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.     

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.     

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.     

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.     

Characterization of two glycoproteins of human pancreatic juice: P35, a truncated protease E and P19, precursor of protein X

Four glycoproteins were separated by SDS-polyacrylamide gel electrophoresis of proteins of human pancreatic juice devoid of free proteolytic activity. The two low molecular weight glycoproteins were isolated and characterized. Protein P19, the precursor family of protein X, was analyzed by its carbohydrate content which seemed to play an important role in protein solubility at pH 8.0. Protein P35 was found to be a Con A-binding protein rich in mannose. Its N-terminal amino acid sequence covering 33 residues revealed a strong homology with human protease E without the dipeptide Val-Val. Is P35 a protein homologous to the subunit III of bovine procarboxypeptidase A?     

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.     

Subunit III of bovine procarboxypeptidase A: its relationship to human pancreatic elastase 1

This paper is a continuation of our study of various animal pancreatic enzymes which are related to human pancreatic elastase 1 (Sziegoleit, A. & Linder, D. (1986) Biol. Chem. Hoppe-Seyler, 367, 527-531). The isolation and immunological analysis of the related protein from bovine pancreas disclosed that the third subunit of the procarboxypeptidase A complex is the antibody-binding component. The similarity of this subunit to elastase 1 is affirmed by comparison of their primary structures. While the complete amino-acid sequence of bovine subunit III recently has been published (Venot, N., Sciaky, M., Puigserver, A., Desnuelle, P. & Laurent, G. (1986) Eur. J. Biochem. 157, 91-99), we here present the amino-acid sequence of the carboxy-terminal tryptic peptide of human pancreatic elastase 1 showing a high degree of homology.     

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.     

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.     

Morphology of the procarboxypeptidase A-S6 complex

Bovine pancreatic procarboxypeptidase A is secreted as a non-covalent association of three different proteins (pro CPA-S6). The free native subunits can be obtained by dissociation of the complex by dimethylmaleylation. Moreover, two specific binary complexes resulting from the high affinity of procarboxypeptidase A (subunit I) for its other two partners (subunits II and III) can also be obtained.In order to better understand the function of the association, an investigation of the morphology of the ternary complex by solution X-ray scattering has been carried out. The radii of gyration of all the molecular species have been obtained and the experimental results have been interpreted in terms of compact objects of simple shape. The various components correspond to globular particles as shown by the value of the ratio Rg/M^1/3. This is confirmed by the moderate anisotropy of the simple geometric shapes determined using an assumed value of 0.3 g H₂O/g protein for the hydration. The distances between the centres of gravity of pairs of species strongly suggest that the components are in the closest distance configuration or close to it. However, the binary complex I–III appears to be more open than the complex I–II. Finally, a model of the interaction between carboxpeptidase A and its activation peptide has been constructed by comparing the hypothetical geometric model of subunit I to the crystallographically determined structure of carboxypeptidase A.Abbreviations pro CPA procarboxypeptidase A - pro CPA-S6 (or T.C.) ternary complex with a sedimentation coefficient of 6S - CPA carboxypeptidase A     

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.     

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.     

Expression of a soluble and activatable form of bovine procarboxypeptidase A in Escherichia coli

Bovine pancreatic procarboxypeptidase A has been overexpressed in a soluble and activatable form in Escherichia coli. When the protein was expressed under the control of bacteriophage T7 promoter in E. coli ADA494 (a thioredoxin reductase deficient bacteria), a thioredoxin fusion protein was produced at relatively high level in the cytoplasm (4 mg/L culture medium). Although the recombinant protein essentially accumulated as inclusion bodies, as much as 30% of the fusion protein was recovered in a soluble form at low growth temperature and could therefore be purified to homogeneity in a single-step procedure by metal-affinity chromatography. The recombinant precursor form of bovine carboxypeptidase A was recognized by a monoclonal antibody directed against purified bovine pancreatic carboxypeptidase A. Moreover, upon tryptic activation it gave rise to an enzyme, the N-terminal sequence, molecular size,and specific activity of which were comparable to those of the enzyme derived from the native precursor purified from bovine pancreas.     

Isolation of a cDNA encoding a human serum marker for acute pancreatitis. Identification of pancreas-specific protein as pancreatic procarboxypeptidase B.

A human pancreas-specific protein (PASP), previously characterized as a serum marker for acute pancreatitis and pancreatic graft rejection, has been identified as pancreatic procarboxypeptidase B (PCPB). cDNAs encoding PASP/PCPB were isolated from a human pancreas cDNA library using a combination of nucleic acid hybridization screening and immunoscreening with antisera raised against native PASP. The deduced amino acid sequence of PASP/PCPB cDNA predicts the translation of a 416-amino acid preproenzyme with a 15-amino acid signal/leader peptide and a 95-amino acid activation peptide. The proenzyme portion of this protein has 76% identity with rat PCPB and 84% identity with bovine carboxypeptidase B. DNA and RNA blot analyses indicate that human PCPB mRNA (1,400 nucleotides) is transcribed from a single locus in the human genome in a tissue-specific fashion. N-terminal sequencing of native PASP and the specific immunoreactivity of bacterially expressed PASP/PCPB with native PASP antibodies confirm the identification of PASP as human pancreatic PCPB.     

Spectrofluorimetric investigation of the interactions between the subunits of bovine pancreatic procarboxypeptidase A-S6

A spectrofluorimetric investigation of the interactions between the subunits of the pancreatic bovine procarboxypeptidase A ternary complex was carried out after covalent insertion of a fluorescent probe at the active center of one of the constituent subunits. The specific insertion of an anthraniloyl group at the active center of subunit II free or bound to subunit I, after its conversion into chymotrypsin II, allowed us to determine the value of the dissociation constant between subunit I and anthraniloyl-chymotrypsin II (Kd = 0.7 +/- 0.1 x 10(-7) M) and between subunit III and the binary complex subunit I-anthraniloyl-chymotrypsin II (Kd = 1.6 +/- 0.3 x 10(-7) M). Moreover, the influence of the association on the flexibility of the active center of chymotrypsin II was deduced from fluorescence polarization measurements and rotational correlation time determination of anthraniloyl-chymotrypsin II free or bound to subunit I. The anthraniloyl group has no motion independently of the whole chymotrypsin II molecule and the binding of subunit I to anthraniloyl-chymotrypsin II results in an increase of the rigidity of the active site in the latter protein.     

Models for the binary complex of bacteriophage T4 gp59 helicase loading protein: gp32 single-stranded DNA-BINDING protein and ternary complex with pseudo-Y junction DNA

Bacteriophage T4 gp59 helicase assembly protein (gp59) is required for loading of gp41 replicative helicase onto DNA protected by gp32 single-stranded DNA-binding protein. The gp59 protein recognizes branched DNA structures found at replication and recombination sites. Binding of gp32 protein (full-length and deletion constructs) to gp59 protein measured by isothermal titration calorimetry demonstrates that the gp32 protein C-terminal A-domain is essential for protein-protein interaction in the absence of DNA. Sedimentation velocity experiments with gp59 protein and gp32ΔB protein (an N-terminal B-domain deletion) show that these proteins are monomers but form a 1:1 complex with a dissociation constant comparable with that determined by isothermal titration calorimetry. Small angle x-ray scattering (SAXS) studies indicate that the gp59 protein is a prolate monomer, consistent with the crystal structure and hydrodynamic properties determined from sedimentation velocity experiments. SAXS experiments also demonstrate that gp32ΔB protein is a prolate monomer with an elongated A-domain protruding from the core. Fitting structures of gp59 protein and the gp32 core into the SAXS-derived molecular envelope supports a model for the gp59 protein-gp32ΔB protein complex. Our earlier work demonstrated that gp59 protein attracts full-length gp32 protein to pseudo-Y junctions. A model of the gp59 protein-DNA complex, modified to accommodate new SAXS data for the binary complex together with mutational analysis of gp59 protein, is presented in the accompanying article (Dolezal, D., Jones, C. E., Lai, X., Brister, J. R., Mueser, T. C., Nossal, N. G., and Hinton, D. M. (2012) J. Biol. Chem. 287, 18596-18607).     

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.