Peanut allergen Ara h 3: Isolation from peanuts and biochemical characterization (1)
Ara h 3 and Ara h 4 are considered to be the same allergen, and are in this paper referred to as Ara h 3.
Peanuts were ground and protein extracts were made by mixing 25 g ground peanut with 250 ml of 20 mM Tris buffer (pH 7.2). After 2 h stirring at room temperature, the aqueous fraction was collected by centrifugation (3000 g, at room temperature for 30 min). The aqueous phase was subsequently centrifuged (10 000 g at room temperature for 30 min) to remove residual traces of fat and insoluble particles.
1.5 g peanut protein was applied on a 300 ml Source Q column previously equilibrated with 20 mM MOPS of pH 7.2 at room temperature. The column was washed with the loading buffer until the A280 of the effluent was less than 0.02. Proteins were eluted using a linear sodium chloride gradient in the loading buffer (up to 1 M in 4000 ml at a flow rate of 100 ml/min).
Ara h 1 was found in pool 1 whereas pool 2 shows a doublet around 21 kDa, indicative for Ara h 2. Pool 3 contained a number of polypeptides and since the relative intensities of these bands were identical for 10 consecutive fractions, the polypeptides are likely associated to form one single hetero-multimeric protein.
With the bands in Pool 3, the two largest polypeptides (45 and 42 kDa) as well as the 14 kDa polypeptide have an N-terminus identical to the recombinant gene-product described previouslyThe bands at 16, 25 and 28 kDa correspond to internal sequences starting at amino acids 217, 326 and 311, respectively. Taken together, these experiments demonstrate that Pool 3 contained a single protein with a hetero-multimeric structure.
Ara h 3 eluted at a slightly higher elution volume corresponding to an apparent Mw of 360– 380 kDa, which indicates an oligomeric structure very similar to glycinin and legumin: a hexameric 11S structure at neutral pH and an ionic strength of 0.2 M. At pH 7.6 and ionic strength of 0.2 M the 11S form of Ara h 3 shows a thermal unfolding characterized by a Tm of 91°C. The thermal unfolding was irreversible.
Patients 22, 40 and 48 have high titers for Ara h 3 while patients 35 and 44 have lower titers. Control individuals (non-allergic and allergic but not peanut allergic) did not react in this assay.
Patients 22 and 35 react intensely with Ara h 3, whereas patients 40, 44 and 48 show a moderate IgE-reactivity towards Ara h 3. Compared to the elisa, patients 40 and 48 react less intensively on immunoblot, whereas the IgE-binding for patient 35 is more pronounced in immunoblot.
Upon unfolding in 6 M urea and reduction of disulfide bonds by DTT the Ara h 3 multimer dissociates into subunits. Two major fractions can be distinguished: a non-bound fraction (fraction 1) and a fraction eluting during the salt gradient; the latter fraction was collected in four different subfractions (fractions 2–5).
The peptide bond between N325 and G326 is proteolytically cleaved by an asparaginyl endopeptidase, analogous to soy glycinin. This gives rise to the major bands observed on SDS-PAGE: the 42–45 kDa acidic subunit and the 25 kDa basic subunit. Most likely the cysteines responsible for the disulfide bond covalently linking the acidic subunit (res 1–325) and the basic subunit (res 326–510) are cys 88 (acidic subunit) and cys 332 (basic subunit).
The major IgE epitope was identified in recombinant Ara h 3 at amino acid position 279–293 that is in 42 and 45 kDa polypeptides.