Proteolytic processing of the peanut allergen Ara h 3 (1)
Ara h 1, a 63 kDa glycoprotein, was identified as the major allergen in peanut with distinct heat-stable immunoglobulin E (IgE) binding sites both on the protein part as well as on the carbohydrate moiety. Ara h 2 was identified as a second important allergen from peanuts and is characterized as a doublet of 17 – 20 kDa on SDS-PAGE. Ara h 3, the third allergen described from peanuts, was first identified as a 14 kDa protein. However, the 14 kDa protein first identified as Ara h 3 is a C-terminally truncated acidic subunit of the mature Ara h 3 protein.
The typical SDS-PAGE pattern for Ara h 3 purified from peanuts is shown as major bands at 45, 42, and 23 kDa and minor bands ranging from 12 – 35 kDa. SDS-PAGE analysis and mass spectrometric analysis give a consistent picture of the Ara h 3 acidic/basic subunit composition linked by a disulfide bridge (acidic subunit C88– basic subunit C338) analogous to the subunit organization of soy glycinin. The basic subunit (band H 23 kDa on SDS-PAGE, 20505 Da by MALDI) appears essentially as a single polypeptide in Ara h 3. For the acidic subunit multiple bands are observed ranging from 13–45 kDa on SDS- PAGE, indicating proteolytic truncation of the (pro)protein in the peanut. Under non reducing conditions, the heterodimers comprised of H–L, H–A, H–B, and H–C are equally abundant and are responsible for 90% of the integrated MALDI signal intensity in the high-molecular-mass range.
The Clustal W multiple sequence alignment of the amino acid sequences of the four Ara h 3 entries in the NCBI protein database, translated from their respective nucleotide sequences, are shown.
Bands A, B, C, (F), G, and L are N-terminal polypeptides of the acidic subunit of the mature Ara h 3 molecule truncated C-terminally at different positions, and are characterized by an apparent molecular weight on SDS-PAGE of 45, 43, 42,27, and 13 kDa, respectively.
The dominant epitope VTVRGGLRILSPDRK (res. 276 – 290, amino acids critical for IgE binding in boldface) is recognized by all Ara h 3-allergic patients.