Mutational Analysis of a Plant Defensin from Radish (Raphanus sativus L.) Reveals Two Adjacent Sites Important for Antifungal Activity

Abstract

Mutational analysis of Rs-AFP2, a radish antifungal peptide belonging to a family of peptides referred to as plant defensins, was performed using polymerase chain reaction-based site-directed mutagenesis and yeast as a system for heterologous expression. The strategy followed to select candidate amino acid residues for substitution was based on sequence comparison of Rs-AFP2 with other plant defensins exhibiting differential antifungal properties. Several mutations giving rise to peptide variants with reduced antifungal activity against Fusarium culmorum were identified. In parallel, an attempt was made to construct variants with enhanced antifungal activity by substituting single amino acids by arginine. Two arginine substitution variants were found to be more active than wild-type Rs-AFP2 in media with high ionic strength. Our data suggest that Rs-AFP2 possesses two adjacent sites that appear to be important for antifungal activity, namely the region around the type VI ␤-turn connecting ␤-strands 2 and 3, on the one hand, and the region formed by residues on the loop connecting ␤-strand 1 and the ␣-helix and contiguous residues on the ␣-helix and ␤-strand 3, on the other hand. When added to F. culmorum in a high ionic strength medium, Rs-AFP2 stimulated Ca 2؉ uptake by up to 20-fold. An arginine substitution variant with enhanced antifungal activity caused increased Ca 2؉ uptake by up to 50-fold, whereas a variant that was virtually devoid of antifungal activity did not stimulate Ca 2؉ uptake.

Defensins from Insects and Plants Interact with Fungal Glucosylceramides

“…1) interaction of RsAFP2 with GlcCer in the fungal plasma membrane (as evidenced by this study) and 2) subsequent permeabilization of the fungal plasma membrane (as demonstrated previously (9)). The finding that RsAFP2(Y38G), an RsAFP2 variant devoid of antifungal activity (21), still interacts to the same extent with fungal GlcCer as native RsAFP2, confirms the hypothesized two-step model for the mode of action of RsAFP2. Indeed, it seems that the mutant RsAFP2(Y38G) is competent for GlcCer binding but is impaired in induction of membrane permeabilization and fungal growth inhibition.…”

“…Materials and Microorganisms-The antifungal peptides DmAMP1, RsAFP2, and RsAFP2(Y38G) were isolated as described previously (6,20,21). Nonimmune and anti-RsAFP2 sera from rabbit were used as described previously (6).…”

Engineering disulfide bridges to dissect antimicrobial and chemotactic activities of human β-defensin 3

“…In fact, this has been shown to be physiologically relevant in cystic fibrosis (CF), where abnormally high NaCl in the lung inactivates hBD1, resulting in the CF-associated defect in bacterial killing (38,39). There appears to be a correlation between the number of positive charges and the ability to retain antimicrobial activity of defensins at higher concentrations of salts (40,41). It is plausible that a greater number of charged residues are less sufficiently screened by salts to diminish the binding to the negatively charged lipid components in membranes.…”

The hydrophobic surface of PaAMP from pokeweed seeds is essential to its interaction with fungal membrane lipids and the antifungal activity

“…The protein concentrations required for 50% growth inhibition (IC 50 ) after 48 h incubation were determined by plotting the growth inhibition versus protein concentrations [3]. A specific antimicrobial activity was defined as 1/IC 50 , and a relative activity of a protein was expressed as described by De Samblanx et al [17]. For microscopic observation of the inhibition of spore germination or hypha growth of F. solani, the fungal spores or hyphae in 1 mL YPG containing PaAMP protein or its derivatives were incubated for 16 h at 25°C with gentle shaking.…”