Latin name: Juglans regia
Source material: rJug r 1 is a CCD-free recombinant protein.
Common names: English walnut, Persian walnut, Common walnut
ImmunoCAP allergen components:
rJug r 1 - f441
rJug r 3 - f442
Allergen: Jug r 1. (1, 2, 3, 4, 5)
Biological function: 2S Albumin, a storage protein.
Mw: 14 - 16 kDa
Walnut allergy is associated with the presence of IgE directed against the major seed-storage proteins in walnut, including the 11S globulin (legumin group) and 2S albumins, both of which represent major allergen classes in several plant seeds. Sensitisation to Jug r 1, a 2S albumin, indicates a primary walnut-nut allergy; and sensitisation to 2S albumins is known to be associated with systemic food reactions. (6, 7, 8)
Jug r 1, has been shown to be a major walnut-nut allergen, with 92% (61 subjects) of patients with walnut allergy displaying raised IgE of 0.35 kUA/L or higher to rJug r 1 and/or rJug r 3. An earlier study of a pool of sera from walnut-allergic patients reported that at least 75% of 20 subjects were positive to an immunodominant linear epitope of Jug r 1. (2) In a study evaluating rJug r 1 reported that rJug r 1-specific IgE was positive in serum of 12 of 16 (75%) walnut-allergic patients. (1)
Recombinant Jug r 1, like other recombinant nut allergens, can be used to assess patient reactivity (in particular to a very stable walnut allergen), characterise IgE binding epitopes, and study the effect of mutations on IgE binding. (9)
A positive walnut f256 with negative Jug r 1 and Jug r 3 results may be explained by sensitisation to:
- Other walnut storage proteins
- Cross-reactivity with pollen proteins such as profilin or PR-10 proteins. Due to the high degree of similarity, markers like Bet v 2 (profilin) and Bet v 1 (PR-10) may be used
- CCD (cross-reacting carbohydrate determinants)
See Walnut, f256
Jug r 1 is a 2S albumin, a storage protein. (3)
Storage proteins are classified into albumins (2S albumins or conglutins) and globulins, which in turn are divided into 7S globulins (vicilins) and 11S globulins (glycinins and legumins). (10)
2S albumins are grouped in the prolamin superfamily; other allergenic proteins included in this superfamily are the non-specific lipid transfer proteins, the alpha-amylase/trypsin inhibitors, and the prolamin storage proteins of cereals. (11)
2S albumins are a major group of seed-storage proteins, widely distributed in both mono- and dicotyledonous plants. They have been reported as major food allergens in the seeds of many plants. They are deposited in the protein bodies of developing seeds, and are used by the plant as a source of nutrients during subsequent germination and seedling growth. (12) 2S albumins have a high sulphur-containing amino-acid content. (13) 2S albumins are thought to sensitise directly via the gastrointestinal tract (GIT); and the high stability of their intrinsic protein structure despite the harsh conditions present in the GIT suggests that these proteins are able to cross the gut mucosal barrier, to sensitise the mucosal immune system and/or elicit an allergic response. (13) The walnut 2S albumin, Jug r 1, shares physicochemical properties similar to other 2S albumins, including cashew, which has been shown to strongly resist heat denaturation (roasting) and hydrolysis by digestive proteases (pepsin, trypsin), and thus displays a high allergenic potency. (14)
2S albumins have been previously identified in Brazil nut (Ber e 1), Oriental mustard (Bra j 1), rapeseed (Bra n 1), black mustard (Bra ni 2S albumin), turnip (Bra r 1), pecan nut (Car i 1), chickpea (Cic a 2S albumin), buckwheat (Fag e 10 kDa, Fag e 16 kDa), soya bean (Gly m 2S albumin), sunflower seed (Hel a 2S albumin), black walnut (Jug n 1), cashew nut (Ana o 3), almond (Pru du 2S albumin), Castor bean (Ric c 1, Ric c 3), sesame seed (Ses i 1, Ses i 2), yellow mustard (Sin a 1), Arabian cotton (CS-1A), and upland cotton (Mat5-D). (13)
2S albumins are also present in pistachio nut (Pis v 1), hazelnut (Cor a 14) and peanut. In peanut, Ara h 2, Ara h 6, and Ara h 7 are all 2S albumins.
Not all 2S albumins should be considered major allergens. In a recent study, none of the patients´ sera from 23 individuals allergic to soya bean was found to have IgE specifically against soya bean 2S albumins, suggesting that these proteins are not major allergens within the patient population analysed. (15)
Despite the skeleton of cysteine residues of 2S albumins being highly conserved, a relatively low amino-acid sequence homology, within and among plant species, has been demonstrated. (13) Overall, the degree of sequence homology of 2S allergens ranges from 14 to 40%, and does not reflect phylogenetic relationships, with the exception of 2S albumins belonging to the Brassicaceae family (e.g. Bra j 1, Bra n 1 and Sin a 1). (13)
Although 2S albumins have high structural homology, cross-reactivity seems to be uncommon in this protein family. (13)
For example, Ber e 1, a 2S albumin, a major allergen of Brazil nut, has been shown to have the following homology with 2S albumins from other tree and ground nuts: Ara h 2 (40%), Ara h 6 (48%), Ana o 3 (61%), Car i 1 (66%) and Jug r 1 (68%). (16) Yet in a study evaluating the ability of Brazil nut to react against other nuts (almond, hazelnut, pecan, cashew, walnut and peanut) or legumes (pea and chickpea), the cross-reactivity of antisera against all protein extracts tested was found to be negligible. (13, 17)
Cross-reactivity between 2S albumins has only been demonstrated in cases of high sequence homology, and seems to be linked to shared linear epitopes between allergens. For example, the 2S allergens from yellow mustard (Sin a 1) and rapeseed (Bra n 1) exhibit the highest sequence homology compared to other 2S proteins, and were recognised by IgE and IgG of sera of mustard-sensitive individuals, as well as by immunoglobulins present in the serum of a rapeseed-hypersensitive patient. (13, 18)
The results of another study suggest that either Ses i 1 or Ses i 2 cross-reacts with a 2S albumin from poppy seed. (19)
A few studies have reported instances in which cross-reactivity between 2S albumins may be clinically relevant.
A high degree of IgE-binding cross-reactivity has been demonstrated between the cashew allergens and the corresponding allergens Pis v 1, Pis v 2 and Pis v 3 of the closely-related pistachio nut. (20, 21) Less frequent cross-reactions also occur with other tree nuts, like almond and hazelnut. Almost no cross-reaction was reported to occur between cashew and peanut. In most cases this cross-reactivity is of no clinical significance, and would simply reflect some phylogenetic relatedness among tree nuts. Cashew and pistachio belong to the same family of Anacardiaceae. (15)
Although no cross-reactivity has been demonstrated between cashew and peanut 2S albumins (15), the peanut allergens Ara h 6 and Ara h 7 (also 2S albumins) show 59 and 35% amino acid sequence identity respectively to Ara h 2, a well-known major food allergen. (22) Inhibition assays have demonstrated significant cross-reactivity between Ara h 2 and Ara h 6 allergens. (23)
Cross-reactivity between 2S albumins of species from the Brassicaceae family, including oilseed rape, turnip rape and mustard, has recently been suggested. (24)
Serum from an individual allergic to mustard was demonstrated to display cross-reactivity between sunflower and mustard 2S albumins. (25)
Cross-reactivity between certain tree-nut pairs has been suggested, specifically cashew-pistachio and walnut-pecan (26); and the pecan 2S albumin Car I 1 has been shown to be cross-reactive with walnut. (27, 28) Importantly, cross-reactivity or sensitisation between walnut and other foods may occur as a result of the presence of other panallergens, e.g. walnut allergen Jug r 2 (a vicilin) (29), Jug r 3 (a lipid transfer protein) (30), etc.
The frequency of walnut allergy in children with IgE-mediated food allergy has been reported as 4.2%. (31) Walnut was most commonly reported to be allergenic by participants in the Food Allergy and Anaphylaxis Network (FAAN) Peanut and Tree Nut registry in the United States. A questionnaire of 5149 patients (mainly children) found that of all allergic reactions to tree nuts, 34% of reactions were to Walnut. (32)
Walnut-allergic patients have high risk of experiencing severe allergic reactions, (33, 34) attributable in particular to heat-labile allergens such as the 2S albumins, and incidents of hypersensitivity reaction to 2S food allergens have been described with increasing frequency. Although symptoms such as mild laryngeal irritation, urticaria and asthma have been reported, severe systemic symptoms including angioedema and anaphylaxis have been described. (13)
A study that expressed and purified recombinant (r) Jug r 1 and Jug r 3 and evaluated these using sera from 66 walnut-sensitised subjects (26 from Spain and Italy (SEU), and 40 from North/Central Europe (NEU) and North America (NA)) found that IgE binding to rJug r 1 and rJug r 3 correlated well with that of their purified natural counterparts, even though residual amounts of other storage proteins in the nJug r 3 preparation had to be accounted for in the comparison. 61 subjects (92%) were shown to have raised serum IgE to rJug r 1 and/or rJug r 3. Of the remaining five subjects, four were sensitised to CCD and one showed IgE binding to Cor a 9 and Cor a 11. Seven subjects (11%) displayed IgE antibody binding to rJug r 1 but not to rJug r 3; 29 (44%) to rJug r 3 but not to rJug r 1; and 25 (38%) to both. Twenty-three of the 66 subjects (35%) displayed CCD-reactive IgE. In 53 of the 66 subjects (80%), the sum of IgE to rJug r 1, rJug r 3 and CCD corresponded to >90% of the IgE to walnut extract. Of the remaining 13 subjects, 12 displayed significant IgE binding to Cor a 9 and/or Cor a 11, suggesting cross-reactive recognition of 11S globulin and vicilin in walnut. Subjects from NEU and NA mainly showed IgE binding to Jug r 1 and CCD, while individuals from SEU displayed dominant sensitisation to Jug r 3. (4) Therefore geographic location may influence the sensitisation profile of patients. Significant correlation in IgE binding between Jug r 3 and Pru p 3, the lipid protein from peach, was observed.
Recently, an Italian study conducted to explore the many unexpected cases of isolated serum positivity to nJug r 2 (vicilin) experienced in an allergy treatment centre evaluated the association of specific-IgE reactivity with clinical symptoms, and the relationship between serum IgE, nJug r 2 and cross-reactive carbohydrate determinants (CCDs). Sera from 320 consecutive allergic outpatients were evaluated utilising the ImmunoCAP ISAC which included native walnut nJug r 1, nJug r 2 (vicilin) and nJug r 3 (lipid transfer protein). Thirty-seven (12%) of 320 sera tested were positive to nJug r 2. Among them 3 (8%) and 8 (22%) respectively were shown to have raised specific IgE for nJug r 1 and nJug r 3 as well. Sera of 73% (27) showed isolated positivity for nJug r 2, and only those with raised serum IgE for nJug r 1 had symptoms related to walnut allergy. Sixty-eight per cent of nJug r 2-positive sera were shown to have concomitant raised serum IgE for nMUXF3; and further evaluation suggested that isolated serum IgE reactivity to nJug r 2 is frequently related to reactivity to CCD epitopes, and therefore lacks clinical significance. (35)
Similarly to cashew-nut-allergic patients sensitised to the 2S albumin Ana o 3, walnut-allergic patients sensitised to Jug r 1 should avoid raw as well as roasted/heated walnuts. (36)
See Walnut f256 for clinical information and further details on Walnut allergy.
Compiled by Dr Harris Steinman,
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