A mutated bovine prochymosin zymogen can be activated without proteolytic processing at low pH

As a first step towards understanding how the zymogen structure of prochymosin contributes to the process by which active enzyme is produced, we altered the nucleotide sequence which encodes the amino-terminal (or propeptide) region of the protein. Of the two sites for autoproteolysis of prochymosin, one where pseudochymosin is formed at a pH of 2 and the other where chymosin is formed at pH 4-5, we changed the former by removing one codon and changing two other codons. This genetically modified prochymosin was proteolytically processed and activated normally at pH 4.5. However, at pH 2.0 we observed only partial activation of the zymogen and found no evidence of proteolytic processing. The properties of this engineered prochymosin suggest that zymogen activation does not require proteolysis and that the two different zymogen processing sites can function independently from one another.     

Investigations on the activation of bovine prochymosin.

Activation of prochymosin at pH below 2.5 results in formation of the active enzyme pseudochymosin by proteolytic cleavage of the bond 27--28. Pseudochymosin is 15 amino acid residues longer than chymosin. It is the final activation product at low pH, whereas chymosin is formed by activation between pH 4 and 5. Pseudochymosin is converted to chymosin when it is brought to pH 5.5. Our present knowledge does not allow quantitative evaluation of the possible reactions involved in formation of pseudochymosin, but the course of activation at pH 2 is in accordance with an intermolecular reaction between two zymogen molecules as the predominant reaction. We find indications of an intramolecular reaction when intermolecular reactions are prevented by immobilization of the zymogen.     

Isolation and partial characterization of prochymosin and chymosin from cat

Extracts of cat gastric mucosa contain a zymogen that after activation shows partial immunochemical identity with chymosin (EC 3.4.23.4) from calf. Cat prochymosin has been purified by column chromatography and gel filtration, and cat chymosin was obtained after acid activation of the zymogen. The enzyme showed the optimum of general proteolytic activity at pH 2.5. The amino acid compositions of cat prochymosin and chymosin were similar to those of the corresponding proteins from calf. The first 27 residues of both cat prochymosin and chymosin have been sequenced. Among these 54 positions only 13 differences have been observed between the proteins from cat and calf. The results support the hypothesis that the chymosins form a group of neonatal gastric proteases with high milk-clotting activity, but with such weak general proteolytic activity that postnatal uptake of IgG is not hindered.     

Activation studies of the multiple forms of prochymosin (prorennin).

Activation of the four separate components of prochymosin (prorennin) at pH 5.0 demonstrated that each zymogen was the precursor to an electrophoretically distinct chymosin (rennin). When the increase in milk-clotting activity with time was analysed, the mechanism of activation of unfractionated prochymosin, individual prochymosin components, and a mixture of the prochymosin fractions at pH 5.0 was shown to follow essentially autocatalytic kinetics. The activation of prochymosin C was completed in 70 h, whereas the other three fractions each required more than 110 h for complete activation under the same conditions. Intact prochymosin, the mixture of four components and prochymosin C were activated at similar rates. Interaction of the individual fractions during activation is suggested to explain the increased rate of the activation for the mixture. Comparison of autocatalytic activation of unfractionated prochymosin purified chromatographically at pH 6.7 and 5.7 demonstrated an increased rate of reaction of the zymogen prepared at the lower pH value. The possibility that prochymosin became susceptible to activation during preparation at pH values slightly below 6.0, as a result of changes in the proportion of the components or a conformational change and exposure of the active site, is discussed.     

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.     

A basic residue at position 36p of the propeptide is not essential for the correct folding and subsequent autocatalytic activation of prochymosin.

Position 36p in the propeptides of gastric aspartic proteinases is generally occupied by lysine or arginine. This has led to the conclusion that a basic residue at this position, which interacts with the active-site aspartates, is essential for folding and activation of the zymogen. Lamb prochymosin has been shown by cDNA cloning to possess glutamic acid at 36p. To investigate the effect of this natural mutation which appears to contradict the proposed role of this residue, calf and lamb prochymosins and their two reciprocal mutants, K36pE and E36pK, respectively, were expressed in Escherichia coli, refolded in vitro, and autoactivated at pH 2 and 4.7. All four zymogens could be activated to active chymosin and, at both pH values, the two proteins with Glu36p showed higher activation rates than the two Lys36p forms. Glu36p was also demonstrated in natural prochymosin isolated from the fourth stomach of lamb, as well as being encoded in the genomes of sheep, goat and mouflon, which belong to the subfamily Caprinae. A conserved basic residue at position 36p of prochymosin is thus not obligatory for its folding or autocatalytic activation. The apparently contradictory results for porcine pepsinogen A [Richter, C., Tanaka, T., Koseki, T. & Yada, R.Y. (1999) Eur. J. Biochem. 261, 746-752] can be reconciled with those for prochymosin. Lys/Arg36p is involved in stabilizing the propeptide-enzyme interaction, along with residues nearer the N-terminus of the propeptide, the sequence of which varies between species. The relative contribution of residue 36p to stability differs between pepsinogen and prochymosin, being larger in the former.     

Activation of spin-labeled chicken pepsinogen

Chicken pepsinogen has been spin-labeled by the attachment of four nitroxides to epsilon-amino groups near the protein's amino terminus. Acidification results in a bond cleavage, generating a nonlabeled, enzymatically active protein. Electron spin resonance spectra of the spin-labeled zymogen, acidified in the presence or absence of pepstatin, are identical and indicate that the nitroxides are quite mobile, compared to the nonacidified zymogen. This mobilization is interpreted as the freeing of the peptide to which the spin-labels are attached, from the protein, subsequent to the acidification that causes a peptide bond cleavage. The rate at which the peptide leaves the protein is 1 order of magnitude slower than the cleavage of the peptide bond, measured by the rate of appearance of milk-clotting activity (first-order rate constants of 0.3 min-1 vs. 6 min-1 at pH 2, 22 degrees C). The inclusion of pepstatin, at molar ratios above 2 during activation, decreases the rate of peptide leaving. These observations, and those previously reported for activation of spin-labeled pig pepsinogen, are incorporated into a model of pepsinogen activation.     

Processing and sorting of the prohormone convertase 2 propeptide

The prohormone convertases (PCs) are synthesized as zymogens whose propeptides contain several multibasic sites. In this study, we investigated the processing of the PC2 propeptide and its function in the regulation of PC2 activity. By using purified pro-PC2 and directed mutagenesis, we found that the propeptide is first cleaved at the multibasic site separating it from the catalytic domain (primary cleavage site); the intact propeptide thus generated is then sequentially processed at two internal sites. Unlike the mechanism described for furin, our mutagenesis studies show that internal cleavage of the propeptide is not required for activation of pro-PC2. In addition, we identified a point mutation in the primary cleavage site that does not prevent the folding nor the processing of the zymogen but nevertheless results in the generation of an inactive PC2 species. These data suggest that the propeptide cleavage site is directly involved in the folding of the catalytic site. By using synthetic peptides, we found that a PC2 propeptide fragment inhibits PC2 activity, and we identified the inhibitory site as the peptide sequence containing basic residues at the extreme carboxyl terminus of the primary cleavage site. Finally, our study supplies information concerning the intracellular fate of a convertase propeptide by providing evidence that the PC2 propeptide is generated and is internally processed within the secretory granules. In agreement with this localization, an internally cleaved propeptide fragment could be released by stimulated secretion.     

Activation profiles of the zymogen of aspergilloglutamic peptidase

Aspergilloglutamic peptidase produced by Aspergillus niger var. macrosporus belongs to the novel glutamic peptidase family. Its zymogen is autocatalytically activated under acidic conditions to the mature enzyme with a two-chain structure. Analyses by SDS-PAGE and mass spectrometry of the activation products of the recombinant zymogen showed that the major pathway of activation includes initial fast cleavage at Glu12-Ala13, followed by stepwise cleavages in the N-terminal and intervening propeptide regions. Essentially the same activation profile was obtained with the recombinant zymogen lacking the N-terminal 12-aa sequence. The missing region includes the most prominent cluster of basic residues of the propeptide, indicating low importance of this cluster for activation and refolding of the zymogen.     

Autoproteolytic cleavage and activation of human acid ceramidase

Herein we report the mechanism of human acid ceramidase (AC; N-acylsphingosine deacylase) cleavage and activation. A highly purified, recombinant human AC precursor underwent self-cleavage into alpha and beta subunits, similar to other members of the N-terminal nucleophile hydrolase superfamily. This reaction proceeded with first order kinetics, characteristic of self-cleavage. AC self-cleavage occurred most rapidly at acidic pH, but also at neutral pH. Site-directed mutagenesis and expression studies demonstrated that Cys-143 was an essential nucleophile that was required at the cleavage site. Other amino acids participating in AC cleavage included Arg-159 and Asp-162. Mutations at these three amino acids prevented AC cleavage and activity, the latter assessed using BODIPY-conjugated ceramide. We propose the following mechanism for AC self-cleavage and activation. Asp-162 likely forms a hydrogen bond with Cys-143, initiating a conformational change that allows Arg-159 to act as a proton acceptor. This, in turn, facilitates an intermediate thioether bond between Cys-143 and Ile-142, the site of AC cleavage. Hydrolysis of this bond is catalyzed by water. Treatment of recombinant AC with the cysteine protease inhibitor, methyl methanethiosulfonate, inhibited both cleavage and enzymatic activity, further indicating that cysteine-mediated self-cleavage is required for ceramide hydrolysis.     

Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation.

We isolated a cDNA encoding a functional human thrombin receptor by direct expression cloning in Xenopus oocytes. mRNA encoding this receptor was detected in human platelets and vascular endothelial cells. The deduced amino acid sequence revealed a new member of the seven transmembrane domain receptor family with a large amino-terminal extracellular extension containing a remarkable feature. A putative thrombin cleavage site (LDPR/S) resembling the activation cleavage site in the zymogen protein C (LDPR/I) was noted 41 amino acids carboxyl to the receptor's start methionine. A peptide mimicking the new amino terminus created by cleavage at R41 was a potent agonist for both thrombin receptor activation and platelet activation. "Uncleavable" mutant thrombin receptors failed to respond to thrombin but were responsive to the new amino-terminal peptide. These data reveal a novel signaling mechanism in which thrombin cleaves its receptor's amino-terminal extension to create a new receptor amino terminus that functions as a tethered ligand and activates the receptor.     

Matrix metalloproteinase 3 (stromelysin) activates the precursor for the human matrix metalloproteinase 9.

Matrix metalloproteinase 9 (MMP-9), also known as 92-kDa gelatinase/type IV collagenase, is secreted from neutrophils, macrophages, and a number of transformed cells in zymogen form. Here we report that matrix metalloproteinase 3 (MMP-3/stromelysin) is an activator of the precursor of matrix metalloproteinase 9 (proMMP-9). MMP-3 initially cleaves proMMP-9 at the Glu40-Met41 bond located in the middle of the propeptide to generate an 86-kDa intermediate. Cleavage of this bond triggers a change in proMMP-9 that renders the Arg87-Phe88 bond susceptible to the second cleavage by MMP-3, resulting in conversion to an 82-kDa form. alpha 2-Macroglobulin binding studies of partially activated MMP-9 demonstrate that the 82-kDa species is proteolytically active, but not the initial intermediate of 86 kDa. This stepwise activation mechanism of proMMP-9 is analogous to those of other members of the MMP family, but the action of MMP-3 on proMMP-9 is the first example of zymogen activation that can be triggered by another member of the MMP family. The results imply that MMP-3 may be an effective activator of proMMP-9 in vivo.     

Studies on the irreversible step of pepsinogen activation

The bond cleavage step of pepsinogen activation has been investigated in a kinetic study in which the denatured products of short-term acidifications were separated on SDS-polyacrylamide gels and the peptide products were quantitated by densitometry. Although several peptide products were observed, under the conditions of the experiments (pH values between 2.0 and 2.8, 22 degrees C), the only one that was a product of an initial bond cleavage was the 44-residue peptide, which upon removal from pepsinogen yields pepsin. The rate constant for this bond cleavage is 0.015 s-1 at pH 2.4, which is the same as that at which the alkali-stable potential activity of pepsinogen had been found to convert to the alkali-labile activity of pepsin. When the conversion of zymogen to enzyme was followed by the change in fluorescence of adsorbed 6-(p-toluidinyl)naphthalene-2-sulfonate (TNS), the rate of change in TNS fluorescence was the same as the conversion to alkali lability. However, pepstatin blocked the bond cleavage of pepsinogen to pepsin, but it permitted the fluorescence change to proceed. In fact, it accelerated the apparent rate of change of TNS fluorescence by shifting the pKa of an essential conjugate acid from 1.7 to 2.6. The conversion to alkali lability, therefore, may be considered to be a composite of a relatively slow conformational change (at the measured rate), followed immediately by a relatively fast bond cleavage.     

Conformational change that accompanies pepsinogen activation observed in real time by fluorescence energy transfer

Pig pepsinogen has been reacted with N-carboxymethylisatoic anhydride to form N-carboxymethyl-anthraniloyl-(CMA-) pepsinogen, derivatized at Lysp18, Lysp23, Lysp27, Lysp30, and Lys320. Conformational change associated with activation was detected by following energy transfer from tryptophan residues of the pepsin moiety, excited at 295 nm, to CMA groups, monitored by emission above 415 nm. Efficiency of this energy transfer is a measure of conformational change. For this zymogen derivative the change in efficiency occurs with a first order rate constant of 0.041 s-1 at pH 2.4, 22 degrees, which equals the rate at which, following acidification, alkali-stable potential activity becomes alkali-labile. For the native zymogen the rate of this conversion had been shown to be identical to the rate of cleavage of the scissile bond of pepsinogen. Therefore, the correspondence in this derivative of the rates of conversion to alkali lability and change in energy transfer demonstrates that a conformational change accompanies the peptide bond cleavage of activation.     

Defective propeptide processing of blood clotting factor IX caused by mutation of arginine to glutamine at position -4

Blood clotting factor IX is synthesized as a precursor polypeptide that would be expected to be proteolytically cleaved in at least two positions during maturation to remove the prepeptide and propeptide regions. We show that a point mutation causing hemophilia B changes the amino acid at position -4 in the propeptide region of factor IX from an arginine to a glutamine, which results in the expression of a stable longer protein with 18 additional amino acids of the N-terminal propeptide region still attached. This suggests that in the normal maturation of factor IX the signal peptidase cleaves the peptide bond between amino acid residues -18 and -19, generating an unstable profactor IX intermediate. Further proteolytic processing to the mature factor IX depends on the arginine residue at -4. The significance of the homologous arginine residue in other processed proteins is discussed.     

Human cationic trypsinogen. Arg(117) is the reactive site of an inhibitory surface loop that controls spontaneous zymogen activation.

Mutation of Arg(117), an autocatalytic cleavage site, is the most frequent amino acid change found in the cationic trypsinogen (Tg) of patients with hereditary pancreatitis. In the present study, the role of Arg(117) was investigated in wild-type cationic Tg and in the activation-resistant Lys(15) --> Gln mutant (K15Q-Tg), in which Tg-specific properties of Arg(117) can be examined selectively. We found that trypsinolytic cleavage of the Arg(117)-Val(118) bond did not proceed to completion, but due to trypsin-catalyzed re-synthesis an equilibrium was established between intact Tg and its cleaved, two-chain form. In the absence of Ca(2+), at pH 8.0, the hydrolysis equilibrium (K(hyd) = [cleaved Tg]/[intact Tg]) was 5.4, whereas 5 mm Ca(2+) reduced the rate of cleavage at Arg(117) at least 20-fold, and shifted K(hyd) to 0.7. These observations indicate that the Arg(117)-Val(118) bond exhibits properties analogous to the reactive site bond of canonical trypsin inhibitors and suggest that this surface loop might serve as a low affinity inhibitor of zymogen activation. Consistent with this notion, autoactivation of cationic Tg was inhibited by the cleaved form of K15Q-Tg, with an estimated K(i) of 80 microm, while no inhibition was observed with K15Q-Tg carrying the Arg(117) --> His mutation. Finally, zymogen breakdown due to other trypsinolytic pathways was shown to proceed almost 2000-fold slower than cleavage at Arg(117). Taken together, the findings suggest two independent, successively functional trypsin-mediated mechanisms against pathological Tg activation in the pancreas. At low trypsin concentrations, cleavage at Arg(117) results in inhibition of trypsin, whereas high trypsin concentrations degrade Tg, thus limiting further zymogen activation. Loss of Arg(117)-dependent trypsin inhibition can contribute to the development of hereditary pancreatitis associated with the Arg(117) --> His mutation.     

Activation of the furin endoprotease is a multiple-step process: requirements for acidification and internal propeptide cleavage.

Activation of furin requires autoproteolytic cleavage of its 83-amino acid propeptide at the consensus furin site, Arg-Thr-Lys-Arg107/. This RER-localized cleavage is necessary, but not sufficient, for enzyme activation. Rather, full activation of furin requires exposure to, and correct routing within, the TGN/endosomal system. Here, we identify the steps in addition to the initial propeptide cleavage necessary for activation of furin. Exposure of membrane preparations containing an inactive RER-localized soluble furin construct to either: (i) an acidic and calcium-containing environment characteristic of the TGN; or (ii) mild trypsinization at neutral pH, resulted in the activation of the endoprotease. Taken together, these results suggest that the pH drop facilitates the removal of a furin inhibitor. Consistent with these findings, following cleavage in the RER, the furin propeptide remains associated with the enzyme and functions as a potent inhibitor of the endoprotease. Co-immunoprecipitation studies coupled with analysis by mass spectrometry show that release of the propeptide at acidic pH, and hence activation of furin, requires a second cleavage within the autoinhibitory domain at a site containing a P6 arginine (-Arg70-Gly-Val-Thr-Lys-Arg75/-). The significance of this cleavage in regulating the compartment-specific activation of furin, and the relationship of the furin activation pathway to those of other serine endoproteases are discussed.     

The activation of matriptase requires its noncatalytic domains,serine protease domain,and its cognate inhibitor

The activation of matriptase requires proteolytic cleavage at a canonical activation motif that converts the enzyme from a one-chain zymogen to an active, two-chain protease. In this study, matriptase bearing a mutation in its catalytic triad was unable to undergo this activational cleavage, suggesting that the activating cleavage occurs via a transactivation mechanism where interaction between matriptase zymogen molecules leads to activation of the protease. Using additional point and deletion mutants, we showed that activation of matriptase requires proteolytic processing at Gly-149 in the SEA domain of the protease, glycosylation of the first CUB domain and the serine protease domain, and intact low density lipoprotein receptor class A domains. Its cognate inhibitor, hepatocyte growth factor activator inhibitor-1, may also participate in the activation of matriptase, based on the observation that matriptase activation did not occur when the protease was co-expressed with hepatocyte growth factor activator inhibitor-1 mutated in its low density lipoprotein receptor class A domain. These results suggest that besides matriptase catalytic activity, matriptase activation requires post-translational modification of the protease, intact noncatalytic domains, and its cognate inhibitor.     

Prochymosin activation by non-aspartic proteinases

Prochymosin can be converted into chymosin by an action of external proteinases. Thus, thermolysin at pH 5.05 converts calf prochymosin into active Phe-chymosin, which is one amino acid longer than chymosin from the N-terminus with a yield of 73%. Even better results were achieved with prochymosin activation by Legionella pneumophila metalloproteinase. Apparently the stretch of prochymosin polypeptide chain adjacent to the normally observed activation point becomes available for an attack by an external proteinase at pH 5.0–6.0. These data indicate that the intermolecular activation pathway might be of physiological importance.     

Furin directly cleaves proMMP-2 in the trans-Golgi network resulting in a nonfunctioning proteinase

Proprotein convertases play an important role in tumorigenesis and invasiveness. Here, we report that a dibasic amino acid convertase, furin, directly cleaves proMMP-2 within the trans-Golgi network leading to an inactive form of matrix metalloproteinase-2 (MMP-2). Co-transfection of COS-1 cells with both proMMP-2 and furin cDNAs resulted in the cleavage of the N-terminal propeptide of proMMP-2. The molecular mass of cleaved MMP-2 (63 kDa), detected in both cell lysates and conditioned medium, is between the intermediate and fully activated forms of MMP-2 induced by membrane type 1-MMP. Furin-cleaved MMP-2 does not possess proteolytic activity as examined in a cell-free assay. Treatment of transfected cells with a furin inhibitor resulted in a dose-dependent inhibition of proMMP-2 cleavage; recombinant tissue inhibitor of metalloproteinase-2, which binds to the active site of membrane type 1-MMP, had no inhibitory effect. Site-directed mutagenesis of amino acids in the furin consensus recognition motif of proMMP-2(R69KPR72) prevented propeptide cleavage, thereby identifying the scissile bond and characterizing the basic amino acids required for cleavage. Other experimental observations were consistent with intracellular furin cleavage of proMMP-2 in the trans-Golgi network. The furin cleavage site in other proMMPs was examined. MMP-3, which contains the RXXR furin consensus sequence, was cleaved in furin co-transfected cells, whereas MMP-1, which lacks an RXXR consensus sequence, was not cleaved. In conclusion, we report the novel observation that furin can directly cleave the RXXR amino acid sequence in the propeptide domain of proMMP-2 leading to inactivation of the enzyme.