Discussion:
In this study we demonstrate that the vicilin LSs from PN and WN vicilins contain a variable number of VBP motifs, which adopt a characteristic a-hairpin fold mediated by disulfides between the conserved CxxxC elements. While similar structures have been described for other VBPs [14, 27, 33], this is the first time such a fold has been observed in major food allergens. This shared structure contains IgE binding sequences that were previously identified by their similarity to dominant epitopes of the 2S albumins, including one major epitope that lies in the loop between the paired disulfides. The structural and IgE binding data shown here adds more evidence to our previous studies [8, 9, 34] for the role of these LSs in mediating cross-reactivity between WN and PN, as IgE in sera from patients with dual allergy to both PN and WN showed particularly strong binding to this region. We also note that similarly located sequences from the LS of cashew (Ana o 1) and pistachio (Pis v 3) vicilins bind IgE from patients with the respective allergies and could contribute to the the clinically significant cross-reactivity between these two foods [35, 36].
While the sequences studied here have similar PCPs, they are quite different in their amino acid sequences. This suggests that the local structure of the area contributes to the recognition by the peptidases responsible for cleaving the pro region or LS from the mature vicilin. The α-hairpin structures are also quite pliable, as indicated by the few long range NOEs and the degree of disordered structure. This suggests that introducing mutations to remove specific epitopes should not greatly alter the structure. For example, while peptide A10, A11 and A12 contain areas around the inter-helical loop region with some of the disordered structure, A11 did not bind IgE from any of the tested sera. Thus, while the ordered hairpin structure can stabilize a variety of peptide sequences, the specific amino acid sequence are ultimately important for antibody binding.
Another consequence of the LS architecture is that its defining α-hairpin scaffold is maintained by only a few conserved interactions, allowing it to accumulate mutations over time without compromising its underlying structure. The resulting sequence diversity could allow LSs to mimic the surface features of unrelated proteins, giving rise to cross-reactivity between different allergen families as observed between J2LS and Ara h 2, as the most conserved region of Ara h 2 contains a similar pattern of cysteine residues separated by variable residues [7, 37].
The disparities in immunogenicity and cross-reactivity among VBPs, despite their shared structures, suggests there are additional factors involved. The differences in IgE binding to the J2LS fragments could be partially explained by examining J2LS trypsin digestion from a previous study which provides some insight into the fragments which may be formed upon gastric digestion [16]. Comparing this data with the NMR structures discussed above, we see that several arginine residues are protected from proteolysis due to their location within the structured CxxxC motif (Figure 1B), potentially contributing their downstream allergic potential. In sum, this illustrates that the mechanisms of cross-reactivity are complicated and involve both structural and amino acid sequence components.