Characterization of inhibitory mechanism and antifungal activity between group-1 and group-2 phytocystatins from taro (Colocasia esculenta)

Abstract

Tarocystatin from Colocasia esculenta, a group-2 phytocystatin, is a defense protein against phytopathogenic nematodes and fungi. It is composed of a highly conserved N-terminal region, which is homological to group-1 cystatin, and a repetitive peptide at the C-terminus. The purified recombinant proteins of tarocystatin, such as full-length (FL), N-terminus (Nt) and C-terminus (Ct) peptides, were produced and their inhibitory activities against papain as well as their antifungal effects were investigated. Kinetic analysis revealed that FL peptide exhibited mixed type inhibition (K(ia) = 0.098 microM and K(ib) = 0.252 microM) and Nt peptide showed competitive inhibition (K(i) = 0.057 microM), whereas Ct peptide possessed weak papain activation properties. A shift in the inhibitory pattern from competitive inhibition of Nt peptide alone to mixed type inhibition of FL peptide implied that the Ct peptide has an regulatory effect on the function of FL peptide. Based on the inhibitory kinetics of FL (group-2) and Nt (group-1) peptides on papain activity, an inhibitory mechanism of group-2 phytocystatins and a regulatory mechanism of extended Ct peptide have each been proposed. By contrast, the antifungal activity of Nt peptide appeared to be greater than that of FL peptide, and the Ct peptide showed no effect on antifungal activity, indicating that the antifungal effect is not related to proteinase inhibitory activity. The results are valid for most phytocystatins with respect to the inhibitory mechanism against cysteine proteinase.

Figure 1

Purification of recombinant proteins and their in‐gel inhibitory activity assay. (A) SDS/PAGE analysis of purified recombinant GST‐fused proteins from bacterial extracts. Lane M, protein standard; FL lane, two bands corresponding to GST‐FL (upper band) and GST (lower band); Nt lane, GST‐Nt peptide (upper band) and GST (lower band); Ct lane, only one band (GST‐Ct peptide). (B) SDS/PAGE analysis of purified recombinant tarocystatin cleaved after thrombin digestion. (C) In‐gel inhibitory activity assay for three different segment recombinant proteins. The band brightness is proportioned to papain activity Samples containing FL or Nt peptides reduce the brightness on the gel, indicating their inhibitory capacity. By contrast, the Ct peptide showed an enhancing capacity. (D) In‐gel inhibitory activity assay for varied concentrations of the Ct peptide. The brightness of the band increased with increasing Ct peptide concentration, confirming its enhancing capacity. Lane 8* indicates a subject containing only Ct peptide recombinant protein, and not containing any papain, where no digestion occurred.

Figure 2

Anti‐fungal activity assay for recombinant proteins of different tarocystatin segments. (A) Five pieces of sclerotia cultured in the presence of recombinant proteins of varied concentrations in a 1‐cm diameter glass tube. Inhibition efficacy is proportional to the clarity of the medium. Additional FL or Nt peptides in the sclerotia culture caused an increase in clarity of the medium, indicating their anti‐fungal activity, whereas Ct peptide did not. (B) The inhibition level was graded from high effective (+++) to null (±) by visual quantification. (C) The different inhibitory strengths of varied FL peptide levels on mycelium growth was observed under high and low magnification. Mildly inhibited mycelium exhibited swelling, less branching and blunt tips. Fully inhibited mycelium exhibited more swelling, no branches, very short tips and fragmentation.

Figure 3

Analysis of inhibitory kinetics of different tarocystatin segments. (A) Plot of papain activity for a single inhibitor concentration (0125 μm) at various substrate concentrations. , Ck (water instead of inhibitor); •, FL peptide; ○, Nt peptide; □, Ct peptide. The y‐axis is the catalytic velocity of papain, expressed as the change in optical density per unit time. The x‐axis is the substrate concentration (mm). Each point represents the mean value of three repeated experiments, with the standard error shown as a bar. (B) Lineweaver–Burk plot for different tarocystatin segments, and also the double reciprocal plot of (A). Ck line crosses lines of the FL, Nt and Ct peptides in the second quadrant, y‐axis and x‐axis, respectively, indicating that the FL peptide behaves with mixed inhibition, the Nt peptide behaves with competitive inhibition and the Ct peptide behaves as an allosteric activator.

Figure 4

Lineweaver–Burk plot for reactions in the presence of two different concentrations of Nt peptide (A) and FL peptide (B). The inhibitor concentrations were 0.125 mm (○) and 0.0625 mm (•) in each case. Water (□) was used as a control.

Figure 5

Sequence alignment of the Nt and Ct peptides of taro and OC‐I (Protein Data Bank: IEQK). The identical residues are shown as a black box and the partially conserved residues are in grey. OC‐I shares 48% identity and 68% positives with the Nt peptide of tarocystatin and 13% identity and 38% positives with the Ct peptide of tarocystatin.

Figure 6

Conjectural structure model of tarocystatin. Flat arrows and helical ribbons represent β‐sheets and α‐helix structures, respectively. The entire structure resembles an earphone comprising two solid masses and a linear structure.

Figure 7

Competition and retrieve test of the Ct peptide to Nt peptide. (A) Competition test: the y‐axis is catalytic velocity of papain, which was measured as the change in optical density over time. Each reaction had the indicated amount of Ct and Nt peptides that reacted with papain. An increasing Ct level did not reduce the inhibitory capacity of the Nt peptide, but instead maintained a steady intensity. (B) Retrieve test: the mixture of Nt and Ct peptides of 625 μm was compared with an equal amount of only FL or Nt peptides in the reaction with varied substrate concentrations. The curve of Nt plus Ct peptides highly overlapped that of the Nt peptide, indicating that the Nt peptide cannot retrieve the inhibition efficacy of the FL peptide when it disconnects from the Ct peptide.