Activation, proteolytic processing, and peptide specificity of recombinant cardosin A.
Cardosins are model plant aspartic proteases, a group of proteases that are involved in cell death events associated with plant senescence and stress responses. They are synthesized as single-chain zymogens, and subsequent conversion into two-chain mature enzymes is a crucial step in the regulation of their activity. Here we describe the activation and proteolytic processing of recombinant procardosin A. The cleavage sites involved in this multi-step autocatalytic process were determined, some of them using a novel method for C-terminal sequence analysis. Even though the two-chain recombinant enzyme displayed similar properties as natural cardosin A, a single-chain mutant form was engineered based on the processing results and produced in Escherichia coli. Determination of its primary specificity using two combinatorial peptide libraries revealed that this mutant form behaved like the natural enzyme. The primary specificity of the enzyme closely resembles those of cathepsin D and plasmepsins, suggesting that cardosin A shares the same peptide scissile bond preferences of its vacuolar/lysosomal mammalian and protozoan homologues.
Castanheira
Pedro
P
Departamento de Bioquímica, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, Apartado 3126, P-3001-401 Coimbra, Portugal.
Samyn
Bart
B
Sergeant
Kjell
K
Clemente
José C
JC
Dunn
Ben M
BM
Pires
Euclides
E
Van Beeumen
Jozef
J
Faro
Carlos
C
D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000502:physiology*; D001665:Binding Sites; D002402:Cathepsin D / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry; D009154:Mutation; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry* / Q000502:physiology*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
D018076:DNA, Complementary; D019151:Peptide Library; D010455:Peptides; D010940:Plant Proteins; D011994:Recombinant Proteins; D016282:Aspartic Acid Endopeptidases; C097455:aspartic proteinases, Cynara cardunculus; D002402:Cathepsin D