Structural, Functional, and Immunological Characterization of Profilin Panallergens Amb a 8, Art v 4, and Bet v 2

Abstract

Ragweed allergens affect several million people in the United States and Canada. To date, only two ragweed allergens, Amb t 5 and Amb a 11, have their structures determined and deposited to the Protein Data Bank. Here, we present structures of methylated ragweed allergen Amb a 8, Amb a 8 in the presence of poly(l-proline), and Art v 4 (mugwort allergen). Amb a 8 and Art v 4 are panallergens belonging to the profilin family of proteins. They share significant sequence and structural similarities, which results in cross-recognition by IgE antibodies. Molecular and immunological properties of Amb a 8 and Art v 4 are compared with those of Bet v 2 (birch pollen allergen) as well as with other allergenic profilins. We purified recombinant allergens that are recognized by patient IgE and are highly cross-reactive. It was determined that the analyzed allergens are relatively unstable. Structures of Amb a 8 in complex with poly(l-proline)10 or poly(l-proline)14 are the first structures of the plant profilin in complex with proline-rich peptides. Amb a 8 binds the poly(l-proline) in a mode similar to that observed in human, mouse, and P. falciparum profilin·peptide complexes. However, only some of the residues that form the peptide binding site are conserved.

Keywords: IgE binding; allergen; allergy; crystal structure; epitope mapping; peptide binding; pollen; protein stability.

FIGURE 1.

Two-dimensional projection of the CLANS clustering results. Proteins are indicated by dots. Lines indicate sequence similarity detectable with BLAST and are colored by a spectrum of shades of gray according to the BLAST p value. Sequences corresponding to structures in the PDB are indicated by blue dots, and sequences of known allergens are indicated by red dots.

FIGURE 2.

A, a structure of Amb a 8·poly(l-Pro)₁₄ complex. Poly(l-Pro) is shown in a stick representation. B, superposition of Amb a 8 and Bet v 2 (PDB entry 1CQA).

FIGURE 3.

Mass spectrometry results for Amb a 8, Art v 4, and Bet v 2. The experiment was performed with a Bruker Ultraflex II MALDI TOF/TOF and processed with flex control software and flexAnalysis version 3.3. Intensity (arbitrary units (a.u.)).

FIGURE 4.

A, interactions between Amb a 8 and poly(l-Pro)₁₄. Amb a 8 residues are shown as yellow sticks, whereas poly(l-Pro) is shown in a ball-and-stick representation and labeled using one-letter codes. B, superposition of profilin complexes with proline-rich peptides. Only side chains of profilin residues involved in binding of proline-rich peptides are shown. The superposition involves Amb a 8 (yellow sticks), human profilin (cyan sticks; PDB entry 3CHW), mouse profilin (gray sticks; PDB entry 2V8F), and profilin from P. falciparum (purple sticks; PDB entry 2JKG). C, superposition of profilin complexes with proline-rich peptides. Only poly(l-Pro) and proline-rich peptide are shown. Poly(l-Pro) from the Amb a 8·poly(l-Pro)₁₄ complex is shown in ball-and-stick representation.

FIGURE 5.

DSF profilin proline screening. Amb a 8, Art v 4, Bet v 2, and their His-tagged counterparts were screened against 10.0 mm l-proline, Pro₆, Pro₁₀, or Pro₁₄ to determine change in protein melting temperature (T_m). The control was profilin stability in gel filtration buffer (10 mm Tris, pH 7.4, 150 mm NaCl). Error bars, S.D.

FIGURE 6.

DSF profilin urea screening. Amb a 8, Art v 4, Bet v 2, and their His-tagged counterparts were screened against different concentrations of urea to determine change in protein melting temperature (T_m). The control was profilin stability in gel filtration buffer (10 mm Tris, pH 7.4, 150 mm NaCl). Error bars, S.D.

FIGURE 7.

A, representative inhibition ELISA results with Bet v 2 used as an inhibiting allergen. Bet v 2, Bet v 2 coated on a plate; Art v 4, Art v 4 coated on a plate; Amb a 8, Amb a 8 coated on a plate; I, inhibition by Bet v 2 (30 μg/ml). B, representative inhibition ELISA results using Art v 4 as an inhibiting allergen. Bet v 2, Bet v 2 coated on a plate; Art v 4, Art v 4 coated on a plate; Amb a 8, Amb a 8 coated on a plate; I, inhibition by Art v 4 (30 μg/ml).

FIGURE 8.

Surface representation of Amb a 8. Residues marked in blue are identical in Amb a 8 and human profilins 1. The poly(l-proline) is shown in a stick representation.

FIGURE 9.

A, sequence conservation of profilins calculated with ConSurf is mapped on the surface of Amb a 8. Actin (pink) was modeled using the structure of human profilin 1 in complex with actin (PDB entry 3CHW). Poly(l-Pro) is shown in a stick representation with carbon atoms marked in yellow. The figure shows three different orientations of the molecules corresponding to 0, 90, and 180° rotation along the y axis. B, residues that are identical in Amb a 8, Art v 4, and Bet v 2 are shown in blue. Residues that are not 100% conserved in these allergens are marked in gray. C, epitopes identified in Cuc m 2. The epitopes are labeled using a convention used in the original paper reporting these epitopes (44, 74). D, epitopes identified in Hel a 2.0101. The epitopes are labeled using a convention used in the original paper reporting these epitopes (43).