Structure and function of plant aspartic proteinases.

Aspartic proteinases of the A1 family are widely distributed among plant species and have been purified from a variety of tissues. They are most active at acidic pH, are specifically inhibited by pepstatin A and contain two aspartic residues indispensible for catalytic activity. The three-dimensional structure of two plant aspartic proteinases has been determined, sharing significant structural similarity with other known structures of mammalian aspartic proteinases. With a few exceptions, the majority of plant aspartic proteinases identified so far are synthesized with a prepro-domain and subsequently converted to mature two-chain enzymes. A characteristic feature of the majority of plant aspartic proteinase precursors is the presence of an extra protein domain of about 100 amino acids known as the plant-specific insert, which is highly similar both in sequence and structure to saposin-like proteins. This insert is usually removed during processing and is absent from the mature form of the enzyme. Its functions are still unclear but a role in the vacuolar targeting of the precursors has been proposed. The biological role of plant aspartic proteinases is also not completely established. Nevertheless, their involvement in protein processing or degradation under different conditions and in different stages of plant development suggests some functional specialization. Based on the recent findings on the diversity of A1 family members in Arabidopsis thaliana, new questions concerning novel structure-function relationships among plant aspartic proteinases are now starting to be addressed.     

Chlapsin, a chloroplastidial aspartic proteinase from the green algae Chlamydomonas reinhardtii

Aspartic proteinases have been extensively characterized in land plants but up to now no evidences for their presence in green algae group have yet been reported in literature. Here we report on the identification of the first (and only) typical aspartic proteinase from Chlamydomonas reinhardtii. This enzyme, named chlapsin, was shown to maintain the primary structure organization of typical plant aspartic proteinases but comprising distinct features, such as similar catalytic motifs DTG/DTG resembling those from animal and microbial counterparts, and an unprecedentedly longer plant specific insert domain with an extra segment of 80 amino acids, rich in alanine residues. Our results also demonstrated that chlapsin accumulates in Chlamydomonas chloroplast bringing this new enzyme to a level of uniqueness among typical plant aspartic proteinases. Chlapsin was successfully expressed in Escherichia coli and it displayed the characteristic enzymatic properties of typical aspartic proteinases, like optimum activity at acidic pH and complete inhibition by pepstatin A. Another difference to plant aspartic proteinases emerged as chlapsin was produced in an active form without its putative prosegment domain. Moreover, recombinant chlapsin showed a restricted enzymatic specificity and a proteolytic activity influenced by the presence of redox agents and nucleotides, further differentiating it from typical plant aspartic proteinases and anticipating a more specialized/regulated function for this Chlamydomonas enzyme. Taken together, our results revealed a pattern of complexity for typical plant aspartic proteinases in what concerns sequence features, localization and biochemical properties, raising new questions on the evolution and function of this vast group of plant enzymes.     

Aspartic proteinases in fishes and aquatic invertebrates

1. The literature on molecular properties and physiological role of aspartic proteinases in fishes and aquatic invertebrates has been reviewed. 2. Pepsins have not been detected in invertebrates, and apparently cathepsin D, as well as other cathepsins, act both as digestive and lysosomal enzymes in many of these animals. The molecular properties of invertebrate cathepsin D correspond with cathepsin D in fishes and mammalians. 3. Fishes with a true stomach have pepsinogen secretion. Fish pepsins have higher pH optimum and are less stable in strong acid conditions than mammalian pepsins. They are very efficient at low temperatures, but less thermostable than mammalian pepsins. 4. Many fishes have two significantly different pepsins: Pepsin I and Pepsin II, which digest haemoglobin at a maximal rate in the pH ranges 3-4 and 2-3 respectively. Usually the pI of Pepsin I is in the range 6.5-7, whereas pI of Pepsin II is about 4. 5. Fish Pepsin I and cathepsin D have very similar molecular properties, and a hypothesis proposing that cathepsin D is the ancestor enzyme of aspartic proteinases in higher animals is presented.     

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.     

Aspartic proteinase genes in the Brassicaceae Arabidopsis thaliana and Brassica napus

Active aspartic proteinase is isolated from Brassica napus seeds and the peptide sequence is used to generate primers for PCR. We present here cDNA and genomic clones for aspartic proteinases from the closely related Brassicaceae Arabidopsis thaliana and Brassica napus. The Arabidopsis cDNA represents a single gene, while Brassica has at least 4 genes. Like other plant aspartic proteases, the two Brassicaceae enzymes contain an extra protein domain of about 100 amino acids relative to the mammalian forms. The intron/exon arrangement in the Brassica genomic clone is significantly different from that in mammalian genes. As the proteinase is isolated from seeds, the same tissue where 2S albumins are processed, this implies expression of one of the aspartic proteinase genes there.     

Plant proteolytic enzymes: possible roles during programmed cell death

Proteolytic enzymes are known to be associated with developmentally programmed cell death during organ senescence and tracheary element differentiation. Recent evidence also links proteinases with some types of pathogen- and stress-induced cell suicide. The precise roles of proteinases in these and other plant programmed cell death processes are not understood, however. To provide a framework for consideration of the importance of proteinases during plant cell suicide, characteristics of the best-known proteinases from plants including subtilisin-type and papain-type enzymes, phytepsins, metalloproteinases and the 26S proteasome are summarized. Examples of serine, cysteine, aspartic, metallo- and threonine proteinases linked to animal programmed cell death are cited and the potential for plant proteinases to act as mediators of signal transduction and as effectors of programmed cell death is discussed.     

Isolation and characterization of a cDNA from flowers of Cynara cardunculus encoding cyprosin (an aspartic proteinase) and its use to study the organ-specific expression of cyprosin

Poly(A)⁺ RNA isolated from flower buds of Cynara cardunculus has been used to prepare a cDNA library. Screening of the cDNA after expression of cloned DNA with antibodies raised against the large subunit of cyprosin 3 resulted in the isolation of six positive clones. One of these clones (cypro1s; a 1.7 kb Eco RI fragment) codes for cyprosin. The nucleotide sequence contain a 1419 bp open reading frame coding for 473 amino acids (aa) including a putative full-length mature protein (440 aa) and a partial prosequence (33 aa). Cypro1s contains a 162 bp 3 non-coding region followed by a poly(A) tail. The deduced amino acid sequence shows high homology to other plant aspartic proteinases. The homology to mammalian and microbial aspartic proteinases is somewhat lower. Plant aspartic proteinases contain an insert of around 100 aa. We are modelling where this plant-specific insert will appear in the structure of cyprosin. Using cypro1s as a probe in northern blot analysis, the expression of cyprosin in developing flowers and other tissues has been studied. The signal on the northern blot increased for RNA samples from early (flower buds 6 mm in length) to later stages of floral development (flower buds up to 40 mm in length). In late stages of floral development (open flowers 50 mm in length and styles from such flowers) no hybridization signal was visualized showing that the synthesis of mRNA encoding the cyprosin starts in early stages of floral development and switches off at maturation of the flower. Southern blot analysis of genomic DNA showed 4–5 strong hybridizing bands and several minor bands indicating that the cyprosin genes are organized as a multi-gene family in C. cardunculus.     

Enzymatic synthesis of chromogenic substrates for Glu,Asp-specific proteinases.

Glu,Asp-specific endopeptidases represent a new subfamily of chymotrypsin-like proteolytic enzymes. These enzymes prefer Glu or Asp residues in the P1 position of the substrates. p-Nitroanilides of N-acylated di-, tri- and tetrapeptides with C-terminal glutamic or aspartic acid residues have been obtained. Acyl peptide p-nitroanilides were synthesized via acylation of glutamic or aspartic acid p-nitroanilides using methyl esters of the respective N-acylated peptides, generally with good yields. The reactions were performed in organic solvents using subtilisin 72 sorbed on silica as a catalyst. The kinetic parameters for the hydrolysis of these p-nitroanilides with proteinases from Bacillus intermedius and Bacillus licheniformis were determined.     

Molecular cloning and characterization of procirsin,an active aspartic protease precursor from Cirsium vulgare (Asteraceae)

Typical aspartic proteinases from plants of the Astereaceae family like cardosins and cyprosins are well-known milk-clotting enzymes. Their effectiveness in cheesemaking has encouraged several studies on other Astereaceae plant species for identification of new vegetable rennets. Here we report on the cloning, expression and characterization of a novel aspartic proteinase precursor from the flowers of Cirsium vulgare (Savi) Ten. The isolated cDNA encoded a protein product with 509 amino acids, termed cirsin, with the characteristic primary structure organization of plant typical aspartic proteinases. The pro form of cirsin was expressed in Escherichia coli and shown to be active without autocatalytically cleaving its pro domain. This contrasts with the acid-triggered autoactivation by pro-segment removal described for several recombinant plant typical aspartic proteinases. Recombinant procirsin displayed all typical proteolytic features of aspartic proteinases as optimum acidic pH, inhibition by pepstatin, cleavage between hydrophobic amino acids and strict dependence on two catalytic Asp residues for activity. Procirsin also displayed a high specificity towards κ-casein and milk-clotting activity, suggesting it might be an effective vegetable rennet.The findings herein described provide additional evidences for the existence of different structural arrangements among plant typical aspartic proteinases.     

The selectivity of action of the aspartic-proteinase inhibitor IA3 from yeast (Saccharomyces cerevisiae).

The ability of the aspartic-proteinase inhibitor IA3 from yeast (Saccharomyces cerevisiae) to affect the activities of a range of mammalian and microbial aspartic proteinases was examined. The inhibitor appeared to be completely selective in that only the aspartic proteinase A from yeast was inhibited to any significant extent. IA3 thus represents the first example of a totally specific, naturally occurring, aspartic-proteinase inhibitor.     

Hydrolysis of ester substrates of trypsin and chymotrypsin by barley carboxypeptidase

Purified barley carboxypeptidase exhibits high activity against a number of N-substituted amino acid esters, which are commonly used as synthetic substrates for mammalian and microbial proteinases. The proteinases of barley, on the contrary, do not hydrolyse these compounds. Because many other plants contain carboxypeptidases closely resembling the barley enzyme, we conclude that synthetic ester substrates should not be used to detect proteinase activity in extracts of higher plants. Plant carboxypeptidases also liberate C-terminal tryptophan from α-casein. Therefore, casein also is an unreliable substrate for plant proteinases.     

The three typical aspartic proteinase genes of Arabidopsis thaliana are differentially expressed.

Genomic sequencing has identified three different typical plant aspartic proteinases in the genome of Arabidopsis thaliana, named Pasp-A1, A2 and A3. A1 is identical to a cDNA we had previously isolated and the two others produce proteins 81 and 63% identical to that predicted protein. Sequencing of the aspartic proteinase protein purified from Arabidopsis seeds showed that the peptides are derived from two of these genes, A1 and A2. Using gene specific probes, we have analyzed RNA from different tissues and found these three genes are differentially expressed. A1 mRNA is detected in all tissues analyzed and more abundant in leaves during the light phase of growth. The other two genes are expressed either primarily in flowers (A3) or in seeds (A2). Insitu hybridization demonstrated that all three genes are expressed in many cells of the seeds and developing seed pods. The A1 and A3 genes are expressed in the sepals and petals of flowers as well as the outer layer of the style, but are not expressed in the transmitting tract or on the stigmatal surface. The A2 gene is weakly expressed only in the transmitting tissue of the style. All three genes are also expressed in the guard cells of sepals. These data suggest multiple roles for aspartic proteinases besides those proposed in seeds.     

Localization and characterization of hemoglobin-degrading aspartic proteinases from the malarial parasite Plasmodium falciparum.

Three hemoglobin-degrading proteinases were partially purified from food vacuoles isolated from trophozoite-stage forms of the malarial parasite Plasmodium falciparum. Two of the proteinases (M1 and M2) were solubilized by repeated sonication. The remaining proteinase (M3) was solubilized by treatment of the particulate fraction with taurocholic acid, suggesting that proteinase M3 is a membrane-bound proteinase whereas proteinases M1 and M2 are weakly associated with parasite membrane. The location of these proteinases suggests that they may participate in the digestion of host cytosolic protein. After partial purification, but not before, proteinases M1, M2 and M3 are highly sensitive to pepstatin, supporting their designation as aspartic proteinases. These aspartic proteinases show broad specificity for protein substrates. Native hemoglobin, acid denatured hemoglobin and oxidatively damaged hemoglobin are comparable substrates. Hemoglobin within the food vacuole was shown to be primarily native hemoglobin. Chemical modification studies indicate that these three aspartic proteinases have similar properties. The peptide maps from degradation of hemoglobin, however, suggest that aspartic proteinases M1, M2 and M3 are distinct proteinases.     

Engineering the substrate specificity of rhizopuspepsin: the role of Asp 77 of fungal aspartic proteinases in facilitating the cleavage of oligopeptide substrates with lysine in P1.

Rhizopuspepsin and other fungal aspartic proteinases are distinct from the mammalian enzymes in that they are able to cleave substrates with lysine in the P1 position. Sequence and structural comparisons suggest that two aspartic acid residues, Asp 30 and Asp 77 (pig pepsin numbering), may be responsible for generating this unique specificity. Asp 30 and Asp 77 were changed to the corresponding residues in porcine pepsin, Ile 30 and Thr 77, to create single and double mutants. The zymogen forms of the wild-type and mutant enzymes were overexpressed in Escherichia coli as inclusion bodies. Following solubilization, denaturation, refolding, activation, and purification to homogeneity, structural and kinetic comparisons were made. The mutant enzymes exhibited a high degree of structural similarity to the wild-type recombinant protein and a native isozyme. The catalytic activities of the recombinant proteins were analyzed with chromogenic substrates containing lysine in the P1, P2, or P3 positions. Mutation of Asp 77 resulted in a loss of 7 kcal mol-1 of transition-state stabilization energy in the hydrolysis of the substrate containing lysine in P1. An inhibitor containing the positively charged P1-lysine side chain inhibited only the enzymes containing Asp 77. Inhibition of the Asp 77 mutants of rhizopuspepsin and several mammalian enzymes was restored upon acetylation of the lysine side chain. These results suggest that an exploitation of the specific electrostatic interaction of Asp 77 in the active site of fungal enzymes may lead to the design of compounds that preferentially inhibit a variety of related Candida proteinases in immunocompromised patients.     

Aspartic proteinases from <Emphasis Type="Italic">Mucor</Emphasis> spp. in cheese manufacturing

Filamentous fungi belonging to the order of Mucorales are well known as producers of aspartic proteinases depicting milk-clotting activity. The biosynthesis level, the biochemical characteristics, and the technological properties of the resulting proteinases are affected by the producer strain and the mode of cultivation. While the milk-clotting enzymes produced by the Rhizomucor spp. have been extensively studied in the past, much less is known on the properties and potential applications of the aspartic proteinases obtained for Mucor spp. Indeed, several Mucor spp. strains have been reported as a potential source of milk-clotting enzymes having unique technological properties. Both submerged fermentation and solid substrate cultivation are proven alternatives for the production of Mucor spp. aspartic proteinases. This review provides an overview on the bioprocessing routes to obtain large amounts of these enzymes, on their structural characteristics as related to their functional properties, and on their industrial applications with focus on cheese manufacturing.     

Structural and evolutionary relationships between retroviral and eucaryotic aspartic proteinases.

Three-dimensional crystal structures of the homologous retroviral proteinases from Rous sarcoma virus (RSV PR) and from human immunodeficiency virus (HIV-1 PR) are to a large extent similar and bear close resemblance to the six known structures of the bilobal fungal and mammalian aspartic proteinases. Systematic three-dimensional structural superpositions were carried out between the retroviral and the eucaryotic aspartic proteinases. Both retroviral enzymes were found to be similarly related to their fungal and mammalian counterparts. The most strongly conserved parts correspond to those regions in the N- and C-domains of the eucaryotic enzymes that are related by the interdomain dyad and consist of a combination of secondary structural elements that form the psi loop-alpha helix motif at the active sites of the aspartic proteinases. The retroviral proteinase monomer exhibits nearly the same degree of structural equivalence to the N- and C-domains of the eucaryotic enzymes. In light of the deduced structural relationships between HIV-1 PR and RSV PR, sequence alignments were performed for a number of retroviral proteinases from different subfamilies. There are three highly conserved amino acid sequence stretches, of which two belong to the psi loop-alpha helix motif, that bear moderate sequence similarity with the eucaryotic enzymes. The third conserved sequence stretch among the retroviral proteinases belongs to the flap and bears no resemblance to the flap sequences in the eucaryotic enzymes. The interdomain antiparallel beta sheet in the cellular enzymes differs from the intersubunit beta sheet in the number, arrangement, and directionality of strands, suggesting the possibility of convergent evolution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aspartic proteinases in Antarctic fish

The present review surveys several recent studies of the aspartic proteinases from Antarctic Notothenioidei, a dominating fish group that has developed a number of adjustments at the molecular level to maintain metabolic function at low temperatures. Given the unique peculiarities of the Antarctic environment, studying the features of Antarctic aspartic proteinases could provide new insights into the role of these proteins in fish physiology. We describe here: (1) the biochemical properties of a cathepsin D purified from the liver of the hemoglobinless icefish Chionodraco hamatus; (2) the biochemical characterization of Trematomus bernacchii pepsins variants A1 and A2 obtained by heterologous expression in bacteria; and (3) the identification of two closely related, novel aspartic proteinases from the liver of the two Antarctic fish species mentioned above. Overall, the results show that Notothenioidei aspartic proteinases display a number of characteristics that are remarkably different from those of mammalian aspartic proteinases, including high turnover number or high catalytic efficiency. We have named the newly identified aspartic proteinases "Nothepsins" and classified them relative to aspartic proteinases from other species.     

The characteristics of cysteine proteinases of parasitic protozoa.

Cysteine proteinases have now been detected in most of the important species of parasitic protozoa. Characterization of the enzymes and sequence determinations have revealed that the enzymes are related to papain and the mammalian cathepsins. All of the protozoan enzymes analyzed to date are members of the cathepsin L/cathepsin H/papain branch of the papain superfamily and are more distantly related to cathepsin B. They thus share some characteristics with the cysteine proteinases of their hosts. Individual enzymes, however, are likely to have sufficient novel features to be potential targets for specific antiprotozoal drugs, and a number of proteinase inhibitors and substrates are currently being tested as possible chemotherapeutic agents.     

Serpins in plants and green algae

Control of proteolysis is important for plant growth, development, responses to stress, and defence against insects and pathogens. Members of the serpin protein family are likely to play a critical role in this control through irreversible inhibition of endogenous and exogenous target proteinases. Serpins have been found in diverse species of the plant kingdom and represent a distinct clade among serpins in multicellular organisms. Serpins are also found in green algae, but the evolutionary relationship between these serpins and those of plants remains unknown. Plant serpins are potent inhibitors of mammalian serine proteinases of the chymotrypsin family in vitro but, intriguingly, plants and green algae lack endogenous members of this proteinase family, the most common targets for animal serpins. An Arabidopsis serpin with a conserved reactive centre is now known to be capable of inhibiting an endogenous cysteine proteinase. Here, knowledge of plant serpins in terms of sequence diversity, inhibitory specificity, gene expression and function is reviewed. This was advanced through a phylogenetic analysis of amino acid sequences of expressed plant serpins, delineation of plant serpin gene structures and prediction of inhibitory specificities based on identification of reactive centres. The review is intended to encourage elucidation of plant serpin functions.