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.     

Existence of ternary complexes of procarboxypeptidase A in the pancreas of some ruminant species

The existence of procarboxypeptidase A, in the form of a non-covalent ternary complex containing the apparently inactive serine protease (subunit III), has so far been observed only in the ox pancreas. Evidence, obtained in the present study, shows that a ternary complex of procarboxypeptidase A, with a subunit III highly homologous with that of the bovine complex, is also present in two other ruminant species, sheep and goat. The biological significance of these complex forms of procarboxypeptidase A and the consistently high biosynthesis level of the apparently inactive subunit III in all three ruminant species is still unknown. Yet the synthesis of subunit III is not related to the animal diet since in the horse, which is a non-ruminant herbivorous animal, the procarboxypeptidase A is monomeric. Reassociation assays between either bovine subunits II or III and monomeric as well as binary forms of procarboxypeptidase A from various species show that, unlike subunit II, the recognition site for subunit III is highly conserved in all the procarboxypeptidases A and that bovine subunit II is different from porcine chymotrypsinogen C with regard to association.     

Purification and properties of five different forms of human procarboxypeptidases

Three different procarboxypeptidases A and two different procarboxypeptidases B have been isolated for the first time, in a pure and native state, from human pancreatic extracts. These proteins were purified in one or two quick steps by anion-exchange HPLC. All these forms have been biochemically characterized. Two of the procarboxypeptidases A, the A1 and A2 forms, are obtained in a monomeric state while the other, the A3 form, is obtained as a binary complex of a procarboxypeptidase A with a proproteinase E. This complex is stable in aqueous buffers at various ionic strengths and develops carboxypeptidase A and proteinase E activities in the presence of trypsin. The A1 and A2 forms show clear differences in electrophoretic mobility in SDS/polyacrylamide gels, isoelectric point, proteolytic activation process with trypsin and susceptibility to thermal denaturation. In contrast, these properties are similar in the A1 and A3 (binary complex) forms. On the other hand, with respect to the properties listed above, the B1 and B2 forms differ from each other mainly in isoelectric point. An overall comparison of the above properties reveals the unusual character of the A2 form, midway between the other A and B forms. N-terminal extended sequence analysis carried out on these proenzymes confirm that they constitute different isologous forms.     

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.     

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.     

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.     

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.     

Prediction of a new class of RNA recognition motif

The observation that activation domains (AD) of procarboxypeptidases are rather long compared to the pro-regions of other zymogens raises the possibility that they could play additional roles apart from precluding enzymatic activity within the proenzyme and helping in its folding process. In the present work, we compared the overall pro-domain tertiary structure with several proteins belonging to the same fold in the structural classification of proteins (SCOP) database by using structure and sequence comparisons. The best score obtained was between the activation domain of human procarboxypeptidase A4 (ADA4h) and the human U1A protein from the U1 snRNP. Structural alignment revealed the existence of RNP1- and RNP2-related sequences in ADA4h. After modeling ADA4h on U1A, the new structure was used to extract a new sequence pattern characteristic for important residues at key positions. The new sequence pattern allowed scanning protein sequences to predict the RNA-binding function for 32 sequences undetected by PFAM. Unspecific RNA electrophoretic mobility shift assays experimentally supported the prediction that ADA4h binds an RNA motif similar to the U1A binding-motif of stem-loop II of U1 small nuclear RNA. The experiments carried out with ADA4h in the present work suggest the sharing of a common ancestor with other RNA recognition motifs. However, the fact that key residues preventing activity within the proenzyme are also key residues for RNA binding might have induced the activation domains of procarboxypeptidases to evolve from the canonical RNP1 and RNP2 sequences.     

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.     

Stereospecific mobilization of intracellular Ca2+ by inositol 1,4,5-triphosphate. Comparison with inositol 1,4,5-trisphosphorothioate and inositol 1,3,4-trisphosphate.

The isolated activation segment of pig procarboxypeptidase A binds two Tb3+ ions in a strong and specific way. In contrast, the binding of Ca2+, Cd2+ and Mg2+ is weak. The binding of Tb3+ increases the resistance of the isolated activation segment against proteolysis and competes for the binding of the carbocyanine dye Stains-All. This dye forms complexes with the activation segment showing spectral properties similar to those observed with EF-hand structures. The presented results support a previous hypothesis on the existence of two regions in the activation segment of pancreatic procarboxypeptidases structurally related to Ca2+-binding domains of the EF-hand protein family.     

Conformational predictive studies on the activation segment of pancreatic procarboxypeptidases

Little is known about the conformation and evolutionary origin of the activation segment of pancreatic procarboxypeptidases. Analysis of the sequence and secondary structure propensities of these propeptide segments indicate that they contain two regions structurally related to the Ca2+-binding sites of the EF-hand protein family. This proposed homology could explain how (and why) carboxypeptidases developed such long (94 residues) activation peptides.     

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.     

Purification of a carboxypeptidase B-like enzyme from the starfish Dermasterias imbricata.

A carboxypeptidase B-like enzyme which catalyses the hydrolysis of synthetic esters of lysine and arginine has been isolated from the starfish Dermasterias imbricata. This carboxypeptidase B-like enzyme has a molecular weight of approximately 34 000 and shares this and other properties with bovine pancreatic carboxypeptidase B. The existence of zymogen for this activity in the pyloric caeca of the starfish is demonstrated. This zymogen has a molecular weight near 40 000 and appears to be analogous to other monomeric procarboxypeptidases B. The zymogen possesses an intrinsic low-level activity toward synthetic substrates of carboxypeptidase B and is activated by trypsin.     

Identification of a procarboxypeptidase A-truncated protease E binary complex in human pancreatic juice

The characterization, in human pancreatic juice, of a binary complex associating procarboxypeptidase A with a 32 kDa inactive glycoprotein (G32) is reported in this paper. Free G32 was isolated after dissociation of the binary complex. N-terminal sequence analysis revealed a complete homology between this protein and human protease E (HPE 1), except for the two strongly hydrophobic N-terminal residues (Val-Val) which are missing in G32. This protein might be a truncated protease E highly analogous to the subunit III of the ruminant procarboxypeptidase A-S6 ternary complex. The analogy with bovine subunit III is further supported by interspecies reassociation experiments showing that bovine procarboxypeptidase A can specifically bind human G32.     

Generation of a subunit III-like protein by autolysis of human and porcine proproteinase e in a binary complex with procarboxypeptidase A

Tryptic treatment of human and porcine proproteinase E, procarboxypeptidase A binary complexes gave rise to active proteinase E after removal of an 11-residue N-terminal activation peptide. By contrast, upon treatment of either complex with active proteinase E, not only was the activation peptide released but also the hydrophobic dipeptide Val12-Val13 of the corresponding enzyme. No serine protease activity on specific synthetic peptide substrates could be detected. The structural homology of inactive proteinase E with subunit III of ruminant procarboxypeptidase A was strengthened by the existence of a functional homology since truncated proteinase E still possessed a weakly functional active site. Thus, subunit III-like proteins are generated by proteinase E-catalyzed limited proteolysis of proproteinase E.     

Intrinsic catalytic activity of procarboxypeptidase A. A kinetic study using fluorine analogues.

Bovine procarboxypeptidase A displays substantial catalytic activity toward halogenated acyl-amino acids, the most active of which is trifluoroacetyl-L-phenylalanine (TFAc-L-Phe). Though this activity is not as great as for the native enzyme, it is quite substantial and far beyond the range of adventitious activation. Both DL-benzylcuccinate and beta-phenylpropionate inhibit zymogen hydrolysis of TFAc-L-Phe, the former with a K1 of 4.1 micrometer and the latter, 900 micrometer (a value much higher than the corresponding enzyme). Apo procarboxypeptidase A will also hydrolyze TFAc-L-Phe, presumably the polarization of the carbonyl carbon being accomplished by the fluorine atoms in the absence of a specific metal ion. That this is not entirely the metal ion function is indicated by the fact that rate enhancements follow the order manganese procarboxypeptidase A approximately zinc procarboxypeptidase greater than apo-procarboxypeptidase. The results indicate considerable similarities for the zymogen-enzyme pair in terms of catalytic groups, pH dependence, specificity and the nature of their transition state binding sites. Some changes in the substrate or inhibitor binding sites are noted.     

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.     

Changes in mRNA levels of rat pancreatic lipase in the early days of consumption of a high-lipid diet

The time-course response of rat pancreatic enzymes to a diet containing 25% sunflower oil was investigated. A 1.2-fold enhancement in lipase specific activity was observed as early as the first day of diet consumption and was further increased up to 1.9-fold on the 5th day. On the other hand, colipase activity was slightly decreased during the first two days of high-lipid diet intake and then increased. An immediate and direct effect was also exerted by the 25% lipid diet on lipase biosynthesis. Both fractional synthetic rate and specific activity of lipase were comparably induced. Due to a 1.6-fold increase in the overall protein synthesis following 5 days of lipid diet consumption, the absolute synthesis of lipase and amylase was increased by 3.5-fold and 0.98-fold, respectively, as compared to control animals. By contrast, the synthesis of procarboxypeptidases and serine proteases did not increase before day 5, probably as the result of a distinct adaptive mechanism. The pancreatic mRNA levels in control and adapted animals, which were determined by dot-blot hybridization with amylase and lipase cDNAs, were consistent with a biphasic induction of lipase synthesis since a first increase in the level of the enzyme-specific mRNA during the first two days of diet intake (4-fold on day 1) was followed by a second increase after the fourth day (6.5-fold on day 5). On the other hand, amylase mRNA level was unchanged during the dietary manipulation. Thus, hyperlipidic diets exerted an both lipase activity and synthesis but a delayed effect on procarboxypeptidase and serine protease synthesis. In a similar manner, the immediate induction of lipase mRNA level by dietary fat, followed by another increase a few days later, suggested that at least two different mechanisms are involved in lipase mRNA induction.     

Further studies on the human pancreatic binary complexes involving procarboxypeptidase A

In contrast to procarboxypeptidase B which has always been reported to be secreted by the pancreas as a monomer, procarboxypeptidase A occurs as a monomer and/or associated to one or two functionally different proteins, depending on the species. Recent studies showed that, in the human pancreatic secretion, procarboxypeptidase A is mainly secreted as a 44 kDa protein involved in at least three different binary complexes. As previously reported, two of these complexes associated procarboxypeptidase A to either a glycosylated truncated protease E or zymogen E. In this paper, we identified proelastase 2 as the partner of procarboxypeptidase A in the third complex, thus reporting for the first time the occurrence of a proelastase 2/procarboxypeptidase A binary complex in vertebrates. Moreover, from N-terminal sequence analyses, the 44 kDa procarboxypeptidase A involved in these complexes was identified as being of the A1 type. Only one type of procarboxypeptidase B, the B1 type, has been detected in the analyzed pancreatic juices, thus emphasizing the previously observed genetic differences between individuals.     

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.