Two new aspartic proteinases have been isolated from stigmas of the cardoon Cyncrrn curdunculus L. by a two-step purificalion procedure including extraction at low pH, gel filtration on Superdex 200, and ion-exchange chromatography o n Mono Q. To follow the conventional nomenclature for aspartic proteinases, we have named these proteinases cardosin A and cardosin B. On SDS/PAGE, cardosin A migrated as two bands with apparent molecular masses of 31 000 Da and 15000 Da whercas the chains of cardosin B migrated as bands of 34000 Da and 14000 Da. The partial amino acid sequences of the two cardosins revealed that they are similar but not identical, and that they differ horn the previously reported cardoon proteinases named cynarases, which were assumed to be derived from a common precursor. Although thc cardosins show somc degree of similarity to each other, we could detect no immunological crossreactivity between theni. Both cardosins were active at low pH and were inhibited by pepslatin, with K, values of 3 nM for cardosin A and 1 nM for cardosin B, jndicaring that they belong to the class of aspartic proteinases. Significant differences between thc two enzymes were also found for thc K,,,,/k,,, values for the hydrolysis of two chromophoric synthetic peptides. The active-sitc ionization constants, pK,, and pKC2, for cardosin A are 2.5 -C 0.2 and 5.3 2 0.2, whci-eas for cardosin R they are 3.73 10.09 and 6.7 5 0.1. The results herein described on the structural and kinetic properties of the cardosins indicate that they are the products of distinct genes which havc probably arisen by gene duplication. A scheme for the proteolytic processing of the two enzymes is also proposed.Ke.ywordx: Cynaru curdunculus L. ; aspartyl proteinascs; milk-clotting enzymes; cardosins.Aspartic proteinases are a group of enzymes that share many features in terms of sequence, three-di rnensional structure and catalytic mechanism [I -31. They are widely distributed in nature and have important roles i n biological systems such as precursor protein processing (retroviral proteases), protein degradation (pepsin, cathepsin D and fungal proteases) and blood-pressure regulation (rcnin) (for reviews, see [3-51).Only a small number of aspartic proteinases have been isolated and partially characterised from plants [6-131. These proteinases, in common with most other aspartic proteinascs, havc an acid pH optimum, are inhibited by pepstatin and preferentially cleave pcptide bonds between hydrophobic residues. Little is known about their biological functions, but it has been suggested that plant aspartic proteinases rrre involved in the hydrolysis of storage and intracellular protcins 11 4-171. of about 100 amino acids which bears no sequence similarity with aspartic proteinnses of inamtnalian or microbial origins.The tlowers of cardoon (genus Cynnm) are traditionally used in Portugal for cheese making and their proteinases are among the few eiizymes from vegctal sources that have been used for this purpose. We have previously reported the isolation ...

The flowers ofcardoon (genus Qmara)are traditionally used in Portugal for cheese making. In this work the vegetable rennet of the species Cynam oan/uncu/us 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 Leul5-Tyrl6, Leul7-Vall8 and Phe2.5-Tyr26. In addition to the bonds mentioned cardosin B cleaves also Glul 3-Ala14, Alal4-Leul5 and Phe24-Phc25 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.

Pollen grains with distinct allergenic abilities release proteases that might be involved in the sensitization to a range of airborne allergens by facilitating allergen delivery across the epithelium and also contribute directly to the inflammation characteristic of allergic diseases.

Plant aspartic proteinases contain a plant-specific insert (PSI) of about 100 amino acids of unknown function with no similarity with the other aspartic proteinases but with significant similarity with saposins, animal sphingolipid activator proteins. PSI has remained elusive at the protein level, suggesting that it may be removed during processing. To understand the molecular relevance of PSI, the proteolytic processing of cardosin A, the major aspartic proteinase from the flowers of cardoon (Cynara cardunculus L.) was studied. Procardosin A, a 64-kDa cardosin A precursor containing PSI and the prosegment was identified by immunoblotting using monospecific antibodies against PSI and the prosegment. Procardosin A undergoes proteolytic processing as the flower matures. PSI was found to be removed before the prosegment, indicating that during processing the enzyme acquires a structure typical of mammalian or microbial aspartic proteinase proforms. In vitro studies showed that processing of PSI occurs at pH 3.0 and is inhibited by pepstatin A and at pH 7.0. Sequence analysis allowed the identification of the cleavage sites, revealing that PSI is removed entirely, probably by an aspartic proteinase. Cleavage of the PSI scissile bonds requires, however, a conformation specific to the precursor since isolated cardosins and pistil extracts were unable to hydrolyse synthetic peptides corresponding to the cleavage sites. In view of these results, a model for the proteolytic processing of cardosin A is proposed and the molecular and physiological relevance of PSI in plant aspartic proteinase is discussed.Keywords : aspartic protease ; milk-clotting enzyme; cardosin ; proteolytic processing; saposin.Aspartic proteinases (AP) are a widely distributed class of kingdom [5]. In the majority of plants AP are located in seeds, endoproteases that share significant similarities at the amino-whether quiescent or germinating. They are believed to particiacid-sequence level and at the structural level [1, 2]. The typical pate in storage-protein cleavage, which is necessary for germinacharacteristics of the family are an acidic pH optimum, inhibi-tion [5,10]. AP have also been found in leaves of some plants. tion by pepstatin A, preference for bonds between hydrophobic In leaves they have been implicated in mechanisms of defense amino acids, and sequence similarities, in particular the conser-against pathogens [11,12]. Species of the genus Cynara contain vation of the catalytic triads Asp-Thr-Gly or Asp-Ser-Gly [1Ϫ considerable amounts of AP in flowers. Recent data indicate that 4]. AP have been implicated in diverse physiological processes, the major AP from cardoon (Cynara cardunculus L.), cardosin such as digestion or blood-pressure regulation, and in some A, may be involved in the sexual reproduction of the plant [6] pathological conditions, including infection by fungi and retro-and may have a defensive role as well [13]. viruses or cancer [5, 6].A few primary structures deduced from the cDNAs of AP Like many other proteases, A...

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.