Detection of LTP in kiwifruit pulp and seed protein extracts
Seed and pulp extracts of green and gold KF were prepared, fractionated by RP-HPLC and the separated protein components were manually collected and analyzed by N-terminal amino acid sequencing. The analysis of a peak eluted at a retention time very similar to that observed for nPru p 3 allowed the identification of the LTP, namely Act c 10 and Act d 10, in the seed extracts from the gold and the green KFs, respectively, rather than from the pulp (see below). Act c 10 was eluted as a single peak, whereas Act d 10 was eluted in three overlapping peaks suggesting the presence of some isoforms. Direct protein sequencing of the purified protein allowed the identification of two different isoforms of Act d 10 (see below). Possible additional isoforms were not identified probably because of the low yield. Conversely, the LTP peak was absent in the RP-HPLC profiles obtained for the pulp extracts of the two KF species.
nAct c 10 and nAct d 10 purification
The recovery of the pure protein was about 0.4 mg per gram of green and gold KF seeds. The pulp was not used for LTP preparation. Protein concentration was estimated on the basis of the molar extinction coefficient at 280 nm (3480 M−1 cm−1). Purity of the protein preparations was checked by SDS-PAGE, RP- HPLC and N-terminal amino acid sequencing.
Full primary structure elucidation of nAct d 10
The direct sequencing of the N-terminal region of native nAct d 10 produced 17 identifiable amino acid residues, AVSCGQVDTALTPCLTY (). A small fraction of the molecules contained the amino acid threonine (T) rather than alanine (A) as first residue. The N-terminal sequence of nAct c 10, comprising 17 amino acid residues, was identical to that of nAct d 10 (AVSCGQVDTALTPCLTY), and no heterogeneity at the first position was observed. The complete amino acid sequence of nAct d 10 was elucidated by automated sequencing of peptides resulting from the enzymatic digestion of denatured and S-pyridilethylated protein. Most of the primary structure was obtained by aligning the amino acid sequence of peptides from trypsin and Asp-N digestions, whereas the regions corresponding to the residues 18–35 and 36–42 were obtained by sequencing peptides from chymotrypsin digestion. shows only the peptides necessary to elucidate the complete amino acid sequence. nAct d 10 comprises 92 amino acids producing a molecular mass of 9,458 Da for the most abundant isoform, having alanine at the N-terminus. The 100% sequence identity in the N-terminal region, together with the observation that nAct d 10 and nAct c 10 have the same chromatographic behavior and very similar molecular masses as estimated by mass spectrometry analyses (see below), suggested a very high structural similarity between the two proteins. Therefore, only the full nAct d 10 primary structure was elucidated. The full amino acid sequence of the nAct d 10 isoforms having alanine or threonine as N-terminal residue, and the partial N-terminal sequence of nAct c 10, have been registered in the UniProt Knowledgebase with the accession numbers P85205, P85206, and P85204, respectively. Act c 10 and Act d 10 are allergen names approved by the WHO-IUIS Allergen Nomenclature Subcommittee (www.allergen.org
), and encoded 5735 and 5737, respectively, in the Allergome database (www.allergome.org
Act d 10 complete primary structure.
Estimation of the molecular mass by mass spectrometry
The analyses by MALDI-TOF mass spectrometry of purified nAct d 10 provided two values, 9.464 (±20) and 9.484 (±20) kDa, in good agreement with the mass values deduced from the amino acid sequence of the two isoforms having alanine (9.458 kDa) or treonine (9.488 kDa) as N-terminal residues, respectively. A molecular mass of 9.460 (±20) kDa was obtained for Act c 10 by mass spectrometry.
Evaluation of the resistance to proteolysis
The analysis by SDS-PAGE and RP-HPLC of the LTP samples subjected to digestion in SGF showed that nAct c 10 and nAct d 10, similarly to nPru p 3, are resistant to gastric digestion. Moreover, in line with the results reported by Cavatorta et al. 
, nPru p 3 was partially digested by trypsin, whereas nAct d 10 and nAct c 10 appeared to be resistant to the same digestion (data not shown).
Sequence identity and alignments among LTPs under study
Homology search in Uniprot protein database carried out using the BLAST algorithm (www.expasy.org
) showed the sequence identity between Act d 10 and other already known allergenic LTPs to be not very high, ranging between 55% with Ara h 9 (isoform Ara h 9.0201) and 35% with Par j 2. Identities among the full length amino acid sequence of the six allergenic LTPs under study (Act d 10, Ara h 9, Art v 3, Cor a 8, Mor n 3 and Pru p 3) are comprised in the 42–70% range (, panel A). The sequence identity values between Act d 10 and other allergenic LTPs, such as Api g 2 (celery stalk), Cit s 3 (orange), Fra a 3 (strawberry), Lac s 1 (lettuce), Len c 3 (lentil), Lyc e 3 (tomato), Mal d 3 (apple), Ory s 14 (rice), Pla or 3 (plane tree pollen), Pru du 3 (almond), Pyr c 3 (pear), Sin a 3 (mustard), Tri a 14 (wheat), Vit v 1 (grape), Zea m 14 (maize), are comprised in the narrow range of 40–55% (data not shown). The alignment of the amino acid sequences of the LTPs analyzed in the present study (, panel B) underlines that the best conserved region is a stretch of 10 contiguous residues (positions 45–54, Act d 10 numbering, , panel B). Twenty-four amino acids (26%) (yellow background) are conserved in all the six aligned sequences, including the eight cysteine residues. Act d 10 shares 18 additional residues with Pru p 3: two of them (brown background) are present only in Act d 10 and Pru p 3, whereas the remaining 16 shared residues (blue background) can be found also in other aligned sequences but not all of them. Twenty-one residues (23%) of Act d 10 are substituted in Pru p 3, whereas they are conserved in at least one of the aligned sequences (green background). A higher number of residues, namely 35 amino acids of Pru p 3 (38%), are substituted in Act d 10, but they are conserved in at least 1 of the other sequences (red background). Act d 10 and Pru p 3 do not share identical residues with any of the other aligned sequences in 27 and 14 sequence positions, respectively (white background).
Amino acid identity table and sequence alignments of Act d 10, Ara h 9, Art v 3, Cor a 8, Mor n 3, and Pru p 3.
Defining the first set of kiwifruit allergic patients being sensitized to kiwifruit LTPs
reports selected patients' diagnostic profiles. All patients reported a reliable clinical history of reactions when eating KF. The IgE screening performed using the ISAC test revealed none of the subjects to be sensitized by other KF allergens, namely nAct d 2, nAct d 5, nAct d 6, nAct d 7, nAct d 11, but one recorded positive to Act d 1, whereas all were recorded IgE positive to nPru p 3, the homologous peach LTP (, panel A). Patients were then tested with a commercial KF extract both by ST and CAP. Results were sometime weak, doubtful or even negative (, panel A). All patients underwent DBPCFC, all stopped because subjective and objective allergic symptoms appeared as reported in , panel A. Thus this selected group of KF allergic patients underwent ST with the purified KF LTPs and IgE detection by mean of the biotinylated KF LTP coupled with the streptavidin-CAP. All ST and CAP testing were scored positive for the two KF LTPs (, panel B), whereas they were negative in 10 allergic subjects used as controls. The same patients were finally tested IgE positive for the two KF LTP preparations, along with the in-house nPru p 3 one, on the ISAC Exp96 (, panel B). The two proteins where thus considered clinically relevant allergens, and the ISAC Exp96 a suitable tool for further investigations.
Diagnostic profiles of seven selected kiwifruit allergic patients used for the initial characterization of kiwifruit LTPs.
To confirm the findings of the previous set of tests and to start a comparative evaluation of LTPs from different sources, a preliminary extended in vivo and in vitro testing has been performed by ST and by detecting IgE on ISAC Exp96, using identical nAct c 10, nAct d 10, and nPru p 3 preparations in the two tests. Forty four patients complaining about symptoms on eating peach, enrolled on the basis of a positive DBPCFC or a reliable history of recent anaphylaxis and tested positive for nPru p 3 on the routine ISAC 103, were evaluated. reports data obtained by ST and IgE detection in this selected cohort of patients. All 44 subjects had a positive ST to nPru p 3 (median wheal area 48.83 mm2, range 1.4–195) confirming the positive IgE result on ISAC 103 used for selection, and replicated on ISAC Exp96 (median 4.68 kU/l, range 0.1–55.6), without statistically significant differences when compared to ISAC 103 results. A slightly not statistically significant different skin reactivity was recorded when considering nAct c 10 (median wheal area 18.0 mm2, range 6.2–148.6) and nAct d 10 (median wheal area 15.0 mm2, range 2.6–159) (). To set the relationship between ST and IgE detection, nPru p 3 results obtained in the two tests were compared; no correlation was found between ST areas and IgE values (, panel A). When the ST was performed using the two KF LTPs 31 subjects reacted to both allergen preparations, whereas positive ST was recorded to one and not to the other in one subject each (, panel B). Similarly, IgE results detected for the two molecules were compared without significant differences (, panel C). Differing from nPru p 3 ST versus IgE data analysis, a non-overlapping reactivity, anyhow not statistically different, has been found when considering nAct c 10 and nAct d 10 ST versus IgE positive subjects, but the correlation between the ST induced wheal areas and the IgE values was statistically significant, though the Spearman r value was just 0.54 and 0.56 for the two LTPs, respectively (, panel D, E). Finally, no statistically significant correlations were found when ST and IgE results were compared between nAct c 10 and nAct d 10 versus nPru p 3 (, panel F, G, H, I).
Skin test and IgE data on 44 peach allergic subjects.
Comparative skin testing and IgE detection using nAct c 10, nAct d 10, and nPru p 3 in 44 peach clinically allergic subjects tested positive for nPru p 3 on ISAC 103.
Overall the findings reported above preliminary marked immunological differences between the LTPs from peach and KFs, thus suggesting that the shared LTP reactivity recorded in the first set of patients and leading to the identification of KF LTPs could not be extended to all nPru p 3 positive subjects.
Defining the magnitude of kiwifruit LTP reactivity by comparative skin testing and IgE detection toward other LTPs
As from the previous section findings, showing heterogeneity between LTPs from two allergenic sources in that small study group, a larger study was set using the routine testing approach by the ISAC microarray, in order to define any possible subsets related to different LTP IgE recognition. A comparative evaluation of seven LTPs was undertaken using the ISAC 103 and ISAC Exp96 microarrays in parallel.
Unless the original two population were defined on the basis of being nPru p 3 IgE positive or negative as tested on ISAC 103, additional nPru p 3 positive subjects were found in the control population using the ISAC Exp96 microarray bearing a different nPru p 3 preparation, thus raising the number of nPru p 3 positive subjects from 259 to 296. The same control population revealed a number of subjects being nPru p 3 IgE negative but positive to at least one of the other LTPs, increasing the overall number of subjects being positive to at least one of the LTPs from 296 to 431. nAct c 10, nAct d 10, rAra h 9, nArt v 3, rCor a 8, and nMor n 3 were positive when nPru p 3 was negative in 48, 41, 17, 56, 43, and 17 cases, respectively. The number of subjects positive to one LTP as listed above and negative to all others was relatively low, being 5, 0, 9, 25, 10, 9, 17. These prevalence were statistically different only when considering nArt v 3 values versus KF LTPs, peanut and mulberry ones. Absence of nAct d 10 exclusively IgE positive subjects statistically differed to all the remaining LTPs rather than nAct c 10 (data not shown). Raw prevalence are reported in , calculated on the subset of 431 patients tested positive to at least one of the seven LTPs. An overall significant p<0.0001 of the χ2 test has been obtained taking together the seven prevalence, whereas not all prevalence were statistically different when compared each other. For instance, nPru p 3 was anyway the most prevalent sensitization with no difference only when compared to nMor n 3. IgE sensitization to nAct c 10 and nAct d 10 had statistically significant higher prevalence than the mugwort and the hazelnut LTPs, but less prevalent than nMor n 3 and nPru p 3, though proven by statistics just for nPru p 3. The less prevalent IgE reactivity have been recorded for the mugwort and the hazelnut LTPs being statistically different from all other LTPs. Detailed statistics are reported in .
IgE prevalence for nAct c 10, nAct d 10, rAra h 9, nArt v 3, rCor a 8, nMor n 3, nPru p 3 on 431 sera.
To visualize exclusive sensitization to one LTP compared to others, a series of Venn diagrams showing all possible logical relations between our finite collections of IgE values obtained by the two microarray testing, have been generated and reported in . The first Venn diagram as reported in , panel A, shows almost overlapping results between the two KF LTPs, and non-overlapping results considering nAct c 10 and nAct d 10 opposed to nPru p 3. Due to the highly similar behavior of the two KF LTPs, the following panels show how each of the other four LTPs under study behaves compared to nAct d 10 and nPru p 3. Venn diagrams change depending on individual LTP features, further showing heterogeneity of the members of this group of molecules. An index of the different reciprocal behavior of the LTPs is the changing number of isolated positivity to the three LTPs used in each graph depending on the third LTP used.
Venn diagram representation of positive IgE results for selected LTPs.
A cluster analysis performed using all subjects having IgE positive results to at least one of the seven LTPs generated the heat map shown in . The overall picture shows the clustering heterogeneity of both allergens and patients. LTP IgE recognition by the sensitized subjects is well depicted for allergens by the presence of the first two clusters segregating nArt v 3 and rCor a 8 reactivity apart from the other LTPs. A second dichotomy is observed, separating the two KF LTPs from the remaining, also representing, as expected, the two molecules to be the closest ones. The remaining three LTPs form an additional cluster further divided in two. Subject clustering is dispersed in a great number of clusters, much reflecting the individual heterogeneity of LTP IgE recognition.
Unsupervised two-way hierarchical clustering analysis of 431 subjects tested for IgE on the seven LTPs.
Beside the evaluation of positive/negative results reported above and clearly showing non-overlapping behavior among LTPs, we sought to verify whether detected IgE value distributions were different comparing the seven LTPs under study. A Kurskal-Wallis test gave statistically significant results (p<0.0001), and, when comparing the paired groups using the Mann-Whitney test several statistically different findings were recorded at different p values (). Differing from prevalence results reported in , nAct c 10 and nAct d 10 showed the lowest median values, having those within the 5–95 percentiles distributed in a quite narrow range (). Both their distributions did not differ significantly from rAra h 9, nArt v 3, and rCor a 8 values, whereas statistically significant differences were obtained when comparison was carried out toward nMor n 3 and nPru p 3 IgE value distributions. rAra h 9 and nArt v 3 behaved almost the same differing only from nPru p 3. Overall evaluating the distributions of IgE values further described the behavioral heterogeneity within LTPs as allergens.
IgE values box-plot representation considering median values and 5–95 percentile distributions.
All the 1,003 paired results were then plotted considering paired allergens as reported in , panels A-I; , panels J-R; , panels S-V. All comparisons were highly statistically significant (p<0.0001). As reference, both for statistical findings and graphical representations, the nAct c 10 versus
nAct d 10 correlation was taken, having the highest Spearman r (0.94) and χ2
(851.6) values (, panel A). Twenty-nine discrepant results out of 258 positive ones (11.2%) for the two KF LTPs were recorded, being the lowest value among the others. Discrepant results are clearly shown along the X and Y axis and reported in the table underneath each graph. Taking advantage of the availability of two different nPru p 3 preparations on the two microarrays in use, we plotted and evaluated their IgE results, giving the Spearman r
0.84 and a χ2
608 values as shown in , panel B. Unless the statistics were very good, a higher number of discrepant results (n
90; 27.9%) were recorded compared to the above reported results obtained with two KF LTPs. A very good correlation was recorded again just when comparing nMor n 3 and nPru p 3 (r
0.91 and a χ2
761.8) (, panel V), showing also the highest number of concordant positive results (n
260), whereas all other correlations showed a quite broad range of values, being the lowest r (0.35) and χ2
(107.2) when comparing nAct c 10 and nArt v 3 (, panel E), and the highest (0.88; 699.5) when rAra h 9 and nMor n 3 IgE results were matched (, panel O). In many cases IgE plotted points showed no linearity at all, being scattered in a broad area (, panels E, F, G, H; , panels M, R; , panel S). In other cases a certain linearity was still present for part of the subjects, but part of the values were anyhow plotted above or below the theoretical central line (, panels C, D, I; , panels J, K, L, N, O, P, Q; , panels T, U). The highest number of discrepant IgE results were recorded for the paired nArt v 3/nPru p 3 results (n
226; 64.2%) (, panel S), whereas those recorded for rAra h 9/nMor n 3 (n
65; 21.8%) were very close to the best ones reported above (, panel O). Overall evaluating IgE correlations among allergens under study shed further light on LTP immunological heterogeneity.
IgE value correlations for paired LTPs. All 1,003 subjects have been plotted in each graph.
IgE value correlations for paired LTPs. All 1,003 subjects have been plotted in each graph.
IgE value correlations for paired LTPs. All 1,003 subjects have been plotted in each graph.
IgE inhibition experiments
To define immunological relationships in terms of IgE binding among LTPs, the SPHIAa assay was run using nAct c 10, nAct d 10, and nPru p 3 as inhibitor on the seven LTPs. The assay was performed using ten individual sera selected for being IgE positive to the three inhibitors as shown in , where IgE values for any given inhibited LTP are given for each of the ten sera. All the positive allergens other than LTPs used for control purposes for each of the samples gave no inhibition values (data not shown). Full autologous IgE inhibitions on the two KF LTPs were achieved, as well as with the homologous nPru p 3 preparation (, panel A and B). As shown in , panel C to F, nPru p 3 fully inhibited all the other LTPs. The two KF LTPs showed a serum/allergen dependent behavior, having IgE inhibition values as follows: greater than 50% for all samples (rAra h 9, , panel C); spread between 0 and 100% (nArt v 3, , panel D); separated in two subgroups, one achieving 100% the other staying below 70% (rCor a 8, , Panel E); hardly reaching 100%, and spread in a wide range, with the lowest value at 29% (nMor n 3, , panel F); not reaching 100%, spread in a wider range, with the lowest value at 19% (nPru p 3, , panel F). All statistics are given in legend. As for some of the other experiments reported above, minor differences between the two KF LTPs were recorded also in the inhibition test. Overall, also IgE inhibition experiments confirmed the immunochemical differences between the two new KF LTPs and the peach one and the different behavior in terms of epitope distribution, recognition and inhibition by the three inhibitors.
Single Point Highest Inhibition Achievable assay (SPHIAa) for LTP IgE inhibition.
Analysis of the LTP distribution within the kiwifruit tissues by biochemical, immunological and clinical methods
To verify whether the heterogeneous immunological behavior between the two KF LTPs and the peach LTP could lead to define a subset of patients who are also not clinically reactive to all LTPs, we first performed a series of experiments to define the LTP distribution within the KF tissues, namely the pulp and the seeds, based on biochemical, immunochemical and clinical methods.
As reported in the first paragraph of the Result
section, using biochemical methods, seed and pulp extracts were fractionated by RP-HPLC (, panel A) and the separated protein components were analyzed by N-terminal amino acid sequencing. The analysis of a peak eluted at a retention time very similar to that observed for nPru p 3 allowed the identification of nAct c 10 and nAct d 10 in the seed extracts from the gold ( A, left) and the green KFs ( A, right), respectively (, panel A, upper parts), whereas that peak was lacking in the RP-HPLC profiles obtained for the pulp extracts of the two KF species (, panel A, lower parts). Nevertheless, the fractions of pulp extracts eluted at the retention time of nAct d 10 and nAct c 10 were collected and analyzed by N-terminal amino acid sequencing. The results obtained confirmed the absence of detectable amounts of nAct d 10 and nAct c 10 in the pulp extracts. As the biochemical findings reported above seemed to be quite conclusive, to increase the confidence with the negative data we approached the issue of LTP distribution in KF tissues using the SPHIAa as well. The inhibition was performed using the same pulp and seed preparations from the green and gold KFs, along with whole KF extracts. A pool of sera having IgE for both the two KF LTPs was used as probe. As shown in , panel B, full or almost full IgE inhibition results were obtained with the whole KF extracts and the seed ones, whereas the pulp gave either no inhibition (green KF pulp on nAct c 10, , panel B) or inhibition values ranging between 18% and 28% (all other pulp/allergen combinations in , panel B). These slightly positive results were replicated and could suggest the presence of minimal amount of LTP in the KF pulp not detected by the biochemical methods. The very low inhibition values were anyhow not considered conclusive, and needing a third discriminating proof. We thus performed in vivo
skin testing with green KF seed and pulp preparations in 21 selected subjects. As reported in , panel C, the comparative analysis showed statistically significant different results between green KF seed and pulp preparations, nevertheless some of the patients did react to the pulp as better shown for individual patients in . As the key values were those obtained on patients having severe clinical reactions on KF ingestion, and high or very high ST and IgE scores when tested with nAct c 10 and nAct d 10 (, lines 15–21), we concluded that tiny amount of LTP are present in the KF pulp. We thus abandoned the idea of challenging KF clinically allergic patients with the separated KF pulp.
Biochemical, immunochemical, and clinical evaluation of LTP distribution in kiwifruit tissues.
Diagnostic and clinical profiles of patients enrolled because of clinical allergy to peach and positive or negative kiwifruit LTP test results.
Defining the tolerability of the green kiwifruit by nPru p 3 positive, nAct c 10/nAct d 10 negative subjects
Opposing to the study starting point, where patients used to identify the new KF LTPs were isolated because of their combined clinical and IgE reactivity to KF and peach, and coherently following all the new findings reported above, showing heterogeneous behavior of single subjects toward one or the other LTP, we sought to define the in vivo clinical reactivity to green KF in well characterized peach allergic patients. We thus recruited patients having different diagnostic profiles. All enrolled ones had to be nPru p 3 positive with either a positive DBPCFC to peach or a recent severe generalized reaction on peach ingestion. All underwent the tests as reported in . Among the enrolled ones we had 15 who passed the DBPCFC eating a full KF at the end, and six who clearly showed symptoms after the challenge. As shown in , the latter were all tested positive for almost all preparations either by in vivo or in in vitro tests, whereas among the KF tolerant in the majority of the cases the KFs LTP were tested with negative results, but with exceptions, unless positive values were in the lowest range for both ISAC and ST (). We thus performed a statistical evaluation of test value distributions between the tolerant and the non-tolerant subsets as reported in . Unless the number of enrolled subjects was not high, we recorded statistically significant differences when nAct d 10 ISAC IgE values and both KF LTPs ST values were compared, finally showing the valuable diagnostic help of adding KF LTP to the testing to identify patients having a inhomogeneous behavior toward LTPs.
IgE and Skin test result evaluation comparing patients being tolerant or not to green kiwifruit ingestion.
To note that the testing had a positive predictive value for those whose KF avoidance remains mandatory. In addition to those reported in , seven subjects, who were invited to participate to the study, underwent tests foreseen in the first part, where their diagnostic profile was recorded very similar to that reported for patients 15–21 in (data not shown). They declined to undergo the DBPCFC. Six of 7 reported a generalized reaction on KF ingestion sometime in the past, thus they found our nAct c 10 and nAct d 10 positive tests “sufficient to prove their clinically relevant allergy to KF”.
In the attempt to explain why the nAct c 10 or nAct d 10 positive results were not accompanied by a clinical reactivity, we simulated physiological digestion of the two KF LTPs as reported above, and, using the SPHIAa, evaluated the retained capability to induce IgE inhibition. nPru p 3 was comparatively evaluated, as it is known to be resistant to digestion. The results showed that also digestion resistant nAct c 10 and nAct d 10 are still active in the IgE inhibition assay (data not shown).