Isolation and characterization of two complete Ara h 2 isoforms cDNA

• Published in: International archives of allergy and immunology Vol. 131; no. 1; p. 14
• Main Authors: Chatel, Jean-Marc, Bernard, Hervé, Orson, Frank M
• Format: Journal Article
• Language: English
• Published: Switzerland 01-05-2003
• Subjects: 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
• ISSN: 1018-2438
• DOI: 10.1159/000070429

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