Cloning and characterization of an 11S legumin, Car i 4, a major allergen in pecan

J Agric Food Chem. 2011 Sep 14;59(17):9542-52. doi: 10.1021/jf2017447. Epub 2011 Aug 4.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Allergens / chemistry
  • Allergens / genetics*
  • Allergens / immunology
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Binding Sites, Antibody
  • Carya* / genetics
  • Carya* / immunology
  • Cloning, Molecular*
  • DNA, Complementary / chemistry
  • Epitopes / chemistry
  • Epitopes / immunology
  • Humans
  • Immunoglobulin E / immunology
  • Molecular Sequence Data
  • Nut Hypersensitivity / immunology
  • Plant Proteins / chemistry
  • Plant Proteins / genetics*
  • Plant Proteins / immunology*
  • Rabbits
  • Recombinant Proteins / immunology
  • Seeds / chemistry*
  • Seeds / genetics
  • Seeds / immunology
  • Sequence Alignment
  • Tandem Mass Spectrometry

Substances

  • Allergens
  • DNA, Complementary
  • Epitopes
  • Plant Proteins
  • Recombinant Proteins
  • legumin protein, plant
  • Immunoglobulin E