**Case 2 (original)**

**Clinical history:** For one year, a 27-year-old male patient suffered from labial edema after ingestion of vanilla ice creams and from cough during the night after ingestion of cow's milk (1 bowl) before bedtime.

Four months ago, an anaphylaxis (generalized urticaria, palpebral edema, dyspnea) occurred after ingestion of a food supplement (100% Whey Ultra) containing a whey protein concentrate.

The reaction started during the morning one hour after ingestion of the food supplement and beginning physical exercise (bodybuilding). He had not ingested anything else since the evening before.

He suffered from asthma since childhood with cat and dust mite allergy. For the last 6 months, due to unemployment, he has been living again with his mother who has a dog.

**Tests with extracts:** Positive aeroallergenic skin prick tests (mm): house dust mites 10, cat 10.5, dog 6, guinea pig 6, rabbit 5, hamster 4, grass pollens 6. Prick-to prick tests (mm): cow's milk 5, goat's milk 8.5, raw pork 12, cooked pork 0, raw lamb 9.5, cooked lamb 2, raw beef 3, cooked beef 1; negative for raw chicken and egg. Specific IgE to cow's milk: 0.45 kU/L

**Tests with molecules:** Specific IgE: Bos d 6: 0.80 kU/L; IgE to casein, beta-lactoglobulin, α-lactalbumin and alpha-gal <0.1 kU/L
IgE Can f 1: 8.66 kU/L, Can f 2: 0.48 kU/L, Can f 3: 34 kU/L
IgE Fel d 1: 5.09 kU/L, Fel d 2: 17 kU/L

**Conclusion:** Results are consistent with a primary sensitization to dog and/or cat and an allergy to milk, triggered by cross-reactivity

between cat and dog serum albumins and cattle serum albumin, present in dairy products.

**Case 3 (published¹⁴⁷¹)**

**Clinical history:** A 38-year-old woman was referred for asthma exacerbation. Upon dog contact, she first experienced oropharyngeal pruritus and rhinitis with sneezing and nasal obstruction, and more recently, cough and wheezing. She reports a previous anaphylactic reaction upon ingestion of horse meat and oropharyngeal pruritus after ingestion of ham.

**Tests with extracts:** Skin prick tests were positive for cat and dog dander as well as for horse meat, pork, and beef (prick-to-prick with raw meat). Allergen-specific IgE were positive for dog dander (67 kU/L), horse dander (0.58 kU/L), and pork (1.61).

**Tests with molecules:** The patient had specific IgE to Can f 1 (2.04 kU/L), Can f 3 (37 kU/L), and Fel d 2 (14.3 kU/L). Specific IgE were negative for alpha-gal, Fel d 1, Can f 2, and Equ c 1. The presence of sIgE directed at Equ c 3 was detected by ELISA. Inhibition experiments confirmed a primary sensitization to Can f 3 and an IgE-cross-reactivity to Equ c 3.

**Conclusion:** Results were consistent with anaphylaxis to horse meat induced by exposure to dog dander and cross-reactivity between dog and horse albumin.

**Allergen nomenclature:** Fel d 2, Bos d 6, Can f 3, Sus s 1, Equ c 3, Gal d 5: cat, bovine, dog, porcine, horse and chicken serum albumins respectively.
**Case 2 (original)**

**Clinical history:** For one year, a 27-year-old male patient suffered from labial edema after ingestion of vanilla ice creams and from cough during the night after ingestion of cow's milk (1 bowl) before bedtime.

Four months ago, an anaphylaxis (generalized urticaria, palpebral edema, dyspnea) occurred after ingestion of a food supplement (100% Whey Ultra) containing a whey protein concentrate.

The reaction started during the morning one hour after ingestion of the food supplement and beginning physical exercise (bodybuilding). He had not ingested anything else since the evening before.

He suffered from asthma since childhood with cat and dust mite allergy. For the last 6 months, due to unemployment, he has been living again with his mother who has a dog.

**Tests with extracts:** Positive aeroallergenic skin prick tests (mm): house dust mites 10, cat 10.5, dog 6, guinea pig 6, rabbit 5, hamster 4, grass pollens 6. Prick-to prick tests (mm): cow's milk 5, goat's milk 8.5, raw pork 12, cooked pork 0, raw lamb 9.5, cooked lamb 2, raw beef 3, cooked beef 1; negative for raw chicken and egg. Specific IgE to cow's milk: 0.45 kU/L

**Tests with molecules:** Specific IgE: Bos d 6: 0.80 kU/L; IgE to casein, beta-lactoglobulin, α-lactalbumin and alpha-gal <0.1 kU/L
IgE Can f 1: 8.66 kU/L, Can f 2: 0.48 kU/L, Can f 3: 34 kU/L
IgE Fel d 1: 5.09 kU/L, Fel d 2: 17 kU/L

**Conclusion:** Results are consistent with a primary sensitization to dog and/or cat and an allergy to milk, triggered by cross-reactivity

between cat and dog serum albumins and cattle serum albumin, present in dairy products.

**Case 3 (published¹⁴⁷¹)**

**Clinical history:** A 38-year-old woman was referred for asthma exacerbation. Upon dog contact, she first experienced oropharyngeal pruritus and rhinitis with sneezing and nasal obstruction, and more recently, cough and wheezing. She reports a previous anaphylactic reaction upon ingestion of horse meat and oropharyngeal pruritus after ingestion of ham.

**Tests with extracts:** Skin prick tests were positive for cat and dog dander as well as for horse meat, pork, and beef (prick-to-prick with raw meat). Allergen-specific IgE were positive for dog dander (67 kU/L), horse dander (0.58 kU/L), and pork (1.61).

**Tests with molecules:** The patient had specific IgE to Can f 1 (2.04 kU/L), Can f 3 (37 kU/L), and Fel d 2 (14.3 kU/L). Specific IgE were negative for alpha-gal, Fel d 1, Can f 2, and Equ c 1. The presence of sIgE directed at Equ c 3 was detected by ELISA. Inhibition experiments confirmed a primary sensitization to Can f 3 and an IgE-cross-reactivity to Equ c 3.

**Conclusion:** Results were consistent with anaphylaxis to horse meat induced by exposure to dog dander and cross-reactivity between dog and horse albumin.

**Allergen nomenclature:** Fel d 2, Bos d 6, Can f 3, Sus s 1, Equ c 3, Gal d 5: cat, bovine, dog, porcine, horse and chicken serum albumins respectively.
# C05 – Tropomyosins

Luis Caraballo, Andreas L. Lopata, Nathalie Acevedo

## Highlights

* Thermostable protein, high allergenicity

* Considered an invertebrate pan-allergen

* High degree of immunological and clinical cross-reactivity

* Seafood allergy, mostly induced by tropomyosins, is frequent in several populations

* Mite and *Ascaris* tropomyosin sensitization may affect asthma symptoms

## 1 – The protein

The shrimp (*Penaeus aztecus*) major allergen, Pen a 1, is one of the most clinically relevant allergenic tropomyosins.<sup>1483–1485</sup> Its basic characteristics are presented in Table 85.

There is no three-dimensional structure available from any allergenic tropomyosin. Still, the predicted models of representative tropomyosins from shrimp, house dust mite and the nematode *Ascaris* are presented in (Figure 160) The secondary structure is a coiled-coil molecule formed by two parallel alpha-helices.

<table><caption>TABLE 85 Protein characteristics of Pen a 1</caption><thead><tr><th colspan="2">Protein characteristics of Pen a 1</th></tr></thead><tbody><tr><td>Allergen source</td><td>Shrimp</td></tr><tr><td>Protein family</td><td>Tropomyosin</td></tr><tr><td>UniProtKB accession</td><td>Q3Y8M6</td></tr><tr><td>Crystal structure</td><td>No</td></tr><tr><td>Molecular structure</td><td>Alpha-helical</td></tr><tr><td>Theoretical molecular weight</td><td>32.8 kDa</td></tr><tr><td>Length</td><td>284 amino acids</td></tr><tr><td>Ligand binding</td><td>Yes</td></tr><tr><td>Dimerization</td><td>Homodimer</td></tr><tr><td>Glycosylation</td><td>No</td></tr><tr><td>Disulfide bonds</td><td>No</td></tr><tr><td>Isoelectric point</td><td>4.72</td></tr><tr><td>Synthesis</td><td>Several isoforms</td></tr><tr><td>Distribution</td><td>Muscle and non-muscle cells</td></tr></tbody></table>

Together with actin and myosin, tropomyosin plays an important role in the muscle contractile activity, and the regulation of cell morphology and motility. Tropomyosins are heat-stable, which partially explains their high allergenic activity; in addition, structural stability,<sup>1486</sup> endolysosomal degradation and subsequent peptide generation explain differences between cellular and humoral responses to some tropomyosins.<sup>1057</sup> Shellfish allergenic tropomyosins were first described in shellfish and shrimp,<sup>1487–1489</sup> but they are important allergens in other sources like house dust mites (HDM) and cockroaches. See also chapters B13 for shellfish/HDM cross-reactivity and B09 for insect cross-reactivity. The relevance of sensitization to tropomyosins varies from low clinical impact to anaphylaxis. In addition, tropomyosin has been found to bind epithelially expressed dectin-1, which suppresses the development of type 2 immune responses through inhibition of the production of IL-33 by bronchial epithelial cells and the subsequent recruitment of IL-13-producing innate lymphoid cells in mice, which in turn regulates dust mite sensitization. This process is genetically controlled since variants of the dectin-1 gene have different levels of expression in the epithelium.<sup>730</sup>

Several IgE-binding epitopes of shrimp tropomyosins have been reported. Ayuso et al. identified five major IgE-binding regions on Pen a 1 cross-reactive epitopes among shrimp, lobster, house dust mite and cockroach.<sup>1490,1678</sup> The sites include eight IgE-binding epitopes: epitope 1 (residues 43 – 55) in region 1; epitope 2 (residues 87 – 101) in region 2; epitopes 3a (residues 137 – 141) and 3b (residues 144 – 151) in region 3; epitope 4 (residues 187 – 197) in region 4; and epitopes 5a (residues 249 – 259), 5b (residues 266 – 273), and 5c (residues 273 – 281) in region 5. The sequence identity of these regions to homologous regions of other tropomyosins varies from 56% (rabbit) to 98% (lobster).

Further analyses of these epitopes and comparing them with other related sequences suggested that they can be classified into three types: epitope 5a that is highly conserved among crustaceans, mollusks, insects and mites. The second type comprised epitopes 2, 3 and 4, that represent all arthropods but not mollusks. The third type includes epitopes 1, 5b, and 5c, specific to crustaceans.<sup>1491</sup> Epitope mapping of other tropomyosins such as Pen j 1, Pen m 1 and Pan b 1 from shrimp, Tur c 1 (snail), Cra g 1 (oyster) and Oct v 1 (octopus) have also been performed. Overall, the C terminal region is the most conserved among invertebrate tropomyosins.<sup>773</sup> The high immunological cross-reactivity among crustacean tropomyosin is probably because 91% of IgE epitopes are conserved, as compared to mites (48%). By contrast, mollusks IgE is epitope-region less than 20% conserved, reflected in low clinical cross-reactivity between

<figure><img src="image_284.png" /><figcaption>FIGURE 160 Molecular models of tropomyosins from shrimp (Pen a 1), house dust mite (Der p 10), and the nematode *Ascaris lumbricoides*, Asc I 3.</figcaption></figure>
# C05 – Tropomyosins

Luis Caraballo, Andreas L. Lopata, Nathalie Acevedo

## Highlights

* Thermostable protein, high allergenicity

* Considered an invertebrate pan-allergen

* High degree of immunological and clinical cross-reactivity

* Seafood allergy, mostly induced by tropomyosins, is frequent in several populations

* Mite and *Ascaris* tropomyosin sensitization may affect asthma symptoms

## 1 – The protein

The shrimp (*Penaeus aztecus*) major allergen, Pen a 1, is one of the most clinically relevant allergenic tropomyosins.<sup>1483-1485</sup> Its basic characteristics are presented in Table 85.

There is no three-dimensional structure available from any allergenic tropomyosin. Still, the predicted models of representative tropomyosins from shrimp, house dust mite and the nematode *Ascaris* are presented in (Figure 160) The secondary structure is a coiled-coil molecule formed by two parallel alpha-helices.

<table><caption>TABLE 85 Protein characteristics of Pen a 1</caption><thead><tr><th colspan="2">Protein characteristics of Pen a 1</th></tr></thead><tbody><tr><td>Allergen source</td><td>Shrimp</td></tr><tr><td>Protein family</td><td>Tropomyosin</td></tr><tr><td>UniProtKB accession</td><td>Q3Y8M6</td></tr><tr><td>Crystal structure</td><td>No</td></tr><tr><td>Molecular structure</td><td>Alpha-helical</td></tr><tr><td>Theoretical molecular weight</td><td>32.8 kDa</td></tr><tr><td>Length</td><td>284 amino acids</td></tr><tr><td>Ligand binding</td><td>Yes</td></tr><tr><td>Dimerization</td><td>Homodimer</td></tr><tr><td>Glycosylation</td><td>No</td></tr><tr><td>Disulfide bonds</td><td>No</td></tr><tr><td>Isoelectric point</td><td>4.72</td></tr><tr><td>Synthesis</td><td>Several isoforms</td></tr><tr><td>Distribution</td><td>Muscle and non-muscle cells</td></tr></tbody></table>

Together with actin and myosin, tropomyosin plays an important role in the muscle contractile activity, and the regulation of cell morphology and motility. Tropomyosins are heat-stable, which partially explains their high allergenic activity; in addition, structural stability,<sup>1486</sup> endolysosomal degradation and subsequent peptide generation explain differences between cellular and humoral responses to some tropomyosins.<sup>1057</sup> Shellfish allergenic tropomyosins were first described in shellfish and shrimp,<sup>1487-1489</sup> but they are important allergens in other sources like house dust mites (HDM) and cockroaches. See also chapters B13 for shellfish/HDM cross-reactivity and B09 for insect cross-reactivity. The relevance of sensitization to tropomyosins varies from low clinical impact to anaphylaxis. In addition, tropomyosin has been found to bind epithelially expressed dectin-1, which suppresses the development of type 2 immune responses through inhibition of the production of IL-33 by bronchial epithelial cells and the subsequent recruitment of IL-13-producing innate lymphoid cells in mice, which in turn regulates dust mite sensitization. This process is genetically controlled since variants of the dectin-1 gene have different levels of expression in the epithelium.<sup>730</sup>

Several IgE-binding epitopes of shrimp tropomyosins have been reported. Ayuso et al. identified five major IgE-binding regions on Pen a 1 cross-reactive epitopes among shrimp, lobster, house dust mite and cockroach.<sup>1490,1678</sup> The sites include eight IgE-binding epitopes: epitope 1 (residues 43 – 55) in region 1; epitope 2 (residues 87 – 101) in region 2; epitopes 3a (residues 137 – 141) and 3b (residues 144 – 151) in region 3; epitope 4 (residues 187 – 197) in region 4; and epitopes 5a (residues 249 – 259), 5b (residues 266 – 273), and 5c (residues 273 – 281) in region 5. The sequence identity of these regions to homologous regions of other tropomyosins varies from 56% (rabbit) to 98% (lobster).

Further analyses of these epitopes and comparing them with other related sequences suggested that they can be classified into three types: epitope 5a that is highly conserved among crustaceans, mollusks, insects and mites. The second type comprised epitopes 2, 3 and 4, that represent all arthropods but not mollusks. The third type includes epitopes 1, 5b, and 5c, specific to crustaceans.<sup>1491</sup> Epitope mapping of other tropomyosins such as Pen j 1, Pen m 1 and Pan b 1 from shrimp, Tur c 1 (snail), Cra g 1 (oyster) and Oct v 1 (octopus) have also been performed. Overall, the C terminal region is the most conserved among invertebrate tropomyosins.<sup>773</sup> The high immunological cross-reactivity among crustacean tropomyosin is probably because 91% of IgE epitopes are conserved, as compared to mites (48%). By contrast, mollusks IgE is epitope-region less than 20% conserved, reflected in low clinical cross-reactivity between

<figure><img src="image_290.png" /><figcaption>FIGURE 160 Molecular models of tropomyosins from shrimp (Pen a 1), house dust mite (Der p 10), and the nematode *Ascaris lumbricoides*, Asc I 3.</figcaption></figure>
<table><caption>TABLE 86 A list of clinically relevant tropomyosins</caption><thead><tr><th>Family</th><th>Allergen source</th><th>Allergen</th></tr></thead><tbody><tr><td rowspan="5">Penaeoidea</td><td>Brown shrimp (Penaeus aztecus)</td><td>Pen a 1</td></tr><tr><td>Northern Red Shrimp (Pandalus borealis)</td><td>Pan b 1</td></tr><tr><td>Giant tiger prawn (Penaeus monodon)</td><td>Pen m 1</td></tr><tr><td>European Shrimp (Litopenaeus vannamei)</td><td>Lit v 1</td></tr><tr><td>Common Shrimp (Crangon Crangon)</td><td>Cra c 1</td></tr><tr><td>Palinuridae</td><td>Spiny lobster (Panulirus stimpsoni)</td><td>Pan s 1</td></tr><tr><td rowspan="2">Cancridae</td><td>Common crab (Charybdis feriatus)</td><td>Cha f 1</td></tr><tr><td>Blue swimmer crab (Portunus pelagicus)</td><td>Por p 1</td></tr><tr><td>Hellixidae</td><td>Brown garden snail (Helix aspersa)</td><td>Hel as 1</td></tr><tr><td>Mytilidae</td><td>Green mussel (Perna viridis)</td><td>Per v 1</td></tr><tr><td>Octopodidae</td><td>Common Octopus (Octopus vulgaris)</td><td>Oct v 1</td></tr><tr><td>Ommastephidae</td><td>Japanese flying squid (Todarodes pacificus)</td><td>Tod p 1</td></tr><tr><td>Osteidae</td><td>Pacific cupped oyster (Crassostrea gigas)</td><td>Cra g 1</td></tr><tr><td>Haliotidea</td><td>Abalone (Haliotis diversicolor)</td><td>Hal d 1</td></tr><tr><td rowspan="2">Pyroglyphidae</td><td>House dust mite (Dermatophagoides farinae)</td><td>Der f 10</td></tr><tr><td>House dust mite (D. pteronyssinus)</td><td>Der p 10</td></tr><tr><td>Echymiopodidae</td><td>Storage mite (Blomia tropicalis)</td><td>Blo t 10</td></tr><tr><td>Blattidae</td><td>American cockroach (Periplaneta americana)</td><td>Per a 7</td></tr><tr><td>Blattellidae</td><td>German cockroach (Blattella germanica)</td><td>Bla g 7</td></tr><tr><td>Anisakidae</td><td>Anisakis (Anisakis simplex)</td><td>Ani s 3</td></tr><tr><td>Ascaridae</td><td>Roundworm (Ascaris lumbricoides)</td><td>Asc l 3</td></tr></tbody></table>

crustacean and mollusks. Specific epitopes in the N- and C-terminal region of tropomysins seem to distinguish between crustacean only and specific crustacean-mollusk cross-reactivity.¹⁰⁸²

In addition, T cell epitopes have been described. Ravkov E et al., using an *in vitro* MHC-peptide biding assay and *ex vivo* proliferation and cytokine release assays, identified and validated 17 T cell epitopes restricted to multiple MHC class II alleles. This finding is potentially useful for designing peptide-based immunotherapy of shrimp allergy. The challenge of further studies is to analyze which tropomyosins epitopes are species-specific markers. For example, common and specific epitopes have been reported among tropomysins from two important HDM.¹⁴⁹²

## 2 – The family

Tropomyosin belongs to a family of proteins (Pfam PF00261) that includes a large number of cross-reactive allergens, most of them from invertebrate sources, such as shrimp, lobster, crab, snail, abalone, whelk, clam, mussels, octopus, house dust mites, cockroaches and helminths (Tables 86 and 87).

Vertebrate tropomyosins have been regarded as nonallergenic, but IgE sensitization to fish tropomyosin¹⁰¹⁰ as well as cross-reactivity between shellfish and fish tropomyosin has been detected.¹⁰¹⁶

A great number of IgE-binding tropomyosins have been described, some of them cloned and expressed as recombinant proteins and tested for allergenicity. However, only few (Pen a 1, Pen m

1, Ani s 3, Bla g 7 and Der p 10) have been included in commercial *in vitro* allergy testing. Tropomyosin amino acid sequence is highly conserved among shellfish and other invertebrates, they share over 70% identity; their comparison with vertebrate tropomyosins reveals 51 to 57% identity.¹⁰¹⁵,¹⁴⁸³

Leung P et al. have shown that serum IgE to shrimp also binds tropomyosin of other shellfish such as greasy back shrimp, spiny lobster, Indo-Pacific swamp crab, abalone, whelk, green mussel, pen shell, scallop, Pacific oyster, cuttlefish, sword tip squid and octopus,¹⁴⁹⁴ which reflects the high cross-reactivity of this family. Still, tropomyosins are not the only shellfish allergens; other cross-reactive clinically relevant allergens have been reported,¹⁴⁹¹ also see Chapter B13.

To compare the protein sequences of allergenic tropomyosins, Leung M et al.¹⁴⁹¹ conducted a phylogenetic analysis. They found that arthropods tropomyosins share 91.7% homology (76.1 - 100%) and mollusks share 77.2% (65.1 - 99.3%), which indicates that, at the whole sequence level, tropomyosins are not species-specific allergy markers.

All tropomyosin pair with a sequence alignment of over 80% could be considered as high and are potentially cross-reactive. Sequence alignments are good primary prediction tools for cross-reactivity, but most importantly, the use of IgE-inhibition studies with sera from clinically well-characterized patients will allow defining clinically relevant cross-reactivity (Figure 161). In fact, the combination of experimental data with bioinformatic tools has been useful for predicting diagnosis-associated tropomyosins cross-reactive epitopes.¹⁰⁸²

A comprehensive phylogenetic tree of allergenic tropomyosins from various taxonomic groups is shown in (Figure 162).
<table><caption>TABLE 86 A list of clinically relevant tropomyosins</caption><thead><tr><th>Family</th><th>Allergen source</th><th>Allergen</th></tr></thead><tbody><tr><td rowspan="5">Penaeoidea</td><td>Brown shrimp (Penaeus aztecus)</td><td>Pen a 1</td></tr><tr><td>Northern Red Shrimp (Pandalus borealis)</td><td>Pan b 1</td></tr><tr><td>Giant tiger prawn (Penaeus monodon)</td><td>Pen m 1</td></tr><tr><td>European Shrimp (Litopenaeus vannamei)</td><td>Lit v 1</td></tr><tr><td>Common Shrimp (Crangon Crangon)</td><td>Cra c 1</td></tr><tr><td>Palinuridae</td><td>Spiny lobster (Panulirus stimpsoni)</td><td>Pan s 1</td></tr><tr><td rowspan="2">Cancridae</td><td>Common crab (Charybdis feriatus)</td><td>Cha f 1</td></tr><tr><td>Blue swimmer crab (Portunus pelagicus)</td><td>Por p 1</td></tr><tr><td>Hellixidae</td><td>Brown garden snail (Helix aspersa)</td><td>Hel as 1</td></tr><tr><td>Mytilidae</td><td>Green mussel (Perna viridis)</td><td>Per v 1</td></tr><tr><td>Octopodidae</td><td>Common Octopus (Octopus vulgaris)</td><td>Oct v 1</td></tr><tr><td>Ommastephidae</td><td>Japanese flying squid (Todarodes pacificus)</td><td>Tod p 1</td></tr><tr><td>Osteidae</td><td>Pacific cupped oyster (Crassostrea gigas)</td><td>Cra g 1</td></tr><tr><td>Haliotidea</td><td>Abalone (Haliotis diversicolor)</td><td>Hal d 1</td></tr><tr><td rowspan="2">Pyroglyphidae</td><td>House dust mite (Dermatophagoides farinae)</td><td>Der f 10</td></tr><tr><td>House dust mite (D. pteronyssinus)</td><td>Der p 10</td></tr><tr><td>Echymiopodidae</td><td>Storage mite (Blomia tropicalis)</td><td>Blo t 10</td></tr><tr><td>Blattidae</td><td>American cockroach (Periplaneta americana)</td><td>Per a 7</td></tr><tr><td>Blattellidae</td><td>German cockroach (Blattella germanica)</td><td>Bla g 7</td></tr><tr><td>Anisakidae</td><td>Anisakis (Anisakis simplex)</td><td>Ani s 3</td></tr><tr><td>Ascaridae</td><td>Roundworm (Ascaris lumbricoides)</td><td>Asc l 3</td></tr></tbody></table>

crustacean and mollusks. Specific epitopes in the N- and C-terminal region of tropomysins seem to distinguish between crustacean only and specific crustacean-mollusk cross-reactivity.¹⁰⁸²

In addition, T cell epitopes have been described. Ravkov E et al., using an *in vitro* MHC-peptide biding assay and *ex vivo* proliferation and cytokine release assays, identified and validated 17 T cell epitopes restricted to multiple MHC class II alleles. This finding is potentially useful for designing peptide-based immunotherapy of shrimp allergy. The challenge of further studies is to analyze which tropomyosins epitopes are species-specific markers. For example, common and specific epitopes have been reported among tropomyosins from two important HDM.¹⁴⁹²

## 2 – The family

Tropomyosin belongs to a family of proteins (Pfam PF00261) that includes a large number of cross-reactive allergens, most of them from invertebrate sources, such as shrimp, lobster, crab, snail, abalone, whelk, clam, mussels, octopus, house dust mites, cockroaches and helminths (Tables 86 and 87).

Vertebrate tropomyosins have been regarded as nonallergenic, but IgE sensitization to fish tropomyosin¹⁰¹⁰ as well as cross-reactivity between shellfish and fish tropomyosin has been detected.¹⁰¹⁶

A great number of IgE-binding tropomyosins have been described, some of them cloned and expressed as recombinant proteins and tested for allergenicity. However, only few (Pen a 1, Pen m

1, Ani s 3, Bla g 7 and Der p 10) have been included in commercial *in vitro* allergy testing. Tropomyosin amino acid sequence is highly conserved among shellfish and other invertebrates, they share over 70% identity; their comparison with vertebrate tropomyosins reveals 51 to 57% identity.¹⁰¹⁵,¹⁴⁸³

Leung P et al. have shown that serum IgE to shrimp also binds tropomyosin of other shellfish such as greasy back shrimp, spiny lobster, Indo-Pacific swamp crab, abalone, whelk, green mussel, pen shell, scallop, Pacific oyster, cuttlefish, sword tip squid and octopus,¹⁴⁹⁴ which reflects the high cross-reactivity of this family. Still, tropomyosins are not the only shellfish allergens; other cross-reactive clinically relevant allergens have been reported,¹⁴⁹¹ also see Chapter B13.

To compare the protein sequences of allergenic tropomyosins, Leung M et al.¹⁴⁹¹ conducted a phylogenetic analysis. They found that arthropods tropomyosins share 91.7% homology (76.1 - 100%) and mollusks share 77.2% (65.1 - 99.3%), which indicates that, at the whole sequence level, tropomyosins are not species-specific allergy markers.

All tropomyosin pair with a sequence alignment of over 80% could be considered as high and are potentially cross-reactive. Sequence alignments are good primary prediction tools for cross-reactivity, but most importantly, the use of IgE-inhibition studies with sera from clinically well-characterized patients will allow defining clinically relevant cross-reactivity (Figure 161). In fact, the combination of experimental data with bioinformatic tools has been useful for predicting diagnosis-associated tropomyosins cross-reactive epitopes.¹⁰⁸²

A comprehensive phylogenetic tree of allergenic tropomyosins from various taxonomic groups is shown in (Figure 162).
<table><caption>TABLE 87 Amino acids identities between some tropomyosins</caption><thead><tr><th rowspan="2">Classification</th><th rowspan="2">Species</th><th rowspan="2">Accesion number</th><th rowspan="2">#</th><th colspan="10">Crustacean</th><th colspan="2">Insect</th><th colspan="2">Mite</th><th colspan="5">Mollusc</th><th rowspan="2">Fish</th><th rowspan="2">Human</th></tr><tr><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th><th>7</th><th>8</th><th>9</th><th>10</th><th>11</th><th>12</th><th>13</th><th>14</th><th>15</th><th>16</th><th>17</th><th>18</th><th>19</th><th>20</th><th>21</th><th>22</th><th>23</th><th>24</th><th>25</th><th>26</th><th>27</th></tr></thead><tbody><tr><td rowspan="12">Crustacean</td><td>Giant tiger prawn</td><td>A1KY22</td><td>1</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Brown shrimp</td><td>Q3Y8M6</td><td>2</td><td>100</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Whiteleg shrimp</td><td>B4YAH6</td><td>3</td><td>100</td><td>100</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Giant fresh water prawn</td><td>D3XNR9</td><td>4</td><td>98</td><td>98</td><td>98</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Blue swimmer crab</td><td>M1H607</td><td>5</td><td>98</td><td>98</td><td>98</td><td>97</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Mud crab</td><td>A7LSV2</td><td>6</td><td>93</td><td>93</td><td>93</td><td>92</td><td>92</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Snow crab</td><td>A2V735</td><td>7</td><td>89</td><td>89</td><td>89</td><td>89</td><td>88</td><td>95</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Common yabby crayfish</td><td>WSRFX0</td><td>8</td><td>98</td><td>98</td><td>98</td><td>98</td><td>99</td><td>93</td><td>89</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>South African spiny lobster</td><td>K9UTQ8</td><td>9</td><td>94</td><td>94</td><td>94</td><td>94</td><td>94</td><td>96</td><td>92</td><td>94</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Red rock lobster</td><td>WSRFX7</td><td>10</td><td>95</td><td>95</td><td>95</td><td>95</td><td>95</td><td>92</td><td>88</td><td>95</td><td>93</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Flathead lobster</td><td>WSRFZ5</td><td>11</td><td>98</td><td>98</td><td>98</td><td>97</td><td>100</td><td>92</td><td>88</td><td>99</td><td>94</td><td>95</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="2">Insect</td><td>American cockroach</td><td>Q9UB83</td><td>12</td><td>82</td><td>82</td><td>82</td><td>82</td><td>81</td><td>83</td><td>80</td><td>82</td><td>83</td><td>81</td><td>82</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>German cockroach</td><td>Q9NG56</td><td>13</td><td>82</td><td>82</td><td>82</td><td>82</td><td>82</td><td>83</td><td>80</td><td>83</td><td>84</td><td>82</td><td>82</td><td>97</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="2">Mite</td><td>European house dust mite</td><td>O18416</td><td>14</td><td>80</td><td>80</td><td>80</td><td>80</td><td>80</td><td>82</td><td>78</td><td>80</td><td>82</td><td>79</td><td>80</td><td>80</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>American house dust mite</td><td>Q23939</td><td>15</td><td>82</td><td>82</td><td>82</td><td>82</td><td>81</td><td>83</td><td>80</td><td>82</td><td>84</td><td>81</td><td>81</td><td>81</td><td>98</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="12">Mollusc</td><td>Bay scallop</td><td>A1KY24</td><td>16</td><td>56</td><td>56</td><td>56</td><td>57</td><td>56</td><td>57</td><td>56</td><td>57</td><td>58</td><td>58</td><td>56</td><td>56</td><td>55</td><td>58</td><td>60</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Japanese scallop</td><td>Q9GUW9</td><td>17</td><td>60</td><td>60</td><td>60</td><td>61</td><td>60</td><td>61</td><td>60</td><td>60</td><td>62</td><td>61</td><td>60</td><td>59</td><td>58</td><td>60</td><td>62</td><td>88</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Blue mussel</td><td>Q25457</td><td>18</td><td>57</td><td>57</td><td>57</td><td>57</td><td>56</td><td>57</td><td>56</td><td>57</td><td>58</td><td>58</td><td>57</td><td>57</td><td>55</td><td>57</td><td>71</td><td>71</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Pacific oyster</td><td>B7XC66</td><td>19</td><td>62</td><td>62</td><td>62</td><td>62</td><td>61</td><td>62</td><td>60</td><td>62</td><td>63</td><td>62</td><td>62</td><td>61</td><td>60</td><td>60</td><td>62</td><td>74</td><td>75</td><td>79</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Common octopus</td><td>Q2VOV0</td><td>20</td><td>64</td><td>64</td><td>64</td><td>64</td><td>63</td><td>64</td><td>63</td><td>64</td><td>65</td><td>64</td><td>64</td><td>62</td><td>62</td><td>63</td><td>65</td><td>71</td><td>74</td><td>71</td><td>76</td><td>100</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Japanese flying squid</td><td>Q2V0V2</td><td>21</td><td>63</td><td>63</td><td>63</td><td>63</td><td>63</td><td>63</td><td>61</td><td>63</td><td>63</td><td>64</td><td>63</td><td>61</td><td>60</td><td>63</td><td>64</td><td>70</td><td>71</td><td>70</td><td>75</td><td>91</td><td>100</td><td></td><td></td><td></td></tr><tr><td>Golden cuttlefish</td><td>Q2V0V4</td><td>22</td><td>63</td><td>63</td><td>63</td><td>64</td><td>63</td><td>64</td><td>63</td><td>64</td><td>64</td><td>64</td><td>62</td><td>61</td><td>64</td><td>65</td><td>71</td><td>73</td><td>71</td><td>76</td><td>92</td><td>98</td><td>100</td><td></td><td></td><td></td></tr><tr><td>Brown garden snail</td><td>O97192</td><td>23</td><td>64</td><td>64</td><td>64</td><td>64</td><td>64</td><td>63</td><td>62</td><td>64</td><td>64</td><td>64</td><td>64</td><td>63</td><td>62</td><td>64</td><td>65</td><td>73</td><td>76</td><td>70</td><td>76</td><td>83</td><td>82</td><td>84</td><td>100</td><td></td></tr><tr><td>Horned turban</td><td>B7XC63</td><td>24</td><td>62</td><td>62</td><td>62</td><td>63</td><td>62</td><td>62</td><td>60</td><td>62</td><td>63</td><td>63</td><td>61</td><td>60</td><td>62</td><td>64</td><td>71</td><td>76</td><td>74</td><td>78</td><td>81</td><td>80</td><td>81</td><td>87</td><td>100</td><td></td></tr><tr><td>Disc abalone</td><td>B7XC62</td><td>25</td><td>63</td><td>63</td><td>63</td><td>64</td><td>63</td><td>63</td><td>61</td><td>63</td><td>64</td><td>64</td><td>63</td><td>62</td><td>62</td><td>63</td><td>64</td><td>71</td><td>77</td><td>75</td><td>79</td><td>83</td><td>81</td><td>81</td><td>87</td><td>97</td><td>100</td></tr><tr><td>Fish</td><td>Mozambique tilapia</td><td>K4PEK4</td><td>26</td><td>57</td><td>57</td><td>57</td><td>58</td><td>57</td><td>58</td><td>57</td><td>60</td><td>58</td><td>57</td><td>54</td><td>54</td><td>57</td><td>58</td><td>52</td><td>54</td><td>52</td><td>55</td><td>56</td><td>55</td><td>55</td><td>55</td><td>55</td><td>55</td><td>100</td></tr><tr><td rowspan="2">Human</td><td>Mozambique tilapia</td><td>P06753</td><td>27</td><td>57</td><td>57</td><td>57</td><td>58</td><td>57</td><td>58</td><td>57</td><td>58</td><td>57</td><td>59</td><td>57</td><td>57</td><td>54</td><td>54</td><td>55</td><td>58</td><td>51</td><td>53</td><td>52</td><td>55</td><td>55</td><td>54</td><td>54</td><td>56</td><td>55</td><td>55</td><td>94</td></tr><tr><td>Human</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody><tfoot><tr><td colspan="24">% identity</td><td>100</td><td>95</td><td>90</td><td>85</td><td>80</td><td>75</td><td>70</td><td>65</td><td>60</td><td>55</td><td>50</td></tr></tfoot></table>

Tropomyosins belonging to the Group 10 allergens of HDM and Group 7 allergens from cockroach cross-react with shrimp tropomyosins and have also been described as clinically relevant allergens. For example, cross-reactivity between purified tropomyosins from mosquito and HDM has been reported recently.¹⁴⁹⁴ In addition, tropomyosins from edible insects, whose consume is increasing in Western countries, can be an important source of cross-reactivity with other tropomyosins, including those from HDM, cockroach and crustaceans.¹⁴⁹⁵,¹⁴⁹⁶

The allergenic activity and cross-reactivity of *Ascaris* lumbricoides (an intestinal helminth) tropomyosin (Asc I 3) has been thoroughly analyzed<sup>773,1497,1498</sup>; further studies have shown an important clinical impact by sensitizing the asthmatic populations in underdeveloped tropical countries,<sup>1499,1500</sup> where helminthiases, together with perennial exposition to mite tropomyosins might increase asthma symptoms and predispose to allergic reactions to shrimp.<sup>775,1501,1502</sup>

Cross-reactivity between HDM allergens and some shellfish has been described and reports suggest that it is clinically significant.<sup>1503</sup> Subcutaneous HDM immunotherapy in patients sensitized to shrimp or snail could increase allergy symptoms after ingestion of these foods. Previous studies demonstrated IgE binding to shrimp tropomyosin in Orthodox Jews, which are prohibited to consume shellfish, most probably due to their house dust mite allergy.<sup>1504</sup> Although cross-reactive tropomyosins are good candidates for explaining these observations, other allergens may be involved. In addition, other authors have obtained opposite results after immunotherapy, suggesting that the adverse side effects are not general and could be influenced by the type of immunotherapy and genetic factors determining the susceptibility to get sensitized by other allergens. Therefore, more research is needed to define

this controversial effect of cross-reactivity among arthropods on immunotherapy.

### 3 – Clinical relevance

Tropomyosins from invertebrates are allergenic for genetically susceptible individuals and due to their extensive cross-reactivity, are considered pan-allergens. Recently the vertebrate tropomyosin, Ore m 4, was described as a major allergen of tilapia (*Oreochromis mosambicus*)<sup>1505</sup> and has subsequently been shown to be a major IgE-binding allergen in salmon and catfish in over 30% of fish allergic children.<sup>1010</sup> Sensitization can occur by ingestion (seafood), inhalation (mites, cockroaches) or parasite infection (ascariasis, anisakiasis) including parasite-infested food sources such as raw fish, e.g., in food sources such as sushi or ceviche (PMID: 26252073, PMID: 29588070). See also Chapter B12. It has been hypothesized that primary inhalant sensitization to HDM tropomyosins might be followed by sensitization to shellfish in regions where HDM exposure is predominant.<sup>1081</sup> However, when the primary sensitizer is a tropomyosin from an inhaled source (Der p 10, Blo t 10 or Bla g 7) the tolerance to crustaceans, mollusks and cephalopods is higher than when Pen a 1 is the primary sensitizer. It remains to be determined how the clinical manifestations can differ depending on the tropomyosin that acts as the primary sensitizer.

Most allergenic tropomyosins are major shellfish allergens. Symptoms may be induced by very low amounts of the offending food and sometimes by inhalation. They include immediate cutaneous reactions (urticaria, angioedema, rash) oral allergy syndrome (swelling in the lips and mouth), digestive symptoms (vomiting, abdominal cramping, and diarrhea), anaphylaxis and asthma.
<table><caption>TABLE 87 Amino acids identities between some tropomyosins</caption><thead><tr><th rowspan="2">Classification</th><th rowspan="2">Species</th><th rowspan="2">Accesion number</th><th rowspan="2">#</th><th colspan="11">Crustacean</th><th colspan="11">Insect</th><th colspan="11">Mollusc</th><th colspan="11">Fish</th><th colspan="11">Human</th></tr><tr><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th><th>7</th><th>8</th><th>9</th><th>10</th><th>11</th><th>12</th><th>13</th><th>14</th><th>15</th><th>16</th><th>17</th><th>18</th><th>19</th><th>20</th><th>21</th><th>22</th><th>23</th><th>24</th><th>25</th><th>26</th><th>27</th></tr></thead><tbody><tr><td rowspan="12">Crustacean</td><td>Giant tiger prawn</td><td>A1KY22</td><td>1</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Brown shrimp</td><td>Q3Y8M6</td><td>2</td><td>100</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Whiteleg shrimp</td><td>B4YAH6</td><td>3</td><td>100</td><td>100</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Giant fresh water prawn</td><td>D3XNR9</td><td>4</td><td>98</td><td>98</td><td>98</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Blue swimmer crab</td><td>M1H607</td><td>5</td><td>98</td><td>98</td><td>98</td><td>97</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Mud crab</td><td>A7LSV2</td><td>6</td><td>93</td><td>93</td><td>93</td><td>92</td><td>92</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Snow crab</td><td>A2V735</td><td>7</td><td>89</td><td>89</td><td>89</td><td>89</td><td>88</td><td>95</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Common yabby crayfish</td><td>WSRFX0</td><td>8</td><td>98</td><td>98</td><td>98</td><td>98</td><td>99</td><td>93</td><td>89</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>South African spiny lobster</td><td>K9UTQ8</td><td>9</td><td>94</td><td>94</td><td>94</td><td>94</td><td>94</td><td>96</td><td>92</td><td>94</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Red rock lobster</td><td>WSRFX7</td><td>10</td><td>95</td><td>95</td><td>95</td><td>95</td><td>95</td><td>92</td><td>88</td><td>95</td><td>93</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Flathead lobster</td><td>WSRFZ5</td><td>11</td><td>98</td><td>98</td><td>98</td><td>97</td><td>100</td><td>92</td><td>88</td><td>99</td><td>94</td><td>95</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="2">Insect</td><td>American cockroach</td><td>Q9UB83</td><td>12</td><td>82</td><td>82</td><td>82</td><td>82</td><td>81</td><td>83</td><td>80</td><td>82</td><td>83</td><td>81</td><td>82</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>German cockroach</td><td>Q9NG56</td><td>13</td><td>82</td><td>82</td><td>82</td><td>82</td><td>82</td><td>83</td><td>80</td><td>83</td><td>84</td><td>82</td><td>82</td><td>97</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="2">Mite</td><td>European house dust mite</td><td>O18416</td><td>14</td><td>80</td><td>80</td><td>80</td><td>80</td><td>80</td><td>82</td><td>78</td><td>80</td><td>82</td><td>79</td><td>80</td><td>80</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>American house dust mite</td><td>Q23939</td><td>15</td><td>82</td><td>82</td><td>82</td><td>82</td><td>81</td><td>83</td><td>80</td><td>82</td><td>84</td><td>81</td><td>81</td><td>81</td><td>98</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="12">Mollusc</td><td>Bay scallop</td><td>A1KY24</td><td>16</td><td>56</td><td>56</td><td>56</td><td>57</td><td>56</td><td>57</td><td>56</td><td>57</td><td>58</td><td>58</td><td>56</td><td>56</td><td>55</td><td>58</td><td>60</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Japanese scallop</td><td>Q9GUW9</td><td>17</td><td>60</td><td>60</td><td>60</td><td>61</td><td>60</td><td>61</td><td>60</td><td>60</td><td>62</td><td>61</td><td>60</td><td>59</td><td>58</td><td>60</td><td>62</td><td>88</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Blue mussel</td><td>Q25457</td><td>18</td><td>57</td><td>57</td><td>57</td><td>57</td><td>56</td><td>57</td><td>56</td><td>57</td><td>58</td><td>58</td><td>57</td><td>57</td><td>55</td><td>57</td><td>71</td><td>71</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Pacific oyster</td><td>B7XC66</td><td>19</td><td>62</td><td>62</td><td>62</td><td>62</td><td>61</td><td>62</td><td>60</td><td>62</td><td>63</td><td>62</td><td>62</td><td>61</td><td>60</td><td>60</td><td>62</td><td>74</td><td>75</td><td>79</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Common octopus</td><td>Q2VOV0</td><td>20</td><td>64</td><td>64</td><td>64</td><td>64</td><td>63</td><td>64</td><td>63</td><td>64</td><td>65</td><td>64</td><td>64</td><td>62</td><td>62</td><td>63</td><td>65</td><td>71</td><td>74</td><td>71</td><td>76</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Japanese flying squid</td><td>Q2V0V2</td><td>21</td><td>63</td><td>63</td><td>63</td><td>63</td><td>63</td><td>63</td><td>61</td><td>63</td><td>63</td><td>64</td><td>63</td><td>61</td><td>60</td><td>63</td><td>64</td><td>70</td><td>71</td><td>70</td><td>75</td><td>91</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Golden cuttlefish</td><td>Q2V0V4</td><td>22</td><td>63</td><td>63</td><td>63</td><td>64</td><td>63</td><td>64</td><td>63</td><td>64</td><td>64</td><td>64</td><td>62</td><td>61</td><td>64</td><td>65</td><td>71</td><td>73</td><td>71</td><td>76</td><td>92</td><td>98</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Brown garden snail</td><td>O97192</td><td>23</td><td>64</td><td>64</td><td>64</td><td>64</td><td>64</td><td>63</td><td>62</td><td>64</td><td>64</td><td>64</td><td>64</td><td>63</td><td>62</td><td>64</td><td>65</td><td>73</td><td>76</td><td>70</td><td>76</td><td>83</td><td>82</td><td>84</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Horned turban</td><td>B7XC63</td><td>24</td><td>62</td><td>62</td><td>62</td><td>63</td><td>62</td><td>62</td><td>60</td><td>62</td><td>63</td><td>63</td><td>62</td><td>61</td><td>60</td><td>62</td><td>64</td><td>71</td><td>76</td><td>74</td><td>78</td><td>81</td><td>80</td><td>81</td><td>87</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Disc abalone</td><td>B7XC62</td><td>25</td><td>63</td><td>63</td><td>63</td><td>64</td><td>63</td><td>63</td><td>61</td><td>63</td><td>64</td><td>64</td><td>63</td><td>62</td><td>62</td><td>63</td><td>64</td><td>71</td><td>77</td><td>75</td><td>79</td><td>83</td><td>81</td><td>81</td><td>87</td><td>97</td><td>100</td><td></td><td></td><td></td><td></td></tr><tr><td>Fish</td><td>Mozambique tilapia</td><td>K4PEK4</td><td>26</td><td>57</td><td>57</td><td>57</td><td>58</td><td>57</td><td>58</td><td>58</td><td>57</td><td>60</td><td>58</td><td>57</td><td>54</td><td>54</td><td>57</td><td>58</td><td>52</td><td>54</td><td>52</td><td>55</td><td>56</td><td>55</td><td>55</td><td>55</td><td>55</td><td>55</td><td>55</td><td>100</td><td></td><td></td><td></td></tr><tr><td>Human</td><td>Mozambique tilapia</td><td>P06753</td><td>27</td><td>57</td><td>57</td><td>57</td><td>58</td><td>57</td><td>58</td><td>58</td><td>57</td><td>59</td><td>57</td><td>57</td><td>54</td><td>55</td><td>58</td><td>58</td><td>51</td><td>53</td><td>52</td><td>55</td><td>55</td><td>54</td><td>54</td><td>56</td><td>55</td><td>55</td><td>55</td><td>55</td><td>94</td><td>100</td><td></td></tr></tbody><tfoot><tr><td colspan="2">% identity</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="2">100</td><td>95</td><td>90</td><td>85</td><td>80</td><td>75</td><td>70</td><td>65</td><td>60</td><td>55</td><td>50</td><td>50</td><td>50</td><td>50</td><td>50</td><td>50</td><td>50</td><td>50</td><td>50</td><td>50</td><td>50</td><td>50</td><td>50</td><td>50</td><td>50</td><td>50</td><td>50</td><td>50</td><td>50</td><td>50</td><td>50</td><td>50</td><td>50</td><td>50</td><td>50</td><td>50</td><td>50</td></tr></tfoot></table>

Tropomyosins belonging to the Group 10 allergens of HDM and Group 7 allergens from cockroach cross-react with shrimp tropomyosins and have also been described as clinically relevant allergens. For example, cross-reactivity between purified tropomyosins from mosquito and HDM has been reported recently.¹⁴⁹⁴ In addition, tropomyosins from edible insects, whose consume is increasing in Western countries, can be an important source of cross-reactivity with other tropomyosins, including those from HDM, cockroach and crustaceans.¹⁴⁹⁵,¹⁴⁹⁶

The allergenic activity and cross-reactivity of *Ascaris* lumbricoides (an intestinal helminth) tropomyosin (Asc I 3) has been thoroughly analyzed<sup>773,1497,1498</sup>; further studies have shown an important clinical impact by sensitizing the asthmatic populations in underdeveloped tropical countries,<sup>1499,1500</sup> where helminthiases, together with perennial exposition to mite tropomyosins might increase asthma symptoms and predispose to allergic reactions to shrimp.<sup>775,1501,1502</sup>

Cross-reactivity between HDM allergens and some shellfish has been described and reports suggest that it is clinically significant.<sup>1503</sup> Subcutaneous HDM immunotherapy in patients sensitized to shrimp or snail could increase allergy symptoms after ingestion of these foods. Previous studies demonstrated IgE binding to shrimp tropomyosin in Orthodox Jews, which are prohibited to consume shellfish, most probably due to their house dust mite allergy.<sup>1504</sup> Although cross-reactive tropomyosins are good candidates for explaining these observations, other allergens may be involved. In addition, other authors have obtained opposite results after immunotherapy, suggesting that the adverse side effects are not general and could be influenced by the type of immunotherapy and genetic factors determining the susceptibility to get sensitized by other allergens. Therefore, more research is needed to define

this controversial effect of cross-reactivity among arthropods on immunotherapy.

### 3 - Clinical relevance

Tropomyosins from invertebrates are allergenic for genetically susceptible individuals and due to their extensive cross-reactivity, are considered pan-allergens. Recently the vertebrate tropomyosin, Ore m 4, was described as a major allergen of tilapia (*Oreochromis mosambicus*)<sup>1505</sup> and has subsequently been shown to be a major IgE-binding allergen in salmon and catfish in over 30% of fish allergic children.<sup>1010</sup> Sensitization can occur by ingestion (seafood), inhalation (mites, cockroaches) or parasite infection (ascariasis, anisakiasis) including parasite-infested food sources such as raw fish, e.g., in food sources such as sushi or ceviche (PMID: 26252073, PMID: 29588070). See also Chapter B12. It has been hypothesized that primary inhalant sensitization to HDM tropomyosins might be followed by sensitization to shellfish in regions where HDM exposure is predominant.<sup>1081</sup> However, when the primary sensitizer is a tropomyosin from an inhaled source (Der p 10, Blo t 10 or Bla g 7) the tolerance to crustaceans, mollusks and cephalopods is higher than when Pen a 1 is the primary sensitizer. It remains to be determined how the clinical manifestations can differ depending on the tropomyosin that acts as the primary sensitizer.

Most allergenic tropomyosins are major shellfish allergens. Symptoms may be induced by very low amounts of the offending food and sometimes by inhalation. They include immediate cutaneous reactions (urticaria, angioedema, rash) oral allergy syndrome (swelling in the lips and mouth), digestive symptoms (vomiting, abdominal cramping, and diarrhea), anaphylaxis and asthma.
<figure><img src="image_285.png" /><figcaption>FIGURE 161 Cross-reactivity among allergenic tropomyosins from several sources. Lines represent documented IgE-cross-reactivity, dotted lines represent potential IgE cross-reactivity based on high sequence identities.</figcaption></figure>

<figure><img src="image_286.png" /><figcaption>FIGURE 162 Phylogenetic tree of allergenic tropomyosins</figcaption></figure>
<figure><img src="image_291.png" /><figcaption>FIGURE 161 Cross-reactivity among allergenic tropomyosins from several sources. Lines represent documented IgE-cross-reactivity, dotted lines represent potential IgE cross-reactivity based on high sequence identities.</figcaption></figure>

<figure><img src="image_292.png" /><figcaption>FIGURE 162 Phylogenetic tree of allergenic tropomyosins</figcaption></figure>
In Europe, sensitization to mite tropomyosin Der p 10 is low (8 -18%¹⁵⁰⁶) and has been considered an effect of cross-reactivity but also a marker for broad sensitization among HDM allergy patients. However, the prevalence of sensitization to Der f 10 was found around 80% in Japan. In addition, sensitization to Der p 10 was found 55% in Zimbabwe and 34% in Colombia,⁷⁷⁵ probably because of perennial exposure to shellfish and helminth infections. Therefore, the clinical impact of nonfood allergenic tropomyosins may be greater than previously thought. It has been suggested that sensitization to tropomyosin from mite,⁷⁷⁵ cockroach, *Ascaris*¹⁷⁷,¹⁵⁰¹ and mosquito¹⁴⁹⁴,¹⁵⁰⁷ could influence the prevalence and severity of asthma in places where co-exposure to several sources of tropomyosin occurs. Also, sensitization to HDM tropomyosin seems to be a risk factor for shrimp allergy in Italian patients.¹⁵⁰⁸ The influence of *Ascaris* tropomyosin sensitization on the outcome of immunotherapy for mite allergy has not been evaluated.

The frequency of IgE sensitization to tropomyosins in shellfish allergic patients ranges from 50 to 100%. In addition, Pen a 1 binds up to 75% of all shrimp-specific IgE antibodies,¹⁴⁸⁴ which is supported by histamine release experiments.¹⁴⁸³ Seafood allergy is common and includes reaction to crustaceans, mollusks and fish.¹⁰¹⁵ In some regions of high consumption such as Singapore and Vietnam, the prevalence of seafood allergy in school children is about 5%. In the USA, shellfish allergy is the most common food allergy among adults with 4% and the third most common food allergy in children.¹⁰⁷⁷ It remains to be evaluated how 62% of nonasthmatic controls are sensitized to *Ascaris* tropomyosin (Asc I 3) without allergic symptoms neither to HDM or shrimp.⁷⁷³

## Family characteristics

* Secondary structure formed by two parallel alpha-helices

* High amino acid identity between sequences of different species

* High degree of immunological and clinical cross-reactivity between different species

* Thermostable proteins, high allergenicity

* Considered invertebrate pan-allergen

## 4 - Clinical management

Clinical history of adverse reaction suggesting allergy after intake of shellfish is crucial for starting diagnosis procedures.¹⁰¹⁸ Whole extracts are beneficial for diagnosing shellfish allergy by ST, although the Prick-by-prick procedure is also useful. Tropomyosin sensitization is very important when evaluating shellfish allergy, but other allergens also play a role.¹⁰¹⁵,¹⁴⁹¹ It has been suggested that *in vitro* determination of IgE antibodies to tropomyosin is more specific and has a higher positive predictive value than the whole

extract in cases of shrimp allergy. In addition, Thalayasingam et al. found that the presence of specific IgE to shrimp has diagnostic test sensitivity of 82% and specificity of 22.2%.¹⁰⁸⁶ This low specificity, mainly due to the high rate of false positives that in turn are a consequence of the high cross-reactivity between shrimp and other arthropods allergens, explains why an allergy to shellfish should often be diagnosed by an oral food challenge. Two shellfish tropomyosins (Pen a 1 and Pen m 1, both from shrimp), Der p 10 (D. pteronyssinus tropomyosin), Ani s 3 from *Anisakis* and Per a 10 from cokroach are commercially available for *in vitro* testing. Diagnostic steps (Figure 163) could be starting with ST with the whole extract, and detecting IgE antibodies to the extract, tropomyosin and other shellfish allergens, such as Pen m 2 (Arginine kinase), Pen m 4 (Sarcoplasmic calcium-binding protein), Pen m 3 (Myosin light chain) and Pen m 6 (Troponin C).

Since most shellfish tropomyosins share sequences and epitopes (cross-sensitization) and there is a great diversity of seafood (cosensitization), it is currently difficult to define the primary allergenic source using component-resolved diagnosis. Then the added value of using single allergens for distinguishing the sensitizing source is limited because there are no species-specific markers of sensitization. However, a panel of tropomyosins from different species (e.g., shrimp, *Anisakis*, house dust mite, could be useful for comparing sensitization patterns from patients with different symptoms or severity of symptoms and identifying clinically useful biomarkers. Pascal M et al. evaluated, in patients from the USA, Brazil and Spain, the efficiency of several allergens to predict shrimp allergy. They found that tropomyosin and sarcoplasmic-calcium-binding protein sensitization is associated with clinical reactivity; in addition, the tropomyosin epitope p51-55 seems to be of good value as a diagnostic test to confirm allergy. The authors present a very interesting flow diagram for shrimp allergy diagnosis.¹⁵

Still, the gold standard for food allergy diagnosis is the double-blind placebo-controlled food challenge. An essential aspect of management is the detection of tropomyosin in food samples to prevent accidental ingestion and anaphylactic reactions. Several approaches and techniques have been proposed.

Management of shellfish allergy is based on strict avoidance based on clinical reactions. In general, if a patient is allergic to shrimp, avoiding of all crustaceans is recommended, although allergy may be limited to a particular crustacean member. Avoidance of mollusks is advised if allergy is demonstrated. However, patients with high IgE reactivity to tropomyosin might be advised to avoid all shellfish. No immunotherapy is currently available for seafood allergy, but experimental approaches to obtain appropriate compounds for specific immunotherapy have been developed. Hypoallergenic Pen a 1, hypoallergenic peptides from Met e 1, pernodate treatment of crab tropomyosin and simulated gastric digestion of the whole shrimp extract are analysed. Animal models for sensitization will help to obtain better reagents for diagnosis and treatment.
In Europe, sensitization to mite tropomyosin Der p 10 is low (8 -18%¹⁵⁰⁶) and has been considered an effect of cross-reactivity but also a marker for broad sensitization among HDM allergy patients. However, the prevalence of sensitization to Der f 10 was found around 80% in Japan. In addition, sensitization to Der p 10 was found 55% in Zimbabwe and 34% in Colombia,⁷⁷⁵ probably because of perennial exposure to shellfish and helminth infections. Therefore, the clinical impact of nonfood allergenic tropomyosins may be greater than previously thought. It has been suggested that sensitization to tropomyosin from mite,⁷⁷⁵ cockroach, *Ascaris*¹⁷⁷,¹⁵⁰¹ and mosquito¹⁴⁹⁴,¹⁵⁰⁷ could influence the prevalence and severity of asthma in places where co-exposure to several sources of tropomyosin occurs. Also, sensitization to HDM tropomyosin seems to be a risk factor for shrimp allergy in Italian patients.¹⁵⁰⁸ The influence of *Ascaris* tropomyosin sensitization on the outcome of immunotherapy for mite allergy has not been evaluated.

The frequency of IgE sensitization to tropomyosins in shellfish allergic patients ranges from 50 to 100%. In addition, Pen a 1 binds up to 75% of all shrimp-specific IgE antibodies,¹⁴⁸⁴ which is supported by histamine release experiments.¹⁴⁸³ Seafood allergy is common and includes reaction to crustaceans, mollusks and fish.¹⁰¹⁵ In some regions of high consumption such as Singapore and Vietnam, the prevalence of seafood allergy in school children is about 5%. In the USA, shellfish allergy is the most common food allergy among adults with 4% and the third most common food allergy in children.¹⁰⁷⁷ It remains to be evaluated how 62% of nonasthmatic controls are sensitized to *Ascaris* tropomyosin (Asc I 3) without allergic symptoms neither to HDM or shrimp.⁷⁷³

## Family characteristics

* Secondary structure formed by two parallel alpha-helices

* High amino acid identity between sequences of different species

* High degree of immunological and clinical cross-reactivity between different species

* Thermostable proteins, high allergenicity

* Considered invertebrate pan-allergen

## 4 - Clinical management

Clinical history of adverse reaction suggesting allergy after intake of shellfish is crucial for starting diagnosis procedures.¹⁰¹⁸ Whole extracts are beneficial for diagnosing shellfish allergy by ST, although the Prick-by-prick procedure is also useful. Tropomyosin sensitization is very important when evaluating shellfish allergy, but other allergens also play a role.¹⁰¹⁵,¹⁴⁹¹ It has been suggested that *in vitro* determination of IgE antibodies to tropomyosin is more specific and has a higher positive predictive value than the whole

extract in cases of shrimp allergy. In addition, Thalayasingam et al. found that the presence of specific IgE to shrimp has diagnostic test sensitivity of 82% and specificity of 22.2%.¹⁰⁸⁶ This low specificity, mainly due to the high rate of false positives that in turn are a consequence of the high cross-reactivity between shrimp and other arthropods allergens, explains why an allergy to shellfish should often be diagnosed by an oral food challenge. Two shellfish tropomyosins (Pen a 1 and Pen m 1, both from shrimp), Der p 10 (D. pteronyssinus tropomyosin), Ani s 3 from *Anisakis* and Per a 10 from cokroach are commercially available for *in vitro* testing. Diagnostic steps (Figure 163) could be starting with ST with the whole extract, and detecting IgE antibodies to the extract, tropomyosin and other shellfish allergens, such as Pen m 2 (Arginine kinase), Pen m 4 (Sarcoplasmic calcium-binding protein), Pen m 3 (Myosin light chain) and Pen m 6 (Troponin C).

Since most shellfish tropomyosins share sequences and epitopes (cross-sensitization) and there is a great diversity of seafood (cosensitization), it is currently difficult to define the primary allergenic source using component-resolved diagnosis. Then the added value of using single allergens for distinguishing the sensitizing source is limited because there are no species-specific markers of sensitization. However, a panel of tropomyosins from different species (e.g., shrimp, *Anisakis*, house dust mite, could be useful for comparing sensitization patterns from patients with different symptoms or severity of symptoms and identifying clinically useful biomarkers. Pascal M et al. evaluated, in patients from the USA, Brazil and Spain, the efficiency of several allergens to predict shrimp allergy. They found that tropomyosin and sarcoplasmic-calcium-binding protein sensitization is associated with clinical reactivity; in addition, the tropomyosin epitope p51-55 seems to be of good value as a diagnostic test to confirm allergy. The authors present a very interesting flow diagram for shrimp allergy diagnosis.¹⁵

Still, the gold standard for food allergy diagnosis is the double-blind placebo-controlled food challenge. An essential aspect of management is the detection of tropomyosin in food samples to prevent accidental ingestion and anaphylactic reactions. Several approaches and techniques have been proposed.

Management of shellfish allergy is based on strict avoidance based on clinical reactions. In general, if a patient is allergic to shrimp, avoiding of all crustaceans is recommended, although allergy may be limited to a particular crustacean member. Avoidance of mollusks is advised if allergy is demonstrated. However, patients with high IgE reactivity to tropomyosin might be advised to avoid all shellfish. No immunotherapy is currently available for seafood allergy, but experimental approaches to obtain appropriate compounds for specific immunotherapy have been developed. Hypoallergenic Pen a 1, hypoallergenic peptides from Met e 1, periodate treatment of crab tropomyosin and simulated gastric digestion of the whole shrimp extract are analysed. Animal models for sensitization will help to obtain better reagents for diagnosis and treatment.
<figure><img src="image_287.png" /><figcaption>**FIGURE 163** Diagnostic algorithm for shellfish allergy. *In vitro* tests for IgE to molecular allergens (CRD) include Pen a 1, Pen m 2, and Pen m 4.

CO6</figcaption></figure>

## 5 - Clinical Cases

### Case 1 (published)¹⁵⁰⁹

**Clinical History:** A 30-year-old man with a 10-year history of mild persistent asthma and allergy to house dust mites and pollen had generalized urticaria, facial erythema, and pharyngeal pruritus after eating shellfish on three separate occasions during two years. He associated the most recent episode with lobster. Since then, he has tolerated some crustaceans, mollusks, and fish, although he has avoided eating shrimp and lobster. No other food or drug allergies were reported, and he has not received immunotherapy for house dust mites.

**Test with extracts:** The patient had positive results using *in vitro* commercial diagnostics for *D. pteronyssinus* (21.4 kU/L) and *D. farinae* extracts (12.6 kU/L). Weak positive SPT to shrimp and IgE to lobster extracts (2.9 kU/L) and PPT to lobster were positive (6 mm).

**Food challenge:** The patient tolerated up to 8 g of cooked shrimp during the challenge (regular servings have been tolerated several times since). The study performed with lobster gave positive results by ImmunoCAP Specific IgE test (2.9 kU/L) and PPT (6 mm). However, the patient refused the oral food challenge with lobster.

**Test with molecules:** The patient had positive results to Der p 1 (4.7 kU/L), Der p 2 (60.9 kU/L), Der f 1 (0.4 kU/L) and Der f 2 (47.2 kU/L). Purified tropomyosins from shrimp (Pen a 1) and *D. pteronyssinus* (Der p 10) were negative.

**Conclusion:** Selective allergy to lobster in a patient with primary sensitization to house dust mite.

## 6 - Research and future perspectives

There are several aspects of tropomyosin allergy that deserve further investigation. Since allergen specific immunotherapy for food allergy
<figure><img src="image_293.png" /><figcaption>**FIGURE 163** Diagnostic algorithm for shellfish allergy. *In vitro* tests for IgE to molecular allergens (CRD) include Pen a 1, Pen m 2, and Pen m 4.

CO6</figcaption></figure>

## 5 - Clinical Cases

**Case 1 (published)¹⁵⁰⁹**

**Clinical History:** A 30-year-old man with a 10-year history of mild persistent asthma and allergy to house dust mites and pollen had generalized urticaria, facial erythema, and pharyngeal pruritus after eating shellfish on three separate occasions during two years. He associated the most recent episode with lobster. Since then, he has tolerated some crustaceans, mollusks, and fish, although he has avoided eating shrimp and lobster. No other food or drug allergies were reported, and he has not received immunotherapy for house dust mites.

**Test with extracts:** The patient had positive results using *in vitro* commercial diagnostics for *D. pteronyssinus* (21.4 kU/L) and *D. farinae* extracts (12.6 kU/L). Weak positive SPT to shrimp and IgE to lobster extracts (2.9 kU/L) and PPT to lobster were positive (6 mm).

**Food challenge:** The patient tolerated up to 8 g of cooked shrimp during the challenge (regular servings have been tolerated several times since). The study performed with lobster gave positive results by ImmunoCAP Specific IgE test (2.9 kU/L) and PPT (6 mm). However, the patient refused the oral food challenge with lobster.

**Test with molecules:** The patient had positive results to Der p 1 (4.7 kU/L), Der p 2 (60.9 kU/L), Der f 1 (0.4 kU/L) and Der f 2 (47.2 kU/L). Purified tropomyosins from shrimp (Pen a 1) and *D. pteronyssinus* (Der p 10) were negative.

**Conclusion:** Selective allergy to lobster in a patient with primary sensitization to house dust mite.

## 6 - Research and future perspectives

There are several aspects of tropomyosin allergy that deserve further investigation. Since allergen specific immunotherapy for food allergy
is increasing, the search for better reagents, both from extracts or isolated tropomyosin molecules should be encouraged. This process involves the identification of specific T and B cell epitopes, specially those associated with clinical manifestations. The molecular evaluation of the role of tropomyosins on allergic reaction following ingestion of edible insects will be essential for managing these problems,

not only in Asia, but in Western countries where the epidemiological impact has to be also analysed. As with other allergens, the search for genetic variants associated with both tropomyosin sensitization and allergic reactions will improve the understanding of basic mechanisms underlying the IgE response and our capacity for managing patients under personalized medicine criteria.
is increasing, the search for better reagents, both from extracts or isolated tropomyosin molecules should be encouraged. This process involves the identification of specific T and B cell epitopes, specially those associated with clinical manifestations. The molecular evaluation of the role of tropomyosins on allergic reaction following ingestion of edible insects will be essential for managing these problems,

not only in Asia, but in Western countries where the epidemiological impact has to be also analysed. As with other allergens, the search for genetic variants associated with both tropomyosin sensitization and allergic reactions will improve the understanding of basic mechanisms underlying the IgE response and our capacity for managing patients under personalized medicine criteria.
# C06 - Polcalcins

## Joaquín Sastre, Marcela Valverde

### Highlights

* Polcalcins are EF-hand calcium-binding proteins.

* Extensive IgE cross-reactivity among pollen polcalcins.

* Specific IgE testing to pollen polcalcin can be performed with any member of the family.

* Can be considered as marker of polysensitization with unknown clinical relevance for respiratory symptoms.

## 1 - The protein

The most representative pollen polcalcin and the first cloned¹⁵¹⁰ is Phl p 7 from *Phleum pratense* (common timothy). Phl p 7 belongs to a subfamily of 2-EF-hand calcium-binding pollen allergens that are preferentially expressed in mature pollen of higher plants including monocotyledonous and dicotyledonous species (Figure 164)

Phl p 7 was detected only in pollen but not in root and leaf extracts. It is completely eluted out of the pollen grains after a few minutes of hydration. It contains 78 amino acids with a molecular weight of 8,677 Da. A summary of the biochemical characteristics of Phl p 7 is shown in Table 88.

The physiological role of Phl p 7 is likely related to regulate the calcium levels in pollen germination and pollen tube growth, as other calcium-binding proteins contained in pollen.

Recombinant Phl p 7 exhibits an allergenic activity and is able to induce basophil histamine release and immediate type skin reactions. Phl p 7 has high stability (thermal and proteolysis)¹⁵¹⁰,¹⁵¹¹ and refolding capacity, a characteristic related to relevant allergens. It contains calcium-modulated conformational IgE epitopes, which

<figure><img src="image_288.png" /><figcaption>FIGURE 164 Ribbon model of the three-dimensional structure of Phl p 7. pdb: 1K9U. February 2022.</figcaption></figure>

<table><caption>TABLE 88 Protein characteristics of Phl p 7</caption><thead><tr><th colspan="2">Protein characteristics of Phl p 7</th></tr></thead><tbody><tr><td>Allergen source</td><td><i>Phleum pratense</i> (common timothy)</td></tr><tr><td>Protein family</td><td>Polcalcin</td></tr><tr><td>UniProtKB accession</td><td>O82040</td></tr><tr><td>Crystal structure</td><td>https://www.rcsb.orgstructure/1K9U</td></tr><tr><td>Molecular structure</td><td>alpha-helical fold</td></tr><tr><td>Theoretical molecular weight</td><td>8,677 Da</td></tr><tr><td>Length</td><td>78 amino acids, mature protein</td></tr><tr><td>Ligand binding</td><td>EF-hand calcium binding allergen</td></tr><tr><td>Dimerization</td><td>Dimer or monomer</td></tr><tr><td>Glycosylation</td><td>no</td></tr><tr><td>Disulfide bonds</td><td>no</td></tr><tr><td>Isoelectric point</td><td>4.39</td></tr><tr><td>Synthesis</td><td>Pollen grains</td></tr><tr><td>Function</td><td>Regulation of pollen germination and pollen tube growth</td></tr></tbody></table>

become accessible only in the calcium-bound form (open conformation), suggesting that IgE recognition is only activated by the calcium-bound conformation (Figure 164)

### Family characteristics

* Common tertiary structure with alpha-helical fold

* Calcium-binding capacity

* High sequence identity (average 77%)

* Small molecules of around 8 kDa

* Conformational IgE epitopes (calcium-modulated)

* Extensive IgE cross-reactivity among pollen polcalcins

## 2 - The family

Polcalcins belong to the 2-EF-hand calcium-binding proteins. Some polcalcins are monomers while others form domain-swapped dimers.

To date, 40 members of this allergen family have been identified in grasses, trees, bushes, weeds, and other flowering plants (Table 89) (see chapters B01, B02, B03) (Figure 165).

The 2-EF-hand allergens share a high degree of sequence similarity (average sequence identity of 77%), which explains the extensive cross-reactivity of allergic patients' IgE with the various members of the family. Although they do not present a complete immunological equivalence, the diagnosis of patients sensitized to polcalcins can be performed with any member of the family.¹⁵¹⁰ The IgE-binding capacity of polcalcins depends on conformational epitopes.¹⁵¹⁰ It has been demonstrated that Phl p 7 and related two EF-hand allergens do not share epitopes with other 3-EF-hand calcium-binding proteins allergens (e.g., Bet v 3, parvalbumins Gad c 1 or carp Cyp c 1) or 4-EF hand calcium-binding proteins allergens (Ole e 8, Jun o 4, Amb a 10).
# C06 - Polcalcins

## Joaquín Sastre, Marcela Valverde

### Highlights

* Polcalcins are EF-hand calcium-binding proteins.

* Extensive IgE cross-reactivity among pollen polcalcins.

* Specific IgE testing to pollen polcalcin can be performed with any member of the family.

* Can be considered as marker of polysensitization with unknown clinical relevance for respiratory symptoms.

## 1 - The protein

The most representative pollen polcalcin and the first cloned¹⁵¹⁰ is Phl p 7 from *Phleum pratense* (common timothy). Phl p 7 belongs to a subfamily of 2-EF-hand calcium-binding pollen allergens that are preferentially expressed in mature pollen of higher plants including monocotyledonous and dicotyledonous species (Figure 164)

Phl p 7 was detected only in pollen but not in root and leaf extracts. It is completely eluted out of the pollen grains after a few minutes of hydration. It contains 78 amino acids with a molecular weight of 8,677 Da. A summary of the biochemical characteristics of Phl p 7 is shown in Table 88.

The physiological role of Phl p 7 is likely related to regulate the calcium levels in pollen germination and pollen tube growth, as other calcium-binding proteins contained in pollen.

Recombinant Phl p 7 exhibits an allergenic activity and is able to induce basophil histamine release and immediate type skin reactions. Phl p 7 has high stability (thermal and proteolysis)¹⁵¹⁰,¹⁵¹¹ and refolding capacity, a characteristic related to relevant allergens. It contains calcium-modulated conformational IgE epitopes, which

<figure><img src="image_294.png" /><figcaption>FIGURE 164 Ribbon model of the three-dimensional structure of Phl p 7. pdb: 1K9U. February 2022.</figcaption></figure>

<table><caption>TABLE 88 Protein characteristics of Phl p 7</caption><thead><tr><th colspan="2">Protein characteristics of Phl p 7</th></tr></thead><tbody><tr><td>Allergen source</td><td><i>Phleum pratense</i> (common timothy)</td></tr><tr><td>Protein family</td><td>Polcalcin</td></tr><tr><td>UniProtKB accession</td><td>O82040</td></tr><tr><td>Crystal structure</td><td>https://www.rcsb.orgstructure/1K9U</td></tr><tr><td>Molecular structure</td><td>alpha-helical fold</td></tr><tr><td>Theoretical molecular weight</td><td>8,677 Da</td></tr><tr><td>Length</td><td>78 amino acids, mature protein</td></tr><tr><td>Ligand binding</td><td>EF-hand calcium binding allergen</td></tr><tr><td>Dimerization</td><td>Dimer or monomer</td></tr><tr><td>Glycosylation</td><td>no</td></tr><tr><td>Disulfide bonds</td><td>no</td></tr><tr><td>Isoelectric point</td><td>4.39</td></tr><tr><td>Synthesis</td><td>Pollen grains</td></tr><tr><td>Function</td><td>Regulation of pollen germination and pollen tube growth</td></tr></tbody></table>

become accessible only in the calcium-bound form (open conformation), suggesting that IgE recognition is only activated by the calcium-bound conformation (Figure 164)

### Family characteristics

* Common tertiary structure with alpha-helical fold

* Calcium-binding capacity

* High sequence identity (average 77%)

* Small molecules of around 8 kDa

* Conformational IgE epitopes (calcium-modulated)

* Extensive IgE cross-reactivity among pollen polcalcins

## 2 - The family

Polcalcins belong to the 2-EF-hand calcium-binding proteins. Some polcalcins are monomers while others form domain-swapped dimers.

To date, 40 members of this allergen family have been identified in grasses, trees, bushes, weeds, and other flowering plants (Table 89) (see chapters B01, B02, B03) (Figure 165).

The 2-EF-hand allergens share a high degree of sequence similarity (average sequence identity of 77%), which explains the extensive cross-reactivity of allergic patients' IgE with the various members of the family. Although they do not present a complete immunological equivalence, the diagnosis of patients sensitized to polcalcins can be performed with any member of the family.¹⁵¹⁰ The IgE-binding capacity of polcalcins depends on conformational epitopes.¹⁵¹⁰ It has been demonstrated that Phl p 7 and related two EF-hand allergens do not share epitopes with other 3-EF-hand calcium-binding proteins allergens (e.g., Bet v 3, parvalbumins Gad c 1 or carp Cyp c 1) or 4-EF hand calcium-binding proteins allergens (Ole e 8, Jun o 4, Amb a 10).
<table><caption>TABLE 89 Allergenic polcalcins described</caption><thead><tr><th>Botanical family</th><th>Allergen source</th><th>Allergen</th></tr></thead><tbody><tr><td rowspan="3">Asteraceae</td><td rowspan="2">Short ragweed <i>Ambrosia artemisiifolia</i></td><td>Amb a 9</td></tr><tr><td>Amb a 10 *</td></tr><tr><td>English mugwort <i>Artemisia vulgaris</i></td><td>Art v 5</td></tr><tr><td>Sieversian wormwood <i>Artemisia sieversiana</i></td><td>Art si 5</td></tr><tr><td rowspan="2">Betulaceae</td><td rowspan="2">Birch <i>Betula verrucosa</i></td><td>Bet v 3 *</td></tr><tr><td>Bet v 4</td></tr><tr><td>Alder <i>Alnus glutinosa</i></td><td>Aln g 4</td></tr><tr><td rowspan="2">Brassicaceae</td><td>Rapeseed <i>Brassica napus</i></td><td>(Bra n 7)</td></tr><tr><td>Block choy Bird rape <i>Brassica rapa</i></td><td>Bra r 5</td></tr><tr><td rowspan="2">Chenopodiaceae</td><td>Goosefoot <i>Chenopodium album</i></td><td>Che a 3</td></tr><tr><td>Russian thistle <i>Salsola kali</i></td><td>Sal k 7</td></tr><tr><td rowspan="2">Cupressaceae</td><td>Arizona cypress <i>Cupressus arizonica</i></td><td>(Cup a 4) *</td></tr><tr><td>Prickly juniper <i>Juniperus oxycedrus</i></td><td>Jun o 4 *</td></tr><tr><td rowspan="3">Oleaceae</td><td>Ash <i>Fraxinus excelsior</i></td><td>(Fra e 3)</td></tr><tr><td rowspan="2">Olive <i>Olea europaea</i></td><td>Ole e 3</td></tr><tr><td>Ole e 8 *</td></tr><tr><td>Common lilac <i>Syringa vulgaris</i></td><td>Syr v 3</td></tr><tr><td rowspan="2">Poaceae</td><td>Bermuda grass <i>Cynodon dactylon</i></td><td>Cyn d 7</td></tr><tr><td>Timothy crass <i>Phleum pratense</i></td><td>Phl p 7</td></tr><tr><td>Urticaceae</td><td>Pellitory <i>Parietaria judaica</i></td><td>Par j 4</td></tr></tbody></table>

<figure><img src="image_289.png" /><figcaption>FIGURE 165 Cross-reactivity among polcalcins from different allergenic sources.</figcaption></figure>
<table><caption>TABLE 89 Allergenic polcalcins described</caption><thead><tr><th>Botanical family</th><th>Allergen source</th><th>Allergen</th></tr></thead><tbody><tr><td rowspan="3">Asteraceae</td><td rowspan="2">Short ragweed <i>Ambrosia artemisiifolia</i></td><td>Amb a 9</td></tr><tr><td>Amb a 10 *</td></tr><tr><td>English mugwort <i>Artemisia vulgaris</i></td><td>Art v 5</td></tr><tr><td>Sieversian wormwood <i>Artemisia sieversiana</i></td><td>Art si 5</td></tr><tr><td rowspan="2">Betulaceae</td><td rowspan="2">Birch <i>Betula verrucosa</i></td><td>Bet v 3 *</td></tr><tr><td>Bet v 4</td></tr><tr><td>Alder <i>Alnus glutinosa</i></td><td>Aln g 4</td></tr><tr><td rowspan="2">Brassicaceae</td><td>Rapeseed <i>Brassica napus</i></td><td>(Bra n 7)</td></tr><tr><td>Block choy Bird rape <i>Brassica rapa</i></td><td>Bra r 5</td></tr><tr><td rowspan="2">Chenopodiaceae</td><td>Goosefoot <i>Chenopodium album</i></td><td>Che a 3</td></tr><tr><td>Russian thistle <i>Salsola kali</i></td><td>Sal k 7</td></tr><tr><td rowspan="2">Cupressaceae</td><td>Arizona cypress <i>Cupressus arizonica</i></td><td>(Cup a 4) *</td></tr><tr><td>Prickly juniper <i>Juniperus oxycedrus</i></td><td>Jun o 4 *</td></tr><tr><td rowspan="3">Oleaceae</td><td>Ash <i>Fraxinus excelsior</i></td><td>(Fra e 3)</td></tr><tr><td rowspan="2">Olive <i>Olea europaea</i></td><td>Ole e 3</td></tr><tr><td>Ole e 8 *</td></tr><tr><td>Common lilac <i>Syringa vulgaris</i></td><td>Syr v 3</td></tr><tr><td rowspan="2">Poaceae</td><td>Bermuda grass <i>Cynodon dactylon</i></td><td>Cyn d 7</td></tr><tr><td>Timothy crass <i>Phleum pratense</i></td><td>Phl p 7</td></tr><tr><td>Urticaceae</td><td>Pellitory <i>Parietaria judaica</i></td><td>Par j 4</td></tr></tbody></table>

<figure><img src="image_295.png" /><figcaption>FIGURE 165 Cross-reactivity among polcalcins from different allergenic sources.</figcaption></figure>
## 3 - Clinical relevance

Members of this protein family have been identified as allergens. However, it is considered a minor allergen in all populations studied, since the reported frequencies of IgE binding to members of this family of proteins varies between 5% and 46%. Che a 3, a polcalcin from Chenopodium album, common in semi-desert areas, is an atypical polcalcin because it showed reactivity up to 46% of sera from individuals with chenopod allergy.¹⁵¹² The high prevalence of this pan-allergen in these patients might have a relationship with their characteristic polysensitization (Table 90). Moverare et al.¹⁵¹³ compared different European populations regarding the reactivity of Bet v 4 and found prevalence values between 5% and 11% for patients from North and Central Europe, and 27% for Italian patients. All these data point to the existence of a certain correlation between the polysensitization degree and geographical area and prevalence of sensitization to minor allergens. In Table 90 the percentage of sensitization to different polcalcins in patients with pollen allergy are shown. Barber et al.²⁸⁰ described that in patients who were simultaneously sensitized to

<table><caption>TABLE 90 Prevalence of polcalcin sensitization in patients with pollen allergy</caption><thead><tr><td>Main sensitisation of the population studied</td><td>Prevalence of sensitisation to Polcalcin</td><td>Reference</td></tr></thead><tbody><tr><td>Birch</td><td>Bet v 4: 5%</td><td>[1518]</td></tr><tr><td>Chenopodium/Salsola</td><td>Che a 3: 46%; Che a 3: 41%</td><td>[1512, 1709]</td></tr><tr><td>Olive</td><td>Ole e 3: 20-30%</td><td>[1514]</td></tr><tr><td>Grass</td><td>Phl p 7: 2-10%</td><td>[85, 86, 1514]</td></tr><tr><td>Alder</td><td>Aln g 4: 18%</td><td>[1710]</td></tr><tr><td>Robinia pseudoacacia</td><td>Che a 3: 33%</td><td>[1397]</td></tr><tr><td>Ash</td><td>Fra a 3: 16%</td><td>[1711]</td></tr><tr><td>Cypress</td><td>Cup a 4: 10%</td><td>[1712]</td></tr><tr><td>Polysensitized to pollen</td><td>Polcalcin: 31%</td><td>[1412]</td></tr><tr><td>Birch, ash, mugwort, grass</td><td>Polcalcin: 10%</td><td>[1515]</td></tr></tbody></table>

polcalcins and profilins, there was a duplication both in the number of sensitizations to major allergens and in the years of disease evolution. Similar findings were obtained by Orovitg et al.¹⁵¹⁴ Moreover, this specific sensitization profile is not linked to any particular pollen.²⁸⁰,¹⁵¹⁵,¹⁵¹⁶ Therefore, sensitization to polcalcin should be considered a marker of a longer duration of symptoms and a more severe respiratory disease. Nevertheless, contrary to profilin,¹³⁹⁷,¹⁴¹² the relevance of polcalcin to induce respiratory symptoms has not been demonstrated.

## 4 - Clinical management

Polcalcins are only expressed in pollen, thus are not related to food allergy, contrary to other pan-allergens such as profilin or nsLTPs. It is considered as a pan-allergen and therefore could be a confounding factor in the diagnosis of polysensitized pollen-allergic patients and may lead to a diagnosis of "allergy mirages"¹³⁹⁷,¹⁵¹⁷ (Figure 166). Through IgE inhibition assays, Asero et al. found important differences in IgE reactivity between rBet v 4 and rPhl p 7, since only grass pollen extract was able to markedly inhibit rPhl p 7. By contrast, other pollen extracts (birch, ragweed, olive, Parietaria) significantly inhibited IgE reactivity to rBet v 4.¹⁴¹²,¹⁴²¹ That suggests that in some areas the primary source of sensitization for polcalcin is grass pollen. Polcalcins are not commercially available for SPT. However, in some research articles, an extract derived from palm pollen has been used and prepared by ALK (Madrid, Spain).¹⁴¹²,¹⁴²¹ Recently a method to purify the olive polcalcin, Ole e 3, has been described.¹⁵¹⁷,¹⁵¹⁸ For specific IgE determinations, there are only three polcalcins available; Phl p 7, Bet v 4 and Aln g 4. The diagnosis of patients sensitized to polcalcins can be performed with specific IgE to Phl p 7 or Bet v 4. The presence of polcalcin sensitization in patients with pollen allergy does not require to change the clinical indications for immunotherapy and does not have to be considered a contraindication. Only grass pollen extracts used in AIT are rich in polcalcin.¹⁴²¹

<figure><img src="image_290.png" /><figcaption>FIGURE 166 Scheme to follow in case of polysensitization to pollen. Added value of the use of specific IgE to species-specific pollen allergens and to pan-allergens</figcaption></figure>
## 3 - Clinical relevance

Members of this protein family have been identified as allergens. However, it is considered a minor allergen in all populations studied, since the reported frequencies of IgE binding to members of this family of proteins varies between 5% and 46%. Che a 3, a polcalcin from Chenopodium album, common in semi-desert areas, is an atypical polcalcin because it showed reactivity up to 46% of sera from individuals with chenopod allergy.¹⁵¹² The high prevalence of this pan-allergen in these patients might have a relationship with their characteristic polysensitization (Table 90). Moverare et al.¹⁵¹³ compared different European populations regarding the reactivity of Bet v 4 and found prevalence values between 5% and 11% for patients from North and Central Europe, and 27% for Italian patients. All these data point to the existence of a certain correlation between the polysensitization degree and geographical area and prevalence of sensitization to minor allergens. In Table 90 the percentage of sensitization to different polcalcins in patients with pollen allergy are shown. Barber et al.²⁸⁰ described that in patients who were simultaneously sensitized to

<table><caption>TABLE 90 Prevalence of polcalcin sensitization in patients with pollen allergy</caption><thead><tr><td>Main sensitisation of the population studied</td><td>Prevalence of sensitisation to Polcalcin</td><td>Reference</td></tr></thead><tbody><tr><td>Birch</td><td>Bet v 4: 5%</td><td>[1518]</td></tr><tr><td>Chenopodium/Salsola</td><td>Che a 3: 46%; Che a 3: 41%</td><td>[1512, 1709]</td></tr><tr><td>Olive</td><td>Ole e 3: 20-30%</td><td>[1514]</td></tr><tr><td>Grass</td><td>Phl p 7: 2-10%</td><td>[85, 86, 1514]</td></tr><tr><td>Alder</td><td>Aln g 4: 18%</td><td>[1710]</td></tr><tr><td>Robinia pseudoacacia</td><td>Che a 3: 33%</td><td>[1397]</td></tr><tr><td>Ash</td><td>Fra a 3: 16%</td><td>[1711]</td></tr><tr><td>Cypress</td><td>Cup a 4: 10%</td><td>[1712]</td></tr><tr><td>Polysensitized to pollen</td><td>Polcalcin: 31%</td><td>[1412]</td></tr><tr><td>Birch, ash, mugwort, grass</td><td>Polcalcin: 10%</td><td>[1515]</td></tr></tbody></table>

polcalcins and profilins, there was a duplication both in the number of sensitizations to major allergens and in the years of disease evolution. Similar findings were obtained by Orovitg et al.¹⁵¹⁴ Moreover, this specific sensitization profile is not linked to any particular pollen.²⁸⁰,¹⁵¹⁵,¹⁵¹⁶ Therefore, sensitization to polcalcin should be considered a marker of a longer duration of symptoms and a more severe respiratory disease. Nevertheless, contrary to profilin,¹³⁹⁷,¹⁴¹² the relevance of polcalcin to induce respiratory symptoms has not been demonstrated.

## 4 - Clinical management

Polcalcins are only expressed in pollen, thus are not related to food allergy, contrary to other pan-allergens such as profilin or nsLTPs. It is considered as a pan-allergen and therefore could be a confounding factor in the diagnosis of polysensitized pollen-allergic patients and may lead to a diagnosis of "allergy mirages"¹³⁹⁷,¹⁵¹⁷ (Figure 166). Through IgE inhibition assays, Asero et al. found important differences in IgE reactivity between rBet v 4 and rPhl p 7, since only grass pollen extract was able to markedly inhibit rPhl p 7. By contrast, other pollen extracts (birch, ragweed, olive, Parietaria) significantly inhibited IgE reactivity to rBet v 4.¹⁴¹²,¹⁴²¹ That suggests that in some areas the primary source of sensitization for polcalcin is grass pollen. Polcalcins are not commercially available for SPT. However, in some research articles, an extract derived from palm pollen has been used and prepared by ALK (Madrid, Spain).¹⁴¹²,¹⁴²¹ Recently a method to purify the olive polcalcin, Ole e 3, has been described.¹⁵¹⁷,¹⁵¹⁸ For specific IgE determinations, there are only three polcalcins available; Phl p 7, Bet v 4 and Aln g 4. The diagnosis of patients sensitized to polcalcins can be performed with specific IgE to Phl p 7 or Bet v 4. The presence of polcalcin sensitization in patients with pollen allergy does not require to change the clinical indications for immunotherapy and does not have to be considered a contraindication. Only grass pollen extracts used in AIT are rich in polcalcin.¹⁴²¹

<figure><img src="image_296.png" /><figcaption>FIGURE 166 Scheme to follow in case of polysensitization to pollen. Added value of the use of specific IgE to species-specific pollen allergens and to pan-allergens</figcaption></figure>
## 5 - Clinical case

**Clinical History:** Male, 26 years, with a 10-year history of rhinitis and asthma during spring. No complaint of adverse reaction to food.

**Test with extracts:** Skin prick test showed positive reaction to grass, olive, cypress and plantain. Due to extensive polysensitization to pollen a molecular diagnosis was performed to give an indication for immunotherapy.

**Test with molecules:** Specific IgE was positive for Phl p 1, Phl p 5, Phl p 12, Phl p 7 and negative for Ole e 1, Cup a 1, Pla I 1.

**Conclusion:** Results indicate a primary sensitization only to grass pollen and pan-allergens (profilin and polcacin), which confirm a longer duration of the respiratory symptoms and the severity of the disease. Immunotherapy with a grass pollen extract was prescribed.
5 - Clinical case

**Clinical History:** Male, 26 years, with a 10-year history of rhinitis and asthma during spring. No complaint of adverse reaction to food.

**Test with extracts:** Skin prick test showed positive reaction to grass, olive, cypress and plantain. Due to extensive polysensitization to pollen a molecular diagnosis was performed to give an indication for immunotherapy.

**Test with molecules:** Specific IgE was positive for Phl p 1, Phl p 5, Phl p 12, Phl p 7 and negative for Ole e 1, Cup a 1, Pla I 1.

**Conclusion:** Results indicate a primary sensitization only to grass pollen and pan-allergens (profilin and polcacin), which confirm a longer duration of the respiratory symptoms and the severity of the disease. Immunotherapy with a grass pollen extract was prescribed.
# C07 - Lipocalins

## Bente Janssen-Weets, Christiane Hilger

### Highlights

* Common tertiary structure with low sequence identity among family members.

* Airborne, easily spreading into indoor environment.

* Sensitization to multiple components is associated with disease severity.

* Cross-reactive subgroup with high sequence identity.

### 1 - The protein

Equ c 1, the major allergen of horse, was one of the first lipocalins to be isolated, cloned and characterized (Table 91).¹⁵¹⁹ The determination of its three-dimensional structure classified it as lipocalin (Figure 167).¹⁵²⁰ The physiological role of Equ c 1 is still under investigation. Lipocalins have diverse functions that are often associated with their ability to transport ligands. Equ c 1 purified from horse sweat contains oleamide, an endogenous bioactive substance, and other small organic molecules. Equ c 1 was found to have surfactant properties; it lowers the surface tension of liquids and could play a role in sweat evaporation.¹⁵²¹ Horse allergens are shed into the air and are passively transported to homes and public places, most likely by sticking to clothes and hair.¹³⁷⁶,¹⁵²² They are detectable in classrooms when many children have regular contact with horses.¹⁵²¹

Equ c 1 has been detected in the majority of air-borne dust samples in small animal veterinary practices and their employees' homes,

<table><caption>TABLE 91 Protein characteristics of Equ c 1</caption><thead><tr><th colspan="2">Protein characteristics of Equ c 1</th></tr></thead><tbody><tr><td>Allergen source</td><td><i>Equus caballus</i>, horse</td></tr><tr><td>Protein family</td><td>lipocalin</td></tr><tr><td>UniProtKB accession</td><td>Q95182</td></tr><tr><td>Crystal structure</td><td>yes</td></tr><tr><td>Molecular structure</td><td>mainly beta-sheet</td></tr><tr><td>Theoretical molecular weight</td><td>20.097 kDa</td></tr><tr><td>Molecular weight measured by mass spectrometry</td><td>22.0 kDa</td></tr><tr><td>Length</td><td>187 amino acids, mature protein 172</td></tr><tr><td>Ligand binding</td><td>yes</td></tr><tr><td>Dimerization</td><td>homodimer</td></tr><tr><td>Glycosylation</td><td>yes</td></tr><tr><td>Disulfide bonds</td><td>1</td></tr><tr><td>Isoelectric point</td><td>4.51</td></tr><tr><td>Synthesis</td><td>sublingual gland, low levels in submaxillary gland and liver, secreted</td></tr><tr><td>Distribution</td><td>fur, saliva, urine</td></tr></tbody></table>

<figure><img src="image_291.png" /><figcaption>FIGURE 167 Ribbon model of the three-dimensional structure of Equ c 1 (PDB: 1EW3). N-terminal end in dark blue, C-terminal end in red.</figcaption></figure>

although horses were not treated there. The highest Equ c 1 concentrations were found in the practices changing rooms, suggesting an important spreading of allergens via passive transfer.⁸²⁰ Gender and castration status seem to influence allergen content in horse hair. Statistically, stallions have higher quantities of Equ c 1 than mares and geldings.⁷⁹⁴

The characterization of B and T cell epitopes is still under investigation. T-cell epitopes seem to cluster in an immunodominant region at the carboxy-terminal end of the molecule¹⁵²⁴. Attempts to develop hypoallergenic Equ c 1 variants for immunotherapy also target its dimerization interface.¹⁵²⁵ Data on horse immunotherapy with extracts are scarce and larger clinical trials are needed for assessing efficacy and safety of the treatment.

### 2 - The family

The majority of the mammalian allergens are lipocalins³⁴⁵ (Table 92). Lipocalins are proteins that are ubiquitous; they are present also in arthropods, plants and bacteria, and have very diverse functions. They are characterized by a common tertiary structure composed of a central β-barrel formed of eight anti-parallel β-strands. The internal binding pocket carries a broad range of small hydrophobic molecules such as retinol, steroids, lipids, pheromones and odorants (see chapter A11). Mammalian allergens isolated so far are mostly odorant and pheromone binding lipocalins. Only few natural ligands have been characterized.

Depending on the individual protein and its concentration, lipocalins exist as monomers or in an oligomeric state. Most of them are glycosylated. Lipocalins are characterized by a weak cellular immune
# C07 - Lipocalins

## Bente Janssen-Weets, Christiane Hilger

### Highlights

* Common tertiary structure with low sequence identity among family members.

* Airborne, easily spreading into indoor environment.

* Sensitization to multiple components is associated with disease severity.

* Cross-reactive subgroup with high sequence identity.

## 1 - The protein

Equ c 1, the major allergen of horse, was one of the first lipocalins to be isolated, cloned and characterized (Table 91).¹⁵¹⁹ The determination of its three-dimensional structure classified it as lipocalin (Figure 167).¹⁵²⁰ The physiological role of Equ c 1 is still under investigation. Lipocalins have diverse functions that are often associated with their ability to transport ligands. Equ c 1 purified from horse sweat contains oleamide, an endogenous bioactive substance, and other small organic molecules. Equ c 1 was found to have surfactant properties; it lowers the surface tension of liquids and could play a role in sweat evaporation.¹⁵²¹ Horse allergens are shed into the air and are passively transported to homes and public places, most likely by sticking to clothes and hair.¹³⁷⁶,¹⁵²² They are detectable in classrooms when many children have regular contact with horses.¹⁵²¹

Equ c 1 has been detected in the majority of air-borne dust samples in small animal veterinary practices and their employees' homes,

<table><caption>TABLE 91 Protein characteristics of Equ c 1</caption><thead><tr><th colspan="2">Protein characteristics of Equ c 1</th></tr></thead><tbody><tr><td>Allergen source</td><td><i>Equus caballus</i>, horse</td></tr><tr><td>Protein family</td><td>lipocalin</td></tr><tr><td>UniProtKB accession</td><td>Q95182</td></tr><tr><td>Crystal structure</td><td>yes</td></tr><tr><td>Molecular structure</td><td>mainly beta-sheet</td></tr><tr><td>Theoretical molecular weight</td><td>20.097 kDa</td></tr><tr><td>Molecular weight measured by mass spectrometry</td><td>22.0 kDa</td></tr><tr><td>Length</td><td>187 amino acids, mature protein 172</td></tr><tr><td>Ligand binding</td><td>yes</td></tr><tr><td>Dimerization</td><td>homodimer</td></tr><tr><td>Glycosylation</td><td>yes</td></tr><tr><td>Disulfide bonds</td><td>1</td></tr><tr><td>Isoelectric point</td><td>4.51</td></tr><tr><td>Synthesis</td><td>sublingual gland, low levels in submaxillary gland and liver, secreted</td></tr><tr><td>Distribution</td><td>fur, saliva, urine</td></tr></tbody></table>

<figure><img src="image_297.png" /><figcaption>FIGURE 167 Ribbon model of the three-dimensional structure of Equ c 1 (PDB: 1EW3). N-terminal end in dark blue, C-terminal end in red.</figcaption></figure>

although horses were not treated there. The highest Equ c 1 concentrations were found in the practices changing rooms, suggesting an important spreading of allergens via passive transfer.⁸²⁰ Gender and castration status seem to influence allergen content in horse hair. Statistically, stallions have higher quantities of Equ c 1 than mares and geldings.⁷⁹⁴

The characterization of B and T cell epitopes is still under investigation. T-cell epitopes seem to cluster in an immunodominant region at the carboxy-terminal end of the molecule¹⁵²⁴. Attempts to develop hypoallergenic Equ c 1 variants for immunotherapy also target its dimerization interface.¹⁵²⁵ Data on horse immunotherapy with extracts are scarce and larger clinical trials are needed for assessing efficacy and safety of the treatment.

## 2 - The family

The majority of the mammalian allergens are lipocalins³⁴⁵ (Table 92). Lipocalins are proteins that are ubiquitous; they are present also in arthropods, plants and bacteria, and have very diverse functions. They are characterized by a common tertiary structure composed of a central β-barrel formed of eight anti-parallel β-strands. The internal binding pocket carries a broad range of small hydrophobic molecules such as retinol, steroids, lipids, pheromones and odorants (see chapter A11). Mammalian allergens isolated so far are mostly odorant and pheromone binding lipocalins. Only few natural ligands have been characterized.

Depending on the individual protein and its concentration, lipocalins exist as monomers or in an oligomeric state. Most of them are glycosylated. Lipocalins are characterized by a weak cellular immune
<table><caption>TABLE 92 Respiratory and food lipocalin allergens from mammals</caption><thead><tr><th>Family</th><th>Allergen source</th><th>Allergen</th></tr></thead><tbody><tr><td rowspan="2">Bovidae</td><td rowspan="2">Domestic cattle (Bos domesticus)</td><td>Bos d 2</td></tr><tr><td>Bos d 5</td></tr><tr><td rowspan="4">Canidae</td><td rowspan="4">Dog (Canis familiaris)</td><td>Can f 1</td></tr><tr><td>Can f 2</td></tr><tr><td>Can f 4</td></tr><tr><td>Can f 6</td></tr><tr><td rowspan="4">Cavidae</td><td rowspan="4">Guinea-pig (Cavia porcellus)</td><td>Cav p 1</td></tr><tr><td>Cav p 2</td></tr><tr><td>Cav p 3</td></tr><tr><td>Cav p 6</td></tr><tr><td rowspan="2">Cricetidae</td><td>Golden hamster (Mesocricetus auratus)</td><td>Mes a 1</td></tr><tr><td>Siberian hamster (Phodopus sungorus)</td><td>Phod s 1</td></tr><tr><td rowspan="2">Equidae</td><td rowspan="2">Domestic horse (Equus caballus)</td><td>Equ c 1</td></tr><tr><td>Equ c 2</td></tr><tr><td rowspan="2">Felidae</td><td rowspan="2">Cat (Felis domesticus)</td><td>Fel d 4</td></tr><tr><td>Fel d 7</td></tr><tr><td rowspan="2">Leporidae</td><td rowspan="2">Rabbit (Oryctolagus cuniculus)</td><td>Ory c 1</td></tr><tr><td>Ory c 4</td></tr><tr><td rowspan="2">Muridae</td><td>Mouse (Mus musculus)</td><td>Mus m 1</td></tr><tr><td>Rat (Rattus norvegicus)</td><td>Rat n 1</td></tr></tbody></table>

response and their mechanism of sensitization remains largely unresolved.²⁶ It has been hypothesized that the binding of ligands might influence their allergenicity. For example, the milk allergen Bos d 5 is able to bind complexed iron. The state of ligand load and the transport of iron to sites of immune activation seem to have a tolerogenic effect.⁴⁶⁶ The dog lipocalin allergen Can f 6 may display immunomodulatory properties when combined with lipopolysaccharide ligands by enhancing Toll-like receptor 4 signaling of the innate immune system.³¹⁸

Lipocalin allergens are present in dander, saliva and urine. They stick to particles, become easily airborne and are transported to public places such as schools or day-care centres.⁸²⁰,¹³⁷⁶,¹⁶⁷² Dog lipocalin allergens Can f 6 and Can f 4 where found to distribute evenly in airborne fractions of different particle size ranging from 0.14 to >8.1 µm, whereas the majority of Can f 1 molecules seem to stick to particles above 8.1 µm.¹⁵²⁶,¹⁵²⁷

Lipocalins are small, secreted molecules of 150-250 amino acids. Among 10 allergen families, lipocalins were ranked lowest according to their propensity for cross-reactivity based on the average of the proteins' sequence similarity and identity.¹⁵²⁹ Despite their highly conserved structure, they display little sequence identity, usually between 20 and 30%. Therefore, lipocalins were considered as species-specific allergy markers. Residues and their positions that are of structural and functional importance are conserved through evolution and can be the potential target for IgE cross-reactivity between homologous allergens (Figure 168). The further isolation of new allergen molecules showed that some lipocalins have much higher sequence identities, which can be as

high as 67%. In inhibition studies, they were able to cross-react at low doses.⁸¹⁴,⁸²¹,¹⁵²⁹ Representatives of this cross-reactive group are Equ c 1, Fel d 4 and Can f 6.⁸²¹ Only lately, Cav p 6 was found to be cross-reactive with Fel d 4 and Can f 6 as well.⁸²² Can f 1 and Fel d 7 also share a high sequence identity (62%) and IgE cross-reactivity was recently confirmed in polysensitized patients.¹⁵³⁰,¹⁵³¹ However, even between molecules of low general sequence identity such as Fel d 4 and Can f 2 (25% identity), single epitopes may have short stretches of sequence identity and lead to patient-dependent IgE cross-reactivity.¹⁵³² Can f 4 has been reported to show some cross-reactivity with a putative bovine allergen sharing only 37% of sequence identity.¹⁵³³ Table 93 displays two-by-two comparisons of amino acid identities between a subgroup of lipocalins.

## Family characteristics

* Common tertiary structure with central barrel

* Divergent sequences with low identity

* Sub-group with high sequence identity

* Small secreted molecules of 16-25 kDa

* Airborne, easily spreading into indoor environment

All pairs with a high sequence identity are potentially cross-reactive. The challenge of further studies is to analyze which lipocalins are adequate species-specific markers and which are
<table><caption>TABLE 92 Respiratory and food lipocalin allergens from mammals</caption><thead><tr><th>Family</th><th>Allergen source</th><th>Allergen</th></tr></thead><tbody><tr><td rowspan="2">Bovidae</td><td rowspan="2">Domestic cattle (Bos domesticus)</td><td>Bos d 2</td></tr><tr><td>Bos d 5</td></tr><tr><td rowspan="4">Canidae</td><td rowspan="4">Dog (Canis familiaris)</td><td>Can f 1</td></tr><tr><td>Can f 2</td></tr><tr><td>Can f 4</td></tr><tr><td>Can f 6</td></tr><tr><td rowspan="4">Cavidae</td><td rowspan="4">Guinea-pig (Cavia porcellus)</td><td>Cav p 1</td></tr><tr><td>Cav p 2</td></tr><tr><td>Cav p 3</td></tr><tr><td>Cav p 6</td></tr><tr><td rowspan="2">Cricetidae</td><td>Golden hamster (Mesocricetus auratus)</td><td>Mes a 1</td></tr><tr><td>Siberian hamster (Phodopus sungorus)</td><td>Phod s 1</td></tr><tr><td rowspan="2">Equidae</td><td rowspan="2">Domestic horse (Equus caballus)</td><td>Equ c 1</td></tr><tr><td>Equ c 2</td></tr><tr><td rowspan="2">Felidae</td><td rowspan="2">Cat (Felis domesticus)</td><td>Fel d 4</td></tr><tr><td>Fel d 7</td></tr><tr><td rowspan="2">Leporidae</td><td rowspan="2">Rabbit (Oryctolagus cuniculus)</td><td>Ory c 1</td></tr><tr><td>Ory c 4</td></tr><tr><td rowspan="2">Muridae</td><td>Mouse (Mus musculus)</td><td>Mus m 1</td></tr><tr><td>Rat (Rattus norvegicus)</td><td>Rat n 1</td></tr></tbody></table>

response and their mechanism of sensitization remains largely unresolved.²⁶ It has been hypothesized that the binding of ligands might influence their allergenicity. For example, the milk allergen Bos d 5 is able to bind complexed iron. The state of ligand load and the transport of iron to sites of immune activation seem to have a tolerogenic effect.⁴⁶⁶ The dog lipocalin allergen Can f 6 may display immunomodulatory properties when combined with lipopolysaccharide ligands by enhancing Toll-like receptor 4 signaling of the innate immune system.³¹⁸

Lipocalin allergens are present in dander, saliva and urine. They stick to particles, become easily airborne and are transported to public places such as schools or day-care centres.⁸²⁰,¹³⁷⁶,¹⁶⁷² Dog lipocalin allergens Can f 6 and Can f 4 where found to distribute evenly in airborne fractions of different particle size ranging from 0.14 to >8.1 µm, whereas the majority of Can f 1 molecules seem to stick to particles above 8.1 µm.¹⁵²⁶,¹⁵²⁷

Lipocalins are small, secreted molecules of 150-250 amino acids. Among 10 allergen families, lipocalins were ranked lowest according to their propensity for cross-reactivity based on the average of the proteins' sequence similarity and identity.¹⁵²⁹ Despite their highly conserved structure, they display little sequence identity, usually between 20 and 30%. Therefore, lipocalins were considered as species-specific allergy markers. Residues and their positions that are of structural and functional importance are conserved through evolution and can be the potential target for IgE cross-reactivity between homologous allergens (Figure 168). The further isolation of new allergen molecules showed that some lipocalins have much higher sequence identities, which can be as

high as 67%. In inhibition studies, they were able to cross-react at low doses.⁸¹⁴,⁸²¹,¹⁵²⁹ Representatives of this cross-reactive group are Equ c 1, Fel d 4 and Can f 6.⁸²¹ Only lately, Cav p 6 was found to be cross-reactive with Fel d 4 and Can f 6 as well.⁸²² Can f 1 and Fel d 7 also share a high sequence identity (62%) and IgE cross-reactivity was recently confirmed in polysensitized patients.¹⁵³⁰,¹⁵³¹ However, even between molecules of low general sequence identity such as Fel d 4 and Can f 2 (25% identity), single epitopes may have short stretches of sequence identity and lead to patient-dependent IgE cross-reactivity.¹⁵³² Can f 4 has been reported to show some cross-reactivity with a putative bovine allergen sharing only 37% of sequence identity.¹⁵³³ Table 93 displays two-by-two comparisons of amino acid identities between a subgroup of lipocalins.

## Family characteristics

* Common tertiary structure with central barrel

* Divergent sequences with low identity

* Sub-group with high sequence identity

* Small secreted molecules of 16-25 kDa

* Airborne, easily spreading into indoor environment

All pairs with a high sequence identity are potentially cross-reactive. The challenge of further studies is to analyze which lipocalins are adequate species-specific markers and which are
<figure><img src="image_298.png" /><figcaption>FIGURE 168 Lipocalins: Highly conserved tertiary structures in a protein family of high amino acid sequence diversity. (A) Phylogenetic tree of mammalian lipocalin allergens (Marked in red: Cross-reactive molecules of high clinical relevance). (B) Superimposition of Equ c 1 (1EW3) and Can f 4 (4ODD) crystal structures (side view and bottom view into the calyx) show a highly similar tertiary structure despite their low amino acid sequence identity (30%). Evolutionary conservation patterns were analyzed using the ConSurf software¹⁶⁷² and based on a multiple sequence alignment (Clustal Omega) comprising 19 identified mammalian derived lipocalin allergens. Slowly evolving, highly conserved amino acid positions are colored in maroon (grade 9), whereas rapidly evolving variable positions are colored in turquoise (grade 1).</figcaption></figure>

<table><caption>TABLE 93 Amino acid identities (%) between lipocalins with high sequence identity</caption><thead><tr><th></th><th>Equ c 1</th><th>Fel d 4</th><th>Fel d 7</th><th>Can f 1</th><th>Cav p 6</th><th>Mus m 1</th><th>Ory c 4</th><th>Rat n 1</th></tr></thead><tbody><tr><th>Equ c 1</th><td></td><td>67</td><td>26</td><td>26</td><td>62</td><td>46</td><td>52</td><td>47</td></tr><tr><th>Fel d 4</th><td></td><td></td><td>26</td><td>26</td><td>67</td><td>49</td><td>63</td><td>55</td></tr><tr><th>Fel d 7</th><td></td><td></td><td></td><td>26</td><td>24</td><td>22</td><td>25</td><td>24</td></tr><tr><th>Can f 1</th><td></td><td></td><td></td><td></td><td></td><td>21</td><td>25</td><td>23</td></tr><tr><th>Can f 6</th><td></td><td></td><td></td><td></td><td></td><td></td><td>24</td><td>23</td></tr><tr><th>Cav p 6</th><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>24</td></tr><tr><th>Mus m 1</th><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>21</td></tr><tr><th>Ory c 4</th><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>24</td></tr><tr><th>Rat n 1</th><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>23</td></tr><tr><th>Equ c 1</th><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>24</td></tr><tr><th>Fel d 4</th><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>26</td></tr><tr><th>Fel d 7</th><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>26</td></tr><tr><th>Can f 1</th><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>24</td></tr><tr><th>Can f 6</th><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>24</td></tr><tr><th>Cav p 6</th><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>24</td></tr><tr><th>Mus m 1</th><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>21</td></tr><tr><th>Ory c 4</th><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>24</td></tr><tr><th>Rat n 1</th><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>23</td></tr></tbody></table>

Blue shaded areas indicate lipocalin pairs with >50% amino acid sequence identity. Figures in bold blue indicate documented IgE cross-reactivity between lipocalins.

markers of cross-reactivity. Sequence alignments are good primary prediction tools for cross-reactivity, even more so when combined with structural information. Nevertheless, the use of IgE-inhibition studies with sera from well-characterized patients is of most importance when defining clinically relevant cross-reactivity.

Figure 169 visualizes how a high amino acid sequence identity combined with a similar tertiary structure can result in large surface areas that form potential cross-reactive IgE epitopes.

The objective of further studies will be the identification of marker molecules for each animal species in order to clearly identify the sensitizing allergen source. Figure 170 shows documented and putative cross-reactive molecules.

3 – Clinical relevance

All mammalian lipocalin allergens are respiratory allergens, with the exception of the β-lactoglobulins (e.g., Bos d 5), which are present in milk.³⁴⁵ They are major allergens of different furry pets and are shed into the environment by animal dander and secretions. They stick to clothes and human hair and are passively transferred to public places.¹³⁷⁶ Allergens quantified in airborne dust in schools have been shown to attain levels that are able to sensitize children or to even stimulate asthma exacerbations. Domestic exposure to high levels of cat and dog allergens was associated with excess asthma attacks in sensitised patients.⁵⁵⁷
<figure><img src="image_292.png" /><figcaption>FIGURE 169 Interspecies cross-reactive lipocalins: The dog allergen Can f 6 shares higher amino acid sequence identities with its homologues derived from cat and horse than with lipocalin allergens within its own species. Surface representation of the dog allergen Can f 6 (6NRE) showing potential cross-reactive IgE epitopes. (A) Amino acid sequence identities between Can f 6, Can f 1 and Can f 2. (B) Amino acid sequence identities between Can f 6, Equ c 1 and Fel d 4. Identical amino acid residues between 3 lipocalin allergens are colored in red. Identical amino acid residues between 2 out of 3 allergens are colored in yellow and non-overlapping residues are colored in gray.</figcaption></figure>

The reduction in pet allergen exposures may significantly de-
crease asthma morbidity.

Up to 50% of the households in industrialized countries have a
pet. Twenty-four percent of European households have a cat, 25%
have a dog and about 6-8% own a small mammal. Particularly fami-
lies with children more frequently own a pet.

Allergy to furry animals is considered a risk factor for develop-
ment of asthma.⁷⁸³ The role of single allergen molecules as markers
of severe or mild disease has been investigated in several studies.⁸⁰³
A general conclusion from those studies is that a polysensitization to
several components of one allergen source and/or polysensitization
to components of several furry animals are associated with a higher
risk of asthma and rhinitis, as well as a predictor of disease severity.
With respect to lipocalins, sensitisation to the dog lipocalins, in par-
ticular to Can f 4 and Can f 6, has shown to be associated with dog
allergy.⁸¹¹ For details, please see chapter B06.

Clinical relevance

* Up to 50% of households have a pet

* Risk factor for respiratory symptoms and asthma

* Sensitization to multiple components is associated with disease severity

* No molecule based therapeutic approach available

For the moment, the best treatment seems to be allergen avoid-
ance. However, this is not always feasible as the allergens are pres-
ent in schools, day-care centres, and public places. Furthermore,

pets are kept in many households. Thus, severely allergic patients
are facing the risk of social exclusion by trying to avoid the allergens.
The only immunotherapies currently available are made of animal
dander extracts. Results of subcutaneous immunotherapy (SCIT)
have shown a benefit in cat-allergic patients with asthma and rhino-
conjunctivitis.⁸⁰² Studies on dog SCIT are limited. Due to the more
complex sensitization pattern in dog allergy and a high variation of
allergen content in allergen extracts,²⁶¹ further efforts are needed
to improve AIT for dog allergy.⁸⁰² Before being able to develop spe-
cific lipocalin vaccine reagents, much more research has to be done
to investigate the mechanism related to their allergenicity.

4 – Clinical management

A careful record of the clinical history such as the presence of pets at home or regular pet contact is of great value. Skin prick test or specific IgE using animal dander will confirm animal sensitization. As animal dander contains cross-reactive molecules such as serum albumins, some of the cross-reactive lipocalins and potentially other cross-reactive molecules, it is important to define the primary allergic source, especially if a specific immunotherapy is intended. Co-sensitization has to be distinguished from cross-sensitization. It is important to acknowledge that IgE cross-reactivity may not always imply clinical cross-reactivity.

At the current state of the art, Fel d 1, Fel d 7, Can f 1, Can f 2, Can f 4, and Can f 5 are commercially available species-specific markers of sensitisation, although sensitisation to Can f 1 is not a specific marker in case of co-sensitisation to cat. Can f 6 is a marker of potential cross-reactivity to cat or horse. Equ c 1 often cross-reacts with Fel
<figure><img src="image_299.png" /><figcaption>FIGURE 169 Interspecies cross-reactive lipocalins: The dog allergen Can f 6 shares higher amino acid sequence identities with its homologues derived from cat and horse than with lipocalin allergens within its own species. Surface representation of the dog allergen Can f 6 (6NRE) showing potential cross-reactive IgE epitopes. (A) Amino acid sequence identities between Can f 6, Can f 1 and Can f 2. (B) Amino acid sequence identities between Can f 6, Equ c 1 and Fel d 4. Identical amino acid residues between 3 lipocalin allergens are colored in red. Identical amino acid residues between 2 out of 3 allergens are colored in yellow and non-overlapping residues are colored in gray.</figcaption></figure>

The reduction in pet allergen exposures may significantly de-
crease asthma morbidity.

Up to 50% of the households in industrialized countries have a
pet. Twenty-four percent of European households have a cat, 25%
have a dog and about 6-8% own a small mammal. Particularly fami-
lies with children more frequently own a pet.

Allergy to furry animals is considered a risk factor for develop-
ment of asthma.⁷⁸³ The role of single allergen molecules as markers
of severe or mild disease has been investigated in several studies.⁸⁰³
A general conclusion from those studies is that a polysensitization to
several components of one allergen source and/or polysensitization
to components of several furry animals are associated with a higher
risk of asthma and rhinitis, as well as a predictor of disease severity.
With respect to lipocalins, sensitisation to the dog lipocalins, in par-
ticular to Can f 4 and Can f 6, has shown to be associated with dog
allergy.⁸¹¹ For details, please see chapter B06.

Clinical relevance

* Up to 50% of households have a pet

* Risk factor for respiratory symptoms and asthma

* Sensitization to multiple components is associated with disease severity

* No molecule based therapeutic approach available

For the moment, the best treatment seems to be allergen avoid-
ance. However, this is not always feasible as the allergens are pres-
ent in schools, day-care centres, and public places. Furthermore,

pets are kept in many households. Thus, severely allergic patients
are facing the risk of social exclusion by trying to avoid the allergens.
The only immunotherapies currently available are made of animal
dander extracts. Results of subcutaneous immunotherapy (SCIT)
have shown a benefit in cat-allergic patients with asthma and rhino-
conjunctivitis.⁸⁰² Studies on dog SCIT are limited. Due to the more
complex sensitization pattern in dog allergy and a high variation of
allergen content in allergen extracts,²⁶¹ further efforts are needed
to improve AIT for dog allergy.⁸⁰² Before being able to develop spe-
cific lipocalin vaccine reagents, much more research has to be done
to investigate the mechanism related to their allergenicity.

4 – Clinical management

A careful record of the clinical history such as the presence of pets at home or regular pet contact is of great value. Skin prick test or specific IgE using animal dander will confirm animal sensitization. As animal dander contains cross-reactive molecules such as serum albumins, some of the cross-reactive lipocalins and potentially other cross-reactive molecules, it is important to define the primary allergic source, especially if a specific immunotherapy is intended. Co-sensitization has to be distinguished from cross-sensitization. It is important to acknowledge that IgE cross-reactivity may not always imply clinical cross-reactivity.

At the current state of the art, Fel d 1, Fel d 7, Can f 1, Can f 2, Can f 4, and Can f 5 are commercially available species-specific markers of sensitisation, although sensitisation to Can f 1 is not a specific marker in case of co-sensitisation to cat. Can f 6 is a marker of potential cross-reactivity to cat or horse. Equ c 1 often cross-reacts with Fel
d 4 and Can f 6, Mus m 1 may cross-react with Rat n 1. If the clinical history does not allow a clear identification of the sensitising animal, the following decision tree (Figures 171-173) will help to orient the use of components. Unfortunately, the number of commercially available components is still limited. The coverage is rather good for cat and dog, but for others, not all allergens are available from all providers. Horse allergens Equ c 1 and Equ c 3 are both cross-reactive, Equ c 4 may be a specific marker, but this needs to be further evaluated. More recently, some allergens of small furry pets became available for component-resolved diagnosis of allergy to hamster, rabbit and guinea-pig.

5 - Clinical cases

**Case 1 (published¹⁵³⁴)**

**Clinical history:** A 24-year-old man presented at the clinic with a 14-year history of rhinitis and asthma when exposed to horses and a 2-year history of rhinitis when exposed to dogs.

**Test with extracts:** Specific IgE to horse dander were elevated (92 kU/L), they were moderate to dog (7.2 kU/L).

**Test with molecules:** Specific IgE were detected to Equ c 1 (18 kU/L). All commercially available dog allergens (Can f 1, 2, 3, 5) were

<figure><img src="image_293.png" /></figure>

**FIGURE 170** Cross-reactivities among allergenic lipocalins. Solid lines represent documented IgE cross-reactivity. Dotted lines represent potential IgE cross-reactivity based on high sequence identities. Allergens depicted in the outer circle (white font) show overall low sequence identities with other family members and are candidates for species-specific marker allergens, but their cross-reactive potential still needs to be investigated.

<figure><img src="image_294.png" /></figure>

**FIGURE 171** Single allergens are of added value to identify the primary sensitisation source in the case of a positive IgE test to cat dander.
d 4 and Can f 6, Mus m 1 may cross-react with Rat n 1. If the clinical history does not allow a clear identification of the sensitising animal, the following decision tree (Figures 171-173) will help to orient the use of components. Unfortunately, the number of commercially available components is still limited. The coverage is rather good for cat and dog, but for others, not all allergens are available from all providers. Horse allergens Equ c 1 and Equ c 3 are both cross-reactive, Equ c 4 may be a specific marker, but this needs to be further evaluated. More recently, some allergens of small furry pets became available for component-resolved diagnosis of allergy to hamster, rabbit and guinea-pig.

5 - Clinical cases

**Case 1 (published<sup>1534</sup>)**

**Clinical history:** A 24-year-old man presented at the clinic with a 14-year history of rhinitis and asthma when exposed to horses and a 2-year history of rhinitis when exposed to dogs.

**Test with extracts:** Specific IgE to horse dander were elevated (92 kU/L), they were moderate to dog (7.2 kU/L).

**Test with molecules:** Specific IgE were detected to Equ c 1 (18 kU/L). All commercially available dog allergens (Can f 1, 2, 3, 5) were

<figure><img src="image_300.png" /></figure>

**FIGURE 170** Cross-reactivities among allergenic lipocalins. Solid lines represent documented IgE cross-reactivity. Dotted lines represent potential IgE cross-reactivity based on high sequence identities. Allergens depicted in the outer circle (white font) show overall low sequence identities with other family members and are candidates for species-specific marker allergens, but their cross-reactive potential still needs to be investigated.

<figure><img src="image_301.png" /></figure>

**FIGURE 171** Single allergens are of added value to identify the primary sensitisation source in the case of a positive IgE test to cat dander.
<figure><img src="image_295.png" /><figcaption>FIGURE 172 Single allergens are of added value to identify the primary sensitisation source in the case of a positive IgE test to dog dander.</figcaption></figure>

<figure><img src="image_296.png" /><figcaption>FIGURE 173 Single allergens are of added value to identify the primary sensitization source in the case of a positive IgE test to horse dander</figcaption></figure>

negative. However IgE to Can f 6 were clearly positive (3.7 kU/L). Inhibition assays showed that IgE recognition of Can f 6 could be totally inhibited by low doses of Equ c 1.

**Conclusion:** In this particular case, clinical symptoms to dog were due to cross-reactivity of Can f 6 with Equ c 1.

**Case 2 (published¹⁵²⁹)**

**Clinical history:** A 30-year-old women had asthma upon exposure to her cat.

**Test with extracts:** Specific IgE were positive for cat (>100 kU/L) and dog dander (9 kU/L).

**Test with molecules:** The patient had specific IgE against Fel d 1 (51 kU/L) and Fel d 4 (51 kU/L), but Fel d 2, Can f 1, Can f 2, and Can f 3 were negative. Specific IgE to the cross-reactive Can f 6 were 18 kU/L. These could be completely inhibited by Fel d 4, suggesting cat as the primary allergen source.

**Conclusion:** Specific IgE were positive to cat and dog, but the presence of specific IgE to the marker allergen Fel d 1 and the absence of specific IgE to Can f 1 or Can f 2 confirmed that cat was the

primary allergen source and that Can f 6 was a IgE-cross-reacting allergen in dog.

**Case 3 (published¹⁵²⁹)**

**Clinical history:** A 53-year-old man presents with respiratory symptoms upon exposure to cat and dog.

**Test with extracts:** Specific IgE were positive for cat (65 kU/L) and dog dander (68 kU/L).

**Test with molecules:** The patient had specific IgE against Fel d 1 (35.8 kU/L), Fel d 2 (0.7 kU/L), Fel d 4 (45 kU/L), Can f 1 (26 kU/L), Can f 2 (13.5 kU/L), Can f 3 (0.2 kU/L) and Can f 6 (33 kU/L).

**Conclusion:** The presence of IgE to the specific markers Fel d 1, Can f 1 and Can f 2 argues for co-sensitization of cat and dog. Inhibition and cross-inhibition studies with Can f 6 and Fel d 4 showed weak inhibition, confirming the hypothesis of co-sensitization.

**Allergen nomenclature:** Fel d 1, cat secretoglobin; Can f 1, Can f 2, Can f 6, Equ c 1, Fel d 4, dog, horse and cat lipocalins; Can f 3, Fel d 2: dog and cat serum albumins; Can f 5, dog kallikrein.
<figure><img src="image_302.png" /><figcaption>FIGURE 172 Single allergens are of added value to identify the primary sensitisation source in the case of a positive IgE test to dog dander.</figcaption></figure>

<figure><img src="image_303.png" /><figcaption>FIGURE 173 Single allergens are of added value to identify the primary sensitization source in the case of a positive IgE test to horse dander</figcaption></figure>

negative. However IgE to Can f 6 were clearly positive (3.7 kU/L). Inhibition assays showed that IgE recognition of Can f 6 could be totally inhibited by low doses of Equ c 1.

**Conclusion:** In this particular case, clinical symptoms to dog were due to cross-reactivity of Can f 6 with Equ c 1.

**Case 2 (published¹⁵²⁹)**

**Clinical history:** A 30-year-old women had asthma upon exposure to her cat.

**Test with extracts:** Specific IgE were positive for cat (>100 kU/L) and dog dander (9 kU/L).

**Test with molecules:** The patient had specific IgE against Fel d 1 (51 kU/L) and Fel d 4 (51 kU/L), but Fel d 2, Can f 1, Can f 2, and Can f 3 were negative. Specific IgE to the cross-reactive Can f 6 were 18 kU/L. These could be completely inhibited by Fel d 4, suggesting cat as the primary allergen source.

**Conclusion:** Specific IgE were positive to cat and dog, but the presence of specific IgE to the marker allergen Fel d 1 and the absence of specific IgE to Can f 1 or Can f 2 confirmed that cat was the

primary allergen source and that Can f 6 was a IgE-cross-reacting allergen in dog.

**Case 3 (published¹⁵²⁹)**

**Clinical history:** A 53-year-old man presents with respiratory symptoms upon exposure to cat and dog.

**Test with extracts:** Specific IgE were positive for cat (65 kU/L) and dog dander (68 kU/L).

**Test with molecules:** The patient had specific IgE against Fel d 1 (35.8 kU/L), Fel d 2 (0.7 kU/L), Fel d 4 (45 kU/L), Can f 1 (26 kU/L), Can f 2 (13.5 kU/L), Can f 3 (0.2 kU/L) and Can f 6 (33 kU/L).

**Conclusion:** The presence of IgE to the specific markers Fel d 1, Can f 1 and Can f 2 argues for co-sensitization of cat and dog. Inhibition and cross-inhibition studies with Can f 6 and Fel d 4 showed weak inhibition, confirming the hypothesis of co-sensitization.

**Allergen nomenclature:** Fel d 1, cat secretoglobin; Can f 1, Can f 2, Can f 6, Equ c 1, Fel d 4, dog, horse and cat lipocalins; Can f 3, Fel d 2: dog and cat serum albumins; Can f 5, dog kallikrein.
# C08 - Seed storage proteins

## Merima Bublin, Christian Radauer, Anna M. Ehlers, Thuy-My Le

### Highlights

* Seed storage proteins (2S albumins, 7S globulins and 11S globulins) are marker allergens for clinically relevant sensitizations to legumes, tree nuts and seeds.

* Not all relevant allergenic seed storage proteins are available for routine diagnosis.

* IgE cross-reactivity occurs between members of the same protein family most with allergens from related plants that show high protein sequence identities.

* IgE cross-reactivity may also occur between allergens from different families of seed storage proteins.

* The clinical relevance of IgE co-sensitization and the impact of cross-reactivity are largely unknown and still have to be studied using well-defined allergens together with primary material (serum, whole blood depending on the test) collected from well-characterized patients.

## 1 - The protein

Seeds comprise the most important constituents of the human diet, but they are also major elicitors of food allergies. Edible seeds are derived from botanically diverse types of plants such as cereals (e.g., wheat, rye, corn, rice see chapter B16), legumes (e.g., peanut, soybean, lentil chapters B17, B18), tree nuts (e.g., walnut, hazelnut) and others falling into neither of those categories (e.g., buckwheat, sesame, mustard chapter B19). Seeds are rich in protein, the most abundant of which are seed storage proteins whose main biological functions are to provide nutrients and energy sources for the germinating plant. Storage proteins of noncereal seeds can be classified into three protein families, the 2S albumins, the 7S globulins, also named vicilins, and the 11S globulins, also named legumins.¹⁵³⁵ All three families contain major allergens from legumes, tree nuts and other seeds. Sensitization to these allergens is often associated with severe reactions. Examples are Ara h 1, 2, 3, 6 and 7 from peanut, Jug r 1, 2, 4 and 6 from walnut and Ses i 1, 2, 3, 6 and 7 from sesame (Table 94). In addition, seed storage proteins may also elicit allergic reactions to certain fruits, such as tomato or kiwifruit, that contain small seeds usually eaten together with the fruit pulp. Cereal grains contain different types of storage proteins, the cereal prolamins, which are discussed in chapter B16.

## 2 - The families

### 2S albumins

2S albumins belong to the prolamin superfamily and are structurally related to the cereal bifunctional amylase and protease inhibitors (chapter B16) and the nonspecific lipid transfer proteins (nsLTPs; see chapter C03). Important members of this family are Ara h 2 and Ara h 6 from peanut, Jug r 1 from walnut, and Ses i 1 and Ses i 2 from sesame (Table 94). Most 2S albumins are composed of two disulfide-linked polypeptide chains that are generated by post-translational cleavage of a single polypeptide (Figure 174A), a small chain of about 4 kDa and a large chain of about 9 kDa (Table 95). Some members of this family consist of a single chain such as Ara h 2 and Ara h 6 from peanut. 2S albumins fold into compact α-helical bundles further stabilized by 4-5 disulfide bonds (Figure 175A). Apart from a conserved pattern of eight cysteine residues, sequences of 2S albumins from unrelated plants show very low sequence identities of less than 40% (Table 96).

#### 2S albumin family characteristics

* Alpha-helical structure stabilized by disulphide bonds

* Low sequence conservation except a conserved cysteine skeleton

* Small, water-soluble molecules of about 15 kDa

* Highly stable against heat and digestion

* Present in all seeds of dicotyledonous plants

### 7S globulins

7S globulins (vicilins) and 11S globulins (legumins) are structurally related and belong to the cupin superfamily. Important allergenic vicilins are Ara h 1 from peanut, Gly m 5 from soybean and Jug r 2 from walnut (Table 94). Vicilins are composed of subunits of about 50 kDa in size (Table 95) folding into structures composed of two so-called cupin β-barrel domains surrounded by surface-exposed α-helices and unstructured loops (Figure 175B). These subunits form stable trimers of 150-190 kDa held together by noncovalent interactions (Figure 175C). In addition, many vicilins contain an N-linked glycan (Figure 174B). Sequences of vicilins from unrelated plants show only low levels of conservation with identities typically between 30% and 50% (Table 97).

During post-translational processing of vicilins, a large N-terminal propeptide is cleaved off and, in many cases, is not degraded but further processed into one or several shorter peptides with anti-microbial activity (Figure 174B). These peptides, known as α-hairpinins, contain a conserved cysteine pattern (CX₃CX₁₀₋₁₂CX₃C) and fold into an α-hairpin structure stabilized by two disulfide
# C08 - Seed storage proteins

## Merima Bublin, Christian Radauer, Anna M. Ehlers, Thuy-My Le

### Highlights

* Seed storage proteins (2S albumins, 7S globulins and 11S globulins) are marker allergens for clinically relevant sensitizations to legumes, tree nuts and seeds.

* Not all relevant allergenic seed storage proteins are available for routine diagnosis.

* IgE cross-reactivity occurs between members of the same protein family most with allergens from related plants that show high protein sequence identities.

* IgE cross-reactivity may also occur between allergens from different families of seed storage proteins.

* The clinical relevance of IgE co-sensitization and the impact of cross-reactivity are largely unknown and still have to be studied using well-defined allergens together with primary material (serum, whole blood depending on the test) collected from well-characterized patients.

## 1 - The protein

Seeds comprise the most important constituents of the human diet, but they are also major elicitors of food allergies. Edible seeds are derived from botanically diverse types of plants such as cereals (e.g., wheat, rye, corn, rice see chapter B16), legumes (e.g., peanut, soybean, lentil chapters B17, B18), tree nuts (e.g., walnut, hazelnut) and others falling into neither of those categories (e.g., buckwheat, sesame, mustard chapter B19). Seeds are rich in protein, the most abundant of which are seed storage proteins whose main biological functions are to provide nutrients and energy sources for the germinating plant. Storage proteins of noncereal seeds can be classified into three protein families, the 2S albumins, the 7S globulins, also named vicilins, and the 11S globulins, also named legumins.¹⁵³⁵ All three families contain major allergens from legumes, tree nuts and other seeds. Sensitization to these allergens is often associated with severe reactions. Examples are Ara h 1, 2, 3, 6 and 7 from peanut, Jug r 1, 2, 4 and 6 from walnut and Ses i 1, 2, 3, 6 and 7 from sesame (Table 94). In addition, seed storage proteins may also elicit allergic reactions to certain fruits, such as tomato or kiwifruit, that contain small seeds usually eaten together with the fruit pulp. Cereal grains contain different types of storage proteins, the cereal prolamins, which are discussed in chapter B16.

## 2 - The families

### 2S albumins

2S albumins belong to the prolamin superfamily and are structurally related to the cereal bifunctional amylase and protease inhibitors (chapter B16) and the nonspecific lipid transfer proteins (nsLTPs; see chapter C03). Important members of this family are Ara h 2 and Ara h 6 from peanut, Jug r 1 from walnut, and Ses i 1 and Ses i 2 from sesame (Table 94). Most 2S albumins are composed of two disulfide-linked polypeptide chains that are generated by post-translational cleavage of a single polypeptide (Figure 174A), a small chain of about 4 kDa and a large chain of about 9 kDa (Table 95). Some members of this family consist of a single chain such as Ara h 2 and Ara h 6 from peanut. 2S albumins fold into compact α-helical bundles further stabilized by 4-5 disulfide bonds (Figure 175A). Apart from a conserved pattern of eight cysteine residues, sequences of 2S albumins from unrelated plants show very low sequence identities of less than 40% (Table 96).

#### 2S albumin family characteristics

* Alpha-helical structure stabilized by disulphide bonds

* Low sequence conservation except a conserved cysteine skeleton

* Small, water-soluble molecules of about 15 kDa

* Highly stable against heat and digestion

* Present in all seeds of dicotyledonous plants

### 7S globulins

7S globulins (vicilins) and 11S globulins (legumins) are structurally related and belong to the cupin superfamily. Important allergenic vicilins are Ara h 1 from peanut, Gly m 5 from soybean and Jug r 2 from walnut (Table 94). Vicilins are composed of subunits of about 50 kDa in size (Table 95) folding into structures composed of two so-called cupin β-barrel domains surrounded by surface-exposed α-helices and unstructured loops (Figure 175B). These subunits form stable trimers of 150-190 kDa held together by noncovalent interactions (Figure 175C). In addition, many vicilins contain an N-linked glycan (Figure 174B). Sequences of vicilins from unrelated plants show only low levels of conservation with identities typically between 30% and 50% (Table 97).

During post-translational processing of vicilins, a large N-terminal propeptide is cleaved off and, in many cases, is not degraded but further processed into one or several shorter peptides with anti-microbial activity (Figure 174B). These peptides, known as α-hairpinins, contain a conserved cysteine pattern (CX₃CX₁₀₋₁₂CX₃C) and fold into an α-hairpin structure stabilized by two disulfide
<table><caption>TABLE 94 Seed storage proteins identified as allergens</caption><thead><tr><th rowspan="2">Plant family</th><th rowspan="2">Allergen source</th><th colspan="3">Allergen name</th></tr><tr><th>2S albumins</th><th>Vicilins (7S globulins)</th><th>Legumins (11S globulins)</th></tr></thead><tbody><tr><td rowspan="6">Legumes</td><td>Fabaceae</td><td>Peanut (Arachis hypogaea)<br>Soybean (Glycine max)<br>Pea (Pisum sativum)<br>Narrow-leaved blue lupine (Lupinus angustifolius)<br>Lentil (Lens culinaris)<br>Mung bean (Vigna radiata)</td><td>Ara h 2, Ara h 6, Ara h 7<br>Gly m 8</td><td>Ara h 1<br>Gly m 5<br>Pis s 1, Pis s 2<br>Lup an 1<br>Len c 1<br>Vig r 2</td><td>Ara h 3<br>Gly m 6</td></tr><tr><td>Tree nuts</td><td>Juglandaceae<br>English walnut (Juglans regia)<br>Black walnut (Juglans nigra)<br>Pecan (Carya illinoinensis)<br>Betulaceae<br>Hazelnut (Corylus avellana)<br>Rosaceae<br>Almond (Prunus dulcis)<br>Anacardiaceae<br>Cashew nut (Anacardium occidentale)<br>Pistachio (Pistacia vera)<br>Lecythidaceae<br>Brazil nut (Bertholletia excelsa)<br>Proteaceae<br>Macadamia nut (Macadamia integrifolia)<br>Pinaceae<br>Korean pine (Pinus koraiensis)<br>Stone pine (Pinus pinea)</td><td>Jug r 1<br>Jug n 1<br>Car i 1<br>Cor a 14</td><td>Jug r 2, Jug r 6<br>Jug n 2<br>Car i 2<br>Cor a 11</td><td>Jug r 4<br>Jug n 4<br>Car i 4<br>Cor a 9<br>Pru du 6<br>Ana o 2<br>Pis v 3<br>Ber e 2<br>Mac i 2<br>Pin k 2<br>Pin p 1</td></tr><tr><td>Other seeds</td><td>Pedaliaceae<br>Sesame (Sesamum indicum)<br>Linaceae<br>Flaxseed (Linum usitatissimum)<br>Brassicaceae<br>Yellow mustard (Sinapis alba)<br>Indian mustard (Brassica juncea)<br>Rapeseed (Brassica napus)<br>Field mustard (Brassica rapa)<br>Rutaceae<br>Actinidaceae<br>Kiwi fruit (Actinidia deliciosa)<br>Cucurbitaceae<br>Pumpkin (Cucurbita maxima)<br>Polygonaceae<br>Common buckwheat (Fagopyrum esculentum)<br>Tartarian buckwheat (Fagopyrum tataricum)<br>Euphorbiaceae<br>Castor bean (Ricinus communis)</td><td>Ses i 1, Ses i 2<br>Lin u 1<br>Sin a 1<br>Bra j 1<br>Bra n 1<br>Bra r 1<br>Zan b 1<br>Act d 13<br>Cuc ma 5<br>Fag e 2<br>Fag t 2<br>Ric c 1, Ric c 3</td><td>Ses i 3</td><td>Ses i 6, Ses i 7<br>Sin a 2<br>Act d 12<br>Cuc ma 4<br>Fag e 3, Fag e 5</td></tr></tbody></table>

bonds.¹⁵³⁶ Some of these peptides, such as those derived from Ara h 1 and Jug r 2, bind IgE from a considerable fraction of patients allergic to peanut or walnut, respectively.¹⁵³⁷ Strikingly, IgE from a subpopulation of these allergic patients does not even bind the respective mature vicilin. Hence, these peptides constitute important allergens distinct from mature vicilins.

### 7S globulin family characteristics

* Trimeric glycoproteins composed of subunits folding into two β-barrels surrounded by α-helical loops

* Moderately conserved sequences

* Trimers of about 150 kDa in size but also containing differently processed molecular forms

* Stable against heat and digestion

* Present in seeds of dicotyledonous plants

### 11S globulins

11S globulins (legumins) are the most abundant seed storage proteins comprising up to 50% of the total protein in some species. Legumins with allergenic properties are Ara h 3 from peanut, Gly m 6 from soybean and Cor a 9 from hazelnut (Table 94). Legumin subunits are 50-60 kDa in size and are composed of a 30-40 kDa acidic chain and a 20 kDa basic chain connected by a conserved disulfide bond (Table 95; Figure 174C). These subunits adopt a fold similar to that of vicilins composed of two cupin β-barrels surrounded by surface-exposed α-helical and unstructured stretches (Figure 175D). Legumins form hexamers of 300-450 kDa in size composed of two trimers one stacked on top of the other (Figure 175E,F). Sequences of legumins are more conserved than those of other storage proteins, with typically 40-60% sequence identity between homologues from nonrelated plants (Table 98). In addition, legumins show considerable sequence similarities with the structurally related vicilins.
<table><caption>TABLE 94 Seed storage proteins identified as allergens</caption><thead><tr><th rowspan="2">Plant family</th><th rowspan="2">Allergen source</th><th colspan="3">Allergen name</th></tr><tr><th>2S albumins</th><th>Vicilins (7S globulins)</th><th>Legumins (11S globulins)</th></tr></thead><tbody><tr><td rowspan="6">Legumes</td><td>Peanut (Arachis hypogaea)</td><td>Ara h 2, Ara h 6, Ara h 7</td><td>Ara h 1</td><td>Ara h 3</td></tr><tr><td>Soybean (Glycine max)</td><td>Gly m 8</td><td>Gly m 5</td><td>Gly m 6</td></tr><tr><td>Pea (Pisum sativum)</td><td></td><td>Pis s 1, Pis s 2</td><td></td></tr><tr><td>Narrow-leaved blue lupine (Lupinus angustifolius)</td><td></td><td>Lup an 1</td><td></td></tr><tr><td>Lentil (Lens culinaris)</td><td></td><td>Len c 1</td><td></td></tr><tr><td>Mung bean (Vigna radiata)</td><td></td><td>Vig r 2</td><td></td></tr><tr><td rowspan="9">Tree nuts</td><td>Juglandaceae</td><td>English walnut (Juglans regia)</td><td>Jug r 1</td><td>Jug r 2, Jug r 6</td><td>Jug r 4</td></tr><tr><td></td><td>Black walnut (Juglans nigra)</td><td>Jug n 1</td><td>Jug n 2</td><td>Jug n 4</td></tr><tr><td></td><td>Pecan (Carya illinoinensis)</td><td>Car i 1</td><td>Car i 2</td><td>Car i 4</td></tr><tr><td>Betulaceae</td><td>Hazelnut (Corylus avellana)</td><td>Cor a 14</td><td>Cor a 11</td><td>Cor a 9</td></tr><tr><td>Rosaceae</td><td>Almond (Prunus dulcis)</td><td></td><td></td><td>Pru du 6</td></tr><tr><td>Anacardiaceae</td><td>Cashew nut (Anacardium occidentale)</td><td>Ana o 3</td><td>Ana o 1</td><td>Ana o 2</td></tr><tr><td></td><td>Pistachio (Pistacia vera)</td><td>Pis v 1</td><td>Pis v 3</td><td>Pis v 2, Pis v 5</td></tr><tr><td>Lecythidaceae</td><td>Brazil nut (Bertholletia excelsa)</td><td>Ber e 1</td><td></td><td>Ber e 2</td></tr><tr><td>Proteaceae</td><td>Macadamia nut (Macadamia integrifolia)</td><td></td><td>Mac i 1</td><td>Mac i 2</td></tr><tr><td rowspan="2">Pinaceae</td><td>Korean pine (Pinus koraiensis)</td><td></td><td>Pin k 2</td><td></td></tr><tr><td>Stone pine (Pinus pinea)</td><td>Pin p 1</td><td></td><td></td></tr><tr><td rowspan="10">Other seeds</td><td>Pedaliaceae</td><td>Sesame (Sesamum indicum)</td><td>Ses i 1, Ses i 2</td><td>Ses i 3</td><td>Ses i 6, Ses i 7</td></tr><tr><td>Linaceae</td><td>Flaxseed (Linum usitatissimum)</td><td>Lin u 1</td><td></td><td></td></tr><tr><td rowspan="4">Brassicaceae</td><td>Yellow mustard (Sinapis alba)</td><td>Sin a 1</td><td></td><td>Sin a 2</td></tr><tr><td>Indian mustard (Brassica juncea)</td><td>Bra j 1</td><td></td><td></td></tr><tr><td>Rapeseed (Brassica napus)</td><td>Bra n 1</td><td></td><td></td></tr><tr><td>Field mustard (Brassica rapa)</td><td>Bra r 1</td><td></td><td></td></tr><tr><td>Rutaceae</td><td>Sichuan pepper (Zanthoxylum bungeanum)</td><td>Zan b 1</td><td></td><td></td></tr><tr><td>Actinidaceae</td><td>Kiwi fruit (Actinidia deliciosa)</td><td>Act d 13</td><td></td><td>Act d 12</td></tr><tr><td>Cucurbitaceae</td><td>Pumpkin (Cucurbita maxima)</td><td>Cuc ma 5</td><td></td><td>Cuc ma 4</td></tr><tr><td rowspan="2">Polygonaceae</td><td>Common buckwheat (Fagopyrum esculentum)</td><td>Fag e 2</td><td>Fag e 3, Fag e 5</td><td></td></tr><tr><td>Tartarian buckwheat (Fagopyrum tataricum)</td><td>Fag t 2</td><td></td><td></td></tr><tr><td>Euphorbiaceae</td><td>Castor bean (Ricinus communis)</td><td>Ric c 1, Ric c 3</td><td></td><td></td></tr></tbody></table>

bonds.1536 Some of these peptides, such as those derived from Ara h 1 and Jug r 2, bind IgE from a considerable fraction of patients allergic to peanut or walnut, respectively.1537 Strikingly, IgE from a subpopulation of these allergic patients does not even bind the respective mature vicilin. Hence, these peptides constitute important allergens distinct from mature vicilins.

### 7S globulin family characteristics

* Trimeric glycoproteins composed of subunits folding into two β-barrels surrounded by α-helical loops

* Moderately conserved sequences

* Trimers of about 150 kDa in size but also containing differently processed molecular forms

* Stable against heat and digestion

* Present in seeds of dicotyledonous plants

### 11S globulins

11S globulins (legumins) are the most abundant seed storage proteins comprising up to 50% of the total protein in some species. Legumins with allergenic properties are Ara h 3 from peanut, Gly m 6 from soybean and Cor a 9 from hazelnut (Table 94). Legumin subunits are 50-60 kDa in size and are composed of a 30-40 kDa acidic chain and a 20 kDa basic chain connected by a conserved disulfide bond (Table 95; Figure 174C). These subunits adopt a fold similar to that of vicilins composed of two cupin β-barrels surrounded by surface-exposed α-helical and unstructured stretches (Figure 175D). Legumins form hexamers of 300-450 kDa in size composed of two trimers one stacked on top of the other (Figure 175E,F). Sequences of legumins are more conserved than those of other storage proteins, with typically 40-60% sequence identity between homologues from nonrelated plants (Table 98). In addition, legumins show considerable sequence similarities with the structurally related vicilins.
<figure><img src="image_297.png" /><figcaption>TABLE 95 Basic protein characteristics of seed storage proteins</figcaption></figure>

<table><thead><tr><th>Porotein characteristics</th><th>2S albumins</th><th>7S globulins</th><th>11S globulins</th></tr></thead><tbody><tr><td>Allergen source</td><td>Legumes, nuts, seeds</td><td>Legumes, nuts, seeds</td><td>Legumes, nuts, seeds</td></tr><tr><td>Molecular structure</td><td>α-helical bundle of 4-5 helices; mostly composed of 2 disulfide-linked chains</td><td>2 β-barrels surrounded by α-helical and unstructured loops</td><td>2 β-barrels surrounded by α-helical and unstructured loops; composed of 2 disulfide-linked chains</td></tr><tr><td>Theoretical molecular weight</td><td>11-18 kDa; small chain: 3-5 kD; large chain: 8-10 kDa</td><td>45-60 kDa</td><td>50-61 kDa; acidic chain: 29-40 kDa; basic chain: 19-22 kDa</td></tr><tr><td>Length</td><td>90-151 aa; small chain: 26-43 aa; large chain: 64-92 aa</td><td>400-522 aa</td><td>438-531 aa; acidic chain: 256-347 aa; basic chain: 172-192 aa</td></tr><tr><td>Ligand binding</td><td>No</td><td>No</td><td>Metal ions, e.g. Mg<sup>2+</sup></td></tr><tr><td>Oligomerization</td><td>No</td><td>Trimers</td><td>Hexamers</td></tr><tr><td>Glycosylation</td><td>No</td><td>Yes</td><td>No</td></tr><tr><td>Disulfide bonds</td><td>4-5</td><td>No</td><td>2</td></tr><tr><td>Isoelectric point</td><td>4.5-7.5; small chain: 5.4-10.1; large chain: 4.6-7.0; Brassicaceae: 9.4-10.8; small chain: 10.3-10.9; large chain: 9.1-9.5</td><td>4.9-7.3</td><td>5.6-7.3; acidic chain: 4.9-6.0; basic chain: 6.1-10.6</td></tr><tr><td>Synthesis</td><td>In seeds after fertilisation until maturation</td><td>In seeds after fertilisation until maturation</td><td>In seeds after fertilisation until maturation</td></tr><tr><td>Distribution</td><td>In seeds of gymnosperms and dicotyledonous plants</td><td>In seeds of mono- and dicotyledonous plants</td><td>In seeds of mono- and dicotyledonous plants</td></tr></tbody></table>

## 11S globulins family characteristics

* Hexameric proteins composed of subunits folding into two β-barrels surrounded by α-helices and unstructured loops

* Moderately conserved sequences

* Hexamers of 300-450 kDa in size

* Stable against heat and digestion

* Highly abundant in seeds of dicotyledonous plants

## Heterogeneity of seed storage proteins

Seed storage proteins purified from their natural sources are heterogeneous mixtures of similar proteins, which presents a challenge for their exact characterization and the production of natural-like recombinant allergens. Several mechanisms contribute to this heterogeneity:

* Isoforms: Many storage proteins are expressed as mixtures of distinct isoforms that differ mainly in their surface-exposed regions and therefore may also have different IgE-binding properties.
<figure><img src="image_304.png" /><figcaption>TABLE 95 Basic protein characteristics of seed storage proteins</figcaption></figure>

<table><thead><tr><th>Porotein characteristics</th><th>2S albumins</th><th>7S globulins</th><th>11S globulins</th></tr></thead><tbody><tr><td>Allergen source</td><td>Legumes, nuts, seeds</td><td>Legumes, nuts, seeds</td><td>Legumes, nuts, seeds</td></tr><tr><td>Molecular structure</td><td>α-helical bundle of 4-5 helices; mostly composed of 2 disulfide-linked chains</td><td>2 β-barrels surrounded by α-helical and unstructured loops</td><td>2 β-barrels surrounded by α-helical and unstructured loops; composed of 2 disulfide-linked chains</td></tr><tr><td>Theoretical molecular weight</td><td>11-18 kDa; small chain: 3-5 kD; large chain: 8-10 kDa</td><td>45-60 kDa</td><td>50-61 kDa; acidic chain: 29-40 kDa; basic chain: 19-22 kDa</td></tr><tr><td>Length</td><td>90-151 aa; small chain: 26-43 aa; large chain: 64-92 aa</td><td>400-522 aa</td><td>438-531 aa; acidic chain: 256-347 aa; basic chain: 172-192 aa</td></tr><tr><td>Ligand binding</td><td>No</td><td>No</td><td>Metal ions, e.g. Mg<sup>2+</sup></td></tr><tr><td>Oligomerization</td><td>No</td><td>Trimers</td><td>Hexamers</td></tr><tr><td>Glycosylation</td><td>No</td><td>Yes</td><td>No</td></tr><tr><td>Disulfide bonds</td><td>4-5</td><td>No</td><td>2</td></tr><tr><td>Isoelectric point</td><td>4.5-7.5; small chain: 5.4-10.1; large chain: 4.6-7.0; Brassicaceae: 9.4-10.8; small chain: 10.3-10.9; large chain: 9.1-9.5</td><td>4.9-7.3</td><td>5.6-7.3; acidic chain: 4.9-6.0; basic chain: 6.1-10.6</td></tr><tr><td>Synthesis</td><td>In seeds after fertilisation until maturation</td><td>In seeds after fertilisation until maturation</td><td>In seeds after fertilisation until maturation</td></tr><tr><td>Distribution</td><td>In seeds of gymnosperms and dicotyledonous plants</td><td>In seeds of mono- and dicotyledonous plants</td><td>In seeds of mono- and dicotyledonous plants</td></tr></tbody></table>

## 11S globulins family characteristics

* Hexameric proteins composed of subunits folding into two β-barrels surrounded by α-helices and unstructured loops

* Moderately conserved sequences

* Hexamers of 300-450 kDa in size

* Stable against heat and digestion

* Highly abundant in seeds of dicotyledonous plants

## Heterogeneity of seed storage proteins

Seed storage proteins purified from their natural sources are heterogeneous mixtures of similar proteins, which presents a challenge for their exact characterization and the production of natural-like recombinant allergens. Several mechanisms contribute to this heterogeneity:

* Isoforms: Many storage proteins are expressed as mixtures of distinct isoforms that differ mainly in their surface-exposed regions and therefore may also have different IgE-binding properties.
<figure><img src="image_298.png" /><figcaption>FIGURE 175 Representative structures of seed storage proteins. A. Ber e 1 from Brazil nut, a 2S albumin (PDB: 2lvf); B, C. Cor a 11 from hazelnut, a 7S globulin (PDB: 6l4c) (B) single subunit and (C) trimer, D-F: Ara h 3 from peanut, a 11S globulin (PDB: 3c3v) (D) single subunit and (E, F) hexamer. Structures in A, B and D are colored by secondary structure elements from blue (N-terminus) to red (C-terminus). In the oligomeric structures in C, E and F, each subunit is colored in a different color. The images were created with UCSF ChimeraX (https://www.rbvi.ucsf.edu/chimerax/).</figcaption></figure>

<figure><img src="image_299.png" /><figcaption>TABLE 96 Amino acid sequence identities (%) between allergenic 2S albumins. Light blue: identities from 35% to 49%, blue: identities from 50% to 74%, dark blue: identities ≥ 75%. Sequence alignments were performed using ClustalX.</figcaption></figure>
<figure><img src="image_305.png" /><figcaption>FIGURE 175 Representative structures of seed storage proteins. A. Ber e 1 from Brazil nut, a 2S albumin (PDB: 2lvf); B, C. Cor a 11 from hazelnut, a 7S globulin (PDB: 6l4c) (B) single subunit and (C) trimer, D-F: Ara h 3 from peanut, a 11S globulin (PDB: 3c3v) (D) single subunit and (E, F) hexamer. Structures in A, B and D are colored by secondary structure elements from blue (N-terminus) to red (C-terminus). In the oligomeric structures in C, E and F, each subunit is colored in a different color. The images were created with UCSF ChimeraX (https://www.rbvi.ucsf.edu/chimerax/).</figcaption></figure>

<figure><img src="image_306.png" /><figcaption>TABLE 96 Amino acid sequence identities (%) between allergenic 2S albumins. Light blue: identities from 35% to 49%, blue: identities from 50% to 74%, dark blue: identities ≥ 75%. Sequence alignments were performed using ClustalX.</figcaption></figure>
<figure><img src="image_300.png" /><figcaption>TABLE 98 Amino acid sequence identities (%) between allergenic 11S globulins. Light blue: identities from 35% to 49%, blue: identities from 50% to 74%, dark blue: identities ≥ 75%. Sequence alignments were performed using ClustalX.</figcaption></figure>

<figure><img src="image_301.png" /></figure>

* Post-translational proteolytic processing: The proteolytic processing described above for all three families further increases the heterogeneity as cleavage may occur at different positions leading to differentially truncated forms of the proteins.

* Glycosylation: Most vicilins harbor a single N-linked glycan. Hence, the existence of different glycoforms adds an additional layer of complexity.

Biochemical and physico-chemical properties

The distinct properties of seed storage proteins explain many of their allergological features

* Abundance: Seeds contain between 10% (cereal grains) and 40% (some legumes and oilseeds) protein; the major part of it is composed of storage proteins.¹⁵³⁵ As a consequence, even minute amounts of seeds contain sufficient quantities of storage proteins to elicit allergic reactions in some highly sensitized individuals. In placebo-controlled oral provocation studies, some patients reacted to as little as 0.1 mg of peanut flour or 0.5 mg of lupine flour.¹⁵³⁸ Small amounts of these proteins are also found as impurities in processed foods. These “hidden” allergens pose a risk to allergic individuals, who may react with dangerously severe symptoms.
<figure><img src="image_307.png" /><figcaption>TABLE 98 Amino acid sequence identities (%) between allergenic 11S globulins. Light blue: identities from 35% to 49%, blue: identities from 50% to 74%, dark blue: identities ≥ 75%. Sequence alignments were performed using ClustalX.</figcaption></figure>

<figure><img src="image_308.png" /></figure>

* Post-translational proteolytic processing: The proteolytic processing described above for all three families further increases the heterogeneity as cleavage may occur at different positions leading to differentially truncated forms of the proteins.

* Glycosylation: Most vicilins harbor a single N-linked glycan. Hence, the existence of different glycoforms adds an additional layer of complexity.

### Biochemical and physico-chemical properties

The distinct properties of seed storage proteins explain many of their allergological features

* Abundance: Seeds contain between 10% (cereal grains) and 40% (some legumes and oilseeds) protein; the major part of it is composed of storage proteins.¹⁵³⁵ As a consequence, even minute amounts of seeds contain sufficient quantities of storage proteins to elicit allergic reactions in some highly sensitized individuals. In placebo-controlled oral provocation studies, some patients reacted to as little as 0.1 mg of peanut flour or 0.5 mg of lupine flour.¹⁵³⁸ Small amounts of these proteins are also found as impurities in processed foods. These “hidden” allergens pose a risk to allergic individuals, who may react with dangerously severe symptoms.
* Stability: Seed storage proteins, especially 2S albumins, are highly stable against food processing and gastro-intestinal digestion.<sup>1539</sup> For instance, it has been shown that roasting of peanuts increased the IgE-binding capacity and stability of the allergens, partially due to covalent cross-linking during the Maillard reaction, which leads to the formation of stable molecular aggregates. This explains why patients sensitized to seed storage proteins usually also react to processed foods, such as roasted peanuts or tree nuts or peanut butter. Their stability against digestive enzymes enables allergenic storage proteins to reach the small intestine and the circulation, thereby causing severe systemic allergic reactions.

## 3 – Clinical relevance

Sensitization to these three allergen families (2S albumins, 7S globulins and 11S globulins) is generally associated with a high risk to develop an allergic reaction upon ingestion. Allergic symptoms, which are elicited by IgE binding to those allergens, can reach from mild (e.g., oral itching) to life-threatening conditions such as anaphylaxis.

### 2S albumins

The extraordinary stable structure of 2S albumins is thought to contribute to its allergenicity and clinical relevance. The measurement of IgE specific to 2S albumins is mostly superior to measuring IgE against total extracts in edible seed allergy diagnosis. In peanut allergy, approximately 90% of patients sensitized to Ara h 2 suffer from a (severe) peanut allergy, while only 70% of patients with IgE to peanut extract are truly allergic.<sup>1540</sup> A comparable clinical importance was described for the other two peanut 2S albumins Ara h 6 and 7.<sup>1540</sup> Consistently, IgE binding to tree nut 2S albumins is also associated with clinically relevant sensitization: hazelnut Cor a 14, cashew nut Ana o 3, Brazil nut Ber e 1 and walnut Jug r 1.<sup>1541,1542</sup> However, it was shown that IgE to Jug r 1 is not significantly more relevant than measuring IgE to walnut extract in adults. 2S albumins are also predominantly recognized by IgE from patients with seed (e.g., sesame Ses i 1) or legume (e.g., soy Gly m 8) allergies.<sup>1543,1544</sup>

### 7S globulins

In tree nut allergy, 7S globulins are clinically less relevant compared with 2S albumins. For example, recognition of the hazelnut 7S globulin Cor a 11 by adults is extremely rare and its clinical relevance is not confirmed.<sup>44</sup> By contrast, the role of 7S globulins appears to be more important in legume allergies. In soybean allergy, 86% of patients with anaphylaxis were sensitized to Gly m 5, while only 33% of patients with subjective symptoms showed IgE to this 7S globulin.<sup>1543</sup> This clear association between sensitization to soybean Gly m 5 and severe symptoms was only confirmed in peadiatric populations. Moreover, 7S globulins were also characterized as major allergens (>50% *in vitro* recognition) in other legumes such as lupine, lentil, pea and chickpea. The lentil 7S globulin Len c 1.01 was recognized by 77% of lentil allergic patients, and 65% of IgE binding

to lentil extract was inhibited by preincubation with Len c 1.01.<sup>1545</sup> Despite their extensive recognition by IgE, the exact clinical relevance of 7S globulins in legume allergy needs still to be determined.

### 11S globulins

The clinical relevance of IgE binding to 11S globulins differs between foods and cannot be assigned to a specific group. While IgE binding to peanut Ara h 3 is less relevant than IgE to Ara h 2 (2S albumin), IgE binding to the hazelnut 11S globulin Cor a 9 has a comparable diagnostic value compared with IgE to the hazelnut 2S albumin Cor a 14<sup>43</sup>. IgE to both allergens is associated with severe symptoms in children. In adults, however, this clear association between IgE binding and allergy or objective symptoms was not found.<sup>1544</sup> In walnut allergy, only a small subpopulation of walnut allergic adults recognizes the walnut 11S globulin Jug r 4<sup>1541</sup> and in cashew nut allergic children, the diagnostic value of IgE to Ana o 2 is comparable to the diagnostic value of IgE to the 2S albumin Ana o 3.<sup>1541</sup> In almond allergy, IgE binding to the almond 11S globulin Pru du 6 shows a high sensitivity (83%) and specificity (78%), which is superior to measuring IgE against almond extract or other almond components.<sup>1547</sup> Strikingly, 2S albumins and 7S globulins—important allergens for other tree nuts—appear to be absent in almond kernels, which makes Pru du 6 an even more important component than 11S globulin in other tree nuts, legumes, and seeds. Although sesame seeds can cause severe allergic reactions, information regarding clinical relevance of IgE to 11S globulins is lacking. Nevertheless, it has been shown that Italian children allergic to sesame strongly recognized the basic subunit of the 11S globulin, which is only accessible after proteolytic cleavage of the acidic subunit.<sup>1548</sup>

#### Clinical relevance of 2S albumins

* Major allergens in peanut and tree nuts such as hazelnut, walnut and cashew nut

* Marker allergens for clinically relevant sensitizations to peanut, seeds and tree nuts

* High risk of cross-reactivity between walnut and pecan nut or cashew nut and pistachio

* IgE to these allergens may elicit severe symptoms

#### Clinical relevance of 7S globulins (vicilins)

* Major allergens of legumes such as soy, pea, lentil and lupine

* Marker allergens for clinically relevant sensitizations to legumes

* Risk of cross-reactivity between peanuts and peas or lupine

* Risk of cross-reactivity between peas and lentils
* Stability: Seed storage proteins, especially 2S albumins, are highly stable against food processing and gastro-intestinal digestion.<sup>1539</sup> For instance, it has been shown that roasting of peanuts increased the IgE-binding capacity and stability of the allergens, partially due to covalent cross-linking during the Maillard reaction, which leads to the formation of stable molecular aggregates. This explains why patients sensitized to seed storage proteins usually also react to processed foods, such as roasted peanuts or tree nuts or peanut butter. Their stability against digestive enzymes enables allergenic storage proteins to reach the small intestine and the circulation, thereby causing severe systemic allergic reactions.

## 3 - Clinical relevance

Sensitization to these three allergen families (2S albumins, 7S globulins and 11S globulins) is generally associated with a high risk to develop an allergic reaction upon ingestion. Allergic symptoms, which are elicited by IgE binding to those allergens, can reach from mild (e.g., oral itching) to life-threatening conditions such as anaphylaxis.

### 2S albumins

The extraordinary stable structure of 2S albumins is thought to contribute to its allergenicity and clinical relevance. The measurement of IgE specific to 2S albumins is mostly superior to measuring IgE against total extracts in edible seed allergy diagnosis. In peanut allergy, approximately 90% of patients sensitized to Ara h 2 suffer from a (severe) peanut allergy, while only 70% of patients with IgE to peanut extract are truly allergic.<sup>1540</sup> A comparable clinical importance was described for the other two peanut 2S albumins Ara h 6 and 7.<sup>1540</sup> Consistently, IgE binding to tree nut 2S albumins is also associated with clinically relevant sensitization: hazelnut Cor a 14, cashew nut Ana o 3, Brazil nut Ber e 1 and walnut Jug r 1.<sup>1541,1542</sup> However, it was shown that IgE to Jug r 1 is not significantly more relevant than measuring IgE to walnut extract in adults. 2S albumins are also predominantly recognized by IgE from patients with seed (e.g., sesame Ses i 1) or legume (e.g., soy Gly m 8) allergies.<sup>1543,1544</sup>

### 7S globulins

In tree nut allergy, 7S globulins are clinically less relevant compared with 2S albumins. For example, recognition of the hazelnut 7S globulin Cor a 11 by adults is extremely rare and its clinical relevance is not confirmed.<sup>44</sup> By contrast, the role of 7S globulins appears to be more important in legume allergies. In soybean allergy, 86% of patients with anaphylaxis were sensitized to Gly m 5, while only 33% of patients with subjective symptoms showed IgE to this 7S globulin.<sup>1543</sup> This clear association between sensitization to soybean Gly m 5 and severe symptoms was only confirmed in peadiatric populations. Moreover, 7S globulins were also characterized as major allergens (>50% *in vitro* recognition) in other legumes such as lupine, lentil, pea and chickpea. The lentil 7S globulin Len c 1.01 was recognized by 77% of lentil allergic patients, and 65% of IgE binding

to lentil extract was inhibited by preincubation with Len c 1.01.<sup>1545</sup> Despite their extensive recognition by IgE, the exact clinical relevance of 7S globulins in legume allergy needs still to be determined.

### 11S globulins

The clinical relevance of IgE binding to 11S globulins differs between foods and cannot be assigned to a specific group. While IgE binding to peanut Ara h 3 is less relevant than IgE to Ara h 2 (2S albumin), IgE binding to the hazelnut 11S globulin Cor a 9 has a comparable diagnostic value compared with IgE to the hazelnut 2S albumin Cor a 14<sup>43</sup>. IgE to both allergens is associated with severe symptoms in children. In adults, however, this clear association between IgE binding and allergy or objective symptoms was not found.<sup>1544</sup> In walnut allergy, only a small subpopulation of walnut allergic adults recognizes the walnut 11S globulin Jug r 4<sup>1541</sup> and in cashew nut allergic children, the diagnostic value of IgE to Ana o 2 is comparable to the diagnostic value of IgE to the 2S albumin Ana o 3.<sup>1541</sup> In almond allergy, IgE binding to the almond 11S globulin Pru du 6 shows a high sensitivity (83%) and specificity (78%), which is superior to measuring IgE against almond extract or other almond components.<sup>1547</sup> Strikingly, 2S albumins and 7S globulins—important allergens for other tree nuts—appear to be absent in almond kernels, which makes Pru du 6 an even more important component than 11S globulin in other tree nuts, legumes, and seeds. Although sesame seeds can cause severe allergic reactions, information regarding clinical relevance of IgE to 11S globulins is lacking. Nevertheless, it has been shown that Italian children allergic to sesame strongly recognized the basic subunit of the 11S globulin, which is only accessible after proteolytic cleavage of the acidic subunit.<sup>1548</sup>

#### Clinical relevance of 2S albumins

* Major allergens in peanut and tree nuts such as hazelnut, walnut and cashew nut

* Marker allergens for clinically relevant sensitizations to peanut, seeds and tree nuts

* High risk of cross-reactivity between walnut and pecan nut or cashew nut and pistachio

* IgE to these allergens may elicit severe symptoms

#### Clinical relevance of 7S globulins (vicilins)

* Major allergens of legumes such as soy, pea, lentil and lupine

* Marker allergens for clinically relevant sensitizations to legumes

* Risk of cross-reactivity between peanuts and peas or lupine

* Risk of cross-reactivity between peas and lentils
## Clinical relevance of 11S globulins (legumins)

* Major allergens in hazelnut and almond

* Marker allergens for clinically relevant sensitizations to almonds, hazelnuts and peanuts

* IgE to these allergens may elicit severe symptoms

## Age-related sensitization patterns

Sensitizations to seed storage proteins are biomarkers for a clinically relevant edible seed allergy in children. For example, IgE to Ara h 2 ≥ 5 kU/l can classify Dutch children as peanut allergic.¹⁵⁴⁹ Additionally, the absence of IgE to Ara h 2 can be used to rule out primary peanut allergy. Such advantageous use of IgE measurements to seed storage proteins (Cor a 9 and 14) was also shown for hazelnut allergy in children suffering from objective symptoms.⁴³ Furthermore, younger children are mostly sensitized to the walnut seed storage proteins Jug r 1 and 4, and their allergies have been shown to be more severe than walnut allergies in older children and adults.¹⁵⁵⁰ By contrast, sensitization patterns of adults, especially in birch-endemic regions (Northern and Central Europe), are more complex due to increased sensitization to the birch pollen PR-10 protein, Bet v 1, and its homologues. So far, this effect has been shown for peanut, hazelnut, and walnut allergies.⁴³,¹⁵⁴⁹,¹⁵⁵⁰ Hence, the absence of IgE binding to seed storage proteins does not necessarily exclude a peanut or tree nut allergy in adults. Nevertheless, PR-10 protein-related allergies are usually less severe.¹⁵⁴⁴

## Geographical differences

The sensitizing allergens in edible seed allergies differ geographically. nsLTP-related food allergies—due to sensitization to, e.g., peanut Ara h 9—are more common in the Mediterranean area, where peanut/tree nut allergies often go along with peach allergy.

However, it was shown that younger peanut allergic children in Spain were predominately sensitized to Ara h 2, while sensitization to Ara h 9 predominated over sensitization to Ara h 2 in older children.¹⁵⁵¹ Nevertheless, seed storage proteins such as Ara h 2 seem to be more important in the clinical setting in the Northern hemisphere.¹⁵⁴⁹

## Cross-reactivity

Although extensive *in vitro* co-sensitization has been shown between homologous seed storage proteins in legumes, tree nuts and seeds, only a small number of such co-sensitization results in clinically relevant co-allergies between tree nuts and peanut. Hence, *in vitro* cross-reactivity does not necessarily imply clinically relevant cross-reactivity. Nevertheless, strong clinically relevant cross-reactivity was described between walnut and pecan nut and between cashew nut and pistachio with walnut and cashew nut as respective primary sensitizers.¹²⁶⁰

Although extensive research has been performed to evaluate *in vitro* and *in vivo* cross-reactivity, several cross-reactivities and potential risks might not have been identified yet. In particular, information on molecular *in vitro* and *in vivo* cross-reactivity of IgE to sesame seed components is lacking despite their known clinical relevance. Moreover, the exact underlying allergens responsible for certain cross-reactions have often not yet been identified.

## Cross-reactivity between 2S albumins

Cross-reactivity between walnut and pecan nut as well as between cashew nut and pistachio can be potentially explained by cross-reactive IgE against the respective 2S albumins. Moreover, IgE to 2S albumins plays a role in clinically relevant cross-reactivity between walnut and hazelnut (Figure 176). Although *in vitro* cross-reactivity has also been shown between peanut Ara h 2 and 2S albumins from lupine (δ-conglutin), Brazil nut Ber e 1 and kiwi seeds, its clinical relevance is still a matter of debate. By contrast, negligible *in vitro* cross-reactivity was shown for IgE to hazelnut Cor a 14 and peanut Ara h 2.⁴³,¹⁵⁴⁴

<figure><img src="image_302.png" /><figcaption>**FIGURE 176** *In vitro* cross-reactivity between 2S albumins from tree nuts and legumes. Strong cross-reactivity has been shown for walnut and pecan nut, cashew and pistachio, and for hazelnut and walnut Cross-reactivity only confirmed *in vitro* and limited knowledge regarding clinical relevance is indicated with a grey arrow.</figcaption></figure>
## Clinical relevance of 11S globulins (legumins)

* Major allergens in hazelnut and almond

* Marker allergens for clinically relevant sensitizations to almonds, hazelnuts and peanuts

* IgE to these allergens may elicit severe symptoms

## Age-related sensitization patterns

Sensitizations to seed storage proteins are biomarkers for a clinically relevant edible seed allergy in children. For example, IgE to Ara h 2 ≥ 5 kU/l can classify Dutch children as peanut allergic.¹⁵⁴⁹ Additionally, the absence of IgE to Ara h 2 can be used to rule out primary peanut allergy. Such advantageous use of IgE measurements to seed storage proteins (Cor a 9 and 14) was also shown for hazelnut allergy in children suffering from objective symptoms.⁴³ Furthermore, younger children are mostly sensitized to the walnut seed storage proteins Jug r 1 and 4, and their allergies have been shown to be more severe than walnut allergies in older children and adults.¹⁵⁵⁰ By contrast, sensitization patterns of adults, especially in birch-endemic regions (Northern and Central Europe), are more complex due to increased sensitization to the birch pollen PR-10 protein, Bet v 1, and its homologues. So far, this effect has been shown for peanut, hazelnut, and walnut allergies.⁴³,¹⁵⁴⁹,¹⁵⁵⁰ Hence, the absence of IgE binding to seed storage proteins does not necessarily exclude a peanut or tree nut allergy in adults. Nevertheless, PR-10 protein-related allergies are usually less severe.¹⁵⁴⁴

## Geographical differences

The sensitizing allergens in edible seed allergies differ geographically. nsLTP-related food allergies—due to sensitization to, e.g., peanut Ara h 9—are more common in the Mediterranean area, where peanut/tree nut allergies often go along with peach allergy.

However, it was shown that younger peanut allergic children in Spain were predominately sensitized to Ara h 2, while sensitization to Ara h 9 predominated over sensitization to Ara h 2 in older children.¹⁵⁵¹ Nevertheless, seed storage proteins such as Ara h 2 seem to be more important in the clinical setting in the Northern hemisphere.¹⁵⁴⁹

## Cross-reactivity

Although extensive *in vitro* co-sensitization has been shown between homologous seed storage proteins in legumes, tree nuts and seeds, only a small number of such co-sensitization results in clinically relevant co-allergies between tree nuts and peanut. Hence, *in vitro* cross-reactivity does not necessarily imply clinically relevant cross-reactivity. Nevertheless, strong clinically relevant cross-reactivity was described between walnut and pecan nut and between cashew nut and pistachio with walnut and cashew nut as respective primary sensitizers.¹²⁶⁰

Although extensive research has been performed to evaluate *in vitro* and *in vivo* cross-reactivity, several cross-reactivities and potential risks might not have been identified yet. In particular, information on molecular *in vitro* and *in vivo* cross-reactivity of IgE to sesame seed components is lacking despite their known clinical relevance. Moreover, the exact underlying allergens responsible for certain cross-reactions have often not yet been identified.

## Cross-reactivity between 2S albumins

Cross-reactivity between walnut and pecan nut as well as between cashew nut and pistachio can be potentially explained by cross-reactive IgE against the respective 2S albumins. Moreover, IgE to 2S albumins plays a role in clinically relevant cross-reactivity between walnut and hazelnut (Figure 176). Although *in vitro* cross-reactivity has also been shown between peanut Ara h 2 and 2S albumins from lupine (δ-conglutin), Brazil nut Ber e 1 and kiwi seeds, its clinical relevance is still a matter of debate. By contrast, negligible *in vitro* cross-reactivity was shown for IgE to hazelnut Cor a 14 and peanut Ara h 2.⁴³,¹⁵⁴⁴

<figure><img src="image_309.png" /><figcaption>**FIGURE 176** *In vitro* cross-reactivity between 2S albumins from tree nuts and legumes. Strong cross-reactivity has been shown for walnut and pecan nut, cashew and pistachio, and for hazelnut and walnut Cross-reactivity only confirmed *in vitro* and limited knowledge regarding clinical relevance is indicated with a grey arrow.</figcaption></figure>
## Cross-reactivity between 7S globulins

Although evidence of (in vitro) cross-reactivity between tree nut 7S globulins is limited, walnut Jug r 6 displays remarkable in vitro cross-reactivity with 7S globulins from hazelnut (Cor a 11), pistachio (Pis v 3) and sesame seed (Ses i 3), which is in contrast to characteristics of IgE to the walnut 7S globulin Jug r 2. Additionally, cross-reactive IgE was also shown for 7S globulins from cashew nut and pistachio.¹⁵⁴⁴ By contrast, cross-reactive IgE to 7S globulins from legumes seem to play a greater role. Patients anaphylactic to pea often suffer from peanut allergy, which can be explained by cross-reactive IgE between pea Pis s 1 and peanut Ara h 1. Comparable cross-reactivity seems also to be present in peanut and lupine allergic patients with IgE to peanut Ara h 1 and lupine β-conglutin. Moreover, IgE binding to pea Pis s 1 was shown to be completely cross-reactive with lentil Len c 1 in vitro without confirmed clinical relevance so far (Figure 177).

## Cross-reactivity between 11S globulins

Limited knowledge is available regarding (in vitro) cross-reactivity between 11S globulins from tree nuts, legumes and seeds. Nevertheless, one study showed that IgE to the 11S globulins from hazelnut (Cor a 9) and walnut (Jug r 4) cross-reacted in hazelnut and walnut allergic patients.¹⁵⁵² Moreover, a certain degree of cross-reactivity was shown between hazelnut Cor a 9 and peanut Ara h 3, while Ara h 3 seems to play no role in cross-reactivity between peanut and lupine.¹⁵⁴³ Another cross-reactive 11S globulin seems to be the yellow mustard Sin a 2 with confirmed in vitro cross-reactivity to 11S globulins of peanut and the tree nuts almond, hazelnut, pistachio and walnut (Figure 178).¹²⁵⁹

## Cross-reactivity between nonhomologous seed storage proteins

Clinically relevant cross-reactivity has been commonly reported between members of the same family. Recently, several lines of evidence demonstrated that IgE cross-reactivity also exists between

members of different protein families of seed storage proteins and can also occur between seed storage proteins and some cow's milk allergens¹⁵⁵³It was demonstrated that IgE cross-reactive to peanut allergens Ara h 1 (vicilin), Ara h 3 (legumin) and Ara h 2 (2S albumin) comprised the major fraction of IgE specific to these allergens.¹⁵⁵⁴ The molecular basis of this cross-reactivity is the presence of highly similar amino acid sequence stretches present on surface-exposed loops. The analysis of antibodies produced by B cells from peanut allergic patients confirmed the presence of the IgE antibodies with high affinity and cross-reactivity to the three major peanut allergens.¹⁵⁵⁵

In addition, cross-reactivity was demonstrated between Ara h 2, almond legumin (Pru du 6), and walnut vicilins (Jug r 2) as well as between non homologous allergens from peanut and lupine.¹⁵⁵³ Future studies will have to address whether the occurrence of such cross-reactive antibodies accounts for the clinically observed co-reactivity to peanut, diverse tree nuts and other seeds.

Similarly, it was shown that IgE cross-reactivity also exists between non related bovine caseins, soy vicilin Gly m 5 and soy legumin Gly m 6 (summarized in¹⁵⁵³). This unexpected cross-reactivity could explain occasionally observed incidents of allergic reaction to a soy protein formula in cow's milk allergic patients, primarily not sensitized to soy.

# 4 – Clinical management

## History

The diagnosis of food allergy always starts with a careful history of the symptoms and the foods that cause the symptoms. Seed storage proteins are stable allergens towards digestion and heating and are therefore found to be associated with severe symptoms. This

<figure><img src="image_303.png" /><figcaption>FIGURE 177 *In vitro* cross-reactivity between 7S globulins from tree nuts, seeds and legumes. Strong cross-reactivity has been shown for cashew and pistachio (black arrow). Cross-reactivity only confirmed *in vitro* and limited knowledge regarding clinical relevance is indicated with a grey arrow.</figcaption></figure>
### Cross-reactivity between 7S globulins

Although evidence of (in vitro) cross-reactivity between tree nut 7S globulins is limited, walnut Jug r 6 displays remarkable in vitro cross-reactivity with 7S globulins from hazelnut (Cor a 11), pistachio (Pis v 3) and sesame seed (Ses i 3), which is in contrast to characteristics of IgE to the walnut 7S globulin Jug r 2. Additionally, cross-reactive IgE was also shown for 7S globulins from cashew nut and pistachio.¹⁵⁴⁴ By contrast, cross-reactive IgE to 7S globulins from legumes seem to play a greater role. Patients anaphylactic to pea often suffer from peanut allergy, which can be explained by cross-reactive IgE between pea Pis s 1 and peanut Ara h 1. Comparable cross-reactivity seems also to be present in peanut and lupine allergic patients with IgE to peanut Ara h 1 and lupine β-conglutin. Moreover, IgE binding to pea Pis s 1 was shown to be completely cross-reactive with lentil Len c 1 in vitro without confirmed clinical relevance so far (Figure 177).

### Cross-reactivity between 11S globulins

Limited knowledge is available regarding (in vitro) cross-reactivity between 11S globulins from tree nuts, legumes and seeds. Nevertheless, one study showed that IgE to the 11S globulins from hazelnut (Cor a 9) and walnut (Jug r 4) cross-reacted in hazelnut and walnut allergic patients.¹⁵⁵² Moreover, a certain degree of cross-reactivity was shown between hazelnut Cor a 9 and peanut Ara h 3, while Ara h 3 seems to play no role in cross-reactivity between peanut and lupine.¹⁵⁴³ Another cross-reactive 11S globulin seems to be the yellow mustard Sin a 2 with confirmed in vitro cross-reactivity to 11S globulins of peanut and the tree nuts almond, hazelnut, pistachio and walnut (Figure 178).¹²⁵⁹

### Cross-reactivity between nonhomologous seed storage proteins

Clinically relevant cross-reactivity has been commonly reported between members of the same family. Recently, several lines of evidence demonstrated that IgE cross-reactivity also exists between

members of different protein families of seed storage proteins and can also occur between seed storage proteins and some cow's milk allergens¹⁵⁵³It was demonstrated that IgE cross-reactive to peanut allergens Ara h 1 (vicilin), Ara h 3 (legumin) and Ara h 2 (2S albumin) comprised the major fraction of IgE specific to these allergens.¹⁵⁵⁴ The molecular basis of this cross-reactivity is the presence of highly similar amino acid sequence stretches present on surface-exposed loops. The analysis of antibodies produced by B cells from peanut allergic patients confirmed the presence of the IgE antibodies with high affinity and cross-reactivity to the three major peanut allergens.¹⁵⁵⁵

In addition, cross-reactivity was demonstrated between Ara h 2, almond legumin (Pru du 6), and walnut vicilins (Jug r 2) as well as between non homologous allergens from peanut and lupine.¹⁵⁵³ Future studies will have to address whether the occurrence of such cross-reactive antibodies accounts for the clinically observed co-reactivity to peanut, diverse tree nuts and other seeds.

Similarly, it was shown that IgE cross-reactivity also exists between non related bovine caseins, soy vicilin Gly m 5 and soy legumin Gly m 6 (summarized in¹⁵⁵³). This unexpected cross-reactivity could explain occasionally observed incidents of allergic reaction to a soy protein formula in cow's milk allergic patients, primarily not sensitized to soy.

## 4 – Clinical management

### History

The diagnosis of food allergy always starts with a careful history of the symptoms and the foods that cause the symptoms. Seed storage proteins are stable allergens towards digestion and heating and are therefore found to be associated with severe symptoms. This

<figure><img src="image_310.png" /><figcaption>FIGURE 177 *In vitro* cross-reactivity between 7S globulins from tree nuts, seeds and legumes. Strong cross-reactivity has been shown for cashew and pistachio (black arrow). Cross-reactivity only confirmed *in vitro* and limited knowledge regarding clinical relevance is indicated with a grey arrow.</figcaption></figure>
<figure><img src="image_304.png" /><figcaption>FIGURE 178 *In vitro* cross-reactivity between 11S globulins from tree nuts and seeds. Strong cross-reactivity has been shown for hazelnut and walnut (black arrow). Cross-reactivity only confirmed *in vitro* and limited knowledge regarding clinical relevance is indicated with a grey arrow.</figcaption></figure>

is in contrast to (secondary) sensitization to pollen-related PR10-proteins and profilins, which are labile proteins and are associated with mild to moderate (usually oropharyngeal) symptoms.

**Diagnostic value of IgE measurements to seed storage proteins**

Skin prick test or allergen specific IgE to whole extracts will confirm sensitization to the respective food. In order to define the primary allergen source (Figure 179), measurement of specific IgE to components can be performed. Not all seed storage proteins are available for routine testing. In the ImmunoCAP Specific IgE test single and multiplex assays (Thermo Fisher Scientific), the following seed storage proteins are currently available for routine in vitro diagnostics: peanut rAra h 1, rAra h 2, rAra h 3, rAra h 6, soy nGly m 5, nGly m 6, hazelnut nCor a 9, rCor a 14, cashew nut rAna o 2, rAna o 3, walnut rJug r 1, nJug r 2, Brazil nut rBer e 1, sesame nSes i 1 and buckwheat nFag e 2.

Several studies demonstrated that seed storage proteins play an important role in the diagnosis of peanut, tree nut and seed allergy. They can support the diagnosis of food allergy, but may also give an indication on the severity of the food allergy. For peanut, it was shown that IgE to Ara h 2 has a better diagnostic accuracy than IgE to peanut extract. Cut-off values for Ara h 2 were defined with positive predictive values (PPV) of up to 100% for diagnosis of peanut allergy.<sup>1540</sup> However, one should keep in mind that PPVs depend on the population, setting and geographic location. Measurement of IgE to Ara h 1, 3 and 6 appears less useful in the diagnosis of peanut allergy when IgE to Ara h 2 is already confirmed. However, one study showed that measurement of Ara h 6 could be useful, because co-sensitization to Ara h 2 and Ara h 6 appeared to be associated with severe reactions distinguishing severe allergy from mild symptoms. IgE to hazelnut Cor a 9 and 14 was found to be predictive for clinical reactivity to hazelnut and both were associated with severe reactions.<sup>1544</sup> IgE to walnut Jug r 1 was found to be superior to IgE

to walnut extract in the diagnosis of walnut allergy in children but appeared not to have an additional value in the diagnosis of walnut allergy in adults. IgE to cashew nut Ana o 3 was highly predictive for cashew nut allergy and discriminated between allergic and tolerant children better than cashew nut extract specific IgE; a cut off was found with 95% PPV for diagnosing cashew nut allergy.<sup>1544,1556</sup> The diagnostic value of Ses i 1 appeared to be better than IgE to sesame extract, to Ses i 2 and to 7S and 11S globulins of sesame.<sup>1264</sup> In conclusion, IgE to seed storage proteins has generally a high predictive value to diagnose an allergy to the respective food, has a higher diagnostic value than measurement of IgE to whole extracts, and for some foods, IgE to these allergens is associated with severe clinical reactions. Therefore, measurement of IgE to seed storage proteins is a useful tool in the diagnosis of peanut, tree nut and seed allergy.

**Clinical relevance of cross-sensitizations**

Cross-sensitization within and between legumes, tree nuts and seeds exists, but clinical relevance of these cross-sensitizations varies and may differ between different geographic regions. With regard to legumes, cross-sensitization of peanut allergic patients with other legumes occurs frequently but mostly does not demonstrate clinical allergy.<sup>1067,1538,1552</sup> Lupine may be the most clinically relevant peanut cross-reactive legume, showing sensitization rates of 34-88% and clinically manifested allergy in 4-88% of peanut allergic patients. In peanut allergic patients, sensitization to soybean occurs frequently (31-58%), while clinical allergy to soybean only ranged from 3% to 15%.

Conversely, one study showed that 88% of soybean allergic patients were also peanut allergic. Co-allergies to other legumes (e.g., lentil, chickpea, and pea) in peanut allergic patients have been reported but are less common, especially for beans. Between other legumes, a high degree of IgE cross-reactivity was demonstrated among lentils, chickpeas and peas. Food challenges confirmed that