Expression, purification, cross-reactivity and homology modeling of peanut profilin.
Plant profilins are known pan-allergens involved in the cross-reactions between pollen and plant foods. Peanut profilin, Ara h 5, is one of the important peanut allergens. Presently, most immunological, biochemical and structural studies on peanut allergens have focused on the three major allergens (Ara h 1, 2 and 3). Here Ara h 5 was cloned, expressed in Escherichia coli, Rosetta2(DE3) (Novagen), purified using a combination of ammonium sulfate fractionation and size-exclusion chromatography and yielded a total of 29 mg/l of culture. IgE reactivity was assayed using multiplexed microarray with other peanut allergens (Ara h 1, 2, 3, and 8) and birch (Bet v 2) and timothy (Phl p 2) profilin using sera from peanut allergic Swedish patients. Using homology modeling, Ara h 5 structure was also generated, compared against other profilins and utilized to predict surface-exposed residues potentially forming epitopes. The allergen was recognized by 3 out of 33 sera (9.1%). IgE reactivity to Ara h 5 also coincided with that of two other profilins, Phl p 12 and Bet v 2, confirming cross-reactivity. Interestingly, IgE reactivity to Ara h 5 was higher than above-mentioned profilins which may be indicating specificity of sera towards peanut profilin. Eight surface-exposed epitopes were predicted and verified against experimentally validated sequential epitopes. Three epitopes (#1, 5 and 7) mostly located at the accessible loops and neutral to relatively electropositive sites were found common among profilins, which should be involved in cross-reactivity. A specific putative epitope (#4) was also found which may explain the relative high IgE reactivity to Ara h 5 as compared to the other profilins. Due to its close relation to other allergenic profilins, Ara h 5 could be used as a model and allergen of choice for profilin allergy diagnosis.
Laboratory of Food Quality Design and Development, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
Cerrone
C
Cabanos
Mary Rose
MR
Tandang-Silvas
Van
V
Odijk
Peter
P
Brostedt
Akira
A
Tanaka
Shigeru
S
Utsumi
Nobuyuki
N
Maruyama
D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000595:Amino Acid Sequence; D010367:Arachis / Q000276:immunology / Q000378:metabolism*; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003429:Cross Reactions; D018985:Epitopes, B-Lymphocyte / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051304:Profilins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D040081:Protein Array Analysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016415:Sequence Alignment
D000485:Allergens; D018985:Epitopes, B-Lymphocyte; D010940:Plant Proteins; D051304:Profilins; D011994:Recombinant Proteins; D007073:Immunoglobulin E