NMR resonance assignments of the four isoforms of the hazelnut allergen Cor a 1.04

Abstract

In large parts of Europe, Northern America and China people are suffering from allergies after consuming certain kinds of fruits and vegetables. Typical allergic symptoms range from scratching and itching of the throat to severe symptoms like rhino conjunctivitis and anaphylaxis. For hazelnuts (Corylus avellana), these allergies result from initial sensitization to the birch (Betula verrucosa) pollen allergen Bet v 1 and subsequent development of allergic cross-reactions to proteins that are similar in their three-dimensional structure to the sensitizing protein Bet v 1. The cross-reactive proteins in hazelnut are the four isoforms Cor a 1.04 with a molecular weight of about 17.5 kDa. Significant differences regarding the immunologic behavior of these proteins have been reported. In this work we assigned backbone and side chain 1 H, 13 C, and 15 N chemical shifts of these four isoforms, Cor a 1.0401, Cor a 1.0402, Cor a 1.0403, and Cor a 1.0404 by solution NMR spectroscopy. The chemical shift data confirm the characteristic Bet v onefold for all four isoforms, consisting of seven β-strands that are separated by two short α-helices, along with a long C-terminal α-helix. These data provide the basis for a comparative structural and dynamic analysis of these proteins by NMR in order to characterize their different immunologic cross-reactivities on a molecular level.

Inverse relation between structural flexibility and IgE reactivity of Cor a 1 hazelnut allergens

“…The NMR chemical shift data clearly show that Pro99, like all other prolines, possesses trans configuration 25 , in accordance with its location in an antiparallel β-sheet. Strand β6 (residues Glu96-Ala106) containing Pro99 and the adjacent strand β7 (Gly112-Thr123) display only slightly reduced β-strand propensities in Cor a 1.0404 when compared to the other three isoforms 26 .

Figure 2 Amphiphilicity of the Cor a 1.04 internal cavity.

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“…Sections of 1 H– 15 N-HSQC spectra of Cor a 1.0401 (top) and Cor a 1.0404 (bottom) directly after purification, as well as 5 and 7 days later. Assignments 26 are indicated by single letter codes and signals labeled with an asterisk indicate aggregation and degradation. …”

“…AAG40330), and Cor a 1.0404 (accession no. AAG40331) and the subsequent expression has been described previously 26 . Plasmid constructs encoding the mutant forms C4S Cor a 1.0401, P99A Cor a 1.0404, and P99T Cor a 1.0404 were created by site-directed mutagenesis using Phusion DNA polymerase (New England Biolabs, Frankfurt am Main, Germany) for C4S Cor a 1.0401 and Platinum SuperFi DNA polymerase (Thermo Fisher Scientific, Vienna, Austria) for P99A Cor a 1.0404, and P99T Cor a 1.0404 according to the protocol of the manufacturers.…”

“…S6 online). Expression of the three mutant proteins was performed as for the wild-type hazelnut isoforms but without using ISOGRO- 15 N powder for the expression 26 . Unlabeled proteins were expressed in M9 minimal medium without isotopically labeled reagents.…”

Structural basis for variable IgE reactivities of Cor a 1 hazelnut allergens

“…AAG40330), and Cor a 1.0404 (accession no. AAG40331) and the subsequent expression has been described previously (Führer et al, 2020). Plasmid constructs encoding the mutant forms C4S Cor a 1.0401, P99A…”