Food Chemistry 210 (2016) 70–77

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Pin p 1 is a major allergen in pine nut and the first food allergen
described in the plant group of gymnosperms
Beatriz Cabanillas a,⇑, Jesus F. Crespo b, Soheila J. Maleki c, Julia Rodriguez b, Natalija Novak a
a
Department of Dermatology and Allergy, University of Bonn Medical Center, Sigmund-Freud-Str. 25, 53127 Bonn, Germany
b
Servicio de Alergia, Hospital Universitario 12 de Octubre, Instituto de Investigación Hospital 12 de Octubre (i+12), Avenida de Córdoba s/n, 28041 Madrid, Spain
c
U.S. Department of Agriculture, Agriculture Research Service, Southern Regional Research Center, 1100 Robert E. Lee Boulevard, New Orleans, LA, USA

a r t i c l e i n f o a b s t r a c t

Article history: This study aimed to report the complete sequence of a 2S albumin purified from pine nut and to analyze
Received 28 November 2015 its allergenic properties. Individual recognition of this protein by serum IgE from pine nut-allergic
Received in revised form 22 March 2016 patients was assessed. IgE cross-linking capacity was analyzed in a basophil activation test. Inhibition
Accepted 18 April 2016
of IgE-binding and stability to heating was also assessed. The complete nucleotide sequence was obtained
Available online 20 April 2016
and a phylogenetic study was carried out. 2S albumin from pine nut (registered as Pin p 1.0101) was rec-
ognized by IgE of 75% of sera. The allergen was heat-stable and had a robust capacity to inhibit IgE-
Keywords:
binding to whole pine nut extract. The IgE cross-linking capacity of Pin p 1 on basophils was also demon-
Albumin
Basophil activation test
strated. Despite the low homology of Pin p 1 sequence with other allergenic 2S albumins from angios-
Pin p 1 perms, Pin p 1 contains the typical skeleton of 8 cysteine residues, important for its a-helixes enriched
Pine nut allergy structure.
Tree nut allergy Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction on blood lipids, lowers blood pressure and serum cholesterol

(Nergiz & Dönmez, 2004). In addition pine nuts contain potassium,
Allergy to tree nuts, in comparison with allergy to other foods, phosphorus, magnesium and other minerals, as well as dietary
has been described as particularly severe. Tree nuts are one of fibres (Nergiz & Dönmez, 2004).
the most frequent cause of fatal anaphylactic reactions (Bock, Allergy to pine nuts has been documented in the scientific liter-
Muñoz-Furlong, & Sampson, 2007). Pine nuts produced in Europe ature since 1958. Of the 45 cases reported in the literature to date,
originate in most cases from Pinus pinea, which belongs to the con- severe anaphylactic reactions after pine nut consumption
ifers division, the largest group of living gymnosperms. Gym- accounted for the majority of the described reactions (76%). More-
nosperms are evolutionally separated from flowering plants over, the number of individuals monosensitized to pine nuts com-
(angiosperms), which produce most of the tree nuts consumed pared to tree nuts from species of angiosperms is high. The
by humans (walnut, hazelnut, cashew, pistachio, Brazil nut, etc). reported reactions have been described to occur after consumption
Pine nuts from Pinus pinea have been consumed in the Mediter- of pine nuts as part of pesto sauce, salads, meatballs or meat, cakes,
ranean region for over 2000 years. Today they are extensively con- candies, cookies as well as after consumption of pine nuts alone
sumed worldwide as an ingredient in different foods, both in raw (Cabanillas & Novak, 2015).
or roasted form. Pine nuts are a source of valuable nutrients. The A few IgE-binding proteins from pine nuts have been studied by
fatty acid composition is characterized by a high content of unsat- means of western blotting so far. However, pine nut allergens have
urated fatty acids, such as linoleic acid, which has beneficial effects not yet been characterized in detail and official allergen names
have not yet been registered by the Allergen Nomenclature Sub-
committee from International Union of Immunological Societies
Abbreviations: AP, adapter primer; AUAP, abridged universal amplification (IUIS).
primer; HRP, Horseradish peroxidase; IUIS, International Union of Immunological In our previous work, we have purified and partially character-
Societies; PBST, PBS plus 0.5% Tween-20; PVDF, polyvinylidene difluoride; TBS, Tris- ized an IgE-binding protein from pine nuts with a molecular
buffered saline; TBST, TBS plus 0.5% Tween-20; TMB, tetramethylbenzidine; OD,
optical density; RT, room temperature.
weight around 15–16 kDa under non-reducing conditions and
⇑ Corresponding author. around 6 kDa under reducing conditions (Cabanillas et al., 2012).
E-mail address: [email protected] (B. Cabanillas). In the present study, we aimed to analyze the allergenic features

http://dx.doi.org/10.1016/j.foodchem.2016.04.068
0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
 B. Cabanillas et al. / Food Chemistry 210 (2016) 70–77 71

and to describe the complete sequence of this protein, which is the were pooled and the pH was adjusted to 7.0 with HCl. The pooled
first allergen described and officially registered for pine nuts. We fractions were loaded on a high S-support fast flow column (strong
also aimed to analyze the evolutionary differences between this cation exchange support, Bio-Rad) and eluted with a 200–400 mM
novel allergen and its allergenic homologues from angiosperms. NaCl linear gradient in 50 mM Tris buffer, pH 7.0. Initial character-
ization, by means of mass spectrometry analysis, showed a high
2. Materials and methods match with the seed storage protein albumin from the species
belonging to the Pinaceae family (Cabanillas et al., 2012).
2.1. Patients and sera Individual recognition of Pin p 1 by serum IgE was assessed by
western blotting. First, SDS-PAGE was performed according to
Sera from 8 patients with IgE-mediated clinical allergy to pine Laemmli’s protocol (Laemmli, 1970). Five micrograms of Pin p 1
nuts, confirmed on the basis of either a history of recent docu- was mixed with Laemmli sample buffer and b-mercaptoethanol,
mented severe anaphylaxis after pine nut ingestion or a positive heated at 95 °C for 10 min, and electrophoresed in a 15% Tris-HCl
double-blind placebo-controlled food challenge (DBPCFC) with gel. The protein was visualized with Coomassie Brilliant Blue
pine nuts, were used in this study (Table 1). Subjects had specific (Bio-Rad, Hercules, CA, USA). For western blotting, Pin p 1 was
IgE levels to pine nut (quantified by the fluorescent enzyme transferred to polyvinylidene difluoride (PVDF) membranes (Merck
immunoassay, CAP-FEIA system, Phadia, Uppsala, Sweden) and/or KGaA, Darmstadt, Germany). Membranes were cut into 3-mm
a positive skin prick test response to pine nut performed by the strips and blocked in Tris-buffered saline (TBS) containing 0.5%
prick-to-prick technique according to standard methods Tween-20 (TBST) and 5% non-fat milk for 1 h at room temperature
(Heinzerling et al., 2013) (Table 1). Written informed consents (RT). Membranes were incubated overnight with individual sera
were obtained from all patients. The study was approved by the from 8 patients with pine nut allergy (1:10 dilution), washed,
Ethics Committee of the Hospital Universitario 12 de Octubre, and then treated with a mouse anti-human IgE monoclonal anti-
Madrid, Spain (Permission No. 0312150129). body (clone 1A2, Abbiotec, San Diego, CA, USA) (stock: 1 mg/ml;
used at 1:5000) for 3 h. Membranes were washed and incubated
2.2. Protein purification and individual recognition by sera’s IgE in with HRP-conjugated goat anti-mouse antibody (stock: 0.4 mg/
western blotting ml; used at 1:5000) (Santa Cruz Biotechnology, Dallas, Texas,
USA) for 1 h. After washing, detection was achieved by means of
Purification of the IgE-binding protein from pine nuts with a enhanced chemiluminescence, according to the manufacturer’s
molecular weight of around 6 kDa under reducing conditions and instructions (Amersham Biosciences, Piscataway, NJ, USA).
around 15–16 kDa under non-reducing conditions (herein Pin p
1) involved a combination of ammonium sulfate precipitation 2.3. IgE-ELISA and ELISA inhibition
and ion exchange chromatography as described in Cabanillas
et al., 2012. Briefly, protein solubility of pine nut extract was tested Specific IgE-binding to Pin p 1 was also determined by ELISA
at different percentages of ammonium sulfate saturation, and it using 8 sera from patients with pine nut allergy. Polystyrene 96-
was found that Pin p 1 precipitated at 100% saturation. The precip- well microtiter plates (BD Falcon 353279, Heidelberg, Germany)
itated proteins were collected by centrifugation and the pellet was were coated with 100 ll/well of Pin p 1 at 8 lg/ml in PBS and incu-
dissolved in 50 mM Tris buffer (pH 8.4). Solubilized proteins were bated at 4 °C overnight. Wells were washed with PBS containing
subjected to ion exchange chromatography. Firstly, the solubilized 0.5% Tween-20 (PBST) and blocked for 1 h with PBS containing
proteins were loaded on a high Q-support fast flow column (strong 3% non-fat milk plus 0.1% Tween-20 (blocking solution). Plates
anion exchange support, Bio-Rad, Richmond, CA, USA). A linear salt were incubated with individual sera (1:7 dilution) for 2 h, washed
gradient from 0 to 1 M NaCl in 50 mM Tris buffer, pH 8.4, was used and incubated with a monoclonal mouse anti-human IgE antibody
to elute bound proteins. The unbound protein fraction, together (Abbiotec) (used at 1:1000) for 1 h. Wells were then washed and
with the protein fractions eluted with NaCl from 0 to 180 mM, incubated with HRP-conjugated goat anti-mouse antibody (Santa
Cruz Biotechnology) (used at 1:1000) for 1 h. After washing, the
peroxidase reaction was developed with tetramethylbenzidine
Table 1 (TMB) substrate (substrate reagent pack, DY999, R&D Systems,
clinical and immunological features of the 8 pine nut-allergic patients whose sera was
Inc., Minneapolis, MN, USA). The reaction was stopped with 2 N
available to be used in this study.
H2SO4, and the optical density (OD) was measured at 470 nm using
Patient SPT CAP- Symptoms Diagnostic Other a Synergy HT multi-detection microplate reader (BioTek, Bad Frie-
No./ (mm) FEIA challenge established
drichshall, Germany). The following negative controls were used:
age/sex (kU/L) food allergies
(i) no Pin p 1 coated-wells: wells coated with non-fat milk (instead
1/30/F 4.5 0.8 OAS DBPCFC Melon
of Pin p 1) and incubated with patients’ sera and the rest of anti-
2/38/F 11.5 0.9 U, AE, P, OAS, a 
DS, TS, A, GM
bodies following the steps described above; (ii) no serum: wells
3/34/M 12 2.27 AE, P, E, OAS, a  coated with Pin p 1 and incubated directly with mouse anti-
GI, DS, TS, GM human IgE and HRP-conjugated goat anti-mouse IgG; and (iii)
4/26/M 17.5 48 U a  non-allergic sera: wells coated with Pin p 1 and incubated with
5/39/F 10 0.02 AE, P, E, OAS, a 
two sera from healthy subjects, followed by the process described
O, TS
6/35/F 11 7.08 U, P, OAS, GI, a  above. All the tests were performed in duplicate. The formula:
GM mean [OD] + 3
SD was calculated for every negative control
7/17/M 5.5 19.3 OAS DBPCFC  and the highest value was considered as cut-off point of positivity
8/18/F 15.5 2.2 AE, DY, DS, a 
(Cabanillas et al., 2015).
OAS, GI
For ELISA inhibition, polystyrene 96-well microtiter plates (BD
AE, angioedema; A, asthma; CAP-FEIA, capsulated hydrolic carrier polymer-fluoro- Falcon 353279) were coated with 100 ll/well of whole pine nut
enzyme immunoassay; DBPCFC, double-blind, plancebo-controlled food challenge; protein extract at 100 lg/ml in PBS and incubated overnight at
DS, difficulty swallowing; DY, dysphonia; E, erythema; GI, gastrointestinal symp-
toms; GM, general malaise; OAS, oral allergy syndrome; O, ocular symptoms; P,
4 °C. In parallel, a serum pool from 6 pine nut allergic patients
pruritus; SPT, skin prick testing; TS, tongue swelling; U, urticaria. a, Not challenged (1:10) that recognized Pin p 1 in western blotting and ELISA was
because of a convincing history of severe reactions to pine nut. pre-incubated with Pin p 1 or whole pine nut protein extract
72 B. Cabanillas et al. / Food Chemistry 210 (2016) 70–77

(inhibitors) (final concentrations: 180, 18, 1.8, and 0.18 lg/ml) poly(C) tail was then added to the 30 -end of the cDNA using TdT
overnight at 4 °C with stirring. Serum pool pre-incubated with and dCTP according to manufacturer’s instructions. The dC-tailed
PBS was also included (non-inhibited serum). Wells were washed cDNA was amplified by PCR using a nested second gene-specific
with PBST and blocked with blocking solution for 1 h at RT. Wells primer GSP2, 50 -GGCTCGGCATCGGCATTGTGGA-30 and the abridged
were then incubated for 3 h with the sera preincubated with the anchor primer provided with the 50 RACE system. The PCR condi-
two inhibitors, or with serum that was not inhibited. After washing tions were as follows: 3 min of denaturation at 95 °C, followed
the wells, a mouse anti-human IgE monoclonal antibody (Abbio- by 31 cycles each of 1 min of denaturation at 95 °C, 1 min of
tec), used at 1:1000 dilution was added and incubated for 1 h. annealing at 70 °C, and 37 s of elongation at 72 °C; and a final
Wells were washed and incubated with a HRP-conjugated goat extension for 10 min at 72 °C. The PCR product was separated on
anti-mouse antibody (Santa Cruz Biotechnology) at 1:1000 dilution a 1% agarose gel. A unique and well-defined band was obtained.
for 1 h. After washing, the peroxidase reaction was developed as The PCR product was purified and cloned into a pCR4-TOPO vector
described above. The percentage of inhibition of the IgE-binding included in the TOPO TA Cloning Kit for Sequencing (Invitrogen)
was calculated as previously described (Cabanillas et al., 2015). which is a one-step cloning strategy for the direct insertion of
All tests were performed in duplicate. Taq polymerase-amplified PCR products into the plasmid pCR4-
TOPO vector for sequencing. The construct was used to transform
2.4. Heat stability of purified Pin p 1 Escherichia coli DH5a competent cells, and the DNAs from several
clones were sequenced using M13 forward and reverse sequencing
Pin p 1 was boiled for 30, 60, and 120 min at 100 °C and ana- primers (GATC Biotech., Konstanz, Germany). To obtain full-length
lyzed by IgE-ELISA using a serum pool from 6 patients with pine cDNA, a 30 RACE system was carried out (30 RACE System for Rapid
nut allergy that recognized Pin p 1 in western blotting and ELISA. Amplification of cDNA Ends, Invitrogen). 1.5 lg of the total RNA
was used for cDNA synthesis using the reverse transcriptase
2.5. Basophil activation assay enzyme (Superscript II) and adapter primer (AP, 50 -GGCCACGCGTC
GACTAGTACTTTTTTTTTTTTTTTT-30 ) provided with the system. The
Basophils from a donor without any clinically relevant allergy to cDNA obtained was amplified by means of PCR with a gene-specific
pine nuts and other nuts, but with specific IgE to hazelnut extract primer 50 -GACGTTGAGATGGGTGACTT-30 which was derived from
(1.33 kU/l) and the recombinant peanut allergen Ara h 8 (1.35 kU/l) the sequence obtained in the 50 RACE experiments. For the reverse
were enriched (>90% purity) by depleting non-basophils using the direction, the 30 primer abridged universal amplification primer
EasySep Human Basophil Enrichment Kit (StemCell Technologies, (AUAP) 50 -GGCCACGCGTCGACTAGTAC-30 , complementary to the
Grenoble, France). IgE was removed from the surface of the baso- adapter sequence, was used to prime first strand cDNA synthesis.
phils by incubation with lactic acid buffer (10 mM lactic acid, Amplification was carried out with Taq DNA polymerase. The PCR
130 mM NaCl, 5 mM KCl, pH 3.9) for 3.5 min on ice (Cabanillas program was: 5 min of denaturation at 95 °C, followed by 31 cycles
et al., 2015). A fraction of basophils was kept unstripped and each of 1 min of denaturation at 95 °C, 1 min of annealing at 60 °C,
directly subjected to allergen challenge. Stripped basophils were and 43 s of elongation at 72 °C; and a final extension for 10 min at
washed and then passively sensitized for 90 min at 37 °C with 72 °C. The obtained PCR product was purified, ligated to pCR4-
the sera (dilution 1:3) from four patients with clinically relevant TOPO and used to transform Escherichia coli as described above.
pine nut allergy (patients No. 2, 3, 6 and 8, Table 1). Before allergen DNAs for several clones were sequenced using M13 forward and
challenge, basophils were allowed to recover in HEPES Tyrodes reverse sequencing primers.
buffer containing 1 mM CaCl2 for 30 min at 37 °C. Stripped and
unstripped basophils were challenged for 20 min with 0.05 mg/ 2.6.2. Sequence comparison and phylogenetic studies
ml of Pin p 1. As controls, basophils were incubated with HEPES Alignment and phylogenetic studies of the amino acid sequence
Tyrodes buffer (negative control) or with a rabbit anti-human IgE of Pin p 1 with albumins from different species of gymnosperms
polyclonal antibody (50 lg/ml) (Dako, Eching, Germany) (positive and angiosperms were carried out with the program CLC Sequence
control). Cell surface staining of the basophil activation marker Viewer, version 7.6.1 (CLC bio, Cambridge, MA). The Neighbor-
CD203c was carried out with anti-human CD203c-PE monoclonal Joining algorithm with 100 replicate bootstraps was used. The
antibodies (clone 97A6, Beckman Coulter, Krefeld, Germany). Cells selected sequences were: Pinus strobus albumin 1 (ID:
were measured with the help of a FACSCanto flow cytometer (BD CAA44298.1), Pinus strobus albumin 2 (ID: CAA44299.1), Pinus stro-
Biosciences, Heidelberg, Germany) and data were analyzed with bus albumin 3 (ID: CAA44300.1), Pinus strobus albumin 4 (ID:
the BD FACSDiva software (BD Biosciences, Heidelberg, Germany). CAA44301.1), Picea glauca albumin-like-protein (ID: AAC34613.1),
A percentage of activation higher than 5% was considered as Pseudotsuga menziesii albumin (ID: AAC27000.1), Anacardium occi-
positive. dentale 2S albumin (Ana o 3) (ID: AAL91665.1), Juglans regia 2S
albumin (Jug r 1) (ID: AAB41308.1), Bertholletia excelsa 2S albumin
2.6. PCR (Ber e 1) (ID: AAA33010.1), Carya illinoinensis 2S albumin (Car i 1)
(ID: AAO32314.1), Juglans nigra 2S albumin (Jug n 1) (ID:
2.6.1. PCR based cloning strategy for the full characterization of Pin p 1 AAM54365.1), Pistacia vera 2S albumin (Pis v 1) (ID:
cDNA ABG73108.1) and Corylus avellana 2S albumin (Cor a 14) (ID:
Pinus pinea seeds were collected in El Álamo, Madrid (Spain). ACO56333.1).
Total RNA was extracted using Spectrum Plant Total RNA Kit Percentages of amino acid identity or similarity between two
(Sigma-Aldrich, St. Louis, MO, USA) and immediately stored at sequences were obtained with the NEEDLE program in the EMBOSS
80 °C to preserve its integrity. 1.8 lg of the total RNA was used package (Rice, Longden, & Bleasby, 2000).
for cDNA synthesis according to the 50 RACE system (50 RACE Sys-
tem for Rapid Amplification of cDNA Ends, Invitrogen) using a 2.7. Identification of potential N-Glycosylation sites in Pin p 1
reverse transcriptase enzyme (Superscript II) and the gene-
specific primer GSP1, 50 -ATCATACGATTGAGATTG-30 which was SDS polyacrylamide gel slices containing Pin p 1 were split and
selected on the basis of the described partial cDNA sequence of this subjected to gel digestion with AspN or GluC (in bicarbonate) (Jeno,
allergen (Cabanillas et al., 2012). The cDNA was purified from unin- Mini, Moes, Hintermann, & Horst, 1995; Rosenfeld, Capdevielle,
corporated dNTPs and GSP1 using S.N.A.P. columnsTM purification. A Guillemot, & Ferrara, 1992). In brief, slices were washed
 B. Cabanillas et al. / Food Chemistry 210 (2016) 70–77 73

consecutively with water, 50% acetonitrile (ACN), and 100% ACN. 95% inhibition was reached using whole pine nut extract as
The protein was reduced with 20 mM DTT in 50 mM ammonium inhibitor.
bicarbonate and alkylated with 40 mM iodoacetamide (in 50 mM
bicarbonate). The slices were washed again and dehydrated with 3.2. Stability of Pin p 1 to heating
ACN. Dried slices were incubated with 400 ng of sequencing grade
protease (Promega, Mannheim, Germany) at 37 °C overnight. The The stability of Pin p 1 to heating was evaluated as well. Similar
peptide extract was separated and the remaining peptides OD values in the IgE-ELISA were obtained for every sample sub-
extracted with 50% ACN. Dried and reconstituted peptides were jected to the different boiling times, indicating that the IgE-
deglycosylated with PNGase F (New England Biolabs, Frankfurt a. binding capacity of purified Pin p 1 remained stable after heating
M., Germany) at 37 °C overnight. Peptides were enriched by C18 (Fig. 1D).
micropurification (ZipTip, Merck, Darmstadt, Germany).
For LC–MS analysis, 1.5 ll were injected onto a C18 trap column 3.3. Basophil activation after challenge with Pin p 1
(20 mm length, 100 lm inner diameter) equilibrated with 0.1%
TFA. Bound peptides were eluted onto a C18 analytical column We next aimed to analyse IgE cross-linking and activation of
(200 mm length, 75 lm inner diameter) equilibrated with 0.1% for- human basophils upon Pin p 1 exposure by measuring the upregu-
mic acid (solvent A). Both columns were made in-house with lation of the specific activation marker CD203c (Boumiza et al.,
ReproSil-Pur 120 C18-AQ, 5 lm or 1.9 lm, respectively (Dr. Maisch 2003). For that purpose, we carried out a stripped basophil activa-
GmbH, Ammerbuch-Entringen, Germany). Peptides were sepa- tion assay using basophils isolated from a donor without any clin-
rated during a linear gradient from 0% to 35% solvent B (80% ical allergy to nuts. The donor’s basophils were subjected to IgE
acetonitrile, 0.1% formic acid) within 60 min at a flow rate of stripping and sensitization, with sera from four pine nut allergic
330 nl/min. The nanoHPLC was coupled online to an LTQ Orbitrap patients (patients Nos. 2, 3, 6, and 8) or they were not stripped
Velos mass spectrometer (Thermo Fisher Scientific, Bremen, (Fig. 2, first column). These basophils, with or without patient sera
Germany). Peptide ions between 330 and 1600 m/z were scanned sensitization, were incubated in the presence of media alone as
in the orbitrap detector with a resolution of 30,000 (maximum fill negative control, Pin p 1 or anti-IgE as positive control. Fig. 2 shows
time 400 ms, AGC target 106). The 25 most intense precursor ions that with this method Pin p 1 was able to induce basophil activa-
(threshold intensity 5000) were subjected to collision induced dis- tion in 75% of the cases (basophils sensitized with sera from
sociation fragments analyzed in the linear ion trap. Fragmented patients Nos. 3, 6, and 8), with percentages of activation ranging
peptide ions were excluded from repeat analysis for 15 s. from 8.1% to 19.7%.
Raw data processing and analysis of database searches were
performed with the Proteome Discoverer software 2.1 (Thermo 3.4. Full characterization of Pin p 1 cDNA
Fisher Scientific). MS2 data was searched against Pinacea
sequences from Trembl (release 2016_01) with Mascot server ver- The full characterization of the nucleotide sequence of Pin p 1
sion 2.5.1 (Matrix Science Ltd, London, UK). Precursor ion m/z tol- was carried out in the present study. Total RNA was obtained from
erance was 7 ppm, fragment ion tolerance 0.5 Da. b- and y-ion Pinus pinea seeds. A well-defined, unique amplification product of
series were included. Peptides with up to two missed cleavages 50 RACE was obtained using gene specific primers designed from
were searched. Carbamidomethylation was set as a static modifica- the partial cDNA sequence previously described for this allergen
tion of cysteines. Oxidation of methionine and deamidation of (Cabanillas et al., 2012). Using the information of the sequence
asparagine were allowed as a dynamic modification. Spectrum derived from the 50 RACE experiment, a gene specific primer was
matches were sent to the percolator algorithm (Kall, Storey, designed in order to characterize the 30 end using the 30 RACE sys-
MacCoss, & Noble, 2008) version 2.05 as implemented in Proteome tem. The sequences derived from 50 and 30 RACE experiments were
Discoverer 2.1.0.81. aligned using Clustal W, and a full length cDNA of 495 bp, which
encoded a protein of 164 amino acids, was obtained (Fig. 3; Gen-
Bank Accession No. LN876267.1). These data were submitted to
3. Results IUIS allergen nomenclature subcommittee, and the official name
of Pin p 1.0101 was given to this allergen (http://www.allergen.
3.1. Pin p 1 purification, individual recognition by sera’s IgE and org/viewallergen.php?aid=842).
inhibition assays The amino acid sequence of Pin p 1 was characterized by a
skeleton of eight cysteine residues which is well-conserved and
The purification of Pin p 1 involved a combination of ammo- characteristic of the members of the prolamin superfamily, to
nium sulfate precipitation and ion exchange chromatography. which the 2S albumins belong (Fig. 3). This skeleton structure is
The elution profiles of the different steps in the purification process found to be conserved in the three-dimensional structure enriched
have been described in our previous work (Cabanillas et al., 2012). in a-helices of 2S albumins.
The purity of the protein was assessed by SDS-PAGE and by mass
spectrometry. Absence of contamination with other proteins was 3.5. Molecular evolution analysis
found in the purified sample.
The recognition of Pin p 1 by the serum IgE from 8 patients with Since Pinus pinea belongs to the group of gymnosperms, sepa-
pine nut allergy was carried out by means of IgE-western blotting rated in the evolution tree from the species that produce other tree
and IgE-ELISA (Figs. 1A and B). The results showed that IgE from 6 nuts that humans consume (all belonging to the angiosperms
out of 8 patients’ sera (75%) recognized Pin p 1 in both western group), we focused on the analysis of the specific features of Pin
blotting and ELISA. ELISA inhibition was performed using a pool p 1 amino acid sequences compared to other 2S albumins. We first
of sera from the 6 patients that recognized Pin p 1 in western blot- carried out a comparison and a phylogenetic study of Pin p 1 with
ting and ELISA (Fig. 1C). The ELISA inhibition was performed with 2S albumins from different species of gymnosperms and angios-
whole pine nut extract coating the solid phase. The same extract perms. The selected sequences were: Pinus strobus albumin 1, Pinus
and Pin p 1 were used as inhibitors. Using Pin p 1 as inhibitor, a strobus albumin 2, Pinus strobus albumin 3, Pinus strobus albumin 4,
progressive increase in the percentage of inhibition of IgE- Picea glauca albumin-like-protein, Pseudotsuga menziesii albumin,
binding to pine nut protein extract up to 83% was observed. A Anacardium occidentale 2S albumin (Ana o 3), Juglans regia 2S
74 B. Cabanillas et al. / Food Chemistry 210 (2016) 70–77

Fig. 1. IgE reactivity, IgE inhibition capacity and thermal resistance of Pin p 1. (A) SDS-PAGE, IgE-western blot and (B) IgE-ELISA analysis of Pin p 1 incubated with sera from
patients with clinical allergy to pine nut (1–8). The cut-off point of positivity for the IgE-ELISA is indicated by the horizontal line in the graph (IgE levels > 0.24, calculated as
indicated in material and methods). (C) ELISA inhibition with pine nut protein extract in the solid phase and Pin p 1 or pine nut protein extract (indicated in the legend) used
as inhibitors at 0, 0.00018, 0.0018, 0.018, and 0.18 mg/ml. A pool of sera from patients allergic to pine nut was used. The percentage of inhibition of the IgE binding was
calculated as previously described (Cabanillas et al., 2015). (D) IgE-ELISA analysis of Pin p 1 heated at 100 °C during 30, 60, or 120 min, or kept it unheated. The IgE-ELISA was
incubated with sera from patients with clinically relevant allergy to pine nut.

Fig. 2. Stripped basophil activation assay. Percentages of activated basophils (CD203c+) (unstripped () or stripped (+) and sensitized with sera from patients with pine nut
allergy (Nos. 2, 3, 6, and 8) measured by flow cytometry after stimulation with medium alone (negative control), Pin p 1 or a anti-human IgE polyclonal antibody (positive
control).

albumin (Jug r 1), Bertholletia excelsa 2S albumin (Ber e 1), Carya similarity and identity of the amino acid sequence of Pin p 1 with
illinoinensis 2S albumin (Car i 1), Juglans nigra 2S albumin (Jug n allergenic 2S albumins from tree nuts belonging to the angiosperm
1), Pistacia vera 2S albumin (Pis v 1) and Corylus avellana 2S albu- group. Results are shown in Fig. 4B. We found that Pin p 1 had the
min (Cor a 14). In order to visually represent the evolutionary clus- lowest percentage of similarity and identity with allergenic 2S
ters of the sequences, a phylogenetic tree was constructed using albumins from tree nuts, with percentages of similarity ranging
the Neighbor-joining method. The phylogenetic tree in Fig. 4A between 36% and 46% and percentages of identity between 22%
shows that 2S albumins from the angiosperms species cluster sep- and 29%.
arately from the 2S albumins from species of gymnosperms, Interestingly, in spite of the low homology between Pin p 1 and
including Pin p 1, highlighting the evolutionary differences the allergenic 2S albumins from tree nuts from angiosperm species,
between the sequences of 2S albumins in both groups of plants. a multiple amino acid sequence alignment of Pin p 1 with seven
Then we were interested in the analysis of the percentages of allergic 2S albumins from angiosperms (Fig. 5), revealed that Pin
 B. Cabanillas et al. / Food Chemistry 210 (2016) 70–77 75

Fig. 3. Nucleotide sequence of the complete cDNA of Pin p 1 and the deduced amino acid sequence. The conserved 8 cysteins are highlighted with squares.

Fig. 4. (A) Phylogenetic analysis of the amino acid sequences of 2S albumins from different species of gymnosperms and angiosperms. The Neighbor-Joining algorithm with
100 replicate bootstraps was used (performed with CLC sequence viewer program). (B) Percentages of identity (%I) and similarity (%S) of amino acid sequences of Pin p 1 and
allergenic 2S albumins from angiosperm species (performed with NEEDLE program in the EMBOSS package).

p 1 shares the placement of 8 cysteine residues at similar positions 4. Discussion

seen in the other sequences. The conserved 8 cysteines in 2S albu-
mins are considered important for the a-bundle structure that is In the present study we have analyzed the allergenic features
typical of these proteins (Fig. 5). and characterized the complete sequence of the first food allergen
described from gymnosperms, the pine nut 2S albumin Pin p 1. A
3.6. First evidence of a N-glycosylation site in Pin p 1 high percentage (75%) of recognition of Pin p 1 by individual sera
from patients with pine nut allergy was found. This indicates that
In order to identify N-glycosylation sites, Pin p 1 was digested Pin p 1 is most likely one of the major allergens of pine nut recog-
by proteinases and then deglycosylated by PNGase F, the digests nized by our study population. Moreover, a common characteristic
were analyzed by LC–MS. PNGase F cleaves N-linked oligosaccha- of the patients whose sera recognized Pin p 1 was a history of sev-
rides from glycoproteins resulting in the conversion of initially gly- ere anaphylaxis after pine nut consumption. ELISA inhibition
cosylated asparagine to aspartic acid. The resulting mass change of showed that Pin p 1 had a robust capacity to inhibit IgE-binding
the peptide can be analyzed in order to identify N-glycosylation to whole pine nut extract, reaching a percentage of inhibition of
sites. Our results showed the first evidence of a potential 83% compared to 95% of inhibition reached by the whole pine
N-glycosylation site at N138 in Pin p 1, in accordance to the nut extract. IgE-cross linking capacity of Pin p 1 in basophils as
ASN-X-Cys motif (Fig. 1, Supplementary material). effector cells of allergic reactions has been also demonstrated.
76 B. Cabanillas et al. / Food Chemistry 210 (2016) 70–77

Fig. 5. Alignment of the amino acid sequences of Pin p 1 with allergenic 2S albumins from angiosperm species: Corylus avellana 2S albumin (Cor a 14), Anacardium occidentale
2S albumin (Ana o 3), Juglans regia 2S albumin (Jug r 1), Bertholletia excelsa 2S albumin (Ber e 1), Carya illinoinensis 2S albumin (Car i 1), Juglans nigra 2S albumin (Jug n 1), and
Pistacia vera 2S albumin (Pis v 1). The level of conservation at each position in the alignment is shown by the grey bars at the bottom of the alignment. The conserved skeleton
of eight cysteines is marked with rectangles and asterisks.

Although no official allergen names for pine nut have been reg- structure of 2S albumins seems to play an important role in keep-
istered by Allergen Nomenclature Subcommittee so far, some IgE- ing the protein intact until they reach the effector cells of our
binding proteins from pine nuts have been detected with western immune system. Upon ingestion, these allergic proteins can resist
blotting in previous studies. A protein from pine nuts with a molec- and survive harsh conditions and can be absorbed in the gut effect-
ular weight of 15–17 kDa under non reducing conditions and ing immunological responses (Moreno & Clemente, 2008).
around 7 kDa under reducing conditions has been described in The nucleotide and amino acid sequence of Pin p 1 was found to
two studies (García-Menaya et al., 2000; Ibáñez, Lombardero, San share high homology with 2S albumins from species of gym-
Ireneo, & Muñoz, 2003). This protein was recognized by the serum nosperms and low homology with 2S albumins from angiosperms.
IgE of 3 patients with severe allergic symptoms after pine nut con- Phylogenetic studies carried out in this study confirmed these find-
sumption. In both studies, authors hypothesized that the strong ings. The group of gymnosperms, to which Pinus pinea belongs, are
recognition of this single IgE-binding protein correlated with the evolutionally separated from flowering plants (angiosperms)
severity of symptoms after pine nut intake. However, no further which produce most of the tree nuts consumed by human beings
analysis besides the IgE-western blotting analysis was undertaken (walnut, hazelnut, cashew, pistachio, etc). This evolutionary differ-
(García-Menaya et al., 2000; Ibáñez et al., 2003). Since Pin p 1 has a ence seems to be important, and has an impact in the clinical find-
molecular weight of 15-16 kDa under non reducing conditions and ings concerning pine nut allergy, in which a high rate of individuals
6 kDa under reducing conditions (Cabanillas et al., 2012), the IgE- monosensitized to pine nuts compared to other tree nuts is found
binding protein pointed out in the two mentioned studies might be (Cabanillas & Novak, 2015). The scientific literature confirms the
Pin p 1. high immunological cross-reactivity among nuts from species of
Several 2S albumins have been characterized as major food gymnosperms, but a low or even missing cross-reactivity between
allergens in tree nuts, such as cashew (Ana o 3), walnut (Jug r 1), nuts from gymnosperms and angiosperms (de las Marinas, Vila, &
Brazil nut (Ber e 1), and have been suggested to be involved in sev- Sanz, 1998; Jansen, Vermeulen, Dieges, & van Toorenenbergen,
ere allergic reactions. The allergen Pin p 1 also seems to be impli- 1996). The unique characteristics of pine nut allergens may have
cated in the severe allergic reactions in patients with pine nut implications in the detection of pine nuts in foodstuff. A recent
allergy. The highly conserved and disulfide-linked compact study has selected marker peptides for the detection of nuts in a
 B. Cabanillas et al. / Food Chemistry 210 (2016) 70–77 77

variety of food products using enzymatic digestion LC-MS/MS. The The study was supported by Cluster of Excellence ImmunoSen-
selection criteria were based on representative marker peptides sation of the German Research Foundation (DFG) (Germany), the
that uniquely represent the specific nut. In the case of pine nuts, Christine Kühne Stiftung CK-CARE and a BONFOR Grant of the
two peptides from pine globulin-1 were selected (Sealey- University of Bonn (Germany).
Voyksner, Zweigenbaum, & Voyksner, 2016). The characteristic The funding sources had no role in the study design; in the col-
low homology of Pin p 1 with other allergenic 2S albumins from lection, analysis and interpretation of data; in the writing of the
angiosperms may also indicate the potential to specifically detect manuscript; and in the decision to submit the article for
the presence of pine nuts in foodstuff at hidden and trace levels. publication.
Interestingly, despite low sequence homologies, Pin p 1 shares
with other allergenic 2S albumins described in angiosperms the Appendix A. Supplementary data
typical skeleton of 8 cysteine residues at a similar position of the
sequence. These cysteines are involved in the formation of four Supplementary data associated with this article can be found, in
intra-chain disulphide bonds. The pattern of 8 cysteines in specific the online version, at http://dx.doi.org/10.1016/j.foodchem.2016.
order appears to be a structural scaffold of conserved helical 04.068.
regions that forms a network of disulfide bridges necessary for
the maintenance of the tertiary structure (Moreno & Clemente, References
2008). Therefore, in spite of the low homology of Pin p 1 with
the sequences of 2S albumins from angiosperms, such as Cor a Bock, S. A., Muñoz-Furlong, A., & Sampson, H. A. (2007). Further fatalities caused by
anaphylactic reactions to food, 2001–2006. The Journal of Allergy and Clinicial
14 from hazelnut, Jug r 1 from walnut or Ana o 3 from cashew, it
Immunology, 119, 1016–1018.
seems that they share a common and compact three-dimensional Boumiza, R., Monneret, G., Forissier, M. F., Savoye, J., Gutowski, M. C., Powell, W. S.,
structure enriched by a-helixes, as observed by circular dichroism et al. (2003). Marked improvement of the basophil activation test by detecting
analysis for Pin p 1 in our previous studies (Cabanillas et al., 2012). CD203c instead of CD63. Clinical & Experimental Allergy, 33, 259–265.
Cabanillas, B., Cheng, H., Grimm, C. C., Hurlburt, B. K., Rodríguez, J., Crespo, J. F., et al.
The highly conserved and compact structure of 2S albumins has (2012). Pine nut allergy: Clinical features and major allergens characterization.
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extreme pH and proteolysis. These features have also been Cabanillas, B., Cuadrado, C., Rodriguez, J., Hart, J., Burbano, C., Crespo, J. F., & Novak,
N. (2015). Potential changes in the allergenicity of three forms of peanut after
observed in Pin p 1, which is characterized by resistance to high thermal processing. Food Chemistry, 183, 18–25.
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We thank Nils Schoof and Juana Alica Hart for their excellent (Part II: Thirty other edible nuts).
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technical support. We also thank Dr. Marc Sylvester from the mass conserved unique peanut and tree nut peptides by LC–MS/MS for multi-allergen
spectrometry unit, IBMB, University of Bonn, for excellent perfor- detection. Food Chemistry, 194, 201–211.
mance of mass spectrometry experiments.