Characterization of glycoprotein antigen (45 kD) of Aspergillus fumigatus

Abstract

An immunodominant glycoprotein antigen (45 kD) from Aspergillus fumigatus was purified and characterized. The purified antigen exhibited strong precipitin reaction with the sera of allergic bronchopulmonary aspergillosis (ABPA) patients. Enzyme-linked immunosorbent assay (ELISA) and Western blot results revealed strong binding of gp 45 with IgG and IgE antibodies of ABPA patients. Three monoclonals of IgM isotype, raised against a glycoprotein fraction of A. fumigatus, recognized 45 and 55 kD antigens. These results suggest the presence of common epitopes in these antigens. The protein to carbohydrate ratio of purified antigen was observed to be 1.4: 1.0. Further analysis by gas liquid chromatography revealed the presence of glucose, mannose and glucosamine residues in a 4 : 3 : 1 ratio. Deglycosylation of N-linked sugars of gp 45 with N-glycosidase-F resulted in a 27 kD band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Neuraminidase treatment destroyed the IgE binding activity of the antigen but IgG binding activity was retained. The IgG binding activity on the other hand was lost on treatment with pronase. The major IgG binding epitope of this glycoprotein antigen may therefore be in the protein part of the antigen. The purified 45 kD antigen displayed protease activity at alkaline pH. It was inhibited by PMSF and ethylene diamine tetraacetic acid (EDTA). Proteolytic activity of the antigen may contribute significantly to pathogenesis by destroying the elastin present in the lung tissue.