Novel ways to prevent proteolysis — prophytepsin and proplasmepsin II

The recently determined crystal structures of two aspartic proteinase zymogens, prophytepsin from barley and proplasmepsin II from the malarial parasite Plasmodium falciparum, have provided new insights into zymogen inactivation. Prophytepsin shows a variation of the mechanism of inhibition used by the well-known gastric aspartic proteinase zymogens, whereas proplasmepsin II uses a completely new mode of inactivation.     

Structural insights into the activation of P. vivax plasmepsin

The malarial aspartic proteinases (plasmepsins) have been discovered in several species of Plasmodium, including all four of the human malarial pathogens. In P.falciparum, plasmepsins I, II, IV and HAP have been directly implicated in hemoglobin degradation during malaria infection, and are now considered targets for anti-malarial drug design. The plasmepsins are produced from inactive zymogens, proplasmepsins, having unusually long N-terminal prosegments of more than 120 amino acids. Structural and biochemical evidence suggests that the conversion process of proplasmepsins to plasmepsins differs substantially from the gastric and plant aspartic proteinases. Instead of blocking substrate access to a pre-formed active site, the prosegment enforces a conformation in which proplasmepsin cannot form a functional active site. We have determined crystal structures of plasmepsin and proplasmepsin from P.vivax. The three-dimensional structure of P.vivax plasmepsin is typical of the monomeric aspartic proteinases, and the structure of P.vivax proplasmepsin is similar to that of P.falciparum proplasmepsin II. A dramatic refolding of the mature N terminus and a large (18 degrees ) reorientation of the N-domain between P.vivax proplasmepsin and plasmepsin results in a severe distortion of the active site region of the zymogen relative to that of the mature enzyme. The present structures confirm that the mode of inactivation observed originally in P.falciparum proplasmepsin II, i.e. an incompletely formed active site, is a true structural feature and likely represents the general mode of inactivation of the related proplasmepsins.     

The aspartic proteinase from the rodent parasite Plasmodium berghei as a potential model for plasmepsins from the human malaria parasite, Plasmodium falciparum

The gene encoding an aspartic proteinase precursor (proplasmepsin) from the rodent malaria parasite Plasmodium berghei has been cloned. Recombinant P. berghei plasmepsin hydrolysed a synthetic peptide substrate and this cleavage was prevented by the general aspartic proteinase inhibitor, isovaleryl pepstatin and by Ro40-4388, a lead compound for the inhibition of plasmepsins from the human malaria parasite Plasmodium falciparum. Southern blotting detected only one proplasmepsin gene in P. berghei. Two plasmepsins have previously been reported in P. falciparum. Here, we describe two further proplasmepsin genes from this species. The suitability of P. berghei as a model for the in vivo evaluation of plasmepsin inhibitors is discussed.     

Kinetic analysis of a general model of activation of aspartic proteinase zymogens

Starting from a simple general reaction mechanism of activation of aspartic proteinase zymogens involving an uni- and a bimolecular simultaneous route, the time course equation of the concentration of the zymogen and of the activated enzyme have been derived. From these equations, an analysis quantifying the relative contribution to the global process of the two routes has been carried out for the first time. This analysis suggests a way to predict the time course of the relative contribution as well as the effect of the initial zymogen and activating enzyme concentrations, on the relative weight. An experimental design and kinetic data analysis is suggested to estimate the kinetic parameters involved in the reaction mechanism proposed. Finally, we apply some of our results to experimental data obtained by other authors in experimental studies of the activation of some aspartic proteinase zymogens.     

Adaptation of the behaviour of an aspartic proteinase inhibitor by relocation of a lysine residue by one helical turn

In addition to self-inhibition of aspartic proteinase zymogens by their intrinsic proparts, the activity of certain members of this enzyme family can be modulated through active-site occupation by extrinsic polypeptides such as the small IA3 protein from Saccharomyces cerevisiae. The unprecedented mechanism by which IA3 helicates to inhibit its sole target aspartic proteinase locates an i, i+4 pair of charged residues (Lys18+Asp22) on an otherwise-hydrophobic face of the amphipathic helix. The nature of these residues is not crucial for effective inhibition, but re-location of the lysine residue by one turn (+4 residues) in the helical IA3 positions its side chain in the mutant IA3-proteinase complex in an orientation essentially identical to that of the key lysine residue in zymogen proparts. The binding of the extrinsic mutant IA3 shows pH dependence reminiscent of that required for the release of intrinsic zymogen proparts so that activation can occur.     

Human renin inhibition by a diazoacyl reagent: Relationship of the enzyme to other proteinases

Human renin is inactivated by a diazoacyl compound (diazoacetylglycine ethyl ester; N₂CHCO-Gly-OEt) in the presence of Cu(II). The mechanism of the inactivation is presumably identical to that which has been determined for pepsin and several other proteinases: esterification of the β-carboxyl of an aspartic acid residue at the active site of the enzyme. Renin's inhibition by the diazoacyl reagent, its specificity toward a hydrophobic sequence, and its inhibition by pepstatin, all suggest a close relationship to the acid proteinases, especially pepsin and cathepsin D. However, renin, a neutral proteinase, would be better classified together with other diazoacyl-inhibited enzymes by active site rather than pH optimum. The term “aspartic proteinase” is suggested for this group of enzymes.     

A kinetic and equilibrium study of the denaturation of aspartic proteinases from the fungi, Endothia parasitica and Mucor miehei

Kinetic and equilibrium analyses of the denaturation of Endothia parasitica and Mucor miehei aspartic proteinases were performed using enzyme activity and ultraviolet absorption as indices of denaturation. Denaturation of these proteinases was shown to be irreversible, suggesting that the conformations of these aspartic proteinases may be predetermined in their zymogens. Thermal and guanidine hydrochloride denaturation of these proteinases produced first-order, two-state, kinetic behaviour. Equilibrium unfolding transitions of these proteinases were highly cooperative but not entirely coincident in the two indices employed, suggesting some deviation from two-state character. Oxidation to remove 37.8% of the carbohydrate of M. miehei glycoproteinase with sodium metaperiodate resulted in a substantial decrease in both kinetic and equilibrium stabilities without modification of the amino acid composition or specific activity. In addition, gel filtration subsequent to equilibrium studies indicated that partial removal of the carbohydrate from M. miehei proteinase promoted autolysis under denaturing conditions.     

An unusual specificity in the activation of neutrophil serine proteinase zymogens

The majority of proteinases exist as zymogens whose activation usually results from a single proteolytic event. Two notable exceptions to this generalization are the serine proteinases neutrophil elastase (HNE) and cathepsin G (cat G), proteolytic enzymes of human neutrophils that are apparently fully active in their storage granules. On the basis of amino acid sequences inferred from the gene and cDNAs encoding these enzymes, it is likely that both are synthesized as precursors containing unusual C-terminal and N-terminal peptide extensions absent from the mature proteins. We have used biosynthetic radiolabeling and radiosequencing techniques to identify the kinetics of activation of both proteinases in the promonocyte-like cell line U937. We find that both N- and C-terminal extensions are removed about 90 min after the onset of synthesis, resulting in the activation of the proteinases. HNE and cat G are, therefore, transiently present as zymogens, presumably to protect the biosynthetic machinery of the cell from adventitious proteolysis. Activation results from cleavage following a glutamic acid residue to give an activation specificity opposite to those of almost all other serine proteinase zymogens, but shared, possibly, by the "granzyme" group of related serine proteinases present in the killer granules of cytotoxic T-lymphocytes and rat mast cell proteinase II.     

Evolution in the structure and function of aspartic proteases

Aspartic proteases (EC3.4.23) are a group of proteolytic enzymes of the pepsin family that share the same catalytic apparatus and usually function in acid solutions. This latter aspect limits the function of aspartic proteases to some specific locations in different organisms; thus the occurrence of aspartic proteases is less abundant than other groups of proteases, such as serine proteases. The best known sources of aspartic proteases are stomach (for pepsin, gastricsin, and chymosin), lysosomes (for cathepsins D and E), kidney (for renin), yeast granules, and fungi (for secreted proteases such as rhizopuspepsin, penicillopepsin, and endothiapepsin). These aspartic proteases have been extensively studied for their structure and function relationships and have been the topics of several reviews or monographs (Tang: Acid Proteases, Structure, Function and Biology. New York: Plenum Press, 1977; Tang: J Mol Cell Biochem 26:93-109, 1979; Kostka: Aspartic Proteinases and Their Inhibitors. Berlin: Walter de Gruyter, 1985). All mammalian aspartic proteases are synthesized as zymogens and are subsequently activated to active proteases. Although a zymogen for a fungal aspartic protease has not been found, the cDNA structure of rhizopuspepsin suggests the presence of a "pro" enzyme (Wong et al: Fed Proc 44:2725, 1985). It is probable that other fungal aspartic proteases are also synthesized as zymogens. It is the aim of this article to summarize the major models of structure-function relationships of aspartic proteases and their zymogens with emphasis on more recent findings. Attempts will also be made to relate these models to other aspartic proteases.     

The expression and function of cathepsin E in dendritic cells

Cathepsin E is an aspartic proteinase that has been implicated in Ag processing within the class II MHC pathway. In this study, we document the presence of cathepsin E message and protein in human myeloid dendritic cells, the preeminent APCs of the immune system. Cathepsin E is found in a perinuclear compartment, which is likely to form part of the endoplasmic reticulum, and also a peripheral compartment just beneath the cell membrane, with a similar distribution to that of Texas Red-dextran within 2 min of endocytosis. To investigate the function of cathepsin E in processing, a new soluble targeted inhibitor was synthesized by linking the microbial aspartic proteinase inhibitor pepstatin to mannosylated BSA via a cleavable disulfide linker. This inhibitor was shown to block cathepsin D/E activity in cell-free assays and within dendritic cells. The inhibitor blocked the ability of dendritic cells from wild-type as well as cathepsin D-deficient mice to present intact OVA, but not an OVA-derived peptide, to cognate T cells. The data therefore support the hypothesis that cathepsin E has an important nonredundant role in the class II MHC Ag processing pathway within dendritic cells.     

Active site specificity of plasmepsin II.

Members of the aspartic proteinase family of enzymes have very similar three-dimensional structures and catalytic mechanisms. Each, however, has unique substrate specificity. These distinctions arise from variations in amino acid residues that line the active site subsites and interact with the side chains of the amino acids of the peptides that bind to the active site. To understand the unique binding preferences of plasmepsin II, an enzyme of the aspartic proteinase class from the malaria parasite, Plasmodium falciparum, chromogenic octapeptides having systematic substitutions at various positions in the sequence were analyzed. This enabled the design of new, improved substrates for this enzyme (Lys-Pro-Ile-Leu-Phe*Nph-Ala/Glu-Leu-Lys, where * indicates the cleavage point). Additionally, the crystal structure of plasmepsin II was analyzed to explain the binding characteristics. Specific amino acids (Met13, Ser77, and Ile287) that were suspected of contributing to active site binding and specificity were chosen for site-directed mutagenesis experiments. The Met13Glu and Ile287Glu single mutants and the Met13Glu/Ile287Glu double mutant gain the ability to cleave substrates containing Lys residues.     

Action of brain cathepsin B,cathepsin D,and high-molecular-weight aspartic proteinase on angiotensins I and II

The action of three previously isolated electrophoretically homogeneous brain proteinases—cathepsin B (EC 3.4.22.1), cathepsin D (EC 3.4.23.5), and high-molecular-weight aspartic proteinase (M_r=90K; EC 3.4.23.−)—on human angiotensins I and II has been investigated. The products of enzymatic hydrolysis have been identified by thin-layer chromatography on Silufol plates using authentic standards and by N-terminal amino acid residue analysis using a dansyl chloride method. Cathepsin D and high-molecular-weight aspartic proteinase did not split angiotensin I or angiotensin II. Cathepsin B hydrolyzed angiotensin I via a dipeptidyl carboxypeptidase mechanism removing His-Leu to form angiotensin II, and it degraded angiotensin II as an endopeptidase at the Val³-Tyr⁴ bond. Cathepsin B did not split off His-Leu from Z-Phe-His-Leu. Brain cathepsin B may have a role in the generation and degradation of angiotensin II in physiological conditions. Special Issue dedicated to Dr. Eugene Kreps.     

cDNA cloning of an aspartic proteinase secreted by Candida albicans.

cDNA of an aspartic proteinase secreted by Candida albicans No. 114 was isolated using the polymerase chain reaction (PCR). The primary structure of the enzyme was deduced from the nucleotide sequence of the cDNA and compared with the structures of Saccharomyces cerevisiae proteinase A and vacuolar aspartyl proteinase of C. albicans. The mature aspartic proteinase consisted of 341 amino acid residues, and was 17.6 and 15.3% identical with the proteinase A and the aspartyl proteinase, respectively. Two active aspartic acid sites and the amino acids near those sites were conserved in the aspartic proteinase. We also showed that there is another gene of aspartic proteinase than that of strain ATCC10231 reported by Hube et al (J. Med. Vet. Mycol. 29 (1991)) in the same C. albicans genome, both in that strain and in No. 114.     

The high-resolution crystal structure of porcine pepsinogen.

The structure of porcine pepsinogen at pH 6.1 has been refined to an R-factor of 0.173 for data extending to 1.65 A. The final model contains 180 solvent molecules and lacks density for residues 157-161. The structure of this aspartic proteinase zymogen possesses many of the characteristics of pepsin, the mature enzyme. The secondary structure of the zymogen consists predominantly of beta-sheet, with an approximate 2-fold axis of symmetry. The activation peptide packs into the active site cleft, and the N-terminus (1P-9P) occupies the position of the mature N-terminus (1-9). Thus changes upon activation include excision of the activation peptide and proper relocation of the mature N-terminus. The activation peptide or residues of the displaced mature N-terminus make specific interactions with the substrate binding subsites. The active site of pepsinogen is intact; thus the lack of activity of pepsinogen is not due to a deformation of the active site. Nine ion pairs in pepsinogen may be important in the advent of activation and involve the activation peptide or regions of the mature N-terminus which are relocated in the mature enzyme. The activation peptide-pepsin junction, 44P-1, is characterized by high thermal parameters and weak density, indicating a flexible structure which would be accessible to cleavage. Pepsinogen is an appropriate model for the structures of other zymogens in the aspartic proteinase family.     

The precursor of secreted aspartic proteinase Sapp1p from Candida parapsilosis can be activated both autocatalytically and by a membrane-bound processing proteinase

Opportunistic pathogens of the genus Candida produce secreted aspartic proteinases (Saps) that play an important role in virulence. Saps are synthesized as zymogens, but cell-free culture supernatants of Candida spp. contain only mature Saps. To study the zymogen conversion, the gene encoding a precursor of C. parapsilosis proteinase Sapp1p was cloned, expressed in E. coli and the product was purified. When placed in acidic conditions, the precursor was autocatalytically processed, yielding an active proteinase. The self-activation proceeded through an intermediate product and the resulting enzyme was one amino acid shorter than the authentic enzyme. This truncation did not cause changes in proteinase activity or secondary structure compared to the authentic Sapp1p. Accurate cleavage of the pro-mature junction, however, required a processing proteinase. A crude membrane fraction prepared from C. parapsilosis cells contained an enzyme with Kex2-like activity, which processed the Sapp1p precursor at the expected site. The pro-segment appeared to be indispensable for Sapp1p to attain an appropriate structure. When expressed without the pro-segment, the Sapp1p mature domain was not active and had a lower content of alpha-helical conformation, as measured by circular dichroism. A similar effect was observed when a His(6)-tag was linked to the C-terminus of Sapp1p or its precursor.     

Characteristics of the Chromaffin Granule Aspartic Proteinase Involved in Proenkephalin Processing

Abstract: Proteolytic processing of neuropeptide precursors is required for production of active neurotransmitters and hormones. In this study, a chromaffin granule (CG) aspartic proteinase of 70 kDa was found to contribute to enkephalin precursor cleaving activity, as assayed with recombinant ([³⁵S]Met)preproenkephalin. The 70-kDa CG aspartic proteinase was purified by concanavalin A-Sepharose, Sephacryl S-200, and pepstatin A agarose affinity chromatography. The proteinase showed optimal activity at pH 5.5. It was potently inhibited by pepstatin A, a selective aspartic proteinase inhibitor, but not by inhibitors of serine, cysteine, or metalloproteinases. Lack of inhibition by Val-d -Leu-Pro-Phe-Val-d -Leu—an inhibitor of pepsin, cathepsin D, and cathepsin E—distinguishes the CG aspartic proteinase from classical members of the aspartic proteinase family. The CG aspartic proteinase cleaved recombinant proenkephalin between the Lys¹⁷²-Arg¹⁷³ pair located at the COOH-terminus of (Met)enkephalin-Arg⁶-Gly⁷-Leu⁸, as assessed by peptide microsequencing. The importance of full-length prohormone as substrate was demonstrated by the enzyme's ability to hydrolyze ³⁵S-labeled proenkephalin and proopiomelanocortin and its inability to cleave tri- and tetrapeptide substrates containing dibasic or monobasic cleavage sites. In this study, results provide evidence for the role of an aspartic proteinase in proenkephalin and prohormone processing.     

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.     

Proteolytic activity in the maize pollen wall

A new protease from maize ( Zea mays L.) pollen is described. It was purified using gel filtration, ion exchange and high performance liquid chromatography. SDS-PAGE and HPLC showed that the enzyme has a dimeric structure of M, ca 60,000. Inhibitor investigations indicated an aspartic acid residue in its active site. The optimum pH for maize pollen aspartic proteinase activity was 5.6, and the optimum temperature was 45°C. The enzyme is easily eluted from the pollen grains and, as confirmed by enzymoblotting after isoelectric focusing, it is located in the pollen wall. Similar to metallo-proteinases, its activity is inhibited by Zn₂₊. The pL value for purified aspartic proteinase, as estimated after IEF, was 5.0. Two-dimensional electrophoresis analysis of proteins eluted from maize pistils suggests that the enzyme digests the proteins and may be involved in pollen-tube germination. The properties of serine and aspartic proteinases from maize pollen are compared.     

Proteolytic enzymes in green wheat-leaves III. Inactivation of acid proteinase II by diazoacetyl-DL-norleucine methyl ester and 1,2-epoxy-3-(p-nitrophenoxy)-propane

Acid proteinase II isolated from green wheat leaves in a purifiedform was rapidly inactivated at pH=5.5 to 6.0 by a 50-fold molarexcess of diazoacetyl-DL-norleucine methyl ester (DAN) in thepresence of cupric ions which were essential for inactivation.The acid proteinase was also inactivated by reaction with 1,2-epoxy-3-(p-nitrophenoxy)-propane(EPNP). The inactivation by EPNP was much slower than by DANand the half-life of the activity was 24 hr. (Received February 6, 1978; )     

Differential localization of two acid proteinases in germinating barley (Hordeum vulgare) seed

A cathepsin D-like aspartic proteinase (EC 3.4.23) is abundant in ungerminated barley ( Hordeum vulgare ) seed while a 30 kDa cysteine endoproteinase (EC 3.4.22) is one of the proteinases synthesized de novo in the germinating seed. In this work, the localization of these two acid proteinases was studied at both the tissue and subcellular levels by immunomicroscopy. The results confirm that they have completely different functions. The aspartic proteinase was present in the ungerminated seed and, during germination, it appeared in all the living tissues of the grain, including the shoot and root. Contrary to previous suggestions, it was not observed in the starchy endosperm. By immunoblotting, the high molecular mass form of the enzyme (32 + 16 kDa) was found in all the living tissues, whereas the low molecular mass form (29 + 11 kDa) was not present in the shoot or root, indicating that the two enzyme forms have different physiological roles. The aspartic proteinase was localized first in the scutellar protein bodies of germinating seed, and later in the vacuoles which are formed by fusion of the protein bodies. In contrast to the aspartic proteinase, the expression of the 30 kDa cysteine proteinase began during the first germination day, and it was secreted into the starchy endosperm; first from the scutellum and later from the aleurone layer. It was not found in either shoots or roots. The 30 kDa cysteine proteinase was detected in the Golgi apparatus and in the putative secretory vesicles of the scutellar epithelium. These results suggest that the aspartic proteinase functions only in the living tissues of the grain, as opposed to the 30 kDa cysteine proteinase which is apparently one of the proteases initiating the hydrolysis of storage proteins in the starchy endosperm.