Figure Legends:
Figure 1: PN and WN VBPaments adopt a common α-hairpin
structure despite low sequence identity. A) NMR structures of J2LS and
A1LS VBPs. Cartoon figures depict representative conformations of each
structure. Inter-helix disulfides shown as sticks and highlighted in
yellow. Complete NMR data tables are available in
<blank>. B) Schematic view illustrating the
disulfide bonding pattern for all three fragments. Trypsin cut sites
identified by Downs et. al. are indicated by the yellow arrows. C)
Identity matrix for the amino acid sequence of the VBPs.
Figure
2: Potential PN cross-reactive regions on A1LS. A) Sequence of
A1LS used in the peptide microarray analysis. Residues depicted in the
solution-NMR structures are shown in capital letters. Residues from the
expression system used to generate the NMR samples but are not present
in the peptide microarrays are denoted with brackets. Residues
highlighted in blue represent alpha-helices identified in the available
NMR structures. Epitopes identified by Burks et. al are indicated in
grey rectangles, whereas cross-reactive peptides identified are
indicated by coloured rectangles. AEP cut site is indicated by the grey
arrow. B) Structure of A1LS coloured by surface similarity (dSIM) values
against J2.1, J2.2, and J2.3 as calculated using the SPADE surface
comparison algorithm. Total dSIM (Σ[ΔSIM] ) for all
residues is indicated. C) IgE-reactive peptides mapped onto the
structure of A1LS. Peptides colour-coded as in (A).
Figure 3: Potential PN cross-reactive regions on J2LS.A) Structure of J2.1, J2.2, and J2.3 coloured by surface similarity
(dSIM) values against A1LS as calculated using the SPADE surface
comparison algorithm. Total dSIM (Σ[ΔSIM] ) for all
residues is indicated. B) Sequence of J2LS, showing the three repeat
areas (Nesbit 2020), with helical areas highlighted in blue, residues
used to obtain the 31 mer consensus sequence underlined, and the central
13mer consensus in bold letters. Residues included in the solution-NMR
structures are capitalized; those included in the NMRsamples but not
present in the peptide microarrays are denoted in parentheses. Epitopes
identified by Maleki et. al 2011 are indicated in grey rectangles, with
cross-reactive peptides identified in this study (Fig. 4B) shown in
coloured rectangles labeled JR5-JR9. The AEP cut site is indicated by
the grey arrow. C) IgE-reactive peptides mapped onto the structure of
J2.1, J2.2, and J2.3. Peptides colour-coded as in (A). For the sake of
simplicity, only JR6-JR9 are shown.
Figure 4: Comparison of IgE binding to the A1LS and J2LS among
walnut (WN), peanut (PN), and dual allergic (PW) patients by microarray
analysis . A) Modified z values
were calculated from the microarray median signal-to-noise ratios (SNRs)
and were used to determine the percentage of IgE binding within an amino
acid range (shown in 2nd column) for each allergy
group. B) The percentage of patients which bind IgE to peptides and
major epitopes of A1LS and J2.1 and J2.2 is shown. A major epitope was
identified as a peptide where 50% or more of the P, W or PW patients
exhibited a positive binding event (z value of 3 or greater) for that
peptide. The epitopes marked with an asterisk were recognized by all
three allergy groups. The shared regions of overlapping peptides are
underlined. Average z values are given as low (3 – 6), medium (6.1 –
9) and high (>9 and in red text). Amino acid composition is
shown as percentage of hydrophobic (H), acidic (A), basic (B) or neutral
(N) amino acids in the peptide sequence.
Figure 5: Property distance (PD)-graph showing the
interrelatedness of the IgE binding epitopes of the LS to those of the
2S albumins of peanuts and tree nuts. The sequences of the LS peptides
identified as IgE binding in this study (Figure 4B) are similar in their
similar PCPs to the consensus and previously identified epitopes from PN
and TN 2S albumins. The PD graph is the graphical depiction of the most
similar sequences, which are
connected by blue lines, and the lines descrease in intensity and width
with increasing PD (lower similarity).
Note that the previously
determined PCP-consensus sequences (Nesbit et al. 2020) lie in the
middle of the graph, and that the LS IgE reactive peptides show
similarity to the 2S albumins.