Thionin Processing

Thionins are antimicrobial peptides which have been isolated from different plant species including cereals and mistletoes. They are usually basic, with a molecular weight around 5 kDa and have a compact structure which is stabilised by 3 or 4 disulfide bridges. Their toxic and antimicrobial activities are probably based on a destruction of cell membranes. Thionins are produced as preproproteins and secreted. The proprotein consists of the thionin and an acidic domain which normally also contains 6 cysteine residues. The aim of this project is to study the processing of the thionin proproteins and the function of the acidic domain. As a first step, the protease responsible for the processing of the proprotein will be purified from barley. The sequence of the protease will then be used to identify the corresponding gene(s) from Arabidopsis (which contains 4 thionin genes). Using mass spectrometry we will study the processing in vitro. Furthermore, mutants or miRNA lines will be used to assess the role of the protease(s) on plant development and on the proprotein in the plant cell. Antibodies will be used to locate the proprotein and the acidic domain in the cell.

Thionins are plant-specific antimicrobial peptides which have been isolated from the endosperm and leaves of cereals, from the leaves of mistletoes and several other plant species. They are generally basic peptides with three or four disulfide bridges and a molecular weight of about 5 kDa. Thionins are produced as preproproteins consisting of a signal peptide, the thionin domain and an acidic domain. Only the mature thionin peptide has 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 the thionin proprotein processing enzyme (TPPE) from barley. The processing was confirmed by isolating and sequencing the native thionin BTH6 from etiolated barley seedlings. The assay included a quenched fluorogenic peptide comprising the naturally occurring amino acid sequence between the thionin and the acidic domain of barley leaf-specific thionins. The TPPE from barley was identified as a serine protease (BAJ93208) and expressed in E. coli. Additionally, the BTH6 proprotein was also expressed in E. coli as a fusion protein. The recombinant TPPE was able to process the BTH6 fusion protein in vitro. Surprisingly, the acidic domain was cleaved at five sites to release the mature thionin TPPE, explains why a protein corresponding to the acidic domain has never been isolated from plants. Furthermore, it was found that the BTH6 thionin domain is also cleaved several times if the three dimensional structure of the proprotein is opened by reducing the disulfide bridges. Thus, the intrinsic three dimensional structure of the BTH6 thionin domain prevents cleavage of the mature BTH6 thionin by TPPE.