Purification and molecular characterization of a truncated-type Ara h1, a major peanut allergen: oligomer structure, antigenicity, and glycoform (1)
Ara h 1 consists of three identical N-glycosylated subunits (63 kDa) and accounts for 12–16% of the total peanut proteins. The molecular characterization of Ara h 1 has nearly been completed, and two isoforms (clone 41B and clone P17) have been identified and characterized. Hydrophobic amino acids play a critical role in trimer formation, and these important amino acids are located at the distal ends, where monomer-monomer interactions occur. The majority of immunoglobulin E (IgE)-binding epitopes are located at these association sites of the Ara h 1 subunit.
The run-through fraction containing the major peanut allergen Ara h 1 with a molecular mass of approximately 63 kDa was pooled.
Four fractions (F-1, −2, −3, and − 4) were obtained by hydrophobic interaction chromatography. F-1 contained the 54 kDa protein, F-2 contained both the 54 kDa and 63 kDa (Ara h 1) proteins, and F-3 contained the 63 kDa (Ara h 1) protein.
The N-terminal sequence of the 63 kDa protein in F-1 and F-2 was R-S/H-P-P-G-E-R-, suggesting that two Ara h 1 molecules encoded by two clone P41B and clone P17 genes are present in F-1. In contrast, the N-terminal sequence of the 54 kDa protein in F-1 and F-2 was E-G-R-E-G-E-Q-, suggesting that this 54 kDa protein resulted from proteolysis of the 63 kDa Ara h 1. A possibility that the 54 kDa molecule was artificially generated in large amount by the action of endogenous peptidase(s) during the purification process cannot be excluded.
The elution profile of 54 kDa Ara h 1 showed a single peak (peak-1) with a molecular mass of approximately 170 kDa, suggesting that the 54 kDa subunit occurs as a trimeric conformation in a native state. In contrast, the elution profile of 63 kDa Ara h 1 was observed as a broad peak (peak- 2).
The IgEs in sera of all nine patients strongly reacted to both subunits of Ara h 1. Some sera from healthy individuals slightly reacted with the 54 kDa Ara h 1 but not 63 kDa.
Five peaks (peak-a, −b, −c, −d, and -e) were detected by NP-HPLC. The structural features of N-glycans from the 54 kDa and 63 kDa Ara h 1 subunits are nearly the same.
The N-glycan structures of both subunits purified were in agreement with those reported previously.
The cross-reactivity of IgE(s) in the serum of one patient (#7) was completely lost by de-N-glycosylation, suggesting the N-glycan, probably β1–2xylosylated N-glycans (Man4- 3GlcNAc2), of the Ara h 1 subunits was the sole epitope for the IgE(s) against the peanut allergen.
