Two Distinct Conformations in Bet v 2 Determine Its Proteolytic Resistance to Cathepsin S

Abstract

Birch pollen allergy affects more than 20% of the European allergic population. On a molecular level, birch pollen allergy can be linked to the two dominant allergens Bet v 1 and Bet v 2. Bet v 2 belongs to the profilin family, which is abundant in the plant kingdom. Importantly, the homologous plant profilins have a conserved cysteine motif with a currently unknown functional relevance. In particular, it is unknown whether the motif is relevant for disulfide formation and to what extent it would affect the profilins’ structural, functional and immunological properties. Here we present crystal structures of Bet v 2 in the reduced and the oxidized state, i.e., without and with a disulfide bridge. Despite overall structural similarity, the two structures distinctly differ at their termini which are stabilized to each other in the oxidized, i.e., disulfide-linked state. These structural differences translate into differences in their proteolytic resistance. Whereas the oxidized Bet v 2 is rather resistant towards the endolysosomal protease cathepsin S, it is rapidly degraded in the reduced form. By contrast, both Bet v 2 forms exhibit similar immunological properties as evidenced by their binding to IgE antibodies from birch pollen allergic patients and by their ability to trigger histamine release in a humanized rat basophilic leukemia cells (RBL) assay, independent of the presence or absence of the disulfide bridge. Taken together our findings suggest that the oxidized Bet v 2 conformation should be the relevant species, with a much longer retention time to trigger immune responses.

Molecular Basis of Plant Profilins’ Cross-Reactivity

“…This structural difference may explain why rPhl p 12 was not recognized by 2D10. Furthermore, Bet v 2 has a clear structural difference in α-helix 1, caused by an internal C13–C115 disulfide bond [ 29 ] that may contribute to a different arrangement of antigenic determinants that could explain its lower binding (54%), as compared to the mAb binding towards rHev b 8 ( Figure 1 ). Westritschnig et al designed a restructured hypoallergenic variant of Timothy grass profilin (Phl p 12-rs) “tail-to-head”, meaning that the C terminus of Phl p 12 was at its N terminus and the N terminus was at its C terminus [ 30 ].…”

Spatiotemporal proteolytic susceptibility of allergens: positive or negative effects on the allergic sensitization?

“…Another birch pollen allergen, Bet v 2 (profilin), was found to exist in both oxidized (with an intra-disulfide bond) and reduced form (without disulfide bond). The oxidized form is more resistant to cathepsin S degradation albeit both forms share similar IgE reactivity and basophil activation capacity ( 37 ). However, whether the proteolytic resistance of oxidized Bet v 2 leads to higher MHC-II presentation remains to be investigated.…”

Crystal structure of timothy grass allergen Phl p 12.0101 reveals an unusual profilin dimer

“…The solventexposed thiol group was shown to be prone to modifications and disulfide bond formation, like in the case of Hev b 8.0102 (Mares-Mejia et al, 2016). Moreover, reducing agents such as 2-mercaptoethanol (β-ME) and dithiothreitol (DTT) were used in purification of these proteins and may have an effect on profilin stability (Soh et al, 2017;Mittermann et al, 1998). Under reducing conditions, profilin exists nearly exclusively in the monomeric form (Cudowska et al, 2020;Kapingidza et al, 2019;Offermann et al, 2016).…”