Digested Ara h 1 Loses Sensitizing Capacity When Separated into Fractions

Abstract

The major peanut allergen Ara h 1 is an easily digestible protein under physiological conditions. The present study revealed that pepsin digestion products of Ara h 1 retained the sensitizing potential in a Brown Norway rat model, while this sensitizing capacity was lost by separating the digest into fractions by gel permeation chromatography. Protein chemical analysis showed that the peptide composition as well as the aggregation profiles of the fractions of Ara h 1 digest differed from that of the whole pool. These results indicate that the sensitizing capacity of digested Ara h 1 is a consequence of the peptides being in an aggregated state resembling the intact molecule or that most peptides of the digests need to be present in the same solution, having a synergistic or adjuvant effect and thereby augmenting the immune response against other peptides.

The Sensitising Capacity of Intact β-Lactoglobulin Is Reduced by Co-Administration with Digested β-Lactoglobulin

“…With fractionation of the peptide fragments by means of GPC it was demonstrated that especially the peptides composed of charged amino acid residues tended to form aggregates. This is in accordance with another study showing likewise that peptide fragments resulting from the digestion process of the peanut allergen Ara h 1 formed the largest complexes when composed mainly of charged and least of hydrophobic amino acid residues [16], indicating that non-covalent interactions other than hydrophobic are the main players in the formation of aggregates.…”

“…Fractionation by preparative gel permeation chromatography (GPC) of gastro-duodenal digests of BLG was performed essentially as described in Bøgh et al [16]. Separated fractions were collected and pooled according to the preparative GPC profile shown in figure 1, resulting in four different pools of BLG digests in the following designation: partially digested BLG, digested BLG, large complexes and small complexes, where partially digested BLG corresponds to the whole pool of BLG digests, containing intact BLG that resisted the digestion process, and where digested BLG corresponds to the combined large and small complexes and thereby the whole pool of digested BLG without any intact BLG.…”

“…For analysis of purity and presence of residual intact BLG and for evaluation of chromatography profiles, RP-HPLC was performed as previously described [16]. …”

“…For analysis of peptide mass distributions in pools of BLG digests matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry (MS) was performed as previously described [16]. …”

“…For analysis of the peptide aggregation profiles in the different pools of BLG digests analytical GPC was performed as previously described [16]. …”