Immunological characterization of Asp f 2, a major allergen from Aspergillus fumigatus associated with allergic bronchopulmonary aspergillosis

Abstract

The 37-kDa recombinant protein Asp f 2, encoding an allergen of Aspergillus fumigatus, was expressed in a prokaryotic expression system and immunologically evaluated for its functional and structural properties. The open reading frame for a 310-amino-acid-long protein was shown to encode a signal peptide of 31 amino acids. A native 37-kDa culture filtrate protein and a 55-kDa mycelial glycoprotein (gp55) exhibited complete N-terminal sequence homology to Asp f 2. A GenBank search for homologous proteins revealed 60 and 44% sequence homologies to the cytosolic protein ASPND1 from Aspergillus nidulans and fibrinogen binding protein from Candida albicans, respectively. The glycosylation sites and cysteine molecules are conserved in all the three proteins. The extracellular matrix protein laminin showed a dose-dependent interaction with Asp f 2. This protein, expressed as a major cell-associated protein within 24 h of in vitro fungal culture, comprises 20 to 40% of total fungal protein. Furthermore, both native and recombinant Asp f 2 exhibited specific immunoglobulin (IgE) binding with allergic bronchopulmonary aspergillosis (ABPA) and cystic fibrosis-ABPA patients, whereas A. fumigatus-sensitized allergic asthma and normal control subjects failed to show IgE binding with Asp f 2. These results indicate that Asp f 2 is a major allergen of A. fumigatus exhibiting IgE antibody binding with sera from patients with ABPA. The antigen should be explored further for its potential role in the differential diagnosis of A. fumigatus-associated allergic diseases.

FIG. 1

Complete nucleotide sequence and the deduced amino acid sequence of the Asp f 2 gene of A. fumigatus. The intron sequences are represented in lowercase. The 20 N-terminal amino acids of the purified native mycelial protein gp55 demonstrate sequence homology from amino acid 32 onward, as represented in italics. The A. fumigatus culture filtrate protein with N-terminal sequence homology from Asp 43 onward are underlined; glycosylation sites are in boldface.

FIG. 2

Conserved sequences of ASPND1 from A. nidulans, a fibrinogen binding protein from C. albicans (C. alb p), and Asp f 2. The cysteine molecules and glycosylation sites are conserved in all the three proteins.

FIG. 3

Inhibition of IgG binding to solid-phase coated nAsp f 2. Polyclonal mouse sera raised against nAsp f 2 AF102 (▵), nAsp f 2 AF104 (□), and rAsp f 2 (○) were preincubated overnight with different amounts of rAsp f 2 as indicated on the x axis. Preincubated serum samples were transferred to nAsp f 2 (AF104)-coated wells, and antibody binding was measured.

FIG. 4

Time kinetics of expression of Asp f 2 in A. fumigatus mycelial extracts. Mycelial proteins (5 μg/ml) at various intervals of growth were separated by SDS-PAGE on a 12% gel and transferred onto nitrocellulose membranes. Separated proteins were evaluated for antibody binding by using polyclonal mouse sera against rAsp f 2. Lane 1, molecular weight markers. Different time interval cultures: lane 2, 24 h; lane 3, 48 h; lane 4, 72 h; lane 5, 96 h; lane 6, 5 days; lane 7, 7 days; lane 8, 14 days; lane 9, 21 days. (B) Time kinetics of expression of Asp f 2 in A. fumigatus culture filtrates. The culture filtrate proteins (5 μg/ml) were separated by SDS-PAGE on a 12% gel and transferred onto nitrocellulose membranes. Separated proteins in lanes 2 to 9 are culture filtrates at the same time intervals as for mycelial extracts (A) and were evaluated for antibody binding by using polyclonal mouse sera against rAsp f 2.

FIG. 5

Western blot analysis of various Aspergillus proteins, using mouse polyclonal sera against rAsp f 2 and rabbit sera against the p40 cytosolic component of A. fumigatus. Lanes: a, molecular weight markers; b to e, treated with anti-rAsp f 2 sera; f to i, treated with anti-p40 sera; b and f, A. fumigatus culture filtrate antigens (AF102); c and g, CFC fraction from A. fumigatus; d and h, rAsp f 2; e and i, ASPND1 from A. nidulans.

FIG. 6

IgE binding of purified rAsp f 2 and ASPND1, using 10 serum samples from each of the indicated groups. Solid bars, ELISA absorbance values at 490 nm for rAsp f 2; shaded bars, absorbance values for ASPND1.

FIG. 7

(A) Specific binding of laminin to Asp f 2. rAsp f 1, rAsp f 12, rAsp f 2, nAsp f 2, and crude A. fumigatus culture filtrates (100 μg/well) were incubated with 10 μg of laminin per ml. The subsequent incubation was with a 1:1,000 dilution of mouse antilaminin antibody. The ELISA IgG absorbance was measured at 490 nm (for details, see Materials and Methods). (B) Dose-dependent binding of laminin (1 ng to 10 μg) to solid-phase coated Asp f 2. Open bars, IgG absorbance of laminin-antilaminin interaction on Asp f 2-coated wells; closed bars, absorbance values for direct laminin-antilaminin binding on laminin-coated wells.

FIG. 8

IgE binding of nAsp f 2, rAsp f 2, and A. fumigatus culture filtrate (AF104) with 10 serum samples from each of the indicated groups.