Digestion of peanut allergens Ara h 1, Ara h 2, Ara h 3, and Ara h 6: A comparative in vitro study and partial characterization of digestion-resistant peptides (1)
Major allergen Ara h 1 was described as a 63.5 kDa protein that occurs naturally in trimeric form of approximately 180 kDa through noncovalent interactions. The trimeric Ara h 1 structures often aggregate, forming multimers of up to 600–700kDa. Ara h 2 migrates as a doublet at approximately 20 kDa. This doublet consists of two isoforms that are nearly identical except for the insertion of the sequence DPYSPS in the higher molecular weight isoform. Ara h 3 is a more complex allergen. Ara h 3 is a proteolytically processed protein consisting of a triplet at approximately 42–45 kDa, a distinct band at approximately 25 kDa, and some less abundant peptide chains in the range of 12–18kDa. Ara h 6 was recently purified by two independent groups with a molecular weight of approximately 15 kDa.
At time point 0, before adding pepsin, a char- acteristic peanut extract pattern was found. By 0.25 min substantial proteolysis of the CPE was observed. Peptides in the molecular weight region of 10–25kDa originate from this proteolysis, and some remained up to an incubation time of 30 min. Under reducing conditions, clear bands at approximately 10 kDa were visible that correspond to a digestion-resistant fragment of Ara h 2. In contrast to the relative stability of the protein bands at approximately 20kDa, the protein bands at higher molecular weights (63.5 kDa, Ara h 1 and 45 kDa, Ara h 3) disappeared rapidly.
A lowering the pepsin concentration resulted in a more gradual breakdown of peptides. With 0.1 U of pepsin per microgram substrate, peptides between approximately 20 and 50 kDa appeared. The analysis on SDS-PAGE did not show differences between reduced and nonreduced samples, as expected based on the fact that Cys residues are not involved in intra or intermolecular disulfide bridges for Ara h 1.
Under non- reducing conditions, the acidic and basic subunits were still associated by a disulfide bridge, giving rise to several bands at approximately 70kDa. Reducing conditions showed dissociation of the subunits. Giving rise to the typical pattern for Ara h 3 with heterogeneity in the N-terminal acidic subunit. At the lowest concentration of pepsin (100-fold lower; 0.1 U of pepsin per microgram Ara h 3), some peptides of inter- mediate weight (10–30 kDa) remained for only 2–4 min. After 10 min, the majority of the protein was found in the range of 7–14 kDa, and after 60 min the majority of the peptides were o7 kDa.
In contrast to Ara h 1 and Ara h 3, Ara h 2 was more stable. The protein band with the highest molecular weight remained intact for up to 4 min. Under non-reducing analysis conditions, virtually no proteolytic breakdown was observed. The necessity of reduc- tion to visualize proteolysis demonstrates that intramolecular disulfide bonds keep the hydrolysis products together as a single molecule with a molecular mass similar to the native Ara h 2. Ara h has a digestion-resistant peptide of 10 kDa.
Ara h 6 showed a digestion pattern which is very similar to that of Ara h 2. Ara h 6 was substantially digested after only 1 min at the highest pepsin concentra- tion. Lowering the pepsin concentration resulted in a more gradual breakdown, and with the lowest pepsin concentra- tion, some Ara h 6 was intact after 30 min. Digestion of Ara h 6 resulted, as visualized on SDS-PAGE under reducing conditions, in a stable peptide of approxi- mately 10 kDa, similar as for Ara h 2. As for Ara h 2, the intramolecular disulfide bridges of Ara h 6 maintain the digestion fragments as a single molecule.
Trypsin treatment revealed a peptide of Ara h 2 of similar sizes same as digestion with pepsin. Two distinct pools were identified, referred to as digestion-resistant peptide-1 (DRP-1) and digestion- resistant peptide-2 (DRP-2). An abundant peptide of approximately 10 kDa with an N-terminus corresponding to the middle part of Ara h 2 (GAGSS), suggesting that the C-terminal part of Ara h 2 is digestion resistant as well.
The sequence underlined should indicate the cleavage site. To explain this, cleavage should have taken place after the arginine residue preceding GAGSS, giving a molecular weight of 8.6kDa for the N-terminal peptide. The deviation between 8.6 and 10kDa can be explained by the imprecision of the analytical method.
