Carlos Faro

The vegetable rennet of Cynara cardunculus L. contains two proteinases with chymosin and pepsin-like specificities

The flowers of cardoon (genus Cynara) are traditionally used in Portugal for cheese making. In this work the vegetable rennet of the species Cynara cardunculus L. was characterized in terms of enzymic composition and proteolytic specificity of its proteinases (cardosin A and cardosin B). Cardosin A was found to cleave insulin B chain at the bonds Leu15-Tyr16, Leu17-Val18 and Phe25-Tyr26. In addition to the bonds mentioned cardosin B cleaves also Glu13-Ala14, Ala14-Leu15 and Phe24-Phe25 indicating that it has a broader specificity. The kinetic parameters for the hydrolysis of the synthetic peptide Leu-Ser-Phe(NO2)-Nle-Ala-Leu-oMe were also determined and compared to those of chymosin and pepsin. The results obtained indicate that in terms of specificity and kinetic parameters cardosin A is similar to chymosin whereas cardosin B is similar to pepsin. It appears therefore that the enzyme composition of cardoon rennet closely resembles that of calf rennet.

Crystal Structure of Cardosin A, a Glycosylated and Arg-Gly-Asp-containing Aspartic Proteinase from the Flowers of Cynara cardunculus L.*

Aspartic proteinases (AP) have been widely studied within the living world, but so far no plant AP have been structurally characterized. The refined cardosin A crystallographic structure includes two molecules, built up by two glycosylated peptide chains (31 and 15 kDa each). The fold of cardosin A is typical within the AP family. The glycosyl content is described by 19 sugar rings attached to Asn-67 and Asn-257. They are localized on the molecular surface away from the conserved active site and show a new glycan of the plant complex type. A hydrogen bond between Gln-126 and Manβ4 renders the monosaccharide oxygen O-2 sterically inaccessible to accept a xylosyl residue, therefore explaining the new type of the identified plant glycan. The Arg-Gly-Asp sequence, which has been shown to be involved in recognition of a putative cardosin A receptor, was found in a loop between two β-strands on the molecular surface opposite the active site cleft. Based on the crystal structure, a possible mechanism whereby cardosin A might be orientated at the cell surface of the style to interact with its putative receptor from pollen is proposed. The biological implications of these findings are also discussed.

Cardosin A, an abundant aspartic proteinase, accumulates in protein storage vacuoles in the stigmatic papillae of Cynara cardunculus L.

Abstract. The function of aspartic proteinases (EC 3.4.23) present in flowers of Cynara species is still unknown. Cardosin A, as a highly abundant aspartic proteinase from Cynara cardunculus L., a relative of the artichoke, is synthesised as a zymogen and subsequently undergoes proteolytic processing, yielding the mature and active enzyme. Here we report the study of the expression and localization of cardosin A, as a first approach to address the question of its physiological relevance. A polyclonal antibody specific for cardosin A was raised against a synthetic peptide corresponding to an amino acid sequence of the enzyme. This antibody was used to study the organ-specific, tissue-specific and subcellular localization of cardosin A by immunoblotting, tissue printing and immunogold electron microscopy. The results showed that expression of cardosin A is highly restricted to the pistils, and that the enzyme accumulates mainly in protein storage vacuoles of the stigmatic papillae. Cardosin A is also present, although much less abundantly, in the vacuoles of the cells of the epidermis of the style. In view of these results, the possible physiological roles of cardosin A are discussed, namely an involvement in defense mechanisms or pollen-pistil interaction, as well as in flower senescence.

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

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.

Specificity of a milk clotting enzyme extracted from the thistleCynara cardunculus L.: Action on oxidised insulin and k-casein

SummaryK-casein and oxidised insulin were digested with an acid protease extracted fromCynara cardunculus L. The fragments produced were isolated and characterised. In k-casein cleavage occured specifically at Phe 105-Met 106 bond. In oxidised insulin seven fragments were obtained and cleavage was found to occur at the carboxylic side of (Phe, Leu, He)-X, where X was preferentially Val or Tyr. The results obtained with insulin B chain suggest thatCynara cardunculus L. protease possesses a greater specificity than other acid proteases reported.

Molecular cloning and characterization of cDNA encoding cardosin B, an aspartic proteinase accumulating extracellularly in the transmitting tissue of Cynara cardunculus L.

Cardosins A and B are related aspartic proteinases from the pistils of Cynara cardunculus L., whose milk-clotting activity has been exploited for the manufacture of cheese. Here we report the cloning of cardosin B cDNA and its organ, tissue and cytological localization. The cDNA-derived amino acid sequence has 73% similarity with that of cardosin A and displays several distinguishing features. Cardosin B mRNA was detected in young inflorescences but not in pistils of fully opened inflorescences, indicating that its expression is developmentally regulated. The proteinase, however, accumulates in the pistil until the later stages of floral development. Immunocytochemistry with a monospecific antibody localized cardosin B to the cell wall and extracellular matrix of the floral transmitting tissue. The location of cardosin B in the pistil is therefore clearly different from that of cardosin A, which was found at protein storage vacuoles of the stigmatic papillae and has been suggested to be involved in RGD-mediated proteolytic mechanisms. In view of these results the possible functions of cardosin B in the transmitting tissue are discussed.

Action on bovine αs1-casein of cardosins A and B, aspartic proteinases from the flowers of the cardoon Cynara cardunculus L.

The cleavage of purified bovine alpha s1-casein separately by cardosin A and cardosin B, two distinct milk-clotting aspartic proteinases (APs) present in the stigmas of the plant Cynara cardunculus L., was studied. Casein digestion peptides were separated either by SDS-PAGE or by reverse-phase HPLC, and their N-terminal amino acid sequences were subsequently determined by automated Edman degradation, thus identifying the cleavage sites. Results showed that both enzymes exert a similar but distinct action on bovine alpha s1-casein. In common they have the preference for the bond Phe23-Phe24, and the cleavage of Trp164-Tyr165 and Phe153-Tyr154. Cardosin A also cleaves the bond Tyr165-Tyr166, whereas Cardosin B cleaves an extra type of bond, Phe150-Arg151, revealing a slightly broader specificity. A model for the action of both enzymes on bovine alpha s1-casein is proposed and discussed. In comparison with the reported action of chymosin on bovine alpha s1-casein, both cardosins proved to have a broader specificity towards this particular substrate due to a higher ability to cleave bonds between residues with large hydrophobic side-chains.

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

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.

A new method for C-terminal sequence analysis in the proteomic era

The overall study of post-translational modifications (PTMs) of proteins is gaining strong interest. Beside phosphorylation and glycosylation, truncations of the nascent polypeptide chain at the amino or carboxy terminus are by far the most common types of PTMs in proteins. In contrast to the analysis of phosphorylation and glycosylation sites, relatively little attention has been paid to the development of approaches for the systematic analysis of proteolytic processing events. Here we present a new mass spectrometry (MS)-based strategy that allows the identification of the C-terminal sequence of proteins. The method can be directly applied to proteins cleaved with cyanogen bromide (CNBr) and purified either by SDS-PAGE, by two-dimensional (2D) PAGE or in solution, and it therefore eliminates the need for specific isolation of the C-terminal peptide. Using Shewanella oneidensis as a model system, we have demonstrated that this approach can be used for C-terminal sequence analysis at a proteomic scale. We also applied the method to study the C-terminal proteolytic processing of procardosin A.

Molecular analysis of the interaction between cardosin A and phospholipase Dα

Cardosin A is an RGD‐containing aspartic proteinase from the stigmatic papillae of Cynara cardunculus L. A putative cardosin A‐binding protein has previously been isolated from pollen suggesting its potential involvement in pollen–pistil interaction [Faro C, Ramalho‐Santos M, Vieira M, Mendes A, Simões I, Andrade R, Verissimo P, Lin X, Tang J & Pires E (1999) J Biol Chem274, 28724–28729]. Here we report the identification of phospholipase Dα as a cardosin A‐binding protein. The interaction was confirmed by coimmunoprecipitation studies and pull‐down assays. To investigate the structural and molecular determinants involved in the interaction, pull‐down assays with cardosin A and various glutathione S‐transferase‐fused phospholipase Dα constructs were performed. Results revealed that the C2 domain of phospholipase Dα contains the cardosin A‐binding activity. Further assays with mutated recombinant forms of cardosin A showed that the RGD motif as well as the unprecedented KGE motif, which is structurally and charge‐wise very similar to RGD, are indispensable for the interaction. Taken together our results indicate that the C2 domain of plant phospholipase Dα can act as a cardosin A‐binding domain and suggest that plant C2 domains may have an additional role as RGD/KGE‐recognition domains.