foods

Review Are Dietary Relevant Allergens in ?

Annick Barre 1 , Els J.M. Van Damme 2 , Mathias Simplicien 1, Hervé Benoist 1 and Pierre Rougé 1,*

1 UMR 152 PharmaDev, Institut de Recherche et Développement, Université Paul Sabatier, Faculté de Pharmacie, 35 Chemin des Maraîchers, 31062 Toulouse, France; [email protected] (A.B.); [email protected] (M.S.); [email protected] (H.B.) 2 Department of Biotechnology, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, B-9000 Ghent, Belgium; [email protected] * Correspondence: [email protected]; Tel.: +33-069-552-0851

 Received: 17 October 2020; Accepted: 21 November 2020; Published: 24 November 2020 

Abstract: Lectins or carbohydrate-binding are widely distributed in 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 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 lectins, type II ribosome-inactivating proteins (RIP-II), (WGA), jacalin-related lectins, GNA (Galanthus nivalis agglutinin)-like lectins, and Nictaba-related lectins. Most of these potentially active allergens belong to the group of storage proteins (legume lectins), pathogenesis-related 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 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.

Keywords: lectin; agglutinin; Ara h agglutinin; legume lectins; soybean lectin; garlic lectin; phytohemagglutinin; chitinase; allergen; IgE-binding epitope; allergenicity; food allergy

1. 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 [4–6]. 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

Foods 2020, 9, 1724; doi:10.3390/foods9121724 www.mdpi.com/journal/foods Foods 2020, 9, 1724 2 of 22 proteins [10], PR-12 defensins [11], and PR-14 lipid transfer proteins (LTP) [12], which all represent abundant proteins in seeds and other vegetative parts of edible . Among the protein allergens occurring in seeds and other vegetative tissues, the widely distributed plant lectins have been poorly investigated. Consequently, their role as relevant food allergens still remains a matter of debate [13]. Nevertheless, different families of lectins with various carbohydrate-binding specificities, have been identified and characterized in detail in all living organisms. Many of these lectins are rather abundant proteins in higher plants [14–16] (Table1). A possible explanation for the lack of data on the allergenic potency of plant lectins relates to their ability to non-specifically bind to the carbohydrate moiety of IgE which most probably hampered the research in this area. However, this shortcoming can be overcome easily by performing the IgE-binding experiments in the presence of a cocktail of inhibiting sugars, e.g., + , that prevent the non-specific binding of plant lectins to the carbohydrate moiety of IgE from sera of allergic patients [17].

Table 1. Lectin content of some edible plants, compared to their protein content.

Lectin Content Edible Plant Lectin Ref. (% of Total Protein) Lentil (Lens culinaris) LcA 2.5% [18] Pea (Pisum sativum) PsA 2.5% [19] Soybean (Glycine max) SBA 2% [20] Peanut (Arachis hypogaea) PNA 1.5% [21] Horse gram (Dolichos biflorus) DbA 10% [22] Kidney (Phaseolus vulgaris) PHA 1% [23] Wheat (Triticum aestivum) WGA 3% [24] Garlic (Allium sativum) ASA 10% [25] Black elderberry (Sambucus nigra) SNA-I 3% [25] (Musa acuminata) Mus a 2 2.3% [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.

2. 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) (Table1). 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 [28–30]. These lectins have been included in Table1 as non-IUIS approved potential food allergens (Table2). Foods 2020, 9, 1724 3 of 22

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.

Allergen Protein Plant Species IUIS Approved Food Allergen Ref. Mus a 2 Class I chitinase Musa acuminata + + [31] Tri a 18 Wheat germ agglutinin Triticum aestivum + + [32] Zea m 8 Class IV chitinase Zea mays + + [33] Bra r 2 Chitin-binding protein Brassica rapa + + [34] Cas s 5 Chitinase Castanea sativa + + * Hev b 6 Hevein precursor Hevea brasiliensis + Contact [35] Hev b 11 Class I chitinase Hevea brasiliensis + Contact * Pers a 1 Class I chitinase Persea americana + + [36] Act c chitinase Class I chitinase Actinidia chinensis + [37] Car p chitinase Class I chitinase Carica papaya + [38] Lyc es chitinase Class I chitinase Lycopersicum esculentum + [39] Ann ch chitinase Class I chitinase Annona cherimola + [39] Pas ed chitinase Class I chitinase Passiflora edulis + [39] Vit v chitinase Class IV chitinase Vitis vinifera + [40] AcA GNA-like Lectin Allium cepa + [41] AsA GNA-like lectin Allium sativum + [42] Ara h agglutinin Arachis hypogaea + [43] DbA Legume lectin Dolichos biflorus + [44] SBA Legume lectin Glycine max + [45] LcA Legume lectin Lens culinaris + [29] PHA-E Legume lectin Phaseolus vulgaris + [29] PHA-L Legume lectin Phaseolus vulgaris + [29] PsA Legume lectin Pisum sativum + [29] Chia lectin Legume lectin Salvia hispanica [29] Ricin RIP-II Ricinus communis + [46] SNA-I RIP-II Sambucus nigra + [29] BanLec Jacalin-related lectin Musa acuminata + [47] Cuc m 17 kDa Nictaba-related lectin Cucumis melo + [48] * No references available at the WHO/IUIS Allergen Nomenclature Sub-Committee. + means yes.

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].

2.1. 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, 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.

2.1.1. 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 Foods 2020, 9, x FOR PEER REVIEW 4 of 23

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].

2.1. 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.

2.1.1. 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- (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 Foods 2020, 9, 1724 4 of 22 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 h agglutininregions recognized and is considered as potential a minorIgE-binding peanut epitopes allergen display [43,50 both,51], Atelectropositively present, PNA (colored is not o ffiblue)cially recognizedand electronegatively as a peanut allergen (colored by red) the charged WHO/IUIS residues, Committee. as shown from the calculation of surface electrostaticNative PNA potentials occurs atas thea homotetramer molecular surface built fromof PNA the ( symmetricalFigure 1C). The non-covalent homology association between ofIgE four-binding identical epitopes protomers accounts of 25 for kDa, the cross exhibiting-reactivity the observed canonical among jelly roll legume fold lectins of legume regarding lectins their ability to interact with sera from peanut-allergic patients. However, this IgE-binding ability consisting of a β-sandwich made of two antiparallel β-sheets of 6 (front face) and 7 β-strands does not depend on the possible interaction of legume lectins with the carbohydrate moiety of IgE, (back face), respectively (Figure1A). Each protomer possesses a carbohydrate-binding site (CBS), since all the binding experiments were conducted in the presence of mannose and galactose, which which specifically recognizes Gal and GalNAc termini from unsialylated N-acetyllactosaminic glycans specifically inhibit the lectin activity of both Gal/GalNAc-specific (PNA, SBA) and man-specific and the T/Tn-antigen expressed at the surface of many cancer cells [52]. (LcA, PsA) legume lectins [53].

(A) (B) (C)

FigureFigure 1. 1.(A ().A) Three-dimensional. Three-dimensional structure structure ofof PNA, showing the the tetrameric tetrameric arrangement arrangement of ofthe the lectin lectin monomersmonomers (PDB (PDB code code 2PEL). 2PEL). Secondary Secondary structurestructure features are are represented represented and and the the molecular molecular surface surface

is shown in transparency. Monomers I, II, III, and IV are colored green, pink, orange, and purple, respectively. Lactose molecules complexed to the carbohydrate-binding sites in each monomer are colored cyan. (B). Localization of the sequential IgE-binding epitopes identified at the molecular surface of the PNA homotetramer. Epitopes 1, 2, 3, 4, 5, and 6 are colored red (1), blue (2), green (3), grey (4), purple (5), and cyan (6), respectively. (C). Electrostatic potentials calculated at the molecular surface of PNA. (A,B) are not centered and thus, are truncated.

Sequential IgE-binding B-cell epitopes identified on the surface of PNA [17] (Figure1B), 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 (Figure1C). 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 (Figure2A). 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. Foods 2020, 9, x FOR PEER REVIEW 5 of 23

is shown in transparency. Monomers I, II, III, and IV are colored green, pink, orange, and purple, respectively. Lactose molecules complexed to the carbohydrate-binding sites in each monomer are colored cyan. (B). Localization of the sequential IgE-binding epitopes identified at the molecular surface of the PNA homotetramer. Epitopes 1, 2, 3, 4, 5, and 6 are colored red (1), blue (2), green (3), grey (4), purple (5), and cyan (6), respectively. (C). Electrostatic potentials calculated at the molecular surface of PNA. (A) and (B) are not centered and thus, are truncated.

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 thatFoods 2020 specifically, 9, 1724 recognizes complex N-glycan chains, but does not recognize simple sugars,5 of 22 probably due to a particular conformation of the CBS.

(A) (B) (C)

Figure 2.2. ((AA).). Three-dimensionalThree-dimensional structure structure of theof the modeled modeled PHA-E PHA from-E blackfrom turtleblack bean turtle (Phaseolus bean (Phaseolus vulgaris), vulgarisshowing), showing the tetrameric the tetrameric arrangement arrangement of the lectinof the monomers.lectin monomers. Secondary Secondary structure structure features features are arerepresented represented and and the the molecular molecular surface surface is shownis shown in in transparency. transparency. Monomers Monomers I,I, II,II, III,III, andand IVIV are colored green, pink, orange,orange, and purple, purple, respectively. respectively. Bisected Bisected glycan glycan chains chains complexed to to the carbohydratecarbohydrate-binding-binding sites sites in each monomer are colored colored cyan. cyan. ( (B)).. Localization of the sequential sequential IgEIgE-binding-binding epitopes identified identified at at the molecular surface of the PHA PHA-E-E homotetramer. Epitopes Epitopes 1, 1, 2, 2, 3, 4, 5, 6 6,, and 7 are colored red (1), blue (2), green (3), grey (4), purple (5) (5) yellow yellow (6) (6),, and and cyan cyan (7), (7), respectively. ((CC).).Electrostatic Electrostatic potentials potentials calculated calculated at the at the surface surface of PHA-E. of PHA (A-E.,B )( areA) notand centered (B) are andnot centeredthus are truncated.and thus are truncated.

Both types of lectins from the white kidney bean [29,54 [29,54–58] and the black turtle bean [59,60] [59,60] have been reported as allergens allergens,, and a detailed bioinformatics study of of the B B-- and T T-cell-cell epitopes of PHAPHA-E-E from black turtl turtlee bean has been performed [59]. [59]. Up Up to to 7 IgE IgE-binding-binding B B-epitopes-epitopes ( (FigureFigure 22B)B) and 3 T T-epitopes-epitopes (Figure(Figure3 3)) occuroccur atat thethe surfacesurface ofof thethe PHA-EPHA-E tetramer.tetramer. SomeSome overlapoverlap waswas identifiedidentified between B-epitope B-epitope #4 # and4 and T-epitope T-epitope #2. Surprisingly, #2. Surprisingly, no B-epitopes no B-epitopes were identified were identifie along thed C-terminal along the Cregion-terminal of the region PHA-E of aminothe PHA acid-E sequenceamino acid despite sequence the pronounced despite the pronounced sequence homologies sequence betweenhomologies this betweenregion and this the region corresponding and the corresponding B-epitope-containing B-epitope region-containing of other region legume of other lectins legume (see Figure lectins 8(see for Figureexplanation). 8 for explanation Similar to PNA,). Similar some to of PNA,the IgE-binding some of the epitopic IgE-binding regions epitopicof PHA-E regions coincide of with PHA the-E coincidepresence with of charged the presence residues of (Figurecharged2C). residues (Figure 2C).

Figure 3. Three-dimensional structure of the modeled PHA-E from black turtle bean (Phaseolus vulgaris), showing the localization of T-cell epitopes 1, 2, and 3 identified by an in silico predictive approach. T-cell epitopes 1, 2, and 3 are colored green (1), red-brown (2), and magenta (3), respectively.

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 (Figures4A and5A). 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). Foods 2020,, 9,, xx FORFOR PEERPEER REVIEWREVIEW 6 of 23

Figure 3. Three-dimensional structure of the modeled PHA-E from black turtle bean (Phaseolus vulgaris), showing the localization of T-cell epitopes 1, 2,, and 3 identified by an in silico predictive approach. T-cell epitopes 1, 2,, and 3 are colored green (1), red-brown (2),, and magenta (3), respectively.

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, Foodsshare2020 strong, 9, 1724 identities/homologies with the epitope-containing regions of other legume lectins,6 of 22 especially the two-chain lectins LcA and PsA (see Figure 8 for explanation).

(A) (B) (C)

Figure 4. (A))... Three-dimensionalThreeThree-dimensional structurestructure of of the the horse horse gram gram lectin lectin DbA DbA (PDB (PDB code code 1LU1), 1LU1), showing showing the thetetramericthe tetramerictetrameric arrangement arrangementarrangement of theofof thethe lectin lectinlectin monomers. monomers.monomers. Secondary SecondarySecondary structure structurestructure features featuresfeatures are areare representedrepresentedrepresented and and thethe molecular surface is is shown in in transparency. Monomers Monomers I, I, II, II, III III,,, and and IV IV are are colored colored pink, pink, green, green, purplepurple,,, and orange, orange, respectively. respectively. Forssman disaccharides complexed to the carbohydrate-bindingcarbohydrate -binding sites in each monomer are colored cyan. ( B))... LocalizationLocalization atat thethe molecular surface of DbA of epitopic regions homologous to to the IgE IgE-binding-binding epitopes identified identified in LcA and PsA. Homologous epitopic regions are are colored colored red red (1), (1), blue blue (2), (2), green green (3), (3), purple purple (4), (4), yellow yellow (5),, (5),and andcyan cyan (6), respectively. (6), respectively. (C).. Electrostatic(C). Electrostatic potentials potentials calculated calculated at the atsurface the surfaceof DbA. of (A DbA.) and ((AB),B are) are not not centered centered and andthus, thus, are truncatedaretruncated truncated..

(A) (B) (C)

Figure 5. ((AA))... Three Three Three-dimensional-dimensional structure structure of of soybean soybean lectin lectin SBA SBA (PDB code 2SBA), showing the tetramerictetrameric arrangementarrangement arrangement ofof of thethe the lectinlectin lectin monomers.monomers. monomers. Secondary Secondary structure structure features features are arerepresented represented and andthe molecularthe molecular surface surface is shown is shown in transparency. in transparency. Monomers Monomers I, II, I, II, II,, II, I and I and IV IV are are colored colored pink, pink, green, green, purplepurple,,, and orange, respectively. Disaccharides complexed to the carbohydrate carbohydrate-binding-binding sites in each monomonomermer are colored cyan. ( (BB))... LocalizationLocalization Localization atat at thethe the surfacesurface ofof thethe SBA SBA tetramer tetramer of of the the IgE-bindingIgEIgE-binding epitopic regions homologous to to the IgE IgE-binding-binding epitopes identified identified in LcA and PsA. Homologous epitopic regions regions are are colored colored red red (1), blue(1), blue (2), green (2), green(3), purple (3), (4), purple yellow (4), (5), yellow and cyan (5),, (6), and respectively. cyan (6), respectively.(C). Electrostatic (C).. potentials ElectrostaticElectrostatic calculated potentialspotentials at the calculatedcalculated surface of atat SBA. thethe (surfacesurfaceA,B) not ofof centered SBA.SBA. (A and) and thus, (B) are not truncated. centered and thus, are truncated. 2.1.2. Homodimeric Two-Chain Lectins 2.1.2.Two Homodimeric homodimeric Two two-chain-Chain Lectins 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]. β Both lectins consist of two protomers with a canonical -sandwich structure, associated by non-covalent bonds in a homodimer which possesses two CBS located at both ends of the dimer (Figures6A and7A). 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 (Figure6B). 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 (Figure7B) [17]. Foods 2020, 9, x FOR PEER REVIEW 7 of 23

Foods 2020, 9, x FOR PEER REVIEW 7 of 23 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 Two homodimeric two-chain lectins have been investigated with respect to their IgE-binding [28,29,62]. Both lectins consist of two protomers with a canonical β-sandwich structure, associated activity, namely the lentil (Lens culinaris) lectin LcA and the garden pea (Pisum sativum) lectin PsA by non-covalent bonds in a homodimer which possesses two CBS located at both ends of the dimer [28,29,62]. Both lectins consist of two protomers with a canonical β-sandwich structure, associated (Figures 6A and 7A). The CBSs readily accommodate mannose, the trimannoside core of by non-covalent bonds in a homodimer which possesses two CBS located at both ends of the dimer N-acetyllactosaminic N-glycans, and more complex high-mannose glycans. (Figures 6A and 7A). The CBSs readily accommodate mannose, the trimannoside core of Nine sequential B-epitopes have been identified at the surface of both the β-subunit (7) and N-acetyllactosaminic N-glycans, and more complex high-mannose glycans. α-subunit (2), in each protomer of lentil lectin LcA (Figure 6B). Similarly, 10 sequential B-epitopes Nine sequential B-epitopes have been identified at the surface of both the β-subunit (7) and were characterized at the surface of the β-subunit (8) and α-subunit (2) of each garden pea lectin PsA α-subunit (2), in each protomer of lentil lectin LcA (Figure 6B). Similarly, 10 sequential B-epitopes protomer (Figure 7B) [17]. wereFoods 2020 characterized, 9, 1724 at the surface of the β-subunit (8) and α-subunit (2) of each garden pea lectin7 PsA of 22 protomer (Figure 7B) [17].

(A) (B) (C)

Figure 6. (A)(.A Three) -dimensional structure of the( Blentil) lectin LcA, showing the dimeric(C) arrangement of the lectin monomers (PDB code 1LES). Secondary structure features of the heavy (pale color) and Figure 6. ((A)).. Three Three-dimensional-dimensional structure of the lentil lectin LcA, showing the dimeric arrangement light (hard color) subunits are represented and the molecular surface is shown in transparency. of the the lectin lectin monomers monomers (PDB (PDB code code 1LES). 1LES). Secondary Secondary structure structure features features of the of heavy the heavy (pale (pale color) color) and Monomers I and II are colored green and pink, respectively. Sucrose molecules complexed to the lightand light (hard (hard color) color) subunits subunits are are represented represented and and the the molecular molecular surface surface is is shown shown in in transparency. transparency. carbohydrate-binding sites in each monomer are colored cyan. (B). Localization of the sequential Monomers I and II are colored green and pink, respectively. Sucrose molecules complexed to to the IgE-binding epitopes identified at the molecular surface of the LcA homodimer. Epitopes 1, 2, 5, 6, 7, carbohydratecarbohydrate-binding-binding sites sites in each monomer are colored cyan. ( (BB)).. Localization of of the sequential 8, and 9 are colored red (1), blue (2), green (5), grey (6), purple (7), cyan (8), and yellow (9), IgEIgE-binding-binding epitopes identifiedidentified atat thethe molecularmolecular surface surface of of the the LcA LcA homodimer. homodimer. Epitopes Epitopes 1, 1, 2, 2, 5, 5, 6, 6, 7, 7, 8, respectively. In each monomer, epitopes 1–7 are located on heavy subunits while epitopes 8 and 9 8and, and 9 are 9 coloredare colored red (1), red blue (1), (2), blue green (2), (5), green grey (5), (6), grey purple (6), (7), purple cyan (8), (7), and cyan yellow (8), and (9), respectively. yellow (9), occur in light subunits. (C). Electrostatic potentials calculated at the surface of LcA. (A) and (B) are respectiveIn each monomer,ly. In each epitopes monomer, 1–7 areepitopes located 1– on7 are heavy located subunits on heavy while subunits epitopes while 8 and epitopes 9 occur 8 in and light 9 not centered. occursubunits. in light (C). subunits. Electrostatic (C) potentials. Electrostatic calculated potentials at the calculated surfaceof atLcA. the surface (A,B) are of notLcA. centered. (A) and (B) are not centered.

(A) (B) (C)

Figure 7. 7. ((A(AA))..) Three Three-dimensional-dimensional structure structure of of (B the the) garden garden pea lectin PsA, showing(C ) the dimeric arrangement of the lectin monomers (PDB code 1BQP). Secondary structure features of the heavy Figure 7. (A). Three-dimensional structure of the garden pea lectin PsA, showing the dimeric (pale color)color) and light (hard color)color) subunits are represented and the molecularmolecular surfacesurface is shown in arrangement of the lectin monomers (PDB code 1BQP). Secondary structure features of the heavy transparency. Monomers Monomers I I and and II are colored colored green green and and pink, pink, respectively. respectively. Mannose molecules (pale color) and light (hard color) subunits are represented and the molecular surface is shown in complexed toto thethe carbohydrate-bindingcarbohydrate-binding sites sites in in each each monomer monomer are are colored colored cyan. cyan. (B). (B Localization). Localization of the of transparency. Monomers I and II are colored green and pink, respectively. Mannose molecules sequentialthe sequential IgE-binding IgE-binding epitopes epitopes identified identified at the molecular at the molecular surface of the surface PsA homodimer. of the PsA Epitopes homodimer. 1–10 complexed to the carbohydrate-binding sites in each monomer are colored cyan. (B). Localization of areEpitopes colored 1– red10 are (1), bluecolored (2), greenred (1), 35), blue magenta (2), green (4), grey 35), (5),magenta orange (4), (6), grey purple (5), (7), orange brown (6), (8), purple yellow (7), (9), the sequential IgE-binding epitopes identified at the molecular surface of the PsA homodimer. andbrown cyan (8), (10) yellow respectively. (9), and In cyan each monomer,(10) respective epitopesly. In 1–7 each are located monomer, onheavy epitopes subunits 1–7 are while located epitopes on Epitopes 1–10 are colored red (1), blue (2), green 35), magenta (4), grey (5), orange (6), purple (7), 8–10heavy occur subun inits light while subunits. epitopes (C 8).–10 Electrostatic occur in light potentials subunits. calculated (C). Electrostatic at the surface potentials of PsA. calculated (A,B) are at brown (8), yellow (9), and cyan (10) respectively. In each monomer, epitopes 1–7 are located on notthe surface centered. of PsA. (A) and (B) are not centered. heavy subunits while epitopes 8–10 occur in light subunits. (C). Electrostatic potentials calculated at the surface of PsA. (A) and (B) are not centered. Due to the high sequential sequential and and structural structural homologies homologies occurring among the closely-relatedclosely-related members of of the the legume legume lectin lectin family, family, most most of the of identified the identified sequential sequential IgE-binding IgE-binding B-epitopes B-epitopes exhibit Due to the high sequential and structural homologies occurring among the closely-related aexhibit very similar a very positioning similar positioning along the along amino the acid amino sequence acid sequenceand at the and surface at the of the surface lectin of protomers, the lectin members of the legume lectin family, most of the identified sequential IgE-binding B-epitopes inprotomers, both homotetrameric in both homotetrameric single-chain single lectins-chain (PNA, lectins PHA-E, (PNA, DbA, PHA SBA)-E, DbA, and homodimeric SBA) and homo two-chaindimeric exhibit a very similar positioning along the amino acid sequence and at the surface of the lectin lectinstwo-chain (LcA, lectins PsA), (LcA, irrespective PsA), irrespective of their structural of their architecture structural architecture and carbohydrate-binding and carbohydrate specificities-binding protomers, in both homotetrameric single-chain lectins (PNA, PHA-E, DbA, SBA) and homodimeric (Figurespecificities8). Most (Figure sequences 8). Most encodingsequences lectins encoding that lectins are abundant that are abundant in other edible in other legumes, edible legumes, such as two-chain lectins (LcA, PsA), irrespective of their structural architecture and carbohydrate-binding lupinesuch as (lupineLupinus (Lupinus albus), fabaalbus bean), faba (Vicia bean faba (Vic),ia winged faba), winged bean ( Psophocarpusbean (Psophocarpus teragonolobus teragonolobus), azuki), azuki bean specificities (Figure 8). Most sequences encoding lectins that are abundant in other edible legumes, (beanVigna (Vigna angularis angularis), and), and many many others, others, look look very very similar similar to to those those of of LcA LcA from from lentil, lentil, PsAPsA from pea, pea, such as lupine (Lupinus albus), faba bean (), winged bean (Psophocarpus teragonolobus), azuki PNA from peanut, and PHA from kidney bean and, especially, at the location of some of the predicted bean (Vigna angularis), and many others, look very similar to those of LcA from lentil, PsA from pea, 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 (Figure9), 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 (Figures1C,2C,4C,5C,6C and7C). Aromatic residues (F,Y,W) also frequently occur in the IgE-binding epitopic regions common to single- and two-chain legume lectins. Foods 2020, 9, x FOR PEER REVIEW 8 of 23

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 and 4–7C). Aromatic residues Foods 2020(F,Y,W), 9, 1724 also frequently occur in the IgE-binding epitopic regions common to single- and two-chain8 of 22

legume lectins.

1 10 20 30 40 50 60 LcA TETTSFSITKFSPDQQNLIFQGDG-YTTKGKLTLTKAVKS------TVGRALYSTPIHIW PsA TETTSFLITKFSPDQQNLIFQGDG-YTTKEKLTLTKAVKN------TVGRALYSSPIHIW PHA-E ATETSFSFQRFN--ETNLILQRDATVSSKGQLRLTNVNDNGEPTLSSLGRAFYSAPIQIW DbA ANIQSFSFKNFN--SPSFILQGDATVSS-GKLQLTKVKENGIPTPSSLGRAFYSSPIQIY SBA AETVSFSWNKFVPKQPNMILQGDAIVTSSGKLQLNKVDENGTPKPSSLGRALYSTPIHIW PNA AETVSFNFNSFSEGNPAINFQGDVTVLSNGNIQLTNLNKVN-----SVGRVLYAMPVRIW

70 80 90 100 110 120 LcA DRDTGNVANFVTSFTFVIDAPSSYNVADGFTFFIAPVDTKPQTGGGYLGVFNSKEYDKTS PsA DRETGNVANFVTSFTFVINAPNSYNVADGFTFFIAPVDTKPQTGGGYLGVFNSAEYDKTT PHA-E DNTTGAVASFATSFTFNIDVPNNSGPADGLAFVLLPVGSEPKDKGGLLGLFNNYKYDSNA DbA DKSTGAVASWATSFTVKISAPSKASFADGIAFALVPVGSEPRRNGGYLGVFDSDVYNNSA SBA DKETGSVASFAASFNFTFYAPDTKRLADGLAFFLAPIDTKPQTHAGYLGLFN--ENESGD PNA SSATGNVASFLTSFSFEMKDIKDYDPADGIIFFIAPEDTQIPAGSIGGGTLGVSDTKGAG

130 140 150 160 170 180 LcA QTVAVEFDTFYNAAWDPSNKERHIGIDVNSIKSVNTKSWNLQNGERANVVIAFNAATNVL PsA QTVAVEFDTFYNAAWDPSNRDRHIGIDVNSIKSVNTKSWKLQNGEEANVVIAFNAATNVL PHA-E HTVAVEFDTLYNVHWDPKP--RHIGIDVNSIKSIKTTTWDFVKGENAEVLITYDSSTKLL DbA QTVAVEFDTLSNSGWDPSM--KHIGIDVNSIKSIATVSWDLANGENAEILITYNAATSLL SBA QVVAVEFDTFRNS-WDPPN--PHIGINVNSIRSIKTTSWDLANNKVAKVLITYDASTSLL PNA HFVGVEFDTYSNSEYNDPP-TDHVGIDVNSVDSVKTVPWNSVSGAVVKVTVIYDSSTKTL

190 200 210 220 230 240 LcA TVTLTYPN-VTSYTLNEVVPLKDVVPEWVRIGFSATTGAEFA---AHEVHSWSFHSQLGH PsA TVSLTYPN-VTSYTLSDVVSLKDVVPEWVRIGFSATTGAEYA---AHEVLSWSFHSELSG PHA-E VASLVYPSLKTSFIVSDTVDLKSVLPEWVIVGFTATTGITKGNVETNDVLSWSFASKLSD DbA VASLVHPSRRTSYILSERVDITNELPEYVSVGFSATTGLSEGYIETHDVLSWSFASKLPD SBA VASLVYPSQRTSNILSDVVDLKTSLPEWVRIGFSAATGLDIP-GESHDVLSWSFASNLPH PNA SVAVTNDN-GDITTIAQVVDLKAKLPERVKFGFSASGSLGGR--QIHLIRSWSFTSTLIT

250 259 LcA TSKS------PsA TSSSKQ------PHA-E GTT-SEALNLANFALNQIL DbA DST-AEPLDLASYLVRNVL SBA ASSNIDPLDLTSFVLHEAI PNA TTRRS------

FigureFigure 8. Multiple 8. Multiple amino amino acid acid sequence sequence alignmentalignment of of two two-chain-chain (LcA, (LcA, PsA) PsA) and and single single-chain-chain (PHA (PHA-E,-E, DbA,DbA, SBA, SBA, PNA) PNA) lectins, lectins, showing showing the localization the localization of B- of and B- T-epitopesand T-epitopes along along the amino the amino acid sequences. acid The IgE-bindingsequences. The B-epitopic IgE-binding regions B-epitopic are indicatedregions are inindicated white lettersin white highlighted letters highlighted blue (LcA), blue (LcA), deep blue (PsA),deep magenta blue (PsA), (PHA-E), magenta and (PHAred (PNA),-E), and respectively. red (PNA), respective Regionsly. of DbA Regions and of SBA DbA homologous and SBA of homologous of B-epitopic regions of LcA and PsA are indicated in white letters highlighted green. B-epitopic regions of LcA and PsA are indicated in white letters highlighted green. The three T-epitopes The three T-epitopes predicted along the amino acid sequence of PHA-E are shown in white letters predicted along the amino acid sequence of PHA-E are shown in white letters highlighted black and highlighted black and burgundy color for T-epitope #2 overlapping the B-epitope #4 region. Foodsburgundy 2020, 9, x FOR color PEER for REVIEW T-epitope #2 overlapping the B-epitope #4 region. 9 of 23

FigureFigure 9. 9Comparison. Comparison ofof somesome sequentialsequential IgE IgE-binding-binding epitopes epitopes identified identified at athomologous homologous positions positions alongalong the the amino amino acidacid sequencesequence of PNA, LcA, LcA, and and PsA. PsA. Identical Identical residues residues are are in inboxed boxed white white letters. letters. StructurallyStructurally homologous homologous residuesresidues according to to Risler’s Risler’s matrix matrix [63] [63 are] are grey grey boxed. boxed. Positively Positively (K,R) (K,R) andand negatively negatively (D,E) (D,E) charged charged residues residues are are colored colored blue blue and and red, red, respectively. respectively. Aromatic Aromatic residues residues (F,Y,W) are(F,Y,W) colored are orange. 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):

Foods 2020, 9, x FOR PEER REVIEW 9 of 23

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 Foods 2020(F,Y,W), 9, 1724 are colored orange. 9 of 22

Obviously, these B-epitope similarities account for their IgE-binding cross-reactivity observed amongObviously, different theselegume B-epitope lectins, similaritiesas previously account reported for theirfor sera IgE-binding from peanut cross-reactivity-allergic patients observed [17]. Asamong shown diff fromerent surface legume plasmon lectins, asresonance previously (SPR) reported measurements, for sera fromboth peanut-allergicPNA, PHA-E, LcA patients, and PsA [17]. Aslectins, shown readily from interactedsurface plasmon with all resonance of the probed (SPR) measurements, sera from peanut both-allergic PNA, PHA-E, patients LcA, (Figure and PsA10). However,lectins, readily a stronger interacted IgE with-binding all of cross the probed-reactivity sera fromwas observed peanut-allergic for two patients-chain (Figurelegume 10 lectins,). However, LcA anda stronger PsA, compared IgE-binding to cross-reactivity that observed wasfor single observed-chain for legume two-chain lectins, legume PNA lectins, and PHA LcA- andE. These PsA, discrepanciescompared to thatamong observed the single for-single-chain and two-chain legume lectins, lectins, would PNA depend and on PHA-E. the way These the protomers discrepancies are assembledamong the single-in both and types two-chain of lectins, lectins, the dimeric would dependassociation on the of protomers way the protomers favoring are a better assembled exposure in both of epitopes,types of lectins, compared the dimeric to the association tetrameric of association protomers favoring in which a better some exposure epitopes of become epitopes, buried compared at the to protomerthe tetrameric interfaces. association in which some epitopes become buried at the protomer interfaces.

Figure 10. SPR measurement, expressed as resonance units (RU), (RU), of the interaction of sera (S1 (S1–S12)–S12) from peanut peanut-allergic-allergic patients with PNA (blue color), LcA (red color), P PsAsA (green color),color), and PHA-EPHA-E (violet color). Each Each value is the mean of three independent measurements [[17].17].

Another homodimeric two-chaintwo-chain lectin from chia (Salvia hispanica ) seeds has been identified identified as a potential IgE-bindingIgE-binding allergen [46]. [46]. The The single single amin aminoo acid acid sequence sequence stretch stretch AVEFDTLYNTNDPNYR AVEFDTLYNTNDPNYR characterized by by mass mass spectrometry spectrometry for for this this IgE-binding IgE-binding allergen allergen exhibits exhibits a high a percentage high percentage of identity of withidentity the withPhaseolus the Phaseolus coccineus coccineus(81.25%) (81.25 and the%) andPhaseolus the Phaseolus vulgaris vulgarisPHA-E (75.0%)PHA-E (75.0 and PHA-L%) and (75.0%)PHA-L Foods (75.0lectins2020, %9, ) (Figurex lectins FOR PEER 11(Figur): REVIEWe 11): 10 of 23

FigureFigure 11. Comparison 11. Comparison of the of the amino amino acid acid sequence sequence stretch stretch ofof SalviaSalvia hispanica hispanica with with the the corresponding corresponding regions of PHA-E, PHA-L and Phaseolus coccineus lectin. regions of PHA-E, PHA-L and Phaseolus coccineus lectin. Sera from people allergic to peanut-allergic people that were available to perform the IgE-binding cross-reactionsSera from people with di ff allergicerent lectins to containedpeanut-allergic specific IgE people that primarily that were recognize available the main to allergenic perform the IgE-bindingproteins fromcross peanut,-reactions the vicillinwith different Ara h 1, thelectins 2S albumins contained Ara specific h 2 and AraIgE hthat 6, and primarily the legumin recognize Ara h the main3. allergenic Therefore, proteins PNA (also from designated peanut, as the Ara vicillin h agglutinin Ara h allergen) 1, the 2S and albumins other IgE-binding Ara h 2 and cross-reacting Ara h 6, and the leguminlectins from Ara other h 3. legume Therefore, seeds PNA (LcA, (also PsA, designatedPHA, SBA), doas notAra actually h agglutinin correspond allergen) to the and main other IgE-bindingcomponents cross of- allergies.reacting Inlectins this respect, from other none oflegume the peanut seeds allergies (LcA, identifiedPsA, PHA, so farSBA), can bedo attributed not actually correspondto the PNA to allergen. the main components of allergies. In this respect, none of the peanut allergies identified so far can be attributed to the PNA allergen.

2.2. 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 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.

Figure 12. (A). Three-dimensional structure of ricin (PDB code 2AAI), showing the arrangement of the catalytic chain A (colored magenta) and the lectin chain B (colored blue), covalently linked by a disulfide bridge (colored red) indicated by a red arrow. Secondary structure features of both chains are represented and their molecular surfaces are shown in transparency. The location of the CBSs in the lectin chain is indicated by a black star (★). (B). Electrostatic potentials calculated at the surface of ricin. (C). Three-dimensional structure of the modeled SNA-I, showing the arrangement of the

Foods 2020, 9, x FOR PEER REVIEW 10 of 23

Figure 11. Comparison of the amino acid sequence stretch of Salvia hispanica with the corresponding regions of PHA-E, PHA-L and Phaseolus coccineus lectin.

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 Foodscorrespond2020, 9, 1724 to the main components of allergies. In this respect, none of the peanut allergies10 of 22 identified so far can be attributed to the PNA allergen.

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

FigureFigure 12. 12(.A ().A) Three-dimensional. Three-dimensional structurestructure of ricin (PDB code code 2AAI), 2AAI), showing showing the the arrangement arrangement of of thethe catalytic catalytic chain chain A A (colored (colored magenta) magenta) andand thethe lectin chain chain B B (colored (colored blue), blue), covalently covalently linked linked by bya a disulfidedisulfide bridge bridge (colored (colored red) red) indicated indicated by by a reda red arrow. arrow. Secondary Secondary structure structure features features of of both both chains chains are representedare represented and their and their molecular molecular surfaces surfaces are shownare shown in transparency.in transparency. The The location location of of the the CBSs CBSs in in the lectinthe lectin chain chain is indicated is indicated by a by black a black star star (F ().★). (B ().B) Electrostatic. Electrostatic potentialspotentials calculated calculated at at the the surface surface of of ricin.ricin. (C ).(C Three-dimensional). Three-dimensional structure structure of the of modeledthe modeled SNA-I, SNA showing-I, showing the arrangement the arrangement of the of catalytic the chain A (colored magenta) and the lectin chain B (colored blue), covalently linked by a disulfide bridge (colored red), indicated by a red arrow. Secondary structure features of both chains are represented and their molecular surfaces are shown in transparency. The location of the CBSs in the lectin chain is indicated by a black star (F). (D). Electrostatic potentials calculated at the surface of the SNA-I model.

2.3. 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). Foods 2020, 9, x FOR PEER REVIEW 11 of 23

catalytic chain A (colored magenta) and the lectin chain B (colored blue), covalently linked by a disulfide bridge (colored red), indicated by a red arrow. Secondary structure features of both chains are represented and their molecular surfaces are shown in transparency. The location of the CBSs in the lectin chain is indicated by a black star (★). (D). Electrostatic potentials calculated at the surface of the SNA-I model.

2.3. 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 haveFoods 2020 been, 9, 1724 identified at the surface of the hevein (Hev b 6.02) polypeptide allergen [70,71]11 (see of 22 Section 2.4).

(A) (B)

Figure 13.13. (A)).. Three-dimensionThree-dimension alal structure structure of of WGA WGA (PDB (PDB code code 2UVO), 2UVO), showing showing the the arrangement arrangement of two of twohevein hevein tetramers, tetramers, colored colored red and red green, and respectively. green, respectively. Secondary Secondary structure features structure of both features hevein of unitsboth heveinare represented units areand their represented molecular and surfaces their are molecular shown in transparency. surfaces are N-acetylglucosamine shown in transpa residuesrency. N(colored-acetylglucosamine cyan) are anchored residues to the (colored CBSs occurring cyan) are in some anchored of the to hevein the CBSsunits. occurring (B). Electrostatic in some potentials of the heveincalculated units. at the(B). surface Electrostatic of WGA. potentials (A) is not calculated centered at and the thus, surface is truncated. of WGA. (A) is not centered and thus, is truncated. 2.4. Chitinases Containing a Hevein Domain 2.4. ChitinasesHevein occurs Containing as a freea Heve polypeptidein Domain allergen Hev b 6.02, which has been identified as a major allergenHevein in the occurs latex fromas a free the rubberpolypeptide tree (Ricinus allergen communis Hev b)[ 6.02,35,72 which,73]. This has isbeen a small identified contact as allergen a major of allergenonly 43 amino in the acids, latex organized from the in rubber a central treeβ-hairpin (Ricinus sandwiched communis) between [35,72,73]. two This short isN a- and smallC-terminal contact allergenα-helices of (Figure only 43 14 aminoA,C), tightlyacids, organized structured in by a four central disulfide β-hairpin bridges. sandwiched Two discontinuous between two B-epitopes short N- andhave C been-terminal identified α-helices at the molecular (Figure 14 surfaceA,C), tightlyof Hev b structured 6.02, corresponding by four todisulfide amino acid bridges. residues Two 2, discontinuous10–12, 14–15, 26, B-epitopes 40, 42 (epitope have been 1) and identified 5–6, 19–21, at the 23, molecular 29, 33–34 surface (epitope of 2),Hev that b 6.02, specifically corresponding interact towith amino two acid moAbs residues scFv G72, 1 and0–12 scFv, 14–15 H7,, 26, respectively 40, 42 (epitope (Figure 1) and 14B) 5– [671, 1].9– T-epitopes21, 23, 29, 3 corresponding3–34 (epitope 2), to thatHLA-DR4, specifically have interactbeen identified with two on moAbsprohevein scFv Hev G7 b and 6.01, scFv the precursor H7, respectively of hevein (Fi whichgure 1 is4B) further [71]. Tpost-transcriptionally-epitopes corresponding cleaved to HLA in two-DR4, Hev have b 6.02 been (hevein) identified and on Hev prohevein b 6.03 proteins, Hev b 6.01, but the none precursor of them ofFoodsoccurs hevein 2020 along, 9 which, x FOR the PEERis Hev further REVIEW b 6.02 post amino -transcriptionally acid sequence cleaved [74]. in two Hev b 6.02 (hevein) and Hev 12b 6.03of 23 proteins, but none of them occurs along the Hev b 6.02 amino acid sequence [74].

(A) (B) (C)

Figure 14.14. (A)).. Three Three-dimensional-dimensional structurestructure ofof HevHev b b 6 6 (PDB (PDB code code 1WKX), 1WKX), showing showing the the arrangement arrangement of ofthe the secondary secondary structure structure features features along along the short the short polypeptide polypeptide chain. chain. The molecular The molecular surface surface is shown is shownin transparency. in transparency. (B). Mapping (B). Mapping of discontinuous of discontinuous B-epitopes B-epitopes on theon the molecular molecul surfacear surface of of Hev Hev b 6,b 6,colored colored green green (epitope (epitope 1) and 1) purple and (epitopepurple (epitope 2), respectively. 2), respectively. (C). Electrostatic (C). Electrostatic potentials calculated potentials at calculatedthe surface at of hevein.the surface (A,B of) are hevein. not centered (A) and (and (B) too are large) not and centered thus, are(and truncated. too large) and thus, are truncated. Hevein occurs as a N-terminal domain in various plant endochitinases belonging essentially to classesHevein I occurs and class as a IVN-terminal chitinases. domain Class in Ivarious chitinase plant allergens endochitinases have been belonging identified essentially in banana to classes(Mus a 2)I and [31, 75class], IV chestnut chitinases. (Castanea Class I chitinase sativa) allergens (Cas s have 5) been [76,77 identified], avocado in banana (Persea (Mus americana a 2)) [31,75],(Per a 1) chestnut [36,76,78 ,(79Castanea], passion sativa fruit) (Cas (Passiflora s 5) [76,77], edulis avocado) (Pas ed (Persea chitinase) americana [39], ) kiwi(Per (aActinidia 1) [36,76,78,79], chinensis) passion(Act c chitinase) fruit (Passiflora [37], papaya edulis (Carica) (Pas papayaed chitinase)) (Car p chitinase)[39], kiwi [ 38(Actinidia], custard chinensis apple (Annona) (Act c cherimola chitinase))[39 [37],,80], papayatomato (Lycopersicum (Carica papaya esculentum) (Car p ) chitinase)(Lyc es chitinase) [38], custard [39,81 ], apple and in (Annona the rubber cherimola tree latex) [39,80], as a contacttomato (allergenLycopersicum Hev besculentum 11 [82]. Similar) (Lyc es to chitinase) Mus a 2 [39,81], from banana, and in classthe rubber I chitinases tree latex comprise as a contact an N-terminal 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].

(A) (B)

Figure 15. (A). Three-dimensional model built for chitinase I allergen Mus a 2 from banana, made of an N-terminal hevein domain (colored magenta) linked by an extended hinge (colored cyan) to the C-terminal chitin-binding glycosyl 19 domain (colored green). (B). Electrostatic potentials calculated at the surface of the Mus a 2 model.

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– 85].

2.5. Jacalin-Related Lectins

Foods 2020, 9, x FOR PEER REVIEW 12 of 23

(A) (B) (C)

Figure 14. (A). Three-dimensional structure of Hev b 6 (PDB code 1WKX), showing the arrangement of the secondary structure features along the short polypeptide chain. The molecular surface is shown in transparency. (B). Mapping of discontinuous B-epitopes on the molecular surface of Hev b 6, colored green (epitope 1) and purple (epitope 2), respectively. (C). Electrostatic potentials calculated at the surface of hevein. (A) and (B) are not centered (and too large) and thus, are truncated.

Hevein occurs as a N-terminal domain in various plant endochitinases belonging essentially to classes I and class IV chitinases. Class I chitinase allergens have been identified in banana (Mus a 2) [31,75], chestnut (Castanea sativa) (Cas s 5) [76,77], avocado (Persea americana) (Per a 1) [36,76,78,79], passion fruit (Passiflora edulis) (Pas ed chitinase) [39], kiwi (Actinidia chinensis) (Act c chitinase) [37], papaya (Carica papaya) (Car p chitinase) [38], custard apple (Annona cherimola) [39,80], tomato (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).

Foods 2020Class, 9, IV 1724 chitinases, which also contain a shortened chitin-binding module, have been identified12 of 22 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 heveincatalytic domain, domain linked expressed by an asextended a recombinant hinge region molecule to a C in-terminal E. coli chitin-bindingstill interacted catalytic with sera domain from (Figurepatients 15 allergic). to maize [33].

(A) (B)

Figure 15.15. (A).). Three-dimensionalThree-dimensional model built for chitinase I allergen Mus a 2 from banana, made of anan N-terminalN-terminal hevein domain (colored magenta) linked by an extended hinge (colored cyan) to the C-terminalC-terminal chitin-bindingchitin-binding glycosyl hydrolase 19 domain (colored green). (B).). Electrostatic potentials calculatedcalculated at the surface of the Mus a 2 model.

ClassDue to IV their chitinases, widespread which also distribution contain a in shortened plants and chitin-binding plant products, module, chitinases have been and identified especially in cornclass (IZea chitinases, mays) (Zea account m 8) [33 for] and IgE in-binding grape ( Vitiscross vinifera-reactivity) (Vit between v chitinase) various [40]. fruits However, and thevegetables allergenicity [83– of85]. 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 maize2.5. Jacalin [33].-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 [83–85].

2.5. 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 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.

2.6. GNA-Like Lectins Food allergic manifestations associated with the consumption of spices like garlic (Allium sativum) and (Allium cepa) have been attributed to the occurrence of lectins belonging to the vast group of GNA-like lectins in these edible bulbs [41,42,90–92]. 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 Foods 2020, 9, x FOR PEER REVIEW 13 of 23

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 Foods 2020homotetrameric, 9, 1724 structures displaying two or four identical CBSs, respectively. In this respect,13 of 22 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 perpendicular to the axis. Each protomer contains three CBSs which specifically recognize mannose antiparallel β-strands arranged into a β-prism structure along a longitudinal axis. Each protomer residues.possesses Two a non-covalently CBSs located at linkedthe top protomersof the β-prism build structure, the hexavalent which specifically homodimeric recognizes structure N-glycans found in the garlicof the lectin high-mannose ASA (Figure type (17Figure). 16) [86].

(A) (B)

FigureFigure 16. 1(A6. ).(A Three-dimensional). Three-dimensional structure structure of the BanLec BanLec dimer dimer (PDB (PDB code code 4PIT), 4PIT), showing showing the the β-prismβ-prism II arrangement II arrangement of of the theβ -sheetsβ-sheets in in each each monomer. monomer. Molecular Molecular surfaces surfaces of both of both monomers, monomers, coloredcolored magenta magenta and and blue, blue, respectively, respectively, are are shown shown in transparency.in transparency. Dimannoside Dimannoside ligands ligands (colored (colored cyan) cyan) anchor to the CBSs located at the top of both monomers. (B). Electrostatic potentials calculated anchor to the CBSs located at the top of both monomers. (B). Electrostatic potentials calculated at the at the surface of BanLec. (A) is not centered and thus, is truncated. surfaceFoods 2020 of, 9 BanLec., x FOR PEER (A )REVIEW is not centered and thus, is truncated. 14 of 23 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. (A) (B) 2.6. GNA-Like Lectins FigureFigure 17. ( A17).. ( Three-dimensionalA). Three-dimensional structure structure ofof the homodimeric garlic garlic lectin lectin ASA ASA (PDB (PDB code code1KJ1), 1KJ1), showingFoodshowing the allergic arrangementthe arrangement manifestations of of both both associated monomers, monomers, with colored the magenta magenta consumption and and blue, blue, of respectively, spices respectively, like garlicthrough through ( Allium a a sativum) and onion (Allium cepa) have been attributed to the occurrence of lectins belonging to the reciprocalreciprocal swapping swapping of theirof their C-terminal C-terminal ends. ends. Secondary structure structure features features of bo ofth both monomers monomers are are vast represented group of andGNA their-like molecular lectins surfacesin these are edible shown bulbs in transparency. [41,42,90– Mannose92]. Being residues members (colored of the represented and their molecular surfaces are shown in transparency. Mannose residues (colored cyan) GNAcyan)-related are lectin anchored group, to theboth three the garlic CBSs occurringlectin ASA in and each the monomer. onion ACA(B). Electrostaticlectin possess potentials protomers are anchored to the three CBSs occurring in each monomer. (B). Electrostatic potentials calculated at exhibitingcalculated the βat- prismthe surface I fold, of ASA.which consists of three bundles of four antiparallel β-strands arranged the surface of ASA. into a β-prism structure perpendicular to the axis. Each protomer contains three CBSs which specificallyIn spite recognize of their mannoseallergenic residues. properties, Two no non B-- covalentlyand T-epitope linked of protomers garlic and build onion the GNA hexavalent-related In spite of their allergenic properties, no B- and T-epitope of garlic and onion GNA-related homodimericlectins have been structure characterized. found in theIn contrastgarlic lectin to the ASA garlic (Figure and 1onion7). lectins, the closely-related taro lectins(Colocasia have been esculenta characterized.) lectin was not In identified contrast as to a the food garlic allergen and [93]. onion lectins, the closely-related taro (Colocasia esculenta) lectin was not identified as a food allergen [93]. 2.7. Nictaba-Related Lectins 2.7. Nictaba-Related Lectins The 17kDa protein in the phloem sap of watermelon (Cucumis melo) belongs to the TheNictaba 17kDa-related protein lectins in the and phloem has been sap identified of watermelon as an allergen (Cucumis involved melo) belongs in systemic to the reactions Nictaba-related to lectinsmelon and and has watermelon been identified consumption as an [48]. allergen Similar to involved the tobacco in ( systemicNicotiana tabacum reactions) lectin to Nictaba, melon and watermelonthe protein consumption is exclusively [48 composed]. Similar toof theβ-stands tobacco arranged (Nicotiana in a β tabacum-sandwich) lectin structure, Nictaba, containing the protein a is exclusivelysingle CBS composed with a GlcNAc of β-stands-specificity arranged (Figure in a1β8)-sandwich [94]. Currently, structure, there are containing no investigations a single CBSon the with a occurrence of B- and T-epitopes in these molecules. GlcNAc-specificity (Figure 18)[94]. Currently, there are no investigations on the occurrence of B- and T-epitopes in these molecules.

(A) (B)

Figure 18. (A). Three-dimensional model built for the 17 kDa Nictaba-related lectin from Cucumis melo, showing the β-sheets arranged in a β-sandwich structure. Secondary structure features are represented and the molecular surface is shown in transparency. GlcNAc residues anchored to the possible CBS are colored cyan. (B). Electrostatic potentials calculated at the surface of the model built for the Nictaba-related lectin from Cucumis melo. (A) is too large and not centered, thus it is truncated.

3. 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

Foods 2020, 9, x FOR PEER REVIEW 14 of 23

(A) (B)

Figure 17. (A). Three-dimensional structure of the homodimeric garlic lectin ASA (PDB code 1KJ1), showing the arrangement of both monomers, colored magenta and blue, respectively, through a reciprocal swapping of their C-terminal ends. Secondary structure features of both monomers are represented and their molecular surfaces are shown in transparency. Mannose residues (colored cyan) are anchored to the three CBSs occurring in each monomer. (B). Electrostatic potentials calculated at the surface of ASA.

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].

2.7. 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 Foods 2020single, 9, CBS 1724 with a GlcNAc-specificity (Figure 18) [94]. Currently, there are no investigations on the14 of 22 occurrence of B- and T-epitopes in these molecules.

(A) (B)

FigureFigure 18. ( A18).. ( Three-dimensionalA). Three-dimensional model model built built for for the the 17 17 kDa kDa Nictaba-related Nictaba-related lectin lectin fromfrom CucumisCucumis melo, showingmelo, the showingβ-sheets the arranged β-sheets arrangedin a β-sandwich in a β-sandwich structure. structure. Secondary Secondary structure structure features features are represented are represented and the molecular surface is shown in transparency. GlcNAc residues anchored to the and the molecular surface is shown in transparency. GlcNAc residues anchored to the possible CBS possible CBS are colored cyan. (B). Electrostatic potentials calculated at the surface of the model built are colored cyan. (B). Electrostatic potentials calculated at the surface of the model built for the for the Nictaba-related lectin from Cucumis melo. (A) is too large and not centered, thus it is truncated. Nictaba-related lectin from Cucumis melo.(A) is too large and not centered, thus it is truncated. 3. Discussion 3. Discussion The group of plant lectins comprise a huge number of proteins which share the common Theproperty group to of specifically plant lectins recognize comprise carbohydrate a huge number structures, of proteins in spite which of very share different the common amino acid property to specificallysequences and recognize structural carbohydrate scaffolds. According structures, to their in spite carbohydrate of very-binding different domain, amino plant acid lectins sequences and structural scaffolds. According to their carbohydrate-binding domain, plant lectins have been divided into twelve distinct families (Table3), which display similar or di fferent carbohydrate-binding specificities towards simple and complex sugars.

Table 3. Classification of plant lectins in twelve families. Families containing allergens are in bold.

Lectin Family Structural Scaffold Simple Sugar Specificity Complex Sugar Specificity Agaricus bisporus lectin family β-sandwich Gal/GalNAc T-antigen Amaranthin family β-trefoil Gal/GalNAc T-antigen Class V chitinase homolog family TIM-barrel (α8β8) Man High-mannose glycans Cyanivirin family β-barrel Man High-mannose glycans Euonymus europaeus lectin family β-trefoil Gal, Man B + H group + high-mannose glycans GNA-like family β-trefoil Man High-mannose glycans Hevein family hevein domain GlcNAc chitin T-antigen, Jacalin-related lectin family β-barrel Gal, GalNAc, Man High-mannose glycans Complex glycans, Legume lectin family β-sandwich Gal, GalNAc, Man High-mannose glycans Chitin, lipochito- Lysin motif domain family αβ GlcNAc , Chitooligosaccharides, Nictaba family β-sandwich GlcNAc, Man high-mannose glycans, complex glycans Ricin B family * β-trefoil Gal Lactose * SNA-I, the RIP-II isolated from Sambucus nigra bark, exhibits an unusual Neu5Acα2,6-Gal/GalNAc- binding specificity [95].

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 Table1, have not yet been o fficially 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 Foods 2020, 9, 1724 15 of 22 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) (Table2). 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 [67–69]. 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 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.

4. Bioinformatics Multiple amino acid sequence comparisons were performed with the CLUSTAL-X program [99]. The atomic coordinates of lectins including PNA from peanut Arachis hypogaea (PDB code 2PEL) [100], DbA from horse gram Dolichos biflorus (PDB code 1LU1) [101], SBA from soybean Foods 2020, 9, 1724 16 of 22

Glycine max (PDB code 2SBA) [102], LcA from lentil Lens culinaris (PDB code 1LES) [103], ricin from castor bean Ricinus communis (PDB code 2AAI) [104], WGA from wheat Triticum aestivum (PDB code 2UVO) [105], Hev b 6 from the rubber tree Hevea brasiliensis (PDB code 1WKX), BanLec from banana Musa acuminata (PDB code 4PIT) [86], and ASA from garlic Allium sativum (PDB code 1KJ1) [106], were taken from the (http://www.rcsb.org/pdb/)[107]. Homology modeling of other lectins including PHA-E from the black turtle bean Phaseolus vulgaris [Acces. AHB17899.1], type II RIP SNA-I from the black elderberry Sambucus nigra [Acces. Q41358.1], the chitinase I Mus a 2 from banana Musa acuminata [Acces. XM_009394597.2], and the Nictaba-related lectin from watermelon Cucumis melo [Acces. NM_001305649.1], was performed with the YASARA Structure program [108] using various protein templates from the PDB, depending on the overall structural scaffold to which they belong (e.g., 1G8W from Phaseolus vulgaris [109], 1LU1 from Dolichos biflorus [101], 3UL2 from Dolichos lablab [110], 3WCS [111] and 5AVA[112] from Phaseolus vulgaris (PHA-E) for modeling the black turtle bean from Phaseolus vulgaris [59], shown in Figures2 and3). PROCHECK [113], ANOLEA [114], and the calculated QMEAN scores [115,116] were used to assess the geometric and thermodynamic qualities of the three-dimensional models. The surface-exposed electrostatic potentials of legume lectins were calculated and displayed (negatively and positively charged regions colored red and blue, respectively, and neutral regions colored grey) at the molecular surface with YASARA, using the classic values for the inner and outer dielectric constants applied to the proteins and the solvent, fixed at 4.0 and 80.0, respectively. Molecular cartoons were drawn with Chimera [117] and YASARA.

Author Contributions: A.B., M.S. and H.B. provided the bibliographic information and analyses. P.R.provided the molecular cartoons and the docking pictures. E.J.M.V.D. and P.R. wrote the review. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

ACA Allium cepa (onion) agglutinin/lectin ASA Allium sativum (garlic) agglutinin/lectin BanLec Banana (Musa acuminata) lectin CBS Carbohydrate-binding site DbA Dolichos biflorus (horse gram) agglutinin/lectin GNA Galanthus nivalis (snowdrop) agglutinin/lectin IgE Immunoglobulin E INFγ Interferon-gamma LcA Lens culinaris (lentil) agglutinin/lectin moAb Monoclonal antibody Nictaba Nicotiana tabacum (tobacco) agglutinin/lectin PHA-E Erythroagglutinin of Phaseolus (kidney bean) agglutinin/lectin PHA-L Leucoagglutinin of Phaseolus agglutinin/lectin PNA Peanut (Arachis hypogaea) agglutinin/lectin (also known as Ara h agglutinin) PsA Pisum sativum (garden pea) agglutinin/lectin RU Resonance unit SBA Soybean (Glycine max) agglutinin/lectin SNA Sambucus nigra (black elderberry) agglutinin/lectin SPR Surface plasmon resonance WGA Wheat (Triticum aestivum) germ agglutinin/lectin (also known as Tri a 18)

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