Campesin, a thermostable antifungal peptide with highly potent antipathogenic activities

Abstract

An 9.4-kDa antifungal peptide designated as campesin was isolated from seeds of the cabbage Brassica campestris. The isolation procedure involved affinity chromatography on Affi-gel blue gel, ion exchange chromatography on Q-Sepharose and Mono S, and gel filtration on Superdex 75 and Superdex Peptide. The peptide was adsorbed on the first three chromatographic media. It exerted an inhibitory action on mycelial growth including Fusarium oxysporum and Mycosphaerella arachidicola, with an IC(50) of 5.1 microM and 4.4 microM, respectively. The peptide was characterized by remarkable thermostability and pH stability. It inhibited proliferation of HepG2 and MCF cancer cells with an IC(50) of 6.4 microM and 1.8 microM, and the activity of HIV-1 reverse transcriptase with an IC(50) of 3.2 microM. It demonstrated lysolecithin binding activity.

Fig. 1

(A) FPLC-ion exchange chromatography on Mono S. Sample: fraction of seed extract previously adsorbed on Q-Sepharose and subsequently adsorbed on Affi-gel blue gel. Fraction S5 with antifungal activity was collected. (B) Gel filtration on a Superdex 75 HR 10/30 column. Fraction SU3 with antifungal activity was collected. (C) Gel filtration on a Superdex Peptide column. Fraction P1 with antifungal activity was collected.

Fig. 2

Tricine-SDS-PAGE of campesin. Left lane: molecular Kaleidoscope prestained molecular standards including carbonic anhydrase, soybean trypsin inhibitor, lysozyme, aprotinin and insulin with molecular mass of 38.8 kDa, 25 kDa, 16.3 kDa, 7.8 kDa and 3.4 kDa, respectively. Right lane: campesin.

Fig. 3

Determination of IC₅₀ of antifungal activity of campesin. The fungi tested were: plate (A) Mycosphaerella arachidicola and (B) Fusarium oxysporum. (C) shows concentration of campesin in each plate.

Fig. 4

Comparison of increase in fluorescence intensity at 329 nm on lysolecithin binding due to campesin and BCLTP. F–F₀ represents the fluorescence intensity of campesin. F is the intensity obtained at each lipid–protein ratio.

Fig. 5

Campesin demonstrated antiproliferative activity (A), and HIV-1 reverse transcriptase inhibitory activity (B), but lacked mitogenic activity (C). Results represent mean ± SD (n = 3).