Complementary DNA cloning and expression of a newly recognized high molecular mass allergen Ph1 p 13 from timothy grass pollen (Phleum pratense)

Background Grass pollen extracts contain a range of different allergenic co mponents that can be classified as having low, middle or high molecular mas s. Almost 75% of patients allergic to grass pollen display immunoglobulin ( Ig) E-reactivity to allergens in the high molecular mass range of 55-60 kDa . These proteins have not yet been fully characterized on the protein and D NA level. Objective The aim of this study was to identify and characterize an allerge n of the high molecular mass fraction of Phleum pratense pollen by N-termin al protein sequencing and molecular cloning. Methods A previously uncharacterized allergen which migrates as a double ba nd with a molecular mass of 55-60 kDa was biochemically purified and invest igated by N-terminal sequencing. Subsequently, a DNA primer was designed to amplify the corresponding cDNA using PCR. The cloned cDNA and deduced amin o acid sequence were compared with sequence data bases. Immunoblots carryin g the recombinant expression product were developed with monoclonal antibod ies and sera derived from allergic subjects. The IgE-binding capacity of na tural and recombinant allergen was determined using EAST. Results The nucleic acid sequence as well as the deduced amino acid sequenc e consisting of 394 amino acids indicated homology with pollen specific pol ygalacturonases. Four potential sites for glycosylation and 16 cysteine res idues were found. The recombinant expression product exhibited the same mol ecular size as the natural allergen and was clearly IgE-reactive. Conclusion The newly characterized allergen Phl p 13, which shows homology with polygalacturonases, is clearly different from the allergen designated as Phl p 4 and therefore the high molecular mass fraction is composed of at least two different allergens. A possible reason why this important allerg en has not been detected until now is that Phl p 13 and Phl p 4 are hardly separable by one dimensional SDS-PAGE.