Natural and recombinant enzymatically active or inactive bee venom phospholipase A2 has the same potency to release histamine from basophils in patients with Hymenoptera allergy.
BACKGROUND
A complementary DNA encoding the major bee venom allergen phospholipase A2 (PLA) has been characterized recently. Recombinant PLA was produced in Escherichia coli and purified to apparent homogeneity. Natural PLA was compared with recombinant PLA in its ability to release histamine from blood basophils.


METHODS
A synthetic gene encoding the mature form of PLA was expressed in E. coli, and the polypeptide was purified to homogeneity by affinity chromatography and refolded, yielding fully enzymatically active PLA. In addition, we have produced a genetically engineered enzymatically inactive variant by substitution of a single amino acid residue in the catalytic center. A standard histamine release assay was used to compare the potency of natural PLA with correctly folded enzymatically active and inactive recombinant PLA to release histamine from blood basophils of nine patients with bee venom allergy.


RESULTS
Recombinant enzymatically active PLA and purified natural protein were equally effective in releasing histamine from sensitized basophils. By comparing the histamine-releasing capacity of enzymatically active and inactive recombinant allergen, we further demonstrate that catalytic activity is not a requirement for allergenicity in the effector phase. Denaturation of natural PLA or incorrect folding of recombinant protein resulted in a total loss of allergenic potency.


CONCLUSIONS
We demonstrate the feasibility of producing native-like recombinant allergens with or without enzymatic activity. We also provide evidence for the requirement of correct three-dimensional structure of PLA to induce histamine release from basophils and thus evidence for its recognition by IgE.
Förster
E
E
Pediatric and First Department of Dermatology, University of Vienna, Austria.
Dudler
T
T
Gmachl
M
M
Aberer
W
W
Urbanek
R
R
Suter
M
M
D001514:Bee Venoms / Q000276:immunology*; D006636:Histamine Release / Q000187:drug effects; D006801:Humans; D006967:Hypersensitivity / Q000097:blood / Q000276:immunology*; D010741:Phospholipases A / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D054467:Phospholipases A2; D017354:Point Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology
D001514:Bee Venoms; D011994:Recombinant Proteins; D010741:Phospholipases A; D054467:Phospholipases A2