der p 2, indicating that epitopes recognized by the three different hIgE mAb are not overlapping and that the 3D-structure of the mutants is conserved. The double 4C8 + 2F10 epitope mutant had a significantly reduced maximal mediator release (48.3%) compared to the WT, in cells sensitized with allergic donor serum. Overall, the area-under-the-curve of mediator release curves induced by the mutants was significantly lower (31-65%) compared to WT. When comparing the EC20, the double 4C8 + 2F10 epitope mutant required a 158-fold higher antigen concentration to induce the same extent of mediator release as WT Der p 2. Conclusion:Der p 2 epitope mutants display significantly reduced allergenicity. Particularly, the double 4C8 + 2F10 epitope mutant demonstrated a strong potential as a novel AIT vaccine candidate.

Kiwifruit allergy was first described over 40 years ago and is becoming increasingly common worldwide. This is most likely related to the fact that kiwifruit production and consumption increased by almost two orders of magnitude during the last 50 years. Currently, there are thirteen officially registered allergens belonging to the species Actinidia deliciosa (green kiwifruit), and three officially registered allergens belonging to the species Actinidia chinensis (golden kiwifruit). The molecular properties of the kiwifruit allergens are summarized, and their features are discussed, considering the protein families to which they belong. At present, kiwifruit allergens are found to belong to 13 protein families. Allergic reactions caused by these molecules can be local, for example, related to the oral cavity, but in some cases systemic responses, such as anaphylaxis, are also observed. Generally, kiwifruit allergy should not be considered as a homogenous disorder, as it was noted that there are distinct groups of patients with different sensitization profiles. Therefore, the diagnostic process may be challenging, as in many cases other food allergies must be considered. Frequently cross-reactivity between kiwifruit allergens and their homologs originating from other organisms has a significant impact on the wellbeing of the affected individuals.

The hole mutagenesis approach was used to interrogate the importance of F337 in Trypanosoma cruzi glucokinase (TcGlcK) in order to understand the complete set of binding interactions that are made by Dglucosamine analogue inhibitors containing aromatic tail groups that can extend to the outer part of the active site. An interesting inhibitor of this analogue class includes 2-N-carboxybenzyl-2-deoxy-Dglucosamine (CBZ-GlcN), which exhibits strong TcGlcK binding with a Ki of 710 nM. The residue F337 is found at the outer part of the active site that stems from the second protein subunit of the homodimeric assembly. In this study, F337 was changed to leucine and alanine so as to diminish phenylalanine's side chain size and attenuate intermolecular interactions in this region of the binding cavity. Results from enzyme - inhibitor assays revealed that the phenyl group of F337 made dominant hydrophobic interactions with the phenyl group of CBZ-GlcN as opposed to 7r - 7r stacking interactions. Moreover, enzymatic activity assays and X-ray crystallographic experiments indicated that each of these sitedirected mutants primarily retained their activity and had high structural similarity of their protein fold. A computed structure model of T. cruzi hexokinase (TcHxK), which was produced by the artificial intelligence system AlphaFold, was compared to an X-ray crystal structure of TcGlcK. Our structural analysis revealed that TcHxK lacked an F337 counterpart residue and probably exists in the monomeric form. We proposed that the D-glucosamine analogue inhibitors that are structurally similar to CBZ-GlcN may not bind as strongly in TcHxK as they do in TcGlcK because of absent van der Waals contact from residue side chains. (c) 2023 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

Structural and allergenic characterization of mite profilins has not been previously pursued to a similar extent as plant profilins. Here, we describe structures of profilins originating from Tyrophagus putrescentiae (registered allergen Tyr p 36.0101) and Dermatophagoides pteronyssinus (here termed Der p profilin), which are the first structures of profilins from Arachnida. Additionally, the thermal stabilities of mite and plant profilins are compared, suggesting that the high number of cysteine residues in mite profilins may play a role in their increased stability. We also examine the cross-reactivity of plant and mite profilins as well as investigate the relevance of these profilins in mite inhalant allergy. Despite their high structural similarity to other profilins, mite profilins have low sequence identity with plant and human profilins. Subsequently, these mite profilins most likely do not display cross-reactivity with plant profilins. At the same time the profilins have highly conserved poly(l-proline) and actin binding sites.

Due to the very low concentration and polyclonal nature of IgE in peripheral blood, the fine molecular detail underlying allergy has been difficult to study. Human monoclonal antibodies (mAbs) obtained from subjects with allergic rhinitis will now allow comprehensive characterization of the major allergens involved in these responses.

Small calcium-binding proteins such as parvalbumins (PVs) are major seafood and fish allergens. However, the impact of structural changes on their capacity to bind IgE has not been studied in detail. Therefore, fish and reptilian PVs, as well as human α-PV, were selected for biochemical, structural, and IgE binding studies. Likely due to their high solubility, crystallization proved difficult, so additional techniques were used to promote crystallization of the proteins. Novel crystal structures were determined for human PV, cod allergen Gad m 1.0201, saltwater crocodile allergen Cro p 1.0101, and the α-PV from thornback ray. β-PVs are considered the major fish allergens, while α-PVs are rarely categorized as allergens. To explain these differences, the results of structural and IgE binding studies were combined. This approach allowed us to provide new insight into IgE binding epitopes present on PVs, focusing on cross-reactivity among the selected α- and β-PVs. In addition, we have shown that these proteins display remarkable thermal stability across a range of pH conditions, which is relevant in the case of food allergens and food processing. Moreover, it is shown that the presence of calcium cations is critical for stability of the studied PVs via their protein folding, which has an impact on the formation of IgE binding epitopes. These studies shows the stability of fish and reptile PV allergens, and it allows for further evaluation of their IgE cross-reactivity.

Identification of IgE epitopes on major dog allergen Can f 1 provides a rationale for the epitope-based design of hypoallergens that may be used for immunotherapy. This process is facilitated by using human IgE monoclonal antibodies (hIgE mAb) from dog-allergic individuals.

Antibodies are widely used in medicinal and scientific research due to their ability to bind to a specific antigen. Most often, antibodies are composed of heavy and light chain domains. Under physiological conditions, light chains are produced in excess, as compared to the heavy chain. It is now known that light chains are not silent partners of the heavy chain and can modulate the immune response independently. In this work, the first crystal structure of a light chain dimer originating from mice is described. It represents the light chain dimer of 6A8, a monoclonal antibody specific to the allergen Der f 1. Building on the unexpected occurrence of this kind of dimer, we have demonstrated that this light chain is stable in solution alone. Moreover, enzyme-linked immunosorbent assays (ELISA) have revealed that, when the light chain is not partnered to its corresponding heavy chain, it interacts non-specifically with a wide range of proteins. Computational studies were used to provide insight on the role of the 6A8 heavy chain domain in the specific binding to Der f 1. Overall, this work demonstrates and supports the ongoing notion that light chains can function by themselves and are not silent partners of heavy chains.

Fish allergy affects up to 3% of the population, with the majority of fish-allergic individuals having IgE recognizing β-parvalbumins. It was shown that fish allergic individuals are at risk of allergic reaction when consuming meat from crocodiles. Therefore, comparative studies of fish and reptilian parvalbumins will help to understand the molecular basis of the cross-reactivity and can improve generation of avoidance guidelines for parvalbumin-allergic patients.

Mites are highly prevalent arthropods that infest diverse ecological niches globally. Approximately 55,000 species of mites have been identified but many more are yet to be discovered. Of the ones we do know about, most go unnoticed by humans and animals. However, there are several species from the Acariformes superorder that exert a significant impact on global human health. House dust mites are a major source of inhaled allergens, affecting 10–20% of the world’s population; storage mites also cause a significant allergy in susceptible individuals; chiggers are the sole vectors for the bacterium that causes scrub typhus; Demodex mites are part of the normal microfauna of humans and their pets, but under certain conditions populations grow out of control and affect the integrity of the integumentary system; and scabies mites cause one of the most common dermatological diseases worldwide. On the other hand, recent genome sequences of mites provide novel tools for mite control and the development of new biomaterial with applications in biomedicine. Despite the palpable disease burden, mites remain understudied in parasitological research. By better understanding mite biology and disease processes, researchers can identify new ways to diagnose, manage, and prevent common mite-induced afflictions. This knowledge can lead to improved clinical outcomes and reduced disease burden from these remarkably widespread yet understudied creatures.