3-trisulfanyl-L-alanine

Abstract

Results: Polysulfide-bridged allergen dimers exhibit different allergenic properties compared with the monomer.

Abstract

Many allergens share several biophysical characteristics, including the capability to undergo oligomerization. The dimerization mechanism in Bet v 1 and its allergenic properties are so far poorly understood. Here, we report crystal structures of dimeric Bet v 1, revealing a noncanonical incorporation of cysteine at position 5 instead of genetically encoded tyrosine. Cysteine polysulfide bridging stabilized different dimeric assemblies, depending on the polysulfide linker length. These dimers represent quaternary arrangements that are frequently observed in related proteins, reflecting their prevalence in unmodified Bet v 1. These conclusions were corroborated by characteristic immunologic properties of monomeric and dimeric allergen variants. Hereby, residue 5 could be identified as an allergenic hot spot in Bet v 1. The presented results refine fundamental principles in protein chemistry and emphasize the importance of protein modifications in understanding the molecular basis of allergenicity.

Abstract

Many allergens share several biophysical characteristics, including the capability to undergo oligomerization. The dimerization mechanism in Bet v 1 and its allergenic properties are so far poorly understood. Here, we report crystal structures of dimeric Bet v 1, revealing a noncanonical incorporation of cysteine at position 5 instead of genetically encoded tyrosine. Cysteine polysulfide bridging stabilized different dimeric assemblies, depending on the polysulfide linker length. These dimers represent quaternary arrangements that are frequently observed in related proteins, reflecting their prevalence in unmodified Bet v 1. These conclusions were corroborated by characteristic immunologic properties of monomeric and dimeric allergen variants. Hereby, residue 5 could be identified as an allergenic hot spot in Bet v 1. The presented results refine fundamental principles in protein chemistry and emphasize the importance of protein modifications in understanding the molecular basis of allergenicity.

Identification of the Recombinant Bet v 1 Dimer

During purification, we observed WT Bet v 1a to elute as a double peak in ion exchange chromatography. Although the dominant peak (∼95% in all analyzed batches) was monomeric wild type Bet v 1a (as evidenced by MS analysis), the high salt fraction contained both monomeric and dimeric Bet v 1a, as judged by size exclusion chromatography (a). This interpretation was confirmed by clear native PAGE and SDS-PAGE (b). The SDS-PAGE analysis also revealed the Bet v 1a dimer to be reduction-sensitive. This observation was surprising, given the lack of cysteine residues in Bet v 1a. More intriguingly, the redox sensitivity of the Bet v 1a dimer can be further distinguished: one dimer species is completely redox stable under native PAGE conditions, whereas a second dimer species dissociates under reducing native PAGE conditions (c). We conclude that the Bet v 1a bridging is inherently redox-sensitive; depending on the quaternary structure, the bridging can be protected from reducing agents by the native dimer structure.