IgE binding to A1LS and J2LS
Peptide microarrays were used to measure IgE binding to J2LS and A1LS
peptides using sera of patients with allergy to PN, WN or both PW.
First, we compared IgE binding (Figure 4A) to the overlapping peptides
of the long isolated VBP repeats characterized by NMR (Figure 1). The
majority of IgE binding in our patient cohort was to the peptides of
A1LS and J2.1 (Figure 4A), consistent with surface similarity values
shown in Figure 3A. IgE from the sera of patients with PW and PN
allergic only bind equally well to J2.1 and A1LS, while IgE from WN only
sera was statistically significantly
lower (i.e., PN = PW
> WN). However, IgE from WN allergic also bound the A1LS,
further supporting the role of leader sequences in allergic
cross-reactivity.
Next, IgE binding to overlapping 15 amino acid peptides from the LSs was
analyzed to identify epitopes recognized at least one allergy group (WN,
PN and PW). Nine immunoactive peptides were identified, three in A1LS
(A4, A12 and A13), five in J2.1 (JR5, JR6, JR7, JR8, and JR9), and one
in J2.2 (JR18) (Figures 2A, 3B and 4B). The binding pattern suggested
that in some cases the peptides represent fragments of longer, and
potentially conformational epitopes. All the IgE binding peptides
identified in A1LS contain previously identified immunodominant epitopes
of Ara h 1 [30, 31]. A4, located in a disordered region of A1LS
(Figure 2C), was only recognized by PN allergic sera. A12 spans the most
structured portion of A1LS and binds IgE from both PN and PW sera, while
A13, contains both ordered and disordered segments, is recognized by all
three serum groups (PN, WN, PW). Both A12 and A13 overlap with the
immunodominant epitope, LEYDPRCVYD (Figure 2A)[32] though this
sequence is truncated in A12, resulting in a reduction in both the
number of sera recognizing it and the intensity of IgE binding based on
average z values (Figure 4B). Although peptides A10 and A11 both contain
the A1LS epitope, QEPDDLKQKAC, the latter has no IgE binding, suggesting
that the surrounding sequences and possibly conformation in reactivity.
As Figure 4 shows, JR6, JR7, JR8 and JR9, bind IgE in sera from all
three patient groups, and all are peptides of JR2.1 that would remain
intact after trypsin digestion (Fig. 1B). This suggests that they could
contribute to cross-reactions between walnut and peanut. We compared the
physicochemical property (PCP) similarity of these peptides to other
known IgE epitopes using a PD-graph, which groups peptides according to
their similarity in PCPs (Figure 5) [26]. Low PD values between
peptides that have been predicted to be similar to known IgE epitopes
have previously been shown to have IgE reactivity [8, 9].
Figure 5 illustrates the most
similar peptide sequences as connected by blue lines, and the lines
descrease in intensity and width with increasing PD value (lower
similarity). These peptides are similar to each other and to the IgE
epitopes of the 2S albumins and the consensus peptides defined
previously [8, 9]. Peptides
JR6, 7, 8, 9 and A13 (shown in red boxes) have some of the highest z
scores and are clustered in the PD graph, which is consistent with the
SPADE similarity parameter results for JR2.1 and A1LS.