Significance in allergy of protein glycosylation in plants and invertebrates

The aim of the present project is to assess the significance of protein-linked carbohydrates in allergy. One strategy will be to elucidate the structures of N-glycans from a variety of relevant allergens - either individual proteins or whole tissues (e.g. pollens) especially from plants but also from e.g. insect venoms and other invertebrates. This should provide a structural basis for an improved understanding of carbohydrate-based cross-reactions. The principle approach here will be mass spectrometric analysis supported, where necessary, by HPLC. We shall also examine an only recently discovered type of protein O-glycosylation which is likewise considered to play a role in IgE binding. Again, mass spectrometry and, additionally, NMR-spectroscopy will be exploited here. Another strategy will be to specify the epitope requirements of anti-glycan antibodies, especially patients` IgE. To this end, the recently cloned and recombinantly expressed core-a1,3-fucosyl- and xylosyltransferases will be used to generate a set of glycoconjugates with defined N-glycan structures, e.g. N-glycans with solely xylose or solely fucose. Neo-glycoproteins containing these structures will be subject to IgG and IgE binding and inhibition studies. The knowledge gained and the materials produced hereby should aid in improving the specificity of routine in vitro allergen diagnosis which is currently often obscured by cross-reactive carbohydrates. In parallel, the IgE-binding potential and the immunogenicity of plant O-glycans of the newly discovered mugwort type shall be investigated. Rabbit antiserum against the mugwort O-glycan shall facilitate the detection of similar modifications in other allergens. A third strategy, again making use of the above mentioned neo-glycoproteins, intends to shed light on the clinical significance of anti-N-glycan IgE. Initially, histamine release experiments shall be performed and, in a later phase, skin tests are envisaged.