# Structural and Stability Analysis of GRP Family Allergens Pru p 7 and Cry j 7, Which Cause Pollen and Food Allergy Syndrome

## TL;DR

This study compares the structural stability and allergenic properties of two GRP family proteins, Cry j 7 and Pru p 7, which are linked to pollen and food allergies.

## Contribution

The novel contribution is the structural and stability analysis of Cry j 7 and Pru p 7 using recombinant expression and biophysical techniques.

## Key findings

- Cry j 7 is more stable in disulfide linkages and retains structure up to 90 °C compared to Pru p 7.
- Pru p 7 shows greater mobility in specific regions, as revealed by NMR analysis.
- Cry j 7 is more susceptible to proteolysis than Pru p 7 in gastrointestinal conditions.

## Abstract

Cry j 7 is a 7 kDa cysteine-rich gibberellin regulatory protein (GRP) with six disulfide bonds. It was isolated from Japanese cedar as the pollen allergen in this study. It exhibits cross-reactivity with food allergens such as Pru p 7 from peach and causes pollen-food allergy syndrome (PFAS). In this work, recombinant Cry j 7 and Pru p 7 were successfully overexpressed using Pichia pastoris in a high-cell-density fermentation culture, and pure proteins were purified by reverse-phase HPLC. The characterization of Cry j 7 and Pru p 7 were performed by MS, CD, and 1H-NMR experiments to confirm the correct native conformation of Cry j 7 as well as Pru p 7. When compared, the results showed that Cry j 7 exhibits excellent stability in disulfide linkages and preserves its original structure up to 90 °C in various pH buffers in comparison to Pru p 7. Notably, NMR analyses indicated the greater mobility in the α-helix and loop regions of S38-C47 in Pru p 7 compared to those of Cry j 7. Furthermore, our results showed that the sensitivity of Cry j 7 to enzyme digestion differed from that of Pru p 7: Cry j 7 was more susceptible to proteolysis, while Pru p 7 displayed better resistance in the gastrointestinal tract. These variations in structural stability and sensitivity to proteolysis provide valuable insights into the allergenicity within the GRP family.