Identification and characterization of the major allergen of the Humulus japonicus pollen.

Abstract

Pollen of Humulus japonicus has been known as one of the important causes of pollinosis in Korea and China. To date, the major allergen of H. japonicus has not been determined. To identify the major allergen of H. japonicus pollen and characterize its biochemical properties. With the sera of 29 patients reactive to H. japonicus, the major allergen of H. japonicus was determined from the results of IgE immunoblotting and ELISA inhibition. The biochemical properties of the major allergen of H. japonicus were evaluated by lectin blotting assay and 2-dimensional PAGE blot. N-terminal amino acid sequences were determined by the Edman degradation method. The suggested major allergen was purified by DEAE anion exchange and gel filtration chromatography. Twenty-nine sera contained IgE bound to the 10, 16, 20, 29 and 42 kDa proteins of H. japonicus in immunoblot analysis. A protein of 10 kDa was the most prevalent allergen in the sera of H. japonicus-reactive patients (72%). The ELISA optical density of H. japonicus-specific IgE was not inhibited by pollen extracts of birch, oak, rye grass and mugwort. The 10-kDa allergen was neither stained with PAS nor bound with ConA and five other lectins. The isoelectric point of the 10-kDa allergen was approximately pH 5.1. We sequenced the N-terminal amino acids of the 10-kDa allergen, which was not homologous with any previously characterized allergen. The 10-kDa allergen could be purified with DEAE anion exchange and gel filtration chromatography. Maximum inhibitions of H. japonicus-specific IgE ELISA by whole extract of H. japonicus and purified 10-kDa allergen were more than 97 and 88%, respectively, while the 50% inhibitory concentration of the whole extract of H. japonicus and purified 10 kDa were 38 and 20 ng/mL, respectively. The 10-kDa peptide could be a major allergen of H. japonicus. Its isoelectric point was 5.1 and it did not bind with lectins. The N-terminal amino acid sequence of the 10-kDa major allergen was also determined.