A novel and exploitable antifungal peptide from kale (Brassica alboglabra) seeds

Abstract

The aim of this study was to purify and characterize antifungal peptides from kale seeds in view of the paucity of information on antifungal peptides from the family Brassicaceae, and to compare its characteristics with those of published Brassica antifungal peptides. A 5907-Da antifungal peptide was isolated from kale seeds. The isolation procedure comprised affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Sepharose and Mono S, and gel filtration on Superdex Peptide. The peptide was adsorbed on the first three chromatographic media. It inhibited mycelial growth in a number of fungal species including Fusarium oxysporum, Helminthosporium maydis, Mycosphaerella arachidicola and Valsa mali, with an IC(50) of 4.3microM, 2.1microM, 2.4microM, and 0.15microM, respectively and exhibited pronounced thermostability and pH stability. It inhibited proliferation of hepatoma (HepG2) and breast cancer (MCF7) cells with an IC(50) of 2.7microM and 3.4microM, and the activity of HIV-1 reverse transcriptase with an IC(50) of 4.9microM. Its N-terminal sequence differed from those of antifungal proteins which have been reported to date.

Fig. 1

Purification of kale (Brassica alboglabra L.H. Bailey) antifungal peptide by chromatography on (A) Affi-gel Blue gel, (B) SP-Sepharose, (C) Mono S and (D) Superdex Peptide. In (A), the extract of kale seeds was applied on an Affi-gel Blue gel column (5 cm × 20 cm). Unadsorbed proteins (fraction BG1) were eluted with the same buffer while adsorbed proteins (fraction BG2) were eluted with 10 mM Tris–HCl buffer (pH 7.8) containing 1 M NaCl as indicated by the arrows. In (B), fraction BG2 from the Affi-gel Blue gel column was dialyzed and applied on an SP-Sepharose column (2.5 cm × 20 cm) in 10 mM NH₄OAc buffer (pH 4.5). After elution of unadsorbed proteins, the column was eluted stepwise with 0.2 M NaCl, 0.5 M NaCl and then with 1 M NaCl added to the buffer as indicated by the arrows. In (C), fraction SP2 from the SP-Sepharose column was loaded on a 1-ml Mono S column. Following elution of unadsorbed proteins with 10 mM NH₄OAc buffer (pH 4.5), adsorbed proteins were eluted sequentially, first with a 0–0.4 M NaCl gradient and then with a 0.4–0.7 M and 0.7–1 M NaCl gradient. In (D), fraction S2 from the Mono S column was subjected to gel filtration on a Superdex Peptide HR 10/30 column in 10 mM NH₄OAc buffer (pH 4.5).

Fig. 2

(A) SDS–PAGE of kale antifungal peptide. (B) Mass spectrometric analysis of kale antifungal peptide.

Fig. 3

The IC₅₀ of antifungal activity of kale antifungal peptide toward (A) Mycosphaerella arachidicola, (B) Fusarium oxysporum, (C) Helminthosporium maydis, and (D) Valsa mali was 2.1 μM, 4.3 μM, 2.4 μM, and 0.15 μM, respectively. The numbers on the plates represent the concentrations of the antifungal peptide in μM.

Fig. 4

(A) Thermostability and (B) pH stability of antifungal activity of kale antifungal peptide. The same amount (2 μg) of peptide was added to each paper disk (except the control disk labeled as (C). The numbers in panel (A) (40–100) and in panel (B) (0–3 and 10–14) near the paper disks represent the various temperatures (panel A) and pH's (panel B) at which the antifungal peptide introduced to the disk had been pretreated for 10 and 30 min, respectively. The antifungal activity of the peptide was found to be stable after exposure to temperatures in the range 20–80 °C for 10 min and to the pH ranges 2–3 and 10–11 for 30 min.

Fig. 5

Kale antifungal peptide demonstrated antiproliferative activity (A and B), and HIV-1 reverse transcriptase inhibitory activity (C).