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Allergy 2002: 57: Suppl. 71: 24–28
Printed in UK. All rights reserved
Copyright © Blackwell Munksgaard 2002
ISSN 0108-1675
Blackwell Science Ltd Ole e 4 and Ole e 5, important allergens of
Olea europaea
J. Carnés,
E. Fernández-Caldas
C.B.F. LETI, S.A. Research Laboratories,
Madrid, Spain
Enrique Fernández-Caldas
C.B.F. LETI, S.A.
Calle del Sol, 5
28760 Tres Cantos
The pollen of Olea europaea is one of the most
important aero-allergens in Mediterranean countries
and elsewhere, including the USA, Argentina
and Japan (1–4). The inhalation of the olive pollen
is one of the most common causes of sensitization
and allergic respiratory symptoms in genetically
susceptible individuals in areas where olive cultivars
are abundant.
Every year the high levels of olive pollen occur
from May to June and the quantities vary every
spring season, depending on the current climatic
conditions and the weather of the first months of the
year. As an example, in the Madrid area, the levels
of olive pollen fluctuated between 150 grains/m3
in 1995 and 2000 grains/m3 in 1999 (Fig. 1, adapted
from www.seaic.es).
In the last years there has been an increasing
interest in the identification, purification, characterization
and quantification of allergens in olive
pollen allergen extracts (5,6) and some allergens
of olive tree have been extensively studied. At the
present time, nine allergens of O. europaea have
been described, and several more await full characterization
and sequencing.
In 1998 Boluda et al. (7), described the allergenic
composition of different batches of olive
pollen collected in Spain and the United States,
and established qualitative and quantitative differences
in the concentration of some allergens, in
particular Ole e 1 and Ole e 4.
O. europaea is a tree belonging to the family
Oleacea. More than 30 genera and 600 species
have been described in this family. The worldwide
distribution of the different members of this family
depends mainly of the specific biological requirements
of the genera. It is therefore, a cosmopolitan
botanical family. Most genera and species belonging
to this botanical family are wind-pollinated trees
or bushes.
An important and interesting aspect of the
study of olive pollen allergy is the high degree of
cross-reactivity that exists among trees and plants
belonging to the same botanical family. Although,
O. europaea is rare in northern Europe, other
plants of the same family are very common in
Nordic regions. Olive tree pollen contains the
majority of the antigens triggering the clinical
response in patients whose primary sensitization
is to O. europaea. Other species, such as Fraxinus
excelsior (ash tree), Ligustrum vulgare (privet) and
Syringa vulgaris (lilac) contain similar allergens
(8,9). More recently we have also described allergenic
cross-reactivity between O. europaea and
Eleagnus angustifolia (Russian olive) (10), an ornamental
tree belonging to the family Eleagnaceae
and closely related to Olive trees. These species
seem to share not only Ole e 1 but also Ole e 4
homologs. E. angustifolia is widely planted in
Spain and other areas of the world, including
central and northern Europe. It is also frequent
in the margins of rivers and other humid areas
and pollinates in the spring. Its allergenicity has
been previously suggested in the USA by Kernerman
et al. (11). In our study conducted in Madrid,
Spain, all patients with a positive skin test to
Russian olive were also positive to olive; but not
all olive-positive patients were positive to Russian
olive. There was a good correlation between specific
IgE levels to Russian olive and olive (r = 0.8) in a
group of patients sensitive to both allergens. By
ELISA inhibition, the Russian olive extract was
not able to inhibit olive, whereas an olive extract
inhibited Russian olive up to 41%. This study
confirms the sensitizing capability of Russian
olive pollen in Spain. A minimal to moderate
cross-reactivity was established, suggesting some
cross-reactivity but not excluding cosensitization.
Antigenic and allergenic profile of O. europaea
pollen extract
The antigenic profiles of several batches of
O. europaea pollen show a wide range of proteins
from less than 10 to more than 100 kDa. Nine of
these proteins have been described as allergenic
and have a variable degree of specific IgE recognition.
Based on the amount of allergens present in
olive, we expect more allergens to be described in
the future. The isoelectric focusing pattern of the
extracts shows bands in the pI range of 4 and
9.5; the majority of the allergens have an acidic
isoelectric point. The majority of the described
allergens have several isoforms.
The allergenic profile of the olive pollen extracts,
as recognized by the specific IgE of olive pollen
sensitized patients shows several IgE binding
proteins/peptides. One of the most recognized
proteins corresponds to Ole e 1, a dimer protein
with a molecular weight of approximately 19 kDa.
A large proportion of allergens can be detected
between 30 and 60 kDa, and also a protein with
low a molecular weight of approximately 10 kDa,
described as Ole e 6. Allergen recognition varies
depending on the geographic location of the patients
and some discrepancies have been described between
patients exposed to large quantities of olive pollen
and patients residing in the Madrid area (12).
An important aspect of the study of Olea pollen
extracts is the allergenic variability of the extracts,
especially between those prepared with pollen
collected in Spain, and in the USA. Recent experiments
conducted in our Laboratory to compare
batches of Olive pollen extracts made with pollen
collected in California and Spain showed signifi-
cantly different allergenic and antigenic patterns.
While Spanish extracts showed a high concentration
of Ole e 1, the most predominant allergen in
American extracts revealed Ole e 4 as one of the
main allergens (Fig. 2). These intraspecies differences
among trees of the same species were
described some years ago by different authors
(13,14). Antigenic and allergenic differences in the
composition of olive pollen extracts have also
been suggested for different cultivars (15). These
variations may be due to the existence of more
than 260 hundred varieties of Olive trees (16). We
have recently conducted a study on the allergenic
variability of six different varieties during 4 years
and have identified important variations in the
allergenic composition of the six varieties and in
the same varieties during the 4 years of the study.
O. europaea pollen is the source of important
allergens. In areas where this plant is abundant,
the rate of sensitization in the total population is
high and comparable to the rate of sensitization
to grasses. Olive pollen is among the group of
pollens whose allergenicity is better characterized.
The degree of characterization of the allergenicity
of olive pollen is comparable to that of other well
studied allergen sources such as Betula verrucosa,
some grasses and Dermatophagoides pteronyssinus.
One of the main allergens of O. europaea is
Ole e 1, which was first described by Villalba et al.
(17,18). This allergen has two main variants. One
of them is nonglycosilated form and has a molecular
weight of 18.5 kDa. The second variant is a
glycosilated polypeptide with a molecular mass of
20 kDa. Both forms are widely recognized by the
sera of sensitized individuals. Ole e 2 is an olive
Figure 2. SDS-PAGE of 12 batches of Olea europaea. From
1 to 6 correspond to pollen collected in Spain and from 7 to
12 correspond to pollen collected in the USA.
Carnés and Fernández-Caldas
pollen profilin with a molecular weight approximately
15 kDa, described by Asturias et al. in 1997
(19) and Ledesma et al. in 1998 (20). Ole e 3 was
described by Batanero et al. (21) in 1996. It is a
protein with a molecular weight of 9.2 kDa and
with capacity to bind calcium. In 1998, Boluda
et al. (22) purified and partial sequenced Ole e 4
and Ole e 5 simultaneously. Ole e 5 shows a high
degree of homology with superoxide dismutase of
A cysteine enriched allergen, designed as Ole e 6,
was described in 1997 by Batanero et al. (23). The
allergen has a molecular weight of approximately
10 kDa and exhibits a high capacity to bind IgE.
Ole e 7 exhibits a high degree of polymorphism.
Its molecular weight varies between 9.8 and
10.3 kDa and has no homology with any known
protein. Ole e 7 was described in 1999 by Tejera
et al. (24). Ole e 8 is a protein around 21 kDa with
capacity to bind calcium. It was described by
Ledesma et al. (25) in 2000. Ole e 9 is the most
recently described allergen of O. europaea pollen.
It is a b 1,3 glucanase and it has been described
by Huecas et al. (26). The allergen has a molecular
weight of approximately 46.4 kDa and is involved
in the allergic response of 65% of patients with
olive tree pollinosis.
Ole e 4 and Ole e 5
Boluda et al. described the allergenic composition
of several batches of olive pollen collected in the
United States using Spanish sera (7). The IgE
binding pattern of these pollen batches differed
from previous reports, especially when Spanish
pollen was used (27,28). A great variability in the
allergenic composition of O. europaea extracts
has been described and it has been demonstrated
that olive tree from different cultivars produce
pollen with different allergenic characteristics. It
has been suggested that the allergenicity of the
olive tree pollen could depend from the variety of
the olive tree (29). This fact may have important
consequences from an allergological point of
view. Since different varieties may produce pollen
with a variable allergen load, olive pollen sensitive
patients may experience a stronger or weaker allergic
response after the inhalation of olive pollen,
depending on the variety of the olive pollen
inhaled. It may also have implications when discussing
pollen counts in different regions, since
some varieties could produce more allergenic
pollen than others, thus, needing smaller amounts
of pollen to induce sensitization and symptoms.
Therefore, exposure to pollen may not be synonymous
of antigen exposure.
Boluda et al. demonstrated, using American
olive pollen, that the frequency of IgE binding to
Ole e 1 was low (40%) (7), in contrast with previous
studies that reported an IgE binding frequency
higher than 80% (27). These differences in the
allergenic composition can be attributed to the
source of the pollen used, the variety of the olive
tree and to the content of specific allergens. The
Ole e 1 content of some of these extracts was
15 mg of Ole e 1/mg of protein. This quantity can
be considered as low, since extracts from other
olive tree pollens may contain more at least three
times more allergen per mg of freeze dried extract.
Extracts prepared using pollen collected in Spain
regularly contain higher concentrations of Ole e 1.
The clinical implications of these finding remain
to be established.
We concentrated our efforts towards the puri-
fication of Ole e 4, because this allergen shows a
high capacity of IgE binding, and Ole e 5, a new
described allergen with a molecular weight of
16 kDa. Ole e 4 has, at least two isoforms in the
pI range of 4.6–5.1. Ole e 5 has, at least five isoforms
with isoelectric points ranging from 5.1 to
6.5, similar to Ole e 1 (30). Thus, Ole e 5 shows a
higher degree of polymorphism than Ole e 4.
IgE binding to Ole e 4 and Ole e 5 was studied
by immunoblotting, direct IgE binding and IgE
inhibition. The allergenicity of Ole e 4 and Ole e 5
was not altered during the chromatographic steps,
because they retained their ability to bind specific
IgE. Ole e 4 fixed higher amounts of IgE than
Ole e 5 by immunoblotting. However, these allergens
captured similar amounts of specific IgE in
solid phase assays. In IgE inhibition experiments,
Ole e 4 exhibited a relatively high inhibition capacity;
the inhibition capacity of Ole e 5 was lower.
The two proteins were sequenced to obtain the
primary structure of the antigens. Ole e 4 was
fragmented with trypsin and the sequences of two
internal fragments of Ole e 4 did not show any
homology with other known proteins, at the time
of publication, based on the Protein Sequence Data
Bank (SWISS-PROT). However, the N-terminal
sequence of Ole e 5 showed a high degree of
homology with superoxide dismutases (SOD) of
several plant species and less homology with
SODs from distant phylogenetic species (Tables 1
Table 1. Amino acid sequence of two internal regions of Ole e 4
Ole e 4 Fragment 1 … AFANTGVEIV …
Ole e 4 Fragment 2 … SIDTYLFSLYDEDK …
Ole e 4 and Ole e 5, allergens of O. europaea
and 2). It is well established that SOD and other
elements implicated in oxidative processes are
implicated in allergic diseases, specially asthma
(31,32). In the last few years, SOD, in particular
MnSOD, has been identified as an allergen in
different organisms, such as Hevea brasilensis
(Hev b 10) (33) or Aspergillus fumigatus (34).
Ole e 5, Hev b 10 and the allergen of A. fumigatus
are the only allergens described so far with SOD
Table 2. N-terminal sequence alignments of Ole e 5 with SOD sequences of different species
10 20 30 %
Ole e 5 ( Olea europaea) VKAVTVLNSSEGPHGIVYFAQEGDGPTTV …
Spinacia oleracea (spinach) M±KAV± VL±SSEG ±± G±VYFAQEGDGPTTV … 79.3
Ipomoea batatas (sweet potato) VKAV± VL± SSEG ±± G± ± ±F± QEGDGPTTV … 72.4
Zea mays (maize) VKAV± VL± SSEG ±± G± ± ±F± QEGDGPTTV … 72.4
Oryza sativa (rice) VKAV± VL± SSEG ±± G± ± ±F± QEGDGPT±V … 68.9
Lycopersicum esculentum (to
VKAV± ± LNSSEG ±± G± ± ±F± Q± G± ± PTTV … 65.5
S. cerevisiae VKAV± VL± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±TV … 27.5
200 210 220
Escherichia coli VKAV± VL± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± G ± ± ± ± ± ± ± … 24.1
320 330 340
Mus musculus (mouse) TVK ± ± TVL ± ± ± ± ± P ± ± ± V ± ± ± ± ± ± ± ± ± ± ± ± ± ± … 24.1
280 290 300
Homo sapiens FVK ± VTVL ± ± ± ± G ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± … 24.1
14 20 30 40
Bos taurus (bovine) V ± A ± ± VL ± S ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± G ± ± ± ± ± ± … 20.7
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