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• W2135229105 abstract "The cDNA of human procarboxypeptidase A2 has been overexpressed in the methylotrophic yeastPichia pastoris and secreted into the culture medium by means of the α-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. The cDNA of human procarboxypeptidase A2 has been overexpressed in the methylotrophic yeastPichia pastoris and secreted into the culture medium by means of the α-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. Pancreatic carboxypeptidases (CPs) 1The abbreviations used are: CPs, carboxypeptidases; pro-CPs, procarboxypeptidases; TLCK, N α-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; α-MF, prepro-α-mating factor; PAGE, polyacrylamide gel electrophoresis. 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 (1Neurath H. J. Cell. Biochem. 1986; 32: 35-49Crossref PubMed Scopus (117) Google Scholar, 2Puigserver A. Chapus C. Kerfelec B. Desnuelle P. Sjöstrom H. Noren O. Molecular and Cellular Basis of Digestion. Elsevier Science Publishers B. V., Amsterdam1986: 235-247Google Scholar, 3Auld D.S. Vallee B.L. New Compr. Biochem. 1987; 16: 201-256Crossref Scopus (32) Google Scholar). 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 (4Avilés F.X. Vendrell J. Guasch A. Coll M. Huber R. Eur. J. Biochem. 1993; 211: 391-399Crossref PubMed Scopus (73) Google Scholar). 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 (4Avilés F.X. Vendrell J. Guasch A. Coll M. Huber R. Eur. J. Biochem. 1993; 211: 391-399Crossref PubMed Scopus (73) Google Scholar, 5Coll M. Guasch A. Avilés F.X. Huber R. EMBO J. 1991; 9: 1-9Crossref Scopus (150) Google Scholar, 6Guasch A. Coll M. Avilés F.X. Huber R. J. Mol. Biol. 1992; 224: 141-157Crossref PubMed Scopus (98) Google Scholar). 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 (4Avilés F.X. Vendrell J. Guasch A. Coll M. Huber R. Eur. J. Biochem. 1993; 211: 391-399Crossref PubMed Scopus (73) Google Scholar, 7Reynolds D.S. Stevens R.L. Gurley D.S. Lane W.S. Austen K.F. Serafin W.E. J. Biol. Chem. 1989; 264: 20094-20099Abstract Full Text PDF PubMed Google Scholar, 8Tan A.K. Eaton D.L. Biochemistry. 1995; 34: 5811-5816Crossref PubMed Scopus (138) Google Scholar, 9Skidgel R.A. Hooper N.M. Zinc Metalloproteases in Health and Disease. Taylor & Francis Ltd., London1996: 241-283Google Scholar, 10Song L. Fricker L.D. J. Biol. Chem. 1997; 272: 10543-10550Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). 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 (11Gardell S.J. Craik C.S. Clauser E. Goldsmith E. Stewart C.-B. Graf M. Rutter W.J. J. Biol. Chem. 1989; 263: 17828-17836Google Scholar, 12Pascual R. Burgos F.J. Salvà M. Soriano F. Méndez E. Avilés F.X. Eur. J. Biochem. 1989; 179: 609-616Crossref PubMed Scopus (49) Google Scholar). 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 (13Famming Z. Kobe B. Stewart C.B. Rutter W.J. Goldsmith E. J. Biol. Chem. 1991; 266: 24606-24612PubMed Google Scholar). Recently, we have reported the cloning and sequence analysis of the human pro-CPA2 cDNA as well as its three-dimensional modeling (14Catasús L. Vendrell J. Avilés F.X. Carreira S. Puigserver A. Billeter M. J. Biol. Chem. 1995; 270: 6651-6657Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). CPA2 isoforms have also been reported in rat extra-pancreatic tissues such as brain, testis, and lung (15Normant E. Gros C. Schwartz J.C. J. Biol. Chem. 1995; 270: 20543-20549Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar); 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 (11Gardell S.J. Craik C.S. Clauser E. Goldsmith E. Stewart C.-B. Graf M. Rutter W.J. J. Biol. Chem. 1989; 263: 17828-17836Google Scholar). 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 (6Guasch A. Coll M. Avilés F.X. Huber R. J. Mol. Biol. 1992; 224: 141-157Crossref PubMed Scopus (98) Google Scholar, 14Catasús L. Vendrell J. Avilés F.X. Carreira S. Puigserver A. Billeter M. J. Biol. Chem. 1995; 270: 6651-6657Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) and significant differences from the corresponding regions in the B form (5Coll M. Guasch A. Avilés F.X. Huber R. EMBO J. 1991; 9: 1-9Crossref Scopus (150) Google Scholar). Accordingly, the A2 proenzyme could be expected to show a bimodal and slow proteolytic activation behavior, as previously reported for the A1 form (16Vendrell J. Cuchillo C.M. Avilés F.X. J. Biol. Chem. 1990; 265: 6949-6953Abstract Full Text PDF PubMed Google Scholar), and to differ from the monotonic and quick activation behavior found for the B proenzyme (17Villegas V. Vendrell J. Avilés F.X. Protein Sci. 1995; 4: 1792-1800Crossref PubMed Scopus (26) Google Scholar). However, earlier proteolytic activation experiments carried out on natural pro-CPA2 isolated from human or rat pancreas (12Pascual R. Burgos F.J. Salvà M. Soriano F. Méndez E. Avilés F.X. Eur. J. Biochem. 1989; 179: 609-616Crossref PubMed Scopus (49) Google Scholar, 18Oppezzo O. Ventura S. Bergman T. Vendrell J. Jörnvall H. Avilés F.X. Eur. J. Biochem. 1994; 222: 55-63Crossref PubMed Scopus (20) Google Scholar) do not fit with these expectations and assumptions. Therefore, this is an issue that requires clarification. In this work, pro-CPA2 has been overexpressed inPichia 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 (19Cregg J.M. Vedvick T.S. Raschke W.C. Bio/Technology. 1993; 11: 905-910Crossref PubMed Scopus (850) Google Scholar). The development of the system reported here to obtain large quantities of fully activable human pro-CPA2should 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 (20Huennekens F.M. Trends Biotechnol. 1994; 12: 234-239Abstract Full Text PDF PubMed Scopus (37) Google Scholar, 21Laethem R.M. Blumenkopf T.A. Cory M. Elwell L. Moxham C.P. Ray P.H. Walton L.M. Smith G.K. Arch. Biochem. Biophys. 1996; 332: 8-18Crossref PubMed Scopus (24) Google Scholar). The efficient expression of human pro-CPA2 inP. 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. 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, N α-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. DNA manipulations were carried out essentially as described by Sambrook et al. (22Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) usingE. 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 byXhoI 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 α-mating factor signal for secretion and theHIS4 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 theEcoRI site of the polylinker. After linearization of the corresponding P. pastorispPIC9-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 (14Catasús L. Vendrell J. Avilés F.X. Carreira S. Puigserver A. Billeter M. J. Biol. Chem. 1995; 270: 6651-6657Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). 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). 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 mmMES, 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. Recombinant human pro-CPA2 at 1 mg/ml in 50 mm Tris-HCl and 1 μmZnCl2, 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 mmTris 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% β-mercaptoethanol, heated at 90 °C for 1 min, and stored at −20 °C until analysis. Electrophoresis was carried out on Tricine-polyacrylamide gels (23Schägger H. Von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10505) Google Scholar). 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. 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. 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 analyses were carried out by the 4-dimethylaminoazobenzene-4′-sulfonyl derivatization method (24Vendrell J. Avilés F.X. J. Chromatogr. 1986; 358: 401-413Crossref Scopus (76) Google Scholar) 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. 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. A 1254-base pair cDNA encoding human preprocarboxypeptidase A2 has been cloned from a pancreatic library by immunological and radioactive approaches (14Catasús L. Vendrell J. Avilés F.X. Carreira S. Puigserver A. Billeter M. J. Biol. Chem. 1995; 270: 6651-6657Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). 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 theEcoRI 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 AOX1gene. The clones transformed with the α-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 (25Ohi H. Ohtani W. Okazaki N. Furuhata N. Ohmura T. Yeast. 1996; 12: 31-40Crossref PubMed Scopus (14) Google Scholar). 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. 1 B) in the lanes corresponding to the extracellular medium. It corresponds to the α-MF-pro-CPA2 fusion product, which containsN-linked glycosylations in the signal peptide (26Kjeldsen T. Brandt J. Andersen A.S. Egel-Mitani M. Hach M. Petterson A.F. Vad K. Gene (Amst.). 1996; 170: 97-112Crossref Scopus (68) Google Scholar) and is not cleaved by the P. pastoris KEX2 processing protease. After purification (see below) and treatment of the sample withN-glycosidase F, conversion of the smear into a lower molecular mass band that corresponds to the α-MF-pro-CPA2fusion 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.Figure 2Deglycosylation and activation with trypsin of the α-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 α-MF-pro-CPA2fusion product; lanes 5 and 6, the α-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.View Large Image Figure ViewerDownload (PPT) As reported above, the human pro-CPA2 gene was cloned behind the α-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 α-MF precursor. The deduced processing targets, located at the boundary between the α-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. 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 (12Pascual R. Burgos F.J. Salvà M. Soriano F. Méndez E. Avilés F.X. Eur. J. Biochem. 1989; 179: 609-616Crossref PubMed Scopus (49) Google Scholar). 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. Analysis of the action of trypsin, chymotrypsin A, and elastase (the major active endoproteolytic counterparts in pancreatic secretion) upon correctly processed recombinant pro-CPA2indicates 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 (3Auld D.S. Vallee B.L. New Compr. Biochem. 1987; 16: 201-256Crossref Scopus (32) Google Scholar). 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 (12Pascual R. Burgos F.J. Salvà M. Soriano F. Méndez E. Avilés F.X. Eur. J. Biochem. 1989; 179: 609-616Crossref PubMed Scopus (49) Google Scholar). 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. 4 A). 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. 4 B). 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. 4 B), although mass spectrometry analysis indicated heterogeneity for this species (see below). 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-CPA2band (Fig. 4 B), 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 (12Pascual R. Burgos F.J. Salvà M. Soriano F. Méndez E. Avilés F.X. Eur. J. Biochem. 1989; 179: 609-616Crossref PubMed Scopus (49) Google Scholar, 16Vendrell J. Cuchillo C.M. Avilés F.X. J. Biol. Chem. 1990; 265: 6949-6953Abstract Full Text PDF PubMed Google Scholar, 27Gomis-Rüth F.X. Gómez M. Bode W. Huber R. Avilés F.X. EMBO J. 1995; 14: 4387-4394Crossref PubMed Scopus (56) Google Scholar). 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 generate" @default.
• W2135229105 created "2016-06-24" @default.
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• W2135229105 creator A5025858582 @default.
• W2135229105 creator A5045647991 @default.
• W2135229105 creator A5056197299 @default.
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• W2135229105 date "1998-02-01" @default.
• W2135229105 modified "2023-10-03" @default.
• W2135229105 title "Overexpression of Human Procarboxypeptidase A2 in Pichia pastoris and Detailed Characterization of Its Activation Pathway" @default.
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