Identification of monkey lung procathepsin D-II as a pepsinogen-C-like acid protease zymogen

Procathepsin D-II (Mr = 37 500) was purified from Japanese monkey lung at pH 7.0, and was shown to be converted to the active form, cathepsin D-II (Mr = 33 000) via an intermediate (Mr = 35 500) upon treatment at pH 3.0 and 14 degrees C. Procathepsin D-II was shown to be the inactive precursor of cathepsin D-II based on the following results: the former was inactive toward heat-denaturated casein at pH 5.4 whereas the latter was active; the former was not inactivated by diazoacetyl-DL-norleucine methyl ester in the presence of Cu2+ ion at pH 6.0 whereas the latter was inactivated rapidly under the same conditions; and the former had no affinity to pepstatin-Sepharose between pH 5 and 7 whereas the latter was adsorbed to it. With a rabbit antiserum against procathepsin D-II, cathepsin D-II, pepsinogen C and pepsin C of Japanese monkey were each found to give a single precipitin line which fused completely with each other on agarose plate. On the other hand, cathepsin D-I purified from the monkey lung, and pepsinogens A (I, II, III-1, III-2 and III-3) obtained from the monkey gastric mucosa failed to precipitate with the antiserum. With the antiserum against the monkey pepsinogen C, the same results were obtained. Further, procathepsin D-II and pepsinogen C were shown to have the same amino-terminal amino acid sequence, Ala-Val-Val-Lys-Val-Pro-Leu-Lys-Lys-Phe-Lys-. All these results indicate a strong similarity of procathepsin D-II and cathepsin D-II to pepsinogen C and pepsin C, respectively.     

Creation of a zymogen

Cells produce proteases as inactive zymogens. Here, we demonstrate that this tactic can extend beyond proteases. By linking the N and C termini of ribonuclease A, we obstruct the active site with the amino acid sequence recognized by plasmepsin II, a highly specific protease from Plasmodium falciparum. We generate new N and C termini by circular permutation. In the presence of plasmepsin II, a ribonuclease zymogen gains approximately 10(3)-fold in catalytic activity and maintains high conformational stability. We conclude that zymogen creation provides a new and versatile strategy for the control of enzymatic activity, as well as the potential development of chemotherapeutic agents.     

Further studies on lipopolysaccharide-sensitive serine protease zymogen (factor C): its isolation from Limulus polyphemus hemocytes and identification as an intracellular zymogen activated by alpha-chymotrypsin, not by trypsin

An intracellular serine protease zymogen, factor C, is an initiator in the hemolymph coagulation system of horseshoe crab. We purified this zymogen from the hemocytes of the American horseshoe crab, Limulus (L.) polyphemus, the objective being to compare its properties with those of the Japanese horseshoe crab, Tachypleus (T.) tridentatus, factor C. The purified zymogen L.-factor C showed similar properties to those of T.-factor C, in terms of molecular mass (123,000), amino acid composition (1,011 residues), subunit structure (two chains), and antigenicity. Like the zymogen T.-factor C, this zymogen was also activated autocatalytically in the presence of bacterial lipopolysaccharide (LPS) and its synthetic lipid A analogue. A most interesting finding is that both protease zymogens are rapidly activated by alpha-chymotrypsin or rat mast cell chymase, but not by trypsin. The active enzyme factor C showed alpha-thrombin-like specificity toward synthetic tripeptide substrates. This factor C was also strongly inhibited by an alpha-thrombin inhibitor, D-Phe-Pro-Arg-chloromethyl ketone. Thus, the enzymatic properties of factor C are similar to those of mammalian alpha-thrombin. On the other hand, the coagulation cascade system present in the hemocyte lysate was activated when chymotrypsin, free from LPS, was added to the lysate used to detect the endotoxins. The implication of our findings is that the chymotrypsin-catalyzed initiation of the horseshoe crab coagulation system is unique, since all known mammalian coagulation, fibrinolysis and complement systems are initiated by trypsin-like enzymes.     

Partial characterization of pepsins and gastricsins and their zymogens from human and toad gastric mucosae.

1. Three zymogens have been isolated from human gastric mucosae and two from the stomachs of the toad Caudiverbera caudiverbera. 2. Human zymogens I and III were immunologically related and cross-reacted with antisera prepared against porcine pepsinogen. The third, (II), showed no cross-reactivity. 3. Human zymogens I and III and toad zymogen ZII gave rise to two human pepsins and to a pepsin-like enzyme, respectively. 4. Human zymogen II (gastricsinogen) and toad zymogen ZI gave rise to human gastricsin and to a gastricsin-like enzyme respectively. 5. The toad enzymes showed much greater stability at neutral and alkaline pH values than the human enzymes.     

A ribonuclease zymogen activated by the NS3 protease of the hepatitis C virus

Translating proteases as inactive precursors, or zymogens, protects cells from the potentially lethal action of unregulated proteolytic activity. Here, we impose this strategy on bovine pancreatic ribonuclease (RNase A) by creating a zymogen in which quiescent ribonucleolytic activity is activated by the NS3 protease of the hepatitis C virus. Connecting the N-terminus and C-terminus of RNase A with a 14-residue linker was found to diminish its ribonucleolytic activity by both occluding an RNA substrate and dislocating active-site residues, which are devices used by natural zymogens. After cleavage of the linker by the NS3 protease, the ribonucleolytic activity of the RNase A zymogen increased 105-fold. Both before and after activation, the RNase A zymogen displayed high conformational stability and evasion of the endogenous ribonuclease inhibitor protein of the mammalian cytosol. Thus, the creation of ribonuclease zymogens provides a means to control ribonucleolytic activity and has the potential to provide a new class of antiviral chemotherapeutic agents.     

Pepsinogen B: the zymogen of pepsin B

1. Pepsinogen B, the precursor of pepsin B, has been isolated by ion-exchange chromatography and gel filtration from neutral extracts of pig gastric mucosa. The material possesses potential activity against acetyl-l-phenylalanyl-l-di-iodotyrosine and against gelatin but has little, if any, potential activity against haemoglobin. 2. The material appears homogeneous in the ultracentrifuge, but on gel filtration and on electrophoresis in starch gel it is shown to be contaminated with a small amount of material having potential activity against haemoglobin. On electrophoresis in starch gel also the material is shown to contain about equal amounts of two major components, both of which have potential activity against the synthetic substrate. Pepsin B has also been shown to contain two active components by electrophoresis under the same conditions. 3. The zymogen is similar to pepsinogen and pepsinogen C in its molecular weight and general physico-chemical properties, but differs from these zymogens in the nature of its N-terminal residues. It is possible that one of the components contains 1 mole of bound phosphate/mole. 4. The material is activated rapidly at pH2 and more slowly at pH4. At both pH values the kinetics of the activation reaction are complex.     

Prediction of the tertiary structure of the beta-secretase zymogen

beta-Secretase, also known as BACE, is a transmembrane aspartyl protease, which generates the N terminus of Alzheimer's disease amyloid beta-peptide. The activity of beta-secretase is the rate-limiting step of brain plaques production in vivo, and hence is a potential target for disease modifying drugs for Alzheimer's disease. To better understand the mechanism of action of beta-secretase and help explore novel strategies for drug discovery for Alzheimer's disease, it is important to elucidate the three-dimensional structure of its zymogen. Based on the X-ray structure of the enzyme's protease domain and the X-ray structure of pepsinogen, a model of the three-dimensional structure of the beta-secretase zymogen has been constructed. Comparison of the computed structure of pro-BACE with X-ray structures of pepsinogen and progastricsin (two other pro-aspartyl proteases) reveals a significant difference in the relationship of the pro-segment to the catalytic aspartates. In both pepsinogen and progastricsin a lysine side-chain in the pro-segment forms a salt bridge to the two catalytic aspartates, occupying the position normally occupied by a catalytic water. In the pro-BACE model there is no salt bridge, and the corresponding residue-a proline-does not interact at all with the catalytic residues. These findings can be used to elucidate the recent observations that the pro-domain of beta-secretase does not suppress activity as in a strict zymogen but does appear to facilitate proper folding of an active protease domain. The predicted three-dimensional structure of beta-secretase zymogen and the relevant findings might also provide useful insights for rational design of effective drugs against Alzheimer's disease.     

Recombinant rhizopuspepsinogen. Expression, purification, and activation properties of recombinant rhizopuspepsinogens

A cDNA clone, which contained the complete rhizopuspepsin structure and the putative proregion, was placed in three different Escherichia coli expression vectors for the synthesis of rhizopuspepsinogen (Rpg). Recombinant Rpgs which were expressed in the cytosol of E. coli as inclusion bodies (cRpg and tRpg) were not active. After solubilization in 6 M urea and refolding by rapid dilution, both of these Rpgs were purified to homogeneity. The third zymogen, pRpg, which was secreted to the periplasmic space of E. coli with an omp leader, was fully active and also was purified. The expression level of pRpg was higher (over 40 mg/liter culture) than that of cRpg (about 1.5 mg/liter culture). Amino-terminal sequence analysis of the zymogens revealed that cRpg and pRpg contain 40 and 51 residues of prosequence, respectively. tRpg, which was expressed under the control of T7 promoter, was synthesized at 500 mg/liter culture and was purified at 50 mg/liter culture. This zymogen contained, in addition to 51 residues of proregion, 16 residues inherited from the expression vector construction. All of these Rpgs spontaneously converted to rhizopuspepsin in solutions of pH less than 5. Each of the conversions was associated with a change of molecular weight as monitored in sodium dodecyl sulfate-polyacrylamide electrophoresis. At least one intermediate of conversion was observed in the pH range of 2 to 3 for both the cRpg and pRpg zymogens. For pRpg and tRpg, kinetic data demonstrated that the Rpg to rhizopuspepsin conversion was accomplished by a first order, unimolecular reaction at pH 2. The first order kinetic constants in this pH at 15 degrees C were 1.1 and 2.4 min-1 for pRpg and tRpg, respectively. The activation rate decreased as pH was raised above pH 2. At pH greater than 3.0, rhizopuspepsin-catalyzed, second-order activation also takes place. Consequently, the recombinant Rpgs are activated by either of two cleavage mechanisms as is the case for pepsinogen. These results also support the hypothesis that Rpg is synthesized in Rhizopus chinensis as a zymogen. Rpg in the host fungus is probably activated by an acid environment of pH less than 5 in the secretory granules to become rhizopuspepsin before secretion.     

Purificaton and characterization of a pepsinogen and its pepsin from proventriculus of the Japanese quail

A crude extract of the proventriculus of the Japanese quail gave at least five bands of peptic activity at pH 2.2 on polyacrylamide gel electrophoresis. The main component, constituting about 40% of the total acid protease activity, was purified to homogeneity by hydroxyapatite and DEAE-Sepharose column chromatographies. At below pH 4.0, the pepsinogen was converted to a pepsin, which had the same electrophoretic mobility as one of the five bands of peptic activity present in the crude extract. The molecular weights of the pepsinogen and the pepsin were 40 000 and 36 000, respectively. Quail pepsin was stable in alkali up to pH 8.5. The optimal pH of the pepsin on hemoglobin was pH 3.0. The pepsin had about half the milk-clotting activity of purified porcine pepsin, but the pepsinogen itself had no activity. The hydrolytic activity of quail pepsin on N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine was about 1% of that of porcine pepsin. Among the various protease inhibitors tested, only pepstatin inhibited the proteolytic activity of the pepsin. The amino acid composition of quail pepsinogen was found to be rather similar to that of chick pepsinogen C, and these two pepsinogens possessed common antigenicity.     

Rearranging the domains of pepsinogen.

Most eukaryotic aspartic protease zymogens are synthesized as a single polypeptide chain that contains two distinct homologous lobes and a pro peptide, which is removed upon activation. In pepsinogen, the pro peptide precedes the N-terminal lobe (designated pep) and the C-terminal lobe (designated sin). Based on the three-dimensional structure of pepsinogen, we have designed a pepsinogen polypeptide with the internal rearrangement of domains from pro-pep-sin (native pepsinogen) to sin-pro-pep. The domain-rearranged zymogen also contains a 10-residue linker designed to connect sin and pro domains. Recombinant sin-pro-pep was synthesized in Escherichia coli, refolded from 8 M urea, and purified. Upon acidification, sin-pro-pep autoactivates to a two-chain enzyme. However, the emergence of activity is much slower than the conversion of the single-chain zymogen to a two-chain intermediate. In the activation of native pepsinogen and sin-pro-pep, the pro region is cleaved at two sites between residues 16P and 17P and 44P and 1 successively, and complete activation of sin-pro-pep requires an additional cleavage at a third site between residues 1P and 2P. In pepsinogen activation, the cleavage of the first site is rate limiting because the second site is cleaved more rapidly to generate activity. In the activation of sin-pro-pep, however, the second site is cleaved slower than the first, and cleavage of the third site is the rate limiting step.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of a pepsin C zymogen produced by human breast tissues.

An aspartic proteinase present in cyst fluid from women with gross cystic breast disease was purified by a procedure involving affinity chromatography on pepstatin-agarose and size-exclusion high performance liquid chromatography. The amino-terminal sequence of the purified breast proteinase was identical to that corresponding to gastric pepsinogen C. Additional data on cleavage specificity, pH optimum, and immunological properties supported the close relationship between both molecules. Northern blot analysis and polymerase chain reaction amplification studies performed on RNAs obtained from normal and pathological breast tissues demonstrated that the protein is produced by mammary carcinomas and cysts, but not by the normal resting mammary gland. Immunohistochemical staining of paraffin-embedded tissue sections confirmed the existence of a subset of tumors that have the ability to synthesize and secrete this pepsin zymogen. On the basis of these results, we suggest that pepsinogen C expression by human mammary epithelium may be involved in the development of breast diseases, being also of potential interest as a biochemical marker of the hormonal imbalance underlying these pathologies.     

A method for isolating human plasma lecithin:cholesterol acyltransferase without using anti-apolipoprotein D, and its characterization

A method for isolating human plasma lecithin:cholesterol acyltransferase (EC 2.3.1.43) purified more than 50 000-fold is described. The crude enzyme obtained by initial ammonium sulfate and citric acid treatment of 21 of human plasma is subjected to repeated DEAE-cellulose chromatography to yield a preparation purified more than 600-fold. Hydroxyapatite chromatography of concentrates from this fraction using 0.5 mM phosphate buffer, pH 6.8, yields enzyme preparations purified more than 50 000-fold. The enzyme isolated by this procedure was free of apolipoprotein D, as shown by the absence of an arc in immunodiffusion with anti-apolipoprotein D. The enzyme showed a single band by polyacrylamide gel electrophoresis in the presence and absence of SDS. Upon analytical isoelectrofocusing the enzyme separated into three iso forms with isoelectric points below that of egg albumin (pI 4.6). The enzyme was characterized by a high content of glutamic acid, leucine and glycine, and a lower content of tyrosine. The enzyme possessed both transferase and phospholipase A2 activities and both activities show absolute requirement for apolipoprotein A-I. The purified enzyme was injected into Balb/c mice and the antiserum reacted both with the purified enzyme and normal human serum in immunodiffusion, giving lines of complete identity. The antiserum gave no precipitation lines with albumin or apolipoprotein D, providing additional evidence for the absence of apolipoprotein D in the purified enzyme. The gamma-globulin isolated from the antiserum inhibited human lecithin:cholesterol acyltransferase activity.     

Autocatalytic processing of the streptococcal cysteine protease zymogen: processing mechanism and characterization of the autoproteolytic cleavage sites.

The autocatalytic processing of the streptococcal cysteine protease zymogen (proSCP) to active streptococcal cysteine protease (SCP) was investigated in vitro using purified protein from Streptococcus pyogenes strain B220. It was found that the autocatalytic maturation of the zymogen proceeds through the sequential appearance of at least six intermediates, five of which were characterized through a combination of N-terminal sequencing and MS. Intermediates were identified as resulting from cleavages after Lys26, Asn41, Lys101, Ala112, and Lys118. Time-course studies of the proSCP processing gave a sigmoidal activity profile and indicated that proSCP catalyses its own transformation, mainly via an intermolecular processing mechanism. A similar sequential appearance of intermediates was observed when inactive Cys192Ser proSCP was treated with native, enzymatically active SCP, thus demonstrating that the maturation can exclusively proceed by a bimolecular mechanism. It was shown that proSCP, but not mature SCP, immobilized on a Sepharose resin is capable of liberating itself from the column, indicating that the zymogen is also capable of intramolecular processing. In order to test whether the amino acid sequences at the processing sites could be used for developing new, specific substrates, 3-amino benzoic acid octapeptide derivatives based on all five characterized amino acid sequences from the autoprocessing cleavage sites were synthesized and tested for activity. The 3-amino benzoic acid derivatives have kcat/KM values ranging from 1200 to 7700.M-1.s-1, making them very good endopeptidase substrates for SCP.     

Antibody production to choline acetyltransferase purified from human brain

Choline acetyltransferase (CAT) was isolated from human caudate and putamen. The enzyme was highly purified by a series of steps involving fractionation by protamine sulfate and ammonium sulfate followed by chromatography on DEAE-Sephadex, hydroxyapatite and carboxymethyl cellulose columns. The isolated CAT gave a single protein band on polyacrylamide gel electrophoresis at pH 8.3 which corresponded with CAT activity. A single band was also obtained at pH 6.8. Rabbit antiserum was prepared to the purified homogeneous CAT from carboxymethyl cellulose columns. It exhibited a single sharp precipitin band on double diffusion tests on Ouchterlony I.D. plates when tested against the partially purified hydroxyapatite enzyme. On preincubation, the antiserum inhibited CAT activity to 50–60% of control independently of the concentration of enzymatic protein. Normal rabbit serum neither produced a precipitin band on double diffusion tests nor inhibited the CAT activity on incubation. The anti-CAT rabbit antibody thus appeared to be specific.     

Seminal pepsinogen C is not identical with, but is very similar to gastric pepsinogen C

Human seminal pepsinogen C has been purified and compared with gastric pepsinogen C. The two zymogens cannot be distinguished by amino acid compositions and sequences of the first 28 N-terminal amino acid residues are identical. Apparent immunological identity is observed with polyclonal antisera. Monoclonal antibodies toward seminal pepsinogen C have been produced. One is able to recognize a non-carbohydrate antigenic determinant only present in seminal pepsinogen C.     

Large-scale purification of calf pancreatic zymogen granule membranes.

A protocol for isolating milligram quantities of highly purified zymogen granule membranes from calf pancreas was developed. The method provides a fivefold enriched zymogen granule fraction that is virtually free from major isodense contaminants, such as mitochondria and erythrocytes. Isolated granules are osmotically stable in isosmotic KCl buffers with half-lives between 90 and 120 min. They display specific ion permeabilities that can be demonstrated using ionophore probes to override intrinsic control mechanisms. A Cl- conductance, a Cl-/anion exchanger, and a K+ conductance are found in the zymogen granule membrane, as previously reported for rat pancreatic, rat parotid zymogen granules, and rabbit pepsinogen granules. Lysis of calf pancreatic secretory granules in hypotonic buffers and subsequent isolation of pure zymogen granule membranes yield about 5-10 mg membrane protein from approximately 1000 ml pancreas homogenate. The purified zymogen granule membranes are a putative candidate for the rapid identification and purification of epithelial Cl- channels and regulatory proteins, since they contain fewer proteins than plasma membranes.     

Circular zymogens of human ribonuclease 1

The endogenous production of enzymes as zymogens provides a means to control catalytic activities. Here, we describe the heterologous production of ribonuclease 1 (RNase 1), which is the most prevalent secretory ribonuclease in humans, as a zymogen. In folded RNase 1, the N and C termini flank the enzymic active site. By using intein‐mediated cis‐splicing, we created circular proteins in which access to the active site of RNase 1 is obstructed by an amino‐acid sequence that is recognized by the HIV‐1 protease. Installing a sequence that does not perturb the RNase 1 fold led to only modest inactivation. In contrast, the ancillary truncation of residues from each terminus led to a substantial decrease in the catalytic activity of the zymogen with the maintenance of thermostability. For optimized zymogens, activation by HIV‐1 protease led to a > 10⁴‐fold increase in ribonucleolytic activity at a rate comparable to that for the cleavage of endogenous viral substrates. Molecular modeling indicated that these zymogens are inactivated by conformational distortion in addition to substrate occlusion. Because protease levels are elevated in many disease states and ribonucleolytic activity can be cytotoxic, RNase 1 zymogens have potential as generalizable prodrugs.     

An improved method of isolation of rat pancreatic prokallikrein: Characterization of the zymogen and of the kallikrein produced by trypsin activation

A prokallikrein was purified 1600-fold from rat pancreatic tissue in an overall yield of 40% by a simple four-stage procedure. The final and crucial step was immunoaffinity chromotography utilizing antibody raised to a very small amount of prokallikrein. Both the pure zymogen and the active kallikrein generated from it by trypsin activation are single chain species with M_r values of 38 400±300 and 35 500±400, respectively. Valine is the N-terminal amino acid residue of prokallikrein. The zymogen Was comparatively stable both to autoactivation and denaturation with respect to temperature and pH. The kallikrein produced by trypsin activation of the zymogen was similar in some of its catalytic properties to the kallikrein purified from autolyzed rat pancreas but the two species differed in their susceptibility to substrate activation.     

Isolation and characterization of pepsinogen from Trimeresurus flavoviridis (Habu snake)

Pepsinogen was isolated from the gastric mucosa of Trimeresurus flavoviridis (Habu snake) by DEAE-cellulose and DEAE-Sepharose ion-exchange chromatographies, and Sephacryl S-200 gel-chromatography. The yield calculated from the crude extract was 29% with 6.2-fold purification. The purified pepsinogen gave a single band on both native- and SDS-PAGE. As no other active enzyme was detected on the chromatographies, it was concluded that the Habu snake has one major pepsinogen. The molecular mass of the pepsinogen was estimated to be 38 kDa by SDS-PAGE. The sequence of the N-terminal 26 amino acid residues was determined and compared with those of other pepsinogens. The N-terminal structure of Habu snake pepsinogen was more homologous with those of mammalian pepsinogens C than those of mammalian pepsinogens A. The pepsinogen was rapidly converted to pepsin by way of an intermediate form induced by acidification. The optimum pH of Habu snake pepsin for bovine hemoglobin was 1.5-2.0, and it retained full activity at pH 6.2 and 30 degrees C on incubation for 30 min. The optimum temperature for the snake pepsin was 50 degrees C and it was stable at 40 degrees C on incubation for 10 min. The proteolytic activity of the pepsin toward bovine hemoglobin was about two times higher than that of porcine pepsin A, however, the activity toward oxidized bovine insulin B-chain was lower than that of porcine pepsin A, and it did not hydrolyze oligopeptides. The specificity for oxidized bovine insulin B-chain of the pepsin was different from that of porcine pepsin A. Habu snake pepsin was inhibited by pepstatin A but not by serine, cysteine, or metallo protease inhibitors.