Isolation and characterization of two complete Ara h 2 isoforms cDNA

Int Arch Allergy Immunol. 2003 May;131(1):14-8. doi: 10.1159/000070429.

Abstract

Background: Ara h 2 is a major peanut allergen recognized by IgE in more than 90% of patients. After electrophoretic separation the purified protein exists as a doublet, and sequences of one incomplete cDNA and one genomic clone for this allergen have been reported.

Methods: Ara h 2 isoforms were purified and analyzed by mass spectroscopy, and PCR amplification products of Ara h 2 were cloned and sequenced.

Results: Mass spectroscopy of purified Ara h 2 clearly identified a molecular doublet of 16,670 and 18,050 Daltons. Amplification of a peanut cDNA library using PCR primer pairs located at the amino- and carboxy-terminus revealed 2 bands separated by 50 base pairs, which we cloned and sequenced. Two types of complete cDNA clones were obtained, Ara h 2.01 and Ara h 2.02. Compared to Ara h 2.01 and the previously reported cDNA sequences, Ara h 2.02 is characterized by a 12 amino acid insertion starting at position 75 that contains a third repeat of the major IgE binding epitope DPYSPS.

Conclusion: We demonstrated the molecular and genetic characteristics of two Ara h 2 isoforms, revealing that one, Ara h 2.02, might be the more potent allergen.

Publication types

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

MeSH terms

  • 2S Albumins, Plant
  • Amino Acid Sequence
  • Antigens, Plant
  • Cloning, Molecular
  • Codon, Initiator / classification
  • Codon, Initiator / genetics
  • Codon, Initiator / isolation & purification
  • Codon, Terminator / classification
  • Codon, Terminator / genetics
  • Codon, Terminator / isolation & purification
  • DNA Primers / classification
  • DNA Primers / genetics
  • DNA Primers / isolation & purification
  • DNA Transposable Elements / genetics
  • DNA Transposable Elements / immunology
  • DNA, Complementary / classification*
  • DNA, Complementary / genetics
  • DNA, Complementary / isolation & purification*
  • Glycoproteins / classification*
  • Glycoproteins / genetics
  • Glycoproteins / isolation & purification*
  • Humans
  • Immunoglobulin E / genetics
  • Immunoglobulin E / immunology
  • Mass Spectrometry
  • Molecular Sequence Data
  • Molecular Weight
  • Open Reading Frames / genetics
  • Plant Proteins
  • Polymerase Chain Reaction
  • Protein Isoforms / classification
  • Protein Isoforms / genetics
  • Protein Isoforms / isolation & purification
  • Terminal Repeat Sequences / genetics
  • Terminal Repeat Sequences / immunology

Substances

  • 2S Albumins, Plant
  • Antigens, Plant
  • Ara h II protein, Arachis hypogaea
  • Codon, Initiator
  • Codon, Terminator
  • DNA Primers
  • DNA Transposable Elements
  • DNA, Complementary
  • Glycoproteins
  • Plant Proteins
  • Protein Isoforms
  • Immunoglobulin E