• Act d 11;
  • allergen;
  • Bet v 1-like;
  • kirola;
  • kiwifruit


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

To cite this article: D’Avino R, Bernardi ML, Wallner M, Palazzo P, Camardella L, Tuppo L, Alessandri C, Breiteneder H, Ferreira F, Ciardiello MA, Mari A. Kiwifruit Act d 11 is the first member of the ripening-related protein family identified as an allergen. Allergy 2011; 66: 870–877.


Background:  Kiwifruit is an important cause of food allergy. A high amount of a protein with a molecular mass compatible with that of Bet v 1 was observed in the kiwifruit extract.

Objective:  To identify and characterize kirola, the 17-kDa protein of green kiwifruit (Act d 11).

Methods:  Act d 11 was purified from green kiwifruit. Its primary structure was obtained by direct protein sequencing. The IgE binding was investigated by skin testing, immunoblotting, inhibition tests, and detection by the ISAC microarray in an Italian cohort and in selected Bet v 1-sensitized Austrian patients. A clinical evaluation of kiwi allergy was carried out.

Results:  Act d 11 was identified as a member of the major latex protein/ripening-related protein (MLP/RRP) family. IgE binding to Act d 11 was shown by all the applied testing. Patients tested positive for Act d 11 and reporting symptoms on kiwifruit exposure were found within the Bet v 1-positive subset rather than within the population selected for highly reliable history of allergic reactions to kiwifruit. Epidemiology of Act d 11 IgE reactivity was documented in the two cohorts. IgE co-recognition of Act d 11 within the Bet v 1-like molecules is documented using the microarray IgE inhibition assay.

Conclusions:  Act d 11 is the first member of the MLP/RRP protein family to be described as an allergen. It displays IgE co-recognition with allergens belonging to the PR-10 family, including Bet v 1.


human serum albumin


major latex protein/ripening-related protein family


root mean square deviation


sodium dodecyl sulfate-polyacrylamide gel electrophoresis


single-point highest inhibition achievable assay


skin prick test

Several IgE-binding proteins have been detected in different species of kiwifruit. The green-fleshed fruit (Actinidia deliciosa) is the most studied and commonly available type of kiwifruit. A number of allergenic proteins were identified in this species, and most of them were officially named by the WHO-IUIS subcommittee for allergen nomenclature. Actinidin (Act d 1) (1); a thaumatin-like protein (Act d 2) (2), and kiwellin (Act d 5) (3, 4) are major allergenic protein components of green kiwifruit extracts. Among other kiwi allergens described so far, recently a member of the pathogenesis-related protein family PR-10 was reported (5).

PR-10 proteins are components of a protein family included in the Bet v 1 superfamily (6), together with the major latex protein/ripening-related protein family (MLP/RRP), the cytokinin-binding proteins from legumes, the norcoclaurine synthases, etc. These proteins share a common fold, whose main feature is the presence of an internal cavity that can accommodate a number of small, mostly hydrophobic ligands (6).

Bet v 1 is the major allergen of birch pollen. Allergenicity of Bet v 1 homologs from many sources, including the recombinant homologs from green and yellow (Actinidia chinensis) kiwifruit, named Act d 8 and Act c 8, was described (5). Probably due to their low amounts in the fruit, isolation of the natural Act d 8 and Act c 8 has not yet been reported. In contrast, a high amount of a protein different from Act d 8, but showing a molecular mass similar to that of Bet v 1-like proteins, was observed in ripe kiwifruit extracts (7).

The new 17-kDa protein, named kirola, has been identified in green kiwifruit and characterized. The relationships of this new kiwifruit allergen with other Bet v 1-like molecules were explored from both the biochemical and the immunological view points. Clinical relevance as symptom trigger was evaluated. On the basis of the data available, the WHO-IUIS subcommittee assigned the name Act d 11 to kirola.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

The extraction and purification of kirola (Act d 11) from green kiwifruit, as well as the mass spectrometry measurement, the amino acid sequencing, the in silico analysis and the computational methods used for statistical evaluation of data are described in Data S1.

Study population, IgE testing

The protocol of the present study was approved by the Institutional Review Board (IDI/CE 2006/192) of IDI-IRCCS (Rome, Italy). All patients gave written informed consent before enrollment in the study.

Thirty-four consecutive patients were selected on the basis of a highly reliable history of allergic reactions to kiwifruit among those attending the outpatient department. After recording preliminary results in this first group, a second selection was set recalling 21 consecutive patients selected among those IgE positive to Bet v 1 by ISAC testing. All the patients included in the study were evaluated by skin prick test (SPT) with a kiwi commercial diagnostic extract (Stallergenes, Antony, France), with an in-house produced solution of kiwi extract (7), by the prick–prick test using fresh kiwifruit, and with the natural purified Act d 11, Act d 1, Act d 2, and Act d 5. For SPT, aliquots of Act d 11, or other natural allergens, at a protein concentration of 1 mg/ml were mixed with sterile glycerin in a 1 : 1 ratio. The solutions were sterilized by membrane filtration through a 0.22-μm filter (Millipore, Bedford, MA, USA), in a sterile horizontal laminar flow hood. The diluent used to preserve the allergens, and a solution of histamine 10 mg/ml, was used as negative and positive control, respectively. Details on SPT are described in Data S1.

Following the SPT, sera were obtained from patients who consented to blood sampling for in vitro procedures.

For immunoblotting, the purified protein was subjected to 15% SDS-PAGE and transferred onto PVDF membranes. IgE immunoblotting was carried out as already described (7) using 50 sera from patients having symptoms on kiwi exposure and a positive SPT to kiwifruit extract.

IgE to kiwi extract was detected by the CAP system (Phadia, Uppsala, Sweden). The prevalence of Act d 11 recognition by sera from the allergic population was established by spotting the molecule on the ISAC system (Phadia-Multiplexing-Diagnostics, Vienna, Austria) for IgE detection (8). ISAC testing was routinely applied to the whole population accessing the outpatient department for IgE routine testing purposes. This population was already described as exposed to different Fagales rather than to birch pollen (9). Four kiwi allergens were available on the chip: Act d 1, Act d 2, Act d 5, and Act d 11. Patient subset definition was carried out considering Bet v 1-like molecules on the chip. Along with Italian samples, a group of 60 Austrian samples, known to be IgE positive to Bet v 1, were tested on the same ISAC103 microarray. For the aim of this study, ISAC IgE data on Act d 11, Bet v 1, Cor a 1, Dau c 1, and Mal d 1 will be reported.

IgE inhibition assay

IgE inhibition experiments were performed as previously reported (10) and detailed in Data S1. Ten Austrian and ten Italian sera were selected on the basis of their IgE recognition of Bet v 1-like allergens.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Protein purification and primary structure elucidation

The recovery of the pure protein was in the range of 0.5–1 mg/100 g of kiwifruit pulp. Amino acid sequence analysis indicated the absence of PR-10 contaminants, even at very low concentrations as 0.1%. Furthermore, results obtained by inhibition assays using recombinant Bet v 1.0101 diluted in human serum albumin indicated that there was no chance to have any PR-10 contamination above 0.1% into our Act d 11 preparation. Details are given in Data S1.

Amino-terminal sequencing indicated that the N-terminus is blocked. The primary structure was elucidated by sequencing the peptides obtained after digestion with Asp-N protease (Fig. 1). Interestingly, Act d 11 sequence is identical to the tentative consensus sequence deduced from the overlapping EST clones FG437290 and FG440357 obtained from kiwifruit (11). The molecular mass deduced from the amino acid sequence of Act d 11 corresponds to the experimental value obtained by mass spectrometry for the major component, assuming that the N-terminal methionine is acetylated. The sequence obtained from a further EST clone, FG445803, has a substitution S[RIGHTWARDS ARROW]T in position 54, which would give a mass value of 17 460, accounting for one of the minor isoforms.


Figure 1.  Amino acid sequence of Act d 11. Arrows indicate sequenced Asp-N peptides. The presence of N-acetylated methionine at the blocked N-terminus was deduced from the molecular mass value determined by mass spectrometry.

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Amino acid sequence analysis

The homology search performed by NCBI-BLASTP revealed a significant similarity of Act d 11 with members of the Bet v 1-related superfamily. A multiple alignment of Act d 11 with some representative sequences of the MLP/RRP family and Bet v 1-like allergens belonging to the PR-10 family (12) is shown in Fig. 2. The sequence identity between Act d 11 and the MLP/RRPs ranges from 45 to 30%, whereas that observed with the PR-10 proteins is never higher than 21%. In line with the sequence identity values, the cladogram built with the same protein sequences includes Act d 11 in the family of MLP/RRP (see Fig E1 in Data S1).


Figure 2.  Alignment of Act d 11 with Bet v 1-like allergens (group 1) and MLP/RRPs (group 2). The sequence accession numbers are on the left. The figure has been drawn by ESPript (25). Residues conserved in all aligned sequences are in white and black shaded. The amino acid numbering of the Bet v 1 sequence is shown on the top. Secondary structure elements of Bet v 1 crystal structure and of Act d 11 model are shown on the top and on the bottom of the figure, respectively. All the sequences shown in the alignment have been used to build the cladogram of Fig E1 (see Data S1).

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Act d 11 displays an overall identity of 17% with Act d 8 and Act c 8, the recently described (5) Bet v 1-like allergens of green and gold kiwifruit. Almost all the conserved amino acids are clustered in three regions corresponding to the residues 35–51, 65–89, and 142–152 of the Bet v 1 numbering. For instance, in the region of residues 35–51, Act d 11 shows 50 and 38% of sequence identity with Act d 8 and Act c 8, respectively. In the C-terminal region (residues 142–152), 45 and 54% of identities are observed with Act d 8 and Act c 8, respectively. Similarly, the comparative analysis of Act d 11 and Bet v 1 primary structures underlines a very low overall sequence identity and the clustering of most of the conserved positions. For example, the region of residues 35–51 displays 44% of conserved positions.

IgE-binding experiments

Sera of 50 subjects allergic to kiwifruit were analyzed by IgE immunoblotting. Act d 11 was recognized by 11 (22%) of tested sera (Fig E2 in Data S1).

Act d 11 IgE epidemiology using ISAC testing

A total of 12 856 Italian subjects complaining about any allergy-related symptom have been screened by using the ISAC test. Eight thousand two hundred and fifty-six subjects (64.21%) were positive to at least one allergen on the chip and 835 of the 8256 (10.11%) were detected IgE positive to at least one of the kiwi allergens spotted on the microarray. Results of epidemiological data are detailed in Data S1 and in Figs E3 and E4.

Specific IgE value correlations as detected by ISAC testing using the whole Italian cohort are shown in Fig. 3. Although all statistical analysis had P < 0.0001 mainly for the number of recorded observations, their r-value varied quite largely among paired allergens under consideration. As expected, the highest correlation was recorded for homologous pollen allergens Bet v 1 and Cor a 1, and for apple allergen Mal d 1, whereas r-values dropped whenever Act d 11 or Dau c 1 was evaluated in relation to the former three allergens and reciprocally, being the r-values ranging between 0.18 and 0.28 (Fig. 3).


Figure 3.  Correlations between IgE values for Bet v 1-like allergens reported in this study measured by the ISAC microarray. Spearman r-values are reported in each panel.

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IgE inhibition experiments

Figure 4 shows the results of IgE inhibition experiments performed with soluble nAct d 11, rBet v 1.0101, and rCor a 1.0104 on the ISAC-spotted allergens nAct d 11, rBet v 1.0101, rCor a 1.0101, rDau c 1.0101, and rMal d 1.0108.


Figure 4.  Single-point highest inhibition achievable assay (SPHIAa) results for defining IgE co-recognition of homologous molecule are reported for ten Italian samples (panel A) and ten Austrian samples (panel B). Percent inhibition values have been calculated by the InterAll software from data received online from the ISAC system. Further experimental details are reported in Data S1, Results section.

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Among the 10 Italian samples, full homologous inhibitions were achieved by using the three inhibitors (Fig. 4A). Act d 11, not at all or only partially inhibited Bet v 1, and Cor a 1, whereas a broad range of values, from 0 to 100%, were recorded for Mal d 1. Only one of three Dau c 1+ sera was inhibited. Soluble Bet v 1 achieved 100% IgE inhibition on almost all Act d 11+ and Mal d 1+ sera. Two of 3 Dau c 1+ sera were fully inhibited, whereas partial or no inhibition was achieved on Cor a 1+. Soluble Cor a 1 achieved an almost full inhibition on almost all Mal d 1+ samples, partial inhibition on several Act d 11+ and Bet v 1+ samples, and three different pattern of inhibition on Dau c 1+. Austrian samples behaved similarly with all three inhibitors (Fig. 4B).

Skin testing and kiwi allergy clinical features

As reported in Table 1, none of 34 kiwi allergic subjects selected on the basis of kiwi-related clinical symptoms had positive SPT to Act d 11, whereas when Act d 11 IgE-positive subjects were tested, 7 (33.3%) were recorded positive in SPT. Some Act d 11 SPT-positive/IgE-positive subjects, without sensitization to other kiwi allergens, underwent double-blind placebo-controlled food challenge (DBPCFC) to detect as objectively as possible a clinical reactivity. Some patients were scored positive by the clinical investigator although always reporting mild symptoms after oral mucosa contact with the kiwifruit.

Table 1.   Demographical, clinical, and diagnostic features of patients selected for skin prick test procedure
 Kiwi allergicsBet v 1-positive
Total34 N.%21 N.
  1. *Symptoms in kiwi allergics (left column) were recorded as reported by patients in a specific questionnaire. Symptoms in Bet v 1-like-positive subjects were recorded after DBPCFC. †Overlapping results were obtained by using the prick–prick test technique with the fresh kiwifruit.

Gender: F/M24/10 17/4 
Age : mean33.17 33.57 
Age : range4–59 16–74 
 Abdominal pain38.80 
 Oral allergy syndrome2264.729.5
 No symptoms on exposure0 1780.9
 Kiwi extract (Phadia)2367.6838.1
 nAct d 1823.50 
 nAct d 110 733.3
 rBet v 125.921100
 rCor a 125.921100
 rDau c 10 29.5
Skin prick test
 Kiwi extract (in house)†2264.7840
 Kiwi extract (Stallergenes)1852.9526.3
 nAct d 11235.30 
 nAct d 2411.8315
 nAct d 5926.5315
 nAct d 1100733.3


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Act d 11 is a 17-kDa protein found in variable amounts in extracts of green kiwifruit. This is a ripening-related protein, the amount of which is influenced by natural ripening and postharvesting treatments, including the exposure to the plant hormone ethylene (7). Here, we describe the molecular and immunological characterization of the natural protein purified from ripe green kiwifruit, but a homolog was identified also in the gold species (data not shown).

As far as a major concern was the possible contamination by any known (Act d 8) and unknown PR-10 in our Act d 11 preparation, we set two different experiments to rule out such risk. Both the amino acid sequence analysis and the rBet v 1 inhibition curves showed that any PR-10 below the threshold of the two assays should not lead to any interference with our study results. Furthermore, as the Act d 8 was cloned from the kiwi buds, we are confident that no contamination could occur in our experimental conditions.

Act d 11 displays the highest sequence identity with members of the MLP/RRP family, which belongs to the Bet v 1 superfamily (12). A lower sequence identity is shared with members of the PR-10 protein family including Bet v 1.

Several antigenic regions of the molecular surface of Bet v 1 and of co-recognized allergens were described following studies based on the mapping of conserved residues (13–16), phage-displayed allergen mimotope technology (17, 18), and X-ray crystallography (14, 19). Most of the residues reported to belong to IgE binding or T-cell epitopes in Bet v 1 or in Bet v 1-related allergens (13, 14, 20–22) are conserved in Act d 11 and cluster mainly in the regions comprising the p-loop motif and the protein C-terminal domain, where a local sequence identity significantly higher than the overall one is observed.

The residue E45 was reported to be critically important in Bet v 1 for IgG and IgE binding (14, 23, 24). Like several MLP/RRPs, Act d 11 shares E45 with Bet v 1 and with most of the homologous allergens. Nevertheless, some of them display a substitution in this position. For instance, Dau c 1 and Api g 1 have a lysine (14) and the non-allergenic Bet v 1-like protein T1 has a glutamine in position 45 (24). The presence of K45 in Dau c 1 may contribute to its lower cross-reactivity with Bet v 1, that is even lower than that displayed by Act d 11, although the former belongs to the same protein family of Bet v 1 and the latter to MLP/RRPs.

The capacity of Act d 11 to inhibit, at least partially, IgE binding to Bet v 1 and to homologs, such as Cor a 1, Dau c 1, and Mal d 1, suggests epitope-sharing regions higher than that inferable from the low overall sequence identity. A value of 35% or more amino acid identity over a sliding window of 80 residues is the criterion used by the FAO/WHO/EFSA/Codex for the assessment of the allergenic risk of novel proteins. Therefore, on the basis of this criterion, Act d 11 was not expected to show co-recognition with the Bet v 1-like allergens, because of the sequence identity generally not higher than 21%. Nevertheless, the observation that in IgE-binding regions, the residues conserved in Act d 11 are generally more than 35%, and sometimes more than 50%, suggests conservation of epitope regions and may explain the detected co-recognitions. Additionally, these results suggest a revision of the criteria adopted by the FAO/WHO/EFSA/Codex to assess an allergenic risk because they take into consideration only the overall sequence identity and pay no attention to the epitope conservation.

Variability in such IgE co-recognition of Bet v 1-like molecules was quite broadly recorded among single patients analyzed by single-point highest inhibition achievable assay (SPHIAa), regardless whether they were from Austria, an area with a great exposure to birch pollen, or from Italy, where taxonomically related pollen induces sensitization in patients. In fact, the overall comparison of IgE prevalence for all the Bet v 1-like molecules considered in the present study shows that, regardless the dominating pollen exposure, Bet v 1 seems to act as the best IgE-detecting in vitro reagent. It seems to bear almost all the IgE epitopes at least for food allergens, unless IgE binding to Cor a 1 that is not fully inhibited in some patients. This phenomenon appears more pronounced for the Italian subset, but the partial inhibition of some Austrian samples suggests the possible role of other Bet v 1-related molecules as sensitizers in area where birch pollen is considered the primary sensitizer. Furthermore, the different epitope sharing among Act d 11 and Bet v 1-like molecules can be deduced by the different dot plot graph distribution of paired ISAC IgE results. Bet v 1-like molecules displaying the highest structural similarity to the birch pollen allergen as detected in a large population show greater correlation values, whereas Act d 11 behaves as Dau c 1.

Interestingly, a different prevalence distribution was recorded between the Austrian and the Italian tested samples when kiwi allergens were considered. This is obviously because of the different selection criteria for patient enrollment. Considering the Bet v 1-like-positive cohorts, differences in prevalence were recorded. Correction for the IgE value for Bet v 1 as recorded by the ISAC testing brought no statistically significant differences between the two populations for Bet v 1 but not for Cor a 1, further confirming the leading role of Bet v 1 as IgE detector, but with the need to carefully study epitopes displayed on other Fagales-related pollen whose allergic populations are exposed to.

Kiwi allergic individuals were enrolled on the basis of clear-cut-reported symptoms. This approach could have biased the selection toward the Act d 11 negative condition. Prick-to-prick testing was applied but did not change the results as reported from skin testing with the two extract preparations (commercial and in-house ones) as the patients were either positive for one or the other or to both of them. The lack of Act d 11 positivity in the kiwi allergic group prompted us toward the second group (Bet v 1-positive subjects) where we found the skin test positivity and the IgE reactivity, and, as expected, some minor clinical symptoms escaped at the first patient observation. We overall consider these findings important to define Act d 11 as a low-risk marker of kiwi allergy when this molecule is scored IgE positive.

In conclusion, we herein report the identification of a new kiwi allergen as a MLP/RRP, having a clinical relevance for causing sometimes symptoms in subjects sensitized to Bet v 1-related allergens. Act d 11 primary structure, its higher prevalence in the Bet v 1-like+ subsets, and its immunological relation to other Bet v 1-like molecules establish this protein as the first nonPR-10 allergen of the Bet v 1-like superfamily.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

We are grateful to Christian Harwanegg, Phadia Multiplexing Diagnostics,Vienna, Austria, for Act d 11 spotting on the ISAC microarray. We thank Chiara Rafaiani for the skillful help during the routine ISAC testing and the IgE inhibition assays and Giorgio Perotti and Silvia Monti for wheal areas measuring and patient data management.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Data S1. Repository.

ALL_2555_sm_DataS1.pdf402KSupporting info item

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