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Due to the wide distribution and heavy pollen production of grasses, approximately 50% of allergic patients are sensitized against grass pollen allergens. cDNAs coding for two isoforms and four fragments of a major timothy grass (Phleum pratense) pollen allergen, Phl p 6, were isolated by IgE immunoscreening from a pollen expression cDNA library. Recombinant Phl p 6 (rPhl p 6), an acidic protein of 11.8 kDa, was purified to homogeneity as assessed by mass spectrometry and exhibited almost exclusive alpha-helical secondary structure as determined by circular dichroism spectroscopy. Phl p 6 reacted with serum IgE from 75% of grass pollen-allergic patients (n = 171). IgE binding experiments with rPhl p 6 fragments indicated that the N terminus of the allergen is required for IgE recognition. Purified rPhl p 6 elicited dose-dependent basophil histamine release and immediate type skin reactions in patients allergic to grass pollen. A rabbit antiserum raised against purified rPhl p 6 identified it as a pollen-specific protein that, by immunogold electron microscopy, was localized on the polysaccharide-containing wall-precursor bodies (P-particles). The association of Phl p 6 with P-particles may facilitate its intrusion into the deeper airways and thus be responsible for the high prevalence of IgE recognition of Phl p 6. Recombinant native-like Phl p 6 can be used for in vitro as well as in vivo diagnoses of grass pollen allergy, whereas N-terminal deletion mutants with reduced IgE binding capacity may represent candidates for immunotherapy of grass pollen allergy with a low risk of anaphylactic side effects.

Atopic allergy is a genetically determined immunodisorder that affects almost 20% of the population worldwide. Immediate symptoms of type I allergy are caused by the release of biologic mediators from effector cells induced by IgE-allergen complexes that cross-link the high-affinity receptor for IgE (FcepsilonRI). Chronic disease manifestations result from allergen-specific T-cell activation, a process that is enhanced when allergens are presented via FcepsilonRI-bound IgE. We report the baculovirus expression, as soluble recombinant proteins, of the minimal units required for human IgE and FcepsilonRI interaction: Cepsilon3 represents the third constant domain of the IgE heavy chain, and alpha2 is the membrane-proximal Ig-like module from FcepsilonRIalpha. Native overlay experiments showed binding of human FcepsilonRIalpha to recombinant Cepsilon3 and of natural or recombinant human IgE to recombinant alpha2. Moreover, recombinant Cepsilon3 inhibited binding of natural IgE antibodies to alpha2, and preincubation of human IgE with alpha2 inhibited anti-IgE-triggered histamine release from human basophils. Isolated Cepsilon3 and alpha2 can now be used for the molecular and structural analysis of the IgE-FcepsilonRI interaction, as well as for diagnostic and therapeutic applications.

Due to the wide distribution and heavy pollen production of grasses, ∼50% of allergic patients are sensitized against grass pollen allergens. cDNAs coding for two isoforms and four fragments of a major timothy grass (Phleum pratense) pollen allergen, Phl p 6, were isolated by IgE immunoscreening from a pollen expression cDNA library. Recombinant Phl p 6 (rPhl p 6), an acidic protein of 11.8 kDa, was purified to homogeneity as assessed by mass spectrometry and exhibited almost exclusive α-helical secondary structure as determined by circular dichroism spectroscopy. Phl p 6 reacted with serum IgE from 75% of grass pollen-allergic patients (n = 171). IgE binding experiments with rPhl p 6 fragments indicated that the N terminus of the allergen is required for IgE recognition. Purified rPhl p 6 elicited dose-dependent basophil histamine release and immediate type skin reactions in patients allergic to grass pollen. A rabbit antiserum raised against purified rPhl p 6 identified it as a pollen-specific protein that, by immunogold electron microscopy, was localized on the polysaccharide-containing wall-precursor bodies (P-particles). The association of Phl p 6 with P-particles may facilitate its intrusion into the deeper airways and thus be responsible for the high prevalence of IgE recognition of Phl p 6. Recombinant native-like Phl p 6 can be used for in vitro as well as in vivo diagnoses of grass pollen allergy, whereas N-terminal deletion mutants with reduced IgE binding capacity may represent candidates for immunotherapy of grass pollen allergy with a low risk of anaphylactic side effects.