Characterization of the Aspergillus nidulans aspnd1 gene demonstrates that the ASPND1 antigen, which it encodes, and several Aspergillus fumigatus immunodominant antigens belong to the same family

Abstract

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 The ASPND1 protein is 53.1% identical to the AspfII allergen from Aspergillus fumigatus and 48% identical to an unpublished Candida albicans antigen.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...

Cloning and expression of Aspergillus fumigatus allergen Asp f 16 mediating both humoral and cell‐mediated immunity in allergic bronchopulmonary aspergillosis (ABPA)

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Cloning and Characterization of PRA1 , a Gene Encoding a Novel pH-Regulated Antigen of Candida albicans

“…Proteins were separated by SDSpolyacrylamide gel electrophoresis (SDS-PAGE) as described previously (27) in 10% (wt/vol) acrylamide gels loaded with 20 g of protein. After transfer to nitrocellulose, proteins were immunodetected with either monospecific Pra1p antibodies (41), polyclonal antibodies against mycelial cell walls (41), or polyclonal antibodies raised against Aspergillus nidulans antigen AspNDI (kindly provided by F. Leal from University of Salamanca [8]). Detection was carried out with an ECL kit (Amersham) according to the manufacturer's instructions.…”

Disruption and basic functional analysis of six novel ORFs of chromosome XV from Saccharomyces cerevisiae

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