Cor a 1.0401

What is Cor a 1.0401 and how does it relate to other hazelnut allergens?

Cor a 1.0401 is the major hazelnut allergen from Corylus avellana, belonging to the pathogenesis-related PR-10 protein family and the Bet v 1 homologues family . It represents one of four variants of Cor a 1.04 (including Cor a 1.0401, Cor a 1.0402, Cor a 1.0403, and Cor a 1.0404) that share 97-99% amino acid sequence identity but exhibit different immunological properties .

Importantly, Cor a 1.04 (hazelnut) shows only 63% sequence identity with Cor a 1.01 (hazel pollen) despite coming from the same plant species . In contrast, Cor a 1.04 shares approximately 83% sequence identity with Bet v 1 from birch pollen, suggesting closer evolutionary relationships between food and pollen allergens across different species than between allergens from different tissues of the same plant .

What is the molecular structure of Cor a 1.0401?

Cor a 1.0401's structure exhibits the characteristic PR-10 protein fold, consisting of a seven-stranded antiparallel β-sheet, two short α-helices arranged in V-shape, and a long C-terminal α-helix that encompasses a hydrophobic pocket . The NMR solution structure (PDB ID: 6Y3H) shows root-mean-square deviation (RMSD) values of 0.4 Å for backbone atoms and 0.5-0.7 Å for heavy atoms, indicating a well-defined structure .

The structure includes a hydrophobic cavity capable of binding specific ligands, similar to other PR-10 proteins. Despite structural similarities with Bet v 1, Cor a 1.0401 binds different ligands with high selectivity, demonstrating that structural homology does not necessarily translate to functional equivalence .

How do the four Cor a 1.04 variants differ structurally and immunologically?

The four Cor a 1.04 variants share very high sequence homology but display significant differences in their immunological properties:

The data reveals an inverse relationship between structural flexibility and allergenicity, with the most rigid variant (Cor a 1.0401) showing the highest IgE binding, while the most flexible variant (Cor a 1.0404) demonstrates significantly reduced allergenicity .

What is the relationship between structural flexibility and IgE reactivity in Cor a 1.04 variants?

Research has established an inverse relationship between structural flexibility and IgE-binding capacity among the four Cor a 1.04 variants . NMR relaxation dispersion (RD) measurements provide site-specific information about conformational heterogeneity, revealing significant differences in protein backbone flexibility:

• Cor a 1.0401: 33% of residues show RD profiles >1 s⁻¹ and 14% >3 s⁻¹ (least flexible)

• Cor a 1.0402: 59% of residues show RD profiles >1 s⁻¹ and 25% >3 s⁻¹

• Cor a 1.0403: 50% of residues show RD profiles >1 s⁻¹ and 15% >3 s⁻¹

• Cor a 1.0404: 68% of residues show RD profiles >1 s⁻¹ and 26% >3 s⁻¹ (most flexible)

This flexibility ranking (Cor a 1.0404 > Cor a 1.0402 > Cor a 1.0403 > Cor a 1.0401) inversely correlates with IgE-binding capacity, suggesting that structural rigidity may preserve conformational epitopes important for antibody recognition . Hydrogen-deuterium exchange experiments further confirm these findings, with Cor a 1.0404 showing remarkably fast exchange rates compared to Cor a 1.0401 .

How does the proline substitution in Cor a 1.0404 affect its immunological properties?

The unique proline residue at position 99 in Cor a 1.0404 (replacing serine found in other variants) appears responsible for dramatically altering its structural and immunological properties . Despite proline's cyclic structure typically restricting backbone conformations, this substitution paradoxically increases flexibility throughout the protein scaffold .

Increased flexibility extends beyond the immediate vicinity of position 99 (in strand β6), affecting residues in adjacent strands β5 and β7 and distributing throughout the protein structure . This widespread conformational heterogeneity correlates with significantly reduced IgE binding, with histamine release assays showing positive reactions in only 2/15 patient sera for Cor a 1.0404, compared to 11-13/15 for other variants .

These findings highlight how a single amino acid substitution can profoundly impact allergenicity by altering protein dynamics rather than just local structure, suggesting potential strategies for designing hypoallergenic variants through targeted mutations affecting flexibility .

What natural ligands does Cor a 1.0401 bind and how does this compare to other PR-10 allergens?

Cor a 1.0401 specifically binds quercetin-3-O-(2"-O-β-D-glucopyranosyl)-β-D-galactopyranoside, as identified through mass spectrometry and NMR spectroscopy . This ligand differs from quercetin-3-O-sophoroside bound by the homologous birch pollen allergen Bet v 1 .

Remarkably, despite the general promiscuity observed for PR-10 allergens in vitro, Cor a 1.0401 and Bet v 1.0101 exhibit highly selective binding for their specific ligands but not for each other's ligands . This selective binding behavior suggests that natural ligands may play roles in the proteins' biological functions that require precise molecular recognition.

The identification of these specific natural ligands provides insights into potential physiological roles of these allergens in plants and may have implications for understanding cross-reactivity patterns in patients with concurrent pollen and food allergies .

What experimental approaches are most effective for studying Cor a 1.0401 structural dynamics?

Several complementary NMR methodologies have proven effective for characterizing the structural dynamics of Cor a 1.0401 and related allergens:

• Relaxation Dispersion (RD) NMR: Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments provide site-specific information about conformational exchange processes occurring on microsecond to millisecond timescales . For Cor a 1.04 variants, these experiments revealed significant differences in conformational heterogeneity, with RD profiles exceeding 1 s⁻¹ for 33-68% of residues depending on the variant .

• Hydrogen-Deuterium (H/D) Exchange: Monitoring the rate of exchange of backbone amide protons with deuterium when the protein is placed in D₂O provides information about solvent accessibility and hydrogen bonding stability . These experiments are particularly valuable for comparing structural stability between variants like Cor a 1.0401 and Cor a 1.0404 .

• Order Parameter Measurements: ¹⁵N relaxation experiments determine S² order parameters, providing information about the amplitude of fast (picosecond to nanosecond) timescale motions of individual backbone N-H bonds .

When designing such experiments, temperature control is critical—lower temperatures (4-15°C) may be necessary to slow exchange rates and observe meaningful differences between variants with different flexibilities . Consistent pH (typically 6.5-7.0) should be maintained across all variant comparisons.

How should researchers assess the IgE-binding capacity of Cor a 1.0401?

Multiple complementary approaches are recommended for comprehensive assessment of Cor a 1.0401's IgE-binding capacity:

• Enzyme Allergo-Sorbent Test (EAST): Studies with sera from patients with positive double-blind, placebo-controlled food challenge (DBPCFC) to hazelnut have shown differential binding to Cor a 1.04 variants, with 95% of sera recognizing Cor a 1.0401 compared to only 74% recognizing Cor a 1.0404 .

• Histamine Release Assays: These functional assays measure the biological activity of allergens by assessing histamine release from basophils. In one study, 13/15 hazelnut-allergic patients showed positive reactions to Cor a 1.0401, while only 2/15 reacted to Cor a 1.0404 .

• Cross-Inhibition Experiments: These are valuable for assessing cross-reactivity between Cor a 1.0401 and related allergens like Bet v 1. Studies have shown only partial IgE cross-reactivity between Cor a 1.0401 and pollen Cor a 1, highlighting the importance of determining specific cross-reactivity patterns .

When comparing IgE reactivity across variants, researchers should use the same panel of patient sera and include appropriate controls. Patient selection is critical, as sensitization patterns vary based on geographic region and exposure to cross-reactive allergens like birch pollen .

What approaches are optimal for producing recombinant Cor a 1.0401 for research purposes?

Recombinant production of Cor a 1.0401 and its variants has been successfully achieved using the following methodology:

• Expression System: E. coli systems provide good yields for PR-10 proteins, which lack post-translational modifications .

• Construct Design: The complete coding sequence of Cor a 1.0401 (GenBank Accession no.: AF136945) should be optimized for bacterial expression with appropriate affinity tags for purification .

• Expression Conditions: Induction parameters should be optimized to maximize soluble protein yield while minimizing inclusion body formation.

• Purification Strategy: Multi-step purification typically involving affinity chromatography followed by size exclusion chromatography has proven effective .

• Quality Control: Recombinant proteins should be verified through:

  • SDS-PAGE and mass spectrometry to confirm purity and molecular weight

  • Circular dichroism to verify correct secondary structure

  • Limited proteolysis to assess proper folding

  • IgE binding assays to confirm immunological activity

For structural studies, especially NMR spectroscopy, isotopic labeling with ¹⁵N and/or ¹³C is required, involving growth in minimal media with appropriate isotope sources .

How should researchers design experiments to investigate cross-reactivity between Cor a 1.0401 and Bet v 1?

Investigating cross-reactivity between Cor a 1.0401 and Bet v 1 requires a multi-faceted experimental approach:

• Patient Selection:

  • Include patients with isolated birch pollen allergy

  • Include patients with hazelnut allergy without birch sensitization

  • Include patients with both birch pollen and hazelnut allergies

  • Carefully document clinical histories and symptoms

• Immunological Approaches:

  • ELISA inhibition assays to determine if pre-incubation with one allergen reduces binding to the other

  • Basophil activation tests to assess functional cross-reactivity

  • IgE epitope mapping to identify shared epitopes

• Structural Comparisons:

  • Overlay of NMR structures of Cor a 1.0401 and Bet v 1

  • Identify conserved surface patches that may serve as shared epitopes

  • Analyze ligand binding sites and their potential role in cross-reactivity

• Controls:

  • Include Cor a 1.01 (hazel pollen allergen) to distinguish between cross-reactivity due to structural similarity versus botanical relationship

  • Include unrelated allergens as negative controls

  • Use recombinant variants with site-directed mutations

Remember that cross-reactivity may be asymmetric and can vary substantially between patients based on their specific sensitization history .

What considerations are important when interpreting studies of Cor a 1.0401 variants' differential IgE binding?

When interpreting studies of differential IgE binding among Cor a 1.04 variants, researchers should consider several important factors:

• Structural Determinants: The inverse relationship between structural flexibility and IgE binding suggests that conformational epitopes dependent on protein rigidity may be critical for IgE recognition . Specific amino acid substitutions, particularly the S99P exchange in Cor a 1.0404, demonstrate how single residue changes can significantly alter allergenicity .

• Methodological Considerations:

  • Different assay systems (EAST, histamine release, ImmunoCAP) may yield variable results

  • Recombinant proteins may differ from natural allergens in post-translational modifications

  • Patient sera selection dramatically influences results

• Clinical Correlations:

  • IgE binding in vitro does not always correlate with clinical reactivity in vivo

  • Sensitization to Cor a 1 has been associated with milder symptoms (primarily oral allergy syndrome) and may be protective against more severe reactions

  • Cor a 1 IgE has low diagnostic accuracy for identifying moderate-to-severe allergic reactions in children

• Geographical Variations:

  • Different patterns of sensitization exist in different regions

  • Birch pollen exposure significantly influences Cor a 1.0401 sensitization patterns

These considerations emphasize the importance of using multiple experimental approaches and carefully selected patient cohorts when studying differential allergenicity of Cor a 1.04 variants .