Identification and proteolytic processing of procardosin A.
Plant aspartic proteinases contain a plant-specific insert (PSI) of about 100 amino acids of unknown function with no similarity with the other aspartic proteinases but with significant similarity with saposins, animal sphingolipid activator proteins. PSI has remained elusive at the protein level, suggesting that it may be removed during processing. To understand the molecular relevance of PSI, the proteolytic processing of cardosin A, the major aspartic proteinase from the flowers of cardoon (Cynara cardunculus L.) was studied. Procardosin A, a 64-kDa cardosin A precursor containing PSI and the prosegment was identified by immunoblotting using monospecific antibodies against PSI and the prosegment. Procardosin A undergoes proteolytic processing as the flower matures. PSI was found to be removed before the prosegment, indicating that during processing the enzyme acquires a structure typical of mammalian or microbial aspartic proteinase proforms. In vitro studies showed that processing of PSI occurs at pH 3.0 and is inhibited by pepstatin A and at pH 7.0. Sequence analysis allowed the identification of the cleavage sites, revealing that PSI is removed entirely, probably by an aspartic proteinase. Cleavage of the PSI scissile bonds requires, however, a conformation specific to the precursor since isolated cardosins and pistil extracts were unable to hydrolyse synthetic peptides corresponding to the cleavage sites. In view of these results, a model for the proteolytic processing of cardosin A is proposed and the molecular and physiological relevance of PSI in plant aspartic proteinase is discussed.
Departamento de Bioquímica and the Centro de Neurociências e Biologia Celular, Universidade de Coimbra, Portugal.
M
M
Ramalho-Santos
P
P
Veríssimo
L
L
Cortes
B
B
Samyn
J
J
Van Beeumen
E
E
Pires
C
C
Faro
D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019659:Asteraceae / Q000201:enzymology*; D002451:Cell Compartmentation; D004792:Enzyme Precursors / Q000302:isolation & purification / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry; D006868:Hydrolysis; D010446:Peptide Fragments / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017421:Sequence Analysis; D014617:Vacuoles / Q000201:enzymology; D014675:Vegetables / Q000201:enzymology*
D004792:Enzyme Precursors; D006023:Glycoproteins; D010446:Peptide Fragments; D016282:Aspartic Acid Endopeptidases; C113804:procardosin A