tailieunhanh - Báo cáo khoa học: Molecular characterization and allergenic activity of Lyc e 2 (b-fructofuranosidase), a glycosylated allergen of tomato

Until now, only a small amount of information is available about tomato allergens. In the present study, a glycosylated allergen of tomato (Lycopersicon esculentum), Lyc e 2, was purified from tomato extract by a two-step FPLC method. The cDNA of two different isoforms of the protein, Lyc e and Lyc e , was cloned into the bacterial expressionvector pET100D. The recombinant proteinswere purified by electroelution and refolded. | Eur. J. Biochem. 270 1327-1337 2003 FEBS 2003 doi Molecular characterization and allergenic activity of Lyc e 2 b-fructofuranosidase a glycosylated allergen of tomato Sandra Westphal1 Daniel Kolarich2 Kav Foetisch1. Iris Lauer1 Friedrich Altmann2 Amedeo Conti3 B F 4 B B F B 4 w B BV B B B B B B B B B w FF B VBB ffB w B FF B B B B B B B BB w B B B B B B fB B B B B B B B B B BB BBBB B B B B B fB B ff BB ff B B B Jesus F. Crespo4. Julia Rodriauez4 Ernesto Enriaue5 Stefan Vieths1 and Stephan Scheurer1 B 1 Department of Allergology Paul-Ehrlich-Institut Langen Germany 2Institute of Chemistry University of Agriculture Vienna Austria 3CNR-ISPA c o Bioindustry Park Colleretto Giacosa Italy 4Servicio de Alergia Hospital Universitario Doce de Octubre Madrid Spain 5Institut Universitari Dexeus Barcelona Spain Until now only a small amount of information is available about tomato allergens. In the present study a glycosylated allergen of tomato Lycopersicon esculentum Lyc e 2 was purified from tomato extract by a two-step FPLC method. The cDNA of two different isoforms of the protein Lyc e and Lyc e was cloned into the bacterial expression vector pET100D. The recombinant proteins were purified by electroelution and refolded. The IgE reactivity of both the recombinant and the natural proteins was investigated with sera of patients with adverse reactions to tomato. IgE-binding to natural Lyc e 2 was completely inhibited by the pineapple stem bromelain glycopeptide MUXF Mana1-6 Xyip1-2 Manp1-4GieNAcp1-4 Fuca1-3 GlcNAc . Accordingly the nonglycosylated recombinant protein isoforms did not bind IgE of tomato allergic patients. Hence we concluded that the IgE reactivity of the natural protein mainly depends on the glycan structure. The amino acid sequences of both isoforms of the allergen contain four possible N-glycosylation sites. By application of MALDI-TOF mass spectrometry the predominant glycan structure of the natural allergen was .