Functional characterization of the N-terminal and C-terminal domains of a sesame group II phytocystatin

Abstract

Background: Phytocystatins are natural inhibitors of cysteine protease, and may regulate endo- or exo-genous proteolytic activities in plants. They are classified into Group I and II differing by the presence of C-terminal extension of Group II. A cDNA fragment encoding a Group II phytosystatin, SiCYS was previously obtained from sesame seeds.

Results: SiCYS as well as its two structural domains, N-terminal and C-terminal domains (SiCYS-N and SiCYS-C), was expressed in Escherichia coli. The recombinant SiCYS and SiCYS-N showed inhibitory activity against papain. The K i values of SiCYS and SiCYS-N were ~1.9 ×10^-8 M and ~7.9 ×10^-8 M, respectively. All the three recombinants possessed comparable ability to inhibit spore germination of Trichoderma reesei, Aspergillus sydowii, and Helminthosporium sesamum. Similar protein profile including proteases in germinating seeds was found in proteins purified by the SiCYS, SiCYS-N or SiCYS-C coupling affinity column.

Conclusion: SiCYS exhibited more effective papain-inhibitory activity than SiCYS-N; while SiCYS-C had almost no inhibitory activity. All displayed similar antifungal activities indicating that there is no correlation between antifungal and papain-inhibitory activities. Structural and sequence analyses suggest that the C-terminal domain of SiCYS may be originated from gene duplication to enhance its inhibitory activity.

Keywords: Antifungal; Cysteine protease; Papain; Phytocystatin; Sesamun indicum.

Figure 1

SDS-PAGE analysis of sesame phytocystatins over-expressed in E. coli . (A) Before or after IPTG induction for 1–5 hours (hr), the total proteins were extracted from E. coli cells carrying recombinant vectors with DNA fragment, SiCYS, SiCYS-N or SiCYS-C, followed by SDS-PAGE analyses. The total proteins (total) extracted from E. coli cells after 3 hr IPTG induction were fractionated into precipitate (ppt.) and supernatant (sup.), and then resolved by SDS-PAGE. The protein bands of over-expressed recombinants on SDS-PAGE with the expected molecular weight, 22 kDa for SiCYS, 10 kDa for SiCYS-N or 14 kDa for His-tagged SiCYS-C were marked by red arrows. (B) The non-fusion recombinants, SiCYS and SiCYS-N were purified via papain-coupled affinity chromatography; while His-tagged SiCYS-C was purified via nickel affinity chromatography. Lane 1, 2 and 3 were the purified recombinant SiCYS, SiCYS-N and His-tagged SiCYS-C on SDS-PAGE analysis, respectively. Lane M represent as protein marker.

Figure 2

Inhibition of papain activity by recombinant sesame phytocystatins. Papain was mixed with various amounts of recombinant SiCYS, SiCYS-N and His-tagged SiCYS-C, respectively. The residual hydrolytic activity was determined and present as percentage of total activity of papain. The final concentration of papain was 1.24 μM in a 350 μl reaction. The added recombinants were depicted as the concentration of cystatin, μM as well. The assay was performed three independent times and presented as mean ± S.E.

Figure 3

Growth arrests of fungi by recombinant sesame phytocystatins. The inhibition of T. reesei, A. sydowii and H. sesamum spore germination by SiCYS, SiCYS-N or His-tagged SiCYS-C at 45 μg/ml of concentration were observed by microscopy comparing to the control, without any recombinant.

Figure 4

SDS-PAGE and zymographic analyses of germinated sesame proteins elute from affinity columns. (A) The proteins extracted from the germinated sesame were collected after the purification with affinity columns, followed by SDS-PAGE analyses. Lane A, B and C were the pull-down proteins/extracted proteins via SiCYS, SiCYS-N and SiCYS-C coupled affinity columns; namely SiCYS CP, SiCYS-N CP and SiCYS-C CP, respectively. The protein bands with molecular weight of ~32, ~45, ~48 and ~52 kDa in SDS-PAGE were marked. (B) SiCYS CP, SiCYS-N CP and SiCYS-C CP were added with or without protease inhibitors, followed by zymographic analyses of protease activity. Lane 2 was added with 20 mM of PMSF; lane 3, 250 μM of E64; lane 4, 5 mM of iodoacetamide; lane 5, 5 mM of N-ethylmaleimide, lane 6, 100 μM of pepstatin A; lane 7, 25 mM of EDTA; while no any inhibitor was added in lane 1. The location of ~45 and ~52 kDa on gel was marked as well.

Figure 5

The predicted structures of SiCYS-N and SiCYS-C, and their sequence alignment with other phytocystatins. (A) The main three dimensional structure of SiCYS-N (2–88 a.a of SiCYS) and SiCYS-C (116–191 a.a. of SiCYS) shown in cartoon diagram was predicted by homology modeling using Orzyacystatin I (PDB: 1EQK) as template. The secondary structure elements corresponding to α-helices and β-sheets were shown in red and yellow. Three conserved motifs (G⁵, Q⁴⁶XV⁴⁸XG⁵⁰, W⁷⁷) were labeled. (B) SiCYS-N (1–88 a.a. of SiCYS) and SiCYS-C (89–199 a.a. of SiCYS) was aligned with Orzyacystatin I (term OC-I), AcCYS and CeCPI. The structures of Orzyacystatin I, AcCYS and CeCPI (2–92 a.a.) but not CeCPI (93–205 a.a.) were currently available in PDB. Amino acid residues identical at least five out of six aligned sequences were boxed in grey. Three conserved motifs were signed in red dot. A specific consensus sequence of phytocystatins, LARFAVXQHN, was marked in green upper line; while SN(S/T)L motif found in Ct extension marked in under line. The sequence corresponding to α-helice and β-sheets were signed in red and blue.