NMR Structure Calculation:
Backbone and side-chain assignments were obtained for over 90% of the
residues for A1LS and the J2LS fragments using standard triple-resonance
experiments. Secondary structure was estimated from the available
chemical shifts using TALOS and found to be consistent with the proposed
structures (S2, S3) [48]. Cystine CB chemical shifts were uniformly
>33 ppm in the oxidized form suggesting that they were all
involved in disulfide bonds, providing further verification of the final
structures (S4).
NOESY spectra in conjunction with the backbone and side chain
assignments were used to calculate the 3D structure via the PONDEROSA
server[49, 50]. All the LS fragments has a paucity of true
long-range NOEs constraints, even relative to other vicilin precursor
fragments (S6). As such, multiple iterations of CYANA were employed to
identify putative long-range NOEs [51], which were verified manually
and resubmitted to the PONDEROSA for further refinement until no new
putative long-range NOEs were suggested by the software. NMR structural
statistics were calculated using the Protein Structure Validation
Software (PSVS) suite are shown in S6 [52].
Statistical analysis of
microarray data
Prior to analysis, we collected the median signal to noise ratio (SNR)
for each peptide spot contained in the J2LS and A1LS for each patient.
We chose SNR as our signal measurement as it corrects the raw signal
intensity for non-specific hybridization and instrument noise
(link).
Each SNR value was then converted into modified z-scores which are
calculated using median and median absolute deviation (MAD) rather than
mean and standard deviation. Z-scores represent a standardized signal
intensity and are frequently used to report immunoglobulin binding to
peptide microarray [53]. Median and MAD were calculated for each
patient and leader sequence combination. MAD was calculated with themad function in R using the constant of 1.4826 to approximate
standard deviation. After calculating patient and LS-specific median and
MAD, z-scores were calculated by subtracting the median from a relevant
spot SNR and dividing this value by the MAD. We defined a true IgE
binding event as a SNR with a converted Z score >= 3.
Statistical analyses were performed using R (version 3.6.3; R
Development Core Team, available at www.r-project.org). A goal of this
study was to assess cross-reactivity among walnut-, peanut- or
co-allergic individuals to regions of A1LS and J2LS. For each LS and
predicted J2LS fragment, we counted the number of significant IgE
binding events (z-value > 3) to their representative
microarray peptides for each allergy type: walnut (WN), peanut (PN), or
co-allergy (PW). For each LS or J2LS fragment we determined the percent
of peptides bound by IgE for each allergy group. For example, JR2.1 is
represented by 11 peptides on each microarray and there are 12 total
walnut allergic individuals meaning that there is a total of 132
possible IgE binding events. As 42 of these peptides exhibited IgE
binding with a z-value >3, we determined that
walnut-allergic patients bound 32% of the possible JR2.1 microarray
peptides. We then used the fisher.test function to perform
Fisher’s exact tests to compare the percent of peptides with significant
IgE binding among the allergy groups for each LS or J2LS fragment.