Boiling down the cysteine-stabilized LTP fold - loss of structural and immunological integrity of allergenic Art v 3 and Pru p 3 as a consequence of irreversible lanthionine formation

Wildner, Sabrina; Griessner, Iris; Stemeseder, Teresa; Regl, Christof; Soh, Wai Tuck; Stock, Lorenz; Völker, Timo; Alessandri, C.; Mari, Adriano; Huber, Christian G.; Stutz, Hanno; Brandstetter, Hans; Gadermaier, Gabriele

doi:10.1016/j.molimm.2019.10.012

Cited by 15 publications

(14 citation statements)

References 63 publications

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“…To generate monoclonal antibodies (mAbs), mice were immunized with recombinant Art v 3.0201 (for simplicity termed Art v 3). We produced the non-tagged allergen using E. coli Rosetta-gami cells enabling disulphide-bond formation relevant for the LTP fold (25). The protein showed the correct identity and high purity (>98%) as verified by gel electrophoresis and mass spectrometry (Fig.…”

Section: Art V 3-specific Murine Monoclonal Antibodies Recognize Strumentioning

confidence: 99%

“…Recombinant Art v 3.0201, was produced as previously described (25). For protein isotope labeling, bacteria were grown in M9 minimal medium with 15 N or 15 N and 13 C as sole nitrogen and carbon source (54).…”

Section: Recombinant Production Of Allergensmentioning

confidence: 99%

“…For protein isotope labeling, bacteria were grown in M9 minimal medium with 15 N or 15 N and 13 C as sole nitrogen and carbon source (54). Proteins were purified as previously described and analyzed by reducing gel electrophoresis (25). Another isoform, i. e. Art v 3.0301 as well as Api g 2, Cor a 8, Pru p 3, Fra a 3, Mal d 3 and Amb a 6 were obtained as recombinant proteins as published and described in the supporting information (33,(55)(56)(57)(58)(59)(60).…”

Section: Recombinant Production Of Allergensmentioning

confidence: 99%

“…This also applies to nonspecific lipid transfer proteins (LTPs) which were identified as allergens in food, pollen and latex (6,24). LTPs are small, non-glycosylated proteins with a typical α-helical fold stabilized by disulphide bonds (25). Due to their high thermal and proteolytic stability, they are able to trigger severe allergic reactions.…”

Section: Introductionmentioning

confidence: 99%

“…The only pollen LTP with IgE cross-reactivity relevant to this syndrome is Art v 3 from mugwort pollen. Our recent study revealed that patients' IgE and murine IgG recognize exclusively conformational antibody binding epitopes (25), suggesting a folded protein during allergic sensitization (37,38). So far, no information on the localization of antibody binding sites accounting for the IgE (cross)reactive epitopes of Art v 3 is available.…”

Section: Introductionmentioning

confidence: 99%

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Hydrogen/deuterium exchange memory NMR reveals structural epitopes involved in IgE cross-reactivity of allergenic lipid transfer proteins

Muzio

Wildner

Huber

et al. 2020

Journal of Biological Chemistry

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Identification of antibody binding epitopes is crucial to understand immunological mechanisms. It is of particular interest for allergenic proteins with high cross-reactivity as observed in the lipid transfer protein (LTP) syndrome that is characterized by severe allergic reactions. Art v 3, a pollen LTP from mugwort is frequently involved in this cross-reactivity, but no antibody binding epitopes have been determined so far. To reveal human IgE binding regions of Art v 3, we produced three murine high-affinity monoclonal antibodies (mAbs), which showed 70-90% coverage of the allergenic epitopes from mugwort pollen allergic patients. As reliable methods to determine structural epitopes with tightly interacting intact antibodies under native conditions are lacking, we developed a straightforward NMR approach termed hydrogen/deuterium exchange memory (HDXMEM). It relies on the slow exchange between the invisible antigen-mAb complex and the free 15N-labeled antigen whose 1H-15N correlations are detected. Due to a memory effect, changes of NH protection during antibody binding are measured. Differences in H/D exchange rates and analyses of mAb reactivity to homologous LTPs revealed three structural epitopes: two partially cross-reactive regions around α-helices 2 and 4, as well as a novel Art v 3-specific epitope at the C-terminus. Protein variants with exchanged epitope residues confirmed the antibody-binding sites and revealed strongly reduced IgE reactivity. Using the novel HDXMEM for NMR epitope mapping allowed identification of the first structural epitopes of an allergenic pollen LTP. This knowledge enables improved cross-reactivity prediction for patients suffering from LTP-allergy and facilitates design of therapeutics.

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Section: Art V 3-specific Murine Monoclonal Antibodies Recognize Strumentioning

confidence: 99%

Section: Recombinant Production Of Allergensmentioning

confidence: 99%