Rs-AFP4

Two novel classes of antifungal proteins were isolated from radish seeds. The first class consists of two homologous proteins (Rs-AFP1 and Rs-AFP2) that were purified to homogeneity. They are highly basic oligomeric proteins composed of small (5-kDa) polypeptides that are rich in cysteine. Both Rs-AFPs have a broad antifungal spectrum and are among the most potent antifungal proteins hitherto characterized. In comparison with many other plant antifungal proteins, the activity of the Rs-AFPs is less sensitive to the presence of cations. Moreover, their antibiotic activity shows a high degree of specificity to filamentous fungi. The amino-terminal regions of the Rs-AFPs show homology with the derived amino acid sequences of two pea genes specifically induced upon fungal attack, to gamma-thionins and to sorghum alpha-amylase inhibitors. The radish 2S storage albumins were identified as the second novel class of antifungal proteins. All isoforms inhibit growth of different plant pathogenic fungi and some bacteria. However, their antimicrobial activities are strongly antagonized by cations.

Plant defensins are a class of cysteine-rich peptides of which several members have been shown to be potent inhibitors of fungal growth. A series of overlapping 15-mer peptides based on the amino acid sequence of the radish antifungal protein Rs-AFP2 have been synthesized. Peptides 6, 7, 8 and 9, comprising the region from cysteine 27 to cysteine 47 of Rs-AFP2 showed substantial antifungal activity against several fungal species (minimal inhibitory concentrations of 30-60 micrograms/mL), but no activity towards bacteria (except peptide 6 at 100 micrograms/mL). The active peptides were shown to be sensitive to the presence of cations in the medium and to the composition and pH of the medium. When present at a subinhibitory concentration (20 micrograms/mL), peptides 1, 7, 8 and 10 potentiated the activity of Rs-AFP2 from 2.3-fold to 2.8-fold. By mapping the characteristics of the active peptide on the structure of Rs-AFP2 as determined by nuclear magnetic resonance, the active region of the antifungal protein appears to involve beta-strands 2 and 3 in combination with the loop connecting those strands. A cyclized synthetic mimic of the loop, cysteine 36 to cysteine 45, was shown to have antifungal activity. Substitution of tyrosine 38 by alanine in the cyclic peptide substantially reduced the antifungal activity, indicating the importance of this residue for the activity of Rs-AFP2 as demonstrated carrier by mutational analysis.

Rs-AFPs are antifungal proteins, isolated from radish (Raphanus sativus) seed or leaves, which consist of 50 or 51 amino acids and belong to the plant defensin family of proteins. Four highly homologous Rs-AFPs have been isolated (Rs-AFP1-4). The structure of Rs-AFP1 consists of three beta-strands and an alpha-helix, and is stabilized by four cystine bridges. Small peptides deduced from the native sequence, still having biological activity, are not only important tools to study structure-function relationships, but may also constitute a commercially interesting target. In an earlier study, we showed that the antifungal activity of Rs-AFP2 is concentrated mainly in the beta2-beta3 loop. In this study, we synthesized linear 19-mer peptides, spanning the entire beta2-beta3 loop, that were found to be almost as potent as Rs-AFP2. Cysteines, highly conserved in the native protein, are essential for maintaining the secondary structure of the protein. Surprisingly, in the 19-mer loop peptides, cysteines can be replaced by alpha-aminobutyric acid, which even improves the antifungal potency of the peptides. Analogous cyclic 19-mer peptides, forced to adopt a hairpin structure by the introduction of one or two non-native disulfide bridges, were also found to possess high antifungal activity. The synthetic 19-mer peptides, like Rs-AFP2 itself, cause increased Ca2+ influx in pregerminated fungal hyphae.