Isoforms of the major peanut allergen Ara h 2: IgE binding in children with peanut allergy.
BACKGROUND
The major peanut allergen Ara h 2 consists of two isoforms, namely Ara h 2.0101 and Ara h 2.0201. The recently identified Ara h 2.0201 isoform contains an extra 12 amino acids including an extra copy of the reported immunodominant epitope DPYSPS. This study aimed to evaluate the IgE binding of the two Ara h 2 isoforms.


METHODS
Ten clones of Ara h 2 were sequenced to assess the relative frequency of the Ara h 2 isoforms and to identify whether there was further variation in the Ara h 2 sequence. IgE binding to Ara h 2.0101 and Ara h 2.0201 was measured for 70 peanut-allergic children using an IgE DELFIA assay to quantitate specific IgE binding. A competition assay was used to measure whether Ara h 2.0201 contained IgE epitopes other than those found for Ara h 2.0101.


RESULTS
The original Ara h 2.0101 sequence was found for 6/10 clones and Ara h 2.0201 was found for 2/10 clones. Ara h 2.0201 had the expected insertion of 12 amino acids as well as substitutions at positions 40 (40G) and 142 (142E). Two new isoforms were identified as different polymorphisms of position 142. One Ara h 2.01 clone (Ara h 2.0102) contained 142E and one Ara h 2.02 clone (Ara h 2.0202) contained 142D. A polymorphism that was previously identified by other investigators at position 77 (77Q or 77R) was not found for any of the 10 sequences. Although the level of IgE binding to Ara h 2.0201 of individual patients was frequently higher than the binding to Ara h 2.0101 (p < 0.01), there was a strong correlation in binding to both isoforms (r = 0.987, p < 0.0001) and when analyzed as a group the means were similar. Ara h 2.0101 was not as efficient at blocking reactivity to Ara h 2.0201 indicating there is an additional IgE specificity for the Ara h 2.0201 isoform.


CONCLUSIONS
Ara h 2.0201 has similar but higher IgE binding than the originally sequenced Ara h 2.0101 isoform and contains other IgE specificities.
Hales
Belinda J
BJ
Centre for Child Health Research, University of Western Australia, Telethon Institute for Child Health Research, West Perth, Australia.
Bosco
Anthony
A
Mills
Kristina L
KL
Hazell
Lee A
LA
Loh
Richard
R
Holt
Patrick G
PG
Thomas
Wayne R
WR
D055516:2S Albumins, Plant; D000293:Adolescent; D000595:Amino Acid Sequence; D000918:Antibody Specificity / Q000276:immunology; D052179:Antigens, Plant; D010367:Arachis / Q000276:immunology*; D001667:Binding, Competitive; D002648:Child; D002675:Child, Preschool; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D007223:Infant; D008969:Molecular Sequence Data; D021183:Peanut Hypersensitivity / Q000276:immunology*; D010940:Plant Proteins; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000276:immunology*
D055516:2S Albumins, Plant; D052179:Antigens, Plant; C078642:Ara h II protein, Arachis hypogaea; D006023:Glycoproteins; D010940:Plant Proteins; D020033:Protein Isoforms; D007073:Immunoglobulin E