Molecular cloning and expression analysis of the main gliadin-degrading cysteine endopeptidase EP8 from triticale

During the development and maturation of cereal grains, storage proteins are accumulated in the starchy endosperm. The enzymes responsible for the mobilisation of stored proteins during seed germination are cysteine endopeptidases and serine carboxypeptidases. In our previous study, we purified the major gliadin-degrading cysteine, endopeptidase EP8, from germinating triticale kernels. We confirmed in an experiment with exogenous gibberellic acid (GA3) that this enzyme is synthesised in aleurone during germination. In this study, the nucleotide sequence of EP8, a barley EP-A (HvEPA) homologue, and the expression pattern during grain development and germination have been analysed. Additionally, by comparing the activity of purified EP8 with the activity pattern of crude enzymatic extracts, the lack of enzyme activity in some tissues and during certain developmental stages has been demonstrated. The correlation of the results of the EP8 expression analysis with the activity pattern of this endopeptidase allowed us to formulate a hypothesis regarding the mechanism of EP8 activity regulation. We believe that the absence of active EP8 until the third day of germination may be associated with high levels of endogenous cystatin TrcC-4, which has the ability to inhibit EP8 in vitro.