Purification and characterization of a soybean hull allergen responsible for the Barcelona asthma outbreaks. II. Purification and sequencing of the Gly m 2 allergen.
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 and 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.


METHODS
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-PAGE/Western blot and IEF/Western 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.


CONCLUSIONS
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.
Codina
R
R
Department of Internal Medicine, University of South Florida, Tampa, USA.
Lockey
R F
RF
Fernández-Caldas
E
E
Rama
R
R
D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000918:Antibody Specificity; D001249:Asthma / Q000453:epidemiology* / Q000209:etiology* / Q000276:immunology; D004196:Disease Outbreaks; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D013025:Glycine max / Q000235:genetics / Q000276:immunology*; D013030:Spain / Q000453:epidemiology
D000485:Allergens; D010940:Plant Proteins; D007073:Immunoglobulin E