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Pomegranate cultivars: identification of the new IgE-binding protein pommaclein and analysis of anti-oxidant variability Lisa Tuppo, Claudia Alessandri, Maria Silvia Pasquariello, Milena Petriccione, Ivana Giangrieco, Maurizio Tamburrini, Adriano Mari, and Maria Antonietta Ciardiello J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00092 • Publication Date (Web): 14 Mar 2017 Downloaded from http://pubs.acs.org on March 22, 2017

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Journal of Agricultural and Food Chemistry

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Pomegranate cultivars: identification of the new IgE-binding protein pommaclein and analysis of anti-oxidant variability

Lisa Tuppoa, Claudia Alessandrib,c, Maria Silvia Pasquariellod, Milena Petriccioned, Ivana Giangriecoa, Maurizio Tamburrinia, Adriano Mari b,c, Maria Antonietta Ciardielloa

a

Institute of Biosciences and BioResources, CNR, I-80131 Naples, Italy.

b

Associated Centers for Molecular Allergology, Rome, Italy.

c

Center for Molecular Allergology, IDI-IRCCS, Rome, Italy.

d

CREA, Fruit Trees Research Unit, I-81100 Caserta, Italy.

Corresponding author: Maria Antonietta Ciardiello, Tel: +39 081 6132573 Fax: +39 0816132646 Email: [email protected]

ACS Paragon Plus Environment


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ABSTRACT 1

The consumption of pomegranate is increasing as it is considered a health-promoting food.

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Nevertheless, it can trigger allergic reactions, sometimes severe. The LTP Pun g 1 is the only

3

pomegranate allergen so far reported. Based on preliminary clinical observations, the main aim of

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this study was the investigation of still unknown allergens contained in this fruit. Pommaclein, a

5

homolog of peamaclein, the peach allergen Pru p 7, was isolated, identified by protein sequencing,

6

and characterized as an IgE-binding protein by different test systems. RP-HPLC protein profiles

7

revealed significant variations of LTP and pommaclein content in the red pulp of selected cultivars

8

and accessions. Conversely, the mesocarp appeared free of proteins and much richer in

9

antioxidants. In conclusion, a new allergen has been identified and it could contribute to

10

improving allergy diagnosis. The study highlights that pomegranate mesocarp could represent a

11

rich and safe source of nutraceuticals also for allergic subjects.

12 13

Keywords: pomegranate, pommaclein, Pun g 1, Pru p 3, Pun g 7, Pru p 7, peamaclein, anti-

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oxidants variability

15 16


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INTRODUCTION

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Pomegranate, Punica granatum L., is a temperate climate species, mainly cultivated in the

19

Mediterranean area, Southern Asia, and in several countries of North and South America. It

20

belongs to the monogeneric family Punicaceae, subclass Rosidae, believed to be native to the

21

region between Iran and northern India.1 It is one of the oldest cultivated species among fruit

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trees. The name pomegranate comes from the Latin “pomum” meaning “apple” and “granatus”

23

meaning “full of seeds”. The edible parts of pomegranate are the arils which are seeds covered by

24

a red pulp, that is a juice sac, called the sarcotesta. The arils are surrounded by the mesocarp, or

25

albedo, a white, fleshy substance separating the arils from the fruit peel.

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During the last few years there has been an increasing interest in the consumption of

27

pomegranate because it is considered a functional food with health-promoting properties

28

effective in risk reduction of diseases such as cancer and coronary and vascular diseases.2-7 The

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arils can be consumed fresh or in the preparation of juices, jellies, jams, and colorings for drinks.

30

They contain considerable amounts of organic acids (including citric, malic and ascorbic), sugars,

31

phenols and important minerals.8 The red pulp is an important source of phenols and tannins,

32

such as punicalin, punicalagin, ellagic acid and a large amount of anthocyanins, such as cyanidin,

33

delphidin and pelargonidin; the corresponding glycosides have been found in genotypes with red

34

arils.9-11 The outer skin and mesocarp are also rich in bioactive components.12

35

In opposition to the health-promoting effects, allergic reactions to this fruit can occur with

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symptoms ranging from mild to severe, including angioedema, urticaria, abdominal pain and

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anaphylactic shock. An allergic reaction to pomegranate was described for the first time in 1991.13

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The first pomegranate allergen was isolated and identified as LTP (Pun g 1) in 2007.14 Zoccatelli et

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al. reported that at least two LTP isoforms had been observed in this fruit displaying different

40

immunological behaviors. Additional LTP isoforms (four) were later detected by 2DACS Paragon Plus Environment


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immunoblotting.15 Food-dependent exercise-induced anaphylaxis (FDEIA) triggered by the

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ingestion of pomegranate can also be found in literature.16 However,attempts to associate FDEIA

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with LTP failed and the reaction was attributed to an unidentified IgE-binding protein with a

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molecular mass higher than that of Pun g 1. In summary, LTP is the only allergen so far identified in

45

the pomegranate.

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Unreported preliminary clinical and immunological data obtained by Centri Associati di

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Allergologia Molecolare allergists (CAAM) suggested the presence of allergens different from LTP

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in the pomegranate. In particular, CAAM allergists noted that one patient allergic to peach was

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IgE-negative to Pru p 3, positive to peamaclein, Pru p 717 and reported an adverse reaction to

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pomegranate. These observations suggested the possible presence of a Pru p 7 homolog in this

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fruit.

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The aim of this study was the investigation of the variability of the amount of IgE-binding

53

proteins, of the physico-chemical properties of the fruit and of some nutraceutical components in

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different cultivars and accessions of this fruit. Taking into consideration the increasing interest in

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the re-evaluation of typical products and ancient flavors, three traditional local cultivars and two

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new accessions have been selected and used for comparative purposes. In addition, we describe

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for the first time the identification and isolation of a protein homologous to Pru p 7 and its

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recognition by specific IgE contained in the sera of patients allergic to pomegranate.

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MATERIALS AND METHODS

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Collection and pomological characterization of fruit samples

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Six different pomegranate (Punica granatum) types were selected for this study: two Italian

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cultivars, Dente di Cavallo and Zanna Bianca; three accessions, CREA-FRC4, CREA-PR1 and CREA-

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PR2; and a commercial Israeli fruit sample that was purchased at a local market and used for some ACS Paragon Plus Environment

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comparative experiments. The samples were collected in 2012, except for a further sample of

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CREA-PR1 collected in 2013. The fruits were grown in the same experimental orchard in Caserta

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(southern Italy), at the CREA Fruit Tree Research Unit, except for pomegranates imported from

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Israel. The samples coming from the experimental local orchard were harvested in October at the

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commercial ripening stage, and screened for uniformity, appearance and the absence of physical

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defects or decay. Ten fruits from each cultivar/accession were used to determine the pomological

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and qualitative traits according to the International Union for the Protection of New Varieties of

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Plants (UPOV 2012). The weight of each fruit was determined on a precision digital balance with

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an accuracy of 0.001 g. The length and width of each fruit, the number of arils per fruit and the

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average size of arils were also determined. Each pomegranate was manually separated into

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mesocarp, pulp (or sarcotesta) and seeds. The mesocarp and pulp samples were separately

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analyzed, whereas seeds were discarded. The chemical harvest indices were estimated as follows:

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(i) the total soluble solid content (TSS, °Brix) was determined in the pulp using a digital

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refractometer (Sinergica Soluzioni, DBR35, Pescara, Italy) and (ii) the total acid content (TA) was

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determined by titrating 10 mL of pulp with 0.1 N NaOH.18 The results are expressed as grams of

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malic acid per liter of pulp. All analyses were performed in triplicate. All reagents, solvents and

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standards were of analytical reagent grade.

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Preparation of protein extracts from pomegranate mesocarp and aril pulp

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The fruit mesocarp and the red pulp were separately homogenized in a blender after the addition

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of 1 M NaCl (1 : 1 w/v or v/w) and stirred at 4°C for 2 h. Then the samples were centrifuged at

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17,300 x g for 45 min and each supernatant, representing the protein-containing extract, was

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collected. The protein concentration of the extracts was determined by the Bradford method BIO-



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RAD Protein Assay (Biorad, Milan, Italy), using a calibration curve made with bovine serum

89

albumin.

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Analysis by RP-HPLC

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The red pulp protein extracts and the proteins isolated from it were analyzed by RP-HPLC on a

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Vydac (Deerfield, IL, USA) C4 column (4.6 x 250 mm), using a Beckman System Gold apparatus

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(Fullerton, CA, USA). The elution was carried out by a multistep linear gradient of eluent B (0.08%

95

TFA in acetonitrile) in eluent A (0.1% TFA) at a flow rate of 1 mL/min. The eluate was monitored at

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220 and 280 nm. The separated fractions were manually collected and analyzed.

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Amino Acid Sequencing

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The direct protein sequencing of the N-terminal region of the purified proteins was obtained using

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a Procise 492 automatic sequencer (Applied Biosystems, Foster City, CA, USA).

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Purification of pommaclein and Pun g 1 from pomegranate pulp

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Two proteins were purified from the pulp of the cultivar PR1, a 9k-LTP (Pun g 1) and pommaclein.

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They were isolated from a protein extract prepared as previously described. During the

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purification procedure, each protein was monitored by RP-HPLC or SDS-PAGE.

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The extract was dialyzed against 10 mM Tris-HCl, pH 7.2, and then loaded on a DE52

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(Whatman, Brentford, UK) column, equilibrated in the same buffer. Both proteins were eluted in

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the DE52 column flow-through that was loaded on a SP-Sepharose column (Amersham

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Biosciences, Uppsala, Sweden), equilibrated in 10 mM sodium acetate, pH 5.0 (buffer A). The

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elution was carried out by a linear gradient from 0% to 100% of buffer B (50 mM sodium acetate,

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pH 5.0, containing 0.5 M NaCl). The eluted fractions were analyzed by RP-HPLC and then pooled. ACS Paragon Plus Environment

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The fractions containing Pun g 1 were desalted by repeated dilutions in water followed by

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ultrafiltration on Amicon Ultra filters (3,000 MWCO, Millipore, Carrigtwohill, Ireland). Further

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purification was achieved by chromatographic separation on a Mono-S HR 10/10 column

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connected to the FPLC system (Amersham-Pharmacia, Uppsala, Sweden). The elution was carried

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out with a linear gradient from 0% to 100% of buffer B. The fractions containing Pun g 1 were

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desalted on a pre-packed PD-10 gel filtration column (GE Healthcare, Little Chalfont,

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Buckinghamshire, UK) equilibrated with 0.1% trifluoroacetic acid (TFA). Then, the sample was

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concentrated by ultrafiltration and subjected to several washes with water to remove traces of

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TFA. The amount of the purified Pun g 1 was estimated on the basis of the molar extinction

121

coefficient at 280 nm (4970 M-1 cm-1), calculated on the basis of the amino acid sequence of the

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clone having the UniProt accession number A0A059STC4.

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The pommaclein preparation obtained from the above described chromatography on the

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SP-Sepharose column contained the protein purified to homogeneity. The sample was dialyzed

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against 2 mM NaCl and concentrated with a rotary evaporator. The protein concentration was

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estimated on the basis of the molar extinction coefficient, at 280 nm (3730 M-1 cm-1), of the

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homologue having the highest sequence identity, that is the one from cotton (UniProt accession

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number M1GN43).

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The purity of each protein preparation was assessed by SDS-PAGE, RP-HPLC and N-terminal amino acid sequencing as previously reported.19

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Analysis by SDS-PAGE

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The pomegranate extracts and purified proteins were analyzed by reducing 15% SDS-PAGE on a

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Bio-Rad Mini Protean apparatus (Biorad, Segrate, Italy). The staining was carried out in 0.05%



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Coomassie R-250 brilliant blue in 40% methanol/10% acetic acid; the rinsing was performed in

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40% methanol/10% acetic acid.

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Specific IgE detection by dot blotting, immunoblotting and FABER test

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To assess the IgE binding of the native and denatured forms, each purified protein was analyzed by

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dot blotting (DB) and immunoblotting (IB), respectively. The experiments were carried out

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following the already reported procedures.20,21 A mouse monoclonal anti-human-IgE conjugated to

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alkaline phosphatase was used (Becton Dickinson Biosciences, San Jose, CA, USA) as a secondary

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antibody. A preliminary evaluation of the allergological impact of new pomegranate allergens in an

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allergic population has been performed by using a novel nanotech-based IgE detection method

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called FABER (manuscript in preparation).22

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Solutions for the skin prick test (SPT)

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Skin tests were carried out with pomegranate pulp extract from PR1 cv, and the peach

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homologous proteins, Pru p 3 and Pru p 7, were used as a reference. Pru p 3 and Pru p 7 were

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purified from the natural source as already reported.17,23 All the proteins were dialyzed against 2

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mM NaCl, concentrated with a centrifugal vacuum concentrator, mixed with sterile glycerin in a

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1:1 ratio and sterilized by membrane filtration through a 0.22-µm filter (Millex; Millipore, Bedford,

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MA, USA), in a sterile horizontal laminar flow hood. The final protein concentration was 0.25

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mg/mL.

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Patients


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The study was approved by the Institutional Review Board of IDI-IRCCS, Rome, Italy (28/CE/2008).

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The patients or caregivers signed an informed consent when the patients were undergoing tests

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not in the routine work up.

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Nineteen sera were extracted from InterAll, the allergy electronic record used by CAAM

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(version 5.0; Allergy Data Laboratories s.c., Latina, Italy) by choosing patients with a reliable clinical

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history of allergic reactions to pomegranate fruit and/or sensitization to Pru p 3 and Pru p 7. The

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sera were stored at -20° C until use. Five out of nineteen patients were tested by skin prick test

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(SPT) with the in-house produced solutions of pomegranate pulp extract, Pru p 3 and Pru p 7. Each

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SPT was performed and recorded as weal areas using a standard methodology as already

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reported.24

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The presence of Pru p 3 specific IgEs were determined in all nineteen sera by means of ISAC

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103 microarray test (Phadia Multiplexing Diagnostics, PMD, Vienna, Austria) and ISAC 112

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(ImmunoCAP ISAC Thermo Fisher Scientific, Phadia AB, Uppsala, Sweden).

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Protein sequence analysis

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Bioinformatics analysis of the protein sequences was performed using the software tools available

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on the ExPASy Bioinformatics Resource Portal (www.expasy.org).The molar extinction coefficient

174

of the purified proteins was calculated using the ProtParam algorithm. The multiple alignments of

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Pun g 1 and pommaclein with the homologues protein sequences were performed using

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AllergomeAligner on the Allergome Platform (www.allergome.org) and ClustalW on the server

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ExPASy, respectively.

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Ascorbic acid content



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The ascorbic acid (AA) content from the pulp and the mesocarp of the pomegranate fruits was

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determined following the method,25 with some modifications. Five g of pulp were homogenized

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with 20 mL 16% (v/v) metaphosphoric acid solution containing 0.18% (w/v) disodium ethylene

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diamine tetraacetic acid. The homogenate was centrifuged at 14,400xg for 10 min. The assay

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mixture contained 400 μL of supernatant 0.3% (v/v) metaphosphoric acid and (5:1, v/v) diluted

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Folin’s reagent, in a final volume of 2 mL. After 10 min, the absorbance of the sample was

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recorded at 760 nm with a UV–vis spectrophotometer (Model V-630, Jasco, Japan). The AA

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concentration was calculated against a 100% (w/v) AA standard curve and was expressed as

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milligrams of AA per 100 g of fresh weight (FW) of fruit.

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Total antioxidant activity

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The antioxidant activity in the fruit pulp and mesocarp was measured with 1,1-diphenyl-2-picryl-

192

hydrazil (DPPH) according to the method of,26 with some modifications. The fruit extracts (75 μL)

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were allowed to react with 1,425 μL of the DPPH solution, monitoring the decrease in absorbance

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for 15 min at 515 nm. The results were expressed in millimolars of the Trolox equivalent (TE) per

195

gram of fresh weight (FW).

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Total Phenol, Monomeric Anthocyanin and Flavonoid Content

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The following determinations were carried out in both the fruit pulp and mesocarp of the analyzed

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pomegranate cultivars/accessions.

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The total phenol content was determined by the Folin-Ciocalteu method.27 A phenolic

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extraction was performed as described by Tomás Barberán & Espín (2001)28 and the total phenol

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content was expressed as milligrams of gallic acid equivalents (GAE) per 100 grams of fresh weight

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(FW). ACS Paragon Plus Environment

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The total monomeric anthocyanin content, estimated only in the pulp, was determined by

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a pH-differential method29 and expressed as cyanidin-3-glucoside equivalent (CGE) per 100 grams

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of fresh weight (FW) (molar extinction coefficient of 26,900 M−1 cm−1). The absorbance

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measurements were conducted at 520 and 700 nm.

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The total flavonoid content was determined by the aluminium chloride colorimetric

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method30 using catechin as a standard. The total flavonoid content was expressed as milligrams of

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catechin equivalent (CE) per 100 grams of fresh weight (FW).

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Statistical analysis

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The data concerning the agro-pomological traits and the level of bioactive compounds were

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analyzed by SPSS software package, version 16.0 (SPSS Inc., Chicago, IL, USA) and expressed as the

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means ± standard deviation. Analyses of Variance (ANOVA) and differences among means were

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determined for significance at p < 0.05 using LSD test. Differences statistically significant are

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indicated with different letters.

218 219

RESULTS

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Pomological features of the fruits

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The pomological traits of the cultivars and accessions Dente di Cavallo, Zanna Bianca, CREA-FRC4,

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CREA-PR1 and CREA-PR2, collected in 2012, are shown in Table S1. The fruit weight varied greatly

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among the cultivars, from a minimum of 330.7 ±13 g (Dente di Cavallo) to a maximum of 535.7±20

225

g (CREA-PR2), although some statistically significant similarities between CREA-FRC4, Zanna Bianca

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and CREA-PR1 were observed. No differences were observed between the cultivars/accessions in



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the aril width values, while statistical differences were highlighted in their length and the number

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of arils per fruit (Table S1).

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Titratable acidity (TA) and total soluble solid (TSS) content are important components of

230

fruit organoleptic quality and their ratio (TSS/TA), also called the Maturity Index, is responsible for

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the taste and flavour of the pomegranate.31 As reported in previous studies, each cultivar with its

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physico-chemical traits, such as total soluble solid content, titratable acidity, pH and TSS/TA ratio,

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may influence the pomegranate fruit pulp quality.32 Significant differences in the total organic acid

234

(TA) content were observed in the analysed pomegranate samples (Table S1). The lowest TA value,

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expressed as the citric acid content, was found in the CREA-PR2 pulp (6.3±0.2 g/L), whereas the

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highest value was measured in the CREA-FRC4 pulp (9.8±0.2 g/L).

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TSS estimates the level of dissolved sugars deriving from the hydrolysis of starch during

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ripening, but also the presence of other soluble compounds, such as acids, salts, water-soluble

239

vitamins and other chemical compounds.33 Their content was lower in the pulp of CREA-PR1,

240

CREA-PR2 and Zanna Bianca than in that of CREA-FRC4 and Dente di Cavallo.

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Ascorbic acid (AA), bioactive compounds and antioxidant activity

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A significant variation of AA, bioactive compounds and antioxidant activity was observed in the

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samples of the different cultivars/accessions (Table 1). Higher values were measured in the

245

mesocarp compared to the pulp. Statistically significant differences were observed between the

246

same tissues of the different cultivars and accessions. For instance, the fruit pulp of the accession

247

CREA-PR1 showed a higher AA, flavonoid and phenol content, as well as antioxidant activity,

248

compared to the other cultivars. The same trend was observed in the mesocarp samples (Table 1).

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The antioxidant activity measured in the mesocarp was higher than that of the pulp and this

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difference seems to be due to the presence of tannins, abundant in this pomegranate tissue. ACS Paragon Plus Environment

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Anthocyanins are components of the phenol chemical family that contribute to the red,

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blue or purple color of many fruits, including pomegranate pulp, and they are well-known for their

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antioxidant activity. A high anthocyanin concentration in highly red colored fruit34 and the deep

254

color formation are among the parameters used in assessing pomegranate fruit aril quality.

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Statistically significant differences in the total content of anthocyanins were observed in the

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pomegranate fruits, with the highest values in Dente di Cavallo and CREA-PR1 (6.43±0.21 and

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7.55±0.13 mg/100 g, respectively).

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Protein profiles of pomegranate extracts

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The protein extracts were prepared from the mesocarp and pulp of six pomegranate samples

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collected in 2012: Dente di Cavallo, Zanna Bianca, CREA-FRC4, CREA-PR1 CREA-PR2, and the Israeli

262

sample. An additional extract was obtained from CREA-PR1 collected in 2013. The protein

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concentration measurement revealed that the mesocarp is devoid of detactable amounts of

264

proteins. A variable amount of proteins, expressed as mg/100mL of pulp, was measured in the

265

pulp of the different cultivars: 3.2 in Dente di Cavallo, 4.2 in CREA-FRC4 5.0 in Zanna Bianca, 5.3 in

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CREA-PR2, 3.6 in CREA-PR1 collected in 2012 and 7.0 in CREA-PR1 collected in 2013. The amount

267

of proteins measured in the pulp of a commercial pomegranate type, imported from Israel, was

268

3.0 mg/100mL.

269

The analysis by SDS-PAGE showed a different protein profile of the pulp extracts with

270

variations in the number and relative abundance of the components (Figure 1, panel (A). A

271

significant variation of the protein component was observed also in the pulp of the same cultivar

272

(CREA-PR1) harvested in two consecutive years (Figure 2 panels F and G).

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In line with the SDS-PAGE results, the separation of the protein components by RP-HPLC

274

chromatography highlighted a different protein profile for each cultivar (Figure 2). The pulp of ACS Paragon Plus Environment


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CREA-PR1 harvested in 2013 had a high amount of two components eluted at times similar to

276

those reported for peamaclein (23 min) and peach 9k-LTP (28 min).20 These two components,

277

indicated by a grey and a black arrow, in the Figure 2 G, were manually collected and analyzed by

278

N-terminal amino acid sequencing

279 280

Pommaclein identification in pomegranate red pulp and homology search

281

The sequencing of the first 20 residues of the protein component contained in the peak eluted at

282

23 min (Figure 2 G) provided two sequences. The most abundant component had the following N-

283

terminus: GSSFCDSKCAVRCSKAGVQD. The minor component (the amount of which was about 10%

284

compared to the major one) had the sequence KDECPCYRDMKNSKGXPKCX. A homology search in

285

data banks allowed the identification of the protein as a homolog of peamaclein.17 In fact, the

286

above two sequences could be nicely aligned with the N-terminal and C-terminal region,

287

respectively, of peamaclein. It is conceivable that the C-terminal domain fragment could have

288

been generated by the action of an endogenous protease and remained bound to the protein by

289

the disulfide bridge connections.

290

In line with the name of the homolog from the peach, we named this new component from

291

pomegranate “pommaclein” and its partial sequence was registered in the UniprotKB under the

292

accession number C0HKC0.

293

The sequence identity between the available (38 residues out of the theoretical 63)

294

pommaclein sequence and peamaclein is 89% (Figure 3). A multiple sequence alignment showed

295

high identities when pommaclein was compared with some homologs found in the database, with

296

values ranging from 84 to 95%. The highest value (95%) was observed with the homolog from

297

cotton, whereas the identity with the homologs from other sources decreases in the following

298

order: coffee (92%) > soybean = peach (Pru p 7) = tomato (89%) > potato (87%) > grape (84%). ACS Paragon Plus Environment

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Detection of Pun g 1.0101 in pomegranate pulp and comparative analysis of the sequence

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The two very close peaks eluted at about 30 min by RP-HPLC, and indicated by a black arrow in

302

Figure 2 G, were separately collected and analyzed. The amino acid sequencing of ten N-terminal

303

residues of both components produced the same sequence: AVTCGQVASS, thus suggesting they

304

were isoforms of the same molecule. A homology search in the Allergome database allowed the

305

identification of this protein as pomegranate 9k-LTP, allergen Pun g 1, having the Allergome code

306

2834.

307

In line with the literature describing different LTP isoforms in pomegranate,15 in the

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Allergome database three isoforms are reported, Pun g 1.0101, Pun g 1.0201 and Pun g 1.0301.

309

The two protein components indicated by a black arrow (Figure 2 G) provided the N-terminal

310

sequence of the isoform Pun g 1.0101, having the Uniprot accession number A0A059STC4. The

311

elution in two peaks by RP-HPLC can reasonably be assumed to be due to sequence substitutions

312

in the protein regions that were not sequenced.

313

A multiple alignment of the Pun g 1.0101 sequence with the most similar LTPs found in the

314

Allergome database is shown in Figure 4. It highlights a significant identity with many homologous

315

food proteins and a lower identity with the pollen LTP Par j 2. The highest value (68%) is observed

316

with the apple LTP (Mal d 3), whereas the identity with the other homologs decreases in the

317

following order Pru du 3 (67%) > Pru p 3 (66%) > Mor n 3 (65%) > Cor a 8 (51% ) > Act d 10 (46%) >

318

Par j 2 (34%).

319 320

Relative abundance of identified proteins in different pomegranate cultivars

321

The two identified proteins are differently expressed in the analyzed cultivars/accessions of the

322

pomegranate (Figure 2). A variation in the relative amounts of the protein components is also ACS Paragon Plus Environment


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323

observed in the fruit of the same cultivar harvested in two different consecutive years (Figure 2 F,

324

G). In fact, pomegranate cv CREA-PR1 collected in 2012 shows a content of the analyzed proteins

325

lower than that of the same fruit harvested in 2013. Pommaclein (grey arrow) was detected in

326

both samples from the CREA-PR1 cv, but it seems not to be present in the other fruits. The

327

amount of Pun g 1 is highly variable in the different cultivars; the highest concentration is

328

observed in the Israeli and in CREA-PR1 (2013) fruits.

329 330

Preparative preparation of purified proteins

331

The proteins identified in pomegranate pulp were purified in amounts sufficient for their

332

characterization using the chromatographic separations described in the Materials and Methods

333

section. The pulp of CREA-PR1 harvested in 2013 was used as the starting material because it was

334

the richest source of those proteins. Amounts corresponding to 0.46 mg of pommaclein and 0.6

335

mg of Pun g 1 were purified to homogeneity from 100 mL of pulp. The purity of the protein

336

preparations was assessed by SDS-PAGE (Figure 1B), RP-HPLC and N-terminal amino acid

337

sequencing.

338 339

Analysis of sensitivity to pomegranate in patients allergic to peach

340

This is a retrospective study performed by selecting electronic records of patients complaining of

341

symptoms after pomegranate ingestion and/or patients positive to Pru p 3 and/or Pru p 7.

342

Pomegranate ingestion was reported in the clinical history of 10 patients, nine of them reacting to

343

it. The main results are in Table 2. Patient 6 complained about oral allergic symptoms after

344

pomegranate ingestion, whilst his ISAC microarray test did not reveal any IgE sensitization towards

345

plant-derived food allergenic proteins known at the time of the test.

346 ACS Paragon Plus Environment

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347

IgE detection by dot blotting, immunoblotting, SPT and FABER testing

348

To investigate the IgE-binding of purified proteins under native and denatured conditions, IgE dot

349

blotting (DB) and immunoblotting (IB) experiments were performed with all 19 sera (Table 2 and

350

Figure S1). Ten patients (53%) displayed IgE binding to pommaclein (Pun g 7) on DB and/or IB,

351

although five of them showed only a weak signal on DB and were negative on IB. Among the nine

352

patients tested by SPT, four of them (patients 8, 9, 12 and 19) resulted positive to Pru p 7: patient

353

8 was positive to Pru p 7 and to pommaclein on DB but he was not sensitized to Pru p 3 (SPT/ISAC)

354

and Pun g 1 (IB/DB); patients 12 and 19 were IgE positive to Pru p 7 (SPT), to pommaclein (on DB

355

and IB) and to Pru p3 (ISAC and SPT); while patient 9 was positive to Pru p 7 (SPT), negative to Pru

356

p 3 (SPT) but positive to Pru p 3 (ISAC) and to Pun g 1 (DB and IB) (Table 2). Only five patients were

357

tested with the pomegranate extract (Pun g) and, among these, one female patient (number 10)

358

was negative to all studied proteins. Fourteen patients displayed an IgE binding to Pun g 1, the

359

pomegranate LTP, by DB and/or IB, seven of them showing a weak signal. The three patients

360

negative to Pru p 3 on ISAC (numbers 6, 8 and 10) were also negative to Pun g 1, whereas two sera

361

positive to Pru p 3 (numbers 13 and 16) were negative to Pun g 1.

362 363

In addition, when IgE were detected in parallel for Pru p 7 and pommaclein by FABER nanotech system22 16 sera out of 1751 were found positive for the former and 6 for the latter.

364 365 366

DISCUSSION

367

The analysis of the protein profile of pomegranate samples led to the identification of a new IgE-

368

binding protein present in this fruit, named pommaclein. It is a homolog of the peach peamaclein,

369

namely allergen Pru p 7.17,20,35 The determined amino acid sequence of pommaclein (about 60%)



Page 18 of 37

18

370

shows a high identity with the peach homolog, although higher values were estimated by means

371

of a comparison with homologous sequences from other sources, such as cotton and coffee.

372

The immunological characterization highlights that, among the patients claiming

373

pomegranate induced symptoms, two of them were monosensitized to pommaclein, whereas four

374

were positive to both pomegranate and peach homologous proteins. A patient (number 19)

375

suffered from peach allergy without any reaction to pomegranate, not customarily consumed.

376

Indeed, it is well known that a positive test result (sensitization) is likely to correspond to a clinical

377

reaction, but this cannot be considered valid in all cases.36,37 Therefore, although the available

378

data are limited, they suggest that the immunological properties of pommaclein are not

379

completely shared with those of Pru p 7. Similar observations can be made for Pun g 1, showing an

380

IgE binding with 17 out of 19 sera positive to Pru p 3, highlighting immunological differences

381

between the two homologous proteins.

382

It is worth noting that the results observed for Pung 1 and pommaclein in DB were not

383

always correlated with those in IB obtained with the same sera and secondary antibody. These

384

discrepancies can be interpreted in line with literature reports describing the influence of

385

experimental conditions on the IgE binding properties of allergens.21,38 It is conceivable that

386

different profiles of conformational and linear sequence epitopes are available for IgE binding in

387

DB and IB. As a matter of fact, the native form of proteins is immobilized and probed with sera in

388

DB experiments, whereas denatured molecules are targeted in the IB procedure. The use of both

389

procedures increases the epitopes available for specific IgE detection. Taking into account the

390

herein reported positive though preliminary IgE results obtained by using the new nanotech-based

391

IgE detection system named FABER,22 a combination of natural and denaturated preparations can

392

be envisage to increase the quality of the diagnostic tests.


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393

A protein sequence similarity search showed very high identities between pommaclein and

394

homologs from many plant sources, such as cotton, coffee, soya bean, tomato, potato, grapes and

395

others, highlighting the wide dissemination of this protein in plants and the high evolutionary

396

conservation of its primary structure. This feature is generally associated with proteins having

397

important roles in the physiology of the living organisms. Pommaclein belongs to the family of the

398

gibberellin-regulated proteins, known also as snakin/GASA proteins, playing key roles in the plant

399

hormone response, defence, development and stress tolerance.39,40 Its concentration in plant

400

tissues depends on inner and environmental factors, including gibberellin administration that

401

could cause the increase of allergenic protein concentrations, such as Pru p 7 and its homologs, in

402

plant tissues.41,42 This treatment is widespread in agricultural practice to control and drive plant

403

development, including growth, fruit size and ripening.43 Recently a new homolog of Pru p 7 from

404

Prunus mume, Pru m 7, has been registered in the IUIS-WHO allergen nomenclature database

405

(www.allergen.org). The sequence available shows 100% identity with the peach Pru p 7.

406

The fruits analysed in this study were cultivated in the same conditions and were not

407

treated with gibberellin. Therefore, the different results obtained suggest that the cultivar can be

408

an additional factor affecting the concentration of this protein in pomegranate. Detectable

409

amounts of pommaclein were identified in the ripe fruit of the cultivar CREA-PR1 only. Similar to

410

pommaclein, the LTP amount detected in different pomegranate samples is not constant. A strong

411

variation was observed as a function of the analysed traditional Italian cultivar and the sample of

412

the Israeli fruit. LTP was detected in significant amounts in CREA-FRC4, Zanna Bianca and CREA-

413

PR1 pomegranates collected in 2013 and in the fruit imported from Israel, whereas it was hardly

414

detected in CREA-PR2, Dente di Cavallo and CREA-PR1 collected in 2012. A high variability of the

415

LTP concentration in different cultivars was reported as well for the apple.44 Although a range of

416

variation of LTP amounts in peach peel is reported,45 its concentration is always high suggesting ACS Paragon Plus Environment


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417

that this protein could be constitutively expressed in high amounts in peach peel. This constant

418

high amount of LTP, independently of the cultivar and other conditions, could be a factor

419

providing Pru p 3 as a marker of LTP sensitization. Something similar is observed in other allergenic

420

sources, for instance Act d 1 and Act d 5 are constitutively expressed in high amounts in kiwifruit

421

and they also represent two markers of sensitization to this plant food.46 Therefore, regarding

422

pomegranate, some LTP allergic patients could tolerate the fruits of cultivars with a low LTP

423

content. This implies that the plant foods available on the market should be labeled with the name

424

of the cultivar and possibly other useful details concerning cultivation features and any chemical

425

or physical treatment. In the near future allergists could provide suitable advice based on this

426

concept. On the other hand, allergic patients should pay more attention to the features of

427

consumed foods with the advantage of avoiding unnecessary deprivations.

428

The analysis of the physico-chemical composition shows high levels of antioxidants in all

429

the analyzed pomegranate samples, although some variations can be observed. Antioxidant

430

activity is influenced principally by phenols, that are secondary metabolites whose content varies

431

in response to several external factors, including environmental ones. Since the three

432

pomegranate cultivars and the two accessions analyzed in this study were cultivated under the

433

same environmental and climatic conditions, the observed differences in nutraceutical and protein

434

components can reasonably be related to the specific adaptive response of each genotype. The

435

availability of data obtained on anti-oxidant and protein concentrations in different cultivars lead

436

us to evaluate the co-variability of the two but no relation was found (data not shown). The major

437

difference is observed when the red pulp is compared to the mesocarp. In line with literature,47

438

the amount of phenols and other antioxidants is much higher in the mesocarp than in the pulp. In

439

contrast, protein components have been detected in the pulp only, whereas the mesocarp seems

440

devoid of proteins, and therefore it should probably be free of allergens. This feature is common ACS Paragon Plus Environment

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441

to all the analysed pomegranate samples. The mesocarp could represent a suitable and safe

442

source of high amounts of antioxidants also for allergic subjects.

443

In conclusion, in the present study we focused on two of the pomegranate tissues,

444

excluding aril seeds, but as it cannot be excluded their ingestion by consumers, it will be worth

445

focusing also on their allergen content in the future. A new IgE-binding protein, pommaclein, has

446

been identified in the pomegranate pulp. Following the IUIS-WHO nomenclature the Pun g 7

447

allergen name is proposed. While all pomegranate samples appear rich in nutraceutical

448

components, the amount of the proteins, including the allergenic ones, displays a great variation

449

in the different pomegranate batches and cultivars. The variability of the allergenic profile within

450

the same plant species is not peculiar to the pomegranate and it is reported for other fruits.44 This

451

is a general and important issue concerning food quality and safety that should receive from the

452

scientific community and the operators in the agricultural sector all the attention it deserves.

453 454

ABBREVIATIONS USED

455

AA, ascorbic acid; CGE, cyanidin-3-glucoside equivalent; CE, catechin equivalent; DB, IgE dot

456

blotting; FW, fresh weight; GAE, gallic acid equivalents; IB, IgE immunoblotting; LTP, lipid transfer

457

protein; SPT, skin prick test; TA, total acid content; TE, trolox equivalent; TSS, total soluble solid

458

content

459 460

Supporting Information: Table S1 and Figure S1

461 462



Page 22 of 37

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463 464 465

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FUNDING

609

The authors MSP and MP received funding from the Progetto MiPAAF "Trattato internazionale sulle

610

risorse fitogenetiche per l'alimentazione e l'agricoltura RGV/FAO" DM 29561 del 18/12/2014 e DM

611

24903 del 24/11/2015. The authors LT, IG, MT, MAC received funding from the International Fund

612

for Advanced Research in Allergy and Immunology (IFARAI) Onlus (Italy).

613


Page 29 of 37


29

614

FIGURE CAPTIONS

615 616

Figure 1. Panel (A): SDS–PAGE of pomegranate pulp extracts. M, molecular mass markers; A,

617

Israeli; B, CREA-FRC4; C, CREA-PR2; D, Dente di Cavallo; E, Zanna Bianca; F, CREA-PR1 (collected in

618

2012); G, CREA-PR1 (collected in 2013). 20 µg of each extract were loaded. Panel (B): SDS–PAGE of

619

the proteins purified from the pulp extract of the cultivar CREA-PR1. M, molecular mass markers;

620

A, Pun g 1 (5 µg); B, pommaclein (5 µg).

621 622

Figure 2. RP-HPLC analysis of the pulp extracts obtained from the Israeli pomegranate (A) and

623

from the Italian samples CREA-FRC4 (B), CREA-PR2 (C), Dente di Cavallo (D), Zanna Bianca (E),

624

CREA-PR1 collected in 2012 (F) and CREA-PR1 collected in 2013 (G). Pun g 1 and pommaclein peaks

625

have been indicated by black and grey arrows, respectively, in the RP-HPLC profile of CREA-PR1

626

collected in 2013.

627 628

Figure 3. Alignment of the available amino acid sequence of pommaclein with selected

629

homologous proteins.

630

The two sequence fragments have been aligned with the homologs from peach, cotton, coffee,

631

soybean, tomato, potato and grapes, whose UniProt accession numbers are P86888, M1GN43,

632

A0A068TY72, C6SY10, K4DF43, M1B5D4 and E0CP56, respectively. The residues conserved in all

633

the aligned homologs are highlighed in grey. The residues of pommaclein conserved only in some

634

homologs are black shadowed. Dots indicate the residues of pommaclein still unknown.

635 636

Figure 4. Alignment of the amino acid sequence of pomegranate LTP, isoform Pun g 1.0101, with

637

selected homologous allergens. ACS Paragon Plus Environment


Page 30 of 37

30

638

The Uniprot accession numbers and sources of the aligned LTP are the following: A0A059STC4,

639

pomegranate (Pun g 1.0101); Q2V6D8, apple (Mal d 3); Q43017, almond (Pru du 3); P81402, peach

640

(Pru p 3); P85894, black mulberry (Mor n 3); Q9ATH2, hazelnut (Cor a 8); P86137, kiwifruit (Act d

641

10); O04403, Parietaria (Par j 2). The residues conserved in all the aligned LTP are highlighted in

642

grey. The residues of Pun g 1 conserved only in some homologs are highlighted in black.


Page 31 of 37


31

Table 1. Total Content of Phenols, Flavonoids, Antioxidant Activity, Ascorbic Acid (AA) and Anthocyanins in the Fruit Mesocarp and pulp at Harvesting Time. Means followed by the same letter do not differ significantly at P=0.05 (LSD test).

Cultivar

Phenols (mg GAE /100g FW)

Flavonoids (mg CE /100gFW)

Antioxidant Activity (mg TE /100gFW)

Ascorbic Acid (mg AA /100gFW)

Anthocyanins (mg CGE /100gFW)

MESOCARP Dente di Cavallo

(1280.2 ±24)e

(119.1±7.2)c

(243.2±10.6)c

(44.8±0.7)e

-

Zanna Bianca

(614±12.6)a

(99.7±4.3)a

(264.0±8.0)d

(34.2±0.6)a

-

CREA-FRC4

(982.3±7)d

(116.3±2.9)bc

(205.9±11.8)b

(42±0.6)c

-

CREA-PR1

(679.3±15.7)b

(112±6.7)bc

(270.9±5.6)d

(43.2±0.8)d

-

CREA-PR2

(869.4±17)c

(107.2±8.5)ab

(176±5.33)a

(39.3±0.2)b

-

RED PULP Dente di Cavallo

(85.9±0.8)c

(9.0±0.9)ab

(13.4±0.6)b

(23.5±0.2)b

(6.4±0.2)c

Zanna Bianca

(50.4±1.4)a

(8.0±0.4)a

(6.6±1.2)a

(21.1±0.2)a

(4.1±0.09)a

CREA-FRC4

(75.6±1.1)b

(8.1±0.9)a

(12.0±1.4)a

(26.9±0.3)d

(6.0±0.3)bc

CREA-PR1

(113.9±0.9)d

(10.8±0.2)c

(7.4±1.2)a

(27.6±0.3)e

(7.5±0.1)d

CREA-PR2

(84.5±1.1)c

(9.9±0.4)bc

(13.8±1.8)b

(24.3±0.2)c

(5.7±0.3)b



Page 32 of 37

32

Table 2. Immunological Data of Patients Involved in the Study

Patients Sera

Pru p 3 Pun g (ISAC) (SPT)

Pru p 3 Pru p 7 Pun g 1 (SPT) (SPT) (IgE DB)

Pun g 1 (IgE IB)

Pun g 7 (IgE DB)

Pun g 7 (IgE IB)

Symptoms

1

34254

1

nd

nd

nd

weak

positive

negative

negative

Ang

2

56684

1

nd

nd

nd

weak

negative

weak

negative

Ang

3

57010

1

nd

nd

nd

weak

negative

negative

negative

OAS

4

61023

1

nd

1

0

negative

weak

negative

negative

OAS

5

64925

1

nd

1

0

negative

positive

weak

positive

OAS, Ang.

6

66653

0

nd

0

0

negative

negative

weak

negative

OAS

7

69024

1

nd

nd

nd

positive

positive

weak

positive

U, Ang

8

58372

0

1

0

1

negative

negative

positive

negative

OAS, U,C

9

59011

1

1

0

1

positive

weak

weak

negative

OAS

10

59095

0

1

0

0

negative

negative

negative

negative

nr

11

EE10

1

1

1

0

weak

weak

negative

negative

nr

12

67646

1

1

1

1

positive

positive

weak

positive

nr

13

58255

1

nd

nd

nd

negative

negative

negative

negative

nr

14

BH63

1

nd

nd

nd

positive

positive

negative

negative

nr

15

CC11

1

nd

nd

nd

weak

negative

weak

negative

nr

16

63927

1

nd

nd

nd

negative

negative

negative

negative

nr

17

68901

1

nd

nd

nd

positive

positive

weak

negative

nr

18

68730

1

nd

nd

nd

positive

weak

negative

negative

nr

19

5961

1

nd

1

1

positive

positive

weak

positive

nr

Pun g : pomegranate pulp extract; Ang:angioedema; Asth: asthma; C: conjunctivitis; OAS: oral allergic syndrome; U:urticaria; nd: not determined; nr: not referred, IgE DB: dot blotting, IgE IB: immunobloting


Page 33 of 37


33

Figure 1.



Page 34 of 37

34

Figure 2


Page 35 of 37


35

Figure 3.



Page 36 of 37

36

Figure 4.


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37

arils

mesocarp

TOC


Pomegranate Cultivars: Identification of the New ... - ACS Publications

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