Ara h 8.0101

What is Ara h 8.0101 and what are its key molecular properties?

Ara h 8.0101 is a Bet v 1-homologous panallergen from peanut (Arachis hypogaea) belonging to the pathogenesis-related protein family 10 (PR-10). It has a calculated molecular mass of 18,192 Dalton when expressed recombinantly . The protein is glycosylated and typically expressed with a 10xHis tag at the N-terminus for purification purposes . It functions as a ribonuclease according to biological function classification . Structurally, it contains the characteristic PR-10 protein fold with a hydrophobic cavity capable of binding various ligands.

What is the stability profile of Ara h 8.0101?

Ara h 8.0101 exhibits low stability to roasting processes and no stability to gastric digestion . This distinct stability profile contrasts with other peanut allergens and explains its primary association with oral allergy syndrome rather than systemic allergic reactions. For laboratory storage, recombinant Ara h 8.0101 can be stored at 4°C if used within 2-4 weeks, or at -20°C for longer periods, avoiding multiple freeze-thaw cycles to maintain protein integrity . The protein is typically supplied in 20mM HEPES buffer (pH 7.9) with 6M Urea for stabilization .

How does the structure of Ara h 8.0101 compare to other allergenic PR-10 proteins?

While the search results don't provide specific structural comparisons for Ara h 8.0101 itself, we can infer structural similarities based on related PR-10 allergens like Pru p 1. The PR-10 fold typically consists of about 43% β-strand and 27% α-helical structure . The protein likely contains a seven-stranded antiparallel β-sheet wrapped around a long C-terminal α-helix, forming a characteristic hydrophobic cavity. This structure is similar to other Bet v 1-homologous allergens, which explains its cross-reactivity patterns with Gly m 4 from soybean and Bet v 1 from silver birch .

What expression systems are optimal for producing recombinant Ara h 8.0101?

Based on the available information, Sf9 insect cells have been successfully used for the expression of recombinant Ara h 8.0101 . This system appears to be effective in producing the glycosylated protein with proper folding. The protein is typically expressed with a 10xHis tag at the N-terminus to facilitate purification through proprietary chromatographic techniques . Alternative expression systems such as E. coli have also been employed for other PR-10 proteins , but may not produce glycosylated forms that fully represent the native protein.

What structural determination methods have been applied to study PR-10 allergens like Ara h 8.0101?

Based on the search results, multiple complementary techniques have been used to elucidate the structure of PR-10 allergens:

• X-ray crystallography: The crystal structure of related peanut allergen Ara h 8 has been determined, as indicated by the crystallographic data and refinement statistics in Table 1 :

• NMR spectroscopy: For related PR-10 allergen Pru p 1, NMR was used to determine the three-dimensional solution structure, providing details about secondary structure elements (43% β strand and 27% α helical) . NMR also allowed investigation of ligand binding through chemical shift perturbation (CSP) experiments .

• Circular dichroism: Used to estimate secondary structure content, which can be compared with values derived from NMR or X-ray structures .

What cross-reactivity patterns does Ara h 8.0101 exhibit with other allergens?

Ara h 8.0101 shows significant cross-reactivity with Gly m 4 from soybean and Bet v 1 from silver birch . This cross-reactivity is based on structural and sequence similarities characteristic of the PR-10 protein family. These cross-reactivity patterns are clinically significant as they explain why patients with birch pollen allergy often develop allergies to certain foods, including peanuts, in what is known as pollen-food allergy syndrome. Interestingly, while the hydroxyl-bearing residues in the β-sheet of related PR-10 allergens like Pru p 1 are almost 80% conserved in Rosaceae allergens (including strawberry Fra a 1 and apple Mal d 1), this conservation drops to 50% or below in Fabaceae (which includes peanut) and Apiaceae .

How does Ara h 8.0101 contribute to oral allergy syndrome?

Ara h 8.0101 is specifically responsible for oral allergy syndrome rather than systemic allergic reactions . This clinical presentation is directly linked to its stability properties: it has low stability to roasting and no stability to gastric digestion . These characteristics mean that the protein can trigger localized allergic symptoms in the oral cavity upon initial contact but is typically degraded before it can cause systemic reactions. This contrasts with other peanut allergens like Ara h 1, Ara h 2, and Ara h 3, which are more stable and associated with severe systemic reactions.

What is the relationship between IgE and IgG4 responses to PR-10 allergens in allergic versus tolerant individuals?

While not specific to Ara h 8.0101, the search results provide insights on related PR-10 allergens: food-tolerant individuals tend to have significantly higher allergen-specific IgG4/IgE ratios compared to individuals with food allergy . Sera from IgG4-positive food-tolerant patients demonstrated IgG-dependent IgE-inhibitory activity, suggesting that specific IgG4 may block IgE binding to food allergens, potentially preventing allergic reactions . This implies that the presence of allergen-specific IgG4 antibodies is not a diagnostic marker for birch pollen-related food allergy but may instead indicate tolerance . This information suggests similar immunological mechanisms might apply to Ara h 8.0101 responses.

What ligands can PR-10 allergens like Ara h 8.0101 bind and with what affinity?

Based on studies of related PR-10 allergens, these proteins typically bind small hydrophobic molecules within their internal cavity. For the related allergen Pru p 1, zeatin (a cytokinin) binding has been studied in detail . NMR analysis revealed a complex dissociation constant (Kd) of 1.9 ± 0.4 mM, which is notably higher (indicating lower affinity) than values derived from isothermal titration calorimetry (ITC) . This discrepancy between measurement techniques appears common for PR-10 proteins, as similar disparities were observed for cytokinin-specific binding protein (CSBP) from Vigna radiata . The rapid binding and release of ligands with residence times in the millisecond range is consistent with the promiscuous binding behavior characteristic of PR-10 proteins .

How can ligand binding to PR-10 allergens be experimentally characterized?

Multiple complementary techniques are used to study ligand binding to PR-10 allergens, as demonstrated with Pru p 1:

• NMR Chemical Shift Perturbation (CSP): Detects changes in chemical environments of protein atoms upon ligand binding .

• Relaxation Dispersion (RD) NMR: Reveals residues involved in rapid ligand binding and release, providing kinetic information about the binding process .

• Paramagnetic Relaxation Enhancement (PRE): Using spin-labeled ligands to identify residues in close proximity to the bound ligand .

• Isothermal Titration Calorimetry (ITC): Provides thermodynamic parameters and binding affinities .

• Pulsed Field Gradient (PFG) NMR: Used to measure the hydrodynamic radius of the protein with and without ligand to detect potential conformational changes .

What structural determinants influence ligand binding specificity in PR-10 allergens?

The internal cavity of PR-10 allergens contains a mix of hydrophobic and polar residues that determine binding specificity. In Pru p 1, several key structural elements have been identified :

• An amphiphilic entrance to the binding cavity, denoted ε1, delimited by the N-terminal end of helix α3 and loops L5, L7, and L9.

• Aromatic residues (phenylalanines and tyrosines) that form part of the internal surface.

• Hydroxyl-bearing residues in the β-sheet that vary in conservation across different PR-10 allergens.

• A single ionizable residue (His69 in Pru p 1) located at the inner end of the cavity.

Similar structural features are likely present in Ara h 8.0101, though with sequence variations that affect specific binding preferences.

How can conflicting binding affinity data from different techniques be reconciled?

The significant discrepancy in binding affinity measurements using different techniques (e.g., NMR vs. ITC showing a 1000-fold difference in Kd values for zeatin binding to Pru p 1 ) presents a methodological challenge. To reconcile such differences, researchers should:

What are the challenges in determining the biological relevance of Ara h 8.0101's ribonuclease activity?

• Distinguishing between native activity and in vitro artifacts requires careful experimental design with appropriate controls.

• Identifying physiological RNA substrates is challenging without knowing the protein's natural cellular context.

• The relatively weak binding affinities observed for other ligands suggest that RNA binding might similarly be promiscuous rather than specific.

• Correlating structural features with catalytic activity requires detailed mechanistic studies that have not been extensively reported for Ara h 8.0101.

• Determining whether the ribonuclease activity is relevant to allergenicity or represents an ancestral function retained from evolutionary history.

How might structural modifications affect the allergenicity of Ara h 8.0101?

Understanding how structural modifications affect allergenicity could lead to potential hypoallergenic variants for therapeutic applications. Key considerations include: