Group 13 grass allergens: Structural variability between different grass species and analysis of proteolytic stability

Background: Determination of the allergen composition of an extract is esse ntial for the improvement of hyposensitization therapy. Surprisingly, altho ugh grass pollen extracts have been studied intensively for 20 years, a fur ther major allergen, Phl p 13, was detected recently in timothy grass polle n. Objectives: We sought to determine the occurrence and importance of grou p 13 allergens in various grass species and to investigate their proteolyti c stability. Methods: The group 13 allergens were determined by means of 2-dimensional P AGE blotting with patient sera and group 13-specific mAbs. The allergens we re isolated chromatographically from several pollen extracts and analyzed b y means of microsequencing. Cross-reactivity among various grass species wa s studied by using Western blots and immunoblot inhibition tests. The stabi lity of the allergens was tested under defined extraction conditions. Results: Group 13 allergens are detectable in all common grasses and show I gE cross-reactivity among them. The allergenic components were identified i n the neutral pH range with molecular masses of 50 to 60 kd, and in the cas e of Phl p 13, maximal binding of the isoforms was observed at 55 kd and at an isoelectric point of 6 to 7.5, Protein sequencing clearly confirms stru ctural identities between different grass species, although individual vari ations are found. If low-molecular-mass components were depleted by means o f gel filtration, a rapid degradation of group 13 allergens was observed. T his is in contrast to other pollen allergens described thus far. Conclusion: Group 13 allergens are widespread and are major allergens in th e grasses. Predicted from their primary structures, these allergens are pol ygalacturonases, This class of enzymes is already known from microorganisms , and these enzymes are recognized as potential inducers of asthma, Our stu dies indicate that the group 13 allergens show a considerable microheteroge neity and degradation, especially after depletion of low-molecular-mass com ponents, One has to be aware of this pivotal fact when soluble grass pollen extracts are prepared for diagnostics and hyposensitization therapy.