Characterization and expression analysis of the aspartic protease gene family of Cynara cardunculus L

Cardosin A and cardosin B are two aspartic proteases mainly found in the pistils of cardoon Cynara cardunculus L., whose flowers are traditionally used in several Mediterranean countries in the manufacture of ewe's cheese. We have been characterizing cardosins at the biochemical, structural and molecular levels. In this study, we show that the cardoon aspartic proteases are encoded by a multigene family. The genes for cardosin A and cardosin B, as well as those for two new cardoon aspartic proteases, designated cardosin C and cardosin D, were characterized, and their expression in C. cardunculus L. was analyzed by RT-PCR. Together with cardosins, a partial clone of the cyprosin B gene was isolated, revealing that cardosin and cyprosin genes coexist in the genome of the same plant. As a first approach to understanding what dictates the flower-specific pattern of cardosin genes, the respective gene 5' regulatory sequences were fused with the reporter beta-glucuronidase and introduced into Arabidopsis thaliana. A subsequent deletion analysis of the promoter region of the cardosin A gene allowed the identification of a region of approximately 500 bp essential for gene expression in transgenic flowers. Additionally, the relevance of the leader intron of the cardosin A and B genes for gene expression was evaluated. Our data showed that the leader intron is essential for cardosin B gene expression in A. thaliana. In silico analysis revealed the presence of potential regulatory motifs that lay within the aforementioned regions and therefore might be important in the regulation of cardosin expression.     

Multiplicity of aspartic proteinases from Cynara cardunculus L.

Aspartic proteinases (AP) play major roles in physiologic and pathologic scenarios in a wide range of organisms from vertebrates to plants or viruses. The present work deals with the purification and characterisation of four new APs from the cardoon Cynara cardunculus L., bringing the number of APs that have been isolated, purified and biochemically characterised from this organism to nine. This is, to our knowledge, one of the highest number of APs purified from a single organism, consistent with a specific and important biological function of these protein within C. cardunculus. These enzymes, cardosins E, F, G and H, are dimeric, glycosylated, pepstatin-sensitive APs, active at acidic pH, with a maximum activity around pH 4.3. Their primary structures were partially determined by N- and C-terminal sequence analysis, peptide mass fingerprint analysis on a MALDI-TOF/TOF instrument and by LC–MS/MS analysis on a Q-TRAP instrument. All four enzymes are present on C. cardunculus L. pistils, along with cyprosins and cardosins A and B. Their micro-heterogeneity was detected by 2D-electrophoresis and mass spectrometry. The enzymes resemble cardosin A more than they resemble cardosin B or cyprosin, with cardosin E and cardosin G being more active than cardosin A, towards the synthetic peptide KPAEFF(NO₂)AL. The specificity of these enzymes was investigated and it is shown that cardosin E, although closely related to cardosin A, exhibits different specificity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Processing and trafficking of a single isoform of the aspartic proteinase cardosin A on the vacuolar pathway

Cardosin A is the major vacuolar aspartic proteinase (APs) (E.C.3.4.23) in pistils of Cynara cardunculus L. (cardoon). Plant APs carry a unique domain, the plant-specific-insert (PSI), and a pro-segment which are separated from the catalytic domains during maturation but the sequence and location of processing steps for cardosins have not been established. Here transient expression in tobacco and inducible expression in Arabidopsis indicate that processing of cardosin A is conserved in heterologous species. Pulse chase analysis in tobacco protoplasts indicated that cleavage at the carboxy-terminus of the PSI could generate a short-lived 50 kDa intermediate which was converted to a more stable 35 kDa intermediate by removal of the PSI. Processing intermediates detected immunologically in tobacco leaves and Arabidopsis seedlings confirmed that cleavage at the amino-terminus of the PSI either preceded or followed quickly after cleavage at its carboxy-terminus. Thus removal of PSI preceded the loss of the prosegment in contrast to the well-characterised barley AP, phytepsin. PreprocardosinA acquired a complex glycan and its processing was inhibited by brefeldin A and dominant-inhibitory AtSAR1 or AtRAB-D2^a mutants indicating that it was transported via the Golgi and that processing followed ER export. The 35 kDa intermediate was present in the cell wall and protoplast culture medium as well as the vacuole but the 31 kDa mature subunit, lacking the amino-terminal prosegment, was detected only in the vacuole. Thus maturation appears to occur only after sorting from the trans-Golgi to the vacuole. Processing or transport of cardosin A was apparently slower in tobacco protoplasts than in whole cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cardosins in postembryonic development of cardoon: towards an elucidation of the biological function of plant aspartic proteinases

Summary. Following on from previous work, the temporal and spatial accumulation of the aspartic proteinases (EC 3.4.23) cardosin A and cardosin B during postembryonic seed development of cardoon (Cynara cardunculus) was studied. mRNA and protein analyses of both cardosins suggested that the proteins accumulate during seed maturation, and that cardosin A is later synthesised de novo at the time of radicle emergence. Immunocytochemistry revealed that the precursor form of cardosin A accumulates in protein bodies and cell walls. This localisation in seeds is different from that previously described for cardoon flowers, suggesting a tissue-dependent targeting of the protein. It is known that procardosins are active and may have a role in proteolysis and processing of storage proteins. However, the presence of procardosin A in seeds could be related to the proposed role of the plant-specific insert in membrane lipid conversion during water uptake and solute leakage in actively growing tissues. This is in accordance with the recently proposed bifunctional role of aspartic proteinase precursor molecules that possess a membrane-destabilising domain in addition to a protease domain. Mature cardosin B, but not its mRNA, was detected in the first hours after seed imbibition and disappeared at the time of radicle emergence. This extracellular aspartic protease has already been implicated in cell wall loosening and remodelling, and its role in seed germination could be related to loosening tissue constraints for radicle protusion. The described pattern of cardosin A and B expression suggests a finely tuned developmental regulation and prompts an analysis of their possible roles in the physiology of postembryonic development. Correspondence: C. S. Pereira, Institute for Molecular and Cell Biology, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.     

Cardosin A contains two vacuolar sorting signals using different vacuolar routes in tobacco epidermal cells

Several vacuolar sorting determinants (VSDs) have been described for protein trafficking to the vacuoles in plant cells. Because of the variety in plant models, cell types and experimental approaches used to decipher vacuolar targeting processes, it is not clear whether the three well‐known groups of VSDs identified so far exhaust all the targeting mechanisms, nor if they reflect certain protein types or families. The vacuolar targeting mechanisms of the aspartic proteinases family, for instance, are not yet fully understood. In previous studies, cardosin A has proven to be a good reporter for studying the vacuolar sorting of aspartic proteinases. We therefore propose to explore the roles of two different cardosin A domains, common to several aspartic proteinases [i.e. the plant‐specific insert (PSI) and the C–terminal peptide VGFAEAA] in vacuolar sorting. Several truncated versions of the protein conjugated with fluorescent protein were made, with and without these putative sorting determinants. These domains were also tested independently, for their ability to sort other proteins, rather than cardosin A, to the vacuole. Fluorescent chimaeras were tracked in vivo, by confocal laser scanning microscopy, in Nicotiana tabacum cells. Results demonstrate that either the PSI or the C terminal was necessary and sufficient to direct fluorescent proteins to the vacuole, confirming that they are indeed vacuolar sorting determinants. Further analysis using blockage experiments of the secretory pathway revealed that these two VSDs mediate two different trafficking pathways.     

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.     

Cardosin A endocytosis mediated by integrin leads to lysosome leakage and apoptosis of epithelial cells

Cardosin A is an aspartic protease present in large amount in the pistils of cardoon flowers. This protease is known to contain an -Arg-Gly-Asp- (RGD) motif located on the molecular surface. In this study, we found that isolated recombinant cardosin A attached to human epithelial cells A549, mediated by the binding of its RGD motif to cell surface integrins. The cell bound cardosin A was internalized to endosomes and lysosomes and triggered the permeability of lysosomal membrane leading to apoptosis of the epithelial cells. These events are identical to those observed for three RGD-containing aspartic proteases, Saps 4-6, secreted by Candida albicans. Such a process, which has been called the Trojan Horse mechanism, is believed to benefit the invasion of C. albican into the epithelium of the host. The location of the RGD motifs of cardosin A and Saps 4-6 are on the opposite ends of the homologous three-dimensional structures, suggesting that the Trojan Horse mechanism is insensitive to the RGD position. Current finding also suggests that cardosin A may have a defensive function against the ingestion of cardoon flowers by human, insects, and other herbivores.     

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.     

GFS9/TT9 contributes to intracellular membrane trafficking and flavonoid accumulation in Arabidopsis thaliana

Flavonoids are the most important pigments for the coloration of flowers and seeds. In plant cells, flavonoids are synthesized by a multi‐enzyme complex located on the cytosolic surface of the endoplasmic reticulum, and they accumulate in vacuoles. Two non‐exclusive pathways have been proposed to mediate flavonoid transport to vacuoles: the membrane transporter‐mediated pathway and the vesicle trafficking‐mediated pathway. No molecules involved in the vesicle trafficking‐mediated pathway have been identified, however. Here, we show that a membrane trafficking factor, GFS9, has a role in flavonoid accumulation in the vacuole. We screened a library of Arabidopsis thaliana mutants with defects in vesicle trafficking, and isolated the gfs9 mutant with abnormal pale tan‐colored seeds caused by low flavonoid accumulation levels. gfs9 is allelic to the unidentified transparent testa mutant tt9. The responsible gene for these phenotypes encodes a previously uncharacterized protein containing a region that is conserved among eukaryotes. GFS9 is a peripheral membrane protein localized at the Golgi apparatus. GFS9 deficiency causes several membrane trafficking defects, including the mis‐sorting of vacuolar proteins, vacuole fragmentation, the aggregation of enlarged vesicles, and the proliferation of autophagosome‐like structures. These results suggest that GFS9 is required for vacuolar development through membrane fusion at vacuoles. Our findings introduce a concept that plants use GFS9‐mediated membrane trafficking machinery for delivery of not only proteins but also phytochemicals, such as flavonoids, to vacuoles.     

N‐linked glycosylation of AtVSR1 is important for vacuolar protein sorting in Arabidopsis

Vacuolar sorting receptors (VSRs) in Arabidopsis mediate the sorting of soluble proteins to vacuoles in the secretory pathway. The VSRs are post‐translationally modified by the attachment of N‐glycans, but the functional significance of such a modification remains unknown. Here we have studied the role(s) of glycosylation in the stability, trafficking and vacuolar protein transport of AtVSR1 in Arabidopsis protoplasts. AtVSR1 harbors three complex‐type N‐glycans, which are located in the N‐terminal ‘PA domain’, the central region and the C‐terminal epidermal growth factor repeat domain, respectively. We have demonstrated that: (i) the N‐glycans do not affect the targeting of AtVSR1 to pre‐vacuolar compartments (PVCs) and its vacuolar degradation; and (ii) N‐glycosylation alters the binding affinity of AtVSR1 to cargo proteins and affects the transport of cargo into the vacuole. Hence, N‐glycosylation of AtVSR1 plays a critical role in its function as a VSR in plants.     

Trafficking and localisation of resident Golgi glycosylation enzymes

The localisation of glycosylation enzymes within the Golgi apparatus is fundamental to the regulation of glycoprotein and glycolipid biosynthesis. Regions responsible for specifying Golgi localisation have been identified in numerous Golgi resident enzymes. The transmembrane domain of Golgi glycosyltransferases provides a dominant localisation signal and in many cases there are also major contributions from the lumenal domain. The mechanism by which these targeting domains function in maintaining an asymmetric distribution of Golgi resident glycosylation enzymes has been intensely debated in recent years. It is now clear that the targeting of Golgi resident enzymes is intimately associated with the organisation of Golgi membranes and the control of protein and lipid traffic in both anterograde and retrograde directions. Here we discuss the recent advances into how Golgi targeting signals of glycosylation enzymes function, and propose a model for maintaining the steady-state localisation of Golgi glycosyltransferases.     

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

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. Received: 10 December 1996 / Accepted: 14 March 1997     

pH-dependent cargo sorting from the Golgi

The vacuolar proton-translocating ATPase (V-ATPase) plays a major role in organelle acidification and works together with other ion transporters to maintain pH homeostasis in eukaryotic cells. We analyzed a requirement for V-ATPase activity in protein trafficking in the yeast secretory pathway. Deficiency of V-ATPase activity caused by subunit deletion or glucose deprivation results in missorting of newly synthesized plasma membrane proteins Pma1 and Can1 directly from the Golgi to the vacuole. Vacuolar mislocalization of Pma1 is dependent on Gga adaptors although no Pma1 ubiquitination was detected. Proper cell surface targeting of Pma1 was rescued in V-ATPase-deficient cells by increasing the pH of the medium, suggesting that missorting is the result of aberrant cytosolic pH. In addition to mislocalization of the plasma membrane proteins, Golgi membrane proteins Kex2 and Vrg4 are also missorted to the vacuole upon loss of V-ATPase activity. Because the missorted cargos have distinct trafficking routes, we suggest a pH dependence for multiple cargo sorting events at the Golgi.     

Toxoplasma gondii resides in a vacuole that avoids fusion with host cell endocytic and exocytic vesicular trafficking pathways.

Toxoplasma gondii actively penetrates its vertebrate host cell to establish a nonfusigenic compartment called the parasitophorous vacuole (PV) that has previously been characterized primarily in phagocytic cells. To determine the fate of this unique compartment in nonphagocytic cells, we examined the trafficking of host cell proteins and lipids in Toxoplasma-infected fibroblasts using quantitative immunofluorescence and immunoelectron microscopy. Toxoplasma-containing vacuoles remained segregated from all levels of the endocytic pathway, as shown by the absence of delivery of transferrin receptors, mannose phosphate receptors, and the lysosomal-associated protein LAMP1 to the vacuole. The PV was also inaccessible to lipids (DiIC16, and GM1) that were internalized from the plasma membrane via the endocytic system. In contrast, vacuoles containing dead parasites or zymosan sequentially acquired both endosomal and lysosomal protein markers and host lipids, reflecting the competency of fibroblasts to process phagocytic vacuoles. The mature PV often lies adjacent to the host cell Golgi, suggesting that it may intersect with vesicles from the exocytic pathway. Despite this proximity, the PV was inaccessible to nitrobenzadiazole-labeled sphingolipids exported from the Golgi and did not contain the host protein markers AP1 or beta-COP. Our results demonstrate that Toxoplasma resides in a compartment that excludes delivery of protein and lipid components from the host cell endocytic and exocytic pathways.     

Allocation of proteolytic activity in the seedling of Cynara cardunculus L. in the initial growth stages

Cynara cardunculus L. seeds were germinated in vitro under environmentally controlled conditions. Seeds showed a 60% germination rate, and three growth stages were established based on the seedling mean relative growth rate (RGR). Root, stem and cotyledons were compared in these stages with respect to the emergence of total proteases and cardosin activity and its allocation in the seedling. In growth stage I (1st-5th post-germinative days), seedlings grew very slowly. Total proteases and cardosins were already active at the onset of seedlings in the stem. Total soluble protein remained constant in cardoon seedlings during stage I, and the content of all free amino acids (aa) but proline (Pro) was equally allocated on the 1st post-germinative day. In growth stage II (5th-10th post-germinative days), seedlings grew intensively and exhibited fully developed cotyledons. A pronounced increase in the content of all free aa up to the middle of growth stage II in both stems and roots was observed. In addition, the allocation of the total proteolytic activity and cardosins followed a gradient from the root to the seedling shoot. However, the whole seedling soluble protein remained constant up to the 7th day in and tended to peak on the 10th post-germinative day, being allocated mainly to the seedling stem. In growth stage III (10th-15th post-germinative days), cardoon seedlings exhibited the lowest mean RGR and the highest R/S growth ratio. An intensive degradation of total soluble protein present in the whole seedling except for cotyledons (ca. 5-fold) was observed. Nevertheless, in growth stage III, both the gradients exhibited by total proteases and cardosins activities between the root and the seedling shoot were enhanced, as were contents of all aa except Pro, exhibiting the highest levels in cotyledons on the 15th post-germinative day.     

The roles of enzyme localisation and complex formation in glycan assembly within the Golgi apparatus

Cell surface glycans govern numerous cell-cell interactions are therefore key determinants of multicellular biology. They originate from biosynthetic pathways comprising an assembly line of glycosyltransferases within the Golgi compartment. Although the mechanisms of Golgi enzyme localisation are still under debate, the distribution of these enzymes among the Golgi cisternae can dictate the overall structures produced by the cell. Fine-tuning of glycan biosynthetic pathways is further accomplished by specific associations among glycosyltransferases. Together, localisation and association govern the assembly of complex glycans and thereby regulate interactions at the cell surface.     

Identification of the protein storage vacuole and protein targeting to the vacuole in leaf cells of three plant species

Protein storage vacuoles (PSVs) are specialized vacuoles devoted to the accumulation of large amounts of protein in the storage tissues of plants. In this study, we investigated the presence of the storage vacuole and protein trafficking to the compartment in cells of tobacco (Nicotiana tabacum), common bean (Phaseolus vulgaris), and Arabidopsis leaf tissue. When we expressed phaseolin, the major storage protein of common bean, or an epitope-tagged version of alpha-tonoplast intrinsic protein (alpha-TIP, a tonoplast aquaporin of PSV), in protoplasts derived from leaf tissues, these proteins were targeted to a compartment ranging in size from 2 to 5 microm in all three plant species. Most Arabidopsis leaf cells have one of these organelles. In contrast, from one to five these organelles occurred in bean and tobacco leaf cells. Also, endogenous alpha-TIP is localized in a similar compartment in untransformed leaf cells of common bean and is colocalized with transiently expressed epitope-tagged alpha-TIP. In Arabidopsis, phaseolin contained N-glycans modified by Golgi enzymes and its traffic was sensitive to brefeldin A. However, trafficking of alpha-TIP was insensitive to brefeldin A treatment and was not affected by the dominant-negative mutant of AtRab1. In addition, a modified alpha-TIP with an insertion of an N-glycosylation site has the endoplasmic reticulum-type glycans. Finally, the early step of phaseolin traffic, from the endoplasmic reticulum to the Golgi complex, required the activity of the small GTPase Sar1p, a key component of coat protein complex II-coated vesicles, independent of the presence of the vacuolar sorting signal in phaseolin. Based on these results, we propose that the proteins we analyzed are targeted to the PSV or equivalent organelle in leaf cells and that proteins can be transported to the PSV by two different pathways, the Golgi-dependent and Golgi-independent pathways, depending on the individual cargo proteins.     

The embryo sac of Cynara cardunculus: ultrastructure of the development and localisation of the aspartic proteinase cardosin B

Cynara cardunculus is a native plant with flowers that are used traditionally in the manufacture of ewe’s cheese in the Iberian Peninsula. Milk clotting ability of the plant is attributed to the high concentrations of aspartic proteinases (APs), named cardosins, found in the flowers. Although these enzymes are well characterised on a molecular and biochemical basis, the biological role of the majority of plant APs is yet unassigned. We suspected APs play an important role in ovule function, and we characterised the maturation of the ovules of C. cardunculus and its Polygonum-type embryo sacs. The internal layer of the integument differentiates into an endothelium as described for other Asteraceae, with differentiation of two nucellar layers, a podium and a hypostase coinciding with the onset of pollen receptivity. In flowering plants, programmed cell death (PCD) events are essential for the success of nucellar maturation and consequent differentiation of a fully functional embryo sac. In C. cardunculus, nucellar PCD is integral to the maturation of the embryo sac, which in turn is closely correlated with the accumulation of the AP cardosin B specifically in the hypostase. The onset of cardosin B expression temporally coincides with the degeneration of nucellar cells. In fully mature embryo sacs, cardosin B is localised in both the hypostase and epistase, two regions that differentiate through PCD. Thus, cardosin B localisations closely correlate with events of PCD in the nucellus of C. cardunculus suggesting involvement in ovule and embryo sac development and further suggest the biological significance of APs like cardosin B, in this particular process. This work contributes new data to the plant AP research field and indicates an involvement of cardosin B in the PCD-dependent degeneration of the nucellus.