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.