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Purification and characterization of a soybean hull allergen responsible for the Barcelona asthma outbreaks. II. Purification and sequencing of the Gly m 2 allergen
Author(s) -
CODINA R.,
LOCKEY R. F.,
EERNÁNDEZCALDAS E.,
RAMA R.
Publication year - 1997
Publication title -
clinical and experimental allergy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.462
H-Index - 154
eISSN - 1365-2222
pISSN - 0954-7894
DOI - 10.1111/j.1365-2222.1997.tb00728.x
Subject(s) - allergen , edman degradation , western blot , gel electrophoresis , peptide sequence , chemistry , chromatography , microbiology and biotechnology , amino acid , polyacrylamide gel electrophoresis , immunoglobulin e , biochemistry , biology , antibody , allergy , immunology , gene , enzyme
Summary Background A low MW allergen from soybean hull, Gly m 1, with two isoallergens, Gly m 1 A and Gly m 1 B, was associated with the asthma outbreaks that occurred in Cartagena, Spain. Using sera of asthmatic epidemic patients (AEP) from Barcelona, three main soybean hull allergens, two of them with MWs atid pIs identical to those reported for Gly m 1 A and Gly m 1 B, were identified. Objective The purpose of this study was to purify and to study the N‐terminal amino acid sequence of the third allergen, which has a MW of 8 kDa. Method The purification procedure combined the double dialysis method and preparative isoelectofocusing (IEF). Specific IgE determination to the fractions obtained demonstrated three peaks, one of them corresponding to the 8 kDa allergen. The pooled fractions containing this allergen were studied by sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), SDS‐PAGEAVestern blot and IEF/Westem blot. Only a band with a MW of 8 kDa and a pI of 6 was obtained. Its allergenic activity was measured and it was demonstrated that the allergenicity of soybean hull correlates with the presence of the 8 kDa allergen. The N‐terminal amino acid sequence of the first 20 amino acids, which was registered at the PIR Data Submission as the N‐terminal partial sequence of Gly m 2, was determined according the Edman degradation method. Results Gly m 2 N‐terminal amino acid sequence lacks homology with that reported for the allergen Gly m 1 but has a homology of 71% with a storage protein from cotyledon of Vigna radiata (cow pea) and 64% with a‘disease response protein’ from Pisum sativum (green pea). These results suggest that Gly m 2 in soybeans could protect against diseases which affect soybean plants. Conclusion This study demonstrates the existence of another soybean hull allergen, Gly m 2, partially responsible for the soybean asthma outbreaks that occurred in Barcelona, Spain.