Comparative structural and thermal stability studies of Cuc m 2.0101, Art v 4.0101 and other allergenic profilins

Abstract

Worldwide, more than one-third of the population suffers from allergies. A significant fraction of officially registered allergens originate from the profilin family of proteins. Profilins are small ubiquitous proteins which are found in plants, viruses and various eukaryotes including mammals. Although they are primarily regarded as minor allergens, profilins are important players in immunoglobulin E (IgE) cross-reactivity. However, in some populations profilins are recognized by IgE from at least 50% of patients allergic to a given allergen source. Cuc m 2.0101 is recognized by IgE in more than 80% of muskmelon-allergic patients. The recombinant isoallergen Cuc m 2.0101 was produced in significant quantities and its X-ray crystal structure was determined. In addition, a new Art v 4.0101 (mugwort profilin) structure was determined. The profilins Cuc m 2.0101 and Art v 4.0101 were compared in terms of their structure and thermal stability. Furthermore, structural similarities and IgE cross-reactivity between profilins from different sources are discussed to explain the molecular basis of various clinical syndromes involving this group of allergens. Special emphasis is placed on discussion of profilins' quaternary structures and their relation to biological function, as well as to protein allergenicity. Moreover, a potential impact of protein purification protocols on the structure of profilins is highlighted.

Keywords: Allergen; Oligomerization; Pollen-food syndrome; Profilin; Thermal stability.

Figure 1.. DSF results.

A) Average melting temperatures (Tm in °C) for Cuc m 2.0101 with and without purification tag. A gradient of salt (0.0−1.0 M NaCl) and pH (4.0−9.5) buffers were used. Yellow, green, and blue represent high, average, and low melting temperatures, respectively. B) Melting temperature (Tm in °C) differences between Cuc m 2.0101 and Art v 4.0101 for proteins with and without purification tag. Yellow, green, and blue represent high, average, and low melting temperature differences respectively. The standard deviation was less than 1°C for all experiments.

Figure 2.. Structure of Art v 4.0101.

Sequence identity and similarity between Art v 4.0101 and Cuc m 2.0101 shown on the Art v 4.0101 structure (PDB code 6B6J). The top images are cartoon representation with the cysteine residues shown in stick representation while the bottom images show surface representation. In Cuc m 2 there is no cysteine that corresponds to Cys95 of Art v 4. This amino acid is replaced by Thr residue and therefore no internal disulfide bridge is present in Cuc m 2. Grey color indicates identical residues, and green color is used to marked residues that are similar, but not identical. Regions displayed in purple display neither identity nor similarity.

Figure 3.. Sequence conservation.

A) Sequence comparison of all allergenic profilins reported in the PDB. Secondary structure elements are marked for Zea m 12.0101 (PDB code 5FEF) and Art v 4.0101 (PDB code 6B6J), respectively. Cysteine residues forming disulfide bridges in Amb a 8.0101 and Art v 4.0101 are marked using green boxes. Initiator methionine residues that are not present in mature forms of the profilins are included. B) Conservation of profilin sequences mapped on structure of Cuc m 2.0101 presented in cartoon representation. Residues that are conserved in all analyzed allergenic profilins herein (Supplementary Table S1 and Figure S3) are marked in red. Residues from profilins that have their structures determined and are completely conserved are shown in red and salmon. It implicates that salmon corresponds to residues that are conserved for profilins shown in this figure, but which are not completely conserved among all analyzed profilins presented in Supplementary Figure 3S. N- and C-terminal residues are marked with corresponding letter. C) Conservation of sequences mapped on molecular surface of Cuc m 2.0101

Figure 4.

Sequence similarities and identities between Cuc m 2.0101 and various plant profilins that are officially registered as allergens by the World Health Organization and International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-committee (www.allergen.org). Proteins are colored according to allergen source. Sequence comparison was performed using Clustal Omega (Sievers et al., 2011) and SIAS (http://imed.med.ucm.es/Tools/sias.html).

Figure 5.. Identified epitopes and mimotope.

A) Cartoon (top) and surface (bottom) representations of Cuc m 2.0101 with mapped IgE epitopes as determined by Lopez-Torrejon et al. (2007). N- and C-terminal residues are marked with corresponding letters. Epitope 1, consisting of residues 66–75 and 81–93, in wheat, epitope 2 (residues 95–99 and 122–131) in brown, epitope 3 (residues 2–10) in light teal, and epitope 4 (residues 35–45) in light pink. B) Mimotope determined by Tordesillas et al. and consisting of residues 2–3, 5–6, 9–10, 111–114, 116–117, as well as 121–122 is shown in light blue (Tordesillas et al., 2010).

Figure 6.. Dimeric and monomeric profilins.

A) Dimeric structure of Hev b 8.0101. B) Cuc m 2.0101 (surface representations) superposed on one of the molecules forming Hev b 8.0101 dimer. Epitope 1 is marked in wheat, epitope 2 in brown, epitope 3 in light teal and epitope 4 in light pink. C) Cuc m 2.0101 with modeled polyproline peptide (shown in sphere representation). The peptide binding site is formed by residues forming epitopes 2 and 3 identified by Lopez-Torrejon et al. (2007). D) Mimotope determined by Tordesillas et al. (2010) (light blue). Cys residues that may be responsible for formation of a dimeric structure is marked in orange. E) Putative Cuc m 2.0101 dimer interface based on structure homology of Hev b 8 (PDB code 5FEG). Residues marked in blue are completely conserved, while residues shown in purple are not conserved.