title: Proteolysis of the peanut allergen Ara h 1 by an endogenous aspartic protease.
abstract: The 7S and 11S globulins of peanuts are subjected to proteolysis two days after seed imbibition, with Ara h 1 and the arachin acidic chains being among the first storage proteins to be mobilized. Proteolytic activity was greatest at pH 2.6-3 and is inhibited by pepstatin A, characteristic of an aspartic protease. This activity persists in seedling cotyledons up to at least 8 days after imbibition. In vitro proteolysis of Ara h 1 at pH 2.6 by extracts of cotyledons from seedlings harvested 24 h after seed imbibition generates newly appearing bands on SDS-PAGE. Partial sequences of Ara h 1 that were obtained through LC-MS/MS analysis of in-gel trypsin digests of those bands, combined with information on fragment size, suggest that proteolysis begins in the region that links the two cupin domains to produce two 33/34 kD fragments, each one encompassing an intact cupin domain. The later appearance of two 18 and 10/11 kD fragments can be explained by proteolysis within an exposed site in the cupin domains of each of the 33/34 kD fragments. The same or similar proteolytic activity was observed in developing seeds, but Ara h 1 remains intact through seed maturation. This is partly explained by the observation that acidification of the protein storage vacuoles, demonstrated by vacuolar accumulation of acridine orange that was dissipated by a membrane-permeable base, occurs only after germination. These findings suggest a method for use of the seed aspartic protease in reducing peanut allergy due to Ara h 1.
authors: Department of Biological Sciences, State University of New York at Binghamton, Binghamton, NY 13902-6000, USA.
authors: Karl A
authors: KA
authors: Wilson
authors: Department of Biological Sciences, State University of New York at Binghamton, Binghamton, NY 13902-6000, USA. Electronic address: annatan@binghamton.edu.
authors: Anna
authors: A
authors: Tan-Wilson
mesh_terms: D000595:Amino Acid Sequence; D052179:Antigens, Plant / Q000737:chemistry / Q000378:metabolism*; D010367:Arachis / Q000254:growth & development / Q000276:immunology; D057055:Aspartic Acid Proteases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D059748:Proteolysis; D017386:Sequence Homology, Amino Acid
chemical_list: D052179:Antigens, Plant; C500889:Ara h 1 protein, Arachis hypogaea; D006023:Glycoproteins; D008565:Membrane Proteins; D010940:Plant Proteins; D057055:Aspartic Acid Proteases
keywords: 7S globulin storage protein; Allergen; Ara h 1; Aspartic protease; Germination; Peanut; Proteolysis