Molecular characterization of timothy grass pollen group V allergens.
Phl p V is the dominant allergen of timothy grass (Phleum pratense) with two isoforms having the apparent molecular weights of 38 (Phl p Va) and 32 kD (Phl p Vb) under Western blot conditions. Two-dimensional electrophoresis/immunoblotting reveals that each isoform is split into at least four isoallergens. Structural differences in the isoforms are shown by N-terminal sequencing (only 60% identity), by reaction patterns of monoclonal antibodies and, more convincingly, by enzymic degradation of purified isoforms followed by immunologic fingerprinting. These findings are confirmed by the deduced primary protein structure of cloned Phl p Va and Phl p Vb. Experiments with IgE--affinity-purified by immobilized recombinant allergens or their fragments--reveal identical epitopes and at least one different epitope between the isoforms. Furthermore, on Phl p Va we can localize different IgE-reactive epitopes at the C terminus as well as the N terminus. By probing serum from 11 patients on recombinant C- or N-terminal fragments, an individual reaction pattern was found. Testing the histamine liberation potency of the fragments, we found the N-terminal fragment of Phl p Va to be superior to that of the C-terminal fragment or the whole molecule. These results give insights into the variability of allergens, the individuality of human reaction patterns to epitopes and the alteration of allergenicity to higher or lower levels by fragmentation.
Becker
W M
WM
Forschungsinstitut Borstel, Germany.
Bufe
A
A
Petersen
A
A
Schlaak
M
M
D000485:Allergens / Q000737:chemistry* / Q000145:classification; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D015180:Electrophoresis, Gel, Two-Dimensional; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D010940:Plant Proteins / Q000737:chemistry* / Q000276:immunology; D006109:Poaceae / Q000276:immunology*; D011058:Pollen / Q000276:immunology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D012260:Ribonucleases / Q000737:chemistry* / Q000276:immunology
D000485:Allergens; D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; C076049:Phl p V protein, Phleum pratense; D010940:Plant Proteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E; C102306:Phl p Vb protein, Phleum pratense; D012260:Ribonucleases