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.

Spotted fever group (SFG) rickettsiae are recognized as important agents of human tick-borne diseases worldwide, such as Mediterranean spotted fever (R. conorii) and Rocky Mountain spotted fever (R. rickettsii). Recent studies in several animal models have provided evidence of non-endothelial parasitism by pathogenic SFG Rickettsia species, suggesting that the interaction of rickettsiae with cells other than the endothelium may play an important role in pathogenesis of rickettsial diseases. These studies raise the hypothesis that the role of macrophages in rickettsial pathogenesis may have been underappreciated. Herein, we evaluated the ability of two SFG rickettsial species, R. conorii (a recognized human pathogen) and R. montanensis (a non-virulent member of SFG) to proliferate in THP-1 macrophage-like cells, or within non-phagocytic cell lines. Our results demonstrate that R. conorii was able to survive and proliferate in both phagocytic and epithelial cells in vitro. In contrast, R. montanensis was able to grow in non-phagocytic cells, but was drastically compromised in the ability to proliferate within both undifferentiated and PMA-differentiated THP-1 cells. Interestingly, association assays revealed that R. montanensis was defective in binding to THP-1-derived macrophages; however, the invasion of the bacteria that are able to adhere did not appear to be affected. We have also demonstrated that R. montanensis which entered into THP-1-derived macrophages were rapidly destroyed and partially co-localized with LAMP-2 and cathepsin D, two markers of lysosomal compartments. In contrast, R. conorii was present as intact bacteria and free in the cytoplasm in both cell types. These findings suggest that a phenotypic difference between a non-pathogenic and a pathogenic SFG member lies in their respective ability to proliferate in macrophage-like cells, and may provide an explanation as to why certain SFG rickettsial species are not associated with disease in mammals.

The Arabidopsis thaliana constitutive disease resistance 1 (CDR1) gene product is an aspartic proteinase that has been implicated in disease resistance signaling (Xia, Y., Suzuki, H., Borevitz, J., Blount, J., Guo, Z., Patel, K., Dixon, R. A., and Lamb, C. (2004) EMBO J. 23, 980–988). This apoplastic enzyme is a member of the group of “atypical” plant aspartic proteinases. As for other enzymes of this subtype, CDR1 has remained elusive until recently as a result of its unusual properties and localization. Here we report on the heterologous expression and characterization of recombinant CDR1, which displays unique enzymatic properties among plant aspartic proteinases. The highly restricted specificity requirements, insensitivity toward the typical aspartic proteinase inhibitor pepstatin A, an unusually high optimal pH of 6.0–6.5, proteinase activity without irreversible prosegment removal, and dependence of catalytic activity on formation of a homo-dimer are some of the unusual properties observed for recombinant CDR1. These findings unveil a pattern of unprecedented functional complexity for Arabidopsis CDR1 and are consistent with a highly specific and regulated biological function.

Members of the pepsin-like family (A1) of aspartic proteases (APs) are widely distributed in plants. A large number of genes encoding putative A1 APs are found in different plant genomes, the vast majority of which exhibit distinct features when compared with the so-called typical APs (and, therefore, grouped as atypical and nucellin-like APs). These features include the absence of the plant-specific insert; an unusually high number of cysteine residues; the nature of the amino acids preceding the first catalytic aspartate; and unexpected localizations. The over-representation of atypical and nucellin-like APs in plants is suggestive of greater diversification of protein functions and a more regulatory role for these APs, as compared with the housekeeping function generally attributed to typical APs. New functions have been uncovered for non-typical APs, with proposed roles in biotic and abiotic stress responses, chloroplast metabolism, and reproductive development, clearly suggesting functional specialization and tight regulation of activity. Furthermore, unusual enzymatic properties have also been documented for some of these proteases. Here, we give an overview of the current knowledge on the distinctive features and functions of both atypical and nucellin-like APs, and discuss this emerging pattern of functional complexity and specialization among plant pepsin-like proteases.