Summary of the sequences used in successive studies.
Abstract
Food allergies due to eating habits, pollution, and other factors are a growing problem in Western nations as well as developing countries. Symptoms of food allergies include changes in the respiratory and digestive systems. Legumes are a potential solution to the enormous demands for healthy, nutritive, and sustainable food. However, legumes also contain families of proteins that can cause food allergies. Some of these legumes include peanut, pea, chickpea, soy, and lupine. It has been shown that processing can alter the allergenicity of legumes since thermic and enzymatic resistance can affect these properties. Cross-reactivity (CR) is an allergy feature of some allergen proteins when the immune system recognizes part of the common share sequences (epitopes) in these allergic proteins. The research about molecular allergy includes comparisons of immunoglobulin E (IgE) and T-cell epitopes, assessment of three-dimensional structure and comparison of secondary structure elements, post-transduction modifications analysis by bioinformatic approach, and post-transduction modifications affecting epitopes properties may facilitate molecular tools to predict protein allergic behavior establishing prevention measurements that could promote the use of legumes and other seeds. This chapter provides an overview of the structural features of the main allergen proteins from legumes and their allergenic potential.
Keywords
- food allergy
- cross allergenicity
- legumes
- allergen proteins
- soy
- lupine
1. Introduction
Legumes are dicotyledon plants in the order Fabales and the family Fabacea. They produce fruit contained in pods and filled with seeds. In this chapter, we discuss three species of legumes in the genus
A food allergy is an immune system reaction that occurs after eating certain types of food. Symptoms are variable and can be caused even by small amounts of allergenic proteins, leading to hives, swollen airways, and digestive problems. Food allergies are a growing concern worldwide. This increase is suspected to be related with industrial production, pollution, additives, and consumption of trash food [1]. There are reports of children of East Asian or African ethnicity in Western nations having an increased risk of developing food allergies compared with Caucasian children. This suggests that adopting Westernized food habits could increase food allergies in African or Asian countries [2, 3].
The research about healthy, low-cost alternative products that can meet the enormous demands of a growing population involve legumes [4]. Legume crops represent a sustainability solution, serving as a fundamental source of high-quality alternative protein, reducing the emission of greenhouse gases, allowing the sequestration of carbon in soils, saving the CO2 print thanks to the nitrogen fertilizer, it free high-quality organic matter that facilitate water retention and perform the soil nutrients circulation among others uses [5]. Despite their advantages, legumes contain proteins that can potentially cause food allergies. Several allergens from different legumes have been identified and characterized as proteins with potential allergic effects. These include lentil, pea, chickpea, soy, peanut, and lupine [6].
Clinically, the absence of sensibilization phase is a reliable indicator of the tolerance to an allergen. In this context, the presence of sensitization to a specific allergen protein has to be proven [7] both, the specific reactivity to a particular allergen protein and the cross-reactivity to other related allergens. The most frequent cross-reactivity process described clinically is that between lupin and peanut [8].
In Spain, consumption of legumes is common because they are an important part of the Mediterranean diet. It is estimated that consumption of legumes in Spain is 4.8 kg per year, with a greater percentage of children eating them as compared to adults. Legume consumption in Spain is greater in girls than in boys [9]. One study in Spain showed that food allergies were detected in 20.8% of children and 14% of adults. In the overall Spanish population, legumes were responsible of the 14.3% of the food allergies [10]. Another study of Spain’s pediatric population found that 10% of children suffered from food allergies caused by lentil and 6.7% of children suffered from food allergies caused by peanuts. Lentil was found to be the most allergenic, causing 78% of reactions, followed by chickpea (72%) and peanut (33%) [9].
In Europe, legumes are the fifth-leading cause of food allergies [11]. A meta-analysis of studies conducted in Europe between January 2000 and September 2012 found that the percentage of the population with symptoms of food allergies plus specific immunoglobulin E (IgE) positivity activation to at least one food allergen was 3%–4.6% in children and 2.2%–2.66% in adults [12]. The same study concluded that the frequency of food allergy is greatest in northwestern European countries compared to southern European countries, which had the lowest prevalence. Some factors related to food allergies include environmental, genetic, and epigenetic factors that could suggest differences between global populations [13].
The general prevalence of food allergies is not clearly defined due to the lack of reliable data and the highly variable allergy patterns in different parts of the world. A selection of mixed developed country data (Allergy, Asthma & Immunology Research 2018) found that some allergies, like those to peanut, demonstrate heritability in Caucasian populations; skin immune responses shows differences between Asians and Caucasians. These types of studies have not yet been conducted in non-White populations, however, there exists some interest data showing that Black South African children present a significantly lower prevalence of peanut allergy compared to children of mixed-race origin (Black and Caucasian) by unknown factors [13].
One interesting fact about cross-reactivity is that it could be caused by proteins that come from species that are taxonomically distant. Examples of these antigens are panallergens, which are proteins conserved by evolution due to their important defense, structural, and storage functions [7]. If a person has an allergy to cow milk proteins, they are also probably allergic to goat milk proteins [14]. In the case of legumes, cross-reactivity to more than one legume is often found in children [9].
Overall, allergic features of allergen proteins could be attenuated by thermic proteolytic denaturalization due to the modification of the quaternary protein structure where superficial epitopes of these proteins’ antigenic regions can still develop some allergenicity reactions. Despite this, there are studies that also show resistance to thermic, chemical, and proteolytic denaturalization, with is a common characteristic in legumes [15]. Some examples of resistance to denaturalization include allergen proteins like Cupins, very stable storage proteins that include legumins (11 S) and vicilins (7 S), both containing two common β-barrel structures in their globular domain. These appear to be a relevant stable structural motif, confirming resistance to denaturation and proteolysis [16]. Lipid transfer proteins (LTPs) have resistance to pepsin and to chemical digestion [17]; PR-proteins have thermostable structure [10] allowing them staying unalterable at physiological temperature. This stability plays an important role in allowing allergen active protein fragments to pass to the gastrointestinal tract, causing a food allergy.
There is a large public database of allergenic legume proteins with several isoforms. The commonly shared partial epitopes and their conservation in the same family of proteins in different species could be helpful in designing possible strategies to prevent cross-reactivity.
The aim of this work is to carry out an exhaustive molecular and structural analysis of the most common allergenic legume proteins through bioinformatic approaches.
2. Materials and methods
2.1 Search of legume proteins sequences
We used the Allergome and UniProt databases to search for allergenic legume proteins for this study. The proteins chosen are characterized by having complete sequences and being in mature form. The search was carried out on the available species of lentil, pea, chickpea, soybean, and lupine (Table 1A-E).
Species | Protein name | Protein type | UniProtKB |
---|---|---|---|
Gly m 5 | Profilin | C6T9L1 (C6T9L1_SOYBN) | |
Gly m 5.0301 | Profilin | P25974 (GLCB1_SOYBN | |
Gly m 8 | 2 s albumin | C6SYA7 (C6SYA7_SOYBN) | |
Gly m 8.0101 | 2 s albumin | P19594 (2SS_SOYBN) | |
Lup a 1 | 7 s vicilin | Q53HY0 (CONB1_LUPAL) | |
Lup a alpha conglutin | 11 s conglutin | Q53I54 (Q53I54_LUPAL) | |
Lup a delta conglutin | 2 s albumin | Q333K7 (Q333K7_LUPAL) | |
Lup a gamma conglutin | Aspartic protease | Q9FEX1 (CONG2_LUPAL) | |
Lup a 4 | PR-protein | O24010 (O24010_LUPAL) | |
Lup an 1 | 7 s vicilin | B0YJF8 (B0YJF8_LUPAN) | |
Lup an 3 | LTP | A0A1J7GK90 (A0A1J7GK90_LUPAN) | |
Lup an 3.0101 | LTP | A0A4P1RWD8 (A0A4P1RWD8_LUPAN) | |
Lup an alpha conglutin | 11 s globulin | F5B8V6 (CONA1_LUPAN) | |
Lup an delta conglutin | 2 s albumin | F5B8W8 (COND1_LUPAN) | |
Lup an gamma conglutin | Aspartic protease | Q42369 (CONG1_LUPAN) | |
Lup l 4 | PR- protein | P52778 (L18A_LUPLU) | |
Pis s 2 | 7 s vicilin | P13915 (CVCA_PEA) | |
Pis s 3 | LTP | A0A158V755 (NLTP2_PEA) | |
Pis s 6 | PR-protein | P13239 (DRR1_PEA) | |
Pis s agglutinin | Agglutinin | B5A8N6 (B5A8N6_PEA) | |
Pis s albumin | Albumin | P08688 (ALB2_PEA) | |
Cic a 1 | 7 s vicilin | Q304D4 (Q304D4_CICAR) | |
Cic a 3 | LTP | O23758 (NLTP_CICAR) | |
Cic a 4 | PR-protein | Q39450 (Q39450_CICAR) | |
Cic a 6 | 11 s globulin | Q9SMJ4 (LEG_CICAR) | |
Ara h 1 | 7 s vicilin | B3IXL2 (B3IXL2_ARAHY) | |
Ara h 1.0101 | 7 s vicilin | P43238 (ALL12_ARAHY) | |
Ara h 2.0101 | 2 s albumin | Q6PSU2–2 (CONG7_ARAHY) | |
Ara h 2.0201 | 2 s albumin | Q6PSU2–3 (CONG7_ARAHY) | |
Ara h 3 | 11 s globulin | A1DZF0 (A1DZF0_ARAHY) | |
Ara h 3.0201 | 11 s globulin | Q9SQH7 (Q9SQH7_ARAHY) | |
Ara h agglutinin | Agglutinin | P02872 (LECG_ARAHY) | |
Ara h 5 | Profilin | D3K177 (D3K177_ARAHY) | |
Ara h 5.0101 | Profilin | Q9SQI9 (PROF_ARAHY) | |
Ara h 6 | 2 s albumin | A1DZE9 (A1DZE9_ARAHY) | |
Ara h 6.0101 | 2S albumin | Q647G9 (CONG_ARAHY) | |
Ara h 7.0101 | 2 s albumin | Q9SQH1 (Q9SQH1_ARAHY9 | |
Ara h 7.0201 | 2 s albumin | B4XID4 (B4XID4_ARAHY) | |
Ara h 7.0301 | 2 s albumin | Q647G8 (Q647G8_ARAHY) | |
Ara h 8 | PR- 10 protein | B1PYZ4 (B1PYZ4_ARAHY) | |
Ara h 8.0101 | PR-10 protein | Q6VT83 (Q6VT83_ARAHY) | |
Ara h 8.0201 | PR- 10 protein | B0YIU5 (B0YIU5_ARAHY) | |
Ara h 9.0101 | 9 k-LPT | B6CEX8 (B6CEX8_ARAHY) | |
Ara h 10.0101 | 16kD protein | Q647G5 (OL101_ARAHY) | |
Ara h 11.0101 | 14KD oleosin | Q45W87 (OL111_ARAHY) | |
Ara h 11.0102 | 14kD oleosin | Q45W86 (OL112_ARAHY) | |
Ara h 13.0102 | Defensine | C0HJZ1 (DEF3_ARAHY) | |
Ara h 14.0101 | 17.5kD oleosin | Q9AXI1 (OL141_ARAHY) | |
Ara h 14.0102 | 17kD oleosin | Q9AXI0 (OL142_ARAHY) | |
Ara h 14.0103 | 17kD oleosin | Q6J1J8 (OL143_ARAHY) | |
Ara h 15.0101 | 17kD oleosin | Q647G3 (OLE15_ARAHY) | |
Ara h 16 | 7 k LPT | A0A445DA28 (A0A445DA28_ARAHY) | |
Ara h 17 | 11 k LTP | A0A445AL51 (A0A445AL51_ARAHY) | |
Ara d 2 | 2 s albumin | A5Z1Q8 (A5Z1Q8_ARADU) | |
Ara d 6 | 2 s albumin | A5Z1Q5 (A5Z1Q5_ARADU) | |
Ara i 2 | 2 s albumin | A5Z1Q9 (A5Z1Q9_ARAIP) | |
Ara i 6 | 2 s albumin | A5Z1Q6 (A5Z1Q6_ARAIP) | |
Len c 3 | LTP | A0AT28 (NLTP1_LENCU) | |
Len c 3.0101 | LTP | A0AT29 (NLTP2_LENCU) | |
Len c agglutinin | Agglutinin | P02870 (LEC_LENCU) |
2.2 Alignment of sequences
The complete and mature sequences of lentil (
2.3 Functional domain analysis
We used the program Pfam v34.0 (http://pfam.xfam.org/) to identify the possible domains present in the isoforms of legume proteins.
2.4 Post-translational modification site prediction
We used the MusiteDeep deep learning framework (https://github.com/duolinwang/MusiteDeep_web) to search for the presence of possible post-translational modifications and identify how they affect the potential allergenicity of the study proteins [18]. The prediction models used are phosphorylation (Y, S, T); N-linked glycosylation (N); O-linked glycosylation (S, T); ubiquitination; N6-acetyllysine (K); Methylarginine (R); Methyllysine (K); Hydroxyproline (P) and Hydroxylysine (K) with a threshold value of 0.8.
S-nitrosylations and T-nitrations were also studied via the iSNO-AAPair tool (Y. Xu et al., 2013), which was used to predict cysteine S-nitrosylation sites (http://app.aporc.org/iSNO-AAPair) with a threshold value greater than 0.8. The GPS-YNO2 tool (Liu et al., 2011) was used to predict tyrosine nitration sites (http://yno2.biocuckoo.org).
2.5 Secondary structure assessment
Secondary structure was assessed using PSIPRED (http://bioinf.cs.ucl.ac.uk/psipred/). Sequence alignment was performed with CLUSTALW (https://www.genome.jp/tools-bin/clustalw), which was visualized with the BioEdit program, and in which the consensus secondary structure was annotated.
2.6 Modeling of three-dimensional structure
The three-dimensional structures of olive ALDH proteins were modeled using the Phyre2 web program (http://www.sbg.bio.ic.ac.uk/phyre2), which is based on Markov algorithms to generate alignments of the problem protein sequences with proteins with experimentally obtained protein crystallographic models (PDB).
2.7 Identification of IgE-binding epitopes
We used the AlgPred server (www.imtech.res.in/raghava/algpred/submission.html), which creates arrays using sequences from known allergens, to identify IgE-binding epitopes and to determine potential allergenicity of proteins based on of their amino acid and dipeptide composition.
2.8 Identification of T cell binding epitopes
We used the ProPred program (Singh et al., 2011) (http://webs.iiitd.edu.in/raghava/propred/) to analyze the protein sequences of legumes in the study. The analysis was performed with a 2% threshold for the most common human HLA-DR alleles among the Caucasian population: [DRB1*0101 (DR1), DRB1*0301 (DR3), DRB1*0401 (DR4), DRB1*0701 (DR7), DRB1*0801 (DR8), DRB1*1101 (DR5), and DRB1*1501 (DR2)].
3. Results and discussion
3.1 Sequences obtained from the Allergome database
We used the Allergome database to retrieve the available sequences of complete proteins of legumes, following the link to UniProt. The legumes included in this study are lentil, lupin, pea, chickpea, and peanut. Only two major allergens (
The reference proteins, soybean major allergens
3.2 Alignment of allergen protein sequences
Sequence alignments were performed to compare the common and differential features between allergen proteins and legumes. Overall, and according to the CODEX Alimentarius Commission in 2003, only proteins with a percentage of identity greater than 50% by local alignment (BLAST) are at risk of allergy or cross-reactivity [22]. Therefore, results obtained from protein–protein alignment beforehand do not show values high enough to make a prediction of possible cross-reactivity between soybean proteins and the rest of the legumes (Table 2).
Arachis duranensis | |||||
---|---|---|---|---|---|
Protein name | Ara d 2 | Ara d 6 | Len c 3 | Len c 3.0101 | Len c agglutinin |
Gly m 5 | 8428 | 6067 | 5239 | 5157 | 11.803 |
Gly m 5.0301 | 9009 | 5909 | 4556 | 5817 | 11.349 |
Gly m 8 | 32.738 | 29.94 | 9942 | 10.465 | 9375 |
Gly m 8.0101 | 33.333 | 29.94 | 9942 | 9884 | 9278 |
Protein name | Lup an 1 | Lup an 10,101 | Lup an 3 | Lup an 30,101 | Lup an alpha conglutin | Lup an delta conglutin | Lup an gamma conglutin |
---|---|---|---|---|---|---|---|
Gly m 5 | 24.463 | 39.739 | 5843 | 4 | 17.304 | 8444 | 15.028 |
Gly m 5.0301 | 24.463 | 39.739 | 4.31 | 4 | 17.304 | 8444 | 14.657 |
Gly m 8 | 8114 | 6209 | 11.561 | 11.243 | 6616 | 35.62 | 5298 |
Gly m 8.0101 | 7877 | 6209 | 12.069 | 11.765 | 6616 | 36.25 | 5066 |
Arachis ipaensis | ||||||
---|---|---|---|---|---|---|
Protein name | Cic a 1 | Cic a 3 | Cic a 4 | Cic a 6 | Ara i 2 | Ara i 6 |
Gly m 5 | 36.759 | 6378 | 8 | 13.587 | 8753 | 6292 |
Gly m 5.0301 | 37.575 | 6378 | 7556 | 8 | 8.85 | 6136 |
Gly m 8 | 7143 | 10.526 | 5021 | 7585 | 31.461 | 29.94 |
Gly m 8.0101 | 6513 | 10.526 | 5021 | 7585 | 31.461 | 30.539 |
Protein name | Lup a 1 | Lup a 4 | Lup a alpha conglutin | Lup a delta conglutin | Lup a gamma conglutin | Lup l 4 |
---|---|---|---|---|---|---|
Gly m 5 | 48.417 | 6.25 | 16.637 | 8036 | 13.645 | 6798 |
Gly m 5.0301 | 48.717 | 6.25 | 16.637 | 8259 | 14.098 | 7456 |
Gly m 8 | 5151 | 10.698 | 6501 | 35.625 | 4425 | 13.115 |
Gly m 8.0101 | 5009 | 10 | 6.18 | 36.25 | 4435 | 13.115 |
Protein name | Pis s 2 | Pis s 3 | Pis s 3.0101 | Pis s 6 | Pis S agglutin | Pis s albumin |
---|---|---|---|---|---|---|
Gly m 5 | 41.638 | 5467 | 5882 | 6798 | 9362 | 6798 |
Gly m 5.0301 | 41.638 | 5145 | 5369 | 6798 | 11.429 | 10.444 |
Gly m 8 | 5759 | 11.765 | 10.588 | 13.402 | 11.273 | 8.98 |
Gly m 8.0101 | 5.41 | 11.176 | 10.588 | 13.402 | 10.204 | 9388 |
Protein name | Ara h 1 | Ara h 1.0101 | Ara h 2 | Ara h 2.0101 | Ara h 2.0201 | Ara h 2.0202 | Ara h 3 | Ara h 3.0201 |
---|---|---|---|---|---|---|---|---|
Gly m 5 | 36.585 | 35.726 | 8753 | 8811 | 8874 | 9031 | 15.412 | 14.685 |
Gly m 5.0301 | 36.748 | 35.885 | 8.85 | 9009 | 9292 | 9234 | 15.762 | 14.86 |
Gly m 8 | 5769 | 8307 | 31.461 | 32.738 | 34.818 | 33.133 | 5.41 | 6015 |
Gly m 8.0101 | 7329 | 7668 | 31.461 | 33.333 | 31.818 | 33.735 | 4.57 | 5636 |
Protein name | Ara h agglutinin | Ara h 5 | Ara h 5.0101 | Ara h 6 | Ara h 6.0101 | Ara h 7.0101 | Ara h 7.0102 | Ara h 7.0301 |
---|---|---|---|---|---|---|---|---|
Gly m 5 | 13.816 | 6349 | 6136 | 6606 | 6292 | 7982 | 7062 | 6292 |
Gly m 5.0301 | 13.717 | 4904 | 5.33 | 6951 | 6136 | 8296 | 6834 | 9131 |
Gly m 8 | 8571 | 10.734 | 6015 | 28.144 | 29.94 | 23.497 | 30.337 | 22,286 |
Gly m 8.0101 | 8571 | 10.674 | 9091 | 28.144 | 30.539 | 23.497 | 30.899 | 22.857 |
Protein name | Ara h 8 | Ara h 8.0101 | Ara h 8.0201 | Ara h 9.0101 | Ara h 10.0101 | Ara h 11.0101 | Ara h 11.0102 | Ara h 13.0102 |
---|---|---|---|---|---|---|---|---|
Gly m 5 | 6181 | 7761 | 6.92 | 3596 | 7761 | 6982 | 7207 | 3139 |
Gly m 5.0301 | 6935 | 7539 | 6.92 | 3.82 | 6828 | 6982 | 7207 | 3139 |
Gly m 8 | 11.429 | 10.233 | 11.64 | 10.405 | 6478 | 6.14 | 6.14 | 9877 |
Gly m 8.0101 | 11.792 | 11.64 | 11.64 | 10.405 | 6883 | 6.14 | 6.14 | 9259 |
Protein name | Ara h 14.0101 | Ara h 14.0102 | Ara h 14.0103 | Ara h 15.0101 | Ara h 16 | Ara h 17 |
---|---|---|---|---|---|---|
Gly m 5 | 8744 | 7848 | 8296 | 7221 | 4698 | 3905 |
Gly m 5.0301 | 8744 | 7848 | 8296 | 7221 | 4698 | 4121 |
Gly m 8 | 5785 | 5859 | 5785 | 5.6 | 11.111 | 11.243 |
Gly m 8.0101 | 5372 | 5859 | 5785 | 5.6 | 11.31 | 11.243 |
The highest percentage of identity was the result of the alignment between the
0,599 (over all) | 5587 (Cic a 6) 3646 (Pis s albumin) | ||
0,468 (over all) | 3076 (Ara h 5.0101) | ||
Max identity values obtained by sequences alignment | |||
48,717 (over all) | |||
Gly m 5.0301 vs. Lup a 1 |
The multiple alignment analysis between
These data show that the percentage of identity of allergens must be kept in mind to compare allergens and to predict potential allergenicity and cross-reactivity, since not only do sequential epitopes have to be taken into account for that purpose, but also 3D and specific structural conformations of particular allergen proteins must be considered.
Using the information obtained by alignment, some of the proteins in the comparative analysis with soybean could be of interest at the molecular allergy level, such as Lup a delta conglutin and Lup an delta conglutin with percentages of identity with
Considering the identity percentages previously indicated, the Ara h 2 identity percentage of 31% at
Interestingly, the percentage of alignment identity between soybean isoforms was low, with values less than 1%, specifically, in the alignment of soybean major allergen
The existence of differences between isoforms of other legume species of the same allergen protein family could open the way for new studies finding significant differences in multiple cross-reactivity candidacy. For example, such as the case of
3.3 Post-translational modification analysis
Post-translational modifications affecting the allergen protein sequences have been defined and involved in processes like alcohol or tiol addition (glycosidations), methyl groups (methylations), phosphates (phosphorylations), carboxyl groups (carboxylations), nitro groups (T-nitrations), or nitroxil groups (S- nitrosylations).
These types of modifications may induce rearrangements in structure, which could indirectly affect lineal and/or conformational epitopes’ influence pm molecular allergy, limiting or favoring immunological recognition as well as generating antigenic diversity [25]. It is interesting to analyze location of where these modifications may occur and the type of modification together with the influence of these modifications in the 2D structural elements.
Phosphorylation is considered a factor of change of molecular pH dynamics [26], generating important alterations in the biophysics of the protein [27]. It has been observed sites of phosphorylation in most of the proteins examined:
Allergen | Post-translational modifications | |||||
---|---|---|---|---|---|---|
Phosphorylation | Glycosylation | Pyrrolidone carboxylic acid | Methylation | Nitration | Nitrosylation | |
Gly m 5 | 232; 234; 235 | 351 | — | — | 158;172 | — |
Gly m 5.0301 | 232; 234; 235 | 351 | — | — | 158; 172 | — |
Gly m 8 | 155; 156 | 120 | — | — | — | 14 |
Gly m 8.0101 | 155; 156 | 120 | 25 | — | — | 14 |
Lup a 1 | 71; 79; 104 | 444 | — | — | 269;316 | — |
Lup a 4 | — | 13; 82 | — | — | 157; 269; 316 | — |
Lup a alpha conglutin | 347 | 403 | 29 | 102 | 199; 448; 497 | 36; 334 |
Lup a delta conglutin | 75;76 | 73; 108 | 27 | — | — | — |
Lup a gama conglutin | — | 133 | 28 | — | 261 | — |
Lup an 1 | 80;82;85 | 152; 434 | 126; 158 | — | 340 | — |
Lup an 1.0101 | 80;82;85; 469; 488 | 434; 519 | 126; 158 | — | 340; 488 | — |
Lup an 3 | — | — | 23 | — | — | 13; 27 |
Lup an 3.0101 | — | — | — | — | 104 | 28; 112 |
Lup an alpha conglutin | 247; 259; 341 | 397; 439 | 24 | 97 | 84; 442; 491 | 31 |
Lup an delta conglutin | 76; 77; 80;83 | — | — | — | — | 42 |
Lup an gamma conglutin | 357 | 130 | — | — | 259 | 350; 391; 440 |
Lup l 4 | 112 | 78; 82 | — | — | 100; 156 | — |
Cic a 6 | 139; 195; 207; 225; 271 | 1; 220 | — | — | 443 | 64; 107 |
Ara h 5.0101 | — | — | — | — | 6; 125 | 115 |
Methylations are quite less abundant modifications. It is observed that their deficiency generates serious alterations in the functioning of proteins, thus having important implications on their three-dimensional structuring as carboxylation [29]. Only two methylation sites were found: one on
Nitrosylation and nitrations generate strong covalent bonds in the protein structure [30, 31]. Nitrations were found on
Post-translational modifications on T-cell epitopes have been found in
Allergen name | Post-translational Modifications | ||||
---|---|---|---|---|---|
Phosphorylation | Glycosylation | Methylation | Nitration | Nitrosylation | |
Gly m 5.0301 | FVVNATSNL(351) | YLQGFDHNI(172) | |||
Lup a alpha conglutin | FGPLRRCN (199) | ||||
YVLNGSAWF (448) | |||||
YVAFKTNDI (497) | |||||
Lup a delta conglutin | LVAALVLVV (76) | ||||
Lup an 3 | VLICMVVVS(13) | ||||
Lup an 3.0101 | YKISTSTNC (104) | YKISTSTNC(112) | |||
Lup an delta conglutin | LVVHTSASR (76) | ||||
Cic a 6 | FGMVFPGCV(107) | ||||
Lup a alpha conglutin | IETWNPNNQEFECAG (102) |
The direct implications of these post-translational modifications may be directly linked to the effects on the variation of the structure of these regions, generating differential epitopes recognition and consequently the allergen response.
Analyzing the location and type of modifications could help to elucidate the relationship of protein structure epitope distribution to the allergen potential of the protein, however, it will not be confirmed whether the different modifications would accentuate or lessen the allergenic impact until a clinical review of the process is carried out. The possibility of inducing post-translational modifications on plant proteins as a therapeutic tool is being examined [27].
3.4 Secondary structure analysis
The combined analysis of secondary structure with multiple alignments allows a direct sequence–structure–functional comparation between different allergen proteins. An interesting analysis has been made to identify the areas of allergens with shared mutual domains as part of structural domains with important implications for cross-reactivity potential.
The
The three allergen proteins include Cupin superfamily domains with a wide variety of representative enzymes, but notably contains the non-enzymatic seed storage proteins [32]. Functional domains that could be candidates to potentially undergo post-translational modifications for
Protein | Functional domain | Alignment amino acid range |
---|---|---|
Lup a 1 | Cupin_1.1 | 332–486 |
Cupin_1 | 137–227 | |
Gly m 5 | Cupin_1 | 240–389 |
Cupin_2 | 86–144 | |
Gly m 5.0301 | Cupin_1 | 240–393 |
Cupin_2 | 86–144 | |
Lup a gamma conglutin | Xylanase inhibitor C-terminal | 271–428 |
Xylanase inhibitor N-terminal | 66–240 | |
Lup an gamma conglutin | Xylanase inhibitor C-terminal | 269–429 |
Xylanase inhibitor N-terminal | 63–237 |
Regarding the predictions of post-translational modifications of these proteins relevant to 2D structural domains, it was found that
3.5 Three-dimensional structure analysis
Analysis of three-dimensional structure of proteins (Figure 4) provides insight into their sequence conformation and epitope arrangement. It also helps to determine the consequences of possible structural changes occurring between protein isoforms with minimal or large number of changes (Table 2) in their sequences [33].
Post-translational modifications over protein domains also may generate changes in their three-dimensional structure, affecting exposure epitopes and increasing or decreasing their allergenic potential.
Some candidates to examine the three-dimensional structure are
The structural differences observed in the consensus structure between the three structures indicate that in
Tridimensional structure comparison between Lup a gamma conglutin and Lup an gamma conglutin result on two principal differences observed between both conglutins, which is an α-helix in the gamma conglutin of
The 3D analysis was useful to determine other cases of interest previously mentioned, such as
3.6 Identification and analysis of T-cell binding epitopes
An epitope is the portion of a macromolecule that is recognized by the immune system, specifically the sequence to which antibodies, B-cell receptors or T-cell receptors, can bind to initiate an immune response. Analysis of the epitopes shared for specific allergen proteins could be relevant to identify potential cross-reactivity. Presence of common T-cell epitopes among different legume species may support cross-reactivity processes; the greater the probability of occurrence, the larger the number of common epitopes.
The data obtained from the analysis of T-cell epitopes allows us to know which epitopes are shared among allergen proteins in the different legume species and to examine possible cases of cross-reactivity. Thus, in the case of soybean
Allergen name | T-cell epitopes | |
---|---|---|
LRSSNSFQT | LRSRNPIYS | |
Gly m 5 | 288–296 | |
Gly m 5.0301 | 36–44 | 242–250 |
Ara h 9.0101 | 21–29 | |
Cic a 1 | 250–258 |
Allergen name | T-cell epitopes | |||||
---|---|---|---|---|---|---|
LVLVLGIVF | MMACNGLTI | YVLHKIEEI | FVLSSSQNS | LVAALVLVV | LVVHTSASR | |
Lup a 1 | 11–19 | |||||
Lup a 4 | 66–75 | |||||
Lup a alpha conglutin | ||||||
Lup a delta conglutin | 67–75 | 73–81 | ||||
Lup a gamma conglutin | 16–24 | 63–71 | ||||
Lup an delta conglutin | 62–70 | 69–77 | ||||
Lup an gamma conglutin | 13–21 | 77% (FVSSSSQD) 69–77 | ||||
Ara d 6 | 13–20 | |||||
Ara h 8.0102 | 77% (YVLHKIDAI) 66–74 | |||||
Cic a 4 | 88% (YVLHKIEAI) 123–132 |
Allergen name | T-cell epitopes | |||||
---|---|---|---|---|---|---|
FQRLNALEP | LRCAGVALS | IRVLERFDQ | FGPLRRCN | VVLNGRATITI | IVRNIKGKN | |
Lup a 1 | 133–138 | 177–190 | ||||
Lup a 4 | ||||||
Lup a alpha conglutin | 83–91 | 112–120 | 192–200 | 279–287 | ||
Lup an 1 | 80% (IRVLERFNQ)204–212 | 248–259 | ||||
Lup an 1.0101 | 80% (IRVLERFNQ)204–213 | 248–260 | ||||
Lup an alpha conglutin | 86–94 | 115–123 | 286–294 | |||
Lup an delta conglutin | 191–198 | |||||
Ara h 1 | 80% (IRVLQRFDQ) 204–212 | |||||
Ara h 1.0101 | 80% (IRVLQRFDQ) 193–201 |
Allergen name | T-cell epitopes | |||
---|---|---|---|---|
IVRVSREQI | IRVNKHM | VRRVRRPH | WRISDEN | |
Lup a 1 | 302–310 | |||
Lup a alpha conglutin | 355–363 | |||
Lup a gamma conglutin | 318–326 | 412–420 | ||
Lup an 1 | 77% (IVRVSKKQI)373–381 | |||
Lup an 1.0101 | 77% (IVRVSKKQI) 373–381 | |||
Lup an 3.0101 | 360–367 | |||
Lup an delta conglutin | 88% (IRVNKHL) 324–332 | 88% (WRISSEN) 421–429 |
Allergen name | T-cell epitopes | |||||
---|---|---|---|---|---|---|
FPILGWLGL | FVIPAGYPI | FVPYYNVNA | YVLNGSAWF | YVAFKTNDI | YKFLVPPPQ | |
Lup a 1 | 433–442 | |||||
Lup a 4 | ||||||
Lup a alpha conglutin | 411–418 | 432–444 | 445–452 | 493–501 | 542–550 | |
Lup an 3.0101 | 88.88% (FPILRWLGL) 413–421 | 434–442 | 447–455 | 495–503 | 544–552 | |
Ara h 3 | 77% (FVPHYNTNA) 404–412 | |||||
Ara h 3.0201 | 77% (FVPHYNTNA) 454–465 |
Allergen name | T-cell epitope |
---|---|
FLLAAHAS | |
Ara d 2 | 13–20 |
Ara h 2 | 13–21 |
Ara h 2.0101 | 13–21 |
Ara h 2.0201 | 13–21 |
Ara h 2.0202 | 13–21 |
On the other hand, the different lupin species show that up to 18 T-cell epitopes are found commonly shared between
Among these allergen proteins, there are also epitopes shared more than one time among more than two species. The same epitope is shared among the allergenic proteins:
Prediction of secondary and tertiary structures allowed us to determine the spatial location of epitopes in proteins and to assess whether they may be affected in their spatial arrangement by post-translational modifications in protein domains over interest proteins.
The T-cell epitopes analyzed on
Therefore, epitopic regions matched between
3.7 Identification and analysis of IgE-binding epitopes
The IgE antibodies are produced by immune B cells, which in turn are stimulated by T cells responsible for recognizing the epitope in a sensitization step. To trigger the allergen inflammatory process, IgE antibodies stimulate the release of histamines. Thus, the recognition of these sequences allows for predicting the recognition capacity of IgE antibodies and whether they will potentially trigger the allergenic response (Figure 6).
The analysis of the allergenic nature of the protein based on amino acid and dipeptide analysis composition has been used for the assessment of the above proteins. It is noticeable that the 30cases with clinically confirmed allergenic epitopes are predicted by their sequence to have an allergenic nature, as is the case of
Allergen name | Based on amino acid composition | Based on dipeptide composition | Allergen name | Based on amin oacid composition | Based on dipeptide composition |
---|---|---|---|---|---|
Lup an 1 | Potential allergen | Potential allergen | Lup a 1 | — | — |
Lup an 1.0101 | — | — | Lup a 4 | Potential allergen | Potential allergen |
Lup an 3 | Potential allergen | Potential allergen | Lup a alpha conglutin | — | — |
Lup an 3.0101 | Potential allergen | Potential allergen | Lup a delta conglutin | Potential allergen | Potential allergen |
Lup an alpha conglutin | — | — | Lup a gama conglutin | — | — |
Lup an delta conglutin | Potential allergen | Potential allergen | |||
Lup an gamma conglutin | — | — | Lup l 4 | Allergen | Potential allergen |
Allergen name | Based on amino acid composition | Based on dipeptide composition | Allergen name | Based on amino acid composition | Based on dipeptide composition |
---|---|---|---|---|---|
Pis s 2 | Potential allergen | Allergen | Gly m 5 | Allergen | Allergen |
Pis s 3 | Potential allergen | Potential allergen | Gly m 5.0301 | Allergen | Allergen |
Pis s 3.0101 | Potential allergen | Potential allergen | Gly m8 | Allergen | Allergen |
Pis s 6 | Potential allergen | Potential allergen | Gly m 8.0101 | Allergen | No allergen |
Pis s aglutin | Potential allergen | Potential allergen | |||
Pis s albumin | Potential allergen | Potential allergen |
Allergen name | Based on amino acid composition | Based on dipeptide composition | Allergen name | Based on amino acid composition | Based on dipeptide composition |
---|---|---|---|---|---|
Cic a 1 | — | — | Ara h 1 | Allergen | Allergen |
Cic a 3 | Potential allergen | Allergen | Ara h 1.0101 | Allergen | Allergen |
Cic a 4 | Potential allergen | Potential allergen | Ara h 2 | — | — |
Cic a 6 | — | — | Ara h 2.0101 | — | — |
Ara h 2.0201 | — | — | |||
Ara d 2 | — | — | Ara h 2.0202 | — | — |
Ara d 6 | — | — | Ara h 3 | — | — |
Ara h 3.0201 | — | — | |||
Ara i 2.0101 | — | — | Ara h 5 | Potential allergen | Potential allergen |
Ara i 6.0101 | — | — | Ara h 5.0101 | Potential allergen | Potential allergen |
Allergen name | Based on amino acid composition | Based on dipeptide composition | Allergen name | Based on amino acid composition | Based on dipeptide composition |
---|---|---|---|---|---|
Ara h 6 | — | — | Ara h 11.0101 | — | — |
Ara h 6.0101 | — | — | Ara h 11.0102 | — | — |
Ara h 7.0101 | Allergen | — | Ara h 13.0102 | Allergen | Allergen |
Ara h 7.0201 | — | — | Ara h 14.0101 | — | — |
Ara h 7.0301 | — | — | Ara h 14.0102 | — | — |
Ara h 8 | Potential allergen | Potential allergen | Ara h 14.0103 | — | — |
Ara h 8.0101 | Potential allergen | Potential allergen | Ara h 15.0101 | Allergen | Allergen |
Ara h 8.0102 | Potential allergen | Potential allergen | Ara h 16 | Allergen | — |
Ara h 9.0101 | Allergen | Allergen | Ara h 17 | Potential allergen | Allergen |
Ara h 10.0101 | — | — | Ara h aglutin | Potential allergen | — |
Other proteins assessed as ambiguous or non-allergenic even though they present bibliographic and clinical antecedents of being allergenic include
Allergen name | IgE epitopes | |||||
---|---|---|---|---|---|---|
HRIFLADKD | NNFGKLFEVK | SYLQEFSRNT | ELHLLGFGIN | KDLAFPGSGE | RRYTARLKEG | |
Gly m 5 | 70% 415-QRNFLAGEKD | 70% 297- NNFGKFFEIT | 70% 217-SYLQGFSHNI | |||
Gly m 5.0301 | 70% 418-QRNFLAGEKD | 70% 300-NNFGKFFEIT | 70% 220-SYLQGFSHNI | |||
Lup a 1 | 70% 286-SYFSGFSRNT | 80% 483-NLRLLGFGIN | 70% 517-KELTFPGSAE | 80% 456-RRYSARLSEG | ||
Lup an 1.0101 | 80% NLRLLGFGIN | 70% KELTFPGSIE | ||||
Ara h 1 | 100% HRIFLADKD | 90% NNFGRLFEVK | 90%SYQGFSRNT | 100% ELHLLGFGIN | 100% KDLAFPGSGE | 100%RRYTARLKEG |
Ara h 1.0101 | 100%HRIFLADKD | 100% NNFGKLFEVK | 100%SYLQEFSRNT | 100% ELHLLGFGIN | 100%KDLAFPGSGE | 100%RRYTARLKEG |
Cic a 1 | 80% DLFLLGFGIN | 70%KEVAFPGSAE |
Allergen name | IgE epitopes | |||||
---|---|---|---|---|---|---|
GNIFSGFTPEFLEQA | IETWNPNNQEFECAG | DRRCQSQLER | HASARQQWEL | KIQRDEDS | KRELRNL | |
Lup a alpha conglutin | 66.67% GNVLSGFDDEFLEEA | 73.34% IETWNPKNDELRCAG | ||||
Lup an alpha conglutin | 66.67% GNVLSGFNDEFLEEA | 73.34% IETWNPKNDQLRCAG | ||||
Ara d 2 | 100% DRRCQSQLER | 100%HASARQQWEL | 100%KIQRDEDS | 100% KRELRNL | ||
Ara d 6 | 85.71% KRELMNL | |||||
Ara h 2 | 100% DRRCQSQLER | 100% HASARQQWEL | 100% KIQRDEDS | 100% KRELRNL | ||
Ara h 2.0101 | 70% DRRCQSQLER | 100% HASARQQWEL | 100% KIQRDEDS | 100% KRELRNL | ||
Ara h 2.0201 | 100% DRRCQSQLER | 100% HASARQQWEL | 100% KIQRDEDS | 100% KRELRNL | ||
Ara h 2.0202 | 100% DRRCQSQLER | 100% HASARQQWEL | 100% KIQRDEDS | 100% KRELRNL | ||
Ara h 3 | 86.67% GNIFSGFTSEFLAQA | 100% IETWNPNNQEFECAG | ||||
Ara h 3.0201 | 100% GNIFSGFTPEFLEQA | 100% IETWNPNNQEFECAG | ||||
Ara h 7.0201 | 85.71% ERELRNL | |||||
Cic a 6 | 73.33% GNIFSGFKRDFLEDA | 73.33% IETWNPSNKQFACAG |
Allergen name | IgE epitopes | ||||||
---|---|---|---|---|---|---|---|
LQGRQQ | LRPCEQHLMQ | QRCDLDVE | QWELQGDR | RDPYSP | RDPYSP | SQDPYSPS | |
Ara d 2 | 100% LQGRQQ | 100% LRPCEQHLMQ | 100% QRCDLDVE | 100% QWELQGDR | 100% RDPYSP | 83.33% RDPYSP | 100% SQDPYSPS |
Ara d 6 | 80% LKPCEQHIMQ | 87.5% QRCDLDVS | |||||
Ara h 2 | 100% LQGRQQ | 100% LRPCEQHLMQ | 87.5% QRCDLEVE | 100% QWELQGDR | 100% RDPYSP | 83.3% QDPYSP | 100% SQDPYSPS |
Ara h 2.0101 | 100% LQGRQQ | 100% LRPCEQHLMQ | 87.5% QRCDLEVE | 100% QWELQGDR | 100% RDPYSP | 83.3% QDPYSP | 100% SQDPYSPS |
Ara h 2.0201 | 100% LQGRQQ | 100% LRPCEQHLMQ | 87.5% QRCDLEVE | 100% QWELQGDR | 100% RDPYSP | 83.3% QDPYSP | 100% SQDPYSPS |
Ara h 2.0202 | 100% LQGRQQ | 100% LRPCEQHLMQ | 87.5% QRCDLEVE | 100% QWELQGDR | 100% RDPYSP | 83.3% QDPYSP | 100% SQDPYSPS |
Ara h 6.0101 | 87.5% QRCDLDVS | ||||||
Ara h 7.0201 | 70% LRPCEEHIRQ | ||||||
Ara h 7.0301 | 70% LRPCEEHIRQ | ||||||
Ara d 2 | 100% LQGRQQ | 100% LRPCEQHLMQ | 100% QRCDLDVE | 100% QWELQGDR | 100% RDPYSP | 83.33% RDPYSP | 100% SQDPYSPS |
Ara d 6 | 80% LKPCEQHIMQ | 87.5% QRCDLDVS | |||||
Ara h 2 | 100% LQGRQQ | 100% LRPCEQHLMQ | 87.5% QRCDLEVE | 100% QWELQGDR | 100% RDPYSP | 83.3% QDPYSP | 100% SQDPYSPS |
Ara h 2.0101 | 100% LQGRQQ | 100% LRPCEQHLMQ | 87.5% QRCDLEVE | 100% QWELQGDR | 100% RDPYSP | 83.3% QDPYSP | 100% SQDPYSPS |
Ara h 2.0201 | 100% LQGRQQ | 100% LRPCEQHLMQ | 87.5% QRCDLEVE | 100% QWELQGDR | 100% RDPYSP | 83.3% QDPYSP | 100% SQDPYSPS |
Ara h 2.0202 | 100% LQGRQQ | 100% LRPCEQHLMQ | 87.5% QRCDLEVE | 100% QWELQGDR | 100% RDPYSP | 83.3% QDPYSP | 100% SQDPYSPS |
Ara h 6.0101 | 87.5% QRCDLDVS | ||||||
Ara h 7.0201 | 70% LRPCEEHIRQ | ||||||
Ara h 7.0301 | 70% LRPCEEHIRQ |
The clinically proven epitopes found in the sequence analysis allowed us to observe how many and to what extent IgE epitopes are shared between proteins of different species and to assess potential cross-reactivity. According to the results, some of the candidate species and proteins for cross-reactivity with soybean (
In addition, shared T-cell epitopes have been found among species that do not include soybean such as
An interesting fact is that different isoforms of the same protein may or may not present the same IgE epitope and, in the case of having it, it does not necessarily have the same degree of similarity. Establishing a relationship with the information obtained in the alignments, we can conclude that the small differences observed in the sequence between isoforms of the same protein can be key to conformation and epitopes presence (Table 10).
Allergen name | IgE epitopes | |||
---|---|---|---|---|
DITNPINLRE | KESHFVSARP | EQEERGQRRW | VTVRGGLRILSPDRK | |
Ara h 1 | 90% DITNPINLRD | 90% RESHFVSARP | 90% EQEERGQRR | |
Ara h 1.0101 | 100%DITNPINLRE | 100%KESHFVSARP | 100%EQEERGQRRW | |
Ara h 3 | 93,345%VTCRGGLRILSPDRK | |||
Ara h 3.0201 | 86.67% VTVRGGLRILSPDRK |
4. Conclusions
This chapter presented a study of functional and allergenic features of legume seed proteins.
Analysis of allergenic legume proteins legume as well as all available isoforms allowed for extracting shared epitopes that can be linked to cross-reactivity processes among the eight studied species (
Small differences in the amino acid sequences (less than 1%) of the same allergen isoforms implied important changes in epitopic conformation and sequences of T-cell and IgE recognizable epitopes. Small differences in amino sequences of isoforms from the same inferred changes over 2D and 3D structure conformation that may affect functional protein domains. Post-translational modifications allowed identification of possible phosphorylation, glycosylation, carboxylation, methylation, nitrosylation, and nitration sites in protein functional domains, near or directly located in different type of epitopes with potential influence in allergenic response.
Primary sequence alignments together with three-dimensional protein modeling allowed to study the conservation of proteins as conglutin gamma proteins among different
The changes described close to the sequence or related to spatial distribution of the epitopes may involve potential alterations on protein allergenicity.
Obtaining reliable clinical data on legume allergies in developing countries could be helpful in clarifying whether the increase in food allergies is actually due to poor dietary habits and increasing industrialization processes.
Further studies on the characterization of more allergenic proteins, including isoforms of major allergens already described, not only sequential but also three-dimensional conformational epitopes, can be a great advancement for the prevention of cross-reactivity and the improvement of knowledge of allergies produced by legumes, which in turn could promote the introduction of this food as a substitute for other foods of lower nutritional quality and with greater environmental impact.
Acknowledgments
This study has been partially funded by The Spanish Ministry of Economy, Industry and Competitiveness through the grants Ref.: RYC-2014-2016,536 (Ramon y Cajal Research Program) to JCJ-L; and Ministry of Health and Families, Andalusian government. Funding for I + D + i in biomedical research and health sciences in Andalusia, grant Ref.: PI-0450-2019.
Abbreviations
LTP | Lipid Transfer Protein |
3D | three-dimensional |
PR | proteins Pathogenesis-related proteins |
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