3.1 Sequence and phylogenetic analyses
Full-length cDNA and amino acid sequences of FtLTP1a and FtLTP1b are shown in Fig. S1. Both FtLTP1a and FtLTP1a contained 117 amino acid residues. FtLTP1a contained a 91-residue mature peptide and a 26-residue putative signal peptide (GenBank accession number: OK340654), whereas FtLTP1b contained a 92-residue mature peptide and a 25-residue signal peptide (GenBank accession number: OK340655). The molecular masses of mature FtLTP1a and FtLTP1b were estimated using DNAMAN software to be 9228.76 Da and 9038.55 Da, respectively. It has been reported that nsLTPs consist of two main subfamilies, namely the 9-kDa nsLTP1 family and the 7-kDa nsLTP2 family [24]. Moreover, both the two purified proteins share high sequence homology with nsLTP1 family. Furthermore, they contain two conserved motifs (T/SXXDR/K and PYXIS), which is also a characteristic of the nsLTP1 family [3]. Therefore, based on the above results, we may conclude that FtLTP1a and FtLTP1b belong to the nsLTP1 family.
The phylogenetic tree is a framework indicating genetic diversities and distances between different biological species and other entities. To explore the phylogenetic relationships between FtLTP1a/b from Tartary buckwheat and those from other species, multiple sequence alignments were analyzed, and a phylogenetic tree of 114 protein sequences was generated using a neighbor-joining method available in MEGA7 software (Fig. 1A). We found that nsLTPs from Tartary buckwheat were grouped with those from Chenopodium quinoa (XP 021723225.1, XP 021766670.1), Spinacia oleracea (XP 021851885.1), and Beta vulgaris subsp. Vulgaris (XP 010690793.1). Additionally, FtLTP1a and FtLTP1b were closely related to many allergens, such as Amb a6 (O04004.1, Ambrosia artemisiifolia), Zea m14 (P19656.1, Zea mays), Tri a14 (P24296.2, Triticum aestivum), Bra o3 (Q9ZSL7, Brassica oleracea var. oleracea), Pla a 3.02 (ALF00099.1, Platanus x hispanic), and Pla o3 (ABY21307.1, Platanus orientalis).
The amino acid sequences of FtLTP1a and FtLTP1b were also compared with three unique proteins, Api g2, Pru p3 and Tri a14, available in the UniProt database (Fig. 1B). The IgE-binding epitopes in Pru p3, Tri a14, and Api g2, the homologous nsLTP from peach, wheat, and celery, respectively, have been identified. FtLTP1a shared 54.9%, 47.3% and 41.1% sequence identity with Api g2, Pru p3, and Tri a14, respectively, and FtLTP1b shared 64.8%, 52.7% and 48.9%, respectively. The comparison of amino acid sequences revealed that FtLTP1b is more similar to Api g2, Pru p3, and Tri a14 than FtLTP1a. Previous studies have identified the IgE binding epitopes in Pru p3 using different methodologies [25–27]. Comparing the amino acid sequences of Pru p3 and FtLTP1a, it was found that there was high degree of diversity in the regions potentially involved in IgE recognition (Fig. 1B). Moreover, FtLTP1b had a high sequence identity with Pru p3, including a highly conserved region (GKCGVSIPYK) that can significantly contribute to IgE binding. This region in Api g2 and Tri a14 is also highly conserved. Therefore, the analysis above suggests that FtLTP1b is more likely to be a potential allergen than FtLTP1a.
3.2 Heterologous expression and purification of FtLTP1s
Recombinants of pET32a-FtLTP1a and pET32a-FtLTP1b were constructed, transformed into E. coli BL21(DE3) cells and induced expression. As shown in Fig. 2, the recombinant Trx-FtLTP1a and Trx-FtLTP1a proteins were expressed in soluble form, and the target proteins were further purified by a Ni2+-NTA column. Using this single-step procedure, the purity of both Trx-FtLTP1a and Trx-FtLTP1b proteins was about 95% and migrated as a single band on SDS-PAGE (Fig. 2). The estimated yield of the FtLTP1a and FtLTP1b was 2.14 mg and 2.46 mg per liter of culture, respectively(Table S3). The purity of FtLTP1a and FtLTP1b were 94.25% and 96.33% by gel scan.
3.3 Both FtLTP1a and FtLTP1b could bind lipid
Two highly conserved regions (T/SXXDR/K and PYXIS) play an essential role in lipid binding, and the fluorescence of tyrosine (Y, in PYXIS) is sensitive to lipid binding. Fluorescence spectroscopy is a tool for investigating the quenching mechanism and the binding properties. Intrinsic fluorescence of FtLTP1a was compared with that of FtLTP1b. As shown in Fig. 3A, the maximal fluorescence emission wavelength of both FtLTP1a and FtLTP1b was 342 nm. However, the intensity of FtLTP1b was dramatically lower than that of FtLTP1a. The difference in intensity may be due to the fact that FtLTP1a contains one tyrosine residue and two phenylalanine residues, while FtLTP1b only contains two tyrosine residues. When the purified FtLTP1a and FtLTP1b were incubated with oleic acid, the fluorescence intensity was associated with the increase in oleic acid concentration (Figs. 3B and 3C). According to the apparent saturation levels, the molar ratio of oleic acid/FtLTP1a or oleic acid/FtLTP1b (Ri) was about 1.0 (Fig. 3D). The fluorescence of FtLTP1a and FtLTPlb with oleic acid was markedly increased by 160% and 144%, respectively, compared to that in the absence of oleic acid. In addition, the binding of FtLTPs with other two types of lipids, stearic acid and linolenic acid, was also studied, from which similar results were observed under the same test conditions (data not shown). The tyr-79 residue of the present nsLTPs and other corresponding residues of nsLTPs from other plant species are involved in increasing of fluorescence intensity when binding to lipids. Tyr-79, located in the C-terminal fragment, a part of the extreme hydrophobic cavity, should be perturbed by lipid-binding [28]. Maize nsLTP-fatty acid complex has shown that the carboxyl group in fatty acid is involved in forming hydrogen bonds with the hydroxyl group of Tyr81 [29]. Consequently, in a lipid-bound state, Tyr-79 in the protein may probably be less mobile, and thus could not be effectively quenched by a possible internal quencher.
3.4 FtLTP1b exhibits weak α-amylase inhibitory activity
According to the protein classification by NCBI, nsLTPs belong to the AAI_LTSS superfamily (protease inhibitor/seed storage/LTP family). However, only a few nsLTPs have been identified to exhibit α-amylase inhibitory activity till now. Thus, the α-amylase inhibitory activity of FtLTP1a and FtLTP1b was further tested. First, the secondary structures of FtLTP1a and FtLTP1b were verified by circular dichroism (CD) spectra. The CD spectra of two proteins showed typical peaks of α-helix, namely two minimum peaks at 208 and 222 nm (Fig.S2). This result is consistent with the CD spectrum of nsLTPs from red wine and grapes [30], peach, cherry, hazelnut [31], and mugwort pollen [32], which are all rich in α-helices. Thus, we conclude that recombinant FtLTP1a and FtLTP1a folded correctly and formed a natural conformation. As shown in Fig. 4, both FtLTP1a and FtLTP1b were unable to inhibit α-amylase from Bacillus licheniformis, and FtLTP1b could weakly inhibit α-amylase from porcine pancreas. It has been reported that lipid transfer proteins from Coffea canephora and Vigna unguiculata L. Walp showed inhibitory activity against α-amylase from human saliva and C. maculatus, respectively [33, 34]. One study has reported that rVu-LTP purified from Vigna unguiculata L. Walp can inhibit the activity of HAS and intestinal α-amylase from C. maculatus, but cannot inhibit intestinal α-amylase from T. maculates[35].
Fragment Asp-Glu-Asp appears in the active site of almost all α-amylases [36]. Some studies have suggested that positively charged (argnine and lysine) amino acids in inhibitors can non-covalent interact with the negatively charged residues of Asp-Glu-Asp, resulting in an ultimate inhibition of the enzyme [37]. Two peptides from Vu-LTP, N29-I47 (NGVKNILNGARTTADRRGI) and αG74-N91 (GVNIPWKISSSTNαNTIN), have been identified and found to be responsible for HAS (human salivary α-amylase) inhibition [21]. The two regions of FtLTP1a did not contain positively charged amino acids, while there was lysine (K) residue in one region (G74-N91) of FtLTP1b. This may explain why FtLTP1b exhibited weak α-amylase inhibitory activity, whereas FtLTP1a did not.
3.5 Both FtLTP1a and FtLTP1b exhibit antimicrobial activity
Plant nsLTPs belong to pathogenesis-related protein, which are reported to display broad-spectrum antimicrobial activity. The antifungal activities of FtLTP1a and FtLTP1b against Aspergillus niger, Aspergillus flavus and Fusarium oxysporum were evaluated. As shown in Fig. 5, the two proteins exhibited inhibitory activity against A. niger. The MIC (mininum Inhibitory Concentration) values of the proteins were determined and tabulated in Table S4. The MIC values of FtLTP1a to Aspergillus niger, Aspergillus flavus and Fusarium oxysporum were 8 µM, 8 µM, and 64 µM, respectively, and those of FtLTP1b were 16 µM, 16 µM, and 64 µM, respectively.
The antimicrobial activities of nsLTPs from different plant species have been studied. Although the nsLTPs can inhibit both bacterial and fungal growths, the inhibition against bacterial growth is more potent than that against fungi growth [38]. Nevertheless, the mechanism of this occurrence remains unclear. The findings to date have indicated the ability to interact with lipids contributes to the antimicrobial activity of plant nsLTPs. Although the peptide Ace-AMP1 (from onion), also characterized as an nsLTP, cannot transfer phospholipid from liposome to mitochondria, it possesses antimicrobial activity [8]. It has been speculated this activity was due to their interaction with cell membranes, causing the loss of membrane integrity and permeabilization [39].
3.6 Immunological characteristics of FtLTP1a and FtLTP1b
Sera from 9 Chinese patients who tested positive for buckwheat allergen were used to screen IgE reactivity of FtLTP1a and FtLTP1b using ELISA. The clinical data of the nine patients who participated in this study are summarized in Table S1. All patients tested positive for IgE antibodies-specific 24-kDa buckwheat allergen and experienced allergic reactions after consuming buckwheat food. The P/N values > 2.1 were used to evaluate the allergenic activity of nsLTPs from buckwheat. As shown in Fig. 6A, all the 2-fold diluted sera from nine individuals (100%) were sensitive to FtLTP1b (P/N values > 2.1) but not FtLTP1a (P/N values < 2.1). In addition, only three 10-fold diluted sera were sensitive to FtLTP1b (Fig. 6B).
The fact that patients who are allergic to a particular allergen tend to be allergic to other agents indicates that the same IgE antibodies may recognize other homologous allergens which possess similar epitopes. As shown in Table S1, eight out of these nine patients were also allergic to the artemisia pollen (W6). FtLTP1a and FtLTP1b were tested on three sera that were sensitive to W6 and polyvalent grass pollen (WX7), to determine whether they share the same IgE epitopes with other allergens and further evaluate their potential cross-reactivity. As shown in Fig. 6C, FtLTP1b was able to bind IgE in these sera (P/N > 2.1). The majority (89%) of Artemisia vulgaris pollen-sensitized patients is also allergic to Art v3, a mugwort allergen [40]. Clinical studies have shown that Art v3 could cross-react with another homologous protein from peach (Pur p3). Patients with peach allergy and are primarily sensitive to Pru p3 can experience allergic response when exposed to mugwort pollen because of cross-reactivity with Art v3 [41]. In this study, certain cross-reactivity occurred between FtLTP1b and pollen allergen (W6 or WX7). However, clinical symptoms caused by this cross-reactivity may not be observable.