Computational Biology and Chemistry 56 (2015) 3032

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Computational Biology and Chemistry

journal homepage: www.elsevier.com/locate/compbiolchem

Brief communication

Pru du 2S albumin or Pru du vicilin?

Cristiano Garino a, * , Angelo De Paolis b , Jean Daniel Cosson a , Marco Arlorio a
a
Dipartimento di Scienze del Farmaco & Drug and Food Biotechnology (DFB) Center, Universit del Piemonte Orientale A. Avogadro, largo Donegani 2,
28100 Novara, Italy
b
Istituto di Scienze delle Produzioni Alimentari (ISPA) CNR, via Prov.le Lecce Monteroni, 73100 Lecce, Italy

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 2 February 2015
Received in revised form 26 March 2015
Accepted 27 March 2015
Available online 30 March 2015

A short partial sequence of 28 amino acids is all the information we have so far about the putative allergen
2S albumin from almond. The aim of this work was to analyze this information using mainly
bioinformatics tools, in order to verify its rightness.
Based on the results reported in the paper describing this allergen from almond, we analyzed the
original data of amino acids sequencing through available software.
The degree of homology of the almond 12 kDa protein with any other known 2S albumin appears to be
much lower than the one reported in the paper that rstly described it. In a publicly available cDNA
library we discovered an expressed sequence tag which translation generates a protein that perfectly
matches both of the sequencing outputs described in the same paper. A further analysis indicated that the
latter protein seems to belong to the vicilin superfamily rather than to the prolamin one. The fact that also
vicilins are seed storage proteins known to be highly allergenic would explain the IgE reactivity originally
observed.
Based on our observations we suggest that the IgE reactive 12 kDa protein from almond currently
known as Pru du 2S albumin is in reality the cleaved N-terminal region of a 7S vicilin like protein.
2015 Elsevier Ltd. All rights reserved.

Keywords:
Almond2S albuminvicilin

Almond (Prunus dulcis or Amygdalus communis L.) is one of the

most commonly consumed nut worldwide. It can be eaten either
raw or processed into a wide variety of foods, particularly in sweets
like pastry, chocolates, and confectionary products. The consumption of almonds has been associated with nutritional benets,
including cholesterol-lowering effects, protection against diabetes
and potential prebiotic properties (Jenkins et al., 2008; Mandalari
et al., 2008). Despite these benets for the health of the consumer,
these fruits represent an allergic threat for sensitized individuals.
Even though almond is commonly included into the tree nuts
group, taxonomically it belongs to the Rosaceae, a family including
a number of allergenic fruits, such as peach, apricot, plum, cherry,
apple, pear, blackberry and strawberry. Unlike allergies to other
foods, such as egg and milk (mostly hitting during childhood and
disappearing in adult age), nut-induced allergies are often
permanent and particularly severe, with multisystemic or
respiratory symptoms in the majority of the cases (Crespo et al.,
2006). Although clinical cross-reactivity is reported to be rare
between tree nuts, a potential clinical allergy to other Rosaceae

* Corresponding author. Tel.: +39 0321 375873; fax: +39 0321 375621.
E-mail addresses: garino@unipmn.it (C. Garino), angelo.depaolis@ispa.cnr.it
(A. De Paolis), coisson@unipmn.it (J.D. Cosson), arlorio@unipmn.it (M. Arlorio).
http://dx.doi.org/10.1016/j.compbiolchem.2015.03.004
1476-9271/ 2015 Elsevier Ltd. All rights reserved.

should not be overlooked by almond allergic patients (Rodriguez

et al., 2000). IgE cross-reactivity has also been reported with
allergens from peanut (de Leon et al., 2005; Holden et al., 2008),
corn (Lee et al., 2005), and lupine (Holden et al., 2008). Despite
their peculiar proximity to so many allergenic fruits (they share
both their phenotypic characteristics with several other tree nuts
and their genetic patrimony with the Rosaceae members), almonds
have never raised a great attention within the food allergy eld,
and comparatively few studies have been carried out and
published in the recent years. Up to date, 8 allergens are present
in the Allergome database (www.allergome.org), but only 4 of
them have been approved by the Allergen Nomenclature SubCommittee of the International Union of Immunological Societies
(www.allergen.org). These are Pru du 3, a non-specic lipid
transfer protein similar to the one from peach, Pru du 4, a prolin
causing mild symptoms limited to oral cavity, Pru du 5, a ribosomal
protein whose allergenicity is still to be dened, and Pru du 6, the
major and best characterized almond allergen, an 11S globulin also
called amandin. Other allergens yet to be elucidated are Pru du 1,
member of the Bet v 1-homologous family, Pru du 2, a thaumatin
like protein, Pru du g-conglutin and Pru du 2S albumin (Costa et al.,
2012).
In 2002 Poltronieris group reported the identication of two
almond IgE-binding proteins, belonging to the albumin fraction.

C. Garino et al. / Computational Biology and Chemistry 56 (2015) 3032

These two proteins, with molecular weights of approximately

12 and 45 kDa, respectively, were subjected to purication and
N-terminal sequencing. While the 25 amino acids sequence
obtained from the 45 kDa protein displayed a convincing degree
of similarity with the mature form of conglutin g from white and
narrow-leafed blue lupine, the 18 amino acids sequence obtained
from 12 kDa protein showed a low degree of similarity with a 2S
albumin from English walnut. In order to validate the homology
with 2S albumin, a new 10 amino acids long sequence was
obtained from a smaller peptide of 2 kDa, generated in vitro in
order to obtain additional internal sequence information. According to the Authors the new sequencing conrmed the rst
statement, and the whole 12 kDa protein was assigned to the 2S
albumin family of seed storage proteins. A new putative allergen
from almond with a partial 28 amino acids sequence, resulting
from the sum of the 18 amino acids peptide and the 10 amino acids
one, is currently reported by the Allergome database and called Pru
du 2S Albumin (Poltronieri et al., 2002).
In order to clarify the real identity of this 12 kDa almond
protein, we decided to carry out an in silico analysis starting
from the 28 amino acids peptide (VTXEEGXYSISDQSKVGEQXIRSPDREM) currently known as Pru du 2S Albumin (UniProt
P82944).
A schematic representation of the results of our analysis is
displayed in Fig. 1. First of all we separated the two original
peptides, and we started by analyzing the rst 18 aa one. With a
sequence identity of 22% and a similarity of 39%, the 2S albumin
from English walnut did not seem to match the almond originated

31

peptide. The sequence was therefore submitted to tBLASTn

analysis (http://blast.st-va.ncbi.nlm.nih.gov/Blast.cgi), and we
looked in the EST sequence database of Prunus dulcis.
Seven EST sequences (GenBank accessions BU574590,
BU573888, BU572660, BU574985, BU575072, BU573652 and
BU573450) displayed 89% identity with our query, with E-values
ranging from 4  10 5 to 9  10 5. All these nucleotide sequences
were then in silico translated into proteins using the ExPASy
Translate tool (http://web.expasy.org/translate), and the obtained
amino acid sequences were subjected to multiple alignment using
ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2). As shown
in Fig. 2, the 7 sequences were different in length, but they all
shared a very high degree of identity. Moreover, both of the
peptides originated from the sequencing of the 12 kDa protein
(Poltronieri et al., 2002) displayed perfect match. By using the
ExPASy Compute pI/Mw tool (http://web.expasy.org/cgi-bin/compute_pi/pi_tool) we calculated the theoretical molecular mass of
the protein portion spanning from the beginning of the 18 aa
peptide to the end of the 10 aa one: the result was 9357.40 Da,
which is compatible (not larger) with the mass of 12 kDa observed
after SDS PAGE separation by Poltronieri et al. Finally, when
looking at the partial sequence of the protein reported in Fig. 1, and
derived from the in silico translation of the corresponding EST
sequence, it is interesting to notice that sulphurated amino acids
account for the 15% of the total, which is one of the main reason,
aside from the similarity degree, why this protein was originally
attributed to the sulphydryl-rich 2S albumin family (Poltronieri
et al., 2002).

Fig. 1. Workow of the in silico analysis performed on Pru du 2S albumin reported amino acid sequence.

32

C. Garino et al. / Computational Biology and Chemistry 56 (2015) 3032

Fig. 2. ClustalW2 multialignment of the 7 Prunus dulcis ESTs (in silico translated into proteins) matching the 18 aa sequence reported in Poltronieri et al. (2002).

In order to investigate the putative role of this new protein, we

performed a new BLAST search with the aim of identifying ortholog
members in other species: rstly, we analyzed it using the BLASTp
suite (search of protein databases using a protein query): although
no conserved domains were detected, the result showed a 90%
identity with 88% of a vicilin C72-like protein from Prunus mume
(GenBank accession XP_008224280), a tree belonging to the same
genus as almond and commonly known as either Chinese plum or
Japanese apricot. Secondly, we decided to run a search starting
from one of the EST sequences and employing the BLASTx suite
(search of protein databases using a translated nucleotide query).
This time the putative conserved domain of the vicilin superfamily
N-terminal region (Fig. 1) was recognized by the CDD tool
(Marchler-Bauer et al., 2013). This region is typical of plant seed
storage proteins (N-terminal end of the Cupin domain), and it has
been shown how in Macadamia integrifolia it is processed into
peptides of approximately 50 amino acids, containing a C-X-X-X-C(10-12)X-C-X-X-X-C motif and exhibiting antimicrobial activity in
vitro (Marcus et al., 1999). In Fig. 1 cysteine residues are marked, in
order to highlight the presence of the above mentioned motif. This
particular motif can be found in the N-terminal region of other
known allergens identied as 7S vicilin, such as Ana o 1 from
cashew (UniProt Q8L5L5), Ara h 1 from peanut (UniProt B3IXL2),
Car i 2 from pecan (UniProt B3STU4), Gly m 5 from soybean
(UniProt C6T9L1), Jug r 2 from English walnut (UniProt Q9SEW4),
Pis v 3 from pistachio (UniProt B4  640), Ses i 3 from sesame
(UniProt Q9AUD0), Sola l vicilin from tomato (UniProt B0JEU3) and
Zea m G1 from corn (UniProt Q03865). Vicilin-like proteins are
commonly large hydrophobic proteins, with a usual molecular
weight of around 60 kDa, and they are known allergens present in
the seeds of several plants commonly employed for human
consumption (Rouge et al., 2011). The C-X-X-X-C-(10-12)X-C-X-XX-C motif is located in a hydrophilic region proximal to the Nterminus, which is present in some, but not all, 7S globulins (Dure,
1990). Despite a recent study suggested that the IgE epitopes of Ara
h 1 are more concentrated in the hydrophobic and less charged
areas (Bgh et al., 2012), an interesting analogy of what could
happen in almond exists in buckwheat, where a small reacting
protein of around 19 kDa and corresponding to the N-terminal
region of a 7S vicilin has been recognized as major allergen (Choi
et al., 2007).
In light of the observations obtained after a brief but detailed in
silico analysis, we can conclude that the IgE binding 12 kDa almond
protein, originally described as a 2S albumin (UniProt P82944),
does not belong to the prolamin superfamily, but is more likely to
correspond to the N-terminal region of a 7S vicilin. Further
investigations will be necessary to characterize and disclose the

real allergenic potential of this new putative almond vicilin in

sensitized individuals.
Conict of interest
All the authors declare no conicts of interest.
References
Bgh, K.L., Nielsen, H., Madsen, C.B., Mills, E.N.C., Rigby, N., Eiwegger, T., Szpfalusi,
Z., Roggen, E.L., 2012. IgE epitopes of intact and digested Ara h 1: a comparative
study in humans and rats. Mol. Immunol. 51, 337346.
Choi, S.-Y., Sohn, J.-H., Lee, Y.-W., Lee, E.-K., Hong, C.-S., Park, J.-W., 2007.
Characterization of buckwheat 19-kD allergen and its application for diagnosing
clinical reactivity. Int. Arch. Allergy Immunol. 144 (4), 267274.
Costa, J., Mafra, I., Carrapatoso, I., Oliveira, M.B.P.P., 2012. Almond allergens:
molecular characterization, detection, and clinical relevance. J. Agric. Food
Chem. 60, 13371349.
Crespo, J.F., James, J.M., Fernandez-Rodriguez, C., Rodriguez, J., 2006. Food allergy:
nuts and tree nuts. Br. J. Nutr. 96, S95S102.
de Leon, M.P., Drew, A.C., Glaspole, I.N., Suphioglu, C., Rolland, J.M., O'Hehir, R.E.,
2005. Functional analysis of cross-reactive immunoglobulin E antibodies:
peanut-specic immunoglobulin E sensitizes basophils to tree nut allergens.
Clin. Exp. Allergy 35, 10561064.
Dure, L., 1990. An unstable domain in the vicilin genes of higher plants. New Biol. 2,
487493.
Holden, L., Sletten, G.B.G., Lindvik, H., Faste, C.K., Dooper, M.M.B.W., 2008.
Characterization of IgE binding to lupin, peanut and almond with sera from
lupin-allergic patients. Int. Arch. Allergy Immunol. 146, 267276.
Jenkins, D.J.A., Kendall, C.W.C., Marchie, A., Josse, A.R., Nguyen, T.H., Faulkner, D.A.,
Lapsley, K.G., Blumberg, J., 2008. Effect of almonds on insulin secretion and
insulin resistance in nondiabetic hyperlipidemic subjects: a randomized
controlled crossover trial. J. Nutr. 138, 908913.
Lee, S.H., Benmoussa, M., Sathe, S.K., Roux, K.H., Teuber, S.S., Hamaker, B.R., 2005. A
50 kDa maize gamma-zein has marked cross-reactivity with the almond major
protein. J. Agric. Food Chem. 53, 79657970.
Mandalari, G., Nueno-Palop, C., Bisignano, G., Wickham, M.S.J., Narbad, A., 2008.
Potential prebiotic properties of almond (Amygdalus communis L.) seeds. Appl.
Environ. Microbiol. 74, 42644270.
Marchler-Bauer, A., Zheng, C., Chitsaz, F., Derbyshire, M.K., Geer, L.Y., Geer, R.C.,
Gonzales, N.R., Gwadz, M., Hurwitz, D.I., Lanczycki, C.J., Lu, F., Lu, S., Marchler, G.
H., Song, J.S., Thanki, N., Yamashita, R.A., Zhang, D., Bryant, S.H., 2013. CDD:
conserved domains and protein three-dimensional structure. Nucleic Acids Res.
41 (D1), D38452.
Marcus, J.P., Green, J.L., Goulter, K.C., Manners, J.M., 1999. A family of antimicrobial
peptides is produced by processing of a 7S globulin protein in Macadamia
integrifolia kernels. Plant J. 19 (6), 699710.
Poltronieri, P., Cappello, M.S., Dohmae, N., Conti, A., Fortunato, D., Pastorello, E.A.,
Ortolani, C., Zacheo, G., 2002. Identication and characterisation of the IgEbinding proteins 2S albumin and conglutin (in almond (Prunus dulcis) seeds. Int.
Arch. Allergy Immunol. 128, 97104.
Rodriguez, J., Crespo, J.F., Lopez-Rubio, A., De La Cruz-Bertolo, J., Ferrando-Vivas, P.,
Vives, R., Daroca, P., 2000. Clinical cross-reactivity among foods of the Rosaceae
family. J. Allergy Clin. Immunol. 106, 183189.
Rouge, P., Brunet, E., Borges, J.P., Jauneau, A., Saggio, B., Bourrier, T., Rance, F., Didier,
A., Barre, A., 2011. Proteins with cupin motif as major seed allergens. Rev. Fr.
Allergol. 51, 3640.