Isolation and Characterization of a Thionin Proprotein-processing Enzyme from Barley

Abstract

Thionins are plant-specific antimicrobial peptides that have been isolated from the endosperm and leaves of cereals, from the leaves of mistletoes, and from several other plant species. They are generally basic peptides with three or four disulfide bridges and a molecular mass of ~5 kDa. Thionins are produced as preproproteins consisting of a signal peptide, the thionin domain, and an acidic domain. Previously, only mature thionin peptides have been isolated from plants, and in addition to removal of the signal peptide, at least one cleavage processing step between the thionin and the acidic domain is necessary to release the mature thionin. In this work, we identified a thionin proprotein-processing enzyme (TPPE) from barley. Purification of the enzyme was guided by an assay that used a quenched fluorogenic peptide comprising the amino acid sequence between the thionin and the acidic domain of barley leaf-specific thionin. The barley TPPE was identified as a serine protease (BAJ93208) and expressed in Escherichia coli as a strep tag-labeled protein. The barley BTH6 thionin proprotein was produced in E. coli using the vector pETtrx1a and used as a substrate. We isolated and sequenced the BTH6 thionin from barley to confirm the N and C terminus of the peptide in planta. Using an in vitro enzymatic assay, the recombinant TPPE was able to process the quenched fluorogenic peptide and to cleave the acidic domain at least at six sites releasing the mature thionin from the proprotein. Moreover, it was found that the intrinsic three-dimensional structure of the BTH6 thionin domain prevents cleavage of the mature BTH6 thionin by the TPPE.

Keywords: antimicrobial peptide; plant defense; proprotein; proprotein convertase subtilisin/kexin type 9 (PCSK9); recombinant protein expression; serine protease; subtilase; thionin.

FIGURE 1.

Alignment of barley leaf thionin proproteins deduced from DNA sequences. Arrows indicate the suggested end of the thionin domain. DB4, DC4, and DG3 are from cDNAs, and BTH6 and BTH7 are from genomic DNA clones. References are given in parentheses.

FIGURE 2.

SDS-PAGE analysis of fractions with protease activity. Protein extracts from etiolated barley seedlings were purified and fractions F8, F14, F24, and F32 were separated on a SDS-polyacrylamide gel. Proteins appearing in bands A–G were extracted from the gel and digested, and peptides were sequenced by LC-ESI-MS/MS.

FIGURE 3.

Sequence and structure of BJ93206. A, schematic domain representation according to the architecture prediction program SMART. B, amino acid sequence of BJ93208. Amino acids forming the catalytic triad His-227, Asn-331, and Ser-556 are highlighted in green. C, domains of BJ93208. Signal peptide, red; inhibitor I9, blue; peptidase domain, black; PA, green; and fibronectin 3 domain, orange.

FIGURE 4.

Predicted structure of TPPE. Mature BAJ93208 was modeled with PHYRE2 (29). Subtilase domain (residues 136–501), PA domain (residues 379–474), and FN3 domain (residues 561–659) are indicated. The catalytic triad consisting of His-227, Asn-331, and Ser-556 is shown in yellow circles and is surrounded by the conserved subtilase domain. The structure has 99% of residues modeled at >90% confidence against subtilisin prosegment (d1scjb), Homo sapiens proprotein convertase (c2p4eP, c2pmwA, and c2w2mP), oyster mushroom proteinase A inhibitor (d1v5ib1, d1itpa), cucumisin (c3vtaB), and tomato subtilase 3 (c3i74B).

FIGURE 5.

MS spectrum of the fluorogenic peptide (ANA)-SDYPKLNLLPK-(MCA) digested with the recombinant BAJ93208 protease. The peptide is cleaved after asparagine.

FIGURE 6.

Production of tagged pro-BTH6. A, expression construct for the His₆-TRX-myc-proBTH6-strep fusion protein. B, SDS-PAGE analysis showing the purification and digestion of the His₆-TRX-myc-proBTH6-strep fusion protein. L, low molecular weight ladder; lane 1) induced Shuffle C3030 cells (F2–F10) fractions from elution of Ni-NTA purification; lane 2) His-TRX-myc-proBTH6-strep fusion protein after digestion with TEV_SH protease; TEV, TEV_SH protease.

FIGURE 7.

Purification of myc-BTH6-strep proprotein. Elution chromatogram of reverse phase purification of the myc-BTH6-strep proprotein on the μRPC C2/C18 column. mAU, milli-absorbance units.

FIGURE 8.

TPPE releases the mature thionin from oxidized pro-BTH6. LC-ESI-MS analysis of the digestion of myc-proBTH6-strep with recombinant TPPE. The acidic domain is cleaved several times, although the thionin is not cleaved. The masses shown correspond to cleavage products of the BTH6 precursor (compare Fig. 9A).

FIGURE 9.

Cleavage of myc-proBTH6-strep by TPPE. Cleavage sites were identified by LC-ESI-MS after incubation of recombinant myc-proBTH6-strep with recombinant activated His₆-BAJ93208-strep protease. A, BTH6 proprotein in oxidized form with intact disulfide bridges. TPPE cleaved the acidic domain and the Myc tag. The preferred cleavage site in the acidic domain corresponds to the cleavage site in the fluorogenic peptide (see Fig. 5), but fragments with the authentic thionin end were also identified. Note that there was no cleavage within the thionin domain. B, precursor in a reduced form with open disulfide bridges. The acidic domain is cleaved at additional sites, and the thionin domain is also cleaved. Green, Myc tag; yellow, acidic domain; red, strep tag.

FIGURE 10.

Amount of mature thionin with correct C terminus increases over time. LC-ESI-MS analysis of the digestion of myc-proBTH6-strep with recombinant TPPE after 30 and 120 min. Note the different scales in the two graphs. The Myc tag at the N terminus is also cleaved by TPPE, giving rise to different peptides with the correct C terminus (indicated by boxed numbers showing the peak heights).

FIGURE 11.

Predicted structure of BTH6. Structure determination by PHYRE (29) shows the typical compact structure of thionin peptides. The structure was modeled against hordothionin, hellethionin D, and viscotoxins A3, A2, B, and C1 with >90% confidence.

FIGURE 12.

TPPE cleaves the reduced pro-BTH6 in the thionin domain. LC-ESI-MS analysis of the digestion of carboxymethylated myc-proBTH6-strep with recombinant TPPE. The acidic domain is cleaved several times with additional sites compared with the oxidized proprotein. The thionin domain is also cleaved at several sites. The masses shown correspond to cleavage products of the BTH6 precursor (compare Fig. 9B).

FIGURE 13.

Sequence alignment of TPPE and SEP-1.