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.
Highlights
Aspartic proteinases [1] are widely distributed in the plant kingdom, and they have been purified and characterized from several species of monocotyledons, dicotyledons, and gymnosperms [2]
Molecular Cloning of Cardosin A cDNA and Characterization of the Deduced Amino Acid Sequence—In the first stage, a 0.8-kilobase internal segment of the cardosin A cDNA was PCR-amplified using degenerated primers encoding two amino acid sequences previously determined by protein sequencing [17]
Based upon the sequence of this fragment, whose nature was confirmed by comparison with the partial amino acid sequence of cardosin A, primers were designed to amplify a fulllength cDNA from a young flower bud cDNA Marathon library
Summary
Aspartic proteinases [1] are widely distributed in the plant kingdom, and they have been purified and characterized from several species of monocotyledons, dicotyledons, and gymnosperms [2]. As will be described a unique feature of cardosin A, among plant aspartic proteinases, is the presence of a functional RGD sequence. We described the cloning of cardosin A cDNA and the identification of a 100-kDa receptor from pollen that interacts with the region of the proteinase that contains an RGD cell attachment sequence.
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