19 Proteomic Analysis of Major and Minor Allergens From Isolated Pollen Cytoplasmic Granules

BACKGROUND: Grass pollen is one of the most important vectors of aeroallergens. Under atmospheric conditions, pollen grains can release pollen cytoplasmic granules (PCGs). The allergens associated with these intrinsic sub-fractions induce, in laboratory animals as well as in asthmatic patients allergic and inflammatory responses. The aims of this study were to characterize and identify the intrinsic allergens of PCGs, to compare them with those of pollen grains. METHODS: PCGs were isolated from Phleum pratense pollen by osmotic shock. The water-soluble proteins were extracted from pollen grains and their PCGs. Nine out of 26 grass sensitized patient sera were selected on the basis of previous ELISA and immunoblotting results showing IgE specific binding to numerous grass pollen allergens. IgE-binding proteins were analyzed by 1- and 2D-immunobloting using grass pollen-sensitized patient sera. Once located, allergens were characterized by mass spectrometry. RESULTS: 2D gels of pollen and PCGs extract revealed about 100 and 40 proteins respectively, with a large spectrum of Mr (10–>94 kDa) and pI (<4.5–10.0). More proteins as well as more allergens in pollen than in PCGs were detected by immunoblotting. Several of the allergens listed in the IUIS nomenclature - Phl p 1, 4, 5, 6, 11 and 12 - were found in pollen and PCGs extracts while Phl p 11 was found only in PCGs and Phl p 2 as well as Phl p 13 only in pollen extract. Some other allergens, not listed in the IUIS nomenclature, were also characterized in both pollen and PCGs extracts. CONCLUSIONS: Since the major grass pollen allergens were found in PCGs and because of their small size, these sub-micronic particles should be considered as very potent sensitizing and challenging respirable vectors of allergens. We demonstrate here that PCGs are at least as much dangerous as pollen grains.