The polyprotein allergens of nematodes (NPAs) - structure at last, but still mysterious

We are engaged in structural and functional studies of several types of lipid binding protein that are only found in nematodes. Amongst these are the nematode polyprotein allergens (NPAs) and we now report the solution structure of ABA-1A (As-NPA-A1), the most repeated unit within the NPA array of Ascaris suum, which is almost identical in amino acid sequence to that of Ascaris lumbricoides. The protein forms a slightly flattened, compact, globular fold consisting of a long central helix that participates in two flanking helical bundles. Two pockets lined with apolar amino acid sidechains are apparent, one in the carboxy-terminal region of the protein, and another smaller one in the amino-terminal region. The former appears to be the main site of fatty acid binding, and the latter may have different, though possibly overlapping, ligand binding propensities. The structure of the binding sites indicates that lipid ligands are anchored within them with their hydrophobic tails oriented towards the core of the protein and their polar headgroups bound to charged sidechains at the mouth of the pockets. The three-dimensional architectures of the amino- and carboxy-terminal halves of ABA-1A are closely similar, thereby strengthening the long-suspected idea that the repeated units of NPAs themselves originate from an ancient duplication event.     

The disulphide mapping,folding and characterisation of recombinant Ber e 1, an allergenic protein,and SFA8, two sulphur-rich 2S plant albumins

We have cloned and expressed genes encoding the allergenic brazil nut 2S albumin (Ber e 1) and the sunflower albumin 8 (SFA8) in the methylotrophic yeast Pichia pastoris. We show that both proteins were secreted at high levels and that the purified proteins were properly folded. We also showed that Ber e 1 is glycosylated during secretion and that the glycan does not interfere with the folding or immunoreactivity. The disulphide map of the Ber e 1 protein was experimentally established and is in agreement with the conserved disulphide structure of other members of the 2S albumin family. A model three-dimensional structure of the allergen was generated. During the expression studies and through mutation we have also shown that alteration of the sequences around the Kex2 endoproteolytic processing site in the expressed fusion protein can compromise the secretion by targeting part of the protein for possible degradation. The secreted production of these properly folded sulphur-rich plant albumins presents an opportunity to delineate the attributes that make an allergen and to facilitate the diagnosis and therapy of type I allergy.     

Cladosporium and respiratory allergy: Diagnostic implications in Saudi Arabia

An allergological study to evaluate allergenicity to Cladosporium, Burkard 7-Day Volumetric Spore Trap and Personal Volumetric air sampler (viable mode) were employed to conduct air sampling for 12 months in three regions of Saudi Arabia. The study was extended for a continuous 3rd year at one site. Skin prick testing (SPT) was also conducted on 605 allergic individuals using commercial extracts of C. herbarum. Cladosporium emerged to be the most prevalent genus in the outdoor environment constituting up to 25% of all fungal spores in the dry region and 37.1 and 41.2% in two coastal cities respectively. Amongst the species C. sphaerospermum, C. macrocarpum, C. cladosporioides and C. herbarum were noted. Maximum hourly concentrations up to 14 x 10(3) m(-3) were recorded in coastal region during winter months. Morning concentrations were higher at both city sites compared to afternoon concentration. SPT result revealed an overall 19.67% positive reactions with majority showing mild reactions.     

Determination of birch pollen allergens in different aerosol sizes

Allergens in fine particles may cause symptoms inallergic asthmatics. In order to assess the exposureof susceptible persons, a method to measure theallergen load in fine and coarse particles wasdeveloped.Aerosols are collected with a high-volume air samplerby multistage impaction. They are separated into fivesize classes, ranging from >10 m to <1 mand sampled on glass fibre filters. After sampling,filters are crushed into a fine powder using ahydraulic press. Allergens are then eluted on a shakerinto Tween-20-containing phosphate buffered saline.After microfiltration, the eluate is ready foranalysis with ELISA-techniques (Enzyme Linked ImmunoSorbens Assay).Two different methods are used for the analysis ofallergens: One is a sandwich-ELISA using monoclonalIgG-antibodies, the other is a competitive ELISA basedon polyclonal IgE-antibodies obtained from patientsallergic to birch pollen. Using the monoclonalantibodies information on the amount of one particularallergen (the major allergen Bet v1) is obtained. Onthe other hand the competitive ELISA using thepolyclonal IgE is much more sensitive and indicatesthe total birch pollen allergens. Data obtained duringspring 1998 show good correlation of pollen counts andallergen content in the coarse particle fractioncontaining intact pollen (>10 m). In smallersized fractions, the allergen load is often close tothe detection limit. When clearly detectable amountsof allergen are present, in the fine size fraction theallergen load shows only a weak correlation to thepollen counts and the allergen concentrations in thecoarse particle fraction.     

Identification and analysis of a conserved immunoglobulin E-binding epitope in soybean G1a and G2a and peanut Ara h 3 glycinins

To identify conserved immunoglobulin E (IgE)-binding epitopes among legume glycinins, we utilized recombinant soybean G2a and G2a-derived polypeptide fragments. All of these fusion polypeptides bound IgE, and the C-terminal 94-residue fragment appeared to bind more IgE. Using synthetic peptides we identified S219-N233 (S(219)GFAPEFLKEAFGVN(233)) as the dominant IgE-binding epitope. Alanine scanning of this epitope indicated that six amino acids (E224, F225, L226, F230, G231, and V232) contributed most to IgE binding. Among these amino acids, only G231 of soybean G2a is not conserved in soybean G1a (S234) and peanut Ara h 3 (Q256). Synthetic peptides corresponding to the equivalent regions in G1a and Ara h 3 bound IgE in the order Ara h 3>/=soybean G2a>soybean G1a. This sequence represents a new IgE-binding epitope that occurs in a highly conserved region present in legume glycinins. Such IgE-binding sites could provide a molecular explanation for the IgE cross-reactivity observed between soybean and peanut proteins.     

A comparative biochemical study of conifer pollen allergens

In the order Coniferales, only the family Cupressaceae is regarded as being a significant source of airborne allergens, withJuniperus ashei characterized as the most significat aeroallergen. Pollen of the closely related speciesJ. virginiana has been shown to cross-react withJ. ashei pollen, however,J. virginiana pollen is not considered an important aeroallergen. Although there have been several reports of allergies toPinus pollen, the pollen of this genus is regarded as hypoallergenic. Our previous studies have shown that pollen extracts ofJ. ashei, J. virginiana, J. pinchotii, Cupressus macrocarpa, Pinus echinata andP. taeda all contained several proteins with the same molecular weights including the reported allergen ofJ. ashei. The present study compared the biochemistry ofJ. ashei, J. virginiana andP. echinata pollen. A time course experiment ofJ. ashei, J. virginiana andP. echinata showed thatJ. ashei released a greater quantity of protein within the first minute of moistening. SDS-PAGE analyses showed that the reported allergen ofJ. ashei pollen extracts was released in large quantities within the first minute of extraction. It was also determined that individual pollen grains ofP. echinata contained a greater quantity of protein than the pollen ofJ. ashei andJ. virginiana, but due to the large size of pine pollen there was less protein per gram of pollen. Lipid analysis of these three taxa showed that the pollen ofP. echinata contained more lipid per grain and per gram of pollen. Results indicate that the rapid release of the reported allergen fromJ. ashei pollen contributes to the allergenicity of this species compared to bothJ. virginiana andP. echinata.     

Intrauterine sensitization of ovalbumin in the third trimester increases the risk of food allergy in progeny

Intrauterine sensitization caused by food allergens plays an important role in the food allergy development in progeny. The aim of our study was to determine the critical period of intrauterine sensitization during pregnancy. Female mice were exposed to ovalbumin (OVA) during different trimesters of pregnancy. Lymphocytes from their offspring were isolated and cultured, and proliferation was evaluated by CCK-8 assay. The levels of IFN-γ and IL-4 in serum were measured using ELISA. In addition, the expressions of IFN-γ and IL-4 mRNAs and proteins were detected by real-time PCR and western blot. The mice were divided into the first trimester pregnancy (FTP1 and FTP2) group, the second trimester pregnancy (STP1 and STP2) group, and the third trimester pregnancy (TTP1 and TTP2) group based on the stages of pregnancy in which their mothers were exposed to OVA and their ages. The OVA-specific lymphocyte proliferation of the TTP1 group was statistically significantly greater that in the FTP1 and STP1 groups. The serum level of IFN-γ in the TTP1 group was significantly decreased, and the serum level of IL-4 in the TTP1 group was significantly increased compared with the levels in the FTP1 and STP1 groups. The mRNA and protein expression levels of IFN-γ in the TTP1 group were significantly decreased and the mRNA and protein expression levels of IL-4 in this group were significantly increased compared with the levels in the FTP1 and STP1 groups. Our results suggest that OVA-induced intrauterine sensitization in the third trimester may increase the risk of food allergy after birth.     

Class B β-expansins are needed for pollen separation and stigma penetration

Group 1 grass pollen allergens comprise a distinctive clade within the β-expansin family of cell wall-loosening proteins and are divided by sequence divergence into two phylogenetically separable classes (A and B). They have been proposed to loosen the walls of the stigma and style. Supporting this idea, we recently showed that a transposon insertion in one of the maize group-1 allergen genes reduces the ability of pollen to effect fertilization under conditions of pollen competition. In this work, we provide additional information on the phenotype of this mutant, showing that pollen deficient in β-expansin gene expression tended to form large aggregates, leading to poor pollen dispersal on anther dehiscence, and that emerging pollen tubes had difficulties entering the silk. In addition, a silencing construct was created to reduce expression of all the class B genes with results that are consistent with those seen with the transposon insertional line, including reduced transgene transmission through the pollen. Our results provide a more detailed understanding of the role of group 1 allergens (pollen β-expansins) in maize pollen development, pollen dispersal, pollen tube penetration into the style, and pollen tube growth through the transmitting tract. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The molecular size of nematode acetylcholinesterases and their separation from nematode allergens

The molecular size of nematode acetylcholinesterases, and their separation from nematode allergens. International Journal for Parasitology 3: 735–741. Acetylcholinesterases and allergens were derived from two parasitic nematodes, Nippostrongylus brasiliensis which parasitises rats and Trichostrongylus colubriformis which infects sheep and guinea pigs.

Chromatographic studies indicated that the mol. wt of nematode acetylcholinesterases lies between 65,000 and 75,000. The acetylcholinesterases of both species were separated from nematode allergens whose mol. wt is in the range of 10,000–50,000.

γG binding antigens of T. colubriformis were located in fractions with a mol. wt range of 30,000–150,000. γE binding activity was confined to allergenic material with a mol. wt of less than 70,000.