We have identified, purified, and characterized structurally and functionally a 90-kDa immunodominant antigen associated with the water-soluble fraction of Aspergillus fumigatus. This antigen is recognized by 90.3% of serum samples from patients with aspergilloma and should be considered either by itself or better in combination with other purified antigens as a candidate for developing a standardized immunoassay for the detection of aspergilloma. p90 is a glycoprotein containing at least two N-linked sugar chains of 2 and 5 kDa, respectively, which are not necessary for its reactivity with aspergilloma serum samples. Using specific anti-p90 rabbit serum, we have demonstrated that under native conditions, p90 exists in oligomeric form and has associated catalase activity. This activity is resistant to extreme temperatures (>60؇C), reducing agents (40 mM dithiothreitol), high concentrations of denaturing agents such as 8 M urea and 8% sodium dodecyl sulfate, and treatments with ethanol-chloroform-water (5:3:10 [vol/vol]) mixtures.

For the first time, an immunodominant Aspergillus nidulans antigen (ASPND1) consistently reactive with serum samples from aspergilloma patients has been purified and characterized, and its coding gene (aspnd1) has been cloned and sequenced. ASPND1 is a glycoprotein with four N-glycosidically-bound sugar chains (around 2.1 kDa each) which are not necessary for reactivity with immune human sera. The polypeptide part is synthesized as a 277-amino-acid precursor of 30.6 kDa that after cleavage of a putative signal peptide of 16 amino acids, affords a mature protein of 261 amino acids with a molecular mass of 29 kDa and a pI of 4.24 (as deduced from the sequence). The ASPND1 protein is 53.1% identical to the AspfII allergen from Aspergillus fumigatus and 48% identical to an unpublished Candida albicans antigen. All of the cysteine residues and most of the glycosylation sites are perfectly conserved in the three proteins, suggesting a similar but yet unknown function. Analysis of the primary structure of the ASPND1 coding gene (aspnd1) has allowed the establishment of a clear relationship between several previously reported A. fumigatus and A. nidulans immunodominant antigens. The increasing incidence of Aspergillus-related diseases (43) has prompted many investigators to search for fungal molecules relevant either for the immunodiagnosis of or for understanding the pathology of the different forms of aspergillosis (ranging from pulmonary aspergilloma to invasive aspergillosis, passing through allergic situations). The role of several putative virulence factors has been investigated without too much success (for a review, see reference 3). In contrast, several antigens of demonstrated value as immunodiagnostic reagents for different Aspergillus infections have been purified, and some of their coding genes have been cloned. The AspfI ribotoxin (27), the AspfII allergen (2), and heat shock protein 1 (HSP-1) (16) from Aspergillus fumigatus have been obtained as recombinant antigens. The CAT1 (the subunit of a catalase) (22) and superoxide dismutase (13) antigens have been purified to homogeneity from A. fumigatus water-soluble extracts. All of these antigens have been shown to be consistently reactive with sera from patients with different forms of aspergillosis but not with sera from control or healthy individuals (2, 10, 24, 26). Certain other purified molecules, such as the 58-kDa (6) and gp55 (40) antigens, or semipurified fractions, such as the CS2 complex (30) or the cytosolic fraction complex (CFC) (24) from A. fumigatus, have been claimed to be useful reagents for immunodiagnosis. In this paper, we attempt to simplify the entangled antigenic array of A. fumigatus antigens by using data obtained from the purification and immunochemical characterization of a previously reported Aspergillus nidulans antigen (4) (now designated ASPND1), cloning of its coding gene, and analysis of its primary structure. The conclusion of this study is that several previously reported antigens or antigenic Aspergillus preparations conta...

Cytosolic fractions of mycelial extracts from Aspergillus nidulans, A. flaws, and three different isolates of A. fumigatus, grown to stationary phase in Czapek-Dox-AOAC medium, were tested by immunoblotting for the presence of antigens reactive to 80 serum samples from aspergilloma patients. Fifty control serum samples were used to determine the specificity of the reactions. In the A. fbmigatus cytosolic fraction a group of four main antigenic bands (p90, p60, p40 and p37) was consistently recognized (in total or partial form) by 90% of the serum samples from the aspergilloma patients. This group of antigens was designated as the 'cytosolic fraction complex' (CFC). As confirmed by two-dimensional electrophoresis followed by immunoblotting with aspergilloma serum samples, each of the four antigenic bands is formed of several isoforms of acidic glycopeptides with slightly different pls. All the isoforms are at least N-glycosylated, as demonstrated by endoglycosidase H removal of a considerable amount of sugar residues. The relationship of these antigens with certain other A. fumigatus antigens previously reported in the literature, and their potential use in the immunodiagnosis of aspergilloma, are discussed.

The influence of culture medium and time and temperature of growth on the appearance of Aspergillus nidulans antigens was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by silver staining or Western blot (immunoblot), of the proteins present in total cellular extracts or culture supernatants. Samples in the exponential, deceleration, and stationary growth phases were selected by