Are Dietary Lectins Relevant Allergens in Plant Food Allergy?

Lectins or carbohydrate-binding proteins are widely distributed in seeds and vegetative parts of edible plant species. A few lectins from different fruits and vegetables have been identified as potential food allergens, including wheat agglutinin, hevein (Hev b 6.02) from the rubber tree and chitinases containing a hevein domain from different fruits and vegetables. However, other well-known lectins from legumes have been demonstrated to behave as potential food allergens taking into account their ability to specifically bind IgE from allergic patients, trigger the degranulation of sensitized basophils, and to elicit interleukin secretion in sensitized people. These allergens include members from the different families of higher plant lectins, including legume lectins, type II ribosome-inactivating proteins (RIP-II), wheat germ agglutinin (WGA), jacalin-related lectins, GNA (Galanthus nivalis agglutinin)-like lectins, and Nictaba-related lectins. Most of these potentially active lectin allergens belong to the group of seed storage proteins (legume lectins), pathogenesis-related protein family PR-3 comprising hevein and class I, II, IV, V, VI, and VII chitinases containing a hevein domain, and type II ribosome-inactivating proteins containing a ricin B-chain domain (RIP-II). In the present review, we present an exhaustive survey of both the structural organization and structural features responsible for the allergenic potency of lectins, with special reference to lectins from dietary plant species/tissues consumed in Western countries.


Introduction
Besides their beneficial effects on human health, the consumption of plant food products may cause some detrimental effects, mainly associated with the presence of toxic molecules and allergenic proteins, which often occur in various plant parts, including leaves, roots, bulbs, fruits, and seeds [1,2]. Furthermore, the consumption of either crude or cooked foods from a variety of edible plant species is likely to trigger more or less severe allergic manifestations in both previously sensitized children and adults [3]. During the two last decades, huge progress has been achieved in terms of the identification and characterization of plant allergens responsible for these food allergies [4][5][6]. However, a lot of research remains to be done to obtain an exhaustive picture of the plant allergen diversity. In this respect, most of the relevant plant allergens consist of seed storage proteins, e.g., vicilins (7S globulin), legumins (11S globulin) and 2S albumins [7], and different families of pathogenesis-related (PR) proteins, e.g., PR-3 endochitinases [8], PR-5 thaumatin-like proteins (TLP) [9], PR-10 Bet v 1-like  [26] At present, a restricted number of higher plant lectins have been recognized unambiguously as potential allergens by the International Union of Immunological Societies (WHO/IUIS) [27]. In addition, many other plant lectins have been scarcely investigated with respect to their allergenic potential. Here, we present an exhaustive review of studies that have been devoted to the allergenicity of edible plant lectins. Furthermore, we propose some ideas for future research.

Allergenic Potential of Lectins from Higher Plants
In addition to their well-known mitogenic properties, essentially on T-lymphocytes, plant lectins are capable of inducing some allergic responses in previously sensitized people, similar to genuine allergens from plant foods and food products.
The list of lectins that have been assigned as potential food allergens by the WHO/IUIS Allergen Nomenclature Sub-Committee [27], comprises a restricted number of proteins including Tri a 18 (wheat germ agglutinin WGA) and a few chitinases with a hevein domain from banana (Mus a 2), turnip (Bra r 2), chestnut (Cas s 5), corn (Zea m 8), and avocado (Per a 1) ( Table 1). Two other contact allergens, Hev b 6 (hevein) and Hev b 11 (class I chitinase containing a hevein domain) from the rubber tree Hevea brasiliensis, have been included in the list because they cross-react with food chitinases of classes I, II, IV, V, VI, and VII, which also possess a hevein domain.
In addition to this official list of lectin allergens, other plant lectins have been identified as potential food allergens following their ability to bind IgE, degranulate mast cells and basophils, and trigger the interleukin responses in various allergic patients [28][29][30]. These lectins have been included in Table 1 as non-IUIS approved potential food allergens (Table 2). Table 2. List of lectins identified as potential food allergens. Lectin allergens approved by the International Union of Immunological Societies (WHO/IUIS Allergen Nomenclature Sub-Committee) [27], are indicated. Lectin allergens Hev b 6 and Hev b 11 from Castor bean (Ricinus communis) latex, are responsible for contact allergies but also for IgE-binding cross-reactivity with hevein-containing food chitinase proteins. The non-IUIS approved lectin allergens consist of members from the different families of plant lectins, namely legume lectins, type II ribosome-inactivating proteins (RIP-II), wheat germ agglutinin, hevein and chitinases with a hevein domain, jacalin-related lectins, GNA-like lectins, and Nictaba-related lectins. All of these lectins exhibit very different structural scaffolds but share the common property to specifically recognize both simple sugars, e.g., mannose or galactose, and complex glycans, e.g., glycans of the N-acetyllactosaminic type or the high-mannose type [49].

Legume Lectins
Depending on the occurrence of a single or two polypeptide chains in their constitutive protomer and their quaternary association as homotetramers or homodimers, legume lectins are classified into two groups of closely-related lectins: the homotetrameric single-chain lectins, comprise both man-specific (e.g., Con A from Jackbean, Canavalia ensiformis, and DgL from Dioclea grandiflora) and Gal/GalNAc-specific lectins (e.g., PNA from peanut, Arachis hypogaea, and SBA from soybean, Glycine max). -the homodimeric two-chain lectins, exclusively comprise man-specific lectins (e.g., LcA from lentil, Lens culinaris, and PsA from garden pea, Pisum sativum).
Both types of lectins have been identified as potential IgE-binding allergens.

Homotetrameric Single-Chain Lectins
The Gal/GalNAc-specific lectin PNA from peanut (Arachis hypogaea), was among the first identified lectin allergens [43]. PNA is present in the protein bodies of the cotyledonary cells similar to other major allergens belonging to the group of seed storage proteins such as Ara h 1 (7S globulin/vicilin), Ara h 2 and Ara h 6 (2S albumin), and Ara h 3 (11S globulin/legumin). The lectin is designated as Ara h agglutinin and is considered a minor peanut allergen [43,50,51], At present, PNA is not officially recognized as a peanut allergen by the WHO/IUIS Committee.
Native PNA occurs as a homotetramer built from the symmetrical non-covalent association of four identical protomers of 25 kDa, exhibiting the canonical jelly roll fold of legume lectins consisting of a β-sandwich made of two antiparallel β-sheets of 6 (front face) and 7 β-strands (back face), respectively ( Figure 1A). Each protomer possesses a carbohydrate-binding site (CBS), which specifically recognizes Gal and GalNAc termini from unsialylated N-acetyllactosaminic glycans and the T/Tn-antigen expressed at the surface of many cancer cells [52].
consisting of a β-sandwich made of two antiparallel β-sheets of 6 (front face) and 7 β-strands (back face), respectively ( Figure 1A). Each protomer possesses a carbohydrate-binding site (CBS), which specifically recognizes Gal and GalNAc termini from unsialylated N-acetyllactosaminic glycans and the T/Tn-antigen expressed at the surface of many cancer cells [52].
Sequential IgE-binding B-cell epitopes identified on the surface of PNA [17] ( Figure 1B), exhibit some amino acid sequence identity to other IgE-binding epitopes occurring at the surface of other legume lectins, such as LcA from lentil, PsA from pea, and SBA from soybean [53]. Most of the regions recognized as potential IgE-binding epitopes display both electropositively (colored blue) and electronegatively (colored red) charged residues, as shown from the calculation of surface electrostatic potentials at the molecular surface of PNA ( Figure 1C). The homology between IgE-binding epitopes accounts for the cross-reactivity observed among legume lectins regarding their ability to interact with sera from peanut-allergic patients. However, this IgE-binding ability does not depend on the possible interaction of legume lectins with the carbohydrate moiety of IgE, since all the binding experiments were conducted in the presence of mannose and galactose, which specifically inhibit the lectin activity of both Gal/GalNAc-specific (PNA, SBA) and man-specific (LcA, PsA) legume lectins [53].   Sequential IgE-binding B-cell epitopes identified on the surface of PNA [17] ( Figure 1B), exhibit some amino acid sequence identity to other IgE-binding epitopes occurring at the surface of other legume lectins, such as LcA from lentil, PsA from pea, and SBA from soybean [53]. Most of the regions recognized as potential IgE-binding epitopes display both electropositively (colored blue) and electronegatively (colored red) charged residues, as shown from the calculation of surface electrostatic potentials at the molecular surface of PNA ( Figure 1C). The homology between IgE-binding epitopes accounts for the cross-reactivity observed among legume lectins regarding their ability to interact with sera from peanut-allergic patients. However, this IgE-binding ability does not depend on the possible interaction of legume lectins with the carbohydrate moiety of IgE, since all the binding experiments were conducted in the presence of mannose and galactose, which specifically inhibit the lectin activity of both Gal/GalNAc-specific (PNA, SBA) and man-specific (LcA, PsA) legume lectins [53].
Kidney bean (Phaseolus vulgaris) seeds contain a mixture of erythroagglutinating (PHA-E) and leucoagglutinating (PHA-L) lectins, which interact with complex glycans of the N-acetyllactosaminic type, but do not recognize simple monosaccharides [53]. Both lectins consist of an arrangement different from that observed in PNA, containing four identical protomers possessing the canonical β-sandwich structure in a homotetrameric configuration (Figure 2A). Each protomer contains a CBS that specifically recognizes complex N-glycan chains, but does not recognize simple sugars, probably due to a particular conformation of the CBS. leucoagglutinating (PHA-L) lectins, which interact with complex glycans of the N-acetyllactosaminic type, but do not recognize simple monosaccharides [53]. Both lectins consist of an arrangement different from that observed in PNA, containing four identical protomers possessing the canonical β-sandwich structure in a homotetrameric configuration (Figure 2A). Each protomer contains a CBS that specifically recognizes complex N-glycan chains, but does not recognize simple sugars, probably due to a particular conformation of the CBS. Both types of lectins from the white kidney bean [29,[54][55][56][57][58] and the black turtle bean [59,60] have been reported as allergens, and a detailed bioinformatics study of the B-and T-cell epitopes of PHA-E from black turtle bean has been performed [59]. Up to 7 IgE-binding B-epitopes ( Figure 2B) and 3 T-epitopes ( Figure 3) occur at the surface of the PHA-E tetramer. Some overlap was identified between B-epitope #4 and T-epitope #2. Surprisingly, no B-epitopes were identified along the C-terminal region of the PHA-E amino acid sequence despite the pronounced sequence homologies between this region and the corresponding B-epitope-containing region of other legume lectins (see Figure 8 for explanation). Similar to PNA, some of the IgE-binding epitopic regions of PHA-E coincide with the presence of charged residues ( Figure 2C). Both types of lectins from the white kidney bean [29,[54][55][56][57][58] and the black turtle bean [59,60] have been reported as allergens, and a detailed bioinformatics study of the B-and T-cell epitopes of PHA-E from black turtle bean has been performed [59]. Up to 7 IgE-binding B-epitopes ( Figure 2B) and 3 T-epitopes ( Figure 3) occur at the surface of the PHA-E tetramer. Some overlap was identified between B-epitope #4 and T-epitope #2. Surprisingly, no B-epitopes were identified along the C-terminal region of the PHA-E amino acid sequence despite the pronounced sequence homologies between this region and the corresponding B-epitope-containing region of other legume lectins (see Figure 8 for explanation). Similar to PNA, some of the IgE-binding epitopic regions of PHA-E coincide with the presence of charged residues ( Figure 2C).
leucoagglutinating (PHA-L) lectins, which interact with complex glycans of the N-acetyllactosaminic type, but do not recognize simple monosaccharides [53]. Both lectins consist of an arrangement different from that observed in PNA, containing four identical protomers possessing the canonical β-sandwich structure in a homotetrameric configuration (Figure 2A). Each protomer contains a CBS that specifically recognizes complex N-glycan chains, but does not recognize simple sugars, probably due to a particular conformation of the CBS. Both types of lectins from the white kidney bean [29,[54][55][56][57][58] and the black turtle bean [59,60] have been reported as allergens, and a detailed bioinformatics study of the B-and T-cell epitopes of PHA-E from black turtle bean has been performed [59]. Up to 7 IgE-binding B-epitopes ( Figure 2B) and 3 T-epitopes ( Figure 3) occur at the surface of the PHA-E tetramer. Some overlap was identified between B-epitope #4 and T-epitope #2. Surprisingly, no B-epitopes were identified along the C-terminal region of the PHA-E amino acid sequence despite the pronounced sequence homologies between this region and the corresponding B-epitope-containing region of other legume lectins (see Figure 8 for explanation). Similar to PNA, some of the IgE-binding epitopic regions of PHA-E coincide with the presence of charged residues ( Figure 2C). Two single-chain Gal/GalNAc-specific lectins, DBA from horse gram (Dolichos biflorus) [44] and SBA from soybean (Glycine max) [45,61], have been identified as potential food allergens. They consist of homotetramers displaying a protomer arrangement very similar to that found in the kidney bean lectins PHA-E and PHA-L ( Figures 4A and 5A). Although no information is available on the occurrence of sequential IgE-binding epitopes along their amino acid sequences, both lectins share strong identities/homologies with the epitope-containing regions of other legume lectins, especially the two-chain lectins LcA and PsA (see Figure 8 for explanation). SBA from soybean (Glycine max) [45,61], have been identified as potential food allergens. They consist of homotetramers displaying a protomer arrangement very similar to that found in the kidney bean lectins PHA-E and PHA-L ( Figures 4A and 5A). Although no information is available on the occurrence of sequential IgE-binding epitopes along their amino acid sequences, both lectins share strong identities/homologies with the epitope-containing regions of other legume lectins, especially the two-chain lectins LcA and PsA (see Figure 8 for explanation).  SBA from soybean (Glycine max) [45,61], have been identified as potential food allergens. They consist of homotetramers displaying a protomer arrangement very similar to that found in the kidney bean lectins PHA-E and PHA-L ( Figures 4A and 5A). Although no information is available on the occurrence of sequential IgE-binding epitopes along their amino acid sequences, both lectins share strong identities/homologies with the epitope-containing regions of other legume lectins, especially the two-chain lectins LcA and PsA (see Figure 8 for explanation).

Homodimeric Two-Chain Lectins
Two homodimeric two-chain lectins have been investigated with respect to their IgE-binding activity, namely the lentil (Lens culinaris) lectin LcA and the garden pea (Pisum sativum) lectin PsA [28,29,62]. Both lectins consist of two protomers with a canonical β-sandwich structure, associated by non-covalent bonds in a homodimer which possesses two CBS located at both ends of the dimer ( Figures 6A and 7A). The CBSs readily accommodate mannose, the trimannoside core of N-acetyllactosaminic N-glycans, and more complex high-mannose glycans.
Nine sequential B-epitopes have been identified at the surface of both the β-subunit (7) and α-subunit (2), in each protomer of lentil lectin LcA ( Figure 6B). Similarly, 10 sequential B-epitopes were characterized at the surface of the β-subunit (8) and α-subunit (2) of each garden pea lectin PsA protomer ( Figure 7B) [17].  (2), green (5), grey (6), purple (7), cyan (8), and yellow (9), respectively. In each monomer, epitopes 1-7 are located on heavy subunits while epitopes 8 and 9 occur in light subunits. Due to the high sequential and structural homologies occurring among the closely-related members of the legume lectin family, most of the identified sequential IgE-binding B-epitopes exhibit a very similar positioning along the amino acid sequence and at the surface of the lectin protomers, in both homotetrameric single-chain lectins (PNA, PHA-E, DbA, SBA) and homodimeric two-chain lectins (LcA, PsA), irrespective of their structural architecture and carbohydrate-binding specificities ( Figure 8). Most sequences encoding lectins that are abundant in other edible legumes, such as lupine (Lupinus albus), faba bean (Vicia faba), winged bean (Psophocarpus teragonolobus), azuki bean (Vigna angularis), and many others, look very similar to those of LcA from lentil, PsA from pea, PNA from peanut, and PHA from kidney bean and, especially, at the location of some of the predicted IgE-binding epitopes. Accordingly, they are suspected to behave as potential allergens even though at present no evidence exists to support their allergenicity.
In addition, most of these predicted IgE-binding epitopic regions contain a high proportion of either electropositively (R,K) or electronegatively (D,E) charged residues (Figure 9), which explains the high electrostatic potentials often calculated for these IgE-binding regions at the molecular surfaces of both single-and two-chain legume lectins ( Figures 1C, 2C, 4C, 5C, 6C and 7C). Aromatic residues (F,Y,W) also frequently occur in the IgE-binding epitopic regions common to single-and two-chain legume lectins. even though at present no evidence exists to support their allergenicity.
In addition, most of these predicted IgE-binding epitopic regions contain a high proportion of either electropositively (R,K) or electronegatively (D,E) charged residues (Figure 9), which explains the high electrostatic potentials often calculated for these IgE-binding regions at the molecular surfaces of both single-and two-chain legume lectins ( Figures 1C, 2C and 4-7C). Aromatic residues (F,Y,W) also frequently occur in the IgE-binding epitopic regions common to single-and two-chain legume lectins.  Obviously, these B-epitope similarities account for their IgE-binding cross-reactivity observed among different legume lectins, as previously reported for sera from peanut-allergic patients [17]. As shown from surface plasmon resonance (SPR) measurements, both PNA, PHA-E, LcA, and PsA lectins, readily interacted with all of the probed sera from peanut-allergic patients ( Figure 10). However, a stronger IgE-binding cross-reactivity was observed for two-chain legume lectins, LcA Figure 9. Comparison of some sequential IgE-binding epitopes identified at homologous positions along the amino acid sequence of PNA, LcA, and PsA. Identical residues are in boxed white letters. Structurally homologous residues according to Risler's matrix [63] are grey boxed. Positively (K,R) and negatively (D,E) charged residues are colored blue and red, respectively. Aromatic residues (F,Y,W) are colored orange.
Obviously, these B-epitope similarities account for their IgE-binding cross-reactivity observed among different legume lectins, as previously reported for sera from peanut-allergic patients [17]. As shown from surface plasmon resonance (SPR) measurements, both PNA, PHA-E, LcA, and PsA lectins, readily interacted with all of the probed sera from peanut-allergic patients ( Figure 10). However, a stronger IgE-binding cross-reactivity was observed for two-chain legume lectins, LcA and PsA, compared to that observed for single-chain legume lectins, PNA and PHA-E. These discrepancies among the single-and two-chain lectins, would depend on the way the protomers are assembled in both types of lectins, the dimeric association of protomers favoring a better exposure of epitopes, compared to the tetrameric association in which some epitopes become buried at the protomer interfaces. Figure 9. Comparison of some sequential IgE-binding epitopes identified at homologous positions along the amino acid sequence of PNA, LcA, and PsA. Identical residues are in boxed white letters. Structurally homologous residues according to Risler's matrix [63] are grey boxed. Positively (K,R) and negatively (D,E) charged residues are colored blue and red, respectively. Aromatic residues (F,Y,W) are colored orange.
Obviously, these B-epitope similarities account for their IgE-binding cross-reactivity observed among different legume lectins, as previously reported for sera from peanut-allergic patients [17]. As shown from surface plasmon resonance (SPR) measurements, both PNA, PHA-E, LcA, and PsA lectins, readily interacted with all of the probed sera from peanut-allergic patients ( Figure 10). However, a stronger IgE-binding cross-reactivity was observed for two-chain legume lectins, LcA and PsA, compared to that observed for single-chain legume lectins, PNA and PHA-E. These discrepancies among the single-and two-chain lectins, would depend on the way the protomers are assembled in both types of lectins, the dimeric association of protomers favoring a better exposure of epitopes, compared to the tetrameric association in which some epitopes become buried at the protomer interfaces. Figure 10. SPR measurement, expressed as resonance units (RU), of the interaction of sera (S1-S12) from peanut-allergic patients with PNA (blue color), LcA (red color), PsA (green color), and PHA-E (violet color). Each value is the mean of three independent measurements [17].
Another homodimeric two-chain lectin from chia (Salvia hispanica) seeds has been identified as a potential IgE-binding allergen [46]. The single amino acid sequence stretch AVEFDTLYNTNDPNYR characterized by mass spectrometry for this IgE-binding allergen exhibits a high percentage of identity with the Phaseolus coccineus (81.25%) and the Phaseolus vulgaris PHA-E (75.0%) and PHA-L (75.0%) lectins ( Figure 11): Figure 10. SPR measurement, expressed as resonance units (RU), of the interaction of sera (S1-S12) from peanut-allergic patients with PNA (blue color), LcA (red color), PsA (green color), and PHA-E (violet color). Each value is the mean of three independent measurements [17].
Another homodimeric two-chain lectin from chia (Salvia hispanica) seeds has been identified as a potential IgE-binding allergen [46]. The single amino acid sequence stretch AVEFDTLYNTNDPNYR characterized by mass spectrometry for this IgE-binding allergen exhibits a high percentage of identity with the Phaseolus coccineus (81.25%) and the Phaseolus vulgaris PHA-E (75.0%) and PHA-L (75.0%) lectins ( Figure 11): Sera from people allergic to peanut-allergic people that were available to perform the IgE-binding cross-reactions with different lectins contained specific IgE that primarily recognize the main allergenic proteins from peanut, the vicillin Ara h 1, the 2S albumins Ara h 2 and Ara h 6, and the legumin Ara h 3. Therefore, PNA (also designated as Ara h agglutinin allergen) and other IgE-binding cross-reacting lectins from other legume seeds (LcA, PsA, PHA, SBA), do not actually correspond to the main components of allergies. In this respect, none of the peanut allergies identified so far can be attributed to the PNA allergen.

Type II Ribosome Inactivating Proteins (RIP-II)
Type II Ribosome-Inactivating Proteins (RIP-II) are chimerolectins built from a toxic A-chain with a catalytic RNA N-glycosidase activity, covalently associated through a disulfide bridge to a two-domain B-chain with lectin activity [14,15]. The B-chain often exhibits Gal/GalNAc-binding Sera from people allergic to peanut-allergic people that were available to perform the IgE-binding cross-reactions with different lectins contained specific IgE that primarily recognize the main allergenic proteins from peanut, the vicillin Ara h 1, the 2S albumins Ara h 2 and Ara h 6, and the legumin Ara h 3. Therefore, PNA (also designated as Ara h agglutinin allergen) and other IgE-binding cross-reacting lectins from other legume seeds (LcA, PsA, PHA, SBA), do not actually correspond to the main components of allergies. In this respect, none of the peanut allergies identified so far can be attributed to the PNA allergen.

Type II Ribosome Inactivating Proteins (RIP-II)
Type II Ribosome-Inactivating Proteins (RIP-II) are chimerolectins built from a toxic A-chain with a catalytic RNA N-glycosidase activity, covalently associated through a disulfide bridge to a two-domain B-chain with lectin activity [14,15]. The B-chain often exhibits Gal/GalNAc-binding specificity. After recognition of Gal/GalNAc-containing glycans on the cell surface by the B-chain, RIP-II molecules are engulfed into the cell and are subsequently cleaved to liberate the toxic A-chain. Through its RNA N-glycosidase activity, the A-chain inactivates the ribosomal 28S RNA via the depurination of an adenine base, which in turn blocks the protein synthesis and provokes cell death [64].
Three RIP-II have been identified as potential food allergens, ricin from castor bean (Ricinus communis) seeds, SNA-I from the black elderberry (Sambucus nigra) [65,66], and the RIP-II from the dwarf elder (Sambucus ebulus) [67]. All these chimerolectins exhibit a similar three-dimensional structure, made of both A-and B-chains covalently linked by a disulfide bridge occurring between two cysteine residues located at the C-terminus of the A-chain and at the N-terminus of the B-chain, respectively ( Figure 12A-D). Toxic lectins from mistletoe (Viscum album) also exhibit a similar organization of their A-and B-chains [68,69]. However, since no IgE-binding B-epitope or T-epitope were identified at the surface of these molecules, we do not know exactly what part of these allergens, either the A-chain or the B-chain or both, triggers occupational allergic manifestations in sensitized people. IgE-binding cross-reactions with different lectins contained specific IgE that primarily recognize the main allergenic proteins from peanut, the vicillin Ara h 1, the 2S albumins Ara h 2 and Ara h 6, and the legumin Ara h 3. Therefore, PNA (also designated as Ara h agglutinin allergen) and other IgE-binding cross-reacting lectins from other legume seeds (LcA, PsA, PHA, SBA), do not actually correspond to the main components of allergies. In this respect, none of the peanut allergies identified so far can be attributed to the PNA allergen.

Type II Ribosome Inactivating Proteins (RIP-II)
Type II Ribosome-Inactivating Proteins (RIP-II) are chimerolectins built from a toxic A-chain with a catalytic RNA N-glycosidase activity, covalently associated through a disulfide bridge to a two-domain B-chain with lectin activity [14,15]. The B-chain often exhibits Gal/GalNAc-binding specificity. After recognition of Gal/GalNAc-containing glycans on the cell surface by the B-chain, RIP-II molecules are engulfed into the cell and are subsequently cleaved to liberate the toxic A-chain. Through its RNA N-glycosidase activity, the A-chain inactivates the ribosomal 28S RNA via the depurination of an adenine base, which in turn blocks the protein synthesis and provokes cell death [64].
Three RIP-II have been identified as potential food allergens, ricin from castor bean (Ricinus communis) seeds, SNA-I from the black elderberry (Sambucus nigra) [65,66], and the RIP-II from the dwarf elder (Sambucus ebulus) [67]. All these chimerolectins exhibit a similar three-dimensional structure, made of both A-and B-chains covalently linked by a disulfide bridge occurring between two cysteine residues located at the C-terminus of the A-chain and at the N-terminus of the B-chain, respectively ( Figure 12A-D). Toxic lectins from mistletoe (Viscum album) also exhibit a similar organization of their A-and B-chains [68,69]. However, since no IgE-binding B-epitope or T-epitope were identified at the surface of these molecules, we do not know exactly what part of these allergens, either the A-chain or the B-chain or both, triggers occupational allergic manifestations in sensitized people.

Wheat Germ Agglutinin (WGA)
Wheat germ agglutinin WGA (Tri a 18 allergen), has been identified as a hevein motif-containing lectin because it results from the association of two tetramers, each comprising four tandemly arrayed hevein motifs that specifically bind to GlcNAc via a network of hydrogen bonds and stacking interactions ( Figure 13). IgE-binding epitopes of WGA consist of the B-epitopes that have been identified at the surface of the hevein (Hev b 6.02) polypeptide allergen [70,71] (see Section 2.4).
Wheat germ agglutinin WGA (Tri a 18 allergen), has been identified as a hevein motif-containing lectin because it results from the association of two tetramers, each comprising four tandemly arrayed hevein motifs that specifically bind to GlcNAc via a network of hydrogen bonds and stacking interactions ( Figure 13). IgE-binding epitopes of WGA consist of the B-epitopes that have been identified at the surface of the hevein (Hev b 6.02) polypeptide allergen [70,71]
(Lycopersicum esculentum) (Lyc es chitinase) [39,81], and in the rubber tree latex as a contact allergen Hev b 11 [82]. Similar to Mus a 2 from banana, class I chitinases comprise an N-terminal hevein domain, linked by an extended hinge region to a C-terminal chitin-binding catalytic domain ( Figure  15).
Class IV chitinases, which also contain a shortened chitin-binding module, have been identified in corn (Zea mays) (Zea m 8) [33] and in grape (Vitis vinifera) (Vit v chitinase) [40]. However, the allergenicity of Zea m 8 is not restricted to the chitin-binding hevein domain since the C-terminal catalytic domain expressed as a recombinant molecule in E. coli still interacted with sera from patients allergic to maize [33]. Due to their widespread distribution in plants and plant products, chitinases and especially class I chitinases, account for IgE-binding cross-reactivity between various fruits and vegetables [83][84][85]. Class IV chitinases, which also contain a shortened chitin-binding module, have been identified in corn (Zea mays) (Zea m 8) [33] and in grape (Vitis vinifera) (Vit v chitinase) [40]. However, the allergenicity of Zea m 8 is not restricted to the chitin-binding hevein domain since the C-terminal catalytic domain expressed as a recombinant molecule in E. coli still interacted with sera from patients allergic to maize [33].

Jacalin-Related Lectins
Due to their widespread distribution in plants and plant products, chitinases and especially class I chitinases, account for IgE-binding cross-reactivity between various fruits and vegetables [83][84][85].

Jacalin-Related Lectins
Jacalin-related lectins consist of a very homogeneous group of plant lectins, exhibiting a β-prism II structure for their constituent protomers, usually organized in either homodimeric or homotetrameric structures displaying two or four identical CBSs, respectively. In this respect, BanLec, the jacalin-related lectin from banana results from the non-covalent association of two protomers with the β-prism II structure. The β-prism II scaffold consists of three bundles of four antiparallel β-strands arranged into a β-prism structure along a longitudinal axis. Each protomer possesses a CBSs located at the top of the β-prism structure, which specifically recognizes N-glycans of the high-mannose type ( Figure 16) [86].
Banana consumption was reported to elicit IgG4 antibody production [47], and BanLec was also reported to induce a non-specific activation of basophils and mast cells in atopic subjects [87]. In vitro treatment of Balb/c and C57 BL/6 splenocytes with the recombinant BanLec resulted in a proliferation of T cells accompanied by an increased secretion of INFγ [88]. Similarly, the man-specific lectin ArtinM lectin from Artocarpus heterophyllus, activated mast cells and induced the release of newly synthesized IL-4 without, however, any co-stimulatory effect on the FcεRI degranulation [89]. Conversely, the Gal/GalNAc-specific jacalin from jackfruit (Artocarpus integrifolia), did not induce any release of IL-4, IL-13, and histamine from activated basophils [29]. At present, no data are available on the identification and localization of B-and T-epitopes at the molecular surface of jacalin-related lectin allergens.

GNA-Like Lectins
Food allergic manifestations associated with the consumption of spices like garlic (Allium sativum) and onion (Allium cepa) have been attributed to the occurrence of lectins belonging to the vast group of GNA-like lectins in these edible bulbs [41,42,[90][91][92]. Being members of the GNA-related lectin group, both the garlic lectin ASA and the onion ACA lectin possess protomers exhibiting the β-prism I fold, which consists of three bundles of four antiparallel β-strands arranged into a β-prism structure perpendicular to the axis. Each protomer contains three CBSs which specifically recognize mannose residues. Two non-covalently linked protomers build the hexavalent homodimeric structure found in the garlic lectin ASA (Figure 17). Jacalin-related lectins consist of a very homogeneous group of plant lectins, exhibiting a β-prism II structure for their constituent protomers, usually organized in either homodimeric or homotetrameric structures displaying two or four identical CBSs, respectively. In this respect, BanLec, the jacalin-related lectin from banana results from the non-covalent association of two protomers with the β-prism II structure. The β-prism II scaffold consists of three bundles of four antiparallel β-strands arranged into a β-prism structure along a longitudinal axis. Each protomer possesses a CBSs located at the top of the β-prism structure, which specifically recognizes N-glycans of the high-mannose type ( Figure 16) [86]. Banana consumption was reported to elicit IgG4 antibody production [47], and BanLec was also reported to induce a non-specific activation of basophils and mast cells in atopic subjects [87]. In vitro treatment of Balb/c and C57 BL/6 splenocytes with the recombinant BanLec resulted in a proliferation of T cells accompanied by an increased secretion of INFγ [88]. Similarly, the man-specific lectin ArtinM lectin from Artocarpus heterophyllus, activated mast cells and induced the release of newly synthesized IL-4 without, however, any co-stimulatory effect on the FcεRI degranulation [89]. Conversely, the Gal/GalNAc-specific jacalin from jackfruit (Artocarpus integrifolia), did not induce any release of IL-4, IL-13, and histamine from activated basophils [29]. At present, no data are available on the identification and localization of B-and T-epitopes at the molecular surface of jacalin-related lectin allergens.

GNA-Like Lectins
Food allergic manifestations associated with the consumption of spices like garlic (Allium sativum) and onion (Allium cepa) have been attributed to the occurrence of lectins belonging to the vast group of GNA-like lectins in these edible bulbs [41,42,[90][91][92]. Being members of the GNA-related lectin group, both the garlic lectin ASA and the onion ACA lectin possess protomers exhibiting the β-prism I fold, which consists of three bundles of four antiparallel β-strands arranged into a β-prism structure perpendicular to the axis. Each protomer contains three CBSs which specifically recognize mannose residues. Two non-covalently linked protomers build the hexavalent homodimeric structure found in the garlic lectin ASA (Figure 17).  In spite of their allergenic properties, no B-and T-epitope of garlic and onion GNA-related lectins have been characterized. In contrast to the garlic and onion lectins, the closely-related taro (Colocasia esculenta) lectin was not identified as a food allergen [93].

Nictaba-Related Lectins
The 17kDa protein in the phloem sap of watermelon (Cucumis melo) belongs to the Nictaba-related lectins and has been identified as an allergen involved in systemic reactions to melon and watermelon consumption [48]. Similar to the tobacco (Nicotiana tabacum) lectin Nictaba, the protein is exclusively composed of β-stands arranged in a β-sandwich structure, containing a single CBS with a GlcNAc-specificity ( Figure 18) [94]. Currently, there are no investigations on the occurrence of B-and T-epitopes in these molecules. In spite of their allergenic properties, no B-and T-epitope of garlic and onion GNA-related lectins have been characterized. In contrast to the garlic and onion lectins, the closely-related taro (Colocasia esculenta) lectin was not identified as a food allergen [93].

Nictaba-Related Lectins
The 17kDa protein in the phloem sap of watermelon (Cucumis melo) belongs to the Nictaba-related lectins and has been identified as an allergen involved in systemic reactions to melon and watermelon consumption [48]. Similar to the tobacco (Nicotiana tabacum) lectin Nictaba, the protein is exclusively composed of β-stands arranged in a β-sandwich structure, containing a single CBS with a GlcNAc-specificity ( Figure 18) [94]. Currently, there are no investigations on the occurrence of B-and T-epitopes in these molecules.
The 17kDa protein in the phloem sap of watermelon (Cucumis melo) belongs to the Nictaba-related lectins and has been identified as an allergen involved in systemic reactions to melon and watermelon consumption [48]. Similar to the tobacco (Nicotiana tabacum) lectin Nictaba, the protein is exclusively composed of β-stands arranged in a β-sandwich structure, containing a single CBS with a GlcNAc-specificity ( Figure 18) [94]. Currently, there are no investigations on the occurrence of B-and T-epitopes in these molecules.

Discussion
The group of plant lectins comprise a huge number of proteins which share the common property to specifically recognize carbohydrate structures, in spite of very different amino acid sequences and structural scaffolds. According to their carbohydrate-binding domain, plant lectins

Discussion
The group of plant lectins comprise a huge number of proteins which share the common property to specifically recognize carbohydrate structures, in spite of very different amino acid sequences and structural scaffolds. According to their carbohydrate-binding domain, plant lectins have been divided into twelve distinct families (Table 3), which display similar or different carbohydrate-binding specificities towards simple and complex sugars. Lectins with allergenic properties have been identified in 6 out of 12 plant lectin families, including the GNA family, the hevein family, the jacalin-related lectin (JRL) family, the legume lectin family, the Nictaba family, and the ricin B family. However, only a restricted number of 8 lectins comprising Mus a 2 from banana, Tri a 18 from wheat, Zea m 8 from corn, Bra r 2 from turnip, Cas s 5 from chestnut, the contact allergens Hev b 6 and Hev b 11 from the rubber tree, and Pers a 1 from avocado, categorize as WHO-IUIS-approved food allergens [27]. Other identified lectin allergens reported in Table 1, have not yet been officially assigned as food allergens in spite of their IgE-binding ability and capacity to trigger some Th2 cytokin/interleukin response in atopic or sensitized people. In addition, other lectins from non-edible plants, such as Con A from jackbean (Canavalia ensiformis), DsA from jimson weed (Datura stramonium), and ricin from the castor bean (Ricinus communis), have also been recognized as potential inducers of interleukin responses [29].
Most of the plant lectins identified as potential food allergens (19 over 26) belong to two families, in particular the family of legume lectins (7 out of 26 proteins) and the group of chitinases with a hevein domain (12 out of 26 proteins) ( Table 2). Legume lectin allergens like the lectins from peanut (Ara h agglutinin/PNA), kidney bean (PHA-E, PHA-L), soybean (SBA), horse gram (DbA), lentil (LcA), and pea (PsA), occur as seed storage proteins sequestered in protein bodies, together with the major cupin allergens, 7S globulins/vicilins and 11S globulins/legumins, and 2S albumin allergens [96]. However, similar to Ara h agglutinin/PNA of peanut and SBA of soybean, they most probably consist of minor allergens [43]. In contrast, class I chitinases are important allergens in many fruits, responsible for oral syndromes often associated with IgE-binding cross-reactivity between different fruits, e.g., between avocado, banana, kiwi, and chestnut [67][68][69]. In addition to allergenic chitinases of types I and IV, which both possess a hevein domain, other allergenic chitinases devoid of hevein domain have been identified in various plant species including Cof a 1 from coffee (Coffea arabica), Hev b 11 and hevamine from the rubber tree Hevea brasiliensis, Pun g 14 from pomegranate (Punica granatum), Trip s 1 from Triplochiton scleroxylon, Zea m 8 from maize (Zea mays), and Ziz m 1 from Ziziphus mauritiana (http://allergome.org). Obviously, their allergenicity is not attributed to the hevein domain, which reinforces the idea that the allergenicity displayed by chitinases containing a hevein domain could also result from the enzyme part of these complex proteins. Similarly, it is predictable that some allergenicity displayed by type II RIPs such as SNA-I also depends on the A domain responsible for the N-glycosidase activity of these complex proteins.
To date, however, the immunogenicity of lectins as allergenic proteins remains poorly known and difficult to foresee, because of the lack of information on the T-cell epitopes in these proteins. Except for PHA-E from black turtle bean [59], no information on the number and distribution of T epitopes is available for other potentially allergenic lectins. However, some information has been collected on the location and distribution of IgE-binding B-cell epitopic regions on the surface of a few lectins, especially legume lectins. In this respect, legume lectins exhibit a B-cell epitope density and diversity comparable to those of other plant allergenic proteins.
Even though plant lectins have been identified as relevant allergens, it is currently not possible to investigate lectins of plant origin as potential food allergens, because except for rHev b 6.02 (recombinant hevein), none of these proteins, e.g., PNA for peanut allergy diagnosis, are available in natural or recombinant form in the allergen microarrays commercially available for the allergen component-resolved diagnosis of food allergy. In addition, the ability of many plant lectins to specifically recognize the carbohydrate moiety of IgE antibodies could generate some unexpected false-positive results, irrespective of the allergic status of the patients [97,98]. Similarly, many plant lectins, e.g., PHA from kidney bean [28] and ArtinM from jackfruit (Artocarpus heterophyllus) [89], can non-specifically degranulate both T lymphocytes and mast cells using an IgE-independent mechanism, and thus interfere with the specific degranulation of IgE-activated cells. To overcome these possible sources of bias, it is absolutely necessary to perform the analyses in the presence of a cocktail of inhibitory sugars, e.g., Man + Gal + Fuc, capable to prevent the non-specific binding of lectins to glycan structures associated with IgE and the cell surface of T-lymphocytes or mast cells.
Due to their widespread distribution in edible seeds and fruits, legume lectins and class I chitinases should be investigated in legume seed allergies (peanut, kidney bean, soybean, lentil, pea) and fruit allergies (banana, avocado, mango, kiwi, chestnut), using natural or recombinant lectins as molecular probes, in addition to rHev b 6.02, which is currently available for the IgE-mediated allergy diagnosis.