Processing, Activity, and Inhibition of Recombinant Cyprosin, an Aspartic Proteinase from Cardoon (Cynara cardunculus)

White, P. Bruce; Cordeiro, M. C. R.; Arnold, Danièle; Brodelius, Peter E.; Kay, John

doi:10.1074/jbc.274.24.16685

Cited by 60 publications

(60 citation statements)

References 42 publications

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“…In vitro activation of recombinant procardosin A leads ultimately to the generation of an active form where the two polypeptide chains still remain associated by a disulfide bond. This has also been reported for cyprosin and the sunflower seed aspartic protease (31,32). The incomplete removal of the PSI described in this work and for cyprosin and sunflower seed aspartic protease suggests therefore that completion of in vivo maturation might require the action of other protease/exopeptidase(s).…”

Section: Discussionmentioning

confidence: 64%

Activation, Proteolytic Processing, and Peptide Specificity of Recombinant Cardosin A

Castanheira

Samyn

Sergeant

et al. 2005

Journal of Biological Chemistry

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Cardosins are model plant aspartic proteases, a group of proteases that are involved in cell death events associated with plant senescence and stress responses. They are synthesized as single-chain zymogens, and subsequent conversion into two-chain mature enzymes is a crucial step in the regulation of their activity. Here we describe the activation and proteolytic processing of recombinant procardosin A. The cleavage sites involved in this multi-step autocatalytic process were determined, some of them using a novel method for C-terminal sequence analysis. Even though the two-chain recombinant enzyme displayed similar properties as natural cardosin A, a single-chain mutant form was engineered based on the processing results and produced in Escherichia coli. Determination of its primary specificity using two combinatorial peptide libraries revealed that this mutant form behaved like the natural enzyme. The primary specificity of the enzyme closely resembles those of cathepsin D and plasmepsins, suggesting that cardosin A shares the same peptide scissile bond preferences of its vacuolar/lysosomal mammalian and protozoan homologues.Sequencing of the Arabidopsis genome unveiled over 50 genes coding for aspartic proteases of the pepsin type (1, 2). These genes have been assigned to three different categories: typical, nucellin-like, and atypical proteases (2). With the exception of the products of cnd41 (3-5) and cdr1 (6) genes, very little is known about the nucellin and the atypical subgroups. In fact, most of the knowledge acquired during the last decade relates to the typical subgroup of plant aspartic proteases and has been generated predominantly from the study of phytepsin from barley seeds and cardosins from the flowers of cardoon (7).Typical plant aspartic proteases constitute a set of enzymes that share many structural and functional features not only among them but also with some of their mammalian and microbial homologues. They display activity at acidic pH and are inhibited by pepstatin A, a natural hexapeptide from Streptomyces. Common features also include the overall three-dimensional structure of mature enzymes, the catalytic apparatus, and a conserved DT/SG motif. A distinguishing feature of typical plant aspartic proteases is the occurrence of an extra 100-amino acid-long internal segment, known as the plant-specific insert (PSI), in the sequence of their precursors. This domain displays structural and functional similarities to saposins (8, 9), sphingolipid-activating proteins from animals, and some antimicrobial peptides such as NK-lysin, granulysin, and amoebapores (10); it folds as an independent domain in the precursor form (11) and is subsequently excised to generate the mature two-chain form (7). Even though the function of PSI 1 is not yet fully elucidated, it seems to play an important role in targeting plant aspartic protease precursors to the vacuole (11,12).Little is known regarding the function of typical plant aspartic proteases. Colocalization studies with putative substrates and thei...

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Section: Discussionmentioning

confidence: 64%

Activation, Proteolytic Processing, and Peptide Specificity of Recombinant Cardosin A

Castanheira

Samyn

Sergeant

et al. 2005

Journal of Biological Chemistry

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“…Clade 3 is closely related to clade 4 which contains the plant APs. The latter are known to contain an additional sequence known as a plant-specific insert or saposin domain, involved in maturation of the active proteinase (White et al, 1999).…”

Section: Phylogeny Of Apsmentioning

confidence: 99%

An aspartic proteinase gene family in the filamentous fungus Botrytis cinerea contains members with novel features

et al. 2004

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Botrytis cinerea, an important fungal plant pathogen, secretes aspartic proteinase (AP) activity in axenic cultures. No cysteine, serine or metalloproteinase activity could be detected. Proteinase activity was higher in culture medium containing BSA or wheat germ extract, as compared to minimal medium. A proportion of the enzyme activity remained in the extracellular glucan sheath. AP was also the only type of proteinase activity in fluid obtained from B. cinerea-infected tissue of apple, pepper, tomato and zucchini. Five B. cinerea genes encoding an AP were cloned and denoted Bcap1–5. Features of the encoded proteins are discussed. BcAP1, especially, has novel characteristics. A phylogenetic analysis was performed comprising sequences originating from different kingdoms. BcAP1 and BcAP5 did not cluster in a bootstrap-supported clade. BcAP2 clusters with vacuolar APs. BcAP3 and BcAP4 cluster with secreted APs in a clade that also contains glycosylphosphatidylinositol-anchored proteinases from Saccharomyces cerevisiae and Candida albicans. All five Bcap genes are expressed in liquid cultures. Transcript levels of Bcap1, Bcap2, Bcap3 and Bcap4 are subject to glucose and peptone repression. Transcripts from all five Bcap genes were detected in infected plant tissue, indicating that at least part of the AP activity in planta originates from the pathogen.

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“…In contrast, peptide 1, at a concentration of 2 M, had no significant ability to inhibit any one of a number of other aspartic proteinases from a wide range of other species (Table III). These have considerable sequence and structural similarities to yeast proteinase A and included yapsin 1 (a membraneattached aspartic proteinase also from S. cerevisiae (15) and other enzymes of fungal, mammalian, parasite (plasmepsin II from Plasmodium falciparum (18) and plant (cyprosin from Cynara cardunculus (19)) origin. Thus, IA 3 is a potent specific inhibitor directed solely against its target enzyme, yeast proteinase A.…”

Section: Interaction Of Protein/peptide Forms Of Ia 3 With Target Andmentioning

confidence: 99%

“…Since cleavage at the mutated ϳVal 29 -Phe 30 ϳ bond was no longer an option, it was found by amino acid analysis that processing had taken place at the adjacent ϳPhe 30 *Lys 31 ϳ bond (data not shown). Similarly, peptides 12 and 13 (Table II) were digested after 72 h incubation with proteinase A at a molar ratio of 10:1, as were peptides 18,19,20, and 26 of the peptides listed in Table IV. Thus, in addition to the L19A mutant (peptide 27) as described above, the other peptides (7,8,12,13,14 (Table II), 18, 19, 20, and 26 (Table IV)) which were not effective as inhibitors of proteinase A, served instead as substrates for the enzyme.…”

Section: Ntd Qqkvs Eifqs Skeka Qgdak Vvsda Fkkmentioning

confidence: 99%

The Potency and Specificity of the Interaction between the IA3 Inhibitor and Its Target Aspartic Proteinase fromSaccharomyces cerevisiae

Phylip¹,

Lees²,

Brownsey³

et al. 2001

Journal of Biological Chemistry

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The yeast IA 3 polypeptide consists of only 68 residues, and the free inhibitor has little intrinsic secondary structure. IA 3 showed subnanomolar potency toward its target, proteinase A from Saccharomyces cerevisiae, and did not inhibit any of a large number of aspartic proteinases with similar sequences/structures from a wide variety of other species. Systematic truncation and mutagenesis of the IA 3 polypeptide revealed that the inhibitory activity is located in the N-terminal half of the sequence. Crystal structures of different forms of IA 3 complexed with proteinase A showed that residues in the N-terminal half of the IA 3 sequence became ordered and formed an almost perfect ␣-helix in the active site of the enzyme. This potent, specific interaction was directed primarily by hydrophobic interactions made by three key features in the inhibitory sequence. Whereas IA 3 was cut as a substrate by the nontarget aspartic proteinases, it was not cleaved by proteinase A. The random coil IA 3 polypeptide escapes cleavage by being stabilized in a helical conformation upon interaction with the active site of proteinase A. This results, paradoxically, in potent selective inhibition of the target enzyme.Aspartic proteinases participate in a variety of physiological processes, and the onset of pathological conditions such as hypertension, gastric ulcers, and neoplastic diseases may be related to changes in the levels of their activity. Members of this proteinase family, e.g. renin, pepsin, cathepsin D, and human immunodeficiency virus-proteinase are generally typecast on the basis of their susceptibility to inhibition by naturally occurring, small molecule inhibitors such as the acylated pentapeptides, isovaleryl-and acetyl-pepstatin. However, the two most recently identified human aspartic proteinases, ␤-site Alzheimer's precursor protein cleavage enzyme and ␤-site Alzheimer's precursor protein cleavage enzyme 2 (1, 2), are not inhibited by this classical type of inhibitor of this family of enzymes. Pepstatins are metabolic products produced by various species of actinomycetes and, as such, are not themselves gene-encoded. Protein inhibitors of aspartic proteinases are relatively uncommon and are found in only a few specialized locations (3). Examples include renin-binding protein in mammalian kidneys which intriguingly has now itself been identified to be the enzyme, N-acetyl-D-glucosamine-2-epimerase (4); a 17-kDa inhibitor of pepsin and cathepsin E from the parasite, Ascaris lumbricoides (5); proteins from plants such as potato, tomato, and squash (6, 7), and a pluripotent inhibitor from sea anemone of cysteine proteinases as well as cathepsin D (8).The IA 3 polypeptide in yeast is an 8-kDa inhibitor of the vacuolar aspartic proteinase (proteinase A or saccharopepsin) that was initially described by Holzer and co-workers (9). The complete sequence of this 68-residue inhibitor has been elucidated (10, 11) and the inhibitory activity of IA 3 has been shown to reside within the N-terminal half of the molecule (10, 12). ...

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Processing, Activity, and Inhibition of Recombinant Cyprosin, an Aspartic Proteinase from Cardoon (Cynara cardunculus)

Cited by 60 publications

References 42 publications

Activation, Proteolytic Processing, and Peptide Specificity of Recombinant Cardosin A

Activation, Proteolytic Processing, and Peptide Specificity of Recombinant Cardosin A

An aspartic proteinase gene family in the filamentous fungus Botrytis cinerea contains members with novel features

The Potency and Specificity of the Interaction between the IA3 Inhibitor and Its Target Aspartic Proteinase fromSaccharomyces cerevisiae

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