Cloning and molecular characterization of the Hevea brasiliensis allergen Hev b 11, a class I chitinase.

BACKGROUND: In the last 10 years type-I allergy against proteins from Hevea brasiliensis latex has become an acknowledged medical issue. Fruit-allergic patients represent one risk group for developing latex allergy. Class I chitinases have been identified from chestnut, avocado and banana as relevant allergens. The chitin binding (hevein) domain from these class I chitinases has been postulated to bear the important IgE binding epitopes. OBJECTIVE: To clone the cDNA of an allergenic latex class I chitinase, to express the recombinant protein and to determine its IgE cross-reactivity with hevein (Hev b 6.02). METHODS: A full-length cDNA coding for a class I chitinase has been isolated from Hevea latex RNA by reverse transcription followed by PCR. The chitinase encoding sequence has been subcloned into the pMAL expression vector and expressed in E. coli as a fusion protein to maltose binding protein. The highly enriched recombinant protein fraction has been tested for its IgE binding capacity in immunoblots and ELISA. Furthermore, the pathogenesis-related function of the recombinant protein was tested in a fungal growth inhibition assay. RESULTS: The Hevea brasiliensis latex chitinase, designated Hev b 11, displays 70% identity to the endochitinase from avocado and its hevein-domain 58% to hevein (Hev b 6.02). The recombinant Hev b 11-maltose binding protein is recognized by latex- and fruit-allergic patients with IgE binding in both, ELISA and immunoblots. Pre-incubation of sera with rHev b 11-maltose binding protein showed an overall 16% inhibition of subsequent binding to rHev b 6.02-maltose binding protein on solid phase. The growth of F. oxysporum was inhibited in a dose dependent manner by addition of rHev b 11-maltose binding protein to the culture. CONCLUSIONS: Hev b 11, a class I chitinase, is another allergen from Hevea latex with a chitin binding domain and displays a different IgE binding capacity compared with hevein.

Characterization of api g 1.0201, a new member of the Api g 1 family of celery allergens.

BACKGROUND: The association of pollinosis with allergy to plant foods occurs in up to 70% of tree pollen-allergic patients. In recent years, some of the relevant cross-reacting proteins have been characterized at the molecular and immunological level. Api g 1 has been identified as the celery homologue of the major birch pollen allergen, Bet v 1. Although a number of Bet v 1 isoforms have been characterized from birch pollen, little is known about isoforms of food allergens and their allergenic features. METHODS: Api g 1.0201, an isoform of Api g 1, was isolated from a cDNA library, cloned and sequenced. The cDNA was expressed in Escherichia coli and the purified recombinant protein was tested in immunoblots. RESULTS: Api g 1.0201 displays 72% sequence similarity to the previously identified Api g 1.0101 and consists of 159 amino acid residues. The sequence of Api g 1.0201 has five additional amino acid residues at the carboxy-terminus as compared to Api g 1.0101. Purified recombinant Api g 1.0201 is recognized by IgE from the sera of celery-allergic patients, as well as by the murine monoclonal anti-Bet v 1 antibody. In general, this isoform displays a weaker IgE-binding capacity than Api g 1.0101, as concluded from immunoblotting experiments. Results from inhibition assays revealed that IgE-binding to Api g 1.0201 is only slightly reduced by preincubation with either purified recombinant Api g 1.0101 or purified recombinant Bet v 1a. Total inhibition was only achieved when using purified natural Bet v 1. CONCLUSIONS: At present, little is known about the IgE-binding capacity of isoforms of Bet v 1 homologues of food allergens. Identification and characterization of such isoforms may help to contribute to a better understanding of food allergy and the observed cross-reactivity to pollen allergy.

New Bet v 1 isoforms including a naturally occurring truncated form of the protein derived from Austrian birch pollen.

Bet v 1, the major pollen allergen from white birch, displays a considerable degree of heterogeneity. Until now, all molecular and immunological characterization studies of Bet v 1 isoforms have been performed with commercially available pollen of Swedish origin. In regard to clinical studies with Austrian birch pollen allergic individuals, knowledge about the isoform repertoire in Austrian birch pollen was necessary. cDNAs coding for Bet v 1 isoforms from Austrian birch pollen were cloned by PCR amplification and sequenced. Besides the Austrian variants of the Swedish isoforms Bet v 1a (62% of the clones), ALK167 (4%), and Bet v 1d/h, Bet v 1g, and Bet v 11 (24%), three sequences with a significantly lower homology to known isoforms and two Bet v 1a-homologous sequences with a 7 bp insertion coding for a truncated protein were detected. No Austrian variants of the majority of the Swedish isoforms were found. The isoforms coding for truncated proteins were expressed in Escherichia coli and tested by immunoblotting. They bound a polyclonal anti-Bet v 1 antibody but did not recognize birch pollen allergic patients' serum IgE and two Bet v 1-specific monoclonal antibodies. The similarity of the Bet v 1 isoform patterns of Swedish and Austrian birch pollen justifies the use of Bet v 1 derived from Swedish pollen for clinical studies with birch pollen allergic individuals from outside Northern Europe.

Molecular characterization of Dau c 1, the Bet v 1 homologous protein from carrot and its cross-reactivity with Bet v 1 and Api g 1.

BACKGROUND: Up to 70% of patients with birch pollen allergy exhibit the so-called oral allergy syndrome, an IgE-mediated food allergy. The most frequent and therefore best characterized pollen-fruit syndrome is apple allergy in patients suffering from tree pollen-induced pollinosis. The occurrence of adverse reactions to proteins present in vegetables such as celery and carrots in patients suffering from pollen allergy has also been reported. cDNAs for Bet v 1 homologous proteins have been cloned from celery, apple and cherry. Objective The aim of the study was to identify Bet v 1 homologues from carrot (Daucus carota), to isolate the respective cDNA, to compare the IgE-binding capacity of the natural protein to the recombinant allergen and determine the cross-reactivity to Api g 1 and Bet v 1. METHODS: Molecular characterization of the carrot allergen was performed using IgE-immunoblotting, cross-inhibition assays, N-terminal sequencing, PCR-based cDNA cloning and expression of the recombinant protein in Escherichia coli. RESULTS: A 16-kDa protein from carrot was identified as a major IgE-binding component and designated Dau c 1. Sequencing corresponding cDNAs revealed three extremely similar sequences (Dau c 1.1, 1.2 and 1.3) with an open reading frame of 462 bp coding for 154 amino acid residues. CONCLUSIONS: Purified recombinant Dau c 1.2 was tested in immunoblots displaying IgE-binding capacity comparable to its natural counterpart. Cross-inhibition assays verified the existence of common B-cell epitopes present on Dau c 1, Api g 1 as well as on Bet v 1.

Biochemical characterization of Pru a 2, a 23-kD thaumatin-like protein representing a potential major allergen in cherry (Prunus avium).

BACKGROUND: The prevalence of allergy to fruits and vegetables increased with pollinosis over the last 10 years. So far, clusters of hypersensitivity have been established and corroborated by the molecular characterization of individual cross-reacting allergens. Several case studies demonstrated the existence of allergic reactions to fruits of the subfamily Prunoideae (apricots, cherries, plums and peaches). Here, we present the characterization of a major allergen in cherry. METHODS: Characterization was performed using IgE immunoblotting and immunoblot inhibition, N-terminal sequencing, mass spectroscopy analysis and PCR-based cDNA cloning. RESULTS: A 23-kD protein was identified as IgE-binding component. As all cherry-extract-reactive sera displayed IgE-binding to this band, it was designated a major allergen from Prunus avium (Pru a 2). Sequencing the corresponding cDNA identified Pru a 2 as a thaumatin-like protein belonging to the group 5 of pathogenesis-related proteins. CONCLUSIONS: A thaumatin-like protein in cherry has been identified as a major allergen (Pru a 2). Homologous proteins from the thaumatin family share sequence similarities and should therefore be checked for the capability to elicit an IgE-mediated allergic reaction.

Molecular characterization of Api g 1, the major allergen of celery (Apium graveolens), and its immunological and structural relationships to a group of 17-kDa tree pollen allergens.

Individuals suffering from immediate hypersensitivity (type-I allergy) to a particular pollen frequently display intolerance to several foods of plant origin. In this respect, individuals sensitized to birch pollen and/or mugwort pollen frequently display type-I allergic symptoms after ingestion of celery. In this study, we expressed the major allergenic protein of celery, Api g 1, which is responsible for the birch-celery syndrome, in the form of a non-fusion protein. The open reading frame of the cDNA of Api g 1 codes for a protein of 153 amino acids with a molecular mass of 16.2 kDa and 40% identity (60% similarity) to the major allergen of birch pollen, Bet v 1. Furthermore, Api g 1 exhibited similar characteristics to (a) two proteins in parsley induced by fungal infection, (b) the major tree pollen allergens and (c) pathogenesis-related and stress-induced proteins in other plant species. The reactivity of recombinant Api g 1 with IgE antibodies present in sera from celery intolerant patients was comparable to that of the natural celery allergen. Cross-reactivity with Bet v 1 was proven by cross-inhibition experiments, which provides further support for the existence of the birch-celery syndrome and for the suggestion that allergies to some vegetable foods are epiphenomena to allergies caused by inhalation of tree pollen.