Cloning and Characterization of cDNA Encoding Cardosin A, an RGD-containing Plant Aspartic Proteinase

Abstract

Cardosin A is an abundant aspartic proteinase from pistils of Cynara cardunculus L. whose milk-clotting activity has been exploited for the manufacture of cheese. Here we report the cloning and characterization of cardosin A cDNA. The deduced amino acid sequence contains the conserved features of plant aspartic proteinases, including the plant-specific insertion (PSI), and revealed the presence of an Arg-Gly-Asp (RGD) motif, which is known to function in cell surface receptor binding by extracellular proteins. Cardosin A mRNA was detected predominantly in young flower buds but not in mature or senescent pistils, suggesting that its expression is likely to be developmentally regulated. Procardosin A, the single chain precursor, was found associated with microsomal membranes of flower buds, whereas the active two-chain enzyme generated upon removal of PSI is soluble. This result implies a role for PSI in promoting the association of plant aspartic proteinase precursors to cell membranes. To get further insights about cardosin A, the functional relevance of the RGD motif was also investigated. A 100-kDa protein that interacts specifically with the RGD sequence was isolated from octyl glucoside pollen extracts by affinity chromatography on cardosin A-Sepharose. This result suggests that the 100-kDa protein is a cardosin A receptor and indicates that the interaction between these two proteins is apparently mediated through RGD recognition. It is possible therefore that cardosin A may have a role in adhesion-mediated proteolytic mechanisms involved in pollen recognition and growth.

High affinity recognition of a Phytophthora protein by Arabidopsis via an RGD motif

“…Finally, the 80 kDa plasma membrane protein is an RGD binding protein capable of strong selectivity towards the RGD sequence. Only few RGD dependent protein-protein interactions have been described in plants [27][28], and their involvement in cell wall-membrane attachments remains to be elucidated. In contrast, the wall-associated kinases (WAKs) are bound to both the plasma membrane and the extracellular matrix [29], but these links are not mediated by an RGD sequence.…”

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

“…The expression vector pET-23a (Novagen, Madison, WI) containing the procardosin A cDNA (pET_pCAϩPSI) has been previously constructed in our lab (14), as well as the PSI deleted procardosin A construct (pET_pCA⌬PSI) (8). The Escherichia coli BL21(DE3) strain was pur-* The costs of publication of this article were defrayed in part by the payment of page charges.…”

“…Upon incubation with carboxypeptidases, both fragments (m/z 1233.68 and 1396.73) started to shift and formed the same ladder (VS(R/Y)). This indicated that both fragments were the same C-terminal peptide with a ragged C terminus R/Y (⌬m ϭ 163.06 Da) being produced by cleavage of the peptide bonds at Tyr 13 -Gly 14 and Arg…”

“…In the particular case of cardosin A, the major aspartic protease from the flowers of cardoon, gene expression occurs specifically at the early stages of flower development, and the protein accumulates in protein storage vacuoles until the later stages of flower senescence (13). As the flower matures, the single-chain precursor form (procardosin A) is converted into a two-chain mature cardosin A by removal of the internal PSI domain and the N-terminal propeptide (14). This event is a crucial step in regulating the activity of cardosin A.…”

Identification of proteins from wild cardoon flowers (Cynara cardunculus L.) by a proteomic approach

“…Cardosin A is a glycosylated aspartic protease (AP) that has been studied in detail and was shown to be similar to chymosin, in terms of kinetic parameters and specificity, by cleaving the same peptide bond (Phe105-Met106) of the casein kappa [7,8]. The most abundant proteolytic activity corresponds to that of cardosin A [9] and at acidic pH, it represents from 75 to 90 % of total extracted enzyme activity [8]. The second aspartic protease studied was cardosin B, which is similar to pepsin, in terms of activity and specificity [7].…”