Microscale Thermophoresis Reveals Oxidized Glutathione as High-Affinity Ligand of Mal d 1

Abstract

Pathogenesis-related (PR)-10 proteins, due to their particular secondary structure, can bind various ligands which could be important for their biological function. Accordingly, the PR-10 protein Mal d 1, the major apple allergen, probably also binds molecules in the hydrophobic cavity of its secondary structure, but it has not yet been investigated in this respect. In this study, various natural products found in apples such as flavonoids, glutathione (GSH), and glutathione disulfide (GSSG) were investigated as possible ligands of Mal d 1 using microscale thermophoresis. Dissociation constants of 16.39 µM, 29.51 µM, 35.79 µM, and 0.157 µM were determined for catechin, quercetin-3-O-rhamnoside, GSH, and GSSG, respectively. Molecular docking was performed to better understand the underlying binding mechanism and revealed hydrophobic interactions that stabilize the ligands within the pocket while hydrophilic interactions determine the binding of both GSH derivatives. The binding of these ligands could be important for the allergenicity of the PR-10 protein and provide further insights into its physiological role.

Multianalytical Approach to Understand Polyphenol-Mal d 1 Interactions to Predict Their Impact on the Allergenic Potential of Apples

“…They determined significantly lower K D values for binding of (+)-CAT than (−)-EC with Mal d 1.02 and also reported different K D values for (+)-EC and (−)-EC, further illustrating the impact of stereo centers on the binding affinity. By docking studies, they identified hydrogen bonds and hydrophobic interactions as the main factors for Mal d 1.02–flavonoid interactions . However, it remains unknown if those generally lower K D values in the μM range are due to the analytical approach or due to the characteristics of the specific isoallergen (Mal d 1.02).…”

“…Chebib et al studied interactions between Mal d 1.02 and PP by applying Microscale Thermophoresis . They determined significantly lower K D values for binding of (+)-CAT than (−)-EC with Mal d 1.02 and also reported different K D values for (+)-EC and (−)-EC, further illustrating the impact of stereo centers on the binding affinity.…”

“…By docking studies, they identified hydrogen bonds and hydrophobic interactions as the main factors for Mal d 1.02−flavonoid interactions. 51 However, it remains unknown if those generally lower K D values in the μM range are due to the analytical approach or due to the characteristics of the specific isoallergen (Mal d 1.02).…”

Ascorbylation of a Reactive Cysteine in the Major Apple Allergen Mal d 1

“…In a first step, we probed the reactivity of Mal d 1 toward L-ascorbate using solution NMR spectroscopy. The addition of L-ascorbate to samples of Mal d 1 did not have an immediate impact on the backbone amide 1 H 15 N-HSQC spectrum of this protein (Figure S2), even at 15-fold excess, indicating that, unlike other amphiphilic compounds of similar size [24], L-ascorbate does not bind to the internal cavity of this protein. However, incubation with L-ascorbate for several days initiated a slow but defined change of the backbone amide 1 H 15 N-HSQC spectrum (Figure 1B).…”

Molecular Approaches for Food Protein Allergenicity Assessment and the Diagnosis and Treatment of Food Allergies

“…In this context, the ability of various natural products found in apples such as flavonoids, glutathione, and glutathione disulfide to bind the major allergen of this fruit Mal d 1 have been studied using microscale thermophoresis by Chebib and Schwab [ 5 ]. Authors found that while flavonoids are bound to Mal d 1 via hydrophobic and polar interactions, glutathione binding to this allergen occurs by hydrophilic hydrogen bonds and van der Waal forces, suggesting differential modification of the allergenicity of Mal d 1 [ 5 ]. Processing food products can induce modifications in the proteins not only by modifying their allergenicity but also by changing their digestibility.…”