Biochemical and clinical studies of putative allergens to assess what distinguishes them from other non-allergenic proteins in the same family

Abstract

Many protein families have numerous members listed in databases as allergens; however, some allergen database entries, herein called "orphan allergens", are members of large families of which all other members are not allergens. These orphan allergens provide an opportunity to assess whether specific structural features render a protein allergenic. Three orphan allergens [Cladosporium herbarum aldehyde dehydrogenase (ChALDH), Alternaria alternata ALDH (AaALDH), and C. herbarum mannitol dehydrogenase (ChMDH)] were recombinantly produced and purified for structure characterization and for clinical skin prick testing (SPT) in mold allergic participants. Examination of the X-ray crystal structures of ChALDH and ChMDH and a homology structure model of AaALDH did not identify any discernable epitopes that distinguish these putative orphan allergens from their non-allergenic protein relatives. SPT results were aligned with ChMDH being an allergen, 53% of the participants were SPT (+). AaALDH did not elicit SPT reactivity above control proteins not in allergen databases (i.e., Psedomonas syringae indole-3-acetaldehyde dehydrogenase and Zea mays ALDH). Although published results showed consequential human IgE reactivity with ChALDH, no SPT reactivity was observed in this study. With only one of these three orphan allergens, ChMDH, eliciting SPT(+) reactions consistent with the protein being included in allergen databases, this underscores the complicated nature of how bioinformatics is used to assess the potential allergenicity of food proteins that could be newly added to human diets and, when needed, the subsequent clinical testing of that bioinformatic assessment.Trial registration number and date of registration AAC-2017-0467, approved as WIRB protocol #20172536 on 07DEC2017 by WIRB-Copernicus (OHRP/FDA Registration #: IRB00000533, organization #: IORG0000432).

Keywords: Allergen; Protein family; Protein structure; Skin prick test.

Fig. 1

Network visualization of the 2020 COMPARE Allergen database. Visualized centrally are clusters (families) of allergens with multiple members. Around the periphery are singlet and small cluster allergens that do not share sufficient sequence similarity with large numbers of other allergens in the database. The three genes utilized in this study are circled and labeled and displayed as diamond symbols. The inset magnifies the cluster of five MDH proteins listed as allergens. From left to right they are identified in the 2020 COMPARE (2022) database as AAO91800.1, P0C0Y4.2, ACB55491.1, the utilized gene AAO91801.1, and COMPARE55. Notably the sequence for ChMDH is present in the 2020 COMPARE (2022) database under the accession AAO91801.1, and its underlying amino acid sequence is identical to that of P0C0Y5.1 which was expressed in this study

Fig. 2

Structural analysis of ChALDH. A The tetrameric structure of ChALDH is shown as a ribbon diagram with each subunit differentially colored with the N- and C- termini labeled. B Pairwise structural comparisons of ChALDH, which is colored white in each overlay, with ALDH from Alternaria alternata (homology model template PDB: 5FHZ), Zea mays (corn/maize; PDB: 4PXL), Spinacia oleracea (spinach; PDB: 4A0M), and Solanum lycopersicum (tomato; PDB: 4I9B). Structurally related proteins were identified using the DALI server (http://ekhidna.biocenter.helsinki.fi/dali_server/). The structural alignment was performed in PyMol (Schrödinger) based on C_α-positions. The statistics of pairwise structural comparison with ChALDH are in Supplementary Table 1A. C Electrostatic surface of each ALDH monomer was generated using the APBS plugin in PyMol (red = acidic; blue = basic). D Hydrophobicity of each ALDH monomer was calculated using the Color-h script based on the Eisenberg hydrophobicity scale in PyMol with darkest red indicating strongest hydrophobicity and white the most polar

Fig. 3

Structural analysis of ChMDH. A The tetrameric structure of ChMDH is shown as a ribbon diagram with each subunit differentially colored. The N- and C- termini are labeled. B Pairwise structural comparisons of ChMDH, which is colored white in each overlay, with structurally related SDR family members from Agaricus bisporus (portobello mushroom; PDB: 1H5Q), Brassica napus (canola; PDB: 1EDO), and Homo sapiens (human; PDB: 4CQM). Structurally related proteins were identified using the DALI server (http://ekhidna.biocenter.helsinki.fi/dali_server/). The structural alignment was performed in PyMol (Schrödinger) based on C_α-positions. The statistics of pairwise structural comparison with ChMDH are in Supplementary Table 1B. C Electrostatic surface of each MDH monomer was generated using the APBS plugin in PyMol with red = acidic and blue = basic. D Hydrophobicity of each MDH monomer was calculated using the Color-h script based on the Eisenberg hydrophobicity scale in PyMol. Darkest red indicates strongest hydrophobicity to white as the most polar