Activation, proteolytic processing, and peptide specificity of recombinant cardosin A

Pedro Castanheira, Bart Samyn, Kjell Sergeant, José C. Clemente, Ben M. Dunn, Euclides Pires, Jozef Van Beeumen, Carlos Faro

Research output: Contribution to journalArticlepeer-review

36 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)13047-13054
Number of pages8
JournalJournal of Biological Chemistry
Volume280
Issue number13
DOIs
StatePublished - 1 Apr 2005
Externally publishedYes

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