Hev b 9, an enolase and a new cross-reactive allergen from Hevea latex andmolds - Purification, characterization, cloning and expression

Natural rubber latex allergy is an IgE-mediated disease that is caused by p roteins that elute from commercial latex products. A complementary DNA (cDN A) coding for Hev b 9, an enolase (2-phospho-D-glycerate hydrolyase) and al lergen from latex of the rubber tree Hevea brasiliensis, was amplified by P CR. The PCR primers were designed according to conserved regions of enolase s from plants. The obtained cDNA amplification product consisted of 1651 bp and encoded a protein of 445 amino-acid residues with a calculated molecul ar mass of 47.6 kDa. Sequence comparisons revealed high similarities of the Hevea latex enolase to mold enolases that have been identified as importan t allergens. In addition, the crucial amino-acid residues that participate in the formation of the catalytic site and the Mg2+ binding site of enolase s were also conserved. Hevea latex enolase was produced as a recombinant pr otein in Escherichia coli with an N-terminal hexahistidyl tag, and purified by affinity chromatography. The yield amounted to 110 mg of purified Hev b 9 per litre of bacterial culture. The recombinant allergen bound IgE from latex, as well as mold-allergic patients, in immunoblot and ELISA experimen ts. The natural enolase was isolated from Hevea latex by (NH4)(2)SO4 precip itation and ion exchange chromatography. The natural and the recombinant (r )Hev b 9 showed equivalent enzymatic activity. Patients' IgE-antibodies pre incubated with rHev b 9 lost their ability to bind to natural (n) Hev b 9, indicating the identity of the B-cell epitopes on both molecules. Cross-rea ctivity with two enolases from Cladosporium herbarum and Alternaria alterna ta was determined by inhibition of IgE-binding to these enolases by rHev b 9. Therefore, enolases may represent another class of highly conserved enzy mes with allergenic potentials.