Analysis of the effects of polymorphism on pollen profilin structural functionality and the generation of conformational, T- and B-cell epitopes

Abstract

An extensive polymorphism analysis of pollen profilin, a fundamental regulator of the actin cytoskeleton dynamics, has been performed with a major focus in 3D-folding maintenance, changes in the 2-D structural elements, surface residues involved in ligands-profilin interactions and functionality, and the generation of conformational and lineal B- and T-cell epitopes variability. Our results revealed that while the general fold is conserved among profilins, substantial structural differences were found, particularly affecting the special distribution and length of different 2-D structural elements (i.e. cysteine residues), characteristic loops and coils, and numerous micro-heterogeneities present in fundamental residues directly involved in the interacting motifs, and to some extension these residues nearby to the ligand-interacting areas. Differential changes as result of polymorphism might contribute to generate functional variability among the plethora of profilin isoforms present in the olive pollen from different genetic background (olive cultivars), and between plant species, since biochemical interacting properties and binding affinities to natural ligands may be affected, particularly the interactions with different actin isoforms and phosphoinositides lipids species. Furthermore, conspicuous variability in lineal and conformational epitopes was found between profilins belonging to the same olive cultivar, and among different cultivars as direct implication of sequences polymorphism. The variability of the residues taking part of IgE-binding epitopes might be the final responsible of the differences in cross-reactivity among olive pollen cultivars, among pollen and plant-derived food allergens, as well as between distantly related pollen species, leading to a variable range of allergy reactions among atopic patients. Identification and analysis of commonly shared and specific epitopes in profilin isoforms is essential to gain knowledge about the interacting surface of these epitopes, and for a better understanding of immune responses, helping design and development of rational and effective immunotherapy strategies for the treatment of allergy diseases.

Figure 1. Surface distribution analysis of the profilin polymorphism.

Different colors were used to highlight the different degree of variability over the surface for the three models used in this study, A) 1cqa, B) 1g5uA, and C) 3nul models. Residues which variability were high (variability index value, viv>3) were depicted in yellow color. Residues with intermediate (13) and low (viv<1) variability were depicted as green and blue, respectively –. Surface residues implicated in ligand-binding domains (actin, PLP and/or PIP) were highlighted with transparent white shadows over the protein surface and discontinues borders lines. Red dotted circles and red arrows pointed a detailed plant specific solvent-filled cavity.

Figure 2. Profilin structure features, ligand-binding domains and electrostatic potential distribution.

Left, central, and right panel represent to 1g5uA, 1cqa, and 3nul models, respectively. A) Solvent accessible surface area (SASA) calculated for the residues of each crystallographic model. Key amino acids implicated in Actin, PLP and PIP interaction are highlighted with orange, blue and purple arrows, respectively. A red dotted line delimited the residues with SASA>25%. B) Three-dimensional structure of profilin models 1g5uA, 1cqa, and 3nul (from left to right panel) showing two views rotated 180°. Different secondary structural elements such as α-helices, β-sheets, and loops are highlighted with letters H, S, respectively. All structures were depicted as a cartoon diagram. C) Surface representation views of the three models of profilins rotated 90°, showing the surface residues involved in the different ligand-binding surfaces such as actin (orange), PLP (light blue), and PIP (purple). Residues belonging to actin-PIP and PLP-PIP binding regions are highlighted with red and deep blue colors, respectively. Red dotted circles and red arrows point a detailed of the plant specific solvent-filled cavity. D) 90° rotated views of the electrostatic potential representation on the three profilin models surface, showing the plant specific solvent-filled cavity highlighted by yellow dotted lines and arrows. The surface colors are clamped at red (−5) or blue (+5). E) Electrostatic potential (isocontour value of ±5 kT/e) surface for the three models of profilins are depicted in 3 rotated 90° views.

Figure 3. Phylogenetic analysis of olive profilin isoforms.

Neighbor-joining (NJ) method was used to perform a phylogenetic analysis of the deduced protein sequences of Olea europaea L. profilin from 24 different cultivars. Each group of proteins are characterized by the 3D structural similarity represented by the PDB models A) 3nul of the Ara t 8 allergen, B) 1g5uA of Hev b 8 allergen, and C) 1cqa of Bet v 2 allergen. Profilin sequences from the same olive cultivar are highlighted with red arrows.

Figure 4. Profilin conservational analysis.

Consurf-conservational analysis of profilin proteins showed in three individual views rotated 90° for the PDB models A) 1g5uA, B) 1cqa, and C) 3nul, respectively. The conserved and variable residues are presented as space-filled models and colored according to the conservation scores. The strictly conserved and variable residues are depicted in purple and blue, respectively. Red dotted circles and red arrows point a detailed of the plant specific solvent-filled. The sequence of the protein is depicted with the evolutionary rates color-coded onto each site. The residues of the query sequence are always numbered starting from 1. The predicted burial status of the site (i.e. “b”-buried vs. “e”-exposed) is annotated under the sequence. Residues predicted to be structurally and functionally important, “s” and “f”, are also pointed out under the sequence. Amino-acid sites categorized as “Insufficient data” are colored in yellow, indicating that the calculation for these sites were generated using only a few of the homologous sequences. Orange, light blue and purple starts highlight the key amino acids implicated in the interaction with actin, PLP and PIP, respectively. Red lines under the sequences represent the profilin characteristic motif, which define this family of proteins. C = conserved, V = variable, U = undefined.

Figure 5. B- and T-cell epitopes superimposition on the surface of the profilin structures.

A) Cartoon representation of profilin model 1cqa two views rotated 180° respectively, showing the localization of 5 B-cell epitopes, 10A4 (red), 5F2 (green), 9A7 (blue), 9G4 (yellow), and 3H8 (pink), in the 2-D structural elements of the protein. Overlapping sequence of 9A7 and 9G4 epitopes are depicted with vertical yellow lines. All epitopes are integrated by final part of two α-helices and its corresponding flanking loops, or a β-sheet. Surface superimposition of epitopes shows a broad distribution. B) Cartoon representation of profilin model 1cqa two views rotated 180° respectively, showing the localization of 5 T-cell specie-specific epitopes, I⁵³ (orange) by Olea europaea L., F⁴¹ (red) for Betula pendula, F⁶⁶ (light blue) for Phleum pratense, and F⁵⁹ (green), for Zea mays, in the 2-D structural elements of the protein. Partial overlapping epitopes are I⁵³ and F⁵⁶. Surface superimposition of epitopes shows the distribution in a specific area of the protein and not overlapping with B-cell epitopes.

Figure 6. Olive cultivars and species specific distribution of T-cell epitopes.

A) Cartoon representation of profilin model 3nul two views rotated 45° respectively two examples of olive cultivars, ‘Picual’ and ‘Loaime’, to compare the localization in the 2-D structural elements of the protein of the common shared T-cell epitopes between both cultivars, and the specific epitopes (V²⁹, I⁵³ and I¹⁰⁶) only present in Loaime cultivar. All epitopes were depicted in orange color. Surface superimposition of both, common and not shared epitopes, are depicted in the same color over the model 3nul of profilin. Red circles were used to highlight the specific epitopes. B) Cartoon representation of profilin model 1cqa of the same view for both species of the Betulaceae genus, Betula pendula and Corylus avellana, showing the specific T-cell epitope F⁴¹, only present in Betula pendula. Presence or absence of the F⁴¹ epitope was located and highlighted in the 2-D structural elements of the protein, as well as over the surface of the model by using red color and red circles. C) Specific epitopes location and comparison between two species of the genus Poaceae, Phleum pratense and Zea mays, by using cartoon representation of 2-D profilin elements or protein surface over the models 3nul and 1g5uA two views rotated 90° or 180°, respectively. Blue color over the model surface and blue circles were used to highlight Phleum pratense specific T-cell epitopes F⁶⁶ and I⁹². Pink circles were used to highlight the absence of Zea mays specific T-cell epitopes V²⁶ and F⁵⁹ over the 3nul model. Reciprocity of colors was used to show the presence or absence of specific epitopes in the model 1g5uA for Zea mays.