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We have recently shown that the majority of allergens can be represented by allergen motifs. This observation prompted us to experimentally investigate the synthesized peptides corresponding to the in silico motifs with regard to potential IgE binding and cross-reactions with allergens. Two motifs were selected as examples to conduct in vitro studies. From the first motif, derived from allergenic MnSOD sequences, the motif stretch of the allergen Asp f 6 was selected and synthesized as a peptide (MnSOD Mot). The corresponding full-length MnSOD was also expressed in Escherichia coli and both were compared for IgE reactivity with sera of patients reacting to the MnSOD of Aspergillus fumigatus or Malassezia sympodialis. For the second motif, the invertebrate tropomyosin sequences were aligned and a motif consensus sequence was expressed as a recombinant protein (Trop Mot). The IgE reactivity of Trop Mot was analyzed in ELISA and compared to that of recombinant tropomyosin from the shrimp Penaeus aztecus (rPen a 1) in ImmunoCAP. MnSOD Mot was weakly recognized by some of the tested sera, suggesting that the IgE binding epitopes of a multimeric globular protein such as MnSOD cannot be fully represented by a motif peptide. In contrast, the motif Trop Mot showed the same IgE reactivity as shrimp full-length tropomyosin, indicating that the major allergenic reactivity of a repetitive structure such as tropomyosin can be covered by a motif peptide. Our results suggest that the motif-generating algorithm may be used for identifying major IgE binding structures of coiled-coil proteins.

Previously it was shown that fusion proteins containing the amino terminus of an apical targeted member of the serpin family fused to the corresponding carboxyl terminus of the non-polarized secreted serpin, antithrombin, are secreted mainly to the apical side of MDCK cells. The present study shows that this is neither due to the transfer of an apical sorting signal from the apically expressed proteins, since a sequence of random amino acids acts the same, nor is it due to the deletion of a conserved signal for correct targeting from the non-polarized secreted protein. Our results suggest that the polarity of secretion is determined by conformational sensitive sorting signals.

We have recently reported a new method based on protein motifs to predict allergenicity by protein sequence. By using the more complete allergen database Allergome (www.allergome.org), as the basis for the motif identification process, we obtained a new set of allergenic motifs. We studied whether an allergenic motif may be used instead of a full length allergen for predicting allergenicity.Sequence accession numbers of allergens were received from A. Mari of the Allergome Project. The sequences were downloaded and integrated in the allergen database, which was used to produce new motifs applicable for allergenicity prediction. The motif containing allergenic tropomyosins of different non vertebrate species was chosen. A consensus sequence of the tropomyosin motif was synthesized and expressed in Escherichia coli (E. coli). The purified peptide was analyzed for specificity with enzyme linked-immunosorbent assay (ELISA).Based on the Allergome dataset, we calculated 69 motifs containing 912 sequences. Prediction performance of this new set of allergenic motifs is increased compared to the original 52 motifs. The IgE immunological reactivity of the recombinant tropomyosin motif was comparable to that obtained with a full length recombinant shrimp tropomyosin (rPen a 1) tested in the ImmunoCAP system.The use of the motif-based allergenicity prediction method is more accurate than the guidelines proposed by FAO/WHO, however continuous updates of the motif dataset are necessary to guarantee a high prediction performance of this method. The experiments performed with the tropomyosin motif showed that peptides based on the consensus sequence of motifs may represent the allergenic epitope, which can be used in future diagnostic or therapeutic approaches.