Background:
Peanut (PN) allergy is among the most commonly reported food allergies, with a global prevalence of 1-3% [1]; a number that is only increasing. Peanut allergy is responsible for over 50% of food-induced anaphylaxis cases, and is rarely outgrown, making it a key health concern [2, 3]. Furthermore, up to 86% of peanut-allergic patients show some level of sensitization to tree-nuts (TN), particularly walnut (WN), with ~30% displaying clinically-relevant levels of IgE cross-reactivity [4, 5]. While allergens from PNs and TNs can be grouped into similar protein families, their overall sequence identity falls well below the 70% threshold previously considered to support cross-reactivity, raising questions regarding the basis for this phenomenon [6]. Dreskin et al. [7] suggest that the highly conserved cysteine motif structure of 2S albumins, which supports intervening amino acids with similar physicochemical properties (PCPs), is essential for IgE cross-reactivity between PN and TNs. The cysteine-rich leader sequences (LS’s) from viclin seed storage proteins could potentially harbour similar interactions, further contruibuting to PN/TN cross-reactivity. Indeed, peptides from the vicilin leader sequences (LS) of walnut and peanut and that have similar PCPs to the 2S-albumin allergens of peanut (Ara h 2, Ara h 6 and Ara h 7) show comparable IgE binding properties [8, 9], supporting this conjecture.
Vicilins are a family of ubiquitous seed storage proteins which are translated as preproproteins consisting of three conserved regions: a short, hydrophobic signal peptide (pre) that is removed upon location to a storage vacuole; an N-terminal, cysteine-rich pro region; and the mature vicilin domain [10-14]. The pro region is processed by an asparaginyl endopeptidase (AEP) [15] to yield the leader sequence (LS). The LS in turn is composed of a contain a variable number of vicilin-buried peptide (VBP) sequences, defined by a common cysteine motif (CxxxC(10-14)CxxxC). The native LSs from both WN vicilin, Jug r 2(J2LS) and PN vicilin, Ara h 1 (A1LS) have been identified in the extracts of mature nuts [16, 17] and shown to bind IgE from patients with allergy to PN or TN [9, 17]. Despite their taxonomical and sequence diversity, VBP’s adopt a common α-hairpin structure mediated by disulfide bonds between the highly conserved CxxxC motifs. While none of the VBP’s described in these studies are known to be immunogenic, this sequence-insensitive scaffold has the potential to elicit an immune response. It is possible that this scaffold contains cross-reactive epitopes with similar PCPs across distantly related, IgE-reactive proteins from different plat species, such as PN and WN.
The contribution of PCPs to IgE cross-reactivity was analyzed by Maleki, et al. [8] in which peptides in the J2LS, with similar PCPs to an immunodominant epitope of the peanut 2S albumin, Ara h 2 (DRRCQSQLER), were shown to correlate with clinically relevant cross-reactivity despite low sequence identity. Indeed, the epitopes identified in the walnut LS bound IgE from PN and TN allergic individuals more intensely than the original immunodominant epitope of Ara h 2. In addition, a consensus peptide (CQRQEQGQRQQQQ) was designed based on the alignment of Ara h 2, Ara h 6 and Ara h 7 with the repeats of CxxxC motif within the J2LS. Using the peptide similarity tool in the Structural Database of Allergenic Proteins (SDAP) [18-21], Nesbit et al. identified IgE binding repeats with PCPs similar to the consensus in a multitude of other nut allergens’ sequences [9]. Anti-consensus peptide antiody was shown to recognize vicilins, glycinins and 2S albumins in different nuts. The J2LS and a 31 mer containing the consensus sequence were both shown to inhibit IgE binding to walnuts and almond extracts. While these efforts establish J2LSs from walnut as potential mediators of cross-reactivity with 2S albumins, its cross-reactivity with LS’s from PN and other distantly related food sources have yet to be explored in a comprehensive manner.
Here, we show by solution NMR that the A1LS and the J2LS adopt a common α-hairpin fold conferred by disulfide bonding between the cysteine residues of the CxxxC motifs. Despite their ordered structure, few inter-helical interactions were observed beyond the conserved disulfide bonds, allowing LSs to tolerate significant sequence variation with minimal structural perturbation. Peptide microarray analysis containing the A1LS and the J2LS reveals the potential for a high degree of IgE cross-reactivity among these LSs [9]. Though these specific immunogenic properties cannot be easily assessed using traditional bioinformatic approaches, a combination of structural, computational and immunological approaches provides insights into the joint contribution of structure and sequence to clinical cross-reactivity.