Fig 1.
Sequence analysis of the wheat TdLTP2.
Pattern and signature as revealed by ScanProsite tool with multiple alignment analysis of closed nsLTPs (A) and Molecular phylogenetic analysis of TdLTP2 sequence, with the reference TdLTP1 protein, by Maximum Likelihood method using MEGA.X program (B). The eight conserved cysteines are highlighted by magenta; the connections between the four disulfide bonds are marked by colored numbers; the two conserved pentapeptides are highlighted by grey.
Table 1.
The physicochemical features of TdLTP2 in comparison with TdLTP1, including protein length, calculated molecular weight, theoretical pHi, stability index and grand average of hydropathicity (GRAVY), were determined using ProtParam tool.
Fig 2.
3D structural modeling of TdLTP2 with the reference model of TdLTP1.
(A) Comparison of the primary and tertiary structures of TdLTP2 and TdLTP1 reveals that nsLTP subfamilies display 71% of sequence homology and share a similar α-helical fold that is stabilized by four disulfide bonds (highlighted in yellow). (B) Pocket size cavity of TdLTPs calculated by DogSite method implemented in the Proteins Plus server.
Fig 3.
Expression profiling by QRT-PCR of TdLTP2 and immunolocalization of nsLTPs in Triticum durum.
(A) QRT-PCR in roots and leaves tissues from durum wheat exposed to different stress conditions (150 mM NaCl, 100 μM ABA and 500 μM SA). The mock control was treated with water solution. Results were shown as means ± standard deviation of three biological replications. Relative gene quantification was calculated by the comparative Ct method. Data were normalized to the expression level of the wheat gene factor CDC (GenBank accession no. Ta54227). (B) Immunolocalization of nsLTPs sections from leaves, roots and seeds fixed with paraformaldehyde 4% and incubated with Alexa 647-labeled LTPs. LTPs detected with anti-Tri a 14 antibody (green) have been added. DAPI (blue) was used as control. Bar: 10 microns. The probability (P) value < 0.05 was used to measure the significant change with unequal variance.
Fig 4.
Identification and characterization of recombinant TdLTP2 (rTdLTP2).
(A) Coomassie stain of rTdLTP2; (B) Western blot analysis of rTdLTP2. All analysis was performed with anti-Tria14 and rTri a 14 as a positive control. (C) MALDI-TOF mass spectra of RP-HPLC purified durum wheat rTdLTP2.
Fig 5.
In vitro lipid binding activity of rLTP2 using different experiments.
(A) Observation of the ligands by TLC in a silica gel plate and Emission of the bands under UV light (365 nm). (B) The binding ability of FAs with (50 μg/ml) for each lipid was tested at varying concentrations (6.6, 3.3, 1.6, 0.8, 0.4, 0.2 and 0.1 μM). Hydrophobic ELISA plates were coated with the indicated lipids. Protein binding was revealed using polyclonal antibody against rTdLTP2. Graphs illustrate the means of the absorbance values at 450 nm ± SEM, n = 3. (C) Molecular dynamics of oleic acid and sitosterol entering the cavity of rTdLTP2.
Fig 6.
Detection of IgE patients’ sera binding to rTdLTP2.
(A) Activation assay using the transfected cell line THP1-XBlue™. Cells were incubated with each ligand in triplicates and the activation was measured using QUANTI-Blue™. Results were represented by the stimulation index, referenced to the negative control (PBS), The threshold was established as the negative control +3SD. (B) Patients were sensitized to Tri a 14 with 76% (23) from a total of 30 sera. Each bar indicates the mean and SD of the inhibition percentage obtained with bakers’ sera from Spanish patients. 60% (18) sera for Tri a 14 were positives to rTdLTP2 and evaluated as significant. Threshold was calculated as the negative control+ 3SD.