Arabinosylated allergens from weed pollens

We plan to study the different aspects of the recently discovered glyco-allergen Art v 1 from mugwort pollen. Topic 1: A glycoprotein allergen from ragweed pollen which shares peptide sequence homology with Art v shall be investigated. Its carbohydrate moiety shall be characterised by mass spectrometry and NMR after degradation of the glycoprotein with proteases or alkali. In parallel, the nucleotide sequence of the gene encoding the ragweed glyco-allergen(s) shall be determined to allow a complete structural elucidation of the ragweed homolog of Art v 1. Topic 2: The immunogenicity of the single ß-arabinose residues on contiguous hydroxyprolines as encountered in Art v 1 and possibly also on homologous allergens shall be investigated in more detail. We will try to characterise the minimal binding requirements for rabbit IgG as well as human IgE antibodies directed against the Art v 1 glyco-moiety by using synthetic glycopeptides with various sequences, varying degree of arabinosylation and varying glycosylation (D-Ara, L-Ara, etc.). Synthetic glycopeptides will facilitate the raising of specific antisera which are not yet available as immunization with purified, natural Art v 1 also generated an anti-N-glycan response due to impurities. These specific sera will be used to reveal the distribution of Ara-Hyp in the plant kingdom and to identify proteins carrying these particular modules. Topic 3: We will determine, how Art v 1 is glycosylated when expressed in heterologous systems belonging to different plant families, i.e. tobacco, a solanaceaeous plant, and lettuce which belongs to the asteraceae just like ragweed and mugwort. Not at least, these experiments shall help to identify a suitable production system for Art v 1 for diagnostic purposes. To this end we have quite recently developed a sensitive micro-analysis method for plant O-glycans. Topic 4: The significance of the ß-Ara-Hyp epitope in allergy shall be estimated. Sera of allergic patients which react (exclusively) with the glycan moiety of Art v 1 will be identified and tested against the different glycopeptides chemically synthesized in order to determine the exact nature of the epitope, i.e., the number of contiguous arabinose residues composing this epitope, the influence of the nature of the amino-acids surrounding the poly(Ara-Hyp) motif, and possibly the number of poly(Hyp) motifs necessary for strong binding. Moreover, granulocyte degranulation studies and affinity determination by surface plasmon resonance shall answer the question as to the possible clinical significance of anti-O-glycan IgE. Topic 5: Finally, we aim to found the molecular biology of protein arabinosylation. Starting from attempts to measure the arabinosyltransferase activity responsible for the generation of ß-Ara-Hyp epitopes, we will go to characterise this enzymatic activity. Ideally, it will be possible to purify the enzyme in order to allow generation of sequence tags useful for cloning of the DNA. However, even the characterisation of any of the arabinosyltransferases involved in protein glycosylation would already constitute a success.

Ragweed is a weed plant, whose pollen constitutes an allergen source of growing relevance in central Europe. In the course of this project, we have characterized a novel allergen from ragweed pollen that may be relevant for allergy diagnosis as well as treatment. This new allergenic protein has received the name Amb a 4. Amb a 4 is a small protein consisting nevertheless of three parts. One is a globule-shaped protein part, which is responsible for most if not all of the allergic potential of Amb a 4. The second part is a rod-shaped protein that carries the third component, i.e. carbohydrate chains. So, natural Amb a 4 was purified from ragweed pollen to allow the structural characterization of the carbohydrate on the one hand and the analysis of the protein backbound on the other. With the help of experts in a special analytical method, we could thus describe the sugar portion of Amb a 4. Ragweed is a close relative of mugwort, which contains as the major allergen a molecule resembling Amb a 4. However, the sugar portions of the two allergens present a difference, which may be of relevance for the differential diagnosis of ragweed and mugwort allergy. The other aim was the identification and cloning of the gene encoding the allergen Amb a 4. This task turned out as unusually difficult and could only be realized after analysis of the natural allergen by sophisticated instrumental methods (mass spectrometry). About a dozen isoforms of Amb a 4 were finally found on the genetic level and one of them was produced in transgenic bacteria. The "artificial" = recombinant allergen used to assess the relevance of Amb a 4 for allergic patients. The sera of 30 % of weed pollen allergic patients reacted with the "artificial" allergen Amb a 4, rendering it a rather important allergen molecule. Arabinoses are a rather common constituent of proteins in plants but nothing is yet known about the enzymes responsible for its attachment. Much energy was alloted to the detection of arabinosyltransferases in various plant tissues - unfortunately in vain. In order to get at the bottom of things, we developed a new sensitive and specific instrumental method, with which we could show the presence of the suspected substrate for arabinosylation in e.g. tobacco leaves, mung bean sprouts and many other plant samples.