{"doi":"10.1111/all.70201","title":"Polymeric α‐Hairpinin Allergens Induce a Functional Response via a Single Antibody","abstract":"Vicilins are abundant seed storage proteins to which peanut and tree nut allergic patients are frequently sensitized. The large C-terminal cupin domain has been extensively characterized, but less well studied are the N-terminal α-hairpinin domains. These domains are characterized by two α-helices held together by two disulfide bonds. Additionally, a skewed amino acid distribution for Arg, Glu, and Gln, coupled with a high structural similarity, suggests α-hairpinins are prone to be cross-reactive across distantly related species and controversially with other protein folds [1, 2]. α-hairpinins occur in variable numbers in different seeds, and occasionally without a cupin domain, e.g., Pru du 8. Prior research suggests that α-hairpinins are cleaved to generate individual low-molecular-weight proteins whose allergenicity is independent of both each other and their cupin domain [3]. However, incomplete cleavage could have important consequences for allergies. In walnuts, Jug r 2.0102 has six α-hairpinins expressed with the central adjacent domains (α3 and α4) exhibiting the highest sequence identity of 90% (Figure 1A and Figure S1A,D). Using hybridoma-based technology we identified a human-derived IgE monoclonal antibody, 6D12, that binds specifically to both recombinant α3 and α4 allergens (Figure S1) [4]. Using X-ray crystallography, we determined a crystal structure that shows 6D12 bound to α4 at the turn between the helices (Figure 1E and Figure S1). Based on this structure, we tested site-directed mutants with ELISA to demonstrate that G30 and R34 of α4 were critical sites for the specificity of 6D12 (Figure S1). These residues are uniquely found in α3 and α4, suggesting why 6D12 is specific for these two α-hairpinins. Furthermore, western blot analysis of walnut extract revealed variable cleavage products of Jug r 2, ranging from one to three α-hairpinin(s) and larger bands still attached to the cupin domain (Figure 1C,D). Consequently, 6D12 antibody alone could potentially elicit an immune response to any cleavage products containing α3-α4 fragments via repeated cross-linking (Figure 2A). A similar scenario was suggested to enhance allergic reactivity to Phl p 5, which has two homologous domains tethered via a flexible linker [5]. To substantiate the functional response to a polymeric allergen with a single antibody, we recombinantly expressed an α3-α4 construct and conducted sandwich ELISA, basophil activation test (BAT), and a mouse model of passive systemic anaphylaxis (PSA). All three assays demonstrated a functional response with 6D12 (Figure 2B–D). However, walnut extract was unable to replicate this response likely due to the presence of cleavage products with individual α3 or α4 monomers. This is analogous to the known hook effect in BAT where high concentrations of antigen inhibit antibody cross-linking. To confirm this assumption, we demonstrated via sandwich ELISA and PSA that monomers of α3 could inhibit the functional response of the α3-α4 construct (Figure 2B,E). This suggests a feasible mechanism to inhibit or enhance activation. Consequently, this presents a uniquely different scenario to that of Phl p 5, whereby repeated cross-linking can be inhibited or activated due to the ratio of inhibitory molecules to activating polymers. The cleavage products of α-hairpinin allergens from other food sources raise concerns. For example, in hazelnut, Cor a 16 contains 12 α-hairpinin repeats with higher aggregate sequence identity (Figure S2A,B). Using single-cell sequencing technologies from allergic donors and ELISA-based screening, we identified a human IgE antibody, IGX-0705, specific for α-hairpinins of Cor a 16 in addition to several α-hairpinins from walnut (Figure S2C,D) [6]. First, this demonstrates that humans can make a cross-reactive antibody to hazelnut α-hairpinins. Second, western blot results indicate partial cleavage products in hazelnut with fewer α-hairpinin monomers (Figure S2E); this ratio could m","journal":"Allergy","year":2025,"id":587639,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9629,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1503681,"name":"Taylor Cosey","orcid":null,"position":1,"is_corresponding":false},{"id":1503682,"name":"Emmanuel R. De Diavoukana","orcid":null,"position":2,"is_corresponding":false},{"id":1220356,"name":"Isabelle R. Lytle","orcid":"0000-0003-3501-3904","position":3,"is_corresponding":false},{"id":658004,"name":"Alexander C. Y. Foo","orcid":"0000-0002-4331-2085","position":4,"is_corresponding":false},{"id":1503683,"name":"L.G. Pedersen","orcid":null,"position":5,"is_corresponding":false},{"id":110872,"name":"Scott A. Gabel","orcid":null,"position":6,"is_corresponding":false},{"id":605486,"name":"Robert M. Petrovich","orcid":"0009-0008-6229-0281","position":7,"is_corresponding":false},{"id":11427,"name":"Min Shi","orcid":"0000-0001-8265-5307","position":8,"is_corresponding":false},{"id":1503684,"name":"Venu Aruva","orcid":null,"position":9,"is_corresponding":false},{"id":1041103,"name":"Paige Creeks","orcid":null,"position":10,"is_corresponding":false},{"id":689344,"name":"Joyce J. W. Wong","orcid":"0000-0002-0521-9146","position":11,"is_corresponding":false},{"id":1059310,"name":"Jian Zhang","orcid":"0000-0001-5552-0132","position":12,"is_corresponding":false},{"id":290007,"name":"R. Stokes Peebles","orcid":"0000-0002-1429-7875","position":13,"is_corresponding":false},{"id":1040506,"name":"Derek Croote","orcid":"0000-0003-4907-1865","position":14,"is_corresponding":false},{"id":875083,"name":"Scott A. Smith","orcid":"0000-0002-5519-8662","position":15,"is_corresponding":false},{"id":334365,"name":"Geoffrey A. Mueller","orcid":"0000-0001-8361-5323","position":16,"is_corresponding":false},{"id":374766,"name":"Gage O. Leighton","orcid":"0000-0003-4636-0370","position":0,"is_corresponding":true}],"reference_count":6,"raw_metadata":null,"created_at":"2026-07-19T02:59:39.958043Z","pmid":"41476027","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}