Abstract:
Background: Vicilin seed storage proteins are translated with
N-terminal leader sequences (LSs) that are cleaved to yield the mature
protein. These LSs were thought to be unstructured and rapidly degraded.
However, Ara h 1 and Jug r 2 LS (A1LS, J2LS) have been identified in
seeds, and immunodominant IgE epitopes detected. Here, common sequences
containing structured CxxxC-repeat motifs were identified as potential
mediators of IgE cross-reactivity despite very low (17%) sequence
identity.
Method: Linear IgE epitopes were identified by peptide
microarrays, in which overlapping 15-mer peptides on glass slides, were
incubated with sera from peanut, walnut or dual allergic individuals.
Similar epitopes were computationally predicted. Peanut A1LS and walnut
J2LS fragments (J2.1, J2.2, J2.3) each with a CxxxC vicilin LS motif
were identified, cloned, expressed, purified and their structures solved
using solution-NMR to locate and assess epitopes on the structure.
Results: A1LS and J2LSs reveal similar helix-turn-helix motifs
connected by disulfide bonds between adjacent CxxxC repeats forming
α-hairpin structures. Peanut-allergic IgE bound more frequently to the
J2LSs, regardless of walnut allergic status or A1LS binding. IgE binding
pattern to peptides from both J2LS and A1LS, along with structure and
computational predictions, suggest that the structure and conserved
amino acid properties of peptides determine cross-reactivity. The
properties of LS IgE epitopes were closely related to epitopes in 2S
albumins.
Conclusion: The shared
α-hairpin structure is a stable
scaffold that contributes to cross-reactivity despite low sequence
identity. Biophysical properties are a better predictor of distant
cross-reactivity than traditional measures of evolutionary conservation.