Structural and Immunological Features of PR-10 Allergens: Focusing on the Major Alder Pollen Allergen Aln g 1.

Today, allergies have become a serious problem. PR-10 proteins are clinically relevant allergens that have the ability to bind hydrophobic ligands, which can significantly increase their allergenicity potential. It has been recently shown that not only the birch pollen allergen Bet v 1 but also the alder pollen allergen Aln g 1, might act as a true sensitizer of the immune system. The current investigation is aimed at the further study of the allergenic and structural features of Aln g 1. By using qPCR, we showed that Aln g 1 was able to upregulate alarmins in epithelial cells, playing an important role in sensitization. With the use of CD-spectroscopy and ELISA assays with the sera of allergic patients, we demonstrated that Aln g 1 did not completely restore its structure after thermal denaturation, which led to a decrease in its IgE-binding capacity. Using site-directed mutagenesis, we revealed that the replacement of two residues (Asp27 and Leu30) in the structure of Aln g 1 led to a decrease in its ability to bind to both IgE from sera of allergic patients and lipid ligands. The obtained data open a prospect for the development of hypoallergenic variants of the major alder allergen Aln g 1 for allergen-specific immunotherapy.

Structural and Immunologic Properties of the Major Soybean Allergen Gly m 4 Causing Anaphylaxis.

Gly m 4 is the major soybean allergen, causing birch pollen cross allergic reactions. In some cases, Gly m 4-mediated anaphylaxis takes place, but the causative factors are still unknown. Here, we studied the structural and immunologic properties of Gly m 4 to shed light on this phenomenon. We showed that Gly m 4 retained its structure and IgE-binding capacity after heating. Gly m 4 was cleaved slowly under nonoptimal gastric conditions mimicking duodenal digestion, and IgE from the sera of allergic patients interacted with the intact allergen rather than with its proteolytic fragments. Similar peptide clusters of Bet v 1 and Gly m 4 were formed during allergen endolysosomal degradation in vitro, but their sequence identity was insignificant. Animal polyclonal anti-Gly m 4 and anti-Bet v 1 IgG weakly cross-reacted with Bet v 1 and Gly m 4, respectively. Thus, we supposed that not only conserved epitopes elicited cross-reactivity with Bet v 1, but also variable epitopes were present in the Gly m 4 structure. Our data suggests that consumption of moderately processed soybean-based drinks may lead to the neutralizing of gastric pH as a result of which intact Gly m 4 can reach the human intestine and cause IgE-mediated system allergic reactions.

Effect of Point Mutations on Structural and Allergenic Properties of the Lentil Allergen Len c 3.

Plant lipid transfer proteins (LTPs) are known to be clinically significant allergens capable of binding various lipid ligands. Recent data showed that lipid ligands affected the allergenic properties of plant LTPs. In this work, we checked the assumption that specific amino acid residues in the Len c 3 structure can play a key role both in the interaction with lipid ligands and IgE-binding capacity of the allergen. The recombinant analogues of Len c 3 with the single or double substitutions of Thr41, Arg45 and/or Tyr80 were obtained by site-directed mutagenesis. All these amino acid residues are located near the "bottom" entrance to the hydrophobic cavity of Len c 3 and are likely included in the IgE-binding epitope of the allergen. Using a bioinformatic approach, circular dichroism and fluorescence spectroscopies, ELISA, and experiments mimicking the allergen Len c 3 gastroduodenal digestion we showed that the substitution of all the three amino acid residues significantly affected structural organization of this region and led both to a change of the ligand-binding capacity and the allergenic potential of Len c 3.

Impact of Different Lipid Ligands on the Stability and IgE-Binding Capacity of the Lentil Allergen Len c 3.

Previously, we isolated the lentil allergen Len c 3, belonging to the class of lipid transfer proteins, cross-reacting with the major peach allergen Pru p 3 and binding lipid ligands. In this work, the allergenic capacity of Len c 3 and effects of different lipid ligands on the protein stability and IgE-binding capacity were investigated. Impacts of pH and heat treating on ligand binding with Len c 3 were also studied. It was shown that the recombinant Len c 3 (rLen c 3) IgE-binding capacity is sensitive to heating and simulating of gastroduodenal digestion. While being heated or digested, the protein showed a considerably lower capacity to bind specific IgE in sera of allergic patients. The presence of lipid ligands increased the thermostability and resistance of rLen c 3 to digestion, but the level of these effects was dependent upon the ligand's nature. The anionic lysolipid LPPG showed the most pronounced protective effect which correlated well with experimental data on ligand binding. Thus, the Len c 3 stability and allergenic capacity can be retained in the conditions of food heat cooking and gastroduodenal digestion due to the presence of certain lipid ligands.