Molecular, immunological, and structural characterization of Phl p 6, a major allergen and P-particle-associated protein from timothy grass (Phleum pratense) pollen

Due to the wide distribution and heavy pollen production of grasses, simila r to 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 immunoscr eening 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 b y mass spectrometry and exhibited almost exclusive cu-helical secondary str ucture as determined by circular dichroism spectroscopy. Phl p 6 reacted wi th serum IgE from 75% of grass pollen-allergic patients (n = 171), IgE bind ing experiments with rPhl p 6 fragments indicated that the N terminus of th e 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 purifi ed rPhl p 6 identified it as a pollen-specific protein that, by immunogold electron microscopy, was localized on the polysaccharide-containing wall-pr ecursor bodies (P-particles), The association of Phl p 6 with P-particles m ay 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 bindi ng capacity may represent candidates for immunotherapy of grass pollen alle rgy with a low risk of anaphylactic side effects.