Purification and characterization of recombinant Bet v I, the major birch pollen allergen. Immunological equivalence to natural Bet v I.

Pollen from trees of the order Fagales (e.g. birch, alder, hazel, oak, and hornbeam) are a major cause of Type I allergies observed in early spring. Previously, we reported the cloning and sequencing of Bet v I, the major birch pollen allergen, which showed high sequence similarities to a family of plant pathogen-activated genes (Breiteneder, H., Pettenburger, K., Bito, A., Valenta, R., Kraft, D., Rumpold, H., Scheiner, O., and Breitenbach, M. (1989) EMBO J. 8, 1935-1938). Here, we present the results on the expression, purification, and characterization of recombinant Bet v I produced in Escherichia coli as fusion and non-fusion protein, respectively. The purified recombinant proteins were analyzed to verify purity and structural integrity, and their immunological properties were compared to those of Bet v I isolated from birch pollen (natural Bet v I). Immunoblot analyses showed that the recombinant proteins are specifically recognized by monoclonal antibodies raised against natural Bet v I as well as by IgE from birch pollen-allergic patients. However, enzyme-linked immunosorbent assays revealed a decreased IgE-binding activity of the recombinant fusion Bet v I compared to the non-fusion and natural Bet v I proteins, which probably results from conformational changes due to the fusion tail. Recombinant non-fusion Bet v I was equivalent to natural Bet v I with respect to IgE-binding properties, the ability to induce in vitro proliferation of allergen-specific T-cell clones, and the ability to release histamine from basophils derived from birch pollen-allergic patients.

Distribution of allergens and allergen-coding mRNAs in various tissues of white birch.

The distribution of allergenic proteins was investigated in various tissues of white birch, Betula verrucosa (pollen, leaves and male inflorescences containing immature pollen). In addition, callus and suspension culture cells were investigated for expression of IgE-binding proteins. Furthermore, RNA was extracted from all these tissues and subjected to in vitro translation in a cell-free wheat germ system. Bet v I, the major birch pollen allergen, could be extracted easily from pollen, and in low amounts from callus and leaves. No Bet v I could be extracted from immature male inflorescences. Minor allergens were expressed in high concentrations in pollen and in low concentrations in immature male inflorescences. No minor allergens could be detected in callus and leaves. In contrast to these observations, RNA from all the tissues as well as from callus could be translated in vitro into Bet v I as well as into minor allergens, in particular birch profilin (Bet v II), an important minor allergen. These data suggest that IgE-binding proteins of B. verrucosa, especially Bet v I, under certain circumstances can readily be synthesized in tissues other than pollen. This concept is corroborated by the recent observation that Bet v I reveals high homology with disease resistance response gene products from other plants, suggesting a similar function of Bet v I for the birch.

Identification of profilin as a novel pollen allergen; IgE autoreactivity in sensitized individuals.

A complementary DNA encoding a pollen allergen from white birch (Betula verrucosa) that was isolated from a pollen complementary DNA library with serum immunoglobulin E from a birch pollen-allergic individual revealed significant sequence homology to profilins. The recombinant protein showed high affinity to poly-L-proline. Immunoglobulin E antibodies from allergic individuals bound to natural and recombinant birch profilin and also to human profilin. In addition, birch and human profilin induced histamine release from blood basophils of profilin-allergic individuals, but not of individuals sensitized to other plant allergens. The structural similarity of conserved proteins might therefore be responsible for maintaining immunoglobulin E antibody titers in type I allergy.

A low molecular weight allergen of white birch (Betula verrucosa) is highly homologous to human profilin.

Cloning of allergens has contributed substantially to the understanding of mechanisms in allergic diseases by providing information about the sequence and hence biological functions of allergens. The major birch pollen allergen, Bet v I [Breiteneder H, et al: EMBO J 1989;8:1935-1938] and the white-faced hornet venom allergen (antigen 5) [Si Yun Fang K, et al: Proc. Natl. Acad. Sc. USA 1988;85:895-899] were shown to be highly homologous to pathogenesis-related proteins of plants. In the case of the major allergen of house dust mite, Der p I, homology to proteases was demonstrated. Therefore, the proposed biological function of these IgE-binding proteins might be related to their allergenic potential. In this paper we tentatively identify a ubiquitous family of low molecular weight allergens as profilins. The identification is based on a sequence homology, (b) binding to poly(L-proline), and (c) immunological cross-reactivity. Recombinant birch profilin was purified to homogeneity and showed the same properties as natural profilins.

The gene coding for the major birch pollen allergen Betv1, is highly homologous to a pea disease resistance response gene.

Pollen of the white birch (Betula verrucosa) is one of the main causes of Type I allergic reactions (allergic rhinoconjunctivitis, allergic bronchial asthma) in Middle and Northern Europe, North America and the USSR. Type I allergies are a major threat to public health in these countries, since 10-15% of the population suffer from these diseases. BetvI, an allergenic protein with an Mr of 17 kd is a constituent of the pollen of white birch and is responsible for IgE binding in more than 95% of birch pollen allergic patients. Here, we report the complete nucleotide sequence and deduced amino acid sequence of a cDNA clone coding for the major pollen allergen (BetvI) of white birch. It is similar to the N-terminal peptide sequences of the allergens of hazel, alder and hornbeam (close relatives) but it has no significant sequence homology to any other known allergens. However, it shows 55% sequence identity with a pea disease resistance response gene, indicating that BetvI may be involved in pathogen resistance of pollen.

Isolation and characterization of messenger RNA from male inflorescences and pollen of the white birch (Betula verrucosa).

A glycoprotein with a molecular weight (MW) of 17 kilodaltons (kD), Bet v I, represents the major allergen of the white birch (Betula verrucosa, BV) and plays an important role in tree-pollen-induced type I allergic reactions. In order to characterize the major and also some minor allergens of BV, we investigated the IgE-binding properties of these allergens using immunoblot techniques. Normal and patients' sera were employed for this study. Furthermore, RNA from male inflorescences and from pollen of BV were isolated and purified by affinity chromatography on oligo-dT-cellulose. Poly(A)+-mRNA thus obtained was translated in vitro in a cell-free wheat germ system and the proteins synthesized were separated by SDS-PAGE and transferred to nitrocellulose. The blots were incubated with normal human sera and with sera from patients allergic to birch pollen. Bound IgE antibodies were detected with 125I-labeled anti-IgE. We observed major IgE binding to a protein of an MW of 12.5 kD, and little IgE binding to a 17-kD protein, presumably Bet v I. Comparing the products of in vitro translation from mRNA preparations of mature pollen and of male inflorescences collected in June, October and February, little seasonal variations could be observed. As the in vitro translation system does not glycosylate proteins, our results show that the majority of IgE in patients' sera is not directed against the carbohydrate moieties of these allergens.