title: Cloning and characterization of an 11S legumin, Car i 4, a major allergen in pecan.
abstract: Among tree nut allergens, pecan allergens remain to be identified and characterized. The objective was to demonstrate the IgE-binding ability of pecan 11S legumin and characterize its sequential IgE-binding epitopes. The 11S legumin gene was amplified from a pecan cDNA library and expressed as a fusion protein in Escherichia coli. The native 11S legumin in pecan extract was identified by mass spectrometry/mass spectrometry (MS/MS). Sequential epitopes were determined by probing the overlapping peptides with three serum pools prepared from different patients' sera. A three-dimensional model was generated using almond legumin as a template and compared with known sequential epitopes on other allergenic tree nut homologues. Of 28 patients tested by dot blot, 16 (57%) bound to 11S legumin, designated Car i 4. MS/MS sequencing of native 11S legumin identified 33 kDa acidic and 20-22 kDa basic subunits. Both pecan and walnut seed protein extracts inhibited IgE binding to recombinant Car i 4, suggesting cross-reactivity with Jug r 4. Sequential epitope mapping results of Car i 4 revealed weak, moderate, and strong reactivity of serum pools against 10, 5, and 4 peptides, respectively. Seven peptides were recognized by all three serum pools, of which two were strongly reactive. The strongly reactive peptides were located in three discrete regions of the Car i 4 acidic subunit sequence (residues 118-132, 208-219, and 238-249). Homology modeling of Car i 4 revealed significant overlapping regions shared in common with other tree nut legumins.
authors: Department of Nutrition, Food and Exercise Sciences, The Florida State University, Tallahassee, Florida 32306, United States.
authors: Girdhari M
authors: GM
authors: Sharma
authors: Andre
authors: A
authors: Irsigler
authors: Pushparani
authors: P
authors: Dhanarajan
authors: Rosalia
authors: R
authors: Ayuso
authors: Luda
authors: L
authors: Bardina
authors: Hugh A
authors: HA
authors: Sampson
authors: Kenneth H
authors: KH
authors: Roux
authors: Shridhar K
authors: SK
authors: Sathe
mesh_terms: D000485:Allergens / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001666:Binding Sites, Antibody; D031325:Carya / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D008969:Molecular Sequence Data; D021184:Nut Hypersensitivity / Q000276:immunology; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology; D012639:Seeds / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D016415:Sequence Alignment; D053719:Tandem Mass Spectrometry; D000097793:Legumins
chemical_list: D000485:Allergens; D018076:DNA, Complementary; D000939:Epitopes; D007073:Immunoglobulin E; D010940:Plant Proteins; D011994:Recombinant Proteins; D000097793:Legumins