Allergenic Diversity among Plant and Animal Food Proteins

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Allergenic Diversity among Plant and
Animal Food Proteins
a
a
a
Sandeep Kumar , Alok Kumar Verma , Mukul Das & Premendra D.
Dwivedi
a
a
Food, Drug and Chemical Toxicology Group, CSIR–Indian Institute of
Toxicology Research (CSIR-IITR), M.G. Marg, Lucknow, India
Available online: 21 Feb 2012
To cite this article: Sandeep Kumar, Alok Kumar Verma, Mukul Das & Premendra D. Dwivedi (2012):
Allergenic Diversity among Plant and Animal Food Proteins, Food Reviews International, 28:3, 277-298
To link to this article: http://dx.doi.org/10.1080/87559129.2011.635391
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Food Reviews International, 28:277–298, 2012
Copyright © Taylor & Francis Group, LLC
ISSN: 8755-9129 print / 1525-6103 online
DOI: 10.1080/87559129.2011.635391
Allergenic Diversity among Plant and Animal
Food Proteins
SANDEEP KUMAR, ALOK KUMAR VERMA, MUKUL DAS,
AND PREMENDRA D. DWIVEDI
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Food, Drug and Chemical Toxicology Group, CSIR–Indian Institute
of Toxicology Research (CSIR-IITR), M.G. Marg, Lucknow, India
A large number of food allergens, usually proteins capable of inducing allergic
symptoms, including severe, even life-threatening reactions in predisposed individuals,
have been identified and characterized. As most of these proteins are from our daily
dietary intake, they are often difficult to avoid. However, the proteins that cause such
immunoglobulin E (IgE)-mediated reactions can be assigned to only a limited number of protein families. Detailed knowledge about the characteristics of food allergens,
their structures, biological activity, and stability, may be helpful in improving diagnosis of food allergy, avoiding unnecessary exclusion of diets, and assessing the risk of
cross-reactive allergies to other food sources. The purpose of this review is to shed light
on the sources and molecular properties of the allergenic proteins, their stability, the
mechanisms of the allergenic responses, and recent findings related to prevention of
this serious issue.
Keywords Anaphylaxis, 2S albumins, IgE-binding proteins, Disulfide bonds, Epitope
mapping, Food allergy
Introduction
Food allergy, a worldwide health problem, is caused by abnormal immunological responses
to certain foods, usually proteins. It is a form of adverse reaction to food in which the cause
is an immunological response. Though diversity of the human diet is enormous, only a
small number of foods account for the majority of food allergies around the world. Milk,
egg, and peanut account for the vast majority of food-induced allergic reactions in children,
whereas peanut, tree nuts, fish, and shellfish account for most of the food-induced allergic
reactions in adults. Food allergies are ranked by the World Health Organization (WHO)
as the sixth problem of human health. Food allergies pose an increasing health risk for
the population of industrialized countries, with around 5–6% of children and up to 2%
of adults(1) being thought to suffer from some type of immunoglobulin E (IgE)-mediated
food allergy. Incidence of food allergy has been increasing in developed as well developing
countries of the world. In the United States, it is estimated that 125–150 people die each
year(2) as the result of food anaphylaxis.
In the last decade, great efforts have been undertaken to identify the characteristics of nontoxic food proteins that evoke IgE-mediated allergic response in predisposed
Address correspondence to Dr. Premendra D. Dwivedi, Scientist, Food Toxicology Division,
CSIR–Indian Institute of Toxicology Research (CSIR-IITR), P.O. Box No. 80, Mahatma Gandhi
Marg, Lucknow-226 001, India. E-mail: pddwivedi@yahoo.com
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individuals and many hundreds of allergens have been identified in a variety of animaland plant-derived foodstuffs. As a consequence, in the recent past a number of allergen
databases have been set up to collect and curate the existing data on allergens, their
physicochemical properties, and their allergenic relevance. Some of these are collections
of allergen sequences, such as AllergenOnline (http://www.allergenonline.org/) and the
International Union of Immunological Societies (IUIS) allergen database (http://www.
allergen.org/). The IUIS database is dedicated to providing a systematic nomenclature for
allergens that allows their unequivocal identification, and upon evidence of IgE binding
activity of the allergenic molecules being provided, the allergen nomenclature subcommittee grants official allergen designations. Other allergen databases are linked to data
on clinical reactivity of foods, such as InformAll (http://www.foodallergens.ifr.ac.uk/),
whereas the Allergome database represents a rapidly updated, nonpeer-reviewed repository of information (http://www.allergome.org/). Another type of database is represented
by Allfam,(3) which groups allergens according to their protein family characteristics
(http://www.meduniwien.ac.at/allergens/allfam/).
Plant Foods with Allergenic Proteins
The Pfam protein family database(4) is a large collection of protein families. The Pfam
database classifies plant protein sequences into families on the basis of sequence homology, which is related to conserved three-dimensional structures and possibly function.
It has become increasingly obvious that almost all plant food allergens are either storage
or defense-related proteins. Strikingly, only three dominating plant food allergen protein
families/superfamilies have been identified, (1) the prolamin superfamily, (2) the cupin
superfamily, and (3) the Bet v 1 family. Common plant foods having allergenic proteins
(Table 1) include peanuts, tree nuts, and soybeans. Peanut (Arachis hypogaea) and soybean (Glycine max) have long been recognized as highly allergenic and are responsible for
IgE-mediated clinical reactions in humans.
The following sections provide details of major plant food allergens families.
The Prolamin Superfamily
This protein superfamily comprises three major groups of plant food allergens: 2S albumins, nonspecific lipid transfer proteins (nsLTPs), and cereal α-amylase/trypsin inhibitors.
These are low-molecular-weight proteins, cysteine rich, have similar three-dimensional
folds that are rich in α-helices, and are stable to thermal processing and proteolysis.
2S albumins. The 2S albumins are a major group of storage proteins present in many
mono- and dicotyledonous plants. 2S albumins can also play a protective role in plants
as defensive weapons against fungal attack. In recent years, some members of this protein family(19) have been described as major food allergens demonstrating their ability
to bind IgE from the sera of allergic patients. Examples include Arabidopsis albumin,
radish albumin, oilseed rape albumin, caster bean albumin, walnut albumin, Brazil nut
albumin, sunflower albumin (SFA8), cotton seed albumin, Arachis hypogea 2 (Ara h2),
Arachis hypogea 6 (Ara h6), soybean albumin 1 (soy alb1) and soybean albumin 3
(soy alb3).
Nonspecific lipid transfer proteins (nsLTPs). The nsLTPs play an important role in plant
defense against fungi and bacteria. These have been identified as major fruit allergens in
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Table 1
Major plants allergenic proteins
Sample
no.
1
2
3
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4
5
6
7
8
9
10
11
12
13
14
Plants (botanical name)
Major allergens
Peanut (Arachis hypogaea)
Ara h 1 (63.5 kDa), Ara h 2 (17–19 kDa), and
Ara h 3 (64 kDa)(5)
Soybean (Glycine max)
Gly m Bd (30 kDa), Gly m Bd (28 kDa), and
Gly m Bd (60 kDa)(6)
Lentil (Lens ensculenta)
Allergens of MW 72, 70, 68, 54, 52, 40, 38, 30,
21, 18, and 16–12 kDa(7)
Chick pea (Cicer arietinum)
Allergens of MW lesser than 20, 70, 64, 62, 53,
51, 36–35, 28–26, and 22–20 kDa(8)
Red gram (Cajanus cajan)
Caj c1 (66 kDa), Caj c2 (45 kDa), Caj c3
(45 kDa), Caj c4 (45 kDa), and Caj c5
(30 kDa)(9)
Green gram (Vigna radiata)
Vig r2 (52 kDa), Vig r3 (50 kDa), Vig r4
(30 kDa), and Vig r5 (18 kDa)(10)
Green bean (Phaseolus spp.)
Proteins of MW 71, 47, and 41 kDa(11)
String bean (Phaseolus spp.)
A protein of MW 35 kDa(11)
Kidney bean (Phaseolus spp.) 170, 100, and 43, 34 and 20 kDa (basic subunit
of legumin)(12,13)
Lima bean (Phaseolus lunatus) 12 proteins of 18–96 kDa(14)
Lupin (Lupinus albus)
Proteins of MW 78, 68, 65, 52, 45, 32, 18,
16.5, and 14 kDa(15)
Melon (Cucumis melo)
A protein of MW 13 kDa(16)
Cherry (Prunus avium)
Pru av 3 (nsLTP), Pru av 1 (Bet v
1 homologue), and Pru av 4 (profilin)(17)
Apple (Malus domestica)
Natural Mal d 1 (nMal d 1)(18)
Note. Major plants allergenic proteins. MW = molecular weight of protein. Ara h = Arachis
hypogea; Gly m = Glycine max; Caj c = Cajanus cajan; Vig r = Vigna radiata; Pru av = Prunus
avium; Mal d = Malus domestica. Superscript numbers are their respective references.
patients from the Mediterranean area. The typical structure of nonspecific LTPs is based
on four disulfide bridges, which contribute to the overall stability of these proteins against
enzymatic digestion or thermal denaturation, although the stability is pH dependent. Under
acidic conditions, thermal denaturation of allergen of peach (Prunus persica) Pru p 3 was
reversible, whereas under neutral conditions Pru p 3 was unable to refold after cooling.(20)
Sensitization to nsLTPs is accompanied by severe reactions, possibly because of specific
biophysical and biochemical properties of this allergen family. The high stability to proteolytic digestion of nsLTPs is thought to be a possible reason for more severe and systemic
reactions(21) compared with other allergens that are digestible in gastrointestinal fluid. They
have a wide distribution in fruits, nuts, seeds, and vegetables and have attracted much
interest for being highly important allergens for almost exclusively Mediterranean atopic
populations. Examples are wheat LTP, rice LTP1, maize LTP.
α-Amylase/trypsin inhibitors. The family of cereal α-amylase and protease inhibitors
mediates a certain degree of resistance to insect pests that feed on plant tissues. Just like
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the 2S albumins and the nsLTPs, the members of this protein family are capable of sensitizing susceptible atopic individuals through ingestion or inhalation. This group of proteins is
found in cereals and sensitize individuals via the lungs, giving rise to occupational allergies
such as bakers’ asthma (wheat, barley, and rye), or via the gastrointestinal tract, resulting
in food allergies (wheat, barley, and rice).
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Cupin Family
Cupins (Latin cupa means small barrel) vary widely in sequence but are characterized by
two short consensus sequence motifs and a core structural feature—a barrel-like, doublestranded β-helix.(22) The cupin superfamily comprises the major globulin storage proteins
mainly from legumes and nuts. The globulins are divided into the 7S vicilin-like globulins
and the 11S legumin-like globulins. Globulins have been found to be highly relevant
allergens in plant foods including peanuts, soybean, lentils, walnut, hazelnut, and sesame.
Despite having very low levels of sequence identity, members of the cupin superfamily
have highly conserved structures.
Bet v 1 Family
Bet v 1 was the first of many allergens published that showed homology to the family
of the pathogenesis-related proteins. Bet v 1–type allergens are rather unstable to heating
and digestion. Consequently, symptoms are mostly restricted to the oral cavity. In general,
Bet v 1 from birch pollen acts as the primary sensitizing agent. The overall high levels
of conserved surface residues between the members of the Bet v 1 family play an important role in conservation of IgE-binding epitopes and underlie the fruit–vegetable-pollen
cross-reactive syndromes. Individuals with pollen allergy frequently suffer from allergic
symptoms after eating certain plant foods. The majority of these reactions are caused
by allergens of Rosaceae fruits (examples include apple, cherry, apricot, and pear) and
Apiaceae vegetables (examples include celery, carrot), which cross-react with allergens
that are present in birch pollen, particularly the major birch pollen allergen Bet v 1.
Other Plant Food Allergen Families
Besides the above-mentioned types, other allergens have been identified belonging to less
widespread allergen families. A few selected protein families are presented in the following
sections.
Profilins. Profilins are present in all eukaryotic cells. These small (12–15 kDa) proteins
are located in the cytosol and act as actin-binding proteins. They may play a key role in
regulating intracellular transport processes and cell morphogenesis and division. Profilinencoding genes form a small gene family as shown for apple profiling.(23) Sequence
similarity among profilins from lower eukaryotes, plants, and animals is low, whereas
profilins of higher plants share 75% and more of their sequence. All profilins have a
compact globular structure(24) consisting of a central seven-stranded, antiparallel β-sheet
enclosed by the N- and C-terminal α-helices on one side and one or two helices on the other
side. The plant homologues are somewhat divergent, notably with a slightly longer solventexposed loop between the N-terminal α-helix and the first β-strand, which is more variable
and represents part of an IgE epitope in the allergenic profilin from birch pollen, Bet v
2.(25) It is generally assumed that 10–20% of tree pollen allergic patients display allergic
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symptoms to profilin. The high sequence and structural similarity of profilins from even
distantly related plants accounts for the IgE cross-reactivity. Although the observed IgE
cross-reactivity does not correlate with clinical symptoms, profilin sensitization is regarded
as a potential risk for developing multiple pollen-associated food allergies. When subjected
to heat treatment, irradiation, or ultrahigh pressure, the IgE binding activity of profilin from
celery was not affected. However, when the pH was lowered and gastric fluid assays were
performed,(26) profilins were readily degraded and displayed no further allergenic activity,
as has been shown for allergens of apple (Malus domestica), Mal d 4, and muskmelon
(Cucumis melo), Cuc m 2. Profilins from various botanical species such as apple, hazelnut,
peanut, celery, and wheat have been included in the EuroPrevall allergen library.
Oleosins. These proteins are considered to contribute towards stabilizing plant lipid
storage bodies and represent the proteinaceous component. Recently, oleosins(27) with
allergenic activity were identified from legumes, nuts, and seeds.
Pathogenesis-related proteins. These proteins are up-regulated within plants upon
pathogen attack or exposure to abiotic stress factors. Surprisingly, a considerable number of food allergens have been identified from various families of pathogenesis-related
proteins,(28) such as the β-1,3-glucanases, various types of chitinases, and the thaumatinlike proteins. Plant (class 1) endochitinases are able to hydrolyze chitin of fungal cell
walls and the exoskeleton of hexapods and can thus play a role in plant protection against
pathogen and pest attack. These glycoside hydrolases (usually 25–35 kDa) contain a
domain binding to chitin subunits, which is called the “hevein-like domain.” This domain,
characteristic for this protein family, is rich in cysteine residues, comprises 40 amino
acid residues, and is located at the N terminus. The enzymatic activity of chitinases(29)
is determined by a catalytic domain of 220–230 amino acid residues. Food allergens of the
pathogenesis-related protein family 5 group have been found in several fruits,(30) such as
cherry (Pru av 2), apple (Mal d 2), kiwi (Act d 2 green kiwi, Act c 2 gold kiwi), and orange,
grape, and bell pepper (Cup a 1).
Glycoproteins. Glycoproteins have been also reported as allergenic. Some of the 23-kDa
peptides were shown to be glycoproteins with an N-linked glycan moiety, binding to IgE
antibodies in the sera of patients sensitive to soybean. The binding of the peptides to IgE
antibodies was suggested to be predominantly dependent on their glycan moiety.(31)
Thaumatin-like proteins. These proteins were named after the intensely sweet tasting protein thaumatin that originated from the African shrub Thaumatococcus daniellii, described
as long ago as 1852. In plants, they are synthesized upon biotic and abiotic stress and in
certain developmental stages, particularly during fruit ripening. Thaumatin-like proteins
have a molecular mass around 20 kDa, form antiparallel β-sheets, and are stabilized via
eight disulfide bonds.(32) Owing to their rigid structure formed by the disulfide bridges,
they are resistant to heat treatment and photolytic degradation.
Animal Foods with Allergenic Proteins
Common animal foods having allergenic proteins are eggs, cow’s milk, and fish. Milk,
often the first food for newborns, can lead to cow’s milk allergy (CMA), defined as
an immunologically mediated reaction against cow’s milk antigens. Childhood CMA is
the third most prevalent food allergy in France,(33) with approximately 9% of the total
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allergies diagnosed. The most abundant milk proteins are αS1 -, αS2 -, β-, and κ-caseins,
α-lactalbumin, and β-lactoglobulin (β-LG) and these are the main allergens in milk.(34)
Other proteins present in milk in lower amounts, such as bovine serum albumin, lactoferrin,
and IgG heavy chain, are also recognized as allergenic by CMA patients.
Hen eggs are one of the most frequent causes of adverse reactions to animal foods.
Study of allergic reactions(35) has shown that they are more frequently caused by egg white
proteins than egg yolk. Approximately two-thirds of children diagnosed with food allergies
are reactive to egg white. The estimated prevalence of egg allergy(36) varies between 1.6%
and 3.2%, making it the second most common cause of food allergies in children. The main
egg allergens are proteins and are mainly present in the egg white (ovalbumin, ovomucoid,
ovotransferrin, and lysozyme). However, egg yolk (for example, R-livetin) also displays
low-level allergenicity. Strict avoidance of the offending food remains the most common
recommendation for egg allergic individuals.
The allergenic potential of shellfish is due to the presence of tropomyosin, which has
been found to be allergenic in nature. It is a heat-stable protein essential for muscle contraction in vertebrates and invertebrates.(37) The molluscan shellfish allergens, such as oyster
(Cra g 1, Cra g 2), abalone (Hal m 1), snail (Tur c 1), and squid (Tod p 1), have been
identified as tropomyosins.(38) Hindered digestion puts patients with fish allergy at risk to
develop severe allergic reactions to even minute amounts of allergens. Fish represents one
of the most important allergenic foods causing severe allergic reactions. Nevertheless, it has
been shown that gastric digestion significantly reduces its allergenic capacity. Incomplete
digestion of codfish(39) represents a risk factor for anaphylaxis in patients with allergy.
The following sections provide details of major animal food allergen families.
Tropomyosins
Tropomyosins in invertebrates are 34–41 kDa proteins that share high homology in their
amino acid sequences(40) and are responsible for the majority of IgE-mediated allergies to
shellfish.(41) Interestingly, tropomyosins in vertebrates are not allergenic. At least 10 different tropomyosins in crustacean shellfish and six different tropomyosins in molluscan
shellfish have been identified. Recently, in addition to tropomyosins, three new classes
of shrimp allergens have been identified, including arginine kinases (Pen m 2 and Lit
v 2), sarcoplasmic calcium-binding proteins (SCP and Lit v 4), and myosin light chain
(Lit v 3).(42)
Caseins
Caseins are mammalian proteins present in milk that bind calcium via a cluster of phosphoserine and phosphothreonine residues of αs1 -casein, αs2 -casein, and β-casein. These types
of caseins form clusters around amorphous calcium phosphate,(43) increasing the calcium
level in milk. κ-Casein stabilizes these nanostructures. αs1 -Casein and αs2 - casein seem to
be the most important allergens, followed by β-casein. The caseins display a random-coil
structure. According to the literature, IgE cross-reactivity between cow’s milk caseins and
caseins from goat and sheep has been demonstrated,(44) whereas mare’s milk caseins seem
to represent a lower risk of IgE cross-reactivity. Since the caseins are all highly phosphorylated and heterogenous, the naturally derived casein fraction from a single genotyped
cow was included in the EuroPrevall allergen library. In parallel, casein from goat’s milk
was purified and the two fractions were subsequently compared regarding folding characteristics and composition. Purified cow’s milk casein consisted primarily of αs1 -casein
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283
and β-casein and only low amounts of αs2 -casein and κ-casein. In contrast, the purified
goat’s milk casein fraction contained primarily β-casein, much lower levels of variants of
αs1 -casein, and only traces of κ-casein and αs2 -casein.
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Other Animal Food Allergen Families
A number of other allergens have been identified belonging to less widespread allergen
families. For example, another milk allergen, β-lactoglobulin (Bos d 5), belongs to
the lipocalin protein family. Members of this protein family share a conserved threedimensional (3D) structure but the overall sequence similarity is low. They serve as carriers
for a range of small molecules such as lipids, steroids, hormones, bilins, and retinoids.(45)
α-Lactalbumin (Bos d 4) is a member of the C-type lysozyme/α-actalbumin family.
It is able to bind calcium and is involved in lactose synthesis in cow’s milk and has a
superimposable 3D structure with hen egg lysozyme.
Other minor allergen families include the Kazal-type protease inhibitors, which are
represented by the hen’s egg white allergen ovomucoid,(46) Gal d 1, a major allergen from
egg white. This protein is extensively glycosylated, which may act to stabilize the protein
against proteolysis.
Allergenic proteins are also reported from the serine protease inhibitors or serpins
(SERine Protease INhibitors) family. One of such examples is hen egg (Gallus domesticus)
allergen ovalbumin, Gal d 2.(47) EF-hand domain (a helix-loop-helix structural domain
protein family) share a conserved domain of a Ca2+ binding residue loop of 12 amino
acid residues in length and two neighboring α-helices of 12 amino acids. Some allergens
from fish and amphibians are EF-domain proteins. Lastly, transferrins, sulfur-rich ironbinding glycoproteins, have been identified as minor allergens in milk (lactoferrin) and in
egg (ovotransferrin, Gal d 3).(48)
Molecular Properties of Proteins and Allergenicity
Foods contain a wide variety of proteins, yet only a few are allergens. The reason why some
proteins are highly allergenic and others are not remains poorly understood, but certain
chemical and physical properties appear to be associated with allergenicity. Most allergens
are said to have a molecular weight of 10–70 kDa and are typically stable to changes in heat
and pH, and to digestion. They generally have an acid isoelectric point (pI) and are soluble
for absorption across the gastrointestinal tract. However, many nonallergenic proteins also
show these properties. Moreover, many allergens do not have these properties; for example,
profilins are not stable to digestion and lipid transfer proteins do not have an acid pI. Several
attempts to categorize plant food allergens on the basis of their three-dimensional structure,
biological function, or protein families have been carried out.
Abundance
Seeds and nuts contain storage proteins that may account for 50% or more of the total
proteins in the organ. Most major food allergens that sensitize via the gastrointestinal tract
are present in at least 10% of the total protein content(49) of plant foods. However, some
proteins that are present in all plants in large quantities, such as the enzyme ribulose-1,5bisphosphate carboxylase, which accounts for 30–40% of total leaf protein, have never
been reported as allergens. In contrast, nonspecific lipid transfer proteins (nsLTPs) are
potent allergens, but are not very abundant. Thus, the amount of protein alone does
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not explain its allergenicity. Although abundance is an important factor, it is probably
secondary to protein stability.
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Stability to Processing and Digestion
A compact three-dimensional structure, ligand binding, disulfide bonds, and glycosylation
contribute to protein stability. These factors are relevant to both the resistance of proteins
to denaturation by food processing and the harsh conditions of the gastrointestinal tract.
Ligand binding can have the overall effect of reducing mobility of the polypeptide backbone, increasing both thermal stability and resistance to proteolysis. Some proteins form
a cavity whereas others possess a tunnel into which a ligand can fit. One of the structural features clearly related to stability is the presence of disulfide bonds. Both inter- and
intrachain disulfide bridges constrain the three-dimensional fold such that perturbation of
the structure by heat or chemicals is limited and frequently reversible. Important plant
food allergens that have high numbers of disulfide bonds include members of the prolamin
superfamily (nsLTPs, 2S albumins, cereal α-amylase/trypsin inhibitors) as well as of the
pathogenesis-related proteins (class I chitinases, thaumatin-like proteins). N-glycosylation
can have a significant stabilizing effect on protein structure.
Digestion assays with simulated gastric fluid have been introduced for characterization of food proteins to predict the effect of stomach proteolysis on dietary compounds in
vitro. Gastric digestion substantially decreases the potential of food proteins to bind IgE,
which increases the threshold dose of allergens required to elicit symptoms in patients with
food allergy. Stability to digestion has been considered by many as one of the properties
shared by food allergens. Resistance of proteins to pepsin digestion has been proposed as
a marker for potential allergenicity because it does appear to be a characteristic shared by
many food allergens. A number of food allergens have been shown to be stable to conditions simulating human gastrointestinal digestion. Examples are β-lactoglobulin A (milk),
β-conglycinin (β-subunit), Gly m1, trypsin inhibitor, soy lectin (soybean), tropomysin
(shrimp), Pn lectin (peanut), ovalbumin and conalbumin (egg), Ara h 1 and Ara h 2 (Arachis
hypogea),(50) red kidney bean,(13) red gram,(9) and green gram.(10)
Thermal processing may alter (increase or decrease) the allergenicity of a protein, but
the overall effect on a complex food allergen cannot be predicted.(51) In addition, interactions with other constituents of the food matrix may occur and have no major effect on the
overall allergenicity of the food. Pastorello et al.(52) did not observe any loss of IgE-binding
capacity in a lipid transfer protein (LTP) of maize after a thermal treatment at 100 ◦ C for
160 minutes. It has been also shown that dry processing at 100 ◦ C for up to 90 minutes had
no effect on the allergenicity of some hazelnut proteins, suggesting the existence of very
heat stable allergenic proteins with molecular weight less than 14 kDa.(53)
Interaction of Protein with Lipid Structures and Aggregation
Many plant food allergens are able to attach with cell membranes or other types of lipid
structures found in food or show a propensity to aggregate as a result of food processing. The allergenic 2S albumin from mustard was shown to interact with phospholipid
vesicles.(54) This led to the proposition that such interactions might affect the uptake and
processing of the allergen in the gastrointestinal tract, indicating that the biologic activity
of these proteins plays a role in attenuating their allergenic potential. Similarly, there is
evidence that nsLTPs can interact with lipid structures as well.(55) A propensity of certain
proteins to aggregate might affect their ability to sensitize by generally enhancing their
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285
immunogenicity. Both 7S and 11S globulins are highly thermostable and it seems that the
cupin barrel remains intact during heating, but the unfolding of other regions of the protein
results in a loss of structure, leading to formation of large aggregates.(54) Peanuts are often
subjected to thermal processing at low water levels such as roasting. Thus, peanut proteins
become more thermo-stable in low water systems, whereas at the same time glycation reactions cross-link individual molecules and increase their allergenic activity. Interaction with
lipids and the formation of larger aggregates contribute to the allergenicity of plant food
proteins in conjunction with the amount of protein ingested and the stability to processing
and digestion.
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Cross-Allergies
The term cross-allergy refers to cross-reaction between different foods and cross-reactions
between foods and nonfood items. Most studies of cross-reactivity are based on skin
prick and IgE antibody test results. In terms of cross-reactivity, patients allergic to lentil
presented skin reactions to chickpea (67–80%), garden pea (22–54%), and green bean
(11%).(56) Patients allergic to chickpea had clinical signs when orally challenged with
lentil (84%), pea (68%), and peanuts (10%).(57) Another protein family known to crossreact with pollen allergens is the profilin family. Fruits, such as cherry (Pru av 4) and pear
(Pyr c 4), and celery (Api g 4) cross-react with the birch pollen Bet v 2 and may cause
allergic symptoms in pollen-sensitized patients.(58) Profilin Mus xp 1 from banana showed
a high IgE cross-reactivity with birch pollen profilin, Bet v 2, and latex profilin, Hev b
8.(59) The marked antigenic similarity between the proteins in the milk of cows, goats,
sheep, and horses means that almost all subjects who are allergic to cow’s milk protein
are allergic to the milks of these other animals. The eggs from turkeys, duck, goose, and
seagull all contain ovalbumin, ovomucoid, and ovotransferrin, the major allergens in hen’s
eggs. Mesquite tree (Prosopis juliflora) and lima bean (Phaseolus lunatus) belong to the
family Leguminosae. There are reports suggesting that lima bean cross-reacts with other
allergenic legumes, such as soya, peanut, and black gram based on skin test reactivity.(60–62)
Cross-reactivity to tropomyosin from other molluscan shellfish species has been observed
with sera from patients allergic to oysters, suggesting that individuals with allergies to
molluscan shellfish should avoid eating all species of molluscan shellfish.(38) The hevein
domain has been identified as a cross-reactive determinant between hevein, from Hevea
brasiliensis latex, and food allergens from avocado (Pers a 1), chestnut (Cas s 5), grape
(Vit v 5), and banana. All these allergens account for the latex-fruit syndrome. The catalytic domain of these proteins displays rather low IgE-binding capacity.(63) Another type
of plant defense is conducted by β-1,3-glucanases. These glycosyl hydrolases share an
(αβ) 8 triosephosphate isomerase (TIM) barrel structure and are usually 25–35 kDa in size.
They catalyze hydrolysis of 1,3-β-D-glucosidic linkages in β-1,3-glucans, and are abundant
in plant cell walls.(64) Allergens from this protein family have been identified from avocado,
banana, chestnut, fig, and kiwi and inhalant allergens are known from olive pollen and latex
contributing to the latex-fruit syndrome.
Mechanism of Food Allergy
Food allergy mainly occurs through IgE-mediated reactions, although non–IgE-mediated
reactions, such as IgE plus IgG–mediated reactions or immune complex–mediated
responses, may also occur (Fig. 1). In non–IgE-mediated responses, adverse reactions
occur through an immune response other than IgE. For example, in some cow milk allergy
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Different mechanisms of food allergies
IgE mediated reactions
Mixed IgE and cell mediated
reactions
Cell mediated reactions
Other reactions
Immediate type-1
hypersensitivity mediated
by IgE, in Th2 dominated
environment.
Mediated by IgG
and IgM along with
IgE. Phagocytosis and
immediate type
reactions occur.
Antibody-dependent cytotoxic
hypersensitivity is both IgG and
IgM mediated. The antibody
binds to cell-bound antigen,
leading to phagocytosis.
Non immunological
mediated reactions
Oral allergy syndrome,
Gastrointestinal
anaphylaxis,
Atopic dermatitis,
Nasobronchial allergy
Edema in uvula
Allergic eosinophilic
esophagitis,
Allergic eosinophilic
gastroenteritis
Atopic dermatitis
Asthma
Enterocolitis,
Proctocolitis,
Enteropathy
syndromes,
Celiac disease,
Contact dermatitis,
Transport deficiency
e.g., glucose/galactose
malabsorption
Enzymatic deficiency
e.g., Lactase
deficiency
Figure 1. The different types of allergic reactions. (color figure available online.)
(CMA) cases, allergic reactions are mediated by IgG. Immune complex–mediated reactions are caused by combination of antigen to IgE, IgG, and/or IgA antibodies. Due to its
predominance, the mechanism of IgE-mediated reactions is given below.
IgE-Mediated Reactions
These responses are the easiest to diagnose among food allergy responses because they are
the most prevalent. IgE-mediated reactions (type I) are also known as immediate hypersensitivity reactions or anaphylaxis because symptoms appear in less than a minute to a
few hours after the ingestion of the offending foods.(65) IgE-mediated reactions occur in a
stepwise manner (Fig. 2), as described below.
Primary exposure of allergens. After primary exposure of allergens in the body, allergens
are captured by antigen-presenting cells (APCs) and are degraded to peptide fragments.
These peptides are then presented by major histocompatibility complex II (MHC-II)
molecules, which are located in the antigen-presenting cells. MHC-II- peptide fragments
are recognized by clusters of differentiation-4 (CD4+) molecules present on the surface
of T helper 2 (Th2) cells. The activated Th2 cells secret cytokines like interleukin-4 (IL4) and interleukin-13 (IL-13). These cytokines and some co-stimulatory factors(66) cause
class switching in the naïve B cells to IgE and memory cells. The abundance of IL-4–
producing cells in bronchial biopsies and in periphery of atopic individuals with specific
IgE for common environmental aeroallergens illustrates the involvement of this functional
subset of CD4+ T cells in allergic diseases.(67,68)
Priming of IgE immunoglobulins on mast cells or basophils. The IgE immunoglobulin
attaches with the FcεR1 receptor of mast cells or basophils. Mast cells are generally
found embedded within the tissues, whereas basophils move freely in the peripheral blood.
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Figure 2. IgE-mediated reaction mechanism. (color figure available online.)
FcεR1, an IgE receptor in humans, is composed of four subunits α (alpha), β (beta), γ1
(gamma 1), and γ2 (gamma 2). The α subunit is found to be involved in attachment of
IgE, whereas β, γ1, and γ2 are involved in the phosphorylation process, which induces a
cascade of signaling events mediated by tyrosine kinase after the secondary exposure by
the same allergens.(69,70)
Secondary exposure of allergens and degranulation of mast cells. Secondary exposure of
the same allergen causes cross-linking to the IgE-FcεR1 complex. This results in nonintrinsic tyrosine kinase-mediated reactions that cause calcium ion mobilization, which
facilitates degranulation of mast cells or basophils.(71)
Mediator release and allergic symptoms. Prostaglandins, cytokines, leukotrienes,
histamine, and other mediators are secreted by degranulated mast cells or basophils. These
mediators may cause smooth muscle dilation, capillary disruption, local swelling, and other
allergic symptoms. In some individuals, these reactions may occur very vigorously leading
to anaphylaxis.(72)
Common Diseases Due To Food Allergy
There are many diseases reported due to food allergy, including allergic rhinitis, bronchial
asthma, atopic dermatitis, and gastrointestinal disorder. Moreover, the allergenic prevalence in each person varies significantly, depending on genetic disposition and environmental factors, which makes a person allergic to one protein but not to another. The different
atopic diseases have been reported due to intake of foods such as peanut, soybeans, red
kidney beans, red grams, green grams, eggs, and fishes.
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Nasobronchial Asthma and Allergic Rhinitis
Nasobronchial asthma and allergic rhinitis are two most common complications that occur
during an allergic attack.(73) There is a clear association between allergic rhinitis and
asthma. Cases of developing asthma and nasal symptoms at or about the same time have
been documented.(74) Patients with above conditions can expect to suffer more severe asthmatic attacks and require stronger medications to treat their asthma. Example of such foods
causing nasobronchial asthma and rhinitis are peanut, soybeans, red gram, green gram, and
red kidney beans.(9,10,13)
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Atopic Dermatitis
Allergic contact eczema is a red, itchy, and weepy reaction where the skin has come into
contact with allergens. Atopic dermatitis is a very common, often chronic, skin disease that
affects a large percentage of the world’s population. It is also called eczema, dermatitis,
or atopy. The term atopic refers to diseases that are hereditary, tend to run in families, and
often occur together. In atopic dermatitis, the skin becomes extremely itchy and inflamed,
causing redness, swelling, cracking, weeping, crusting, and scaling. Atopic dermatitis often
accompanies asthma, allergies or hay fever, and eczema. Examples of some food causing
atopic dermatitis are egg, wheat, milk, and soy.(75)
Oral Allergy Syndrome (OAS)
OAS is an allergic reaction to food that is limited to the lips, mouth, and throat. The major
symptoms of OAS include itching and swelling of the lip or throat. These symptoms generally start within minutes of eating and settle down within an hour. OAS is caused by
cross-reactivity between proteins in fresh fruits and vegetables and pollens. The proteins
in the fruits and vegetables causing OAS are easily broken down with cooking or processing. Therefore, OAS typically does not occur from eating cooked or baked fruits and
vegetables, or processed fruits. Kiwi fruit is now one of the most common causes of OAS.
Although patients often present with mild OAS, severe systemic reactions are not uncommon, particularly in children.(76) Examples of some foods causing OAS are raw fruits such
as cherry and vegetables.
Eosinophilic Esophagitis
Eosinophilic esophagitis is inflammatory condition in which the wall of the esophagus is
filled by large numbers of eosinophils. Although, the cause of eosinophilic esophagitis is
unknown, allergic responses including food allergies have been implicated. The esophagus is a muscular tube utilized for propelling swallowed food from the mouth into the
stomach. Esophagitis refers to inflammation of the esophagus. The most common cause of
esophagitis is acid reflux, which most frequently results in heartburn, although acid reflux
also can cause ulcers in the inner lining of the esophagus. Examples of foods involved
in induction of eosinophilic oesophagitis are egg, milk, and soy. These foods were identified most frequently with skin prick testing. Corn, soy, and wheat were identified most
frequently with atopy patch testing.(77)
Edema of the Uvula
The uvula is a tiny organ in the oral cavity attached to the soft palate. It is involved in
the articulation of human voice, swallowing, and prevents the entry of foods into the nasal
Allergenic Food Proteins
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cavity. Swelling of the uvula occurs in different medical conditions including food allergy.
Usually, edema manifests as fullness of the oropharynx, difficulty in talking, difficulty in
breathing, and, since it affects the vocal cords, dysphonia.(78) Seafoods, hazelnuts, and
walnuts are some examples of foods that can induce edema of the uvula.
Reccurent Aphthous Stomatitis (RAS)
RAS is one of the most common oral lesions induced by milk, gluten, and other allergens.
RAS can occur either in single or multiple forms in oral mucosa. This chronic, incurable
condition can be painful to the patient, making it uncomfortable to speak, eat, or drink.(79)
RAS can be caused by multiple foods.
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Food Protein-Induced Enterocolitis Syndrome (FPIES)
FPIES is a non–IgE-mediated, gastrointestinal food-mediated hypersensitivity.(80)
Vomiting, followed by an elevation of the peripheral blood polymorphonuclear leukocyte
number, diarrhea, and possibly lethargy and hypotension are characteristic of FPIES.(81)
Rice is the most common solid food causing FPIES.
Neonatal Diarrhea
Diarrhea represents a major condition responsible for pediatric mortality worldwide.
Diarrhea in children due to cow milk and other foods have been very frequently reported.
The onset of neonatal diarrhea may rapidly lead to life-threatening dehydration and
malnutrition.(82) Milk, soy, and grains can cause diarrhea in children.
Diagnosis of Food Allergy
There are several diagnostic methods for determination of food allergies. A brief overview
of each different diagnostic method for detection of food allergy is given below.
Skin Prick Test (SPT)
Skin prick test can be performed with pollens, insects, danders, dust, fungi, and food
extracts in bronchial asthma, allergic rhinitis, allergic dermatitis, and other atopic patients.
Glycerinated buffer saline and glycerol histamine acid phosphate can be used as negative
and positive controls, respectively. Comparison against a histamine-induced weal determines the allergic reaction. If the SPT weal is smaller than the histamine-induced weal, a
score of +1 is given. If the weals are equal, the score is +2. If the test weal is larger than
the histamine weal, the score is +3, and if it is larger with pseudopodia, then the score
is +4. The results are read 20 minutes after the skin prick.(13) The presence of allergenspecific IgE on cutaneous mast cells results in a positive skin test in the form of a transient
“weal-and-flare” reaction.(83) When possible, allergy skin testing is the preferred method
in comparison to various in vitro tests for assessing the presence of specific IgE antibodies
because it is more sensitive and specific, simpler to use, and less expensive.(84)
Oral Food Challenge
A placebo control food challenge is a very common test for allergy, often considered
the gold standard in the diagnosis of food allergy.(85,86) The aim of a food challenge
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is to study the consequences of a food or food additive ingestion. In a double-blind,
placebo-controlled, oral food challenge, the specific food is masked in a vehicle food
and then administered in a graded fashion. The active food and an equivalent amount
of placebo are given in random order and both tests are performed in a controlled
manner. A single-blinded challenge is when the patient is unaware but the physician is aware of the content of the challenge. It is sufficient as a screening tool for
reactivity.
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RAST (Radioallergosorbent Test)
The RAST is a radioimmunoassay test to detect specific IgE antibodies to suspected or
known allergens. This in vitro test using the blood of a susceptible individual is useful
for detection of allergy with good reproducibility.(87) One of the major advantages of this
test is that it is not necessary to stop antihistamine medications. It is also used if skin
conditions (such as eczema) are so widespread that allergy skin testing cannot be done.
A commercially available, radiolabeled anti-human (an antibody directed against human)
IgE antibody can be used to detect reactivity. The amount of radioactivity is proportional
to the serum IgE for the allergen.
In Vitro Specific Immunoglobulin Test
In vitro tests for food-specific antibodies may also be used to screen patients suspected of
IgE-mediated food allergies.(88) Enzyme-linked immunosorbant assay (ELISA) has been
used as quantification tool to determine the IgG1, IgG2a, and IgE levels in the serum of
allergic patients. This in vitro test is very sensitive and requires only a small amount (4–5
μL) of serum.(13)
Bryan’s Test
Cytotoxic food testing, also known as “Bryan’s Test,” involves observing changes in the
shape of white cells when a specific antigen is added to whole blood. It is prone to bias as
it depends on subjective interpretation.(89)
Sublingual/Intradermal Provocation Tests
Here, the allergen is applied sublingually or intradermally, followed by an observation
period for a local response. The application of allergen is progressively increased until a
weal appears on the skin (intradermal provocation dose), and the dosage is then decreased
until the weal disappears.(90)
Western Blotting
Western blotting can be used as a diagnostic method for identification of a culprit food
using human serum. With this method, the interaction between IgE presented in the
serum of the patient and food proteins transferred on the polyvinyl difluoride membrane can be easily observed. Western blotting also determines allergenic proteins among
whole-food proteins, since the IgE only binds to the proteins having an epitope on their
surface.
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Recent Advances
Our understanding of the allergenicity of food proteins has increased substantially in
the past few years. Several allergenic proteins have been identified and characterized at
the molecular level, which improves our understanding of the immunopathogenesis of
many allergic disorders and might soon lead to novel diagnostic and immunotherapeutic
approaches. Advancement in the assessment of allergenicity potential of foods, epitope
mapping, anti-IgE therapy, allergen-specific immunotherapy, production of monoclonal
antibodies, bioinformatics approaches, detection of trace amounts of allergens in commercial foods, and the concept of allergen-free genetically modified (GM) foods using
biotechnology and genetic engineering are a few examples.
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Assessment of Allergenicity Potential of Foods
Several new methods have been reported for the assessment of allergenicity potential of different foods.(9,10,13) These methods include bioinformatics approaches, pepsin digestibility
assay, thermal stability, pancreatin digestibility assay, IgE immunoblotting, and specific
IgE level. An outline of assessment of allergenicity potential has been given in Fig. 3.
Epitope Mapping
An allergenic epitope is a localized region on the surface of an allergen that is capable of providing an allergic response and combining with a specific IgE to counter
that response.(91) Production of hypoallergenic allergens can be used as the vaccine.
Epitope mapping may help in therapeutic aspects such as production of hypoallergenic
molecules. Molecular cloning and epitope analysis of the peanut allergen Ara h 3 has been
documented.(92)
Anti-IgE Therapy
Treatment with a class of drugs derived from proteins that work by binding to and blocking
the action of immunoglobulin E (IgE) is known as anti-IgE therapy. Omalizumab is one
Assessment of allergenic proteins
Source of protein
Amino acid
sequences
homology
Pepsin resistant
Immunoblotting
Serum screening
Additional tests
Knowledge of
source of protein is
first requirement,
whether the proteins
are from plants or
animals having
allergenic proteins.
Bioinformatics
sources may be
use to predict
allergenicity of
proteins with
known
sequences.
Pepsin digestiblity
of protein may be an
important criteria to
check out stability
of proteins in
stomach and
intestine to elicite
allergic responses.
IgE immunoblotting
may be performed
to point out IgE
binding proteins.
Specific IgE
level can be
used to observe
allergenicity of
specific
protein.
Several other tests
like thermal
stability, pancreatin
digestibility assay
may be used to
observe the heat
stability, intestinal
stability.
Figure 3. Different methods of assessment of allergenicity potential of food proteins. (color figure
available online.)
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such example of an anti-IgE drug.(93) Successful clinical trials of anti-IgE therapy in
allergic patients have been performed.(94)
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Allergen-Specific Immunotherapy
Specific immunotherapy (SIT) is in the lime light these days because of its superiority over
the administration of total crude protein extracts (CPEs). In traditional immunotherapy
CPE of an allergic food is given to patients. The CPEs contain many proteins and susceptible patients may be prone to anaphylactic reactions. In allergen SIT, the major allergen
from a food is purified and its epitope has been deleted to make it hypoallergenic. Here,
the patients are treated with hypoallergic proteins only. For selected patients, allergen SIT
offers the prospect of long lasting clinical efficacy. SIT involves the administration of the
allergen extract using a standardized regimen, usually subcutaneously or increasingly sublingually. However, application of this potentially curative treatment is restricted, largely
due to the risk of serious adverse events, especially in asthmatics and for potent allergens
such as peanut, seafood, and latex. It is currently inappropriate and potentially dangerous to
advocate deliberate exposure of foods involved in serious reactions against current recommendations and particularly so among food allergic children until more basic and positive
clinical research outcome becomes available.(95)
Production of Monoclonal Antibodies
Production of monoclonal antibodies against allergens is a major step towards combating
food allergies. The development of animal models for developing specific IgE presenting
the same specificity as human IgE and similar clinical symptoms as those observed in
allergic patients are of great interest for the understanding of mechanisms involved in the
induction and regulation of food allergy.(96)
Bioinformatics Approaches for Identification of Novel Allergenic Proteins
Bioinformatics approaches to classifying allergens and predicting cross-reactivity have
been very beneficial for acquiring knowledge about the allergens in a limited time span.(97)
Bioinformatics helps identify novel allergenic proteins through amino acid sequence
matching and phylogenic analysis. This can be done by using sequence similarity searching programs such as FASTA. There are many allergic protein databases, including the
structural database of allergenic proteins (SDAP) and AllergenOnline.org. The purified
allergenic proteins can be sequenced and characterized by liquid chromatography tandem mass spectrometry (LC-MS/MS), which determines the amino acid sequence of the
proteins. Allergenic proteins from red gram(9) and green gram(10) have been characterized recently using this method. Bioinformatics approaches are also used in predictive
allergenicity of GM foods/crops.
Trace Allergens in Processed Foods
Processed foods often may contain trace amounts of allergenic proteins at such a low level
that they cannot be easily measured. The development of new detection methods, such as
real-time polymerase chain reaction (PCR) and a sandwich ELISA, for detection of trace
amounts of potential allergens in processed food now allows detection down to very low
levels (<10 ppm).(98) For example, the presence of peanut proteins in processed foods
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can be easily quantified with the help of such techniques.(98) In addition, the determination of allergenic proteins by liquid chromatography(99) and mass spectrometry(99,100)
has greatly advanced in recent years. These methods are very helpful for allergic patients
suffering from food allergies. Once it has been confirmed that certain packaged food contains some trace amount of food proteins from which individual is allergic, they can easily
avoid it.
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Allergenless Genetically Modified (GM) Crops
The production of allergen-less GM crop has potential to reduce food allergies.
An allergenic reaction to proteins expressed in GM crops has been one of the prominent
concerns among biotechnology critics and a concern of regulatory agencies. However,
genetic engineering can be used to turn off specific gene products that cause allergic
reactions.(101) Thus, biotechnology may be used to eliminate allergens naturally present
in crops. For example, gene silencing has been used to remove a major allergen, Gly m
Bd 30 K, in soybean,(102) demonstrating that genetic modification can be used to reduce
allergenicity of food and feed. This provides a model for further use of genetic modification
approaches to eliminate allergens.(103)
Conclusions
Food allergy incidences are increasing globally and serious endeavors on many aspects
need attention. A diverse range of plant and animal food allergens have been identified
and characterized. The allergenicity of any protein primarily depends on its structure and
resistance to digestion, although other factors may also be important. Furthermore, crossallergenicity may be a major problem because one can consume a variety of proteins
in a single day from different sources, which may cross-react. Diagnostic approaches of
food allergy are continually being improved and new methods to detect and counter food
allergies are being developed. Specific immune therapies, anti-IgE therapy, and production of monoclonal antibodies are very important milestones in the therapeutics of food
allergy. Real-time PCR and ELISA in detection of allergenic proteins in processed and
packaged foods are extremely important for screening of allergenic proteins. This can
prevent susceptible individuals, especially small children and the elderly, from consuming allergen-containing foods. Multidisciplinary approaches to the problem are needed to
combat the effects of food allergies.
Acknowledgments
This work was supported by Supra Institutional Project-08 (SIP-08) of Council of Scientific and
Industrial Research (CSIR), New Delhi. S.K. and A.K.V. are thankful to CSIR, New Delhi, for the
award of their Senior Research Fellowships. This is CSIR-IITR paper number 2953.
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