Molecular and biochemical characterization of subtilisin-like proteases in Arabidopsis thaliana
Subtilisin-like proteases form a large group of serine proteases with diverse functions, including the specific processing of a variety of proproteins and prohormones, and are found in prokaryotic and eukaryotic organisms. The work in this thesis focuses mainly on the Aral2 subtilisin-like protease following its discovery in the filtrate of Arabidopsis cell suspension cultures. Evidence obtained by Southern blotting and database searching is presented for the existence of a large gene family encoding subtilisin-like proteases in the model plant Arabidopsis thaliana. There may be more than fifty members in this gene family. Three of the corresponding DNA sequences have been cloned by RT-PCR and used as probes in Northern analysis to investigate the tissue specificity of the gene transcripts. These three genes appear to be expressed to varying degrees in Arabidopsis leaf, stem, root and silique tissues. A 650bp cDNA fragment encoding the C-terminal portion of the Aral2 protease has been obtained by RT-PCR, ligated to the malE gene and overexpressed as a fusion protein in E. coli cells. Polyclonal antisera have been raised against a combination of the fusion protein and the Aral2 C-terminal protein purified after cleavage from the fusion protein using Factor Xa protease. Aral2 protein has been detected in Arabidopsis tissues, particularly in siliques and stems, by Western blotting using these antibodies. An apoplastic location has been ascribed to Aral2 protease by immunocytochemistry using electron microscopy. The mature Aral2 subtilisin-like protease has been purified to homogeneity from Arabidopsis cell suspension culture filtrate by ion exchange chromatography and hydrophobic interaction chromatography. The purified enzyme has an acidic pH optimum of approximately pH5.5, which is unusual for a plant subtilisin-like protease. Aral2 protease is relatively thermostable and is activated in the presence of Ca(^2+) ions. The known serine protease inhibitors phenylmethanesulphonyl fluoride (PMSF), 4-(2- aminoethyl) benzenesulphonyl fluoride (AEBSF) and diisopropyl fluorophosphates (DFP) have an inhibitory effect on the proteolytic activity of Aral2. Substrate specificity studies have been performed using artificial peptide substrates, native proteins and cell wall protein extracts from Arabidopsis cells.