Analysis of the activation process of porcine procarboxypeptidase B and determination of the sequence of its activation segment

The molecular events which lead to the proteolytic transformation of porcine procarboxypeptidase B (PCPB) in carboxypeptidase B (CPB) have been determined. Among pancreatic and other tested proteinases, trypsin is the only one capable of generating carboxypeptidase B activity from the zymogen, in vitro. In the first step of this process, trypsin produces cleavage at the boundary between the activation region and the CPB region. Subsequently, a definite sequence of cleavages occurs at the C-terminal end of the released activation segment of 95 residues, giving rise to characteristic intermediates and to a proteolytically resistant activation fragment of 81 residues. In this process, the newly formed CPB participates in the quick-trimming of the released activation peptides. Only a single CPB species is formed in the activation process. This fact and the inability of the released activation peptides to inhibit CPB--and, therefore, their inability to slow down the kinetics of appearance of CPB activity--are two important characteristics differentiating between the activation processes of procarboxypeptidases A and B. The sequence of the 95 residues (MW = 12,835) of the activation region of porcine PCPB has also been deduced, largely from the information obtained by Edman degradation of its fragments and in part by considerations of homology with the rat precursor. The porcine PCPB activation region contains a high percentage of acidic residues, lacks cysteines, methionines, and side-chain posttranslational modifications, and presents a low but significant homology (31%) with the corresponding sequence of porcine procarboxypeptidase A.     

Mapping the pro-region of carboxypeptidase B by protein engineering. Cloning, overexpression, and mutagenesis of the porcine proenzyme.

The proteolytic processing of pancreatic procarboxypeptidase B to a mature and functional enzyme is much faster than that of procarboxypeptidase A1. This different behavior has been proposed to depend on specific conformational features at the region that connects the globular domain of the pro-segment to the enzyme and at the contacting surfaces on both moieties. A cDNA coding for porcine procarboxypeptidase B was cloned, sequenced, and expressed at high yield (250 mg/liter) in the methylotrophic yeast Pichia pastoris. To test the previous hypothesis, different mutants of the pro-segment at the putative tryptic targets in its connecting region and at some of the residues contacting the active enzyme were obtained. Moreover, the complete connecting region was replaced by the homologous sequence in procarboxypeptidase A1. The detailed study of the tryptic processing of the mutants shows that limited proteolysis of procarboxypeptidase B is a very specific process, as Arg-95 is the only residue accessible to tryptic attack in the proenzyme. A fast destabilization of the connecting region after the first tryptic cut allows subsequent proteolytic processing and the expression of carboxypeptidase B activity. Although all pancreatic procarboxypeptidases have a preformed active site, only the A forms show intrinsic activity. Mutational substitution of Asp-41 in the globular activation domain, located at the interface with the enzyme moiety, as well as removal of the adjacent 310 helix allow the appearance of residual activity in the mutated procarboxypeptidase B, indicating that the interaction of both structural elements with the enzyme moiety prevents the binding of substrates and promotes enzyme inhibition. In addition, the poor heterologous expression of such mutants indicates that the mutated region is important for the folding of the whole proenzyme.     

Three-dimensional structure of porcine procarboxypeptidase B: a structural basis of its inactivity.

Procarboxypeptidase B is converted to enzymatically active carboxypeptidase B by limited proteolysis catalysed by trypsin, removing the long N-terminal activation segment of 95 amino acids. The three-dimensional crystal structure of procarboxypeptidase B from porcine pancreas has been determined at 2.3 A resolution and refined to a crystallographic R-factor of 0.169. The functional determinants of its enzymatic inactivity and of its activation by limited proteolysis have thus been unveiled. The activation segment folds in a globular region with an open sandwich antiparallel-alpha antiparallel-beta topology and in a C terminal alpha-helix which connects it to the enzyme moiety. The globular region (A7-A82) shields the preformed active site, and establishes specific interactions with residues important for substrate recognition. AspA41 forms a salt bridge with Arg145, which in active carboxypeptidase binds the C-terminal carboxyl group of substrate molecules. The connecting region occupies the putative extended substrate binding site. The scissile peptide bond cleaved by trypsin during activation is very exposed. Its cleavage leads to the release of the activation segment and to exposure of the substrate binding site. An open-sandwich folding has been observed in a number of other proteins and protein domains. One of them is the C-terminal fragment of L7/L12, a ribosomal protein from Escherichia coli that displays a topology similar to the activation domain of procarboxypeptidase.     

Primary structure of the activation peptide from bovine pancreatic procarboxypeptidase A

The complete sequence of the 94 residues composing the activation peptide of bovine procarboxypeptidase A has been determined by automated analysis of the intact activation segment and of three peptides resulting from enzymatic cleavages of the isolated peptide. The sequencing of a CNBr peptide isolated from procarboxypeptidase A allowed to connect the activation peptide with alpha-carboxypeptidase A (peptidylprolyl-L-amino-acid hydrolase, EC 3.4.17.1). The activation segment has a high content of acidic residues and a proline-rich region. Conformational prediction studies show that the bovine peptide, as the porcine and rat peptides, contains a high proportion of secondary structure and that the structural disposition of the regions in secondary structure is similar in the three peptides. The comparison of the sequence of the bovine, porcine and rat peptides, although exhibiting a striking homology, clearly shows that 40% of the substitutions have led to a charge change.     

Membrane glycoprotein synthesis: cleavage of the signal sequence in the absence of glycosylation

The controlled action of trypsin on porcine pancreatic procarboxypeptidase A releases a large activation peptide which contains the activation segment of the proenzyme. Circular dichroism studies indicate that the isolated activation peptide contains a high percentage of residues in ordered secondary structures (mainly α-helix). This result agrees with predictions of secondary structure carried out on the published amino acid sequence of the homologous rat proenzyme. Moreover, proton magnetic resonance spectroscopy shows that the peptide adopts a thermostable tertiary structure with characteristics typical of globular proteins. The results as a whole indicate that the activation segment of porcine pancreatic procarboxypeptidase A constitutes a folded structural domain.     

The three-dimensional structure of the native ternary complex of bovine pancreatic procarboxypeptidase A with proproteinase E and chymotrypsinogen C.

The metalloexozymogen procarboxypeptidase A is mainly secreted in ruminants as a ternary complex with zymogens of two serine endoproteinases, chymotrypsinogen C and proproteinase E. The bovine complex has been crystallized, and its molecular structure analysed and refined at 2.6 A resolution to an R factor of 0.198. In this heterotrimer, the activation segment of procarboxypeptidase A essentially clamps the other two subunits, which shield the activation sites of the former from tryptic attack. In contrast, the propeptides of both serine proproteinases are freely accessible to trypsin. This arrangement explains the sequential and delayed activation of the constituent zymogens. Procarboxypeptidase A is virtually identical to the homologous monomeric porcine form. Chymotrypsinogen C displays structural features characteristic for chymotrypsins as well as elastases, except for its activation domain; similar to bovine chymotrypsinogen A, its binding site is not properly formed, while its surface located activation segment is disordered. The proproteinase E structure is fully ordered and strikingly similar to active porcine elastase; its specificity pocket is occluded, while the activation segment is fixed to the molecular surface. This first structure of a native zymogen from the proteinase E/elastase family does not fundamentally differ from the serine proproteinases known so far.     

Comparison of the NMR solution structure with the X-ray crystal structure of the activation domain from procarboxypeptidase B

Summary The NMR solution structure of the activation domain isolated from porcine procarboxypeptidase B is compared with the X-ray crystal structure of the corresponding segment in the intact proenzyme. For the region of the polypeptide chain that has a well-defined three-dimensional structure in solution, i.e., the backbone atoms of residues 11–76 and 25 amino acid side chains in this segment that form a hydrophobic core in the activation domain, the root-mean-square distance between the two structures is 1.1 Å. There are no significant differences in average atom positions between the two structures, but only the NMR structure shows increased structural disorder in three outlying loops located along the same edge of the activation domain. These regions of increased structural disorder in the free domain coincide only partially with the interface to the enzyme domain in the proenzyme.     

Three-dimensional structure of porcine pancreatic procarboxypeptidase A. A comparison of the A and B zymogens and their determinants for inhibition and activation.

The three-dimensional structure of procarboxypeptidase A (PCPA) from porcine pancreas has been determined at 2 A resolution and refined to a crystallographic R-factor of 0.198, with a root-mean-square deviation from ideal values for bond lengths of 0.015 A and for angles of 2.1 degrees. It is compared with procarboxypeptidase B (PCPB) from the same tissue. The 94/95 residue activation segments of PCPA/PCPB have equivalent folds: an N-terminal globular region with an open sandwich antiparallel alpha/antiparallel beta topology, followed by an extended alpha-helical segment, the connection to the enzyme. Alignment of the secondary structures of the activation segments of PCPA and PCPB (residues A1 to A99) indicates a two residue insertion between residues A34 and A35 and a C-terminal helix that is two turns longer in PCPA compared to PCPB. A deletion is observed between residues A43 to A45, the region containing the short 3(10) helix that covers the active site in PCPB. The globular region (A4 to A80) shields the preformed active center of carboxypeptidase A (CPA), but none of the residues involved in catalysis makes direct contacts with the activation segment. In contrast, subsites S2, S3 and S4 of the enzyme, involved in the binding of peptidic substrates, are blocked by specific contacts with residues AspA36, TrpA38, ArgA47, AspA53 and GluA86 of the activation segment. It has been described that several residues of CPA exhibit different conformations in the free enzyme compared to when substrate is bound: Arg127, Arg145, Glu270 and Tyr248. In PCPA all of these residues are found in the "active" conformation, as if substrate were actually bound. The presence of a ligand, tentatively interpreted as a free amino acid (Val) in the active center could explain this fact. The connecting region (A80 to A99), the target for proteolytic activation, establishes fewer contacts with the enzyme in PCPA than in PCPB. The activation segment of PCPA (A4 to A99) remains bound to the enzyme after the first trypsin cleavage between ArgA99-Ala1 probably due to the stability conferred on it by the alpha-helix (alpha 3) of the connecting segment. These and other structural features may explain the differences in intrinsic activity and different rates or proteolytic activation of each zymogen.     

Chymotrypsin C is a co-activator of human pancreatic procarboxypeptidases A1 and A2

Human digestive carboxypeptidases CPA1, CPA2, and CPB1 are secreted by the pancreas as inactive proenzymes containing a 94-96-amino acid-long propeptide. Activation of procarboxypeptidases is initiated by proteolytic cleavage at the C-terminal end of the propeptide by trypsin. Here, we demonstrate that subsequent cleavage of the propeptide by chymotrypsin C (CTRC) induces a nearly 10-fold increase in the activity of trypsin-activated CPA1 and CPA2, whereas CPB1 activity is unaffected. Other human pancreatic proteases such as chymotrypsin B1, chymotrypsin B2, chymotrypsin-like enzyme-1, elastase 2A, elastase 3A, or elastase 3B are inactive or markedly less effective at promoting procarboxypeptidase activation. On the basis of these observations, we propose that CTRC is a physiological co-activator of proCPA1 and proCPA2. Furthermore, the results confirm and extend the notion that CTRC is a key regulator of digestive zymogen activation.     

Analysis of the thermal unfolding of porcine procarboxypeptidase A and its functional pieces by differential scanning calorimetry

Porcine pancreatic procarboxypeptidase A and its tryptic peptides, carboxypeptidase A and the activation segment, have been studied by high-sensitivity differential scanning calorimetry (DSC). The thermal denaturation of the zymogen and the active enzyme has been carried out at two pH values, 7.5 and 9.0, at different ionic strengths and at different scan rates. The endothermic transitions for these two proteins were always irreversible under all conditions investigated. The denaturation behaviour of both proteins seems to fit very well with the kinetic model for the DSC study of irreversible unfolding of proteins recently proposed by one of our groups. From this model, the activation energies obtained for the denaturation of the pro- and carboxypeptidase were 300 +/- 20 kJ mol-1 and 250 +/- 14 kJ mol-1 respectively. On the other hand, the isolated activation segment appears as a thermostable piece with a highly reversible thermal unfolding which follows a two-state process. The denaturation temperature observed for the isolated segment was always at least 15 K higher than those of the zymogen and the active enzyme.     

Primary structure of the activation segment of procarboxypeptidase A from porcine pancreas

The complete primary structure of the activation segment of monomeric procarboxypeptidase A from porcine pancreas has been determined by automated and manual Edman-like degradation methods performed on its fragments generated by enzymatic cleavage. The polypeptide consists of 94 residues, with a molecular mass of 10,768, and presents a high proportion of acidic and hydrophobic residues and a proline-rich region in the center of the molecule. Comparison of this sequence with the already reported equivalent sequence deduced from rat procarboxypeptidase A cDNA reveals a very high degree of homology between the two propeptides (up to a 81% of identities), which is even higher in certain large zones of the molecule.     

The tryptic activation pathway of monomeric procarboxypeptidase A

Procarboxypeptidases are the remaining major digestive zymogens the activation process of which remains unsolved. Here it is shown that in the tryptic activation of monomeric procarboxypeptidase A from porcine pancreas, the generation of carboxypeptidase A (CPA) activity parallels the limited proteolysis of the 94-residue activation segment. This degradation proceeds from the COOH-terminal end of the molecule, and CPA itself makes an important and unexpected contribution by excising the COOH-terminal arginine residue of the released primary activation fragment. Successive cleavages at some of the peptide bonds of the activation segment nearest to the COOH terminus were found to be of prime importance in eliciting CPA activity, particularly those involving the carbonyl groups of Arg94 and Gly93 which were first cleaved. It is also shown that the rate of activation does not depend directly upon the generation of CPA-alpha and its conversion to CPA-beta.     

Sequence-specific 1H NMR assignments and determination of the secondary structure for the activation domain isolated from pancreatic procarboxypeptidase B

Nearly complete sequence-specific 1H NMR assignments are presented for amino acid residues 3-81 in the 81-residue globular activation domain of porcine pancreatic procarboxypeptidase B isolated after limited tryptic proteolysis of the zymogen. These resonance assignments are consistent with the chemically determined amino acid sequence. Regular secondary structure elements were identified from nuclear Overhauser effects and the sequence locations of slowly exchanging backbone amide protons. The molecule contains two alpha-helices, including residues 20-30 and approximately residues 58-72, and a three-stranded antiparallel beta-sheet with the individual strands extending approximately from 12 to 17, 50 to 55, and 75 to 77. The identification of these secondary structures and a preliminary analysis of additional long-range NOE distance constraints show that isolated activation domain B forms a stable structure with the typical traits of a globular protein. The data presented here are the basis for the determination of the complete three-dimensional structure of activation domain B, which is currently in progress.     

Sequential homologies between procarboxypeptidases A and B from porcine pancreas

Automated Edman degradation of monomeric procarboxypeptidases A and B from porcine pancreas shows that their N-terminal regions (from residue 1 to 34-37) present a high degree of sequential homology to each other as well as to other related procarboxypeptidases. Conformational predictions based on these sequences confirm their structural homology and indicate the probable existence of two beta-turns, one beta-chain and a long alpha-helix in them. On the other hand, tryptic peptide maps on a reverse-phase column indicate great sequential similarities (if not identity) between monomeric procarboxypeptidase A and the procarboxypeptidase A subunit isolated from its binary complex with proproteinase E.     

Limited proteol ysis of coupling factor-latent ATPase from Mycobacterium phlei. Effects of different enzymes and modifying agents

The activation of the coupling factor-latent ATPase enzyme by tryptic proteolysis may resemble the activation of many proenzymes by limited proteolysis. The beta (53 000 dalton) subunit of solubilized coupling factor-latent ATPase from Mycobacterium phlei was selectively lost in some trypsin-treated samples. Since a concomitant loss of ATPase activity was not observed, the beta subunit may not be essential for ATPase catalytic activity. Treatment of solublized coupling factor with chymotrypsin rapidly produced an A′-type (61 000 dalton) species from the native alpha (64 000 dalton) subunits with partial activation of the ATPase enzyme. Secondary chymotryptic cleavage yielded an A″-type (58 000 dalton) species and a less-active enzyme. Storage of fresh coupling factor samples at ?20°C in the presence of 4 mM MgCl₂ with several freeze-thaw cycles resulted in loss of ATPase activity without apparent change in alpha subunit structure. Storage at 4°C in the presence or absence of MgCl₂ both decreased ATPase activity and generated A′-type alpha subunit species. Since presence of phenylmethylsulfonyl fluoride prevented these changes, an unknown protease was suspected. The peptide bonds first cleaved by trypsin, chymotrypsin, and the unknown protease are all apparently located within the same small segment of alpha subunit polypeptide chain.     

The isolation and partial characterization of precursor forms of ostrich carboxypeptidase

Ostrich carboxypeptidases A and B were recently purified and characterized. The aim of this study was to isolate and purify, and partially characterize in terms of molecular weight, pI, amino acid composition and N-terminal sequencing, the precursor forms of carboxypeptidases from the ostrich pancreas. Inhibition studies with soybean trypsin inhibitor and activation studies with three proteases (bovine trypsin, bovine chymotrypsin and porcine elastase) were performed on crude ostrich acetone powder and the carboxypeptidase A and B activities were determined. SDS-PAGE was carried out after every incubation to monitor the rate and degree of conversion of a M(r) 66K component to procarboxypeptidase and carboxypeptidase A and B. The precursor forms were purified by Toyopearl Super Q and Pharmacia Mono Q chromatography. All three proteases converted the M(r) 66K component to procarboxypeptidases and carboxypeptidases over a set time interval, with carboxypeptidase A and B activities being detected in the acetone powder. Chymotrypsin was the preferred protease since it exhibited a more controlled activation of the procarboxypeptidases. The amino acid composition of procarboxypeptidase A revealed 525 residues. The N-terminal sequence of procarboxypeptidase A showed considerable homology when compared with several other mammalian sequences. M(r) and pI values of 52K and 5.23 were obtained for procarboxypeptidase A, respectively. This study indicated that ostrich procarboxypeptidase A is closely related to other mammalian procarboxypeptidase A molecules in terms of physicochemical properties.     

The three-dimensional structure of human procarboxypeptidase A2. Deciphering the basis of the inhibition, activation and intrinsic activity of the zymogen.

The three-dimensional structure of human procarboxypeptidase A2 has been determined using X-ray crystallography at 1.8 A resolution. This is the first detailed structural report of a human pancreatic carboxypeptidase and of its zymogen. Human procarboxypeptidase A2 is formed by a pro-segment of 96 residues, which inhibits the enzyme, and a carboxypeptidase moiety of 305 residues. The pro-enzyme maintains the general fold when compared with other non-human counterparts. The globular part of the pro-segment docks into the enzyme moiety and shields the S2-S4 substrate binding sites, promoting inhibition. Interestingly, important differences are found in the pro-segment which allow the identification of the structural determinants of the diverse activation behaviours of procarboxypeptidases A1, B and A2, particularly of the latter. The benzylsuccinic inhibitor is able to diffuse into the active site of procarboxypeptidase A2 in the crystals. The structure of the zymogen-inhibitor complex has been solved at 2.2 A resolution. The inhibitor enters the active site through a channel formed at the interface between the pro-segment and the enzyme regions and interacts with important elements of the active site. The derived structural features explain the intrinsic activity of A1/A2 pro-enzymes for small substrates.     

Human procarboxypeptidase B: three-dimensional structure and implications for thrombin-activatable fibrinolysis inhibitor (TAFI)

Besides their classical role in alimentary protein degradation, zinc-dependant carboxypeptidases also participate in more selective regulatory processes like prohormone and neuropeptide processing or fibrinolysis inhibition in blood plasma. Human pancreatic procarboxypeptidase B (PCPB) is the prototype for those human exopeptidases that cleave off basic C-terminal residues and are secreted as inactive zymogens. One such protein is thrombin-activatable fibrinolysis inhibitor (TAFI), also known as plasma PCPB, which circulates in human plasma as a zymogen bound to plasminogen. The structure of human pancreatic PCPB displays a 95-residue pro-segment consisting of a globular region with an open-sandwich antiparallel-alpha antiparallel-beta topology and a C-terminal alpha-helix, which connects to the enzyme moiety. The latter is a 309-amino acid residue catalytic domain with alpha/beta hydrolase topology and a preformed active site, which is shielded by the globular domain of the pro-segment. The fold of the proenzyme is similar to previously reported procarboxypeptidase structures, also in that the most variable region is the connecting segment that links both globular moieties. However, the empty active site of human procarboxypeptidase B has two alternate conformations in one of the zinc-binding residues, which account for subtle differences in some of the key residues for substrate binding. The reported crystal structure, refined with data to 1.6A resolution, permits in the absence of an experimental structure, accurate homology modelling of TAFI, which may help to explain its properties.     

Intracellular proteolysis of pancreatic zymogens.

Activation of pancreatic digestive zymogens within the pancreatic acinar cell may be an early event in the development of pancreatitis. To detect such activation, an immunoblot assay has been developed that measures the relative amounts of inactive zymogens and their respective active enzyme forms. Using this assay, high doses of cholecystokinin or carbachol were found to stimulate the intracellular conversion of at least three zymogens (procarboxypeptidase A1, procarboxypeptidase B, and chymotrypsinogen 2) to their active forms. Thus, this conversion may be a generalized phenomenon of pancreatic zymogens. The conversion is detected within ten minutes of treatment and is not associated with changes in acinar cell morphology; it has been predicted that the lysosomal thiol protease, cathepsin B, may initiate this conversion. Small amounts of cathepsin B are found in the secretory pathway, and cathepsin B can activate trypsinogen in vitro; however, exposure of acini to a thiol protease inhibitor (E64) did not block this conversion. Conversion was inhibited by the serine protease inhibitor, benzamidine, and by raising the intracellular pH, using chloroquine or monensin. This limited proteolytic conversion appears to require a low pH compartment and a serine protease activity. After long periods of treatment (60 minutes), the amounts of the active enzyme forms began to decrease; this observation suggested that the active enzyme forms were being degraded. Treatment of acini with E64 reduced this late decrease in active enzyme forms, suggesting that thiol proteases, including lysosomal hydrolases, may be involved in the degradation of the active enzyme forms. These findings indicate that pathways for zymogen activation as well as degradation of active enzyme forms are present within the pancreatic acinar cell.