Domains and functional motives of nsLTPs.
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 B-cell 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,
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].
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.europa.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
Plant nsLTPs are small extracellular proteins, which includes a significant number of allergens [20, 21, 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
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 (
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.php), and reprOlive (http://www.scbi.uma.es/olivodb/).
We retrieved 25 sequences as follow: The sequences and their access number are:
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.ebi.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 N-glycosylations, 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 post-translational 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://cello.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
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,
Scientific name and accession number | Number of aminoacids | Prosite (ID) | |
---|---|---|---|
120 | Tryp_alpha_amyl (Inhibidor proteasa/almacenamiento semillas/familia LTP) Clan: Prolamina (CL0482) | PLANT_LTP (PS00597) | |
116 | Tryp_alpha_amyl (Inhibidor proteasa/almacenamiento semillas/familia LTP) Clan: Prolamina (CL0482) | PLANT_LTP (PS00597) [94-115] | |
116 | Tryp_alpha_amyl (Inhibidor proteasa/almacenamiento semillas/familia LTP) Clan: Prolamina (CL0482) | PLANT_LTP (PS00597) [93-114] | |
115 | Tryp_alpha_amyl (Inhibidor proteasa/almacenamiento semillas/familia LTP) Clan: Prolamina (CL0482) | PLANT_LTP (PS00597) [93-114] | |
104 | Tryp_alpha_amyl (Inhibidor proteasa/almacenamiento semillas/familia LTP) Clan: Prolamina (CL0482 | PLANT_LTP (PS00597) [82-103] | |
117 | Tryp_alpha_amyl (Inhibidor proteasa/almacenamiento semillas/familia LTP) Clan: Prolamina (CL0482) | PLANT_LTP (PS00597) [95-116] | |
132 | Tryp_alpha_amyl (Inhibidor proteasa/almacenamiento semillas/familia LTP) Clan: Prolamina (CL0482) | — | |
131 | Tryp_alpha_amyl (Inhibidor proteasa/almacenamiento semillas/familia LTP) Clan: Prolamina (CL0482) | PLANT_LTP (PS00597) | |
117 | Tryp_alpha_amyl (Inhibidor proteasa/almacenamiento semillas/familia LTP) Clan: Prolamina (CL0482) | PLANT_LTP (PS00597) [95-116] | |
129 | Tryp_alpha_amyl (Inhibidor proteasa/almacenamiento semillas/familia LTP) Clan: Prolamina (CL0482) | PLANT_LTP (PS00597) [95-116] | |
121 | Tryp_alpha_amyl (Inhibidor proteasa/almacenamiento semillas/familia LTP) Clan: Prolamina (CL0482) | PLANT_LTP (PS00597) [99-120] | |
115 | Tryp_alpha_amyl (Inhibidor proteasa/almacenamiento semillas/familia LTP) Clan: Prolamina (CL0482) | PLANT_LTP (PS00597) [93-114] | |
115 | Tryp_alpha_amyl (Inhibidor proteasa/almacenamiento semillas/familia LTP) Clan: Prolamina (CL0482) | — | |
110 | Tryp_alpha_amyl (Inhibidor proteasa/almacenamiento semillas/familia LTP) Clan: Prolamina (CL0482) | PLANT_LTP (PS00597) [87-108] | |
130 | Tryp_alpha_amyl (Inhibidor proteasa/almacenamiento semillas/familia LTP) Clan: Prolamina (CL0482) | LEUCINE_ZIPPER (PS00029) [84-105] | |
165 | Tryp_alpha_amyl (Inhibidor proteasa/almacenamiento semillas/familia LTP) Clan: Prolamina (CL0482) | LEUCINE_ZIPPER (PS00029) [6-27; 13-34] | |
116 | Tryp_alpha_amyl (Inhibidor proteasa/almacenamiento semillas/familia LTP) Clan: Prolamina (CL0482) | PLANT_LTP (PS00597) [93-114] | |
117 | Tryp_alpha_amyl (Inhibidor proteasa/almacenamiento semillas/familia LTP) Clan: Prolamina (CL0482) | PLANT_LTP (PS00597) [95-116] | |
124 | Tryp_alpha_amyl (Inhibidor proteasa/almacenamiento semillas/familia LTP) Clan: Prolamina (CL0482) | PLANT_LTP (PS00597) [102-123] | |
118 | Tryp_alpha_amyl (Inhibidor proteasa/almacenamiento semillas/familia LTP) Clan: Prolamina (CL0482) | PLANT_LTP (PS00597) [95-116] | |
123 | Tryp_alpha_amyl (Inhibidor proteasa/almacenamiento semillas/familia LTP) Clan: Prolamina (CL0482) | — | |
117 | Tryp_alpha_amyl (Inhibidor proteasa/almacenamiento semillas/familia LTP) Clan: Prolamina (CL0482) | PLANT_LTP (PS00597) [94-115] | |
122 | Tryp_alpha_amyl (Inhibidor proteasa/almacenamiento semillas/familia LTP) Clan: Prolamina (CL0482) | — | |
117 | Tryp_alpha_amyl (Inhibidor proteasa/almacenamiento semillas/familia LTP) Clan: Prolamina (CL0482) | PLANT_LTP (PS00597) [95-116] | |
120 | Tryp_alpha_amyl (Inhibidor proteasa/almacenamiento semillas/familia LTP) Clan: Prolamina (CL0482) | PLANT_LTP (PS00597) [97-118] |
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,
Interestingly, it is also observed that the most related species based on nsLTPs comparisons are the species of the genus
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
Regarding
3.2 nsLTPs physical and chemical proprieties analysis
The physical and chemical properties analysed were described in Table 2. The longest sequence is
Sequence | Number of aminoacids | MW (Da) | Ip | Aliphatic index | Stability index |
---|---|---|---|---|---|
120 | 12022.25 | 8.88 | 99.17 | 33.21 | |
116 | 11719.91 | 9.38 | 98.53 | 39.47 | |
116 | 11381.51 | 9.04 | 89.40 | 31.06 | |
115 | 11691.97 | 9.04 | 85.74 | 42.46 | |
104 | 10993.72 | 11.00 | 77.12 | 23.91 | |
117 | 11872.06 | 9.14 | 95.04 | 27.12 | |
132 | 13209.80 | 9.43 | 102.20 | 34.59 | |
131 | 13691.95 | 8.62 | 84.89 | 42.53 | |
117 | 12219.40 | 9.69 | 87.52 | 32.17 | |
129 | 13284.65 | 8.79 | 92.17 | 52.63 | |
121 | 12733.98 | 9.24 | 98.18 | 21.54 | |
115 | 11778.77 | 9.23 | 83.91 | 34.12 | |
115 | 11487.36 | 9.10 | 84.17 | 17.21 | |
110 | 11024.96 | 8.75 | 86.09 | 30.02 | |
130 | 13792.45 | 9.02 | 108.08 | 39.97 | |
165 | 17354.17 | 8.84 | 87.70 | 31.69 | |
116 | 11587.65 | 9.07 | 93.53 | 29.50 | |
117 | 12125.32 | 10.46 | 88.46 | 37.20 | |
124 | 13061.24 | 9.04 | 92.74 | 22.15 | |
118 | 12009.36 | 9.30 | 97.54 | 37.16 | |
123 | 12995.34 | 9.28 | 99.19 | 45.62 | |
117 | 11910.02 | 8.76 | 99.32 | 45.59 | |
122 | 12744.89 | 9.21 | 81.48 | 36.50 | |
117 | 11777.91 | 9.24 | 90.94 | 15.46 | |
120 | 12095.29 | 8.89 | 85.58 | 34.45 |
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
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
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
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,
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 (
3.4 nsLTPs subcellular location
The subcellular location of the 25 nsLTP proteins has been identified and the results are shown in Table 3.
Secuencia | CELLO | WOLF PSORT | PSORT |
---|---|---|---|
Lup an 3 | Ec | Ec | Ec |
Lup an 3.0101 | Ec | Ec | Ec/Vac |
Ec | Ec | Ec/Vac | |
Ec | Ec | Ec/Vac | |
Ec | Ec | Ec | |
Ole e 7 | Ec | Ec | Ec/Vac |
Ec | Ec | Ec | |
Ec | Ec | MP | |
Ec | Ec | MP | |
Ec | Ec | Ec | |
Ec | Ec | Ec | |
Ec | Ec | Ec | |
Ec | Ec | Ec | |
Ec | Ec | Ec/Vac | |
Ec | Ec | Ec | |
Ec | Ec | MP | |
Ec | Ec | Ec | |
Ec | Ec | Ec | |
Ec | Ec | Ec | |
Ec | Ec | Ec | |
Ec | Ec | Ec/Vac | |
Ec | Ec | Ec | |
Ec | Ec | Ec | |
Ec | Ec | Ec/Vac | |
Ec | Ec | MP/MRE |
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,
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, 21, 22]. In this regard, the secondary structure of LTPs is very important to maintain the binding 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 α-helices 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, 21, 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,
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
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 (
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,
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
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,
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 transported by Lup an 3.0101 and the analysed LTP of
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,
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.
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
An example of an interaction network is the case of
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 (
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 cross-reactivity 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
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
Regarding the B-cell epitopes (Table A8), B1 and B4 epitopes are present in most of the analysed sequences, with the exception of
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
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
Acknowledgments
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.
Sequence | N-Glycosilation (start-end): ‘modification’ (PS00001) | Phosphorilation (start-end): ‘modification’ | N-Myristoilation (start-end): ‘modification’ (PS00008) | Posttranslational modification – REDOX metabolism | ||||
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MAPK (NethPhos) | PKC (PS00005) | CK2 (PS00006) | PKA | S-nitrosylation (C) | T-nitration (Y) | |||
Lup an 3 (Uniprot: A0A1J7GK90) | - | - |
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Lup an 3.0101 (Uniprot: A0A4P1RWD8) | - |
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Medicago truncatula (Uniprot: A0A072UTH7) | - |
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Ole e 7 (NCBI: XP_022893508.1) |
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Sequence | T | S | Y |
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Sequence | Carbonylations | |||
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Protein name and accesión number | ligad | Residues involved in the interactino |
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Acid esteárico (STE) | V31, L34, A35, C37, I38, V55, L58, V59, A62, L75, I82, L93, V101, Y103, I105 | |
Ácid 10-oxo-12-octadecenoic (ASY) | V31, L34, A35, L75, S77, A78, V79, A81, I82, I105 | |
Lauroil (LAP) | V55, V59, K68, A90, S100, V101, P102, Y103 | |
Prostaglandine B2 (E2P) | L35, C38, L42, C52, I56, I59, A63, C75, L76, A79, L94, I102, P103, Y104, K105, I106 | |
1-Miristoil-SN-glicerol-3-fosfocolina (LPC) | L60, R69, C73, L76, A80, A91, N101, K105, S107, T108, I115 | |
Ácido 10-oxo-12-octadecenoico (ASY) | T32, L35, A36, L76, A78, A79, A80, N82, T83, I106 | |
Ácido esteárico (STE) | T31, L34, A35, C37, I38, V55, L58, N59, A62, L75, I82, L93, I101, Y103, I105 | |
Ácido 10-oxo-12-octadecenoico (ASY) | T31, L34, A35, L75, S77, A78, A79, A81, I82, I105 | |
1-Miristoil-SN-glicerol-3-fosfocolina (LPC) | N59, R68, C72, L75, A79, A90, A100, P104, K106, C107, K114 | |
Ácido esteárico (STE) | A31, L34, A35, C37, A38, V55, L58, N59, A62, I75, I82, L93, I101, Y103, I105 | |
Ácido 10-oxo-12-octadecenoico (ASY) | A31, L34, A35, I75, S77, T78, A79, S81, I82, I105 | |
1-Miristoil-SN-glicerol-3-fosfocolina (LPC) | N59, R68, C72, I75, A79, A90, S100, P104, P106, M107, I114 | |
Ácido esteárico (STE) | T19, L22, G23, C25, Y26, V43, L46, N47, A50, I63, L70, L82, I90, Y92, I94 | |
Ácido 10-oxo-12-octadecenoico (ASY) | T19, L22, G23, I63, G65, A66, A67, A69, L70, I94 | |
1-Miristoil-SN-glicerol-3-fosfocolina (LPC) | N47, R56, C60, I63, A67, A79, R89, P93, S95, A96, V103 | |
Ácido palmítico (PLM) | V32, L36, V43, I57, L60, Y61, I73, L77, A92, L95, P96, V101, V103, Y105 | |
Ácido 10-oxo-12-octadecenoico (ASY) | V33, L36, K37, L77, S79, L80, A81, S83, F84, I107 | |
1-Miristoil-SN-glicerol-3-fosfocolina (LPC) | L36, K37, L40, I57, L80, S83, F84 | |
Stearic acid (STE) | T32, L35, A36, C38, I39, V56, L59, V60, A63, L76, I83, L93 , I101, Y103, I105 | |
Ácid 10-oxo-12-octadecenoic (ASY) | T32, L35, A36, L76, S78, A79, A80, A82, I83, I105 | |
Trifluoroacetil (TFA) | L59, V60, A63, A72, R100, I101, P102, Y103 | |
Palmitic acid (PLM) | V31, L35, L42, V56, L59, V60, A72, L76, A90, L93, P94, V99, L101, Y103, I105 | |
Ácid 10-oxo-12-octadecenoico (ASY) | V32, L35, A36, L76, S78, V79, A80, S82, T83, I105 | |
Stearic acid (STE) | V32, L35, A36, C38, L39, V56, L59, V60, A63, L76, T83, L93, L101, Y103, I105 | |
Palmitic acid (PLM) | V32, L36, L43, V57, I60, V61, V73, L77, A92, L95, P96, V101, I103, Y105, I107 | |
1-Miristoil-sn-glicerol-3-fosfocoline (LPC) | V61, R70, C74, L77, A81, A92, N102, K106, S108, T109, I116 | |
Ácid 10-oxo-12-octadecenoic (ASY) | Q33, L36, A37, L77, I79, A80, A81, A83, V84, I107 | |
Stearic acid (STE) | T33, L36, T37, C39, L40, V57, I60, L61, A64, L77, V84, L95, I103, Y105, I107 | |
Ácid 10-oxo-12-octadecenoic (ASY) | T33, L36, T37, L77, S79, S80, A81, Q83, V84, I107 | |
1-Miristoil-SN-glicerol-3-fosfocoline (LPC) | L61, R70, C74, L77, A81, A92, N102, K106, S108, P109, I116 | |
Palmitic acid (PLM) | V33, L37,V44, I59, L62, Y63, V75, I79, A96, L99, P100, V105, I107, Y109 | |
Ácid 10-oxo-12-octadecenoic (ASY) | V34, L37, T38, I79, N81, A82, I83, A85, I86, I111 | |
1-Miristoil-sn-glicerol-3-fosfocoline (LPC) | Y63, R72, C76, I79, I83, A96, N106, K110, S112, P113, V120 | |
Prostaglandine B2 (E2P) | L34, C37, L41, C51, V55, I58, A62, C74, L75, A78, L93, I101, P102, Y103, K104, I105 | |
Ácid 10-oxo-12-octadecenoic (ASY) | Q31, L34, V35, L75, T77, A78, A79, A81, V82, I105 | |
1-Miristoil-SN-glicerol-3-fosfocoline (LPC) | M59, I68, C72, L75, A79, A90, N100, K104, S106, T107, I114 | |
Stearic acid (STE) | T33, L36, I37, C39, I40, V57, L60, N61, A64, L77, I84, L95, I103, Y105, I107 | |
Ácid 10-oxo-12-octadecenoic (ASY) | T33, L36, I37, L77, S79, A80, A81, Q83, I84, I107 | |
1-Miristoil-SN-glicerol-3-fosfocoline (LPC) | N61, R70, C74, L77, A81, A92, S102, K106, S108, T109 | |
Stearic acid (STE) | T25, L28, S29, C31, L32, V49, L52, L53, A56, L69, I76, L87, I95, Y97, I99 | |
Ácid 10-oxo-12-octadecenoic (ASY) | T25, L28, S29, L69, S71, A72, A73, S75, I76, I99 | |
1-Miristoil-SN-glicerol-3-fosfocoline (LPC) | L53, R62, A66, L69, A73, A84, N94, K98, S100, T101, V108 | |
Palmitic acid (PLM) | V33, L37, L44, I59, L62, N63, V75, L79, A94, I97, L98, V103, L105, Y107, L130 | |
Ácido 10-oxo-12-octadecenoico (ASY) | Q34, L37, T38, L79, S81, T82, A83, S85, L86, I109 | |
Group trifluoroacetil (TFA) | L62, N63, A66, V75, N104, L105, P106, Y107 | |
Stearic acid (STE) | E59, L62, A63, C65, I66, V83, I86, L87, A90, L103, V110, L121, I129, Y131, I133 | |
Ácid 10-oxo-12-octadecenoic (ASY) | E59, L62, A63, L103, T105, A106, A107, A109, V110, I133 | |
Myristic acid (MYR) | V83, L87, A90, R96 | |
Stearic acid (STE) | S31, L34, A35, C37, L38, V55, L58, N59, A62, L75, I82, S93, I101, Y103, I105 | |
Ácid 10-oxo-12-octadecenoic (ASY) | S31, L34, A35, L75, S77, A78, A79, S81, I82, I105 | |
1-Miristoil-SN-glicerol-3-fosfocolina (LPC) | N59, R68, C72, L75, A79, A90, N100, K104, S106, T107, I114 | |
Ácido esteárico (STE) | T32, I35, S36, C38, I39, V57, L60, N61, A64, L77, F84, L95, I103, Y105, I107 | |
1-Miristoil-SN-glicerol-3-fosfocolina (LPC) | N61, R70, C74, L77, A81, A92, R102, R106, S108, P109, I116 | |
Ácido 10-oxo-12-octadecenoico (ASY) | T32, I35, S36, L77, S79, L80, A81, S83, F84, I107 | |
Ácido palmítico (PLM) | V34, L38, V45, V60, L63, N64, V76, I80, A99, L102, P103, V108, I110, Y112, I114 | |
Ácido 10-oxo-12-octadecenoico (ASY) | V35, L38, T39, I80, N82, A83, V84, N86, S87, I114 | |
Grupo trifluoroacetil (TFA) | L63, N64, A67, V76, N109, I110, P111, Y112 | |
Ácido palmítico (PLM) | V32, L36, L43, V57, V60, L61, V73, L77, A92, L95, P96, V101, I103, Y105, I107 | |
Ácido 10-oxo-12-octadecenoico (ASY) | T33, L36, A37, L77, T79, S80, A81, Q83, V84, I107 | |
1-Miristoil-SN-glicerol-3-fosfocolina (LPC) | L61, R70, C74, L77, A81, A92, N102, K106, S108, P109, I116 | |
Ácido palmítico (PLM) | I33, V37, L44, L59, L62, N63, G75, L79, L94, T97, P98, I103, L105, Y107, I109 | |
Ácido 10-oxo-12-octadecenoico (ASY) | Q34, V37, A38, L79, S81, T82, I83, S85, L86, I109 | |
1-Miristoil-SN-glicerol-3-fosfocolina (LPC) | N63, R72, C76, L79, I83, L94, N104, K108, S110, P111, Y118 | |
Ácido esteárico (STE) | T32, L35, A36, C38, L39, V56, L59, L60, A63, L76, L83, L94, I102, Y104, I106 | |
Ácido 10-oxo-12-octadecenoico (ASY) | T32, L35, A36, L76, G78, A79, A80, Q82, L83, I106 | |
1-Miristoil-SN-glicerol-3-fosfocolina (LPC) | L60, K69, C73, L76, A80, A91, N101, S107, T108, I115 | |
Ácido esteárico (STE) | Q31, L34, A35, C37, I38, V55, I58, L59, S62, L75, V82, L93, L101, Y103, I105 | |
Ácido 10-oxo-12-octadecenoico (ASY) | Q31, L34, A35, L75, A77, A78, A79, A81, V82, I105 | |
1-Miristoil-SN-glicerol-3-fosfocolina (LPC) | L59, R68, C72, L75, A79, A90, N100, K104, S106, A107, Y114 | |
Ácido esteárico (STE) | T32, I35, A36, C38, F39, I56, I59, N60, A63, L76, L83, L95, I103, Y105, I107 | |
Ácido 10-oxo-12-octadecenoico (ASY) | T32, I35, A36, L76, S78, V79, A80, S82, L83, I107 | |
1-Miristoil-SN-glicerol-3-fosfocolina (LPC) | N60, R69, C73, L76, A80, A92, S102, K106, S108, T109, I116 | |
Ácido esteárico (STE) | S33, L36, A37, C39, L40, V59, L62, L63, A66, L79, I86, L97, I105, Y107, I109 | |
1-Miristoil-SN-glicerol-3-fosfocolina (LPC) | L36, A37, L40, V59, A82, S85, I86 | |
Lauroil (LAP) | V59, L63, R72, A94, S104, I105, P106, Y107 |
Sequences | Protein ligands | Type of interaction | Score |
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T | 0,592 | ||
T | 0,592 | ||
T | 0,592 | ||
T | 0,52 | ||
T | 0,52 | ||
T | 0,453 | ||
T | 0,453 | ||
T, H, C | 0,866 | ||
T | 0,812 | ||
T | 0,669 | ||
T | 0,66 | ||
T | 0,625 | ||
T | 0,612 | ||
T | 0,611 | ||
T | 0,602 | ||
T | 0,597 | ||
T | 0,573 | ||
T | 0,895 | ||
T | 0,695 | ||
T | 0,677 | ||
T | 0,643 | ||
T | 0,643 | ||
T | 0,637 | ||
T | 0,623 | ||
T | 0,593 | ||
T | 0,568 | ||
T | 0,567 | ||
T, H, C | 0,866 | ||
T | 0,67 | ||
T | 0,664 | ||
T | 0,649 | ||
T | 0,649 | ||
T | 0,642 | ||
T | 0,628 | ||
T | 0,624 | ||
T | 0,616 | ||
T | 0,58 | ||
T | 0,592 | ||
T | 0,592 | ||
T | 0,592 | ||
T | 0,52 | ||
T | 0,52 | ||
T | 0,453 | ||
T | 0,453 | ||
T | 0,592 | ||
T | 0,592 | ||
T | 0,592 | ||
T | 0,52 | ||
T | 0,52 | ||
T | 0,453 | ||
T | 0,453 | ||
Lup an 3 (Uniprot: A0A1J7GK90) | T | 0,592 | |
T | 0,592 | ||
T | 0,592 | ||
T | 0,52 | ||
T | 0,52 | ||
T | 0,453 | ||
T | 0,453 | ||
Lup an 3.0101 (Uniprot: A0A4P1RWD8) | T | 0,592 | |
T | 0,592 | ||
T | 0,592 | ||
T | 0,52 | ||
T | 0,52 | ||
T | 0,453 | ||
T | 0,453 |
Protein name | Mapped IgE and PID epitopes | Motives MEME/MAST | SVM – aminoacid composition | SVM-dipeptide composition | Blast ARPs |
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- | - | Potential allergen | Potential allergen | GSISGVNPNNAAGLPGKCGVNVPY | |
- | - | Potential allergen | Potential allergen | SNLAPCINYVKGGGAVPPACCNGI | |
- | - | Potential allergen | Potential allergen | CCNGIRNVNNLARTTPDRRTACNC | |
- | - | Potential allergen | Alérgeno potencial | VPPACCNGIRNVNNLARTTADRR | |
- | - | allergen | Potential allergen | SGVKNLNSIAKTTPDRQQACNCIQ | |
- | - | Potential allergen | Potential allergen | CNGVRTINNAAKTTADRRTACQCL | |
- | - | Potential allergen | Potential allergen | AGIPGKCGVNIPYAISQGTDCSK | |
- | - | allergen | Potential allergen | CIAYVRGGGAVPPACCNGIRNI | |
- | - | Potential allergen | Potential allergen | NGIRNVNNLARTTPDRQAACNCLK | |
- | - | Potential allergen | Allergen | AASIPSKCNVNVPYTISPDIDCS | |
- | - | Potential allergen | Potential allergen | NLVAGIPGKCGVNIPYAISQGT | |
- | - | Potential allergen | Potential allergen | CCNGIRNVNNLARTTPDRRTACNC | |
- | - | Potential allergen | Potential allergen | CCNGIRNVNNLARTTPDRRTACNC | |
- | - | Potential allergen | Potential allergen | LNLNNAASIPSKCNVNVPYTIS | |
- | - | allergen | Allergen | KTTADRQTACNCLKQLSASVPGVN | |
- | - | Potential allergen | No allergen | VSSSLAPCIGYVRGGGAVPPACCN | |
- | - | Potential allergen | Potential allergen | LKQLSGSISGVNPNNAAALPGKCG | |
- | - | allergen | No Allergen | GVKNLNSIAKTTPDRQQACNCIQ | |
- | - | Potential allergen | Potential allergen | LNLNNAASIPSKCNVNVPYTISPD | |
- | - | Potential allergen | Potential allergen | GSISGVNPNNAAGLPGKCGVNVPY | |
- | - | Allergen | Allergen | CCNGVTNLKNMASTTPDRQQACRC | |
- | - | Potential allergen | Potential allergen | SNLAPCINYVKGGGAVPPACCNGI | |
- | - | Potential allergen | Allergen | TCGQVSSSLAPCIGYVRGGGAVPP | |
- | - | Potential allergen | Potential allergen | SGVKNLNSIAKTTPDRQQACNCIQ | |
- | - | Potential allergen | Potential allergen | AGIPGKCGVNIPYAISQGTDCSKV |
Proteína | T1 | T2 | T3 | T4 | T5 | T6 | T7 | T8 | T9 |
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Lup an 3 | IKVACVVLMCMAVVAA | YKISVSTNC | |||||||
Lup an 3.0101 | IVKLACAVLICMVVVSAPLTK | YKISTSTNC | LRSGGAVPA | ||||||
MKVACVLLMMCIIVAPM | YKISTSTNC | ||||||||
LRFFTCLVLTVCIVAS | |||||||||
MLVTAPMAS | VRISYPISA | VQRLNSLAR | |||||||
Ole e 7 | VVKATCFVLIAVALVA | ||||||||
LVLMCMAVVAA | VRIPYKISTSTNC | FLRFGGPVS | FNPTNAAAL | VRALVAAAQ | VLFALPLQI | ||||
MVLMCMVVVGAPIAQA | YKISTSTNS | VKGLVALAQ | YRHHSKFLV | VVSSLAPCL | |||||
FTKLACMVLACMVVMVAHNTV | YKISTSTNC | VRKLNPYNA | VRSIVNNAR | ||||||
IRVTCVVLMVCMALLSA | YKISPSTNC | LNLANAGSLPS | |||||||
VVKLVLMATVWVAVLSPKA | YKISPSTDC | YVLNGGKTV | VVSNLTPCVS | ||||||
VKFACVVVLCMVVVGAHTAQG | YKISTSTNC | FVPAGCCNG | VRGLNPNNA | VRNIMNSAR | LVPCVTFLQ | ||||
VLCMVVVSAPMAH | YKISTSTNC | ||||||||
VVLVMCMVVIAPMAE | YKISTSTNC | ||||||||
LVKVTCFAMICLVLGIPLAD | YKISPSIDC | YISLNQLSI | |||||||
FKLACAVLVCMAAVGAH | VRGLNPSNA | LHNHFPLRM | |||||||
MKVVCVALIMCIVIAPMAES | YKISTSTNC | VRNLNSAAV | |||||||
LKLASVVAVMCMVLVTAPLT | MRIPYRISPSTNC | VRRLNSAAR | |||||||
IRLVCLAIVCL | YQISPNTDC | YVVYGGNMVPAQ | FNLNLAAGL | VKNLNSMAR | VVNNLTPCIS | ||||
IRVTCVVLMVCMALLSAPMV | YKISPSTNC | LAYLQRGGAPPL | INLANAGSLPS | ||||||
LVKVTCLAL---LVLNIPLAN | YKISPSINC | IFSLPGINL | |||||||
LIKVACMVVLCVALVA | YKISTSTNC | VRSLLSAAQ | |||||||
VKFACVVVMFMVVVGSHS | YKISASTNC | VRGINPNNA | |||||||
VVLMLCMAIVGAPIAKA | YKISTSTNC | IQCSFVTKSIAPC | |||||||
MKLACVALVMCMVVIAPMAE | YKISTSTNC |
Protein | B1 | B2 | B3 | B4 | B5 | B6 |
---|---|---|---|---|---|---|
Lup an 3 | SSAQTTADKRT | PNYNDANA | ||||
Lup an 3.0101 | AKTTPDRRTACN | GLNPSNAG | YKISTSTN | |||
IGYLKGGSGP | PYKISTSTN | |||||
SMAKTTPDRQQACR | ||||||
SLARTTRDRQQACR | ||||||
Ole e 7 | TSAKTTADRRS | PYKIDPSTDC | ||||
PYKISTSTN | ||||||
YKISTSTNSSSSEE | MWWERYRHHSKF | |||||
NNARTTGDRRA | PYKISTSTNCNS | |||||
SARTPADRRT | PYKISPSTNCNT | |||||
AHNTPDRQT | YKISPSTDCSR | |||||
NSARSTADRRG | GLNPNNAQA | PYKISTSTN | ||||
AKTTADRQTACN | LQNGGTPPSG | PYKISTSTN | ||||
AANTTPDRQAA | KLNTNNAAA | YLTGGPGPS | YKISTSTNCNT | |||
NNQAKTTPDRQS | GYLRRPGPS | |||||
ARTTADRRA | GLNPSNAQA | PYKISTSTN | NLLHNHFPLR | |||
YLQGGPGS | PYKISTSTN | |||||
NSAARTTGDRRTACN | YRISPSTNCNR | |||||
MARTTPDRQT | YQISPNTDCSR | |||||
ARTPADRKT | YKISPSTNCNT | |||||
RNLNNQAKSTPDRRSGCR | ||||||
QTTVDKQTVCN | PYKISTSTN | |||||
LNSSRTTPDRRA | GINPNNAEA | ISASTNCNR | ||||
NGTAKTTSDRQA | YKISTSTNCSS | |||||
RLNTNNAAA | QAPNNASPP | YKISTSTNCNT |
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