Purification, cloning and autoproteolytic processing of an aspartic proteinase from Centaurea calcitrapa.
Plant aspartic proteinases (APs) have been isolated from several seed and leaf sources but the only well characterized enzymes from flowers are cardosins and cyprosins from cardoon, Cynara cardunculus L. Here we report a full-length cDNA clone encoding an AP named cenprosin from the flowers of Centaurea calcitrapa L., a thistle related to cardoon. As found for all eukaryotic APs, the deduced primary sequence consists of a signal sequence, a propart and a mature enzyme. In addition, an internal sequence region of 104 residues typical only of plant APs (a plant-specific insert) is present in the primary structure. Northern analysis revealed that the strongest expression 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 corresponding enzyme was purified in its precursor form from fresh flowers using ammonium-sulfate precipitation followed by ion-exchange and hydrophobic-interaction chromatography. The processing of the precursor into its mature form 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 obtained, suggesting that autoproteolytic activation of procenprosin in vivo occurs mainly during drying of the flowers. This may indicate a specific degradative role for the enzyme during senescence of the flowers.
Domingos
A
A
Departamento de Biotecnologia, Instituto Nacional de Engenharia e Tecnologia Industrial, Lisboa, Portugal. ana.domingos@mail.ineti.pt
Cardoso
P C
PC
Xue
Z T
ZT
Clemente
A
A
Brodelius
P E
PE
Pais
M S
MS
D000595:Amino Acid Sequence; D000645:Ammonium Sulfate / Q000494:pharmacology; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification*; D019659:Asteraceae / Q000201:enzymology*; D015152:Blotting, Northern; D002384:Catalysis; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003208:Concanavalin A / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000378:metabolism; D037121:Plant Lectins; D011480:Protease Inhibitors / Q000494:pharmacology; D012639:Seeds / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014105:Tosyl Compounds / Q000494:pharmacology
D018076:DNA, Complementary; D037121:Plant Lectins; D011480:Protease Inhibitors; D014105:Tosyl Compounds; D003208:Concanavalin A; C003614:4-toluenesulfonyl fluoride; D016282:Aspartic Acid Endopeptidases; D000645:Ammonium Sulfate