Fine structural recognition specificities of IgE antibodies distinguishing amoxicilloyl and amoxicillanyl determinants in allergic subjects.

IgE antibodies in the sera of subjects allergic to beta-lactam antibiotics detect a spectrum of specificities ranging from side-chain groups to an entire penicillin or cephalosporin molecule. In addition to such structural heterogeneity of allergenic determinants, IgE antibodies in the sera of different allergic subjects show heterogeneous recognition responses. Detailed immunochemical studies were carried out on the sera of penicillin-allergic subjects that showed selective and unexpected reactions with the frequently prescribed penicillin, amoxicillin. Antibodies from one subject reacted only with the amoxicilloyl determinant while IgE from another subject showed multiple reactivity with penicilloyl and penicillanyl determinants of different penicillins but not with the amoxicilloyl determinant. Quantitative hapten inhibition studies revealed that the combining sites of the former antibodies were complementary to amoxicillin in a form that permits binding to the hydroxyaminobenzyl side-chain and the thiazolidine ring carboxyl. These conditions are satisfied with the drug in the '-oyl' but not in the '-anyl' form which involves linkage through the 2-carboxyl of the thiazolidine ring. With the second serum, adsorption studies showed that the wide-ranging reactivity of IgE was due to a single population of antibodies that detected a common specificity on the different penicillins. Combining site studies revealed clear recognition of the benzyl portion of the side-chain of benzylpenicilloyl, benzylpenicillanyl, ampicilloyl, ampicillanyl and amoxicillanyl determinants when free antibody access to the side-chain was possible but little or no recognition of the ring hydroxyl of amoxicillin. Such uninhibited access may not occur, however, when amoxicillin is conjugated in the '-oyl' form since opening the beta-lactam ring allows increased flexibility and rotation of the molecule and the possibility of close association of the hydroxyaminobenzyl side-chain of amoxicillin with the linked peptide carrier. In such close steric association, H-bonding involving the ring hydroxyl and amino acids of the carrier may prevent antibody access to the side-chain region of the amoxicilloyl determinant.     

Pollen allergens: development and function

Pollen allergens interact with the human immune system and the resulting IgE antibodies provide specific probes for their identification and characterisation. In one case, grass allergenic proteins are expressed late in pollen development coincident with the laying down of reserves. Sequence similarity of allergens has indicated possible functions for some allergens. The major birch pollen allergen shows sequence similarity with pathogenesis-related proteins, which form a secondary response in plant host-pathogen interactions and show anti-microbial activity. Some allergens of unknown function are cysteine-rich proteins, while some others have cysteine-rich regions; for example, the major allergen from rye-grass pollen, Lol p 1, has a cysteine-rich N-terminal region, while at the C-terminal region four tryptophan residues together with tyrosine and phenylalanine residues resemble those of cellulose- or sugar-binding domains of other proteins. Several pollen allergens show sequence similarity to cell wall-associated enzymes, while others show hydrolytic enzyme activity often associated with cell walls.     

Modelling and Bioinformatics Analysis of the Dimeric Structure of House Dust Mite Allergens from Families 5 and 21: Der f 5 Could Dimerize as Der p 5

Abstract

Allergy represents an increasing thread to public health in both developed and emerging countries and the dust mites Dermatophagoides pteronyssinus (Der p), Blomia tropicalis (Blo t), Dermatophagoides farinae (Der f), Lepidoglyphus destructor (Lep d) and Suidasia medanensis (Sui m) strongly contribute to this problem. Their allergens are classified in several families among which families 5 and 21 which are the subject of this work. Indeed, their biological function as well as the mechanism or epitopes by which they are contributing to the allergic response remain unknown and their tridimensional structures have not been resolved experimentally except for Blo t 5 and Der p 5. Blo t 5 is a monomeric three helical bundle, whereas Der p 5 shows a three helical bundle with a kinked N-terminal helix that assembles in an entangled dimeric structure with a large hydrophobic cavity. This cavity could be involved in the binding of hydrophobic ligands, which in turn could be responsible for the shift of the immune response from tolerance to allergic inflammation. We used molecular modelling approaches to bring out if other house dust mite allergens of families 5 and 21 (Der f 5, Sui m 5, Lep d 5, Der p 21 and Der f 21) could dimerize and form a large cavity in the same way as Der p 5. Monomeric models were first performed with MODELLER using the experimental structures of Der p 5 and Blo t 5 as templates. The ClusPro server processed the selected monomers in order to assess their capacity to form dimeric structures with a positive result for Der p 5 and Der f 5 only. The other allergens (Blo t 5, Sui m 5, Lep d 5, Der p 21 and Der f 21) did not present such a propensity. Moreover, we identified mutations that should destabilize and/or prevent the formation of the Der p 5 dimeric structure. The production of these mutated proteins could help us to understand the role of the dimerization process in the allergic response induced by Der p 5, and if Der p 5 and Der f 5 behave similarly.     

Crystal structures of two homologous pathogenesis-related proteins from yellow lupine

Pathogenesis-related class 10 (PR10) proteins are restricted to the plant kingdom where they are coded by multigene families and occur at high levels. In spite of their abundance, their physiological role is obscure although members of a distantly related subclass (cytokinin-specific binding proteins) are known to bind plant hormones. PR10 proteins are of special significance in legume plants where their expression patterns are related to infection by the symbiotic, nitrogen-fixing bacteria. Here we present the first crystal structures of classic PR10 proteins representing two homologues from one subclass in yellow lupine. The general fold is similar and, as in a birch pollen allergen, consists of a seven-stranded beta-sheet wrapped around a long C-terminal helix. The mouth of a large pocket formed between the beta-sheet and the helix seems a likely site for ligand binding. The shape of the pocket varies because, in variance with the rigid beta-sheet, the helix shows unusual conformational variability consisting in bending, disorder, and axial shifting. A surface loop, proximal to the entrance to the internal cavity, shows an unusual structural conservation and rigidity in contrast to the high glycine content in its sequence. The loop is different from the so-called glycine-rich P-loops that bind phosphate groups of nucleotides, but it is very likely that it does play a role in ligand binding in PR10 proteins.     

Evolutionary divergence of β–expansin structure and function in grasses parallels emergence of distinctive primary cell wall traits

Expansins are wall‐loosening proteins that promote the extension of primary cell walls without the hydrolysis of major structural components. Previously, proteins from the EXPA (α–expansin) family were found to loosen eudicot cell walls but to be less effective on grass cell walls, whereas the reverse pattern was found for EXPB (β–expansin) proteins obtained from grass pollen. To understand the evolutionary and structural bases for the selectivity of EXPB action, we assessed the extension (creep) response of cell walls from diverse monocot families to EXPA and EXPB treatments. Cell walls from Cyperaceae and Juncaceae (families closely related to grasses) displayed a typical grass response (‘β–response’). Walls from more distant monocots, including some species that share with grasses high levels of arabinoxylan, responded preferentially to α–expansins (‘α–response’), behaving in this regard like eudicots. An expansin with selective activity for grass cell walls was detected in Cyperaceae pollen, coinciding with the expression of genes from the divergent EXPB–I branch that includes grass pollen β–expansins. The evolutionary origin of this branch was located within Poales on the basis of phylogenetic analyses and its association with the ‘sigma’ whole‐genome duplication. Accelerated evolution in this branch has remodeled the protein surface in contact with the substrate, potentially for binding highly substituted arabinoxylan. We propose that the evolution of the divergent EXPB–I group made a fundamental change in the target and mechanism of wall loosening in the grass lineage possible, involving a new structural role for xylans and the expansins that target them.     

Ani s 10, a new Anisakis simplex allergen: cloning and heterologous expression

Anisakiasis is a human disease caused by accidental ingestion of larval nematodes belonging to the Anisakidae family. Anisakiasis is often associated with a strong allergic response. Diagnosis of A. simplex allergy is currently carried out by test based on the IgE reactivity to a complete extract of L3 Anisakis larvae although the specificity of these diagnostic tests is poor. Improving the specificity of the diagnostic test is possible using purified recombinant allergens. A new Anisakis allergen, named Ani s 10, was detected by immunoscreening an expression cDNA library constructed from L3 Anisakis simplex larvae. The new allergen was overproduced in Escherichia coli; it is a protein of 212 amino acids and it was localized as a 22 kDa protein band in an ethanol fractionated extract from the parasite. Ani s 10 has no homology with any other described protein, and its sequence is composed by seven almost identical repetitions of 29 amino acids each. A total of 30 of 77 Anisakis allergic patients (39%) were positive both to rAni s 10 and natural Ani s 10 by immunoblotting. The new allergen could be useful in a component-resolved diagnosis system for Anisakis allergy.     

Unconventional method based on circular dichroism to detect peanut DNA in food by means of a PNA probe and a cyanine dye

In this paper we report an innovative and unconventional method based on circular dichroism for the identification of peanut DNA in food, which can be detected after PCR amplification at the nanomolar level by using an achiral PNA probe complementary to a tract of the peanut Ara h 2 gene and an achiral 3,3'-diethylthiadicarbocyanine dye [DiSC(2)(5)]. Peanuts are one of the most common causes of severe allergic reactions to foods and are particularly dangerous when they are "hidden" (undeclared) in food. For better protection of consumers, detection methods are required to specifically detect the presence of hidden allergens in a wide variety of food items. Alternative to the detection of the proteins is the determination of species-specific DNA, which is more resistant to technological treatments. PNAs are very specific probes able to recognize DNA sequences with high affinity and evidence for the binding can be obtained by using the DiSC(2)(5) dye, which aggregates onto the PNA-DNA duplex giving rise to a characteristic visibile band at 540 nm. Because the PNA-DNA duplex is in a right-handed helical conformation, the aggregation of the dye to the duplex gives also rise to a strong CD signal in the 500-600 nm region with a strong exciton coupling due to the formation of multimeric species, since the handedness of the helix is transferred to the dye aggregate. The dye does not interact with the free single-stranded DNA and although aggregating on the achiral PNA, this interaction is obviously not detectable by circular dichroism. Thus, only the formation of the PNA-DNA duplex, which takes place only upon specific Watson-Crick hydrogen binding between the PNA and the DNA bases, is detected, ensuring a very high specificity and sensitivity. The method has been optimized in a model system by using a synthetic oligonucleotide complementary to the PNA probe, showing that the intensity of the signal is linearly related to the amount of the DNA. The optimized method has been applied to the identification and quantitation of DNA extracted and amplified by PCR from peanuts and from peanut-containing foods, allowing for a very sensitive detection at a very low level (few pmol).     

IgE binding to proteins from sesame and assessment of allergenicity: implications for biotechnology?

Successful prediction of the potential allergenicity of a protein may be a key factor in the development of novel, genetically modified foods. The use of the decision tree approach for the prediction of allergenicity is discussed. The methods currently used for identifying allergenic proteins (including use of IgE from patient sera for recognition of proteins) are reviewed. Finally, a specific review of the literature concerning identification of allergens from sesame leads to the conclusion that in the absence of validated animal models, identification of allergenicity (and, consequently, prediction of allergenicity) may be problematic.     

Proteomic analysis of wheat proteins recognized by IgE antibodies of allergic patients

Wheat belongs to six major food allergens inducing IgE-mediated hypersensitivity reaction manifesting as cutaneous, gastrointestinal, and respiratory symptoms. Although cereals are a staple food item in most diets, only a few wheat proteins causing hypersensitivity have been identified. To characterize wheat allergens, salt-soluble wheat extracts were separated by 1-DE and 2-DE and IgE-binding proteins were detected by immunoblotting using sera of patients with allergy to ingested wheat. Proteins, frequently recognized by IgE on 2-DE were analyzed by MALDI-TOF and QTOF and their spectrum was completed by 1-DE and LCQ(DECA) nLC-MS/MS IT technique. Using all three techniques we identified 19 potential wheat allergens such as alpha-amylase inhibitors, beta-amylase, profilin, serpin, beta-D-glucan exohydrolase, and 27K protein. Employing newly developed ELISA, levels of IgE Abs against Sulamit wheat extract and alpha-amylase inhibitors type 1 and 3 were quantified and shown to be significantly elevated in sera of allergic patients compared to those of healthy controls. The level of IgE Abs against alpha-amylase inhibitor type 3 was lower, slightly above the cut-off value in the majority of patients' sera. Our findings contribute to the identification of wheat allergens aimed to increase the specificity of serum IgE and cell activation diagnostic assays.     

Allergenic evaluation of Malassezia furfur crude extracts

Crude extracts of the lipophilic yeast Malassezia furfur were obtained from 2, 6, 10 and 28 day old cultures. The in vitro cultivation periods corresponded, respectively, to the lag phase, middle of the log phase, end of log phase and the decline phase of the growth curve, which was based on viable cell counts obtained with a fluorescent viability test. Biochemical analyses showed that the protein and carbohydrate contents were greater in day 10 extracts. Seventy patients with different allergic manifestations and 30 healthy volunteers were skin prick tested using the extracts. Of these, thirteen (18.57%) patients gave positive responses. SDS PAGE gradient electrophoretic profiles of the preparations indicated that the 28 day extracts contained the greatest number of protein bands with molecular weights ranging mostly between 30 and 94 kDa. Immunoblots incubated with individual patient sera showed that four IgE binding M. furfur allergens of approximately 88, 61, 52 and 39 kDa were present in the 28 day extracts. The components identified could be used for detecting IgE mediated responses to M. furfur among individuals affected with different allergic conditions.This revised version was published online in October 2005 with corrections to the Cover Date.