Pollen-related food allergy: cloning and immunological analysis of isoforms and mutants of Mal d 1, the major apple allergen, and Bet v 1, the major birch pollen allergen.

BACKGROUND: Mal d 1, the major apple allergen, cross-reacts with IgE specific for the major birch pollen allergen, Bet v 1, and is responsible for birch pollen related food allergy to apple. Isoforms of Bet v 1 showing minor sequence variations display different binding capacity for specific IgE antibodies from allergic patients. Moreover, strain-dependent variation of allergenicity has been reported for apples. OBJECTIVE: To investigate the occurrence of strain-dependent isoforms of Mal d 1 which may differ in their allergenic potential, to obtain data on structures essential for binding of Mal d 1 to the antibody, and to gain insights into the structures responsible for its IgE cross-reactivity to Bet v 1. METHODS: The cDNA of Mal d 1 from various apple strains was amplified by a PCR strategy based on conserved regions of known Mal d 1-sequences, and sequenced. Two major isoforms of Mal d 1 were expressed as recombinant proteins and purified, as were different variants of the major birch pollen allergen, Bet v 1. Together with already existing recombinant birch pollen and apple allergens, these were subjected to allergenicity testing by IgE-immunoblotting, enzyme allergo sorbent test and dose related mediator release. "Hot-spots" for IgE-reactivity were identified by site-directed mutagenesis. RESULTS: Twelve Mal d 1-clones were sequenced from 7 apple varieties and compared to 3 known Mal d 1 sequences. The clones were clustered into two groups, each showing a high degree of sequence identity to one of the known sequences and specific differences to the third sequence. No strain-specific sequences were identified. In contrast, apple strains with reported differences in allergenicity showed different expression levels of the major allergen. Immunologic testing of recombinant allergens revealed high IgE binding capacity of 2 major isoforms, named GD26 and GS29, with a slightly higher IgE binding capacity of GD26. Moreover, the allergenicity was similar to another r Mal d 1 reported in the literature, representing the isoform divergent from our clones. Mutational analysis of our Mal d 1 allergens identified serine in position 111 as essential for IgE binding. Allergenicity was almost depleted by changing this residue into a proline. Moreover, the corresponding serine residue, present in position 112 of Bet v 1, was in a similar manner crucial for the allergenicity of the birch pollen allergen. CONCLUSION: We conclude that divergent allergenicity of apple strains mainly depends on different expression levels of the major allergen. Introduction of a proline residue in position 111 of Mal d 1 and in position 112 of Bet v 1 led to a drastic reduction of allergenicity of both the pollen and the food allergen, obviously also removing the cross-reactive epitope. Mutants with reduced IgE-reactivity but maintained T-cell reactivity may represent new candidates for a safer specific immunotherapy with reduced side-effects.

Cross-reactivity and epitope analysis of Pru a 1, the major cherry allergen.

A high percentage of birch pollen allergic patients experiences food hypersensivity after ingestion of fresh fruits and vegetables. The cross-reactivity of the major allergens of sweet cherry (Pru a 1), apple (Mal d 1), pear (Pyr c 1), celery tuber (Api g 1) and carrot (Dau c 1) is due to structural similarities which are reflected by high amino acid sequence identities with Bet v 1a, the major birch pollen allergen. Apart from a strong cross-reactivity to Bet v 1a, IgE inhibition experiments with Mal d 1, Pru a 1 and Api g 1 demonstrated the presence of common and different epitopes among the tested food allergens. Secondary structure prediction of all investigated allergens indicated the presence of almost identical structural elements. In particular, the 'P-loop' region is a common domain of the pollen related food allergens and of pathogenesis related proteins. To identify the IgE binding epitopes, five overlapping recombinant Pru a 1 fragments representing the entire amino acid sequence with lengths of approximately 60-120 residues were investigated. Weak IgE binding capacity was measured exclusively with Pru a IF4 (1-120) by immunoblotting, whereas none of the fragments showed allergenicity in the rat basophil leukaemia cell mediator release assay. Site-directed mutagenesis experiments with Pru a 1 revealed that amino acid S112 is critical for IgE binding of almost all patients sera tested. This reduced IgE binding was also observed with a single point mutant of Bet v 1a (S112P) and thus indicated serine 112 as an essential residue for preserving the structure of a cross-reactive IgE epitope. Moreover, two Pru a 1 mutants with an altered 'P-loop' region, showed a lowered IgE binding capacity for IgE from a subgroup of allergic patients. The investigation of essential features for preserving cross-reactive IgE-epitopes provides the structural basis for understanding the clinically observed cross-allergenicity between pollen and fruits. Moreover, non-anaphylactic allergen fragments or variants derived from the IgE-inducing pollen allergens may serve as useful tools for a new strategy of specific immunotherapy.

Kitasatospora cheerisanensis sp. nov., a new species of the genus Kitasatospora that produces an antifungal agent.

An actinomycete, strain YC75T, which produced bafilomycin-like antifungal compounds, was identified as a member of the genus Kitasatospora on the basis of morphological and chemotaxonomic characteristics. The strain produced the aerial and fragmenting vegetative mycelia consisting of straight chains of 20 or more smooth-surfaced spores. Submerged spores were formed in tryptic soy broth. No soluble pigments were formed. Whole-cell hydrolysates contained glucose and mannose, but not galactose. The 16S rDNA sequence of YC75T was compared with those of the other representative kitasatosporae and streptomycetes. Strain YC75T formed a significant monophyletic clade with Kitasatospora phosalacinea. The levels of DNA relatedness between strain YC75T and representatives of the genus Kitasatospora ranged from 16 to 59% including K. phosalacinea (28 and 40%). It is clear from polyphasic evidence that the isolate should be classified as Kitasatospora cheerisanensis sp. nov., whose type strain is YC75T (= KCTC 2395T). The presence of galactose in whole-cell hydrolysates may not be a stable chemical marker for the genus Kitasatospora.