Molecular and Functional Characterisation of Allergenic Non-specific Lipid Transfer Proteins of Sweet Lupin Seed Species

*Maria Rodrigo-Garcia, Esther Rodriguez-de Haro, Salvador Priego-Poyato, Elena Lima-Cabello, Sonia Morales-Santana and Jose C. Jimenez-Lopez*

## **Abstract**

Non-specific lipid transfer proteins (nsLTPs) are small proteins abundant in plants, which function in transferring phospholipids and galactolipids across the membrane. nsLTPs also play a key role in plant resistance to biotic and abiotic stresses, growth and development, as well as in sexual reproduction, seed development, and germination. In addition, these proteins have previously been identified as food allergens. In the present study, we carried out a molecular and functional comparative characterisation of 25 sequences of nsLTPs of lupin legumes and other species. Extensive analysis was carried out; including comparison of databases, phylogeny, physical–chemical properties, functional properties of post-translational modifications, protein structure conservation, 2-D and 3D modelling, functional interaction analysis, and allergenicity including identification of IgE, T-cell, and Bcell binding epitopes. The results indicated that particular structural features of nsLTPs are essential to the functionality of these proteins, high level of structural stability and conservation. Information about different functional interactions between nsLTPs and ligands showed that nsLTPs can accommodate several of them with different structure; and that the relationship between structure and allergenicity was investigated through the identification of epitopes susceptible of being involved in cross-reactivity between species of the *Fabaceae* family.

**Keywords:** *Lupinus angustifolius*, PULSE, nsLTP, legume, seed allergenic proteins, food allergies, cross-reactivity

### **1. Introduction**

Sweet lupin group has four lupin species currently used for food, namely, *L. angustifolius*, *L. albus*, *L. luteus*, and *L. mutabilis*. Lupin seed contain large amount of proteins ranging between 38 and 52%, depending of the species and cultivar [1]. The protein content of sweet lupin is usually higher compared to other legumes, i.e. pea, soya, or lentil. Main protein content of lupine seed belongs to two families called globulins (80–94%) and albumins (5–15.4%) [2, 3], while other proteins (glutelins and prolamins) are in low quantities [4].

Globulins are the most abundant proteins in sweet lupin group seeds and the most polymorphic family in terms of gene and protein sequence [5]. Globulins comprise different families of seed storage proteins (SSPs): α-conglutins (legumins or 11S type globulins), β-conglutins (vicilins or type 7S globulins), γ-conglutins (basic 7S type globulins); and δ-conglutins, and others in much more less amount as 2S sulphur-rich albumins, LTPs, profilin, PRP [3, 5].

*L. angustifolius* and *L. albus* are particularly suitable for food because their nutritional and nutraceutical properties, help in preventing diseases such as diabetes, digestive tract and cardiovascular diseases, overweight, obesity or cancer, while reducing celiac disease problems as lupine does not contain gluten [4, 6].

Currently, products based on lupine proteins are gaining more attention in the food industry, due to their low cost, and the high demand for sustainable foods [4, 7, 8]. Besides important techno-functional (physical and chemical) properties, such as high water retention capacity and great emulsifying and foaming capacity, lupine flours or lupine protein concentrates have been used to formulate and substitute technological agents in baked, meat, and dairy products by the industry food [4].

Interestingly, and despite the great health benefits of lupin seeds, they are also a source of anti-nutritional factors such as phytic acids, saponins, phenolic compounds, enzyme inhibitors, lectins and hemagglutinins. The most problematic factors are the alkaloids because their bitter taste provided to the food [9, 10]. Fortunately, recent alkaloid content [3, 7, 11]. Some of these anti-nutritional factors an cause adverse physiological effects if they are consumed by animals while others (i.e. polyphenols and oxalates) limit the bioavailability of minerals from foods [9, 10].

Nevertheless, lupine was labelled in 2008 as an allergen in packaged foods, as recommended by the European Food Safety Authority (EFSA, http://www.efsa.e uropa.eu/) [7, 11]. According to the list of allergens provided in the databases of the Allergen Nomenclature Subcommittee of the World Health Organisation, the International Union of Immune Societies and Allergome (WHO; UISI, http://www. allergen.org/; http://www.allergome.org/), where the main lupine allergen is β globulins and other minor fractions such as non-specific lipid transfer proteins (nsLTP) (Lup an 3) has high relevance because their cross-reactivity [4].

Lupine allergy is normally mediated by Immunoglobulin E (IgE) and allergic reactions to lupine can occur *via* ingestion, inhalation, or occupational exposure [7]. For example, exposure to lupine *via* the respiratory tract can be considered as the primary reason for allergic sensitization in food industry workers [4, 12–14]. Co-sensitization *via* inhalation has also been proposed as a common cause of lupine and wheat allergy among bakers [4, 14]. Indirect sensitization to lupine proteins can also occur through crossreactivity with other legumes and particularly in previous peanut allergy patients [4, 15–18]. Clinical symptoms can vary in intensity and severity, including asthma, allergic rhinitis, urticaria, nausea or gastrointestinal pain, and anaphylaxis [4, 19].

Plant nsLTPs are small extracellular proteins, which includes a significant number of allergens [20–22]. They are usually located in the outer layers of the shell of fruits and seeds and their allergenic potency can be reduced when are removed [4, 21, 23]. It has been observed that its molecular characteristics, such as its great stability against proteolysis, thermal denaturation and cross-reactivity, are linked to its allergenicity [20]. Sensitization to nsLTPs may depend on geographic differences, sensitization pathways, type of diet, and is often associated with severe symptoms [24]. In this regard, lupine β and γ conglutins may correlate with the severity of clinical reactions [4, 16], although more families may be involved.

Recently, an nsLTP was identified and included by the WHO/IUIS as an allergenic food protein in *L. angustifolius* (Lup an 3) [4].

Structural homologies of lupine allergens or commonly shared epitopes with other legume allergens lead to support cross-reactivity reactions between them [4]. The present study carries out the molecular and functional characterisation of proteins of the non-specific lipid transfer proteins (nsLTPs) family of the lupine seed (*Lupinus angustifolius* L.), compared to other of legumes and plant species as olive tree (*Olea europaea* L). For this purpose, we identified nsLTP sequences expressed in *L. angostifolius* seed, classifying and analysing phylogenetic relationships among them, the functional and the molecular processes that they are involved; we also analysed the proteins at a structural level, identifying potential motifs implicated in functional differences; and we established the potential allergenicity of the nsLTPs through identification and analysis of different epitopes involved in allergy phenomenon.

## **2. Material and methods**

#### **2.1 nsLTPs sequences of lupine, legumes and other plant species**

Different gen and protein databases were used to search and retrieved nsLTPs from legume species and other model plants: NCBI (https://www.ncbi.nlm.nih.gov/), Uniprot (https://www.uniprot.org/), Allergome (http://www.allergome.org/index.ph p), and reprOlive (http://www.scbi.uma.es/olivodb/).

We retrieved 25 sequences as follow: The sequences and their access number are: *Lupinus angustifolius* (Lup an 3) Uniprot: A0A1J7GK90, *Lupinus angustifolius* (Lup an 3.0101) (Uniprot: A0A4P1RWD8), *Medicago truncatula* (Uniprot: A0A072UTH7), *Arabidopsis thaliana* (nsLTP-3) (Uniprot: Q9LLR7), *Arabidopsis thaliana* (nsLTP-5) (Uniprot: Q9XFS7), *Olea europaea* (Ole e 7) (NCBI: XP\_022893508.1), *Lupinus albus* (Uniprot: A0A6A5MQ88), *Lupinus angustifolius* (Uniprot: A0A4P1RV83), *Glycine max* (Uniprot: I1J7M1), *Arachis hypogaea* (NCBI: XP\_025656480.1), *Cajanus cajan* (NCBI: XP\_020237462), *Phaseolus vulgaris* (Uniprot: D3W146), *Glycine soja* (Uniprot: A0A445M2F4), *Lens culinaris* (Uniprot: A0AT33),*Trifolium pratense* (Uniprot: A0A2K3M7A7), *Spatholubus suberectus* (NCBI: TKY63608.1), *Cicer arietinum* (Uniprot: O23758), *Vigna ungiculata* (Uniprot: UPI0010170F74), *Abrus precatorus* (Uniprot: UPI000F7C313B), *Arachis ipaensis* (NCBI: XP\_020971907.1),*Trifolium subterraneum* (NCBI: GAU29990.1), *Prosopis alba* (NCBI: XP\_028808641.1), *Vigna angularis* (NCBI: KOM57753.1), *Arachis duranensis* (NCBI: XP\_015950831.1), *Pisum sativum* (NCBI: A0A158V755.1).

#### **2.2 Multiple alignments of nsLTPs sequences of lupine and other species**

We carried out multiple alignments with the 25 amino acid sequences previously obtained with the Clustal Omega program (https://www.ebi.ac.uk/Tools/msa/clustalo/). In addition, partial alignments with different number of sequences were also performed to be sure that reproducibility of these analysis was covered. The alignment was verified manually with Bioedit v7.2.5 (http://www.mbio.ncsu.edu/ bioedit/bioedit.html) and Jalview 2.11.1.4.

#### **2.3 Phylogenetic analysis of the 25 nsLTPs sequences of lupine and other species**

Different simulations of the phylogenetic analysis of the sequences were carried out with the multiple amino acid alignments, assuring accuracy and reproducibility. It was analysed using the MEGA-X software, with the neighbour-joining method, including bootstrap defined by the software, following the Poisson model, with Uniform Rates¸ Pairwise Deletion and using 4 threads.

## **2.4 Physical and chemical properties analysis of nsLTPs**

We used the tool Protparam (https://web.expasy.org/protparam/). We analysed isoelectric point (pI), aliphatic index (AI), and instability index (II) among others.

## **2.5 Functional motifs analysis**

Domains and functional motifs were analysed using PfamScan (https://www.eb i.ac.uk/Tools/pfa/pfamscan/), Pfam (http://pfam.xfam.org/search#tabview=tab0), and ScanProsite (https://prosite.expasy.org/scanprosite/). The use of all these tools assured accuracy and reproducibility in the analysis.

## **2.6 Post-translational (functional) modifications of the nsLTPs proteins**

We identified different post-translational modifications such as Nglycosylations, N-myristoylation, and phosphorylation sites for casein kinase (CK2), protein kinase C (PKC), and cAMP-dependent protein kinase (PKA) using ScanProsite (https://prosite.expasy.org/scanprosite/). We also identified posttranslational modifications related to stress and REDOX regulation such as S-nitrosylation of cysteine using iSNOAAPair (http://app.aporc.org/iSNO-AAPair), and N-nitrations of tyrosine with GPS-YNO2 (http://yno2.biocuckoo.org) [25]. Carbonylation sites were identified by iCarPS (http://lin-group.cn/server/iCarPS/ webServer.html). NetPhos 3.1 (http://www.cbs.dtu.dk/services/NetPhos/) was used to predict phosphorylation sites. NetAcet-1.0 was used to check acetylations (https://services.healthtech.dtu.dk/service.php?NetAcet-1.0). The use of all these tools assured accuracy and reproducibility in the analysis.

#### **2.7 Subcellular location of nsLTPs proteins**

The subcellular localization was identified using pSORT (https://www.genscript. com/psort.html, http://psort1.hgc.jp/form.html), WoLF SORT (https://wolfpsort. hgc.jp/, https://www.genscript.com/wolf-psort.html) and CELLO V 2.5 (http://ce llo.life.nctu.edu.tw/). Subsequently, verification of the extracellular, mitochondrial, and chloroplastidial localization was made by the TargetP (http://www.cbs.dtu.dk/ services/TargetP/) tool. The use of all these comparative tools assured accuracy and reproducibility in the analysis.

#### **2.8 Secondary structure (2D) prediction of nsLTPs**

The prediction of the secondary structure of nsLTPs was carried out using the PSIPRED program (http://bioinf.cs.ucl.ac.uk/psipred/.

#### **2.9 3D structure of nsLTPs**

To build the 3D structure, we used the bioinformatics tools I-TASSER (https:// zhanglab.dcmb.med.umich.edu/I-TASSER/) and Phyre2 (http://www.sbg.bio.ic.ac. uk/phyre2/html/page.cgi?id=index). The figures were drawn using the PyMOL program.

## **2.10 Conservational study of nsLTPs proteins in different species**

The Consurf server tool (https://consurf.tau.ac.il/) was used for this purpose.

### **2.11 Functional interactomics analysis of nsLTPs**

To carry out the interactomics analysis, the STRING tool was used to predict the interactomics analysis (https://string-db.org/cgi/input?sessionId= bwP3HkaoJSDc&input\_page\_show\_search=on) using *Medicago truncatula* as a model species for the lupine sequences, and *Arabidopsis thaliana* for the olive sequence.

#### **2.12 nsLTPs and multiple ligands binding analysis study**

I-TASSER tool (https://zhanglab.dcmb.med.umich.edu/I-TASSER/) was used to identify the multiple ligands of nsLTPs.

### **2.13 Allergenicity study and identification of allergenic epitopes from nsLTPs**

The selected allergen families of LTPs were obtained in the Allergome database (http://www.allergome.org/index.php). AlgPred tool (https://webs.iiitd. edu.in/raghava/algpred/submission.html) was used to carry out the study of IgE binding epitopes. It was analysed whether the protein sequences present experimentally tested IgE binding epitopes as allergen representative peptides (ARPs); if they present epitope motifs, with the MEME / MAST tool that forms matrices from sequences of known allergens; and the allergenicity potential of the 25 protein sequences was determined, based on the amino acid and dipeptide composition.

#### **2.14 T-cell epitopes identification and analysis in nsLTPs**

To carry out these T-cell binding epitope identification studies, we used the tool ProPred (https://webs.iiitd.edu.in/raghava/propred/). Identification of MHC II binding regions was carried out for the 25 amino acid sequences from lupine, olive, and other legumes using quantitative matrices. A threshold of 3% was set 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). The epitope sequences shared by three or more HLA II analysed were annotated.

#### **2.15 B-cell epitopes identification and analysis in nsLTPs**

For the identification of B-cell binding epitopes, we used the tool Bcepred (https://webs.iiitd.edu.in/raghava/bcepred/bcepred\_submission.html). The 25 protein sequences of lupine, olive, and other legumes were analysed. Regarding the values for the identification of B cell epitopes, we used predetermined threshold values, being the most suitable for the study that we carried out for each of the analysed characteristics: hydrophilicity, accessibility, surface exposure, antigenic propensity, flexibility, turns, polarity, and the combination of all.

## **3. Results and discussion**

## **3.1 Multiple alignments of nsLTPs proteins and phylogenetic analysis**

**Table 1** shows the list of nsLTPs sequences analysed with their functional domains. **Figure 1** shows the multiple alignments of 8 representative protein sequences of nsLTPs of such as Lup an 3, Lup an 3.0101, *Medicago truncatula nsLTP*, *A. thaliana* (nsLTP-3), *A. thaliana* (nsLTP-5), *L. angustifolius*, and *L. albus*. A large representative number of nsLTPs in a general alignment (**Figure A1**). The conservation of each residue in the alignment is shown with bars. Overall, the most conserved amino acids were found in the regions between the position 30 to 60 and in the N-terminal regions of the protein.



#### **Table 1.**

*Domains and functional motives of nsLTPs.*

nsLTPs can be considered as basic proteins with high identities among their sequences [20]. The sequences sharing high identity within the alignment are Lup an 3, *M. truncatula*, and Lup an 3.0101, as well as the sequences of nsLTPs from *L. albus* and *L. angustifolius*. The two *A. thaliana* sequences (nsLTP-3 and nsLTP-5) are also very similar each other, even though nsLTP-3 has a longer ORF. These similarities between nsLTPs are also observed in **Figure 2**, where clusters of sequences are grouped, except for Lup an 3.0101. The bootstrap values indicate the probability that the sequences are grouped by similarity. Thus, a bootstrap value of

#### **Figure 1.**

*Multiple alignment of nsLTPs. Eighth main nsLTP protein sequences have been aligned. The similarity index (0-10) between the aligned sequences. The conservation index is shown as yellow bars, and has values ranged from 0 to 10. Lup an 3 (Uniprot: A0A1J7GK90), Lup an 3.0101 (Uniprot: A0A4P1RWD8),* Medicago truncatula *(Uniprot: A0A072UTH7),* Arabidopsis thaliana *3 (Uniprot: Q9LLR7),* Arabidopsis thaliana *5 (Uniprot: Q9XFS7), Ole e 7 (NCBI: XP\_022893508.1),* Lupinus albus *(Uniprot: A0A6A5MQ88),* Lupinus angustifolius *(Uniprot: A0A4P1RV83).*

60 (in base 100) indicates that the probability that the sequences have not been randomly grouped is 60%, being the overall limit 70%.

Interestingly, it is also observed that the most related species based on nsLTPs comparisons are the species of the genus *Trifolium* (*T. pratense* and *T. subterraneum*), with a bootstrap value of 100 (**Figure 2**). Since they belong to the same genus; they appear more related to each other than to the rest of the analysed species. Similarly, the species of the genus *Arachis* (*A. ipaensis* and *A. hypogaea*), *Cajanus cajan* and *Abrus precatorius* have also grouped, with a very high bootstrap value (99.4). This similarity could be related to the original geographic regions since both arise in India and Africa and their current distributions are also very similar.

On the other hand, although Lup an 3 and Lup an 3.0101 are quite similar, in **Figure 1**, they are phylogenetically distant from each other (see **Figure 2**). In the case of Lup an 3, it has been grouped with the *L. albus* sequence, with 68.2 bootstrap values. These two species are related to *L. angustifolius*, with a bootstrap greater than 70. Lup an 3.0101, it is grouped with *Glycine max*, *Spatholobus suberectus*, *Phaseolus vulgaris*, and *Vigna angularis*, but with a very low bootstrap value. As a result, Lup an 3.0101 maybe an isoform of Lup an 3 with differences in key aminoacids since they are phylogenetically more distant.

Regarding *A. thaliana* sequences (nsLTP-3 and nsLTP-5), they appear grouped, with a high bootstrap (87.2), since they belong to the same species, and are the same kind of protein (nsLTP). Ole e 7 is grouped with *Trifolium* sequences (*T. pratense* and *T. subterraneum*) with a bootstrap value of 62.6. *Arachis duranensis* in **Figure 2** is shown as an outgroup, despite belonging to the same genus as other species included in this analysis (*A. ipaensis* and *A. hypogaea*). Although the origin of *A. hypogaea* is the hybridization of *A. duranensis* x *A. ipaensis*, the largest set of chromosomes of its karyotype comes from *A. ipaensis*, thus this protein probably comes from this set of chromosomes [26]. Furthermore, *A. duranensis* and *A. ipaensis* separated 3 million years ago [26]. Hence, *A. ipaensis* and *A. hypogaea* group have a bootstrap of 100, while *A. duranensis* has been identified as an outgroup.

#### **Figure 2.**

*Phylogenetic analysis of nsLTPs. Twenty-five representative nsLTPs sequences were used for clustering analysis. The bootstrap value (in base 100) is indicated in the nodes. The different colours indicate the groups formed.*

#### **3.2 nsLTPs physical and chemical proprieties analysis**

The physical and chemical properties analysed were described in **Table 2**. The longest sequence is *Spatholobus suberectus* with 165 aa and 17354.17 Da of MW, and the shortest analysed in *A. thaliana* (nsLTP-5) with 104 aa and 10993.72 Da of MW.

Regarding Lup an 3 and Lup an 3.0101, they are 120 aas and 116 aa long, respectively, and with comparable MW such as 120. 22 kDa and 117.20 kDa, respectively.

Stability of the protein is shown as aliphatic (AI) and instability (II) indexes. II values lower than 40 proteins are stable. Most of the sequences were stable except for *A. thaliana* (nsLTP-3) (42, 46), *L. angustifolius* (42.53), *A. hypogaea* (52, 63),*T. subterraneum* (45.62) and *P. alba* (45.59). Although their values are higher than 40, they are close to the limit value. The AI has an important role in thermal stability, the higher the value of the AI, the more thermally stable a protein is. All the proteins analysed were highly stable, being the lowest value of 77.12. The stability (thermal and proteolytic) of these proteins is important at a molecular level to improve transport and defence function, as well as in their allergenic capacity even in processed and cooked foods [20].


*Table shows the protein molecular weight (MW), the isoelectric point (pI), the aliphatic index and the instability index. If the instability index value is less than 40 the protein is classified as stable, and if the value is greater than 40 it is classified as unstable.*

#### **Table 2.**

*Physic-chemical properties.*

#### **3.3 nsLTPs functional motifs and post-translational modification analysis**

Analysis of functional motifs and post-translational modifications were carried out on 25 protein sequences of lupine, other legumes, olive trees, and model plants showed in **Table 1**, **Tables A1**–**A3**.

**Table 1** shows that all the sequences have a comparable length, where the shortest sequence contains 104 aa in *A. thaliana* (nsLTP-5), while the longest contains 165 aa in case of *Spatholobus suberectus*.

Pfam functional motifs reveal that the sequences present the protease inhibitor and seed storage motif of the nsLTP family (prolamin family). The prolamin clan was integrated by trypsin-alpha amylase inhibitors, reserve proteins in seeds, and lipid transfer proteins in plants [27]. nsLTP family is a group highly conserved of

7–9 kDa proteins found in higher plant tissues, which function transfering lipids, and is divided into 2 structurally related subfamilies: LTP1 (9 kDa) and LTP2 (7 kDa).

Prosite functional motifs show that most of the sequences contain a motif belonging to the LTPs of plants as it is expected, except for *L. albus*, *G. soja*, *T. subterraneum*, and *V. angularis*.

Post-translational modifications are described in **Tables A1**–**A3**. Phosphorylations, N-myristoylation, glycosilations, N-nitrosylation (cysteine), N-nitrations, and carbonylations, were the most commonly found in the studied nsLTPs.

Phosphorylation (**Tables A1** and **A2**) is common and reversible in proteins, and generally fulfil a regulatory activity of the function of the protein (activate or inhibit its function) in processes such as growth, development of immunity, and responses to stress [28], so it regulate the nsLTPs functional roles. Furthermore, it has previously been observed that Ser and/or Thr residues in seed storage proteins are extensively phosphorylated improving the transport mechanism of these storage proteins [28].

No abundant glycosylation modifications were found while N-myristoylation are quite abundant (**Table A2**) which may indicate that snLTPs membrane location is well regulated under variable stresses conditions. N-glycosylations have also been found not abundant in nsLTPs, only found in Ole e 7, *L. angustifolius*,*T. pratense*, *V. angularis*, *and A. duranensis*. Glycosylation have been previously mentioned as markers of allergenicity and may be related to allergenic properties, due to interaction with the innate immune system [29].

Post-translational redox modifications, such as N-nitrosylation and T-nitration, and carbonylation were involved in the defence function, and coping to biotic and abiotic stresses, and redox signalling (**Tables S2** and **S3**).

#### **3.4 nsLTPs subcellular location**

The subcellular location of the 25 nsLTP proteins has been identified and the results are shown in **Table 3**.

Bioinformatic tools, CELLO and Wolf PSort, both show that all proteins are found in the extracellular environment. PSort tool also indicates that in some cases (Lup an 3.0101, *M. truncatula*, *A. thaliana* (nsLTP-3), Ole e 7, *L. culinaris*, *T. subterraneum*, and *A. duranensis*) the location of these proteins are vascular as well as extracellular. *L. angustifolius*, *G. max*, *S. suberectus*, and *P. sativum* are in the plasma membrane; and in the endoplasmic reticulum membrane as well as *P. sativum*.

Structural, biochemical, and physiological features of nsLTPs confirms that these proteins are involved in lipid transport in the vacuolar - plasma membrane secretion pathway to the extracellular space [20]. Thus, the subcellular location of the proteins analysed confirm the nsLTPs functional properties.

#### **3.5 Secondary structure of nsLTP proteins**

Secondary structures of analysed nsLTPs of lupin, Arabidopsis, Medicago and olive species are shown in **Figure 3**. α-helix structures present in the nsLTPs are shown in red, and the conserved eight cysteine motif is shown with yellow arrows, which is present in all nsLTPs [20, 22]. This conserved motif integrates four disulphide bridges making a hydrophobic environment inside the protein, where the lipids are transported, while keeping a hydrophilic external environment, maintaining the water-soluble characteristics of these proteins [20–22]. In this regard, the secondary structure of LTPs is very important to maintain the binding


*The table shows the subcelular location of each nsLTP protein assessed by the software CELLO, WOLF PSORT and PSORT described in material and methods. Ec: Extracellular; Vac: Vacuolar; MP: Plasmatic membrane; MPI: internal Plasmatic membrane; MRE: Endoplasmic reticulum membrane.*

#### **Table 3.**

*Subcellular location of nsLTPs.*

stability of their structure to carry out their functional properties of transporting hydrophobic macromolecules [22].

Regarding the 2-D structures as α-helix, most of them are integrated by 5 αhelices and no β sheets have been found. This structure is typical in nsLTPs, and comparison with other species have shown a conserved 4 α-helices [20–22].

Interestingly, despite the low sequence identity shown in the alignment of **Figure 3**, 2D structural features among different species are conserved.

#### **3.6 3D structure analysis of nsLTPs sequences**

3D structure of 8 main nsLTP proteins analysed are shown in **Figure 4** (Lup an 3, *Lupinus albus* and Ole e 7) and **Figure A2** (*Lupinus angustigolius*, Lup an 3.0101, *Medicago truncatula*, *Arabidopsis thaliana* nsLTP-3 and *Arabidopsis thaliana* nsLTP-5).


#### **Figure 3.**

*Secondary structure assessment of nsLTPs. The amino acids involved in the α-helix are highlighted with red bars. Residues that are part of an α-helix and β-sheet are highlighted in red and yellow colour, respectively. The blue arrows indicate the most conserved residues (a value of 9 on the Consurf bioinformatics tool scale). The yellow arrows indicate the cysteines involved in the 8 cysteine motif (C-Xn-C-Xn-CC-Xn-CXC-Xn-C -Xn-C), where Xn is an aminoadic repeated n times.*

Overall, no specific differences have been shown in the proteins modelling 3D structures. However, a detailed analysis shows differences at local level such as length of α-helices, special location of the 2-D structures. Noticeable differences in protein size as nsLTP-3 or nsLTP-5 are the smaller proteins, leading to the maintenance of a more compact structures compared to large nsLTPs such as *L. albus* or *L. angustifolius*, being more open structures to the solvent to the outside, which can affect the type of lipid they can carry.

#### **3.7 Conservational analysis of nsLTPs**

The primary and 3D structures of the nsLTPs proteins were used to analyse the conservational features of nsLTPs. The results are shown in **Figure 5** (Lup an 3) and **Figure A3** (*Lupinus angustifolius*, Lup an 3.0101, *Medicago truncatula*, *Arabidopsis thaliana* nsLTP-3, *Arabidopsis thaliana* nsLTP-5, *Lupinus albus* and Ole e 7). Most conserved residues of the proteins are found relatively close to the 8-cysteine motif. Most of the highly conserved residues are buried residues and placed around interacting locations with lipids, thus functionality is maintained over time.

**Figure 4.**

*3D structure of nsLTPs. 3D structures modelling of Lup an 3 (Uniprot: A0A1J7GK90), Ole e 7 (NCBI: XP\_022893508.1) and* Lupinus albus *(Uniprot: A0A6A5MQ88). Cartoon mode representation were build using Phymol software. A-helices are depicted in red colour.*

#### **3.8 Functional interaction analysis of nsLTPs with their ligands**

The analyses carried out using I-TASSER identified the main ligands of Lup an 3, Lup an 3.0101, *M. truncatula*, *A. thaliana* (nsLTP-3), *A. thaliana* (nsLTP-5), Ole e 7, *L. albus*, and *L. angustifolius*, they are shown in **Figure 6** and **Figure A4**. **Table A4** summarise the main ligands of lipid nature that can located in the nsLTPs cysteine functional motif, and **Table A5** summarise the functional interaction with other proteins.

**Figure 6** shows the interaction of the Lup an 3 protein with stearic acid, its main ligand, and the hydrophilic environment of the nsLTP that has to maintain inside of the protein [20], fundamental for the carrying lipid function and interaction of Lup an 3 with stearic acid.

The conserved motif cysteines and disulfide bridges have considerable plasticity, allowing the ability to accommodate different ligands [20]. The plasticity of the disulphide bridge pattern can also be observed in **Figure A4**, where the *L. angustifolius* sequence maintain the hydrophobic environment only with 7 cysteines. However, the 3D and function of the protein is maintained and therefore is capable of binding to different ligands such as stearic acid or palmitic acid, among others. Notably, some of the nsLTPs can decrease specificity for ligands, which can be

#### **Figure 5.**

*Conservation analysis of nsLTPs of Lup an 3. Conservational analysis of Lup an 3 (Uniprot: A0A1J7GK90). the conservation values of Consurf was used to show the amino acids conservational index according with the colours scale (from purple – conserved to green – no conserved residues; yellow indicates no information found about this residues). Below the sequence, (e) indicates residue exposed; (b) indicated buried residue, according to the neural network algorithm in both cases; (f) highly conserved and exposed functional residue; (s) highly conserved and buried. The arrows (blue and yellow) indicate the highly conserved residues (with a value of 9) in all the sequences analysed. The yellow arrows indicate the cysteines of the conserved 8 cysteine motif and the blue arrows other representative conserved residues in the analysed sequences. Three-dimensional representation of proteins is depicted as spheres.*

attributed to the flexibility of the van der Waals volume of the internal hydrophobic cavities sufficient to accommodate single or double chain lipids [30].

**Table A4** shows examples of nsLTPs transport ligands of diverse nature: stearic acid (STE), 10-oxo-12-octadecenoic acid (ASY), prostaglandin B2 (E2P), 1-myristoyl-SN -glycerol-3-phosphocholine (LPC), and palmitic acid (PLM). Fatty acids are the main constituents of cellular membranes, in addition to their role as a source of energy, signalling and mediation in cellular transport. They also accumulate in the seeds of vegetables, such as palmitic acid, transported by Ole e 7, *L. angustifolius*, *G. max*, *C. cajan*,*T. pratense. A. precatorus*, *A. ipaensis*, and *T. subterraneum* (**Table A5**), which is also involved in the lipogenesis pathway. Therefore, LTPs make an important class of proteins performing membraneassociated signalling processes under different environmental stresses and an important function in lipids storage in seeds.

Prostaglandins are lipids derived from arachidonic acid that have an effect similar to gibberellins in the endosperm and maintain homeostasis and mediate pathogenesis in animals [31]. For example, prostaglandin B2, which can be

#### **Figure 6.**

*Protein-ligand interaction assessment for Lup an 3. Lup an 3 (Uniprot: A0A1J7GK90) interaction with stearic acid (STE). A) Lup an 3 protein in cartoon model with STE ligand in sphere model. Motif 8 cysteine pinpointed conserved cysteines that allow the hydrophobic environment for the lipid interaction. B) Lup an 3 protein interacting with STE ligand with disulphide bridges, in purple colour, created between the cysteines of the conserved 8 cysteine motif. C) Lup an 3 protein with STE ligand. Pink colour depicted the sites of interaction of the protein with the ligand. D) Interaction pocket of the STE ligand with the Lup an 3 protein.*

transported by Lup an 3.0101 and the analysed LTP of *P. vulgaris* (**Table A5**), plays a key role in the generation of the inflammatory response in animals [32], thus it could be involved in responses to allergies to LTPs.

The structural interaction between lipid ligands and nsLTP, as well as functional interaction with a plethora of proteins show the diversity of bound ligands and the heterogeneity of the binding and functionality. However, it is clear that the type and mode of lipid binding and proteins interactions with nsLTPs determine the biological function and if it affects the allergenic properties of nsLTPs.

#### **3.9 Protein interactions study of lupine and other species nsLTPs**

Potential functional pathways and molecular interactions of nsLTPs are shown in **Table A5**.

Among all proteins analysed, Lup an 3, Lup an 3.0101, *Medicago truncatula*, *L. albus*, and *L. angustifolius* interact with calmodulin-binding heat shock proteins and

with ATP-dependent DEAD-box RNA helicase (DDX1). The DDX1 family comprises enzymes that participate in RNA metabolism, and are associated with different cellular functions, including abiotic stress in plants, and regulation of cell maturation, growth, and differentiation [25]. It is observed that DDX1 interact directly with profilin present in the cytosol of all eukaryotic cells modifying the actin cytoskeleton dynamics in response to external signals or stimuli and protecting the cell from oxidative damage maintaining a redox state in the cytoplasm [33–35]. It seems that these LTPs are involved in the response to abiotic stress and cellular regulation processes, participating in the signalling pathways.

nsLTP-5 appears to interact primarily with other LTPs, such as several LTPs that belong to seed storage 2S albumin superfamily protein and are bifunctional inhibitors; or with LTPG1 protein, a glycosylphosphatidylinositol-bound LTP1 involved in the export of cuticular lipids and resistance against fungal pathogens [36]. It also interacts with the protein AT1G10770, which has an inhibitory role for pectin methyl-esterase participating in the growth of the pollen tube. Thus, it appears that nsLTP is primarily is associated with the seed storage function.

*Arabidopsis thaliana* (nsLTP-3), in addition to interacting with several seed storage proteins and with LTP4, also interacts with MYB96, a transcription factor that activates cuticular wax biosynthesis under drought stress, it is involved in the regulation of ABA (abscisic acid) biosynthesis, regulates seed germination and activates LTP3, or *A. thaliana* (nsLTP-3) in our case, in response to drought or frost [37]. *A. thaliana* (nsLTP-3) also interacts with ELP, a protein involved in transcriptional elongation and involved in oxidative stress signalling. In addition, LTP3 from *A. thaliana* is involved both in transport and storage in seeds, as well as in response to abiotic stresses, such as droughts or frosts, transporting lipids during the cuticle.

Ole e 7 interacts with other LTPs of seed storage 2S albumin superfamily and with LTP3. In addition, it interacts with AT3G58690, a protein kinase that may be involved in the post-translational modifications suffered by LTPs. It also interacts with an ELP, as does *A. thaliana* (nsLTP-3).

An example of an interaction network is the case of *Arabidopsis thaliana* (nsLTP-3 an nsLTP-4). The interactions with seed storage proteins and other LTPs are observed. Considering the interaction between LTP3 and LTP4 of *A. thaliana* as a model, we can observe that both proteins (LTP3 and LTP4) interact with some common proteins, which could be related to their functional roles, underlying the possibility that these proteins could transport lipids together.

Therefore, it can be concluded that nsLTPs are involved in signalling pathways in response to abiotic stress, such as drought or cool, response to pathogens such as fungi, and the storage of proteins and lipids in seeds and maintaining seed dormancy, as well as in many other functions.

#### **3.10 Analysis of potential allergenicity nsLTPs**

The nsLTPs sequences used in this study were comparatively analysed using databases such as Allergome, as described in the material and methods section. The analysis of the nsLTPs allergenicity assessment were based on primary structure of the protein, 2D and 3D, oligomerization state of proteins, functional features, as well as experimental results.

These analyses confirm the allergenic character of most of the nsLTPs sequences. These nsLTP sequences analysed are the following: All c 3 (*Allium cepa*), Ara h 17 (*Arachis hypogaea*), Aspa o 1 (*Asparagus officinalis*), Cas s 8 (*Castanea sativa*), Cit l 3 (*Citrus limonum*), Dau c 3 (*Daucus carota*), Len c 3 (*Lens culinaris*), Lup an 3 (*Lupinus angustifolius* L.), Lup an 3.0101 (*Lupinus angustifolius* L.), Mal d 3 (*Malus*

*domestica*), Mus a 3 (*Musa acuminata*), Ole e 7 (*Olea europaea*), Pha v 3.0201 (*Phaseolus vulgaris*), Sola l 7 (*Solanum lycopersicum*), Tri a 14 *(Triticum aestivum*) y Zea m 14 (*Zea mays*). nsLTPs have allergenic nature, which will help to continue the study of these proteins at molecular and functional level.

#### **3.11 IgE-binding epitope assessment**

Legumes contain proteins that share epitopes (full or partially), which would make possible to develop cross-reactivity between them. However, the similarity between sequences does not ensure cross-reactivity, since cases of atopic individuals have been observed occur no cross-allergenicity, even when both species share large similarity in proteins such as lupine and peanut vicilin (Ara h 1 and Lup an 1). In addition, none of the clinically studied lupine allergic individuals reacted to peanuts [4, 38, 39]. Recent studies have also shown clinically relevant crossreactivity of lupine with other legumes, such as lentils, beans, chickpeas, peas, soybeans, and almonds [4, 15, 18, 19, 40].

The IgE results from binding epitopes analysis (**Table A6**) reveal that all the proteins analysed present Allergen Representative Peptide (ARPs) sequences highlighted in red, representing residues that share the analysed sequences and the ARPs. The SVM analysis based on amino acid and dipeptide composition show that all sequences are allergenic or potentially allergenic. Considering that all the sequences are present in seeds, the relationship between these proteins and food allergies seems to have relationship.

It can also be observed in **Table A6** that Lup and 3 and *A. ipaensis* ARPs are comparable, pointing out that their IgE binding epitopes are very similar. Furthermore, cross-reactivity between lupine and *A. ipaensis* (wild peanut) seems to have clinical importance, especially considering different cases reported of crossreactivity between lupine and peanut [4]. Therefore, Lup an 3 appears susceptible to cross-reaction with *A. ipaensis*, based on their epitopes. The same situation occurs with the sequences Lup an 3.0101 and *P. alba; M. truncatula*, *P. vulgaris* and *G. soja; A. thaliana* (nsLTP-5), *V. unguiculata and A. duranensis; L. albus and P. sativum;* and, *L. culinaris and A. precatorus*. Members of the same protein families, in this case, nsLTPs, share IgE epitopes as depicted by our analysis, which can potentially lead to an allergic reaction due to cross-reactivity [41].

## **3.12 T-cell and B-cell binding epitope analysis**

Hypersensitivity reactions are mediated by IgE, T- and B- cells, and these cells play important roles contributing to the pathophysiology of a wide range of allergic reactions [42]. Analysis of T- and B-cell binding epitopes (**Tables A7** and **A8**) reveals up to nine T-cell and up to six B-cell epitopes, with significant differences between species. Cross-reactivity at the T-cell level depends on homologies between amino acid sequences. Regarding the T-cell epitopes found in the analysed sequences (**Table A7**), it can be observed that epitope T1 is present in all the analysed sequences and located in the same region of the analysed proteins, also containing comparable number and sequence of residues. T2 epitope is present in most the analysed sequences with the exception of nsLTP-3, Ole e 7, and *S. suberectus*. This suggests that T2 is also highly conserved among species and is involved in cross-allergenicity among them.

Regarding the B-cell epitopes (**Table A8**), B1 and B4 epitopes are present in most of the analysed sequences, with the exception of *M. truncatula*, *L. albus*, *L. angustifolius*, *C. arientinum*, and *P. sativum* for B1 epitope; and Lup an 3,

*A. thaliana* (nsLTP-3), *A. thaliana* (nsLTP-5),*T. pratense* and *T. subterraneum* for B4 epitope.

It is also important to note that the T2 epitope and the B4 epitope are the same, which could be relevant when it comes to the primary sensitization process to the nsLTPs sharing these epitopes.

Furthermore, it has been observed that B5 and B6 epitopes are unique for *Lupinus angustifolius* and *Spatholobus suberectus*, respectively. Interestingly, the B3 epitope was also present in the species widely used in food worldwide such as *M. truncatula*, *G. soja* (soybean), *L. culinaris* (lentil),*T. pratense*, *C arientinum* (chickpea), and *P. sativum* (pea).

## **4. Conclusions**

The functional analysis of nsLTPs proteins show comparable motifs in their primary sequence with prolamin storage proteins family and trypsin-alpha amylase inhibitors, involved in lipid transfer, biotic and abiotic stress response, and defence against pathogens. Differential post-translational modifications showed nsLTPs involvement in the regulation of nsLTP in multiple functional roles, beside lipid transfer. LTPs may also suffer redox-related modifications that would be related to copping different environmental stresses and signalling functions. The LTPs analysed sequences where primarily located close related to different membranes in the secretion pathway. This location is tightly related to LTPs signalling physiological functions, and the relative lipid abundance depending of the subcellular specific organelle locations.

Structural analysis and ligand interaction analysis of LTPs show the importance of the functional 8 cysteine motif (4 disulphide bridges), that are highly conserved and brings stability to nsLTPs, and maintaining the adequate hydrophobic environment for nsLTP-lipid of different nature interaction and transport, i.e. stearic acid or palmitic acid, among others.

nsLTPs has been identified as main allergens. The identification of binding IgE, T-cells, and B-cells epitopes allows us to confirm the potential allergenicity of these studied proteins such in the case of *L. angustifolius* and comparatively nsLTPs of other related and unrelated species, as well as the possibility of cross-allergenicity between some of them. This study has great application potential in the development of molecular tools for the diagnosis and allergy therapies to nsLTPs.

#### **Acknowledgements**

This study has been partially funded by The Spanish Ministry of Economy, Industry and Competitiveness through the grants Ref.: RYC-2014-16536 (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.

#### **Conflicts of interest**

The authors have declared that no competing interests exist.

## **A. Appendix**

#### **Figure A1.**

*Multiple alignment of nsLTPs. Eighth main nsLTP protein sequences have been aligned. The similarity index (0-10) between the aligned sequences. The conservation index is shown as yellow bars, and has values ranged from 0 to 10.* Lupinus angustifolius *(Lup an 3) Uniprot: A0A1J7GK90,* Lupinus angustifolius *(Lup an 3.0101) (Uniprot: A0A4P1RWD8),* Medicago truncatula *(Uniprot: A0A072UTH7),* Arabidopsis thaliana *(nsLTP-3) (Uniprot: Q9LLR7),* Arabidopsis thaliana *(nsLTP-5) (Uniprot: Q9XFS7),* Olea *europaea (Ole e 7) (NCBI: XP\_022893508.1),* Lupinus albus *(Uniprot: A0A6A5MQ88),* Lupinus angustifolius *(Uniprot: A0A4P1RV83),* Glycine max *(Uniprot: I1J7M1),* Arachis hypogaea *(NCBI: XP\_025656480.1),* Cajanus cajan *(NCBI: XP\_020237462),* Phaseolus vulgaris *(Uniprot: D3W146),* Glycine soja *(Uniprot: A0A445M2F4),* Lens culinaris *(Uniprot: A0AT33),* Trifolium pratense *(Uniprot: A0A2K3M7A7),* Spatholubus suberectus *(NCBI: TKY63608.1),* Cicer arietinum *(Uniprot: O23758),* Vigna ungiculata *(Uniprot: UPI0010170F74),* Abrus precatorus *(Uniprot: UPI000F7C313B),* Arachis ipaensis *(NCBI: XP\_020971907.1),* Trifolium subterraneum *(NCBI: GAU29990.1),* Prosopis alba *(NCBI: XP\_028808641.1),* Vigna angularis *(NCBI: KOM57753.1),* Arachis duranensis *(NCBI: XP\_015950831.1),* Pisum sativum *(NCBI: A0A158V755.1).*

#### **Figure A2.**

*3D structure of nsLTPs. 3D structures modeling of Lup an 3.0101 (Uniprot: A0A4P1RWD8), Medicago truncatula (Uniprot: A0A072UTH7), Arabidopsis thaliana 3 (Uniprot: Q9LLR7), Arabidopsis thaliana 5 (Uniprot: Q9XFS7), Lupinus angustifolius (Uniprot: A0A4P1RV83). Cartoon mode representation were build using Phymol software. A-helices are depicted in red color.*

#### **Figure A3.**

*Conservation analysis of nsLTPs of nsLTPs. Conservational analysis of Lup an 3.0101 (Uniprot: A0A4P1RWD8), Medicago truncatula (Uniprot: A0A072UTH7),* Arabidopsis thaliana *3 (Uniprot: Q9LLR7),* Arabidopsis thaliana *5 (Uniprot: Q9XFS7),* Lupinus angustifolius *(Uniprot: A0A4P1RV83), Ole e 7 (NCBI: XP\_022893508.1) and* Lupinus albus *(Uniprot: A0A6A5MQ88). The conservation values of Consurf was used to show the amino acids conservational index according with the colours scale (from purple – conserved to green – no conserved residues; yellow indicates no information found about this residues). Below the sequence, (e) indicates residue exposed; (b) indicated buried residue, according to the neural network algorithm in both cases; (f) highly conserved and exposed functional residue; (s) highly conserved and buried. The arrows (blue and yellow) indicate the highly conserved residues (with a value of 9) in all the sequences analysed. The yellow arrows indicate the cysteines of the conserved 8 cysteine motif and the blue arrows other representative conserved residues in the analysed sequences. Three-dimensional representation of proteins is depicted as spheres.*

#### **Figure A4.**

*Protein-ligand interaction assessment for nsLTP. Lup an 3.0101 (Uniprot: A0A4P1RWD8), Medicago truncatula (Uniprot: A0A072UTH7),* Arabidopsis thaliana *3 (Uniprot: Q9LLR7),* Arabidopsis thaliana *5 (Uniprot: Q9XFS7),* Lupinus angustifolius *(Uniprot: A0A4P1RV83), Ole e 7 (NCBI: XP\_022893508.1) and* Lupinus albus *(Uniprot: A0A6A5MQ88) interaction with their respective ligands. A) Lup an 3 protein in cartoon model and ligand in sphere model. Motif 8 cysteine pinpointed conserved cysteines that allow the hydrophobic environment for the lipid interaction. B) Disulphide bridges representation in purple colour, created between the cysteines of the conserved 8 cysteine motif. C) Pink colour depicted the sites of interaction of the protein with the ligand. D) Interaction pocket of the ligand with the nsLTP protein.*






**Table A1.** *Post-translational modifications: glycosilations, phosphorylations, myristoilations and post-translational modifications related to redox metabolism.*


**Sequence T S Y** • 108: PYKISTSTN (cdc2) *Lens culinaris* - • 26: GAVTSDLSP (cdc2) • 29: TSDLSPCLT (cdk5) • 42: GPGPSPQCC (cdk5) • 42: GPGPSPQCC (GSK3) - *Trifolium pratense* • 38: QLTLTPCLG (cdk5) • 4: -MASSMLVK (cdc2)) • 85: STALSLPGL (CKI) • 96: PAAASILAK (cdc2) • 112: KISPSIDCN (cdc2) • 107: VNLPYKISP (unsp) • 118: DCNTYISLN (unsp) • 118: DCNTYISLN (INSR) *Spatholobus suberectus* - • 149: LMLSSFLCI (cdc2) - *Cicer arientinum* • 65: AAVTTPDRQ (cdk5) • 106: PYKISTSTN (cdc2) • 41: PCLGYLQGG (unsp) *Vigna unguiculata* • 72: GDRRTACNC (PKG) • 72: GDRRTACNC (CKI) • 36: TSAISPCIG (cdk5) • 108: PYRISPSTN (cdk5) • 108: PYRISPSTN (cdc2) • 45: PCIGYLRGG (unsp) *Abrus precatorius* • 39: VNNLTPCIS (GSK3) • 58: AQCCSGVKN (cdc2) • 93: FTYTSFNLN (cdc2) • 115: PYQISPNTD (cdk5) • 44: PCISYVVYG (unsp) • 91: SGFTYTSFN (unsp) • 91: SGFTYTSFN (INSR) • 112: VNIPYQISP (unsp) *Arachis ipaensis* • 66: AGARTPADR (cdk5) • 66: AGARTPADR (GSK3) • 80: CLKTSAGQV (PKG) • 94: ANAGSLPSK (CKI) • 94: ANAGSLPSK (DNAPK) • 94: ANAGSLPSK (cdc2) • 105: VNIPYKISP (unsp) *Trifolium subterraneum* • 69: QAKSTPDRR (cdk5) • 97: ALASTPTKC (cdk5) • 4: -MASSMLVK (cdc2) • 85: STIFSLPGI (DNAPK) • 85: STIFSLPGI (CKI) • 85: STIFSLPGI (cdc2) • 43: PCLGYLRNP (unsp) • 107: INLPYKISP (unsp) *Prosopis alba* • 33: GQVTTSLAP (cdc2) • 34: QVTTSLAPC (DNAPK) • 41: PCLSYLQSG (unsp) *Vigna angularis* • 65: SSRTTPDRR (cdk5) - • 103: VNLPYKISA (unsp) *Arachis duranensis* - • 82: SVAGSLGSQ (CKI) • 85: GSLGSQINL (DNAPK) • 85: GSLGSQINL (ATM) • 108: PYKISTSTN (cdc2) • 41: PCFGYLKSG (unsp) *Pisum sativum* • 69: AATTTPDRQ (cdk5) • 91: SRLNTNNAA (RSK) • 4: -MATSMKLA (cdc2) • 28: EAALSCGTV (CKI) • 50: PNNASPPPP (cdK5) • 42: PCLTYLQAP (unsp)

#### *Molecular and Functional Characterisation of Allergenic Non-specific Lipid Transfer… DOI: http://dx.doi.org/10.5772/intechopen.102889*

*Phosphorylations are classified by modified residues. T: threonine; S: serine; Y: tyrosine.*

#### **Table A2.**

*Post-translational modifications. phosphorylations.*








**Table A3.**

*Post-translational modifications. carbonylations.*





*This table presents the main ligands of each nsLTP analyzed and the number of residues involved in the protein-ligand binding site. The three highest scoring ligands are shown for each nsLTP.*

#### **Table A4.**

*Interaction motives in nsLTPs for ligands of lipidic nature.*




#### **Table A5.**

*Analysis of functional interaction between nsLTPs and protein ligands.*



 **A6.**

**Table***IgEbindingepitopes.*


## *Molecular and Functional Characterisation of Allergenic Non-specific Lipid Transfer…*


**Table A7.** *Identification*

 *of T-cell binding epitopes.*

## *Legumes Research - Volume 1*



## **Table A8.**

*Identification of B-cell binding epitopes.*

## **Author details**

Maria Rodrigo-Garcia<sup>1</sup> , Esther Rodriguez-de Haro<sup>1</sup> , Salvador Priego-Poyato<sup>1</sup> , Elena Lima-Cabello<sup>1</sup> , Sonia Morales-Santana<sup>2</sup> and Jose C. Jimenez-Lopez1,3\*

1 Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Spanish National Research Council (CSIC), Granada, Spain

2 Proteomic Research Department, San Cecilio University Hospital, Biosanitary Research Institute of Granada (Ibs.GRANADA), Granada, Spain

3 The UWA Institute of Agriculture and School of Agriculture and Environment, The University of Western Australia, CRAWLEY Perth, WA, Australia

\*Address all correspondence to: josecarlos.jimenez@eez.csic.es

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **References**

[1] Kohajdová Z, Karovičová J, Schmidt Š. Lupin composition and possible use in bakery: A review. Czech Journal of Food Sciences. 2011;**29**:203-211

[2] Carvajal-Larenas FE, Linnemann AR, Nout MJR, Koziol M, van Boekel MAJS. *Lupinus mutabilis*: Composition, uses, toxicology, and debittering. Crit Rev Food Sci Nutr. 2016;**56**(9): 1454-1487

[3] Jimenez-Lopez JC. Narrow-leafed lupin (*Lupinus angustifolius* L.) βconglutin: A multifunctional family of proteins with roles in plant defence, human health benefits, and potential uses as functional food. Legume Science. 2020;**2**(2):1-8

[4] Villa C, Costa J, Mafra I. Lupine allergens: Clinical relevance, molecular characterization, cross-reactivity, and detection strategies. Comprehensive Reviews in Food Science and Food Safety. 2020;**19**(6):3886-3915

[5] Lima-Cabello E, Robles-Bolivar P, Alché JD, Jimenez-Lopez JC. Narrow leafed lupin beta-conglutin proteins epitopes identification and molecular features analysis involved in crossallergenicity to peanut and other legumes. Genomics and Computational Biology. 2016;**2**(1):29

[6] Priego-Poyato S, Rodrigo-Garcia M, Escudero-Feliu J, Garcia-Costela M, Lima-Cabello E, Carazo-Gallego A, et al. Current advances research in nutraceutical compounds of legumes, Pseudocereals and cereals. In: Grain and Seed Proteins Functionality. IntechOpen; 2021

[7] Jimenez-Lopez JC, Foley RC, Brear E, Clarke VC, Lima-Cabello E, Florido JF, et al. Characterization of narrow-leaf lupin (*Lupinus angustifolius* L.) recombinant major allergen IgE-binding proteins and the natural β-conglutin

counterparts in sweet lupin seed species. Food Chemistry. 2018:60-70

[8] James AP, Jayasena V. Effect of germination on the nutritional and protein profile of Australian Sweet Lupin (*Lupinus angustifolius* L.). Food and Nutrition Science. 2012;**3**:621-626

[9] Mohan VR, Tresina PS, Daffodil ED. Antinutritional factors in legume seeds: Characteristics and determination. Encyclopedia of Food and Health. 2016; **1**:211-220

[10] Samtiya M, Aluko RE, Dhewa T. Plant food anti-nutritional factors and their reduction strategies: An overview. Food Production, Processing and Nutrition. 2020;**2**(1):1-14

[11] Jimenez-Lopez JC, Lima-Cabello E, Melser S, Foley RC, Singh KB, Alché JD. Lupin allergy: Uncovering structural features and epitopes of β-conglutin proteins in Lupinus Angustifolius L. with a focus on cross-allergenic reactivity to peanut and other legumes. Lecture Notes in Computer Science. 2015;**9043**:96-107

[12] Campbell CP, Jackson AS, Johnson AR, Thomas PS, Yates DH. Occupational sensitization to lupin in the workplace: Occupational asthma, rhinitis, and work-aggravated asthma. Journal of Allergy and Clinical Immunology. 2007;**119**(5):1133-1139

[13] Crespo JF, Rodríguez J, Vives R, James JM, Burbano C, Muzquiz M. Occupational IgE-mediated allergy after exposure to lupine seed flour. Journal of Allergy and Clinical Immunology. 2001; **108**(2):295-297

[14] Van Kampen V, Sander I, Quirce S, Brüning T, Merget R, Raulf M. IgE sensitization to lupine in bakers-Crossreactivity or co-sensitization to wheat

flour? International Archives of Allergy and Immunology. 2015;**166**:63-70

[15] Guillamón E, Rodríguez J, Burbano C, Muzquiz M, Pedrosa MM, Cabanillas B, et al. Characterization of lupin major allergens (*Lupinus albus* L.). Molecular Nutrition and Food Research. 2010;**54** (11):1668-1676

[16] Sanz ML, De Las Marinas MD, Fernández J, Gamboa PM. Lupin allergy: A hidden killer in the home. Clinical and Experimental Allergy. 2010;**40**: 1461-1466

[17] Verma AK, Kumar S, Das M, Dwivedi PD. A comprehensive review of legume allergy. Clinical Reviews in Allergy and Immunology. 2013;**45**: 30-46

[18] Peeters KABM, Koppelman SJ, Penninks AH, Lebens A, Bruijnzeel-Koomen CAFM, Hefle SL, et al. Clinical relevance of sensitization to lupine in peanut-sensitized adults. Allergy: European Journal of Allergy & Clinical Immunology. 2009;**64**(4):549-555

[19] Jappe U, Vieths S. Lupine, a source of new as well as hidden food allergens. Molecular Nutrition and Food Research. 2010;**54**:113-126

[20] Breiteneder H, Mills C. Nonspecific lipid-transfer proteins in plant foods and pollens: An important allergen class. Current Opinion in Allergy and Clinical Immunology. 2005;**5**:275-279

[21] Salcedo G, Sánchez-Monge R, Barber D, Díaz-Perales A. Plant nonspecific lipid transfer proteins: An interface between plant defence and human allergy. Biochimica et Biophysica Acta—Molecular and Cell Biology Lipids. 2007;**1771**(6):781-791

[22] Liu F, Zhang X, Lu C, Zeng X, Li Y, Fu D, et al. Non-specific lipid transfer proteins in plants: Presenting new advances and an integrated functional

analysis. Journal of Experimental Botany. 2015;**66**:5663-5681

[23] Zuidmeer L, Van Ree R. Lipid transfer protein allergy: Primary food allergy or pollen/food syndrome in some cases. Current Opinion in Allergy and Clinical Immunology. 2007;**7**:269-273

[24] Martín PL. Proteínas Tranferidoras de Lípidos (nsLTP): una familia de proteínas implicada en la alergia al tomate. Universidad Complutense de Madrid; 2019

[25] Macovei A, Vaid N, Tula S, Tuteja N. A new DEAD-box helicase ATP-binding protein (OsABP) from rice is responsive to abiotic stress. Plant Signalling Behavior. 2012;**7**(9):1138-1143

[26] Bertioli DJ, Cannon SB, Froenicke L, Huang G, Farmer AD, Cannon EKS, et al. The genome sequences of Arachis duranensis and Arachis ipaensis, the diploid ancestors of cultivated peanut. Nature Genetics. 2016;**48**(4):438-446

[27] Rico M, Bruix M, González C, Monsalve RI, Rodríguez R. 1H NMR assignment and global fold of napin BnIb, a representative 2S albumin seed protein. Biochemistry. 1996;**35**(49): 15672-15682

[28] Freire JEC, Moreno FBMB, Monteiro-Júnior JE, Sousa AJS, Vasconcelos IM, Oliveira JTA, et al. Mo-CBP3, a 2S albumin from *Moringa oleifera*, is a complex mixture of isoforms that arise from different posttranslational modifications. Plant Physiology and Biochemistry. 2019;**140**: 68-77

[29] van Ree R. Allergens: Structure and function. In: Akdis CA, Agache I, editors. EAACI Global Atlas of Allergy. Zurich: European Academy of Allergy and Clinical Immunology; 2014. pp. 6-8

[30] Scheurer S, Schülke S. Interaction of non-specific lipid-transfer proteins with plant-derived lipids and its impact on allergic sensitization. Frontiers in Immunology. 2018

[31] Dooper MM, Plassen C, Holden L, Lindvik H, Faeste CK. Immunoglobulin E cross-reactivity between lupine conglutins and peanut allergens in serum of lupine-allergic individuals. Journal of Investigational Allergology and Clinical Immunology. 2009;**19**(4): 283-291

[32] Ricciotti E, Fitzgerald GA. ATVB in Focus Inflammation Prostaglandins and Inflammation 2011

[33] Zuidmeer L, Salentijn E, Rivas MF, Mancebo EG, Asero R, Matos CI, et al. The role of profilin and lipid transfer protein in strawberry allergy in the Mediterranean area. Clinical and Experimental Allergy. 2006;**36**(5):666- 675

[34] Andersen M-BS, Hall S, Dragsted LO. Identification of European allergy patterns to the Allergen Families PR-10, LTP, and Profilin from Rosaceae Fruits. Clinical Reviews in Allergy and Immunology. 2009; **41**(1):4-19

[35] Pan JL, Bardwell JC. The origami of thioredoxin-like folds. Protein Sci. 2006;**15**(10):2217-2227

[36] Fahlberg P, Buhot N, Johansson AN, Ats M, Andersson X. Involvement of lipid transfer proteins in resistance against a non-host powdery mildew in *Arabidopsis thaliana*. Molecular Plant Pathology. 2019;**20**

[37] Seo PJ, Lee SB, Suh MC, Park MJ, Go YS, Park CM. The MYB96 transcription factor regulates cuticular wax biosynthesis under drought conditions in Arabidopsis. Plant Cell. 2011;**23**(3): 1138-1152

[38] Sirtori E, Resta D, Brambilla F, Zacherl C, Arnoldi A. The effects of various processing conditions on a protein isolate from *Lupinus angustifolius*. Food Chemistry. 2010; **120**(2):496-504

[39] Goggin DE, Mir G, Smith WB, Stuckey M, Smith PMC. Proteomic analysis of lupin seed proteins to identify conglutin β as an allergen, Lup an 1. Journal of Agricultural and Food Chemistry. 2008;**56**(15):6370-6377

[40] Holden L, Sletten GBG, Lindvik H, Fæste CK, Dooper MMBW. Characterization of IgE binding to lupin, peanut and almond with sera from lupin-allergic patients. International Archives of Allergy and Immunology. 2008;**146**(4):267-276

[41] Bohle B. Allergens and crossreactivity. In: Akdis C, Agache I, editors. EAACI Global Atlas of Allergy. Zurich: European Academy of Allergy and Clinical Immunology; 2014. pp. 11-12

[42] Abdel-Gadir A, Chatila T. B Cells. In: Akdis C, Agache I, editors. EAACI Global Atlas of Allergy. European Academy of Allergy and Clinical Immunology; 2014. pp. 62-64

## **Chapter 9**
