The Antifungal Protein AFP of Aspergillus giganteusIs an Oligonucleotide/Oligosaccharide Binding (OB) Fold-containing Protein That Produces Condensation of DNA

Abstract

The antifungal protein AFP is a small polypeptide of 51 amino acid residues secreted by Aspergillus giganteus. Its potent activity against phytopathogenic fungi converts AFP in a promising tool in plant protection. However, no data have been reported regarding the mode of action of AFP. The three-dimensional structure of this protein, a small and compact ␤ barrel composed of five highly twisted antiparallel ␤ strands, displays the characteristic features of the oligonucleotide/oligosaccharide binding (OB fold) structural motif. A comparison of the structures of AFP and OB fold-containing proteins shows this structural similarity despite the absence of any significant sequence similarity. AFP, like most OB fold-containing proteins, binds nucleic acids. The protein promotes charge neutralization and condensation of DNA as demonstrated by electrophoretic mobility shift and ethidium bromide displacement assays. Nucleic acid produces quenching of the protein fluorescence emission. This nucleic acid interacting ability of AFP may be related to the antifungal activity of this small polypeptide.The mold Aspergillus giganteus produces two major extracellular proteins, ␣-sarcin, a potent cytotoxic ribonuclease, and the so-called antifungal protein (AFP) 1 (1-3). AFP is a small polypeptide of 51 amino acid residues that is secreted under the form of a rather inactive larger precursor containing 6 extra amino acid residues at the NH 2 -terminal end, which is processed in the extracellular medium (4). The amino acid and cDNA sequences of AFP have been reported, revealing a high content of disulfide bridges (fours bonds) and tyrosines and lysines (6 and 12 residues, respectively) (5-7). AFP shows a significant degree of sequence similarity only with both the abundantly secreted antifungal PAF protein (55 amino acid residues) from Penicillium chrysogenum (47% sequence identity) (8) and the antifungal peptide Anafp (56 amino acid residues) secreted by Aspergillus niger (31% sequence identity) (9). AFP has been tested, at concentrations as large as 0.2 mM, against a wide variety of microorganisms, including prokaryotes and eukaryotes (3). This protein inhibits the growth of some filamentous fungi, the minimal protein concentration for total inhibition being in the range of 6 -25 M (3), but it does not promote any effect on the producing mold or on bacteria or yeast. AFP is not active against P. chrysogenum or A. niger (3), the organisms producing PAF and Anapf proteins, respectively. Although these three proteins have been described as antifungal molecules, no data regarding their mode of action have been so far reported. We herein report the interaction of AFP with DNA. EXPERIMENTAL PROCEDURESUnless otherwise stated, all materials and reagents were molecular biology grade. Calf thymus double-stranded DNA (dsDNA) was purified as described previously (10, 11). The DNA fraction selected for the experiments described in this work (kept at Ϫ20°C until used) was that containing 200 bp dsDNA on average according to their electrophoret...

Survival Strategies of Yeast and Filamentous Fungi against the Antifungal Protein AFP

“…It comprises a chitin-binding domain and inhibits chitin synthesis in sensitive filamentous fungi (13). In collapsed and dead cells, AFP can also be found intracellularly (14,15), where it might bind via its oligonucleotide/oligosaccharide-binding fold to anionic molecules, such as nucleic acids (16). A shorter version of AFP exhibiting the chitin-binding domain but lacking the hydrophobic domain (sAFP) does not disturb the cell wall/ cell membrane integrity of AFP-sensitive filamentous fungi, although it is able to bind to chitin and nucleic acids under in vitro conditions (13).…”

Intron requirement for AFP gene expression in Trichoderma viride

“…By comparison of the intron/exon structures of various alcohol dehydrogenase genes and related enzyme genes, Duester et al (1986) showed that the evolution of the oligonucleotide-binding domain was intron-dependent. A recent study indicated that AFP has the characteristic features of an oligonucleotide-binding domain (Martinez Del Pozo et al, 2002). In view of our current study and the evidence summarized above, it is tempting to speculate that the evolution of the AFP gene might be intron-dependent.…”

Production of the biotechnologically relevant AFP from Aspergillus giganteus in the yeast Pichia pastoris

“…Mean residue weight ellipticities were expressed in units of degree x cm 2 x dmol -1 . All these determinations were performed under conditions described elsewhere [11,20,27].…”

“…It displays the characteristic features of an oligonucleotide/oligosaccharide binding (OB-fold) structural motif [20] being a small and compact β-barrel composed of five highly twisted antiparallel β-strands. It should be also mentioned that cysteine pairing isomerism exists within its four disulfide bridges [19].…”

“…Apparently, the AFP protein enters into the fungal cell and targets the nucleus, as revealed by co-localization experiments of Alexa-labeled AFP with the SYTOX Green dye [15]. AFP would bind fungal nucleic acids promoting charge neutralization and condensation of DNA [15,20], maybe triggering apoptosis [23]. The available data suggest that it is this combination of fungal cell permeabilization, cell-penetrating ability and nucleic acid-binding activity of AFP that determines its potent antifungal activity.…”