Glucosidase II, a glycoprotein of the endoplasmic reticulum membrane. Proteolytic cleavage into enzymatically active fragments

Glucosidase II removes the inner two alpha-linked glucose residues from freshly transferred Asn-linked oligosaccharide chains in the endoplasmic reticulum. This enzyme, whose activity could be measured by the hydrolysis of an artificial substrate (p-nitrophenyl alpha-D-glucopyranoside), was purified 240-fold from a rat liver microsome fraction by DEAE-cellulose, concanavalin A-Sepharose 4B, and hydroxylapatite chromatography. The apparent molecular weight of the active polypeptide was 123 000 as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Glucosidase II has at least one high-mannose oligosaccharide chain that can be cleaved by endoglycosidase H. Trypsin readily cleaved the 123-kilodalton (kDa) form of glucosidase II into a fully active 73-kDa core. The pattern of this cleavage suggests a domain structure for this enzyme. We demonstrate that trypsin first removes a glycosylated 25-kDa domain to yield an apparently unglycosylated 98-kDa product which is further cleaved to yield the active 73-kDa core.     

Mitochondrial import of rat pre-ornithine transcarbamylase: accurate processing of the precursor form is not required for uptake into mitochondria, nor assembly into catalytically active enzyme

Mitochondrial uptake of the cytoplasmically synthesized precursor of the mammalian enzyme ornithine transcarbamylase is mediated by an N-terminal leader sequence of 32 amino acids. In the mitochondrial matrix, the precursor form is processed to the mature subunit by proteolytic removal of this pre-sequence and in the enzyme from rat liver it has been suggested that this occurs in a two-step process which involves an intermediate cleavage at residue 24. We show that deletion of residues 20-26 spanning this intermediate cleavage site prevents correct processing to the mature subunit but it does not prevent mitochondrial targeting and internalization or assembly of the incorrectly processed product into a catalytically active enzyme. The incorrectly processed enzyme, which is larger than the normal mature enzyme, is nevertheless more susceptible to proteolytic degradation in permanently transfected human cells than the correctly processed enzyme.     

Efficient production of active Vibrio proteolyticus aminopeptidase in Escherichia coli by co-expression with engineered vibriolysin

The Vibrio proteolyticus aminopeptidase is synthesized as a preproprotein and then converted into an active enzyme by cleavage of the N-terminal propeptide. In recombinant Escherichia coli, however, the aminopeptidase is not processed correctly and the less-active form that has the N-terminal propeptide accumulates in the culture medium. Recently, we isolated a novel vibriolysin that was expressed as an active form in E. coli by random mutagenesis; this enzyme shows potential as a candidate enzyme for the processing of aminopeptidase. The E. coli cells were engineered to co-express the novel vibriolysin along with aminopeptidase. Co-expression of vibriolysin resulted in an approximately 13-fold increase in aminopeptidase activity, and a further increase was observed in the form lacking its C-terminal propeptide. The active aminopeptidase was purified from the culture supernatant including the recombinant vibriolysin by heat treatment and ion exchange and hydroxyapatite chromatography with high purity and 35% recovery rate. This purified aminopeptidase effectively converted methionyl-human growth hormone (Met-hGH) to hGH. Thus, this co-expression system provides an efficient method for producing active recombinant V. proteolyticus aminopeptidase.     

Cathepsin L1, the major protease involved in liver fluke (Fasciola hepatica) virulence: propetide cleavage sites and autoactivation of the zymogen secreted from gastrodermal cells

The secretion and activation of the major cathepsin L1 cysteine protease involved in the virulence of the helminth pathogen Fasciola hepatica was investigated. Only the fully processed and active mature enzyme can be detected in medium in which adult F. hepatica are cultured. However, immunocytochemical studies revealed that the inactive procathepsin L1 is packaged in secretory vesicles of epithelial cells that line the parasite gut. These observations suggest that processing and activation of procathepsin L1 occurs following secretion from these cells into the acidic gut lumen. Expression of the 37-kDa procathepsin L1 in Pichia pastoris showed that an intermolecular processing event within a conserved GXNXFXD motif in the propeptide generates an active 30-kDa intermediate form. Further activation of the enzyme was initiated by decreasing the pH to 5.0 and involved the progressive processing of the 37 and 30-kDa forms to other intermediates and finally to a fully mature 24.5 kDa cathepsin L with an additional 1 or 2 amino acids. An active site mutant procathepsin L, constructed by replacing the Cys(26) with Gly(26), failed to autoprocess. However, [Gly(26)]procathepsin L was processed by exogenous wild-type cathepsin L to a mature enzyme plus 10 amino acids attached to the N terminus. This exogenous processing occurred without the formation of a 30-kDa intermediate form. The results indicate that activation of procathepsin L1 by removal of the propeptide can occur by different pathways, and that this takes place within the parasite gut where the protease functions in food digestion and from where it is liberated as an active enzyme for additional extracorporeal roles.     

myo-Inositol oxygenase from rat kidneys. I: Purification by affinity chromatography; physical and catalytic properties.

Using the technique of affinity chromatography on a myo-inositol-substituted Sepharose, the myo-inositol oxygenase from rat kidneys was purified to homogeneity. The active enzyme contains iron, most probably in its divalent form. Electrophoresis on polyacrylamide gel containing sodium dodecylsulphate causes the cleavage of the enzyme protein into apparently identical subunits with a molecular weight of approximately 17,000. The smallest active unit consists of 4 subunits, and is in a pH-dependent equilibium with species consisting of 8, 12, and 16 subunits, respectively, which all show the same specific enzyme activity. In the presence of oxygen the enzyme is highly unstable; at the early stages of inactivation it can be reactivated by reducing agents like NaBH4. Under anaerobic conditions or under the influence of Fe2-chelating agents, the enzyme is also inactivated; this inactivation is caused by the loss of iron and concomitant cleavage into the subunits. It can be reversed by incubation with FeSO4 in the presence of air. If myo-inositol and FeSO4 are present, the reactivation involves an oligomerization to the species with 16 subunits with the uptake of 8 gram-atoms of iron per mole of this species. The enzyme reaction follows Michaelis-Menten kinetics; the Michaelis constants are 4.5 x 10(-2)M for myo-inositol and 9.5 x 10(-6)M for oxygen.     

Transport of proteins into chloroplasts. The precursor of small subunit of ribulose bisphosphate carboxylase is processed to the mature size in two steps

The precursor of the small subunit of ribulose bisphosphate carboxylase in Pisum sativum (relative molecular mass 20 000) is processed to the mature size (relative molecular mass 14 000) by the purified processing enzyme in two steps. The maturation proceeds via an intermediate of Mr 18 000. Both processing reactions may be carried out by the same enzyme although different residues are involved in the two cleavage sites. The second cleavage is inhibited if the precursor is pre-incubated with iodoacetate. The processing intermediate cannot be detected during the uptake of the precursor by intact isolated chloroplasts but iodoacetate-treated precursor is taken up and converted to a number of polypeptides of Mr 18 000 and below.     

Characterisation of Fasciola hepatica cytochrome c peroxidase as an enzyme with potential antioxidant activity in vitro.

Cytochrome c peroxidase oxidises hydrogen peroxide using cytochrome c as the electron donor. This enzyme is found in yeast and bacteria and has been also described in the trematodes Fasciola hepatica and Schistosoma mansoni. Using partially purified cytochrome c peroxidase samples from Fasciola hepatica we evaluated its role as an antioxidant enzyme via the investigation of its ability to protect against oxidative damage to deoxyribose in vitro. A system containing FeIII-EDTA plus ascorbate was used to generate reactive oxygen species superoxide radical, H2O2 as well as the hydroxyl radical. Fasciola hepatica cytochrome c peroxidase effectively protected deoxyribose against oxidative damage in the presence of its substrate cytochrome c. This protection was proportional to the amount of enzyme added and occurred only in the presence of cytochrome c. Due to the low specific activity of the final partially purified sample the effects of ascorbate and calcium chloride on cytochrome c peroxidase were investigated. The activity of the partially purified enzyme was found to increase between 10 and 37% upon reduction with ascorbate. However, incubation of the partially purified enzyme with 1 mM calcium chloride did not have any effect on enzyme activity. Our results showed that Fasciola hepatica CcP can protect deoxyribose from oxidative damage in vitro by blocking the formation of the highly toxic hydroxyl radical (.OH). We suggest that the capacity of CcP to inhibit .OH-formation, by efficiently removing H2O2 from the in vitro oxidative system, may extend the biological role of CcP in response to oxidative stress in Fasciola hepatica.     

Role of the prosegment of Fasciola hepatica cathepsin L1 in folding of the catalytic domain

The N-terminal propeptides of cysteine proteinases play regulatory roles in the folding and stability of their catalytic domains, as well as being potent and highly specific inhibitors of their parental mature enzymes. Cysteine proteinases play a major role in the biology of the parasitic trematode Fasciola hepatica; in particular, this organism secretes significant amounts of cathepsin L enzymes. The isolated propeptide of F. hepatica cathepsin L1 functioned as a chaperone for the mature enzyme in renaturation experiments. A double point mutation (N701/F721) within the GxNxFxD motif of the propeptide affected its conformation and markedly decreased its affinity for the mature enzyme. When this mutation was introduced into preprocathepsin L1 expressed in yeast, the secretion of active enzyme dropped dramatically. However, significant enzyme activity was recovered from the culture supernatants after denaturation and renaturation in the presence of native propeptide. Thus, the variant prosegment gave rise to an enzyme with altered conformation, which could be refolded to the active form with the assistance of the native propeptide.     

Isolation and some properties of an acid protease from Fasciola hepatica.

A new protease, detected in an extract of Fasciola hepatica, was isolated and partly purified. The pH optimum for the cleavage of denaturated haemoglobin by the enzyme is pH 3.0. This proteolytic activity is inhibited by diazoacetylnorleucine methyl ester, pepstatin, the pepsin inhibitor from Ascaris suum, and phenylalanine. The cathepsin D inhibitor from potatoes, EDTA, mercaptoethanol and the inorganic salts tested have no inhibitory effect. The cleavage of the B-chain of oxidized insulin by enzyme was studied and compared with the digestion of the same substrate by chicken and pig pepsin. The protease from Fasciola hepatica belongs to the carboxyl group of proteases and probably plays an important role in helminth nutrition.     

Identification of membrane-bound serine proteinase matriptase as processing enzyme of insulin-like growth factor binding protein-related protein-1 (IGFBP-rP1/angiomodulin/mac25)

Insulin-like growth factor (IGF) binding protein-related protein-1 (IGFBP-rP1) modulates cellular adhesion and growth in an IGF/insulin-dependent or independent manner. It also shows tumor-suppressive activity in vivo. We recently found that a single-chain IGFB-rP1 is proteolytically cleaved to a two-chain form by a trypsin-like, endogenous serine proteinase, changing its biological activities. In this study, we attempted to identify the IGFBP-rP1-processing enzyme. Of nine human cell lines tested, seven cell lines secreted IGFBP-rP1 at high levels, and two of them, ovarian clear cell adenocarcinoma (OVISE) and gastric carcinoma (MKN-45), highly produced the cleaved IGFBP-rP1. Serine proteinase inhibitors effectively blocked the IGFBP-rP1 cleavage in the OVISE cell culture. The conditioned medium of OVISE cells did not cleave purified IGFBP-rP1, but their membrane fraction had an IGFBP-rP1-cleaving activity. The membrane fraction contained an 80-kDa gelatinolytic enzyme, which was identified as the membrane-type serine proteinase matriptase (MT-SP1) by immunoblotting. When the membrane fraction was separated by SDS/PAGE, the IGFBP-rP1-cleaving activity comigrated with matriptase. A soluble form of matriptase purified in an inhibitor-free form efficiently cleaved IGFBP-rP1 at the same site as that found in a naturally cleaved IGFBP-rP1. Furthermore, small interfering RNAs for matriptase efficiently blocked both the matriptase expression and the cleavage of IGBP-rP1 in OVISE cells. These results demonstrate that IGFBP-rP1 is processed to the two-chain form by matriptase on the cell surface.     

Characterization of endothelin-converting enzyme-2. Implication for a role in the nonclassical processing of regulatory peptides

Most neuroendocrine peptides are generated by proteolysis of the precursors at basic residue cleavage sites. Prohormone convertases belonging to the subtilisin family of serine proteases are primarily responsible for processing at these "classical sites." In addition to the classical cleavages, a subset of bioactive peptides is generated by processing at "nonclassical" sites. The proteases responsible for these cleavages have not been well explored. Members of several metalloprotease families have been proposed to be involved in nonclassical processing. Among them, endothelin-converting enzyme-2 (ECE-2) is a good candidate because it exhibits a neuroendocrine distribution and an acidic pH optimum. To examine the involvement of this protease in neuropeptide processing, we purified the recombinant enzyme and characterized its catalytic activity. Purified ECE-2 efficiently processes big endothelin-1 to endothelin-1 by cleavage between Trp(21) and Val(22) at acidic pH. To characterize the substrate specificity of ECE-2, we used mass spectrometry with a panel of 42 peptides as substrates to identify the products. Only 10 of these 42 peptides were processed by ECE-2. A comparison of residues around the cleavage site revealed that ECE-2 exhibits a unique cleavage site selectivity that is related to but distinct from that of ECE-1. ECE-2 tolerates a wide range of amino acids in the P1-position and prefers aliphatic/aromatic residues in the P1'-position. However, only a small fraction of the aliphatic/aromatic amino acid-containing sites were cleaved, indicating that there are additional constraints beyond the P1- and P1'-positions. The enzyme is able to generate a number of biologically active peptides from peptide intermediates, suggesting an important role for this enzyme in the biosynthesis of regulatory peptides. Also, ECE-2 processes proenkephalin-derived bovine adrenal medulla peptides, and this processing leads to peptide products known to have differential receptor selectivity. Finally, ECE-2 processes PEN-LEN, an endogenous inhibitor of prohormone convertase 1, into products that do not inhibit the enzyme. Taken together, these results are consistent with an important role for ECE-2 in the processing of regulatory peptides at nonclassical sites.     

Cathepsin L is involved in proteolytic processing of the Hendra virus fusion protein

Proteolytic processing of paramyxovirus fusion (F) proteins is essential for the generation of a mature and fusogenic form of the F protein. Although many paramyxovirus F proteins are proteolytically processed by the cellular protease furin at a multibasic cleavage motif, cleavage of the newly emerged Hendra virus F protein occurs by a previously unidentified cellular protease following a single lysine at residue 109. We demonstrate here that the cellular protease cathepsin L is involved in converting the Hendra virus precursor F protein (F(0)) to the active F(1) + F(2) disulfide-linked heterodimer. To initially identify the class of protease involved in Hendra virus F protein cleavage, Vero cells transfected with pCAGGS-Hendra F or pCAGGS-SV5 F (known to be proteolytically processed by furin) were metabolically labeled and chased in the absence or presence of serine, cysteine, aspartyl, and metalloprotease inhibitors. Nonspecific and specific protease inhibitors known to decrease cathepsin activity inhibited proteolytic processing of Hendra virus F but had no effect on simian virus 5 F processing. We next designed shRNA oligonucleotides to cathepsin L which dramatically reduced cathepsin L protein expression and enzyme activity. Cathepsin L shRNA-expressing Vero cells transfected with pCAGGS-Hendra F demonstrated a nondetectable amount of cleavage of the Hendra virus F protein and significantly decreased membrane fusion activity. Additionally, we found that purified human cathepsin L processed immunopurified Hendra virus F(0) into F(1) and F(2) fragments. These studies introduce a novel mechanism for primary proteolytic processing of viral glycoproteins and also suggest a previously unreported biological role for cathepsin L.     

Post-translational processing of the highly processed,secreted periplasmic carbonic anhydrase of Chlamydomonas is largely conserved in transgenic tobacco

The periplasmic carbonic anhydrase (CA) gene CAH1 of Chlamydomonas reinhardtii codes for a highly processed secreted glycoprotein. The primary translation product of the CAH1 gene is targeted to the ER, where it is proteolytically processed to yield two different subunits, glycosylated, assembled into an active heterotetramer, and secreted. After replacing the target leader sequence with that from tobacco anionic peroxidase, expression of this gene in transgenic tobacco plants was investigated. SDS-PAGE gels of the purified protein from tobacco, showed that it migrated as a series of discrete bands (two large and one small) with slightly faster mobility than the comparable bands in the purified algal protein. The expressed protein in the plant was active, and staining with thymol and sulfuric acid confirmed that it was also glycosylated. The periplasmic CA1 (peri-CA1) also was found to be enriched in the intercellular fluid of transgenic tobacco, indicating it was secreted. The specific activity of the enzyme and its sensitivity to sulfonamide inhibitors were similar to that of the native algal enzyme. These results suggest that the post translational processing of Chlamydomonas peri-CA1 is largely conserved in a higher plant.     

Overexpression, purification, and partial characterization of Saccharomyces cerevisiae processing alpha glucosidase I

The gene encoding yeast processing alpha glucosidase I, CWH41, was overexpressed in Saccharomyces cerevisiae AH22, resulting in a 28-fold increase in expression of the soluble form of the enzyme. The soluble enzyme results from proteolytic cleavage between residues Ala 24 and Thr 25 of the transmembrane sequence of the membrane-bound form of the enzyme. This cleavage could be partially inhibited by addition of leupeptin and pepstatin during the enzyme isolation. The enzyme was purified to a final specific activity of 8550 U/mg protein using a combination of ammonium sulfate precipitation, anion exchange, concanavalin A, and gel filtration chromatography. The soluble form of the enzyme is a monomer with a molecular weight of 98 kDa by SDS-PAGE, and 89 kDa by gel filtration. The molecular weight decreased by approximately 5 kDa after treatment with N-glycosidase F, indicating that it is a glycoprotein. Soluble glucosidase I was sensitive to diethyl pyrocarbonate and not affected by N-ethylmaleimide, suggesting that mechanistically it is more similar to the plant than the mammalian form of the enzyme.     

Hetero- and autoprocessing of the extracellular metalloprotease (Mpr) in Bacillus subtilis

Most proteases are synthesized as inactive precursors which are processed by proteolytic cleavage into a mature active form, allowing regulation of their proteolytic activity. The activation of the glutamic-acid-specific extracellular metalloprotease (Mpr) of Bacillus subtilis has been examined. Analysis of Mpr processing in defined protease-deficient mutants by activity assay and Western blotting revealed that the extracellular protease Bpr is required for Mpr processing. pro-Mpr remained a precursor form in bpr-deficient strains, and glutamic-acid-specific proteolytic activity conferred by Mpr was not activated in bpr-deficient strains. Further, purified pro-Mpr was processed to an active form by purified Bpr protease in vitro. We conclude that Mpr is activated by Bpr in vivo, and that heteroprocessing, rather than autoprocessing, is the major mechanism of Mpr processing in vivo. Exchange of glutamic acid for serine in the cleavage site of Mpr (S93E) allowed processing of Mpr into its mature form, regardless of the presence of other extracellular proteases, including Bpr. Thus, a single amino acid change is sufficient to convert the Mpr processing mechanism from heteroprocessing to autoprocessing.     

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.     

Patterns of prohormone processing. Order revealed by a new procholecystokinin-derived peptide.

An 83-amino acid cholecystokinin peptide with a sulfated tyrosine and an amidated carboxyl terminus (CCK-83) was purified from human intestinal mucosa. The purified peptide was chemically characterized, and its bioactivity was compared to CCK-8. Several post-translational processing steps such as cleavage at basic residues, sulfation, and amidation are necessary to form biologically active cholecystokinin from its nascent prepropeptide. The discovery of CCK-83 gives new insight into the order of preprohormone processing. The processing of prepro-CCK appears to be in the order of: 1) signal peptidase cleavage, 2) tyrosine sulfation, 3) cleavage after a carboxyl-terminal pair of basic residues, 4) carboxypeptidase B-like cleavage of these basic residues, 5) amidation (which results in the formation of CCK-83), and 6) cleavage at monobasic residues by endopeptidases (which results in the smaller molecular forms of cholecystokinin). The characterization of biologically active CCK-83 with a sulfated tyrosine and an amidated carboxyl terminus establishes the site of signal peptidase action and suggests an order of post-translational modifications that give rise to the various molecular forms of cholecystokinin.     

Proteolytic cleavage of single-chain pro-urokinase induces conformational change which follows activation of the zymogen and reduction of its high affinity for fibrin

A plasminogen activator secreted from human kidney cells was highly purified by affinity chromatography on an anti-urokinase IgG-Sepharose column. The purified plasminogen activator was inactive and had a single-chain structure and a Mr of 50,000. It not only did not incorporate diisopropyl fluorophosphate, which reacts with active site serine residue in urokinase, but also did not bind to p-aminobenzamidine-immobilized CH-Sepharose, to which urokinase bind via its side-chain binding pocket present in active center. The plasminogen activator was converted to the active two-chain form with the same Mr by catalytic amounts of plasmin. Its potential enzymatic activity was quenched completely by anti-urokinase IgG, but not by anti-tissue plasminogen activator Ig. These results indicate that the plasminogen activator is an inactive proenzyme form of human urokinase. Therefore, the plasminogen activator was termed single-chain pro-urokinase. The cleavage of single-chain pro-urokinase by plasmin induced conformational change which followed the generation of reactive serine residue at active site, the increase enzyme activity and the reduction of its high affinity for fibrin. These findings suggest that conformational change occurs in both regions responsible for enzyme activity and affinity for fibrin upon activation of single-chain pro-urokinase.     

Vacuolar processing enzyme is self-catalytically activated by sequential removal of the C-terminal and N-terminal propeptides

A vacuolar processing enzyme (VPE) responsible for maturation of various vacuolar proteins is synthesized as an inactive precursor. To clarify how to convert the VPE precursor into the active enzyme, we expressed point mutated VPE precursors of castor bean in the pep4 strain of Saccharomyces cerevisiae. A VPE with a substitution of the active site Cys with Gly showed no ability to convert itself into the mature form, although a wild VPE had the ability. The mutated VPE was converted by the action of the VPE that had been purified from castor bean. Substitution of the conserved Asp-Asp at the putative cleavage site of the C-terminal propeptide with Gly-Gly abolished both the conversion into the mature form and the activation of the mutated VPE. In vitro assay with synthetic peptides demonstrated that a VPE exhibited activity towards Asp residues and that a VPE cleaved an Asp-Gln bond to remove the N-terminal propeptide. Taken together, the results indicate that the VPE is self-catalytically maturated to be converted into the active enzyme by removal of the C-terminal propeptide and subsequent removal of the N-terminal one.