Comparative studies of seafood and reptile α- and β-parvalbumins

Abstract

Small calcium-binding proteins such as parvalbumins (PVs) are major seafood and fish allergens. However, the impact of structural changes on their capacity to bind IgE has not been studied in detail. Therefore, fish and reptilian PVs, as well as human α-PV, were selected for biochemical, structural, and IgE binding studies. Likely due to their high solubility, crystallization proved difficult, so additional techniques were used to promote crystallization of the proteins. Novel crystal structures were determined for human PV, cod allergen Gad m 1.0201, saltwater crocodile allergen Cro p 1.0101, and the α-PV from thornback ray. β-PVs are considered the major fish allergens, while α-PVs are rarely categorized as allergens. To explain these differences, the results of structural and IgE binding studies were combined. This approach allowed us to provide new insight into IgE binding epitopes present on PVs, focusing on cross-reactivity among the selected α- and β-PVs. In addition, we have shown that these proteins display remarkable thermal stability across a range of pH conditions, which is relevant in the case of food allergens and food processing. Moreover, it is shown that the presence of calcium cations is critical for stability of the studied PVs via their protein folding, which has an impact on the formation of IgE binding epitopes. These studies shows the stability of fish and reptile PV allergens, and it allows for further evaluation of their IgE cross-reactivity.

Keywords: IgE binding; allergy; calcium‐binding protein; crystal structure; parvalbumin; seafood allergy.

FIGURE 1

Differential scanning fluorimetry results for PVs discussed in this work.

FIGURE 2

2D‐NMR results for ray PV and WT‐Cro p 1.0101. The spectra for native protein are shown in blue, 3.0 mM EDTA in green, and 30 mM EDTA in red. (a) Native ray PV. (b) Native WT‐Cro p 1.0101. (c) Ray PV with EDTA added to 3.0 and 30 mM. (d) WT‐Cro p 1.0101 with EDTA added to 3.0 and 30 mM.

FIGURE 3

Crystal structure of ray PV. (a) Full protein with α‐helices labeled. The AB motif is shown in pink, the CD in light yellow, and the EF in aquamarine. (b) Calcium binding sites with coordinating residues labeled and bonds shown with yellow dashes.

FIGURE 4

Additional crystal structures described in this work. (a) Gad m 1.0201 (calcium binding residues shown), (b) Cro p 1.0101‐GFP (Cro p 1.0101 in blue; linker in red; GFP in green), and (c) human α‐PV (lysine side chains shown in pink). Calcium cations are shown as green spheres and lanthanum cations are shown as blue spheres.

FIGURE 5

Immunological evaluation of selected PVs using ELISA. (a) Comparison of the reactions of fish‐allergic patients to the PVs versus the negative control sera. (b) Reaction of each patient to each of the five PVs. (c) IgE binding to immobilized PVs with increasing concentrations of EDTA (0–10 mM). The results of ELISA are presented as means with error bars indicating range (minimum–maximum) of the results. The inhibition results are presented as means of three experiments with standard errors.

FIGURE 6

IgE binding epitopes suggested by patches on the surfaces of the proteins that seem surface‐exposed and easily accessible, and are conserved between PVs studied in this work. (a) Ray PV with two potential cross‐reactive epitopes for α‐PV, colored in light gray and dark gray. (b) Gad m 1.0201 with one potential cross‐reactive epitope for β‐PV, colored in light aqua.