Activation, Proteolytic Processing, and Peptide Specificity of Recombinant Cardosin A

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.Sequencing of the Arabidopsis genome unveiled over 50 genes coding for aspartic proteases of the pepsin type (1, 2). These genes have been assigned to three different categories: typical, nucellin-like, and atypical proteases (2). With the exception of the products of cnd41 (3-5) and cdr1 (6) genes, very little is known about the nucellin and the atypical subgroups. In fact, most of the knowledge acquired during the last decade relates to the typical subgroup of plant aspartic proteases and has been generated predominantly from the study of phytepsin from barley seeds and cardosins from the flowers of cardoon (7).Typical plant aspartic proteases constitute a set of enzymes that share many structural and functional features not only among them but also with some of their mammalian and microbial homologues. They display activity at acidic pH and are inhibited by pepstatin A, a natural hexapeptide from Streptomyces. Common features also include the overall three-dimensional structure of mature enzymes, the catalytic apparatus, and a conserved DT/SG motif. A distinguishing feature of typical plant aspartic proteases is the occurrence of an extra 100-amino acid-long internal segment, known as the plant-specific insert (PSI), in the sequence of their precursors. This domain displays structural and functional similarities to saposins (8, 9), sphingolipid-activating proteins from animals, and some antimicrobial peptides such as NK-lysin, granulysin, and amoebapores (10); it folds as an independent domain in the precursor form (11) and is subsequently excised to generate the mature two-chain form (7). Even though the function of PSI 1 is not yet fully elucidated, it seems to play an important role in targeting plant aspartic protease precursors to the vacuole (11,12).Little is known regarding the function of typical plant aspartic proteases. Colocalization studies with putative substrates and thei...

Processing and trafficking of a single isoform of the aspartic proteinase cardosin A on the vacuolar pathway

“…In fact, it was already described that for recombinant cardosin A to be active, all it takes is the partial removal of the prosegment (Castanheira et al 2005). Our results, obtained in vivo, diVer however to the ones described by Castanheira et al (2005) on the processing of recombinant procardosin A in vitro, that show the partial excision of the pro segment prior to the incomplete removal of the PSI. The authors suggest that complete maturation of cardosin A in vivo requires the action of additional proteases or exopeptidases (Castanheira et al 2005).…”

“…Nevertheless, the late removal of the prosegment in cardosin A, presumably upon arrival to vacuole, suggests that this region may also take part in an inactivation strategy in cardosin A. In fact, it was already described that for recombinant cardosin A to be active, all it takes is the partial removal of the prosegment (Castanheira et al 2005). Our results, obtained in vivo, diVer however to the ones described by Castanheira et al (2005) on the processing of recombinant procardosin A in vitro, that show the partial excision of the pro segment prior to the incomplete removal of the PSI.…”

Characterization of Recombinant CDR1, an Arabidopsis Aspartic Proteinase Involved in Disease Resistance

“…Both constructs, CDR1 wild-type (wtCDR1) and the mutant CDR1(D108A), were transformed into the E. coli BL21(DE3) strain. The method of recombinant protein purification was adapted from Castanheira et al (16). Briefly, after growth of the cells at 37°C to D 600 of 0.6, protein expression was induced by the addition of isopropyl 1-thio-␤-D-galactopyranoside (0.5 mM final concentration).…”

“…Rather unexpectedly, and in contrast to other aspartic proteinases, wtCDR1 cleaved the Leu 15 -Phe 16 bond of the insulin B chain in a selective manner at an optimal pH of 6.0. Under these conditions, addition of pepstatin A only partially inhibited this cleavage activity.…”

Recombinant Laforin for Structural Studies

“…As previously reported, laforin expression in E. coli is associated with the appearance of inclusion bodies (Girard et al, 2006), with the CBM behaving in a similar way . Our expertise in refolding proteins expressed in the form of inclusion bodies was crucial for the successful production of high amounts of correctly folded protein (Castanheira et al, 2005;Turner et al, 2001;Simões et al, 2007). After cell pellet disruption, the inclusion bodies were washed in 50 mM Tris-HCl, 50 mM NaCl, pH 7.4, followed by a second washing step in 50 mM Tris-HCl, 50 mM NaCl, pH 7.4, 0.1% Triton X-100 (v/v).…”