Precision engineering for localization, validation, and modification of allergenic epitopes.

The allergen-IgE interaction is essential for the genesis of allergic responses, yet investigation of the molecular basis of these interactions is in its infancy. Precision engineering has unveiled the molecular features of allergen-antibody interactions at the atomic level. High-resolution technologies, including x-ray crystallography, nuclear magnetic resonance spectroscopy, and cryo-electron microscopy, determine allergen-antibody structures. X-ray crystallography of an allergen-antibody complex localizes in detail amino acid residues and interactions that define the epitope-paratope interface. Multiple structures involving murine IgG mAbs have recently been resolved. The number of amino acids forming the epitope broadly correlates with the epitope area. The production of human IgE mAbs from B cells of allergic subjects is an exciting recent development that has for the first time enabled an actual IgE epitope to be defined. The biologic activity of defined IgE epitopes can be validated in vivo in animal models or by measuring mediator release from engineered basophilic cell lines. Finally, gene-editing approaches using the Clustered Regularly Interspaced Short Palindromic Repeats technology to either remove allergen genes or make targeted epitope engineering at the source are on the horizon. This review presents an overview of the identification and validation of allergenic epitopes by precision engineering.

Structural analysis of human IgE monoclonal antibody epitopes on dust mite allergen Der p 2.

BACKGROUND: Human IgE (hIgE) mAbs against major mite allergen Der p 2 developed using human hybridoma technology were used for IgE epitope mapping and analysis of epitopes associated with the hIgE repertoire. OBJECTIVE: We sought to elucidate the new hIgE mAb 4C8 epitope on Der p 2 and compare it to the hIgE mAb 2F10 epitope in the context of the allergenic structure of Der p 2. METHODS: X-ray crystallography was used to determine the epitope of anti-Der p 2 hIgE mAb 4C8. Epitope mutants created by targeted mutagenesis were analyzed by immunoassays and in vivo using a human high-affinity IgE receptor (FcεRIα)-transgenic mouse model of passive systemic anaphylaxis. RESULTS: The structure of recombinant Der p 2 with hIgE mAb 4C8 Fab was determined at 3.05 Å. The newly identified epitope region does not overlap with the hIgE mAb 2F10 epitope or the region recognized by 3 overlapping hIgE mAbs (1B8, 5D10, and 2G1). Compared with wild-type Der p 2, single or double 4C8 and 2F10 epitope mutants bound less IgE antibodies from allergic patients by as much as 93%. Human FcεRIα-transgenic mice sensitized by hIgE mAbs, which were susceptible to anaphylaxis when challenged with wild-type Der p 2, could no longer cross-link FcεRI to induce anaphylaxis when challenged with the epitope mutants. CONCLUSIONS: These data establish the structural basis of allergenicity of 2 hIgE mAb nonoverlapping epitopes on Der p 2, which appear to make important contributions to the hIgE repertoire against Der p 2 and provide molecular targets for future design of allergy therapeutics.

Structural and functional studies reveal the molecular basis of substrate promiscuity of a glycosyltransferase originating from a major agricultural pest.

The two-spotted spider mite, Tetranychus urticae, is a major cosmopolitan pest that feeds on more than 1100 plant species. Its genome contains an unprecedentedly large number of genes involved in detoxifying and transporting xenobiotics, including 80 genes that code for UDP glycosyltransferases (UGTs). These enzymes were acquired via horizontal gene transfer from bacteria after loss in the Chelicerata lineage. UGTs are well-known for their role in phase II metabolism; however, their contribution to host adaptation and acaricide resistance in arthropods, such as T. urticae, is not yet resolved. TuUGT202A2 (Tetur22g00270) has been linked to the ability of this pest to adapt to tomato plants. Moreover, it was shown that this enzyme can glycosylate a wide range of flavonoids. To understand this relationship at the molecular level, structural, functional, and computational studies were performed. Structural studies provided specific snapshots of the enzyme in different catalytically relevant stages. The crystal structure of TuUGT202A2 in complex with UDP-glucose was obtained and site-directed mutagenesis paired with molecular dynamic simulations revealed a novel lid-like mechanism involved in the binding of the activated sugar donor. Two additional TuUGT202A2 crystal complexes, UDP-(S)-naringenin and UDP-naringin, demonstrated that this enzyme has a highly plastic and open-ended acceptor-binding site. Overall, this work reveals the molecular basis of substrate promiscuity of TuUGT202A2 and provides novel insights into the structural mechanism of UGTs catalysis.

Structural homology of mite profilins to plant profilins is not indicative of allergic cross-reactivity.

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.

Microscopic Menaces: The Impact of Mites on Human Health.

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.

Structural, Biophysical, and Computational Studies of a Murine Light Chain Dimer.

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.

ß-Cyanoalanine synthase protects mites against Arabidopsis defenses.

Glucosinolates are antiherbivory chemical defense compounds in Arabidopsis (Arabidopsis thaliana). Specialist herbivores that feed on brassicaceous plants have evolved various mechanisms aimed at preventing the formation of toxic isothiocyanates. In contrast, generalist herbivores typically detoxify isothiocyanates through glutathione conjugation upon exposure. Here, we examined the response of an extreme generalist herbivore, the two-spotted spider mite Tetranychus urticae (Koch), to indole glucosinolates. Tetranychus urticae is a composite generalist whose individual populations have a restricted host range but have an ability to rapidly adapt to initially unfavorable plant hosts. Through comparative transcriptomic analysis of mite populations that have differential susceptibilities to Arabidopsis defenses, we identified ß-cyanoalanine synthase of T. urticae (TuCAS), which encodes an enzyme with dual cysteine and ß-cyanoalanine synthase activities. We combined Arabidopsis genetics, chemical complementation and mite reverse genetics to show that TuCAS is required for mite adaptation to Arabidopsis through its ß-cyanoalanine synthase activity. Consistent with the ß-cyanoalanine synthase role in detoxification of hydrogen cyanide (HCN), we discovered that upon mite herbivory, Arabidopsis plants release HCN. We further demonstrated that indole glucosinolates are sufficient for cyanide formation. Overall, our study uncovered Arabidopsis defenses that rely on indole glucosinolate-dependent cyanide for protection against mite herbivory. In response, Arabidopsis-adapted mites utilize the ß-cyanoalanine synthase activity of TuCAS to counter cyanide toxicity, highlighting the mite's ability to activate resistant traits that enable this extreme polyphagous herbivore to exploit cyanogenic host plants.

EAACI Molecular Allergology User's Guide 2.0.

Since the discovery of immunoglobulin E (IgE) as a mediator of allergic diseases in 1967, our knowledge about the immunological mechanisms of IgE-mediated allergies has remarkably increased. In addition to understanding the immune response and clinical symptoms, allergy diagnosis and management depend strongly on the precise identification of the elicitors of the IgE-mediated allergic reaction. In the past four decades, innovations in bioscience and technology have facilitated the identification and production of well-defined, highly pure molecules for component-resolved diagnosis (CRD), allowing a personalized diagnosis and management of the allergic disease for individual patients. The first edition of the "EAACI Molecular Allergology User's Guide" (MAUG) in 2016 rapidly became a key reference for clinicians, scientists, and interested readers with a background in allergology, immunology, biology, and medicine. Nevertheless, the field of molecular allergology is moving fast, and after 6 years, a new EAACI Taskforce was established to provide an updated document. The Molecular Allergology User's Guide 2.0 summarizes state-of-the-art information on allergen molecules, their clinical relevance, and their application in diagnostic algorithms for clinical practice. It is designed for both, clinicians and scientists, guiding health care professionals through the overwhelming list of different allergen molecules available for testing. Further, it provides diagnostic algorithms on the clinical relevance of allergenic molecules and gives an overview of their biology, the basic mechanisms of test formats, and the application of tests to measure allergen exposure.

Human Monoclonal IgE Antibodies-a Major Milestone in Allergy.

PURPOSE OF REVIEW: Bound to its high affinity receptor on mast cells and basophils, the IgE antibody molecule plays an integral role in the allergic reaction. Through interactions with the allergen, it provides the sensitivity and specificity parameters for cell activation and mediator release that produce allergic symptoms. Advancements in human hybridoma technologies allow for the generation and molecular definition of naturally occurring allergen-specific human IgE monoclonal antibodies. RECENT FINDINGS: A high-resolution structure of dust mite allergen Der p 2 in complex with Fab of the human IgE mAb 2F10 was recently determined using X-ray crystallography. The structure reveals the fine molecular details of IgE 2F10 binding its 750 Å2 conformational epitope on Der p 2. This review provides an overview of this major milestone in allergy, the first atomic resolution structure of an authentic human IgE epitope. The molecular insights that IgE epitopes provide will allow for structure-based design approaches to the development of novel diagnostics, antibody therapeutics, and immunotherapies.

Role of Small Molecule Ligands in IgE-Mediated Allergy.

PURPOSE OF REVIEW: A significant fraction of allergens bind small molecular ligands, and many of these compounds are classified as lipids. However, in most cases, we do not know the role that is played by the ligands in the allergic sensitization or allergic effector phases. RECENT FINDINGS: More effort is dedicated toward identification of allergens' ligands. This resulted in identification of some lipidic compounds that can play active immunomodulatory roles or impact allergens' molecular and allergic properties. Four allergen families (lipocalins, NPC2, nsLTP, and PR-10) are among the best characterized in terms of their ligand-binding properties. Allergens from these four families are able to bind many chemically diverse molecules. These molecules can directly interact with human immune system and/or affect conformation and stability of allergens. While there is more data on the allergens and their small molecular ligands, we are just starting to understand their role in allergy.

Development of fluorophore labeled or biotinylated anticancer small molecule NSC243928.

Small molecule NSC243928 binds with LY6K, a potential target for the treatment of triple-negative breast cancer, and induces cancer cell death with an unclear mechanism. We have developed chemical tools to identify the molecular mechanisms of NSC243928-LY6K interaction. Herein, we report on the development and synthesis of biotinylated and fluorophore-tethered derivatives of NSC243928 guided by docking studies and molecular dynamics. Surface plasmon resonance assay indicates that these derivatives retained a direct binding with LY6K protein. Confocal analysis revealed that nitrobenzoxadiazole (NBD) fluorophore tagged NSC243928 is retained in LY6K expressing cancer cells. These novel modified compounds will be employed in future in vitro and in vivo studies to understand the molecular mechanisms of NSC243928 mediated cancer cell death. These studies will pave the path for developing novel targeted therapeutics and understanding any potential side-effects of these treatments for hard-to-treat cancers such as triple-negative breast cancer or other cancers with high expression of LY6K.

Intradiol ring cleavage dioxygenases from herbivorous spider mites as a new detoxification enzyme family in animals.

BACKGROUND: Generalist herbivores such as the two-spotted spider mite Tetranychus urticae thrive on a wide variety of plants and can rapidly adapt to novel hosts. What traits enable polyphagous herbivores to cope with the diversity of secondary metabolites in their variable plant diet is unclear. Genome sequencing of T. urticae revealed the presence of 17 genes that code for secreted proteins with strong homology to "intradiol ring cleavage dioxygenases (DOGs)" from bacteria and fungi, and phylogenetic analyses show that they have been acquired by horizontal gene transfer from fungi. In bacteria and fungi, DOGs have been well characterized and cleave aromatic rings in catecholic compounds between adjacent hydroxyl groups. Such compounds are found in high amounts in solanaceous plants like tomato, where they protect against herbivory. To better understand the role of this gene family in spider mites, we used a multi-disciplinary approach to functionally characterize the various T. urticae DOG genes. RESULTS: We confirmed that DOG genes were present in the T. urticae genome and performed a phylogenetic reconstruction using transcriptomic and genomic data to advance our understanding of the evolutionary history of spider mite DOG genes. We found that DOG expression differed between mites from different plant hosts and was induced in response to jasmonic acid defense signaling. In consonance with a presumed role in detoxification, expression was localized in the mite's gut region. Silencing selected DOGs expression by dsRNA injection reduced the mites' survival rate on tomato, further supporting a role in mitigating the plant defense response. Recombinant purified DOGs displayed a broad substrate promiscuity, cleaving a surprisingly wide array of aromatic plant metabolites, greatly exceeding the metabolic capacity of previously characterized microbial DOGs. CONCLUSION: Our findings suggest that the laterally acquired spider mite DOGs function as detoxification enzymes in the gut, disarming plant metabolites before they reach toxic levels. We provide experimental evidence to support the hypothesis that this proliferated gene family in T. urticae is causally linked to its ability to feed on an extremely wide range of host plants.

Experimental and Computational Studies Reveal Novel Interaction of Lymphocytes Antigen 6K to TGF-ß Receptor Complex.

TGF-ß signaling promotes migration, invasion, and distant colonization of cancer cells in advanced metastatic cancers. TGF-ß signaling suppresses the anti-tumor immune response in a tumor microenvironment, allowing sustained tumor growth. TGF-ß plays an important role in normal physiology; thus it is no surprise that the clinical development of effective and safe TGF-ß inhibitors has been hampered due to their high toxicity. We discovered that increased expression of LY6K in cancer cells led to increased TGF-ß signaling and that inhibition of LY6K could lead to reduced TGF-ß signaling and reduced in vivo tumor growth. LY6K is a highly cancer-specific protein, and it is not expressed in normal organs except in the testes. Thus, LY6K is a valid target for developing therapeutic strategies to inhibit TGF-ß signaling in cancer cells. We employed in vitro pull-down assays and molecular dynamics simulations to understand the structural determinants of the TGF-ß receptor complex with LY6K. This combined approach allowed us to identify the critical residues and dynamics of the LY6K interaction with the TGF-ß receptor complex. These data are critical in designing novel drugs for the inhibition of TGF-ß in LY6K expressing cancer, induction of anti-tumor immune response, and inhibition of tumor growth and metastatic spread.

The first reptilian allergen and major allergen for fish-allergic patients: Crocodile ß-parvalbumin.

BACKGROUND: Clinical cross-reactivity between bony fish, cartilaginous fish, frog, and chicken muscle has previously been demonstrated in fish-allergic patients. In indicative studies, two reports of anaphylaxis following the consumption of crocodile meat and IgE-cross-binding were linked to the major fish allergen parvalbumin (PV). This study investigates IgE-binding proteins in crocodile meat with a focus on PV and their clinical relevance. METHODS: Proteins were extracted from muscle tissue of crocodile, three bony fish, and two cartilaginous fish. A cohort of fish-allergic pediatric patients (n = 77) underwent allergen skin prick testing (SPT) to three fish preparations (n = 77) and crocodile (n = 12). IgE-binding proteins were identified and quantified by SDS-PAGE, mass spectrometric analyses, and immunoblotting using commercial and in-house antibodies, as well as individual and pooled patients' serum. PV isoforms were purified or recombinantly expressed before immunological analyses, including human mast cell degranulation assay. RESULTS: Of the tissues analyzed, PV was most abundant in heated crocodile preparation, triggering an SPT of ≥3 mm in 8 of 12 (67%) fish-allergic patients. Seventy percent (31 of 44) of fish PV-sensitized patients demonstrated IgE-binding to crocodile PV. Crocodile ß-PV was the major IgE-binding protein but 20-fold less abundant than α-PV. Cellular reactivity was demonstrated for ß-PV and epitopes predicted, explaining frequent IgE-cross-binding of ß-PVs. Both PV isoforms are now registered as the first reptile allergens with the WHO/IUIS (ß-PV as Cro p 1 and α-PV as Cro p 2). CONCLUSION: Fish-allergic individuals may be at risk of an allergy to crocodile and should seek specialist advice before consuming crocodilian meat.

Evaluation of the theoretical risk of cross-reactivity among recently identified food allergens for dogs.

BACKGROUND: There is increasing evidence of cross-reactivity between allergens of close or distant species. The A-RISC (Allergens'-Relative Identity, Similarity and Cross-reactivity) index helps evaluate the risk of theoretical cross-reactivity between proteins of the same family among different species. OBJECTIVES: To report the A-RISC indices for several food allergens of dogs between multiple food sources. MATERIALS AND METHODS: We selected several recently characterised food allergens for dogs from fish and chicken (ACTA1, ALDOA, CKM, ENO3, GAPDH, PKM and TPI1), fish (TPM1/2), beef/lamb (PGM1) and corn/potato (WAXY). When quality sequence data were available, A-RISC indices were calculated between multiple animal and plant species that can be used as food sources. For the TPM subunits, A-RISC indices also were calculated with the environmental allergens Bla g 4 and Der f 10, and the Toxocara canis nematode. RESULTS: The A-RISC indices suggest a substantial theoretical risk of cross-reactivity between species for all allergens considered. For TPM, this risk also extends to the environmental and nematode allergens. CONCLUSIONS AND CLINICAL RELEVANCE: There is a high theoretical risk of cross-reactivity between allergens of different species used as food sources. The clinical relevance of these elevated A-RISC indices should be studied further.

Allergens and their associated small molecule ligands-their dual role in sensitization.

Many allergens feature hydrophobic cavities that allow the binding of primarily hydrophobic small-molecule ligands. Ligand-binding specificities can be strict or promiscuous. Serum albumins from mammals and birds can assume multiple conformations that facilitate the binding of a broad spectrum of compounds. Pollen and plant food allergens of the family 10 of pathogenesis-related proteins bind a variety of small molecules such as glycosylated flavonoid derivatives, flavonoids, cytokinins, and steroids in vitro. However, their natural ligand binding was reported to be highly specific. Insect and mammalian lipocalins transport odorants, pheromones, catecholamines, and fatty acids with a similar level of specificity, while the food allergen ß-lactoglobulin from cow's milk is notably more promiscuous. Non-specific lipid transfer proteins from pollen and plant foods bind a wide variety of lipids, from phospholipids to fatty acids, as well as sterols and prostaglandin B2, aided by the high plasticity and flexibility displayed by their lipid-binding cavities. Ligands increase the stability of allergens to thermal and/or proteolytic degradation. They can also act as immunomodulatory agents that favor a Th2 polarization. In summary, ligand-binding allergens expose the immune system to a variety of biologically active compounds whose impact on the sensitization process has not been well studied thus far.

A Human IgE Antibody Binding Site on Der p 2 for the Design of a Recombinant Allergen for Immunotherapy.

Der p 2 is one of the most important allergens from the house dust mite Dermatophagoides pteronyssinus Identification of human IgE Ab binding epitopes can be used for rational design of allergens with reduced IgE reactivity for therapy. Antigenic analysis of Der p 2 was performed by site-directed mutagenesis based on the x-ray crystal structure of the allergen in complex with a Fab from the murine IgG mAb 7A1 that binds an epitope overlapping with human IgE binding sites. Conformational changes upon Ab binding were confirmed by nuclear magnetic resonance using a 7A1-single-chain variable fragment. In addition, a human IgE Ab construct that interferes with mAb 7A1 binding was isolated from a combinatorial phage-display library constructed from a mite-allergic patient and expressed as two recombinant forms (single-chain Fab in Pichia pastoris and Fab in Escherichia coli). These two IgE Ab constructs and the mAb 7A1 failed to recognize two Der p 2 epitope double mutants designed to abolish the allergen-Ab interaction while preserving the fold necessary to bind Abs at other sites of the allergen surface. A 10-100-fold reduction in binding of IgE from allergic subjects to the mutants additionally showed that the residues mutated were involved in IgE Ab binding. In summary, mutagenesis of a Der p 2 epitope defined by x-ray crystallography revealed an IgE Ab binding site that will be considered for the design of hypoallergens for immunotherapy.

Antigen-Antibody Complexes.

In vertebrates, immunoglobulins (Igs), commonly known as antibodies, play an integral role in the armamentarium of immune defense against various pathogens. After an antigenic challenge, antibodies are secreted by differentiated B cells called plasma cells. Antibodies have two predominant roles that involve specific binding to antigens to launch an immune response, along with activation of other components of the immune system to fight pathogens. The ability of immunoglobulins to fight against innumerable and diverse pathogens lies in their intrinsic ability to discriminate between different antigens. Due to this specificity and high affinity for their antigens, antibodies have been a valuable and indispensable tool in research, diagnostics and therapy. Although seemingly a simple maneuver, the association between an antibody and its antigen, to make an antigen-antibody complex, is comprised of myriads of non-covalent interactions. Amino acid residues on the antigen binding site, the epitope, and on the antibody binding site, the paratope, intimately contribute to the energetics needed for the antigen-antibody complex stability. Structural biology methods to study antigen-antibody complexes are extremely valuable tools to visualize antigen-antibody interactions in detail; this helps to elucidate the basis of molecular recognition between an antibody and its specific antigen. The main scope of this chapter is to discuss the structure and function of different classes of antibodies and the various aspects of antigen-antibody interactions including antigen-antibody interfaces-with a special focus on paratopes, complementarity determining regions (CDRs) and other non-CDR residues important for antigen binding and recognition. Herein, we also discuss methods used to study antigen-antibody complexes, antigen recognition by antibodies, types of antigens in complexes, and how antigen-antibody complexes play a role in modern day medicine and human health. Understanding the molecular basis of antigen binding and recognition by antibodies helps to facilitate the production of better and more potent antibodies for immunotherapy, vaccines and various other applications.

Delta class glutathione S-transferase (TuGSTd01) from the two-spotted spider mite Tetranychus urticae is inhibited by abamectin.

GSTs (Glutathione S-transferases) are known to catalyze the nucleophilic attack of the sulfhydryl group of reduced glutathione (GSH) on electrophilic centers of xenobiotic compounds, including insecticides and acaricides. Genome analyses of the polyphagous spider mite herbivore Tetranychus urticae (two-spotted spider mite) revealed the presence of a set of 32 genes that code for secreted proteins belonging to the GST family of enzymes. To better understand the role of these proteins in T. urticae, we have functionally characterized TuGSTd01. Moreover, we have modeled the structure of the enzyme in apo form, as well as in the form with bound inhibitor. We demonstrated that this protein is a glutathione S-transferase that can conjugate glutathione to 1-chloro-2,4-dinitrobenzene (CDNB). We have tested TuGSTd01 activity with a range of potential substrates such as cinnamic acid, cumene hydroperoxide, and allyl isothiocyanate; however, the enzyme was unable to process these compounds. Using mutagenesis, we showed that putative active site variants S11A, E66A, S67A, and R68A mutants, which were residues predicted to interact directly with GSH, have no measurable activity, and these residues are required for the enzymatic activity of TuGSTd01. There are several reports that associate some T. urticae acaricide resistance with increased activity of GSTs . However, we found that TuGSTd01 is not able to detoxify abamectin; in fact, the acaricide inhibits the enzyme with Ki = 101 µM. Therefore, we suggest that the increased GST activity observed in abamectin resistant T. urticae field populations is a part of the compensatory feedback loop. In this case, the increased production of GSTs and relatively high concentration of GSH in cells allow GSTs to maintain physiological functions despite the presence of the acaricide.

Structural Characterization of Act c 10.0101 and Pun g 1.0101-Allergens from the Non-Specific Lipid Transfer Protein Family.

(1) Background: Non-specific lipid transfer proteins (nsLTPs), which belong to the prolamin superfamily, are potent allergens. While the biological role of LTPs is still not well understood, it is known that these proteins bind lipids. Allergen nsLTPs are characterized by significant stability and resistance to digestion. (2) Methods: nsLTPs from gold kiwifruit (Act c 10.0101) and pomegranate (Pun g 1.0101) were isolated from their natural sources and structurally characterized using X-ray crystallography (3) Results: Both proteins crystallized and their crystal structures were determined. The proteins have a very similar overall fold with characteristic compact, mainly α-helical structures. The C-terminal sequence of Act c 10.0101 was updated based on our structural and mass spectrometry analysis. Information on proteins' sequences and structures was used to estimate the risk of cross-reactive reactions between Act c 10.0101 or Pun g 1.0101 and other allergens from this family of proteins. (4) Conclusions: Structural studies indicate a conformational flexibility of allergens from the nsLTP family and suggest that immunoglobulin E binding to some surface regions of these allergens may depend on ligand binding. Both Act c 10.0101 and Pun g 1.0101 are likely to be involved in cross-reactive reactions involving other proteins from the nsLTP family.