Cloning and Characterization of an 11S Legumin, Car i 4, a Major Allergen in Pecan
| Autor: | Andre Irsigler, Luda Bardina, Girdhari M. Sharma, Shridhar K. Sathe, Hugh A. Sampson, Pushparani Dhanarajan, Rosalia Ayuso, Kenneth H. Roux |
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| Rok vydání: | 2011 |
| Předmět: |
DNA
Complementary Protein subunit Molecular Sequence Data Dot blot Sequence alignment Epitope Epitopes Tandem Mass Spectrometry Botany Animals Humans Legumin Amino Acid Sequence Cloning Molecular Peptide sequence Carya Plant Proteins Base Sequence cDNA library Chemistry food and beverages General Chemistry Allergens Immunoglobulin E Recombinant Proteins Epitope mapping Biochemistry Seeds Binding Sites Antibody Nut Hypersensitivity Rabbits General Agricultural and Biological Sciences Sequence Alignment |
| Zdroj: | Journal of Agricultural and Food Chemistry. 59:9542-9552 |
| ISSN: | 1520-5118 0021-8561 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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