Crystal structure of bovine procarboxypeptidase A-S6 subunit III, a highly structured truncated zymogen E.

Subunit III, a defective serine endopeptidase lacking the typical N-terminal hydrophobic dipeptide is secreted by the pancreas of ruminant species as part of the bovine ternary complex procarboxypeptidase A-S6. Two monoclinic crystal forms were obtained and subsequently used to solve its X-ray structure. The highest resolution model of subunit III was refined at 1.7 A resolution to a crystallographic R-factor of 18.4%, with r.m.s. bond deviations from ideality of 0.012 A. About 80% of the model presents the characteristic architecture of trypsin-like proteases. The remaining zones, however, have well-defined, unique conformations. The regions from residues 70 to 80 and from 140 to 155 present maximum distances of 16 and 18 A relative to serine proteases and zymogens. Comparisons with the structures of porcine elastase 1 and chymotrypsinogen A indicate that the specific binding pocket of subunit III adopts a zymogen-like conformation and thus provide a basis for its inactivity. In general, the structural analysis of subunit III strongly suggests that it corresponds to a truncated version of a new class of highly structured elastase-like zymogen molecules. Based on the structures of subunit III and elastase 1, it is concluded that large concerted movements are necessary for the activation of zymogen E.     

Autolysis of proproteinase E in bovine procarboxypeptidase A ternary complex gives rise to subunit III

Extracts of bovine pancreatic tissue are shown by HPLC to contain two distinct ternary complexes of procarboxypeptidase A (subunit I), chymotrypsinogen C (subunit II) and either proproteinase E or subunit III. It is shown that proproteinase E in the complex generates subunit III by removal of 13 N-terminal residues when the former is allowed to autolyze in solution or when catalytic amounts of isolated active proteinase E are added to it. Autolysis of proproteinase E was accompanied by the loss of potential activity towards specific synthetic substrates and occurred at a higher rate in pancreatic juice than in pancreatic tissue extracts, even when both were processed in the presence of serine protease inhibitors. We conclude that subunit III (also called truncated protease E) is an autolytic product of proproteinase E and not an ab initio component of the native ternary complex.     

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.     

Subunit III of ruminant procarboxypeptidase A-S6 complexes and pancreatic proteases E. A new family of pancreatic serine endopeptidases?

Subunit III (BSIII) of the bovine ternary complex of procarboxypeptidase A-S6 (PCPA-S6), a defective serine endopeptidase-like protein, actively synthesized by the pancreas of some ruminant species, is highly homologous to human protease E (HPE). Both proteins possess the same atypical disulfide bridge in position 98-99b. They are structurally related to porcine elastase 1 and human elastase 2 (about 56% identity). However, in contrast to those two enzymes which have an overall positive net charge, BSIII and HPE are negatively charged. Three-dimensional models of BSIII and HPE have been constructed from the crystallographic structure of porcine pancreatic elastase 1. The inhibitor-binding site for TFAI in these three proteins seems to be very similar; the atypical disulfide bridge does not seem to be involved in this binding site. The specific structural features of BSIII and HPE strongly support the assumption that BSIII is a truncated protease E and that both proteins belong to a separate serine endopeptidase family.     

Amino acid sequence and disulfide bridges of subunit III, a defective endopeptidase present in the bovine pancreatic 6 S procarboxypeptidase A complex

The sequence of the 240 amino acids and the position of the five S-S bridges of subunit III of the bovine pancreatic 6 S procarboxypeptidase A complex have been determined thus confirming its phylogenetic filiation with the pancreatic serine endopeptidase group. The subunit contains at equivalent positions all the elements of the catalytic site of these enzymes. The elements of a binding pocket very similar to that of porcine elastase I are also present in the protein thus accounting for its zymogen-like activity. The most obvious difference is the absence in the subunit of the two strongly hydrophobic amino acids (16 and 17 in the chymotrypsinogen numbering), which are known to participate in the stabilization of a fully functional binding pocket in active endopeptidases. Four of the five disulfide bridges of subunit III are homologous with those common to all pancreatic endopeptidases. In contrast the fifth bridge forms a very small loop of only four amino acids, which is not encountered in active endopeptidases. Other potentially lethal modifications in the structure of the subunit are not excluded.     

The inactive subunit of ruminant procarboxypeptidase A-S6 complexes. Structural basis of inactivity and physiological role

Subunit III has so far been found only in the pancreas of ruminants in a non-covalent association (procarboxypeptidase A-S6) with two different proteins: the procarboxypeptidase A itself (subunit I) and a C-type chymotrypsinogen (subunit II). In contrast with these latter two proteins, which are zymogens of pancreatic proteases, subunit III seems to be devoid of any activity towards specific substrates of pancreatic proteases. However, it possesses a weakly functional active site which allows it to hydrolyze a non-specific ester, p-nitrophenyl acetate, and to react with several active-site titrants. The binding of proflavin to subunit III shows that this protein owns a non-polar binding site with a very high Kd compared to that of chymotrypsin. The comparison of the amino acid sequences of subunit III and some serine proteases showed that subunit III is closely related to an elastase. Models of the tertiary structure of subunit III suggest a conformational modification that affects the substrate binding and could explain the lack of specific enzymatic activity. The presence of subunit III in the ternary complex is not related to an enzymatic function. This protein does not participate in the activation process of subunit I but prevents the denaturation of this subunit at low pH. This may represent its biological role in the acidic environment of the duodenum in ruminants.     

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.     

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.     

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.     

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.     

The activation peptide of pancreatic procarboxypeptidase A is the keystone of the bovine procarboxypeptidase A-S6 ternary complex

In some ruminant species, pancreatic procarboxypeptidase A is the central element of a ternary complex involving two other components, a C-type chymotrypsinogen and an inactive protease E. Although the complex is devoted to protein digestion, the fate of this system upon activation of its constituent subunits has, as yet, not been clearly established. In this paper, the activation peptide of procarboxypeptidase A is shown to play a key role in the association of the three subunits and a model is proposed for the in vivo function of the complex.     

Two-step dissociation of bovine 6S procarboxypeptidase A by dimethylmaleylation

Reversible condensation of the ternary complex form of bovine pancreatic procarboxypeptidase A with 2,3-dimethyl maleic anhydride was investigated at pH 9.0 and low concentration of reagent over the acylable amino groups. After subsequent modification of only a few lysyl residues, subunit III was found to have been released from the quaternary structure leading to the separation of an apparently native protein devoid of any contaminating subunit II, while dissociation of the remaining binary complex occurred upon further addition of the anhydride. This observation suggests that the electrostatic interactions existing between subunits I and III are more rapidly weakened than those between subunits I and II, probably because fewer lysyl residues are involved and/or there is greater accessibility to the chemical reagant. Although completely inactive on the specific substrates of trypsin, chymotrypsin and elastase, subunit III hydrolyzed p-nitrophenyl acetate at a rate similar to that of chymotrypsin but without any burst of p-nitrophenol, which indicates that the weakly functional active site of the subunit is not quite comparable to that of serine protease zymogens. Subunit III already has some of the functional characteristics of the corresponding active enzymes.     

牙鲆弹性蛋白酶cDNA全长和蛋白质结构分析

为研究牙鲆丝氨酸蛋白酶家族的功能和及其家族的分子进化规律,从本实验室已构建的牙鲆肝胰脏cDNA文库进行了部分测序,从而筛选出一个弹性蛋白酶新成员：弹性蛋白酶5。在此基础上,结合Genbank数据库中已经提交的胰凝乳蛋白酶和胰蛋白酶,对三者蛋白质进行了序列分析和三维结构的比较。牙鲆弹性蛋白酶cDNA包含一个完整的读码框（提交Genbank的登录号为EU873084）。其编码区平均GC含量为54％,推测编码的蛋白质包含296个氨基酸,分子量为29．04KD,等电点为6．14。蛋白序列比较表明它和牙鲆弹性蛋白酶3相似性最高。通过同源建模得到弹性蛋白酶5的三维结构和牛胰凝乳蛋白酶结构相似,包含了2个α螺旋、β个8折叠和13个转角结构。牙鲆弹性蛋白酶、胰凝乳蛋白酶和胰蛋白酶中底物结合区的3个关键氨基酸有明显的区别,这些氨基酸的变化改变了底物结合位点开口的大小,胰凝乳蛋白酶2的三个关键氨基酸和牛胰凝乳蛋白酶相同,该区域能接受结构较大的芳香族氨基酸;胰蛋白酶3能更好的结合阳性氨基酸Lys或Arg;而弹性蛋白酶开口很小,只能结合小的残基。上述结果证明了牙鲆丝氨酸蛋白酶家族中的弹性蛋白酶、胰凝乳蛋白酶和胰蛋白酶底物结合位点的结构差异决定了其对底物选择的特异性。     

Zymogen activation in a reconstituted pancreatic acinar cell system

Pathological activation of digestive zymogens within the pancreatic acinar cell initiates acute pancreatitis. Cytosolic events regulate this activation within intracellular compartments of unclear identity. In an in vivo model of acute pancreatitis, zymogen activation was detected in both zymogen granule-enriched and microsomal cellular fractions. To examine the mechanism of this activation in vitro, a reconstituted system was developed using pancreatic cytosol, a zymogen granule-enriched fraction, and a microsomal fraction. Addition of cytosol to either particulate fraction resulted in a prominent increase in both trypsin and chymotrypsin activities. The percentage of the pool of trypsinogen and chymotrypsinogen activated was about twofold and sixfold greater, respectively, in the microsomal than in the zymogen granule-enriched fraction. Activation of chymotrypsinogen but not trypsinogen was significantly enhanced by ATP (5 mM) but not by the inactive ATP analog AMP-PNP. The processing of procarboxypeptidase B to its mature form also demonstrated a requirement for ATP and cytosol. E64d, an inhibitor of cathepsin B, a thiol protease that can activate trypsin, completely inhibited trypsin activity but did not affect chymotrypsin activity or carboxypeptidase B generation. These studies demonstrate that both zymogen granule-enriched and microsomal fractions from the pancreas can support cytosol-dependent zymogen activation. A component of the activation of some zymogens, such as chymotrypsinogen and procarboxypeptidase, may depend on ATP but not on trypsin or cathepsin B.     

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 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.     

Modeling zymogen protein C

A solution structure for the complete zymogen form of human coagulation protein C is modeled. The initial core structure is based on the x-ray crystallographic structure of the gamma-carboxyglutamic acid (Gla)-domainless activated form. The Gla domain (residues 1-48) is modeled from the x-ray crystal coordinates of the factor VII(a)/tissue factor complex and oriented with the epidermal growth factor-1 domain to yield an initial orientation consistent with the x-ray crystal structure of porcine factor IX(a). The missing C-terminal residues in the light chain (residues 147-157) and the activation peptide residues 158-169 were introduced using homology modeling so that the activation peptide residues directly interact with the residues in the calcium binding loop. Molecular dynamics simulations (Amber-particle-mesh-Ewald) are used to obtain the complete calcium-complexed solution structure. The individual domain structures of protein C in solution are largely unaffected by solvation, whereas the Gla-epidermal growth factor-1 orientation evolves to a form different from both factors VII(a) and IX(a). The solution structure of the zymogen protein C is compared with the crystal structures of the existing zymogen serine proteases: chymotrypsinogen, proproteinase, and prethrombin-2. Calculated electrostatic potential surfaces support the involvement of the serine protease calcium ion binding loop in providing a suitable electrostatic environment around the scissile bond for II(a)/thrombomodulin interaction.     

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.     

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.     

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.