Purification, cloning and autoproteolytic processing of an aspartic proteinase from Centaurea calcitrapa

Plant aspartic proteinases (APs) have been isolated from several seed and l eaf sources but the only well characterized enzymes from flowers are cardos ins and cyprosins from cardoon, Cynara cardunculus L. Here we report a full -length cDNA clone encoding an AP named cenprosin from the flowers of Centa urea calcitrapa L., a thistle related to cardoon. As found for all eukaryot ic APs, the deduced primary sequence consists of a signal sequence, a propa rt and a mature enzyme. In addition, an internal sequence region of 104 res idues typical only of plant APs (a plant-specific insert) is present in the primary structure. Northern analysis revealed that the strongest expressio n is in fresh flowers. The enzyme is also expressed in fairly high amounts in seeds and in leaves, a feature not detected for cardoon APs. The corresp onding enzyme was purified in its precursor form from fresh flowers using a mmonium-sulfate precipitation followed by ion-exchange and hydrophobic-inte raction chromatography. The processing of the precursor into its mature for m was studied in vitro. The enzyme underwent autocatalytic processing at pH 3.0 resulting in two chains of 16 and 30 kDa. When dried flowers were used as a starting material for purification, only 16- and 30-kDa chains were o btained, suggesting that autoproteolytic activation of procenprosin in vivo occurs mainly during drying of the flowers. This may indicate a specific d egradative role for the enzyme during senescence of the flowers.