Proteolytic processing of the peanut allergen Ara h 3.
The allergen Ara h 3 has been purified recently from peanuts. In contrast to recombinant Ara h 3, a 60 kDa single-chain polypeptide, the allergen isolated from its native source is extensively proteolytically processed. The characteristic proteolytic processing for 11S plant storage proteins of the glycinin family is observed for Ara h 3 yielding an acidic and a basic subunit, bound by a disulfide bridge. In addition to this, proteolytic truncation is observed for the acidic subunit but not for the basic subunit of Ara h 3. A series of Ara h 3 polypeptides ranging from 13-45 kDa was separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and each band was digested by trypsin. Peptides related to the bands were identified and a scheme positioning the different polypeptides in the Ara h 3 sequence has been constructed. Peptide analysis showed sequence heterogeneity at two positions indicating the presence of multiple genes encoding variant, but highly homologous Ara h 3 proteins. The pool of Ara h 3 polypeptides from its native source illustrated that allergen from the peanut is much more complex than the recombinant protein used for epitope mapping experiments. From several Ara h 3 truncation products one or more immunoglobulin E (IgE) binding sites had been removed. Characterization of the allergenicity of Ara h 3 should therefore also include IgE-binding studies with peanut-derived Ara h 3, providing the high degree of variation in the Ara h 3 protein structure, as this is what peanut-allergic individuals are confronted with.
FOM Institute for Atomic and Molecular Physics, Amsterdam, The Netherlands. Piersma@amolf.nl
Sander R
SR
Piersma
Marco
M
Gaspari
Susan L
SL
Hefle
Stef J
SJ
Koppelman
D000485:Allergens / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins; D021122:Protein Subunits / Q000737:chemistry; D055314:Seed Storage Proteins; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism*
D000485:Allergens; D052179:Antigens, Plant; D010446:Peptide Fragments; D010940:Plant Proteins; D021122:Protein Subunits; D055314:Seed Storage Proteins; C118207:allergen Ara h3; D014357:Trypsin