Overexpression of Human Procarboxypeptidase A2 in Pichia pastoris and Detailed Characterization of Its Activation Pathway*

David Reverter, Salvador Ventura, Virtudes Villegas, Josep Vendrell, and Francesc X. AvilésDagger

From the Departament de Bioquímica i Biologia Molecular, Unitat de Ciències and the Institut de Biologia Fonamental, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The cDNA of human procarboxypeptidase A2 has been overexpressed in the methylotrophic yeast Pichia pastoris and secreted into the culture medium by means of the alpha -mating factor signal sequence, yielding a major protein of identical size and N-terminal sequence as the wild-type form. Two other forms containing the proenzyme have also been overexpressed: one of them resulted from an incomplete processing of the signal peptide, whereas the other was a glycosylated derivative. Recombinant procarboxypeptidase A2 was purified to homogeneity, and it was shown that its mature active form displays functional properties similar to those of the enzyme directly isolated from human pancreas. The overall yield was ~250 mg of proenzyme or 180 mg of mature enzyme/liter of cell culture. The proteolysis-promoted activation process of the recombinant proenzyme has been studied in detail. During maturation by trypsin, the increase in activity of the enzyme is a rapid and monotonic event, which reflects the rate of the proteolytic release of the inhibitory pro-segment and the weaker nature of its interactions with the enzyme moiety compared with procarboxypeptidases of the A1 type. Three main forms of the pro-segment (96, 94, and 92 amino acids), with no inhibitory capability in the severed state, and a single mature carboxypeptidase A2 are produced during this process. No further proteolysis of these pro-segments by the generated carboxypeptidase A2 occurs, in contrast with observations made in other procarboxypeptidases (A1 and B). This differential behavior is a result of the extreme specificity of carboxypeptidase A2.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Pancreatic carboxypeptidases (CPs)1 are digestive metalloenzymes involved in the hydrolysis of alimentary proteins and peptides from their C-terminal end. Their participation as proenzymes in the digestive cascade (promoted by limited proteolysis) is a well documented process (1-3). Also, their specificity classification between the A forms (CPA, with preference for apolar C-terminal residues) and the B forms (CPB, with preference for basic C-terminal residues) and the tertiary structures of both forms are well known (4). In recent years, there has been an increasing interest in the study of the synthesis, storage, activation, and three-dimensional structure of procarboxypeptidases (pro-CPs), the precursors of such proenzymes (4-6).

The classification of metallocarboxypeptidases has been widened in the last few years with reports about new non-digestive pancreatic-like carboxypeptidases in different extra-pancreatic tissues and biological fluids, with the same evolutionary ancestors as pancreatic carboxypeptidases (4, 7-10). Additionally, the traditional classification of pancreatic carboxypeptidases and their zymogens into the A and B forms has been expanded with the identification of the A1 and A2 isoforms in rat and humans (11, 12). CPA1 and CPA2 differ in specificity for peptide substrates: the former (assignable to the traditional A form) shows a wider preference for aliphatic and aromatic residues, whereas the latter is more restrictive for aromatic residues; this reflects significant differences in the specificity pocket of the enzymes (13). Recently, we have reported the cloning and sequence analysis of the human pro-CPA2 cDNA as well as its three-dimensional modeling (14). CPA2 isoforms have also been reported in rat extra-pancreatic tissues such as brain, testis, and lung (15); these CPA2 isoforms are shorter and have a distinct role from the pancreatic isoform. The high sequence identity found between human pro-CPA2 and rat pro-CPA2 (89% homology) as compared with human pro-CPA1 (64% homology) corroborates the proposal that locates the appearance of the two isoforms by gene duplication before speciation of mammals (11).

Comparison of the prodomain structures in the family of pancreatic proenzymes shows close similarities in conformation between the A1 and A2 forms in regions assumed to be critical for their inhibition and proteolytic activation (6, 14) and significant differences from the corresponding regions in the B form (5). Accordingly, the A2 proenzyme could be expected to show a bimodal and slow proteolytic activation behavior, as previously reported for the A1 form (16), and to differ from the monotonic and quick activation behavior found for the B proenzyme (17). However, earlier proteolytic activation experiments carried out on natural pro-CPA2 isolated from human or rat pancreas (12, 18) do not fit with these expectations and assumptions. Therefore, this is an issue that requires clarification.

In this work, pro-CPA2 has been overexpressed in Pichia pastoris to produce the protein in quantities amenable to the study of the structural and functional determinants of its behavior and activation. The methylotrophic yeast P. pastoris was chosen because of its high yield and capacity of secreting heterologous proteins when linked to the appropriate secretion signal (19). The development of the system reported here to obtain large quantities of fully activable human pro-CPA2 should facilitate not only the characterization of this form, but probably also that of other structurally related forms to which the same procedure could be applied. It could also facilitate its potential biotechnological use, such as the large-scale production of carboxypeptidases able to act as activators of antitumoral prodrugs (20, 21). The efficient expression of human pro-CPA2 in P. pastoris has allowed us to investigate the different events in the proteolytic activation and processing of this proenzyme in detail and to compare them with the processes described in other pancreatic procarboxypeptidases. An overall maturation scheme of such zymogens emerges from this study.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Restriction endonucleases, T4 DNA ligase, Vent polymerase, deoxynucleotide stocks, and N-glycosidase F were purchased from Boehringer Mannheim. Salts and media for Escherichia coli and P. pastoris growth were purchased from Difco. The P. pastoris expression kit was purchased from Invitrogen. Trypsin (treated with tosylphenylalanyl chloromethyl ketone) was from Worthington. Trifluoroacetic acid, Nalpha -p-tosyl-L-lysine chloromethyl ketone (TLCK), and N-(3-(2-furyl)acryloyl)-L-phenylalanyl-L-phenylalanine (FAPP) were from Sigma. Electrophoretic studies were carried out in a Bio-Rad Mini-Protean system. HPLC studies were carried out in a Waters chromatograph.

Plasmid Constructs-- DNA manipulations were carried out essentially as described by Sambrook et al. (22) using E. coli strains JM83 and MC1061 as hosts. The cDNA of human pro-CPA2 was amplified by polymerase chain reaction from a pUC9 vector to introduce an XhoI site at the 5'-end and an EcoRI site at the 3'-end of the cDNA using the following primers: sense primer, 5'-GTATCTCTCGAGAAAAGACTAGAAACATTTGTGGGAGA-3'; and antisense primer, 5'-CTAGAATTCATGGCTCTTGTTTCTTCC-3'. After restriction enzyme digestion of the polymerase chain reaction product, the cDNA was cloned and subcloned into M13 pBluescript to confirm the entire sequence and the changes made in the polymerase chain reaction. pBluescript-pro-CPA2 was digested by XhoI and EcoRI, and the cDNA was ligated to the P. pastoris shuttle vector pPIC9 between the 5'-promoter and the 3'-terminator of the alcohol oxidase gene (AOX1). pPIC9 provides the alpha -mating factor signal for secretion and the HIS4 gene for selection of the recombinant yeast clones. pHIL-D2 was also used for the expression and secretion of pro-CPA2 using its own signal sequence. In the latter case, the original pUC9-pro-CPA2 clone was digested and ligated to the P. pastoris pHIL-D2 shuttle vector using the EcoRI site of the polylinker.

Transformation and Selection of the Productive Clones-- After linearization of the corresponding P. pastoris pPIC9-pro-CPA2 and pHIL-D2-prepro-CPA2 vectors with BglII and NotI, respectively, the P. pastoris GS115 (his4) strain was transformed either by electroporation or by the spheroplast method. After simultaneously plating the transformants in MM and MD agar (1.34% yeast nitrogen base, 0.00004% biotin, and 0.5% methanol or 1% dextrose, respectively), those clones that suffered homologous recombination with the AOX1 sequence (slow growing in MM agar) were selected. To test for the most productive clones, colonies were grown in 10 ml of buffered liquid BMGY medium (1% yeast extract, 2% peptone, 90 mM potassium phosphate, pH 6.0, 1.34% yeast nitrogen base, 0.00004% biotin, and 1% glycerol) at 30 °C for 4 days. Cells were collected by centrifugation and gently resuspended in 2 ml of buffered liquid BMMY medium (same as BMGY medium but containing 0.5% methanol instead of 1% glycerol) and cultured for another 2 days to induce the expression of the recombinant protein. The production of the clones was monitored after 6, 24, and 48 h by electrophoretic analysis of the supernatant on SDS-12% polyacrylamide gels. Western blotting was carried out as described previously using 1:500 anti-human pancreatic procarboxypeptidase antiserum (14). The functionality of the expressed protein was analyzed with the synthetic substrate FAPP after activation of the proenzyme with trypsin (at a 40:1 ratio by weight).

Expression and Purification of Recombinant Human Pro-CPA2-- 1-Liter shake-flask cultures were grown at 30 °C for 4 days in buffered BMGY medium (20-30 A units at 600 nm; 20 g of cells (dry weight)/liter). Cells were collected by centrifugation at 1500 g, gently resuspended in 200 ml of BMMY medium, and cultured for another 2 days to induce the production of pro-CPA2. The culture medium was separated from the cells by centrifugation, and after equilibrating its ionic strength, it was processed through a two-step chromatographic purification: hydrophobic interaction chromatography on a butyl-Toyopearl 650M column eluted with a decreasing gradient of ammonium sulfate and fast protein liquid chromatography on a preparative anion-exchange column (TSK-DEAE 5PW) eluted with a gradient of ammonium acetate in 30 mM MES, pH 5.7. The elution of recombinant pro-CPA2 was observed at 9% ammonium sulfate and 6 mM ammonium acetate, respectively. The identity of recombinant pro-CPA2 was confirmed after automated Edman degradation analysis of its N-terminal sequence and MALDI-TOF spectrometry analysis.

Activation Studies of Recombinant Human Pro-CPA2-- Recombinant human pro-CPA2 at 1 mg/ml in 50 mM Tris-HCl and 1 µM ZnCl2, pH 8.0, was treated with trypsin at 40:1 and 400:1 (w/w) ratios at 0 °C. At given times after trypsin addition, aliquots were removed for activity measurements, for electrophoretic reverse-phase HPLC and mass spectrometry analyses, and for quantification of the released amino acids. For activity measurements, 10 µl of the activation mixture were added to 190 µl of aprotinin (bovine pancreas trypsin inhibitor) at 0.1 mg/ml in 20 mM Tris and 0.1 M NaCl, pH 7.5, and 10 µl of this new mixture were used to carry out spectrophotometric activity measurements with the FAPP substrate at 330 nm. For electrophoretic analysis, 20 µl of the activation mixture were mixed with 2 µl of 22 mM TLCK in water to reach a final trypsin inhibitor concentration of 2 mM. Each sample was immediately mixed with electrophoretic loading buffer containing 1% SDS and 3% beta -mercaptoethanol, heated at 90 °C for 1 min, and stored at -20 °C until analysis. Electrophoresis was carried out on Tricine-polyacrylamide gels (23). For HPLC and mass spectrometry analyses, 90-µl samples were removed from the activation mixture, made 0.5% in trifluoroacetic acid to inhibit proteolysis, and immediately chromatographed or kept at -20 °C for subsequent analysis. For quantitation of the amino acids released into the activation mixture, 90-µl samples were taken (2 nmol of initial pro-CPA2) and mixed with trifluoroacetic acid up to a final concentration of 0.5%. 1.5 nmol of norleucine were added as a quantitative reference before the addition of 3 volumes of ethanol to precipitate proteins and large peptides. The supernatant was lyophilized and analyzed for amino acid composition.

Chromatographic Analysis by Reverse-phase HPLC-- Samples removed from the activation mixtures were analyzed by reverse-phase HPLC on Vydac C4 supports. A 214TP54 column (250 × 4.6 mm, 5-µm particle size, 0.3 µm-pore) was used, and elution was followed at 214 nm. Chromatographies were performed in 0.1% trifluoroacetic acid with an eluting linear gradient between water (solvent A) and acetonitrile (solvent B) according to the following steps: 10% solvent B from 0 to 10 min, 10-32% solvent B from 10 to 30 min, and 32-52% solvent B from 30 to 130 min.

Mass Spectrometry-- The activation mixtures were analyzed by mass spectrometry with a MALDI-TOF spectrometer (Biflex with Reflectron, Bruker). 50 pmol of each sample in 1 µl were mixed with 1 µl of 50% synapinic acid as a matrix and loaded.

Amino Acid Analysis-- Amino acid analyses were carried out by the 4-dimethylaminoazobenzene-4'-sulfonyl derivatization method (24) using materials and protocols from Beckman. A reverse-phase HPLC NovaPak C18 column was used to separate the amino acids and oligopeptides produced during the activation.

N-terminal Sequence Analysis-- After analysis by SDS-PAGE or analysis and purification by HPLC, N-terminal sequence analysis of pro-CPA2 and the activation products was performed by blotting the samples on polyvinylidene difluoride membranes, followed by direct analysis on a Beckman LF3000 Protein Sequencer.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Construction of the Expression Vector and Transformation into P. pastoris-- A 1254-base pair cDNA encoding human preprocarboxypeptidase A2 has been cloned from a pancreatic library by immunological and radioactive approaches (14). This cDNA codes for a short leader signal peptide of 16 amino acids, a pro-segment of 96 amino acids, and an active enzyme of 305 amino acids (see below). In this work, this cDNA was modified by polymerase chain reaction to add an XhoI site at the 5'-end and an EcoRI site at the 3'-end to amplify only the proenzyme. The complete human prepro-CPA2 cDNA was also cloned into the EcoRI site of the P. pastoris pHIL-D2 expression vector to test whether the native signal sequence of pro-CPA2 could release the proenzyme into the extracellular medium.

Transformation of the P. pastoris GS115 (his4) strain with the linearized vectors was carried out by the spheroplast and electroporation methods. Both gave a similar number of transformants, which were screened for histidinol dehydrogenase (His+) phototrophy by plating on a dextrose-based medium without histidine supplementation. Nearly 20% of the His+ clones from the transformation showed reduced growth on methanol as the sole carbon source (slow growing, Mut-), indicating the integration of the expression cassette into the AOX1 gene.

Overexpression and Purification of Recombinant Human Pro-CPA2-- The clones transformed with the alpha -MF-pro-CPA2 fusion product secreted a dominant 45-kDa protein in the P. pastoris supernatant upon induction by methanol. Those transformed with pHIL-D2-prepro-CPA2 did not secrete any protein into the medium. The 45-kDa protein was found to correspond to human pro-CPA2 by Western blot analysis. Upon induction with 0.5% methanol, the Mut- phenotypes expressed more protein than the Mut+ phenotypes. However, a 3-fold increase in protein secretion was observed in the Mut+ phenotypes when induction was assayed with 5% methanol (data not shown). One of these high productivity clones was selected for large-scale production of human pro-CPA2.

Analysis of the production of human pro-CPA2 in the above system by SDS-PAGE and Western blotting is shown in Fig. 1. After 2 days of induction by methanol, a strong band of the proenzyme and a faint band corresponding to the 34-kDa active form were detected in the intracellular soluble part of the culture. This observation is in accordance with previous reports about the existence of proteases with trypsin-like activity in P. pastoris (25). After 30 h of methanol induction, in addition to the dominant 45-kDa pro-CPA2 protein, a smear of high relative molecular mass, immunodetected by human procarboxypeptidase antiserum, was visible (Fig. 1B) in the lanes corresponding to the extracellular medium. It corresponds to the alpha -MF-pro-CPA2 fusion product, which contains N-linked glycosylations in the signal peptide (26) and is not cleaved by the P. pastoris KEX2 processing protease. After purification (see below) and treatment of the sample with N-glycosidase F, conversion of the smear into a lower molecular mass band that corresponds to the alpha -MF-pro-CPA2 fusion product was observed (Fig. 2). The different protein products was finally identified by N-terminal sequencing of the blotted SDS-polyacrylamide bands (data not shown). Trypsin treatment gave rise in all cases to a CPA2 (Fig. 2) and to activation peptides (not shown in the figure) with the expected molecular masses, i.e. 34 and 10-11 kDa, respectively.


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Fig. 1.   Production of recombinant human pro-CPA2 in P. pastoris. Shown are the results from analysis of the production of the recombinant proenzyme throughout the course of fermentation by SDS-polyacrylamide gel electrophoresis. Parallel 12% polyacrylamide gels were directly stained with Coomassie Blue (A) or immunoblotted (B) and stained. The samples were as follows: Cg, cell extract; M, cell-free medium after 4-day growth in 1% glycerol; Cm, cell extract after 48 h of 0.5% methanol induction; M1, M2, and M3, cell-free medium after 6, 24, and 48 h of 0.5% methanol induction, respectively. The cell extract samples contained the protein solubilized after treating the cells with electrophoresis loading buffer (62 mM Tris, 10% glycerol, 2% SDS, 0.7% beta -mercaptoethanol, and bromphenol blue) for 1 min at 100 °C. In the case of the cell-free medium samples, a volume equivalent to 5 µl of the fermentation medium was loaded in the electrophoresis wells. A control sample (st) containing 1.5 µg of pro-CPA2 isolated from human pancreas was included.


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Fig. 2.   Deglycosylation and activation with trypsin of the alpha -MF-pro-CPA2 fusion protein. SDS-8% polyacrylamide gel electrophoresis was used to analyze the different products containing pro-CPA2 purified from the P. pastoris culture. Samples were analyzed before (lanes 1, 3, and 5) and after (lanes 2, 4, and 6) trypsin treatment. Lanes 1 and 2, correctly processed recombinant human pro-CPA2; lanes 3 and 4, samples containing a glycosylated derivative of the alpha -MF-pro-CPA2 fusion product; lanes 5 and 6, the alpha -MF-pro-CPA2 fusion product after deglycosylation. 2 µg of CPA2 purified from correctly processed recombinant pro-CPA2 were loaded in lane 7 as a control.

As reported above, the human pro-CPA2 gene was cloned behind the alpha -MF peptide to accomplish secretion of the recombinant protein. N-terminal sequence analysis of secreted recombinant human pro-CPA2 showed the occurrence of heterogeneity in the processing of the alpha -MF precursor. The deduced processing targets, located at the boundary between the alpha -MF precursor and pro-CPA2, are indicated in Fig. 3. Native pro-CPA2 was the major form found in all cultures, but different unspecific cleavage targets for endopeptidases were found in every induction performed on the same clone. Since the system used has been pushed to overexpress the recombinant protein at >300 mg/liter, the processing machinery might be unable to properly process all the material.


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Fig. 3.   Processing of the recombinant alpha -MF-pro-CPA2 fusion product. The nucleotide and amino acid sequences at the boundary of the alpha -MF-pro-CPA2 fusion product are shown. XhoI (underlined sequence) indicates the cloning site for pro-CPA2 cDNA. The KEX2 cleavage site (I) after the dibasic Lys-Arg peptide produces a correct N terminus for recombinant pro-CPA2 (amino acid sequence in italics). Unspecific processing sites (II) found by N-terminal sequencing of recombinant products are indicated with dashed arrows.

A chromatographic approach was developed to separate the differently processed forms of human recombinant pro-CPA2 and to isolate the native form. It was based upon a two-step purification scheme, with atmospheric hydrophobic interaction chromatography on a butyl column applied first, followed by fast protein liquid anion-exchange chromatography to separate the different forms (see "Experimental Procedures"). Starting from a cell culture of a Mut+ clone that achieved a cell density of 20 g of cells (dry weight)/liter, an overall yield of 250 mg of total pure pro-CPA2 was normally obtained. The correctly processed pro-CPA2 form was generally the principal one, with yields varying from 80 to 200 mg/liter, although in certain cultures, the glycosylated and incorrectly processed forms could account for as much as 60% of the purified material. In contrast, treatment of total pro-CPA2 with trypsin before the anion-exchange chromatography yielded ~180 mg of homogeneous, correctly processed CPA2/liter of cell culture. Both the purified recombinant pro-CPA2 and CPA2 forms were fully functional, with enzymatic properties similar to those previously reported for the natural forms isolated from human pancreas (12). Recombinant CPA2 and the enzyme isolated from pancreas showed the same maximum specific activity of ~85 µmol of FAPP substrate hydrolyzed per min/mg of protein.

Activation of Recombinant Human Pro-CPA2 with Different Proteases-- Analysis of the action of trypsin, chymotrypsin A, and elastase (the major active endoproteolytic counterparts in pancreatic secretion) upon correctly processed recombinant pro-CPA2 indicates that trypsin is, by far, the most efficient activator (data not shown). At the pro-CPA2/endoprotease ratio used, 90% activation of the former was achieved by trypsin in ~10 min, whereas the same level of activation was only achieved after 125 min of treatment with elastase; in the latter time span, chymotrypsin was able to generate only 10% of active CPA2. This is in agreement with previous studies that reported trypsin as the main enzyme responsible for the activation of pancreatic procarboxypeptidases (3).

Trypsin Activation of Recombinant Human Pro-CPA2-- The action of trypsin at 37 °C on recombinant pro-CPA2 at a 40:1 (w/w) pro-CPA2/trypsin ratio is excessively quick for detailed mechanistic analysis, in agreement with previous studies on natural human proenzymes (12). The study of the activation process was therefore carried out at 0 °C and at a 400:1 ratio. Under these conditions, the appearance of carboxypeptidase activity was measured with the synthetic substrate FAPP; it followed a quick and monotonic activation curve, which can be fitted to a pseudo first-order kinetics (Fig. 4A). Using Tricine/SDS-PAGE, the rapid proteolytic severing of the pro-segment, the concomitant appearance of CPA2, and a straight correlation of this proteolysis with the monotonic activity curve were observed (Fig. 4B). Nearly all pro-CPA2 was converted into its active form in 20 min, and a maximum value in the activity curve was achieved. The generated pro-segment appeared as a single band on Tricine/SDS-PAGE (Fig. 4B), although mass spectrometry analysis indicated heterogeneity for this species (see below).


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Fig. 4.   Generation of the mature enzyme from human pro-CPA2 by the action of trypsin. Recombinant human pro-CPA2 at 1 mg/ml in 50 mM Tris-HCl and 0.01 mM ZnCl2, pH 8.0, was treated with trypsin at 0 °C and at a ratio of 400:1 (w/w). At controlled times, aliquots were withdrawn and analyzed for carboxypeptidase A2 appearance by activity measurements and SDS-PAGE. A, carboxypeptidase activity values obtained with 0.2 mM FAPP as substrate; units are expressed as µmol of substrate hydrolyzed per min/mg of protein. B, quantitation of the relative amounts of pro-CPA2 (black-square) and CPA2 (black-triangle) by densitometry after SDS-PAGE and Coomassie Blue staining. The inset shows the electrophoretic follow-up of the activation process on Tricine/SDS-polyacrylamide gel. The electrophoretic band corresponding to the activation sequence of pro-CPA2 is denoted by asA2.

The N-terminal sequence of the electrophoretic CPA2 band, obtained from different samples at different activation times and transferred to a polyvinylidene difluoride membrane, was always Ser-Gly-Asn-, a fact that indicates that the Arg96-Ser97 peptide bond is the first point of cleavage of the proenzyme by trypsin. From this result and from the increase in the intensity of the CPA2 band in parallel with the appearance of CPA2 activity in the medium and the corresponding decrease in the intensity of the pro-CPA2 band (Fig. 4B), it can be assumed that the cleavage of the Arg96-Ser97 peptide bond is sufficient to generate all the carboxypeptidase activity. As a consequence, it can be concluded that the severed pro-segment does not inhibit CPA2, in contrast to what has been previously reported for the pro-CPA1 forms (12, 16, 27).

To study the maturation process in more detail, the time course of degradation of the pro-segment was followed by reverse-phase HPLC. It was expected that this degradation would take place only at the C-terminal arginine-rich end of the pro-segment since N-terminal sequence analyses of the activation mixtures indicated the appearance of only two N termini throughout the process, one corresponding to the original proenzyme and one from the generated mature enzyme. The chromatographic analyses of the activation mixtures at different times are represented in Fig. 5, where the appearance and disappearance of three different protein fragments arising from the proteolysis of the pro-segment (labeled alpha , beta , and gamma ) can be observed. Mass spectrometry (MALDI-TOF) was used for the characterization of the molecular masses of these fragments (Table I). Their identification was facilitated by the knowledge of the sequence of pro-CPA2 and by analysis of peptides and amino acids that were released into the medium by the action of trypsin and CPA2. In particular, it is worth mentioning that two dipeptides (Glu-Arg and Arg-Arg) were detected by mass spectrometry at early and late activation times and that no significant free amino acids were detected by parallel amino acid analysis of the protein-free activation medium. All of this information was used to establish and confirm the sequence of the cleavages during the proteolytic processing.


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Fig. 5.   Analysis by reverse-phase HPLC of the fragments generated during tryptic activation of human pro-CPA2. At different times, aliquots were taken from the activation mixture under the same conditions as described in the legend to Fig. 4 and analyzed by reverse-phase chromatography in an HPLC system. The action of trypsin was stopped by adding 0.1% trifluoroacetic acid. 100 µl of each sample were loaded on a Vydac C4 reverse-phase column, and elution was followed at 214 nm. The alpha -fragment corresponds to residues 1-96, the beta -fragment to residues 1-94, and the gamma -fragment to residues 1-92. PCPA2, pro-CPA2.

                              
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Table I
Analysis of the tryptic activation fragments of human pro-CPA2 by mass spectrometry
Values are the relative molecular masses derived by MALDI-TOF mass spectrometry analysis of samples taken at different times of the activation process, carried out under the same conditions as described in the legend to Fig. 4. The action of trypsin was stopped by adding 0.1% trifluoroacetic acid. The alpha -fragment corresponds to residues 1-96, the beta -fragment to residues 1-94, and the gamma -fragment to residues 1-92.

According to the above information, during the tryptic maturation of pro-CPA2, the first cleavage is at the C terminus of Arg96, producing the alpha -fragment (residues 1-96), as shown in Fig. 6. Subsequently and while some proenzyme molecules are still intact, a rapid cleavage occurs at Arg94 of the alpha -fragment, giving rise to the beta -fragment (residues 1-94) and to the release of a Glu-Arg dipeptide. The third cleavage (also attributable to trypsin, as are the former cleavages) occurs slowly at Arg92 of the beta -fragment, giving rise to the gamma -fragment (residues 1-92) and to the release of a second dipeptide, Arg-Arg. The lack of detection of free amino acids in the medium indicates the null action of generated CPA2 on the alpha -, beta -, or gamma -fragment. The gamma -fragment is resistant to further proteolysis under these conditions. Only a single CPA2 species was detected throughout the proteolytic process by HPLC, N-terminal, and mass spectrometry analyses.


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Fig. 6.   Schematic representation of the cleavage points in the activation process of human pro-CPA2 promoted by trypsin. The scheme shows the sequence of the alpha -helical connecting region between the globular activation domain and the active enzyme. Arrows indicate the tryptic cleavage points observed under the same conditions as described in the legend to Fig. 4, which released three activation fragments (alpha , beta , and gamma ) of 96, 94, and 92 residues, respectively, as deduced by reverse-phase HPLC, N-terminal sequencing, and mass spectrometry studies. No cleavages were detected in other parts of the molecule. The activation sequence of pro-CPA2 is denoted by asA2.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

One of the aims of this study was the development of a highly efficient recombinant expression system for a native and activable form of human procarboxypeptidase A2 in the P. pastoris heterologous system to study its proteolytic activation and maturation pathway in vitro. It is worth mentioning that pancreatic carboxypeptidases and their precursors are difficult to express in native and soluble forms in E. coli (28) and that reports about the expression of their precursor forms in eukaryotic cells (i.e. Saccharomyces cerevisiae) have indicated a moderate yield (<10 mg/liter) until now (21, 29). In fact, the primary production of the proenzymes by recombinant approaches followed by proteolytic activation is probably the best strategy to obtain active carboxypeptidases given that it takes advantage of the high folding capability of the pro-segment in heterologous systems (29, 30). The recent biotechnological interest in metallocarboxypeptidases as prodrug activators for cancer therapy (21, 31, 32) makes the availability of large-scale procedures to obtain pro- and carboxypeptidases, particularly the human forms, very useful. The efficient heterologous production of extra-pancreatic metalloprocarboxypeptidases, with many of them involved in important biological functions and some with a tertiary structure or domains structurally related to the pancreatic ones (4), could also facilitate their detailed characterization. Hence, the development of efficient heterologous expression systems for enzymes and proenzymes such as the one reported here is highly desirable.

The experiments designed to establish an easy and robust expression system capable of providing large amounts of soluble human pro-CPA2 were restricted to shake-flask cultures, which rendered a production of ~250 mg of recombinant proenzyme/liter or 180 mg of active enzyme/liter. This production is more than enough for studies such as those presented here and for subsequent structural determinations as well as for other analytical and semipreparative purposes. However, P. pastoris can increase this production 10-fold by scaling up from shake-flask to high density fermentation (19).

The original target for the removal of the alpha -MF propeptide is the sequence KREAEAEA, which is cleaved by the yeast endopeptidase KEX2 after the dibasic peptide and undergoes subsequent elimination of Glu-Ala dipeptides by dipeptidyl aminopeptidase A (33). Thus, according to the design of the pPIC9-pro-CPA2 expression plasmid, the pro-segment of the alpha -mating factor was expected to be cleaved after the dibasic residues Lys-Arg by a single KEX2 endopeptidase action. However, the N-terminal sequence of expressed recombinant pro-CPA2 displayed microheterogeneity at the amino terminus, indicating that cleavage by the KEX2 endopeptidase is only partial and that other endoproteinases might be responsible for the unspecific cleavages (see Fig. 3). The microheterogeneity observed did not become an important problem since the principal product found in all of the cultures was native pro-CPA2. Part of the secreted fusion product appeared glycosylated; however, this product was converted to normal pro-CPA2 after deglycosylation. In any case, all of these products can generate fully active CPA2 by limited proteolysis.

A correctly processed alpha -MF propeptide has been reported for recombinant proteins expressed in P. pastoris without the C-terminal Glu-Ala extension (34, 35); other reports indicated heterogeneity at the N terminus of the secreted proteins (i.e. aprotinin and coffee bean galactosidase), also resulting from different cleavage points in the processing of the alpha -MF pro-segment (36, 37). Thus, the microheterogeneity observed in some cases could be dependent on the nature of the recombinant protein expressed (33).

The trypsin-promoted maturation mechanism of human pro-CPA2, as derived from our studies, is shown in Fig. 6. Due to the presence of several arginines at the boundary region between the pro-segment and the enzyme moiety, a rapid tryptic cleavage at the most exposed arginine of this region and subsequent cleavages at the remaining trypsin targets should be expected. Alternatively, simultaneous cleavages could occur, but our results indicate that it is the first mechanism that takes place. In contrast to other procarboxypeptidases studied, the Arg96-Ser97 peptide bond is the first target for trypsin action observed in pro-CPA2, releasing a pro-segment of 96 residues into the medium (alpha -fragment in Figs. 5 and 6). From sequence alignments and structure comparisons (14), this peptide bond is considered to belong to the enzyme moiety in porcine pro-CPA1 and pro-CPB, where the cleavage occurs two residues farther along N-terminally (16, 17). The released primary activation fragment and mature enzyme are therefore two residues larger and shorter, respectively, in pro-CPA2.

The second step in the proteolytic processing is the rapid elimination of the C-terminal Glu-Arg dipeptide end from the alpha -fragment. The product of this cleavage (beta -fragment in Figs. 5 and 6) is due to a tryptic action on arginine 94 of the alpha -fragment, as shown by mass spectrometry analysis and by the lack of release of free amino acids. The action of trypsin in this conversion is not as fast as in the first cleavage, probably due to the less efficient tryptic endoproteinase activity near the C-terminal end of proteins, and takes place while some proenzyme molecules are still intact. The third and last sequence of 92 residues (gamma -fragment in Figs. 5 and 6), resistant to further proteolysis under these conditions, is generated by trypsin action in the long run, releasing an Arg-Arg dipeptide into the activation medium. It is also worth commenting upon that no release of free amino acids into the medium is observed in the course of activation, in contrast to previous observations in the corresponding activation processes of pro-CPA1 and pro-CPB (16, 17, 38). This indicates the high specificity shown by human CPA2, which is unable to trim the C-terminal arginines from its activation peptides, even at the high concentration of these species in the activation medium.

According to this study, it seems clear that the pro-segments of pro-CPA2 released into the medium do not inhibit the activity of the active enzyme, even in its longer (primary) form (96 amino acids), since CPA2 reaches total activity following a rapid hyperbolic activation curve at activation times when a substantial concentration of the alpha -fragment sequence is still present in solution. In contrast to this behavior, porcine pro-CPA1, which shows a slower and biphasic activation process, needs subsequent trimmings at the C-terminal end and a second tryptic cleavage inside the globular domain of the pro-sequence to achieve full carboxypeptidase activity. In this sense, pro-CPA2 behaves in a way that is more similar to pro-CPB (17).

X-ray crystallography and modeling studies have shown the structural similarities between pro-CPA1 and pro-CPA2, both sharing a long C-terminal alpha -helix at the region connecting the globular activation domain with the active enzyme, and the differences of these two proenzymes from pro-CPB, whose corresponding connecting region has a much shorter alpha -helix and is less structured overall (5, 6, 14). These differences were proposed to be the primary determinant responsible for the diversity observed in the rates of activation of pro-CPA1 and pro-CPB, arguing that the larger regular structure of the connecting region in pro-CPA1 would render the interactions with the enzyme more stable and make the structural relaxation needed for full release slower (4, 39). Modeling studies with pro-CPA2 (14) have shown that such a connecting region is also structured in a long alpha -helix, at least the same size as in porcine pro-CPA1. Taking into account the former evidence and the observation of a rapid and monotonic activation curve for pro-CPA2, it can be concluded that the folding of the connecting region of procarboxypeptidases in a long alpha -helix does not give rise to inhibitory activation fragments and to slow activation processes by itself. Other structural determinants, such as surface complementarity and electrostatic and Van der Waals interactions, should be considered to evaluate the stability of the bimolecular complex between the pro-segment and the enzyme moiety once the first proteolytic cleavage has taken place.

Preliminary x-ray diffraction studies2 on human pro-CPA2 show that the connecting region is highly structured and that extensive interactions take place between the enzyme moiety and both the globular domain region and the connecting region of the pro-segment. These observations confirm that the latter two regions are responsible for the activation behavior of pro-CPA2 and for its functional differences from other procarboxypeptidases.

    ACKNOWLEDGEMENTS

We thank Dr. S. Bartolomé (Laboratory for Image Analysis) for help in the elaboration of the figures and Dr. F. Canals (Servei de Seqüenciació i Biocomputació) for performing the N-terminal sequence analysis of the proteins.

    FOOTNOTES

* This work was supported by Grant BIO95-0848 from the Comisión Interministerial de Ciencia y Tecnología (Ministerio de Educación y Ciencia, Spain) and by the Center de Referencia de Biotecnologia (Generalitat de Catalunya).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 34-3-5811315; Fax: 34-3-5812011; E-mail: FX.Aviles{at}blues.uab.es.

1 The abbreviations used are: CPs, carboxypeptidases; pro-CPs, procarboxypeptidases; TLCK, Nalpha -p-tosyl-L-lysine chloromethyl ketone; FAPP, N-(3-(2-furyl)acryloyl)-L-phenylalanyl-L-phenylalanine; HPLC, high pressure liquid chromatography; MES, 4-morpholineethanesulfonic acid; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; alpha -MF, prepro-alpha -mating factor; PAGE, polyacrylamide gel electrophoresis.

2 I. García-Sáez, personal communication.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Neurath, H. (1986) J. Cell. Biochem. 32, 35-49[Medline] [Order article via Infotrieve]
  2. Puigserver, A., Chapus, C., and Kerfelec, B. (1986) in Molecular and Cellular Basis of Digestion (Desnuelle, P., Sjöstrom, H., and Noren, O., eds), pp. 235-247, Elsevier Science Publishers B. V., Amsterdam
  3. Auld, D. S., and Vallee, B. L. (1987) New Compr. Biochem. 16, 201-256
  4. Avilés, F. X., Vendrell, J., Guasch, A., Coll, M., and Huber, R. (1993) Eur. J. Biochem. 211, 391-399[Abstract]
  5. Coll, M., Guasch, A., Avilés, F. X., Huber, R. (1991) EMBO J. 9, 1-9[Abstract]
  6. Guasch, A., Coll, M., Avilés, F. X., Huber, R. (1992) J. Mol. Biol. 224, 141-157[Medline] [Order article via Infotrieve]
  7. Reynolds, D. S., Stevens, R. L., Gurley, D. S., Lane, W. S., Austen, K. F., Serafin, W. E. (1989) J. Biol. Chem. 264, 20094-20099[Abstract/Free Full Text]
  8. Tan, A. K., and Eaton, D. L. (1995) Biochemistry 34, 5811-5816[Medline] [Order article via Infotrieve]
  9. Skidgel, R. A. (1996) in Zinc Metalloproteases in Health and Disease (Hooper, N. M., ed), pp. 241-283, Taylor & Francis Ltd., London
  10. Song, L., and Fricker, L. D. (1997) J. Biol. Chem. 272, 10543-10550[Abstract/Free Full Text]
  11. Gardell, S. J., Craik, C. S., Clauser, E., Goldsmith, E., Stewart, C.-B., Graf, M., and Rutter, W. J. (1989) J. Biol. Chem. 263, 17828-17836[Abstract/Free Full Text]
  12. Pascual, R., Burgos, F. J., Salvà, M., Soriano, F., Méndez, E., and Avilés, F. X. (1989) Eur. J. Biochem. 179, 609-616[Abstract]
  13. Famming, Z., Kobe, B., Stewart, C. B., Rutter, W. J., Goldsmith, E. (1991) J. Biol. Chem. 266, 24606-24612[Abstract/Free Full Text]
  14. Catasús, L., Vendrell, J., Avilés, F. X., Carreira, S., Puigserver, A., Billeter, M. (1995) J. Biol. Chem. 270, 6651-6657[Abstract/Free Full Text]
  15. Normant, E., Gros, C., and Schwartz, J. C. (1995) J. Biol. Chem. 270, 20543-20549[Abstract/Free Full Text]
  16. Vendrell, J., Cuchillo, C. M., and Avilés, F. X. (1990) J. Biol. Chem. 265, 6949-6953[Abstract/Free Full Text]
  17. Villegas, V., Vendrell, J., and Avilés, F. X. (1995) Protein Sci. 4, 1792-1800[Abstract/Free Full Text]
  18. Oppezzo, O., Ventura, S., Bergman, T., Vendrell, J., Jörnvall, H., and Avilés, F. X. (1994) Eur. J. Biochem. 222, 55-63[Abstract]
  19. Cregg, J. M., Vedvick, T. S., and Raschke, W. C. (1993) Bio/Technology 11, 905-910[Medline] [Order article via Infotrieve]
  20. Huennekens, F. M. (1994) Trends Biotechnol. 12, 234-239[Medline] [Order article via Infotrieve]
  21. Laethem, R. M., Blumenkopf, T. A., Cory, M., Elwell, L., Moxham, C. P., Ray, P. H., Walton, L. M., Smith, G. K. (1996) Arch. Biochem. Biophys. 332, 8-18[CrossRef][Medline] [Order article via Infotrieve]
  22. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  23. Schägger, H., and Von Jagow, G. (1987) Anal. Biochem. 166, 368-379[Medline] [Order article via Infotrieve]
  24. Vendrell, J., and Avilés, F. X. (1986) J. Chromatogr. 358, 401-413[CrossRef]
  25. Ohi, H., Ohtani, W., Okazaki, N., Furuhata, N., and Ohmura, T. (1996) Yeast 12, 31-40[CrossRef][Medline] [Order article via Infotrieve]
  26. Kjeldsen, T., Brandt, J., Andersen, A. S., Egel-Mitani, M., Hach, M., Petterson, A. F., Vad, K. (1996) Gene (Amst.) 170, 97-112
  27. Gomis-Rüth, F. X., Gómez, M., Bode, W., Huber, R., and Avilés, F. X. (1995) EMBO J. 14, 4387-4394[Abstract]
  28. Villegas, V. (1995) Detailed Characterization of the Activation Process of Procarboxypeptidase B through the Use of Inhibitors: Design and Construction of Mutants of the Activation Segment. Ph.D. thesis, Universitat Autònoma de Barcelona
  29. Phillips, M. A., and Rutter, W. J. (1996) Biochemistry 35, 6771-6776[CrossRef][Medline] [Order article via Infotrieve]
  30. Villegas, V., Azuaga, A., Catasús, L., Reverter, D., Mateo, P. L., Avilés, F. X., Serrano, L. (1995) Biochemistry 34, 15105-15110[Medline] [Order article via Infotrieve]
  31. Vitols, K. L., Haag-Zeino, B., Baer, T., Montejano, Y. D., Huennekens, F. M. (1995) Cancer Res. 55, 478-481[Abstract]
  32. Rowsell, S., Pauptit, R. A., Tucker, A. D., Melton, R. G., Blow, D. M., Brick, P. (1997) Structure 15, 337-347
  33. Brake, A. J., Merryweather, J. P., Coit, D. G., Heberlein, V. A., Masiarz, F. R., Mullenbach, G. T., Urdea, M. S., Valenzuela, P., Barr, P. J. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 4642-4646[Abstract]
  34. Wagner, S. L., Siegel, R. S., Vedvick, T. S., Raschke, W. C., Van Nostrand, W. E. (1992) Biochem. Biophys. Res. Commun. 196, 1139-1145
  35. Vozza, L. A., Wittwer, L., Higgins, D. R., Purcell, T. J., Bergseid, M., Collins-Raci, L. A., LaVallie, E. R., Hoeffler, J. P. (1996) Bio/Technology 14, 77-91[Medline] [Order article via Infotrieve]
  36. Vedvick, T., Buckholz, R. G., Engel, M., Urcan, M., Kinney, J., Provow, S., Siegel, R., and Thill, G. P. (1991) J. Ind. Microbiol. 7, 197-202[Medline] [Order article via Infotrieve]
  37. Zhu, A., Monahan, C., Zhang, Z., Hurst, R., Leng, L., and Goldstein, J. (1995) Arch. Biochem. Biophys. 324, 65-70[CrossRef][Medline] [Order article via Infotrieve]
  38. Burgos, F. J., Salvà, M., Villegas, V., Soriano, F., Méndez, E., and Avilés, F. X. (1991) Biochemistry 30, 4092-4099
  39. Vendrell, J., Catasús, L., Oppezzo, O., Ventura, S., Villegas, V., and Avilés, F. X. (1993) in Innovations in Proteases and Their Inhibitors (Avilés, F. X., ed), pp. 279-297, Walter de Gruyter & Co., Berlin


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