Gloria Garcia-Casado

Identification of IgE-binding epitopes of the major peach allergen Pru p 3

BACKGROUND Lipid transfer proteins (LTPs) are clinically relevant plant food panallergens and have been proposed as ideal tools to study true food allergy. Pru p 3, the major peach allergen in the Mediterranean area, is among the best-characterized allergenic members of the LTP family. Its diagnostic value for Rosaceae fruit allergy has been demonstrated both in vivo and in vitro. OBJECTIVE We sought to locate major IgE-binding epitopes of Pru p 3. METHODS A serum pool and individual sera from patients with peach allergy and positive skin prick test results to Pru p 3 were used. Three-dimensional modeling was achieved by using experimentally available structures of Pru p 3 homologues as templates. Theoretical prediction of potential IgE-binding regions was performed by selecting specific residues on the molecular surface displaying prominent electrostatic potential features. Point mutants of Pru p 3 were constructed by standard polymerase chain reaction procedures with the appropriate primers. Mutants were expressed in P pastoris by means of the pPIC 9 vector and purified from the corresponding supernatants by gel-filtration chromatography followed by RP-HPLC. IgE binding by Pru p 3 mutants was tested by immunodetection and quantified by ELISA and ELISA inhibition assays. Synthetic peptides (10 mer; 5 amino acids overlapping) covering the full Pru p 3 sequence were used to detect IgE epitopes by (125)I-anti-IgE immunodetection. RESULTS Pru p 3 showed a 3-dimensional structure comprising 4 alpha-helixes and a nonstructured C-terminal coil (residues 73 to 91). Regions around amino acids in positions 23 to 36, 39 to 44, and 80 to 91, particularly residues R39, T40, and R44, K80 and K91, were predicted as potential antibody recognition sites according to their relevant surface and electrostatic properties. Point mutants K80A and K91A were found to have an IgE-binding capacity similar to that of recombinant Pru p 3, but the triple mutant R39A/T40A/R44A showed a substantial decrease (approximately 5 times) of IgE binding. IgE immunodetection of synthetic peptides led to the identification of Pru p 3 sequence regions 11 to 25, 31 to 45, and 71 to 80 as major IgE epitopes. CONCLUSIONS Main IgE-binding regions of the Pru p 3 amino acid sequence were identified. The three major ones comprised the end of an alpha-helix and some residues of the following interhelix loop. These data can help to search for Pru p 3 hypoallergenic forms.

Wheat and barley inhibitors active towards α-amylase and trypsin-like activities from Spodoptera frugiperda

The α-amylase activity was determined throughout the larval development of Spodoptera frugiperda. Maximal activities with optimal pH in the range 8.5–9.5 were found in last instars. Protein preparations enriched in heterotetrameric inhibitors from wheat flour were active towards gut amylases from last instars, while those corresponding to homodimeric and monomeric inhibitors showed low inhibition levels. These results were further supported by testing purified members of each inhibitor type and by analyzing the effects of the inhibitors on the amylase isoenzyme pattern from native PAGE. High levels of trypsin-like activity were also found in gut extracts from last instars. Different genetic variants of the major barley trypsin inhibitor were active against this gut enzyme. None of the other larval digestive protease activities (chymotrypsin-like, elastase-like, leucine aminopeptidase-like, and carboxypeptidase A and B-like) were inhibited, indicating that the barley inhibitor is specific towards trypsin-like enzymes.