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.