Isolation and characterization of two complete Ara h 2 isoforms cDNA.
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.


CONCLUSIONS
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.
Chatel
Jean-Marc
JM
Veterans Affairs Medical Center, Baylor College of Medicine, Houston, Tex., USA. chatel@dsvidf.cea.fr
Bernard
Hervé
H
Orson
Frank M
FM
D055516:2S Albumins, Plant; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D003001:Cloning, Molecular; D018387:Codon, Initiator / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D018388:Codon, Terminator / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D004251:DNA Transposable Elements / Q000235:genetics / Q000276:immunology; D018076:DNA, Complementary / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D006023:Glycoproteins / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007073:Immunoglobulin E / Q000235:genetics / Q000276:immunology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames / Q000235:genetics; D010940:Plant Proteins; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D020079:Terminal Repeat Sequences / Q000235:genetics / Q000276:immunology
D055516:2S Albumins, Plant; D052179:Antigens, Plant; C078642:Ara h II protein, Arachis hypogaea; D018387:Codon, Initiator; D018388:Codon, Terminator; D017931:DNA Primers; D004251:DNA Transposable Elements; D018076:DNA, Complementary; D006023:Glycoproteins; D010940:Plant Proteins; D020033:Protein Isoforms; D007073:Immunoglobulin E