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Gewählte Publikation:

Plattner, S; Gruber, C; Stadlmann, J; Widmann, S; Gruber, CW; Altmann, F; Bohlmann, H; .
(2015): Isolation and Characterization of a Thionin Proprotein-processing Enzyme from Barley.
J Biol Chem. 2015; 290(29):1805-1867 FullText FullText_BOKU

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 similar to 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.
Autor*innen der BOKU Wien:
Altmann Friedrich
Bohlmann Holger
Grünwald-Gruber Clemens
Plattner Stephan
Stadlmann Johannes
Widmann Stefan
BOKU Gendermonitor:

Find related publications in this database (using NML MeSH Indexing)
Amino Acid Sequence
Hordeum/chemistry
Hordeum/enzymology*
Hordeum/metabolism
Models, Molecular
Molecular Sequence Data
Plant Proteins/chemistry
Plant Proteins/isolation & purification
Plant Proteins/metabolism*
Protein Conformation
Proteolysis
Sequence Alignment
Serine Proteases/chemistry
Serine Proteases/isolation & purification
Serine Proteases/metabolism*
Thionins/chemistry
Thionins/metabolism*



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