Biologically Active Recombinant Human Progastrin6

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 276, No. 11, Issue of March 16, pp. 7791–7796, 2001 Printed in U.S.A.

Biologically Active Recombinant Human Progastrin6 – 80 Contains a Tightly Bound Calcium Ion* Received for publication, November 2, 2000 Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M009985200

Graham S. Baldwin‡§, Fre´de´ric Hollande¶, Zhiyu Yang‡, Yulia Karelina‡, Adrienne Paterson‡, Rosslyn Strang‡储, Daniel Fourmy**, Greg Neumann‡‡, and Arthur Shulkes‡ From the ‡University Department of Surgery, Austin Hospital, Heidelberg, Victoria 3084, the 储Russell Grimwade School of Biochemistry, University of Melbourne, Melbourne, Parkville, Victoria 3052, and the ‡‡Department of Biochemistry, Latrobe University, Bundoora, Victoria 3083, Australia and the ¶Faculte´ de Pharmacie, Universite´ de Montpellier, Montpellier, and the **INSERM U 151, CHU Rangeuil, Toulouse, France

Gastrin is a classical gut peptide hormone that was identified originally as a stimulant of gastric acid secretion. Like many other peptide hormones, gastrin is synthesized as a large precursor molecule of 101 amino acids (Fig. 1), which is converted to progastrin (80 amino acids) by cleavage of the Nterminal signal peptide. Progastrin is processed further by endo- and carboxypeptidases and by C-terminal amidation to yield the final end products glycine-extended gastrin17 and amidated gastrin17 (1). Although amidated gastrins were thought originally to be the only forms of the hormone with

* This work was supported in part by the Austin Hospital Medical Research Foundation and by Grants 940924 and 980625 (to G. B.) and 960258 and 114123 (to A. S.) from the National Health and Medical Research Council of Australia. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § To whom correspondence should be addressed at the following address: Dept. of Surgery, Austin Campus, A&RMC, Studley Rd., Heidelberg, Victoria 3084, Australia. Tel.: 613-9496-5592; Fax: 613-94581650; E-mail: [email protected]. This paper is available on line at http://www.jbc.org

biological activity, glycine-extended gastrin17 has been shown to stimulate the proliferation of several cell lines (2– 4). Progastrin itself appears to act as a growth factor for normal colon, because transgenic mice expressing progastrin in the liver have increased concentrations of serum progastrin and a hyperplastic colonic mucosa (5). In addition, the observation of increased numbers of aberrant crypt foci (6) and tumors (7) in the colonic mucosa of transgenic mice overexpressing progastrin in comparison with wild-type mice following treatment with azoxymethane suggests that progastrin may act as a co-carcinogen in the development of colorectal carcinoma. However, the possibility should be borne in mind that progastrin, or a breakdown product, might have been acting indirectly on a tissue other than the colonic mucosa to release a second growth factor responsible for the effects observed in the colon. The possibility that colorectal carcinoma cells might utilize progastrin or progastrin-derived peptides as autocrine growth factors has recently received considerable attention (8). The autocrine model predicts that a cell synthesizes a particular growth factor, which, after release into the surrounding medium, binds to specific receptors on the surface of the same cell and stimulates the proliferation of that cell. The observation that expression of antisense gastrin mRNA inhibits proliferation of colon-derived cell lines in vitro and in vivo (3, 4) provides strong evidence that progastrin or progastrin-derived peptides may act as autocrine growth factors in colorectal carcinoma. As predicted by the autocrine model, most colon carcinomas and derived cell lines synthesize gastrin mRNA and progastrinderived peptides (see Ref. 8 for review) and increased concentrations of progastrin-derived peptides have been detected in the sera of patients with colorectal carcinoma (9). However, the identity of gastrin receptors on colorectal carcinomas is still unclear (see Ref. 8 for review). Experiments on the role of progastrin and its receptors in the development of colorectal carcinoma have been limited by the scarcity of the prohormone. Small amounts of progastrin1– 80 (less than 1 nmol/gm tissue) have been isolated from human tissues (10), but the largest progastrin-derived peptide available in bulk to date via organic synthesis is progastrin20 –71 (10). The related prohormone procholecystokinin (pro-CCK)1 has been expressed with an N-terminal histidine tag and purified from baculovirusinfected insect cells (11). We have now developed a method for expression and purification of progastrin6 – 80 from Escherichia coli, to test directly its biological activities on colon-derived cell lines in vitro, to measure its affinity for gastrin and CCK receptors, and to investigate its structure. 1 The abbreviations used are: CCK, cholecystokinin; GST, glutathione S-transferase; HPLC, high pressure liquid chromatography; PBS, phosphate-buffered saline.

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Evidence is accumulating that gastrin precursors may act as growth factors for the colonic mucosa in vivo. The aims of this study were to prepare recombinant human progastrin6 – 80 and to investigate its structure and biological activities in vitro. Human progastrin6 – 80 was expressed in Escherichia coli as a glutathione S-transferase fusion protein. After thrombin cleavage progastrin6 – 80 was purified by reverse phase high pressure liquid chromatography and characterized by radioimmunoassay, amino acid sequencing, and mass spectrometry. Assays for metal ions by atomic emission spectroscopy revealed the presence of a single tightly bound calcium ion. Progastrin6 – 80 at concentrations in the pM to nM range stimulated proliferation of the conditionally transformed mouse colon cell line YAMC. The observations that progastrin6 – 80 did not bind to either the cholecystokinin (CCK)-A or the gastrin/CCK-B receptor expressed in COS cells and that antagonists selective for either receptor did not reverse the proliferative effects of progastrin6 – 80 suggested that progastrin6 – 80 stimulated proliferation independently of either the CCK-A or the gastrin/CCK-B receptor. We conclude that recombinant human progastrin6 – 80 is biologically active and contains a single calcium ion. With the exception of the well known zinc-dependent polymerization of insulin and proinsulin, this is the first report of selective, high affinity binding of metal ions to a prohormone.

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Biologically Active Recombinant Progastrin

FIG. 1. Structures of progastrin-derived peptides. The structure of the GST-progastrin fusion protein is compared with the structures of naturally occurring progastrin-derived peptides. Amino acids are shown in the one-letter codes with progastrin sequences in capital letters and linker sequences in lowercase letters. Numbering commences at the N terminus of mature progastrin (10). Thrombin cleavage sites are indicated by vertical arrows.

EXPERIMENTAL PROCEDURES

Chemicals and Cell Lines—Gastrin17gly was custom synthesized by Auspep (Melbourne, Australia). The conditionally transformed mouse colon cell line YAMC (16) was generously provided by Dr. R. H. Whitehead (Ludwig Institute for Cancer Research, Melbourne, Australia). Synthesis of Progastrin Fusion Protein in E. coli—A HindIII-HindIII fragment of human gastrin cDNA, corresponding to nucleotides 59 –325 of the sequence reported by Boel and coworkers (17) and hence encoding the entire sequence of mature human progastrin1– 80 (10), was subcloned into HindIII-cut and dephosphorylated pGEX-2TH (18). Clones with the insert in the correct orientation were selected by restriction mapping. The predicted sequence of the fusion protein, which was confirmed by nucleotide sequencing, consisted of glutathione S-transferase (GST) joined to progastrin1– 80 by a 6-amino acid linker (GSEFQA) arising from the multiple cloning site. The GST-progastrin fusion protein was purified from sarkosyl lysates of E. coli by binding to glutathione-agarose as described by Frangioni and Neel (19). Briefly, E. coli strain NM522 was transformed with the plasmid of interest and grown overnight at 37 °C with shaking in LB medium containing 100 ␮g/ml of ampicillin. The overnight culture (40 ml) was used to inoculate the same medium (360 ml). When an absorbance at 600 nm of 0.8 was reached, the expression of the GSTprogastrin fusion protein was induced by treatment with 0.1 mM isopropylthiogalactoside for 6 h. The bacterial cells were harvested by centrifugation at 2500 ⫻ g for 10 min. The cell pellet was washed in cold STE buffer (10 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA), and resuspended in 24 ml of STE buffer containing 100 ␮g/ml lysozyme. After incubation on ice for 15 min dithiothreitol was added to 5 mM, and proteins were solubilized with 1.5% Sarkosyl (Sigma). After vortexing for 15 s, cells were sonicated for 2 ⫻ 30 s (power level, 4; duty cycle, 50%) in a model 250 sonifier (Branson Sonic Power Co., Danbury, CT). The lysate was clarified by centrifugation at 2500 ⫻ g for 5 min at 4 °C. The supernatant was transferred to a new tube, and Triton X-100 was added to 2%. After vortexing for 10 s, washed glutathione-agarose beads (2 ml/10 ml lysate, 50% (v/v) suspension in phosphate-buffered saline (PBS)) were added, and the suspension was gently mixed by rotation at


Previous structural studies on progastrin-derived peptides have been limited to investigation of the conformation of gastrin17 and shorter fragments by circular dichroism and ultraviolet and NMR spectroscopy (Refs. 12–14 and references therein). Binding of three Mg2⫹ or Ca2⫹ ions to human [Nle11]gastrin13 and [Nle15]-gastrin17 was observed in trifluoroethanol (12–13), but the affinities are presumably considerably lower in aqueous solution because no binding was detected by either circular dichroism (12) or NMR spectroscopy (14). In trifluoroethanol the dissociation constants for binding of Ca2⫹ to the three sites in [Nle15]-gastrin17 were K1 ⫽ 0.29 ␮M, K2 ⫽ 0.29 ␮M, and K3 ⫽ 7.1 ␮M. To determine whether the N- and C-terminal extensions of progastrin6 – 80 increased its affinity for metal ions, samples of recombinant human progastrin6 – 80 were analyzed by inductively coupled plasma atomic emission spectroscopy. Here we report that progastrin6 – 80 is biologically active and contains a single tightly bound calcium ion. With the exception of the well known zinc-dependent polymerization of insulin and proinsulin (15), this is the first report of selective, high affinity binding of a metal ion to a prohormone.

4 °C for 1 h. The beads were then washed three times with ice-cold PBS by repeated low speed centrifugation and resuspension in PBS. Finally the beads with the GST-progastrin fusion protein attached were stored at ⫺70 °C in storage buffer (1 ml/1 ml of beads, 50 mM Na⫹ Hepes, pH 7.4, 150 mM NaCl, 5 mM dithiothreitol, 10% (v/v) glycerol). The protein content of samples at various stages of the purification was analyzed by SDS-polyacrylamide gel electrophoresis on 10% gels with a buffer system designed for peptides and proteins in the molecular mass range 5–70 kDa (20). Thrombin Cleavage—Progastrin was cleaved from the GST-progastrin fusion protein bound to glutathione-agarose beads by incubation with thrombin (8 units, Sigma) in cleavage buffer (1 ml/1 ml of beads, 50 mM Hepes, pH 8.0, 150 mM NaCl, 2.5 mM CaCl2) for 1 h at 37 °C. Following cleavage, the supernatant containing progastrin was separated from the beads by centrifugation. The beads were washed with elution buffer (1 ml/1 ml of beads, 1 M urea, 50 mM Hepes, pH 7.5), and the washes were combined with the initial supernatant. Reverse Phase High Performance Liquid Chromatography—The recombinant human progastrin prepared above was applied to a C18 column (8 ⫻ 100 mm, Waters Associates, Milford, MA), which had been equilibrated with 50 mM ammonium bicarbonate, 20% acetonitrile. The progastrin was eluted with a gradient from 20 –50% acetonitrile in 50 mM ammonium bicarbonate at a flow rate of 1 ml/min. Fractions of 0.5 ml were collected and dried on a Speed Vac (Savant, Hicksville, NY) for radioimmunoassay, mass spectrometry, and amino acid sequencing. Radioimmunoassay—The concentrations of recombinant human progastrin in chromatographic fractions were measured by radioimmunoassay as previously described (9). A polyclonal antiserum (1137) was raised in rabbits against an undecapeptide consisting of the C-terminal gastrin decapeptide with an additional tyrosine residue at the N terminus for iodination (9). A C-terminal flanking peptide standard curve was constructed with 125I-C-terminal flanking peptide as label. The ID50 was 1.3 ⫾ 0.2 fmol/tube, and the intra-assay variation was ⬍7%. Mass Spectrometry—Electrospray ionization mass spectrometry was performed on a Sciex API-300 triple quadrupole mass spectrometer (PerkinElmer Life Sciences) fitted with a micro-ionspray ion source (flow rate, 0.2 ␮l/min), previously calibrated to an accuracy of ⫾0.01% using singly charged poly(propylene glycol) reference ions. Samples (2– 4 ␮l) from reverse phase HPLC fractions were mixed with 1:1 acetonitrile, 0.2% formic acid prior to analysis. Signal-averaged mass spectra obtained from 50 –100 scans over 5–10 min using an m/z scan range of 100 –3000 daltons/unit charge (Da/z) (half-height peak width, 0.6 Da/z) were subsequently analyzed using Sciex BioMultiview software (PerkinElmer Life Sciences). Amino Acid Sequencing—N-terminal amino acid sequences were obtained by sequential Edman degradation using a Hewlett-Packard G1005A automated protein sequencing system, calibrated with phenylthiohydantoin-derivative standards prior to each sequencing run. Inductively Coupled Plasma Atomic Emission Spectroscopy—Samples of recombinant human progastrin from reverse phase HPLC were concentrated on a Speed Vac to remove acetonitrile, treated with 10 mM EDTA in 8 M urea for 16 h at 4 °C, transferred to SpectraPor dialysis tubing (molecular mass cut-off, 3.5 kDa; Spectrum Medical Industries, Houston, TX), and dialyzed against 10 mM Na-Hepes, pH 7.6, containing 10 ␮M EDTA and 0.005% Tween 20 for 6 days at 4 °C. Control samples were treated in parallel except that the EDTA/urea treatment was omitted. Samples of dialyzed progastrin and of the dialysis buffers were analyzed in triplicate for the presence of aluminum, calcium, cobalt, chromium, copper, iron, magnesium, manganese, nickel, scandium, titanium, vanadium, and zinc by inductively coupled plasma atomic emission spectroscopy using a Varian Liberty Series II spectroscope fitted with an axial torch (Varian, Mulgrave, Australia). Concentrations of progastrin were calculated from the absorbance at 280 nm, which was determined on a Cary 5 spectrophotometer (Varian, Mulgrave, Australia). Proliferation Assays—Cell proliferation was measured by incorporation of bromodeoxyuridine. The conditionally transformed mouse colon cell line YAMC (16) was plated onto sterile 14-mm coverslips in 24-well plates at a density of 25,000 cells/well in RPMI 1640 medium containing 5% fetal calf serum and 1 unit/ml ␥-interferon and grown overnight at 33 °C. The cells were then transferred to 39 °C and washed once in PBS, and the medium was replaced with RPMI 1640 containing L-glutamine but without fetal calf serum or interferon. After 24 h cells were incubated for a further 18 h in the same medium containing 1% fetal calf serum and 100 ␮M bromodeoxyuridine, with or without the factors to be tested. The cells were then rinsed once with PBS, fixed for 5 min in ice-cold methanol at 4 °C, and washed three times in PBS. The cells were permeabilized in PBS containing 0.5% HCl for 10 min, washed


FIG. 2. Expression of progastrin fusion protein. Human progastrin was expressed in E. coli as a fusion protein with glutathione S-transferase. The fusion protein was purified from bacterial lysates by chromatography on glutathione-agarose and cleaved by treatment with thrombin, as described under “Experimental Procedures.” Samples from the indicated stages of the purification were electrophoresed on 10% SDS-polyacrylamide gels with a buffer system designed for peptides and proteins in the molecular mass range of 5–70 kDa (20) and visualized by staining with Coomassie Blue. A band in the position expected for progastrin6 – 80 was apparent in track 5. Lane 1, bacterial lysate; lane 2, glutathione-agarose run through; lane 3, glutathioneagarose bound material; lane 4, glutathione-agarose bound material after thrombin cleavage; lane 5, glutathione-agarose supernatant after thrombin cleavage; lane 6, glutathione S-transferase; lane 7, molecular mass markers (size in kDa).

RESULTS

Expression of Human Progastrin in E. coli—Human progastrin was expressed in E. coli as a fusion protein with glutathione S-transferase (Fig. 2). The fusion protein was isolated by binding to glutathione-agarose beads according to Frangioni and Neel (19). Recombinant human progastrin was cleaved from the fusion protein bound to glutathione-agarose beads by treatment with thrombin (Fig. 2). Purification and Characterization of Recombinant Human Progastrin6 – 80—Recombinant human progastrin was purified by reverse phase HPLC (Fig. 3). The absorbance peak at fraction 21–22 matched very well with the peak of immunoreactivity observed with antiserum 1137, which was raised against an undecapeptide consisting of the C-terminal decapeptide of progastrin (residues 71– 80) with an additional tyrosine residue at the N terminus for iodination (9). The conclusion that the recombinant human progastrin contained the C terminus of progastrin was confirmed by electrospray ionization mass spectrometry. The molecular mass of HPLC-purified recombinant human progastrin was 8427.1 ⫾ 0.7 Da, which is in excellent agreement with the mass of 8427.1 Da predicted for human progastrin6 – 80. The N-terminal amino acid sequence of HPLC-purified recombinant human progastrin determined by Edman analysis was SQQPDAPL, which corresponded precisely to residues 6 –13 of human progastrin. We conclude that the HPLC-purified recombinant human progastrin consists of residues 6 – 80 inclusive of human progastrin. Because the N-terminal sequence of human progas-

FIG. 3. Purification of recombinant human progastrin. The glutathione S-transferase-progastrin fusion protein was digested with thrombin while bound to glutathione-agarose. The supernatant, which contained human progastrin, was separated from the agarose beads by centrifugation and subjected to reverse phase HPLC as described under “Experimental Procedures.” The peak of progastrin immunoreactivity (B) detected by radioimmunoassay with antibody 1137, which recognizes the C-terminal decapeptide, coincided precisely with the protein peak (A) detected in fractions 21–22 by absorbance at 214 nm.

trin is SWKPRSQQPDAPL, it appears that thrombin has cleaved the peptide bond between the arginine residue at position 5 and the serine residue at position 6. Metal Analyses—Previous reports have indicated that human [Nle11]-gastrin13 and [Nle15]-gastrin17 bind three Mg2⫹ or Ca2⫹ ions in trifluoroethanol (12–13). The presence of metal ions in recombinant human progastrin6 – 80 was therefore investigated by inductively coupled plasma atomic emission spectroscopy. The analysis revealed that recombinant human


once in PBS, and incubated for 1 h with a mouse anti-bromodeoxyuridine antibody. After three rinses in PBS, the cells were incubated with FITC-labeled goat anti-mouse IgG for 30 min. After three further rinses the coverslips were mounted on slides with cytifluor and observed on a fluorescence microscope. Transient Transfection of COS Cells—COS7 cells were transiently transfected by the DEAE-dextran method as described previously (21). One day before transfection, 0.7–1.0 ⫻ 106 COS cells were seeded in 10-cm plates in Dulbecco’s modified Eagle’s medium and grown in 5% CO2 such that on the day of transfection the cells were 60% confluent. On the day of transfection, a DNA/DEAE-dextran solution was prepared by dropwise addition of 0.5 ml 2 mg/ml DEAE-dextran in PBS to 0.5 ml of 0.1% glucose in PBS containing 3.5 ␮g/ml pRFNeo plasmid DNA encoding either the human CCK-A or the human CCK-B receptor (22). The medium was aspirated, and the cells were washed once with PBS and gently rocked at 37 °C for 20 min in the DNA/DEAE-dextran solution. The solution was then replaced with 10 ml of 100 ␮M chloroquine, and the cells were incubated at 37 °C for 3.5 h. After incubation, the solution was aspirated, and the cells were washed twice with serum-free Dulbecco’s modified Eagle’s medium and grown in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum overnight. On the next day, the transfected cells were dislodged with 0.02% EDTA, replated onto a 24-well dish (20,000 –50,000/well) and grown for a further 48 h prior to the receptor binding assay. Receptor Binding Assays—Binding of progastrin to either the human CCK-A receptor or the human gastrin/CCK-B receptor was measured by competition for 125I-labeled Bolton and Hunter CCK8 binding as described by Kopin and coworkers (23). Transfected COS7 cells were grown to 60 –70% confluence as described above, washed once with PBS, and then incubated for 80 min at 37 °C in 150 ␮l of Dulbecco’s modified Eagle’s medium containing 125I-CCK8 (50,000 cpm, 14.5 fmol; Amersham Pharmacia Biotech), 150 ␮M phenylmethylsulfonyl fluoride, 0.05% bacitracin, and 0.1% BSA. Cells were then washed twice with PBS and lysed with 300 ␮l of 1 M NaOH. Lysates were counted in a ␥-counter (LKB-Wallac, Turku, Finland) at 77% efficiency. Estimates of IC50 values and of the levels of 125I-CCK8 bound in the absence of competitor were fitted as previously described (24). Statistics—Results are expressed as the means ⫾ S.E., except where otherwise stated. Parametric and nonparametric data sets were analyzed by one-way analysis of variance and by Kruskal-Wallis one-way analysis of variance on ranks, respectively. If there was a statistically significant difference in the mean or median values of each set, the values were individually compared with the control value by Dunnett’s or Dunn’s methods, respectively. Differences with p values of ⬍ 0.05 were considered significant.

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progastrin6 – 80 contained 1.06 ⫾ 0.08 mol calcium ions/mol (mean ⫾ S.E., n ⫽ 3) (Fig. 4). No other cations were detected consistently. The calcium ion was not removed by extensive dialysis at pH 7.6 against 10 ␮M EDTA (Fig. 4) or 100 ␮M EDTA (data not shown), by treatment with 8 M urea containing 10 mM EDTA (Fig. 4), or by extensive dialysis at pH 5.5 against 100 ␮M EDTA (data not shown). Proliferation Studies—Recombinant human progastrin6 – 80 stimulated proliferation of the conditionally transformed mouse colon cell line YAMC in a dose-dependent manner with maximal stimulatory effects seen in the range 10 to 100 pM (Fig. 5A). The stimulatory effect of recombinant human progastrin6 – 80 on YAMC cells was unaffected by either the CCK-A receptor-selective antagonist L364,718 or the gastrin/CCK-B receptor-selective antagonist L365,260 at concentrations as high as 10 ␮M (Fig. 5B). Gastrin17gly also stimulated proliferation of YAMC cells in the concentration range 1 pM to 1 nM, as has been reported previously with a colorimetric assay (4). Receptor Binding—Studies of progastrin binding were confined to CCK-A and gastrin/CCK-B receptors, which have both been fully characterized at the nucleotide sequence level. Binding of recombinant human progastrin6 – 80 to either the human CCK-A or human gastrin/CCK-B receptor was investigated by competition for the binding of 125I-CCK8 to transiently transfected COS cells as described under “Experimental Procedures.” Recombinant human progastrin6 – 80 had no effect on the binding of 125I-CCK8 to the gastrin/CCK-B receptor even at concentrations as high as 100 nM (Fig. 6) and consistently stimulated the binding of 125I-CCK8 to the CCK-A receptor. CCK8 and gastrin17 were used as positive controls for measurement of binding to the CCK-A and gastrin/CCK-B receptors. In the absence of progastrin dose-dependent displacement of 125 I-CCK8 from specific binding sites on COS cells transfected with plasmids encoding either the CCK-A receptor or the gastrin/CCK-B receptor was observed in the presence of unlabeled CCK8 or gastrin17, respectively. The IC50 values determined by computer fitting of the data to a single site model were 12 ⫾ 5 nM for the CCK-A receptor and 36 ⫾ 15 nM for the gastrin/ CCK-B receptor. These values are higher than previously reported values for the affinities of the human CCK-A receptor (3 nM for CCK8SO4 (25)) and human gastrin/CCK-B receptor (6 nM for gastrin17 (26)) for their preferred ligands. The discrep-

FIG. 5. Recombinant human progastrin stimulates YAMC cell proliferation. Proliferation of synchronized YAMC mouse colon cells after incubation with increasing concentrations of human recombinant progastrin6 – 80 (open circles) or gastrin17gly (closed circles) was measured as described under “Experimental Procedures” by bromodeoxyuridine incorporation. Significant (p ⬍ 0.05) stimulation of YAMC cell proliferation was observed in the presence of progastrin6 – 80 at concentrations between 10 pM and 1 nM (A). Gastrin17gly had a similar potency. Neither the gastrin/CCK-B receptor-selective antagonist L365,260 nor the CCK-A receptor-selective antagonist L364,718 reversed the stimulation of YAMC cell proliferation by recombinant human progastrin6 – 80 (control without progastrin, closed bars; with 0.1 nM progastrin, open bars) at concentrations of either 10 nM (hatched bars) or 10 ␮M (cross-hatched bars) (B). Results are expressed as the percentages of cells incorporating bromodeoxyuridine, error bars represent the standard error of the mean from at least two separate experiments, and statistical significance compared with controls without progastrin6 – 80 or gastrin17gly was assessed by Student’s t test.

ancy may be due in part to the presence of more than one class of binding sites (22). DISCUSSION

In this paper the production and purification of recombinant human progastrin6 – 80 from E. coli is reported for the first time. Progastrin was synthesized as a fusion protein with GST (Fig. 1) and partially purified by utilizing the affinity of GST for glutathione-agarose (Fig. 2). After treatment of the bound fusion protein with thrombin, progastrin6 – 80 was released into the supernatant, which was separated from the glutathioneagarose-bound GST by centrifugation. Final purification of the supernatant by reverse phase HPLC resulted in preparations of recombinant human progastrin that were homogeneous by gel electrophoresis (Fig. 2) and mass spectrometry. The molecular mass of recombinant human progastrin determined by electrospray ionization mass spectrometry was 8427.1 ⫾ 0.7 Da, in excellent agreement with the value of 8427.1 Da expected for human progastrin6 – 80. This peptide would be generated by cleavage between Arg5 and Ser6 in the progastrin sequence Trp-Lys-Pro-Arg-Ser-Gln, which is consistent with the preferred thrombin recognition sequence P4-P3-Pro-Arg/Lys-P1⬘P2⬘, where P3 and P4 are hydrophobic amino acids, and P1⬘ and P2⬘ are nonacidic amino acids (27). Isolation of progastrin1–35 and progastrin6 –35 from a human gastrinoma by Reeve and co-workers (28) had previously revealed that the signal peptide of preprogastrin was 21 amino acids long and that an additional cleav-


FIG. 4. Recombinant human progastrin contains a tightly bound calcium ion. Samples of recombinant human progastrin6 – 80 were treated with 10 mM EDTA in 8 M urea for 16 h at 4 °C, dialyzed for 6 days at 4 °C against 10 mM Na-Hepes, pH 7.6, containing 10 ␮M EDTA and 0.005% Tween 20 and assayed for the indicated metal ions by inductively coupled plasma atomic emission spectroscopy. Control samples were treated in parallel except that the EDTA/urea treatment was omitted. The concentration of metal ion in urea-treated (open bars) and control samples (closed bars) was determined by comparison with appropriate standards, and the concentration of progastrin was determined by measurement of absorbance at 280 nm. Bars represent the means of triplicates, and error bars represent the S.E.; similar results were obtained in a second experiment.


age occurred between Arg5 and Ser6. Subsequently Rehfeld and Johnsen (10) purified progastrin1– 80, progastrin1–71, progastrin1–35, progastrin6 –35, progastrin20 –35, and progastrin20 –36 from human antral tissue. Progastrin6 –35 and progastrin1–35 were present in similar amounts and were approximately four times more abundant than progastrin1– 80 or progastrin1–71. Hence, the cleavage between Arg5 and Ser6 observed in our experiments in vitro also occurs in human tissue. Analysis of recombinant human progastrin6 – 80 by inductively coupled plasma atomic emission spectroscopy indicated the presence of a single calcium ion (Fig. 4). With the exception of the well known zinc-dependent polymerization of insulin and proinsulin (15), this is the first report of selective, high affinity binding of a metal ion to a prohormone. Although the insulin monomer is biologically active, Zn2⫹ or other divalent metal ions promote the assembly of insulin and proinsulin dimers into hexamers. In the “2Zn-insulin hexamer” each of two Zn2⫹ ions is octahedrally coordinated by three water molecules and by N⑀ of the imidazole ring of histidine B10 from three insulin monomers (29). The binding site for the calcium ion in progastrin has not yet been defined. The affinity of progastrin6 – 80 for the calcium ion is high, because the metal ion was not removed by extensive dialysis against EDTA at pH 7.6. or 5.5, or by treatment with 8 M urea

in the presence of EDTA. Binding of three Mg2⫹ or Ca2⫹ ions to human [Nle11]-gastrin13 and [Nle15]-gastrin17 in trifluoroethanol has been reported previously (12–13), but no binding has been detected in aqueous solution by circular dichroism (12) or by NMR spectroscopy (14). The dissociation constants for binding of Ca2⫹ to the three sites in [Nle15]-gastrin17 in trifluoroethanol were K1 ⫽ 0.29 ␮M, K2 ⫽ 0.29 ␮M, and K3 ⫽ 7.1 ␮M. The apparent increase in affinity for Ca2⫹ ions between human [Nle15]-gastrin17 and human progastrin6 – 80 indicates either that the structure of the pentaglutamate sequence of gastrin13 is altered by the addition of the N- and C-terminal extensions of progastrin6 – 80, with a consequent increase in affinity, or that a new binding site is created by the additional amino acids. The decrease in the stoichiometry of calcium binding from 3 in gastrin17 to 1 in progastrin6 – 80 favors the latter explanation. The observation that there are several acidic residues that are conserved across species (30) in both N- and C-terminal extensions is also consistent with the existence of a different binding site. However, the absence of previously described motifs such as the E-F hand from both N- and Cterminal extensions suggests that the calcium binding site may be different from known binding sites. A number of possible roles for the tightly bound Ca2⫹ ion can be envisaged. Firstly, the Ca2⫹ ion might contribute to the thermostability of progastrin, as has been reported previously for binding of Ca2⫹ ions to trypsin (31). Secondly, the Ca2⫹ ion might alter the ability of progastrin to polymerize, as has been reported previously for the binding of Zn2⫹ ions to proinsulin (29). For example, during biosynthesis and storage in the pancreatic ␤-cell proinsulin assembles into dimers, which in the presence of Zn2⫹ or other divalent metal ions further assemble into hexamers. Thirdly, the Ca2⫹ ion might enhance the solubility of progastrin, as has been reported previously for the binding of Zn2⫹ ions to proinsulin (32). Fourthly, the Ca2⫹ ion might redirect the processing of progastrin by preventing cleavage at some dibasic sites, in the same way that phosphorylation of Ser75 prevents cleavage of the Arg73–Ser74 bond (33). The observation that the calcium remained bound to progastrin even after extensive dialysis at pH 5.5 (data not shown), which is the pH within the secretory granule where processing occurs (34), is consistent with a role in processing. The fact that amidated and nonamidated gastrins act on different receptors to generate different effects (8) suggests that such modification of the processing pathways could profoundly effect biological activity. Experimental testing of all of the above hypotheses will require the development of methods for removal of the Ca2⫹ ion from progastrin. Proliferation of the conditionally transformed mouse colon cell line YAMC (16) was stimulated by concentrations of recombinant human progastrin6 – 80 in the pM to nM range (Fig. 5A). As well as demonstrating that recombinant human progastrin6 – 80 is correctly folded when synthesized as a fusion protein in E. coli and is not denatured during purification, the proliferation data provide the first evidence that progastrin has a direct effect on cells of colonic origin. Previous reports that progastrin acts as a growth factor for normal colon in transgenic mice expressing progastrin in the liver (5) and that such mice have increased numbers of aberrant crypt foci (6) and tumors (7) in the colonic mucosa following treatment with azoxymethane in comparison with wild-type mice may be subjected to the criticism that the observed effects on the colonic mucosa may not reflect a direct effect of progastrin itself. For example progastrin synthesized from the liver transgene could have been acting on an unidentified cell type, in a tissue other than the colon, to release a second hormone active on colonic cells. In addition, in the currently available mouse models the


FIG. 6. Recombinant human progastrin does not bind to the gastrin/CCK-B receptor. Binding of [125I]CCK8 (30 pM, 20,000 cpm) to COS cells expressing the human CCK-A (A) or gastrin/CCK-B (B) receptors was measured in the presence of increasing concentrations of recombinant human progastrin6 – 80 (closed squares) as described under “Experimental Procedures.” Binding in the presence of increasing concentrations of CCK8 (A, open circles) and gastrin17 (B, open squares) was measured as a control. Values are expressed as percentages of the value obtained in the absence of competitor. Points are the means ⫾ S.E. of triplicates from three experiments, and the line of best fit was obtained by nonlinear regression to a single site model as described previously (24). The values for IC50 and for the predicted ordinate intercept were 12 ⫾ 5 nM and 84 ⫾ 6% for the CCK-A receptor and 36 ⫾ 15 nM and 81 ⫾ 7% for the gastrin/CCK-B receptor, respectively. Statistical significance compared with controls without unlabeled peptide was assessed as described under “Experimental Procedures.” *, p ⬍ 0.05. Progastrin6 – 80 did not compete with [125I]CCK8 for binding to the gastrin/CCK-B receptor (B). Binding of [125I]CCK8 to the CCK-A receptor (A) was increased in the presence of progastrin6 – 80.

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Biologically Active Recombinant Progastrin indicate that the proliferative effects of progastrin are independent of either the CCK-A or the gastrin/CCK-B receptors. Recombinant human progastrin6 – 80 contains a single calcium ion, but the high binding affinity has so far prevented analysis of the role, if any, of the calcium ion in biological activity. With the exception of the well known zinc-dependent polymerization of insulin and proinsulin (15), this is the first report of selective, high affinity binding of metal ions to a prohormone. It is anticipated that recombinant human progastrin6 – 80 will be an essential tool with which to investigate the biological effects of progastrin in vivo, the nature of the receptors involved, the role of the tightly bound calcium ion in biological activity, and the structure of progastrin itself. Acknowledgments—We gratefully thank Rosemary Condron (Department of Biochemistry, Latrobe University) for the amino acid sequencing, Dr. Peter Curtis (Commonwealth Scientific and Industrial Research Organisation Division of Manufacturing Science and Technology) for metal analyses, and Professor R. J. Wettenhall (Russell Grimwade School of Biochemistry, University of Melbourne) for many helpful discussions. REFERENCES 1. Dockray, G. J., Varro, A., and Dimaline, R. (1996) Physiol. Rev. 76, 767–798 2. Seva, C., Dickinson, C. J., and Yamada, T. (1994) Science 265, 410 – 412 3. Singh, P., Owlia, A., Varro, A., Dai, B., Rajaraman, S., and Wood, T. (1996) Cancer Res. 56, 4111– 4115 4. Hollande, F., Imdahl, A., Mantamadiotis, T., Ciccotosto, G. D., Shulkes, A., and Baldwin, G. S. (1997) Gastroenterology 113, 1576 –1588 5. Wang, T. C., Koh, T. J., Varro, A., Cahill, R. J., Dangler, C. A., Fox, J. G., and Dockray, G. J. (1996) J. Clin. Invest. 98, 1918 –1929 6. Singh, P., Velasco, M., Given, R., Wargovich, M., Varro, A., and Wang, T. C. (2000) Am. J. Physiol. 278, G390 –G399 7. Singh, P., Velasco, M., Given, R., Varro, A., and Wang, T. C. (2000) Gastroenterology 119, 162–171 8. Baldwin, G. S., and Shulkes, A. (1998) Gut 42, 581–584 9. Ciccotosto, G. D., McLeish, A., Hardy, K. J., and Shulkes, A. (1995) Gastroenterology 109, 1142–1153 10. Rehfeld, J. F., and Johnsen, A. H. (1994) Eur. J. Biochem. 223, 765–773 11. Wang, W., Yum, L., and Beinfeld, M. C. (1997) Peptides. 18, 1295–1299 12. Peggion, E., Mammi, S., Palumbo, M., Moroder, L, and Wunsch, E. (1983) Biopolymers 22, 2443–2457 13. Peggion, E., Mammi, S., Palumbo, M., Moroder, L, and Wunsch, E. (1984) Biopolymers 23, 1225–1240 14. Torda, A. E., Baldwin, G. S., and Norton, R. S. (1985) Biochemistry 24, 1720 –1727 15. Blundell, T. L., Dodson, G. G., Dodson, E., Hodgkin, D. C., and Vijayan, M. (1971) Recent Prog. Horm. Res. 27, 1– 40 16. Whitehead, R. H., VanEeden, P. E., Noble, M. D., Ataliotis, P., and Jat, P. S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 587–591 17. Boel, E., Vuust, J., Norris, F., Norris, K., Wind, A., Rehfeld, J. F., and Marcker, K. A. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2866 –2869 18. Smith, D. B., and Johnson, K. S. (1988) Gene (Amst.) 67, 31– 40 19. Frangioni, J. V., and Neel, B. G. (1993) Anal. Biochem. 210, 179 –187 20. Gallagher, S. R. (1988) in Current Protocols in Protein Science (Coligan, J. E., Dunn, B. M., Ploegh, H. L., Speicher, D. W., and Wingfield, P. T., eds) pp. 10.1.11–10.1.12. John Wiley & Sons, New York 21. Mantamadiotis, T., and Baldwin, G. S. (1994) Biochem. Biophys. Res. Commun. 201, 1382–1389 22. Kennedy, K., Escrieut, C., Dufresne, M., Clerc, P., Vaysse, N., and Fourmy, D. (1995) Biochem. Biophys. Res. Commun. 213, 845– 852 23. Kopin, A. S., Lee, Y.-M., McBride, E. W., Miller, L. J., Lu, M., Lin, H. Y., Kolakowski, L. F., and Beinborn, M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 3605–3609 24. Yang, Z., Hollande, F., and Baldwin, G. S. (1998) Cancer Lett. 124, 187–191 25. de Weerth, A., Pisegna, J. R., Huppi, K., and Wank, S. A. (1993) Biochem. Biophys. Res. Commun. 194, 811– 818 26. Pisegna, J. R., de Weerth, A., Huppi, K., Wank, S. A. (1992) Biochem. Biophys. Res. Commun. 189, 296 –303 27. Chang, J.-Y. (1985) Eur. J. Biochem. 151, 217–224 28. Huebner, V. D., Jiang, R., Lee, T. D., Legesse, K., Walsh, J. H., Shively, J. E., Chew, P., Azumi, T., and Reeve, J. R. (1991) J. Biol. Chem. 266, 12223–12227 29. Derewenda, U., Derewenda, Z., Dodson, G. G., Hubbard, R. E., and Korber, F. (1989) Br. Med. Bull. 45, 4 –18 30. Moore, C., Jie, R., Shulkes, A., and Baldwin, G. S. (1997) DNA Sequence 8, 39 – 44 31. Sipos, T., and Merkel, J. R. (1970) Biochemistry 9, 2766 –2775 32. Emdin, S. O., Dodson, G. G., Cutfield, J. M., and Cutfield, S. M. (1980) Diabetologia 19, 174 –182 33. Bishop, L., Dimaline, R., Blackmore, C., Deavall, D., Dockray, G. J., and Varro, A. 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duration of exposure to progastrin-derived peptides and the serum concentrations of progastrin-derived peptides were not controlled (5, 6), so that the transgenic mice were exposed to widely varying concentrations of progastrin-derived peptides both in utero and throughout their adult life. The stimulation of YAMC cell proliferation in the presence of progastrin6 – 80 reported herein (Fig. 5) clearly demonstrates that progastrin6 – 80 itself has short term direct effects on cells of colonic origin. Attempts to define the binding properties of the progastrin receptor responsible for mediating the proliferative effects of progastrin on YAMC cells have been unsuccessful. As yet we have been unable to iodinate progastrin reproducibly by either the chloramine T or iodogen methods, possibly because the single tyrosine residue is buried within the progastrin structure. We have succeeded in labeling the GST-progastrin fusion protein, presumably on one or more of the 14 tyrosines in the GST sequence. However the presence of the GST appears to prevent receptor binding, because no binding of the iodinated fusion protein to YAMC cells has been detected. The following observations suggest that the biological effects of progastrin are not mediated by either the CCK-A or gastrin/ CCK-B receptor. Firstly recombinant human progastrin6 – 80 does not bind to the gastrin/CCK-B receptor at concentrations as high as 100 nM (Fig. 6). Secondly the stimulatory effect of recombinant human progastrin on YAMC cells was unaffected by either the CCK-A receptor-selective antagonist L364,718 or the gastrin/CCK-B receptor-selective antagonist L365,260, at concentrations as high as 10 ␮M (Fig. 5B). Thirdly previous studies have not detected high affinity binding sites for 125Igastrin17 on YAMC cells, and amidated gastrin17 has no effect on their proliferation (4). Surprisingly, binding of 125I-CCK8 to the CCK-A receptor was consistently higher in the presence of recombinant human progastrin6 – 80 (mean percentage of control ⫾ S.E. ⫽ 144 ⫾ 7) (Fig. 6). One possible explanation for the increase is that progastrin6 – 80 is binding to the CCK-A receptor at a site distinct from the CCK binding site and that the binding of progastrin6 – 80 increases the affinity of the CCK binding site for 125 I-CCK8. The absence of detectable competition between progastrin6 – 80 and 125I-CCK8 for binding to either the CCK-A or gastrin/CCK-B receptor is in agreement with previous reports that removal of the C-terminal amide group from CCK (35) or gastrin (36) results in a substantial reduction in affinity for the CCK-A and gastrin/CCK-B receptor, respectively. The receptor binding data presented herein have significant implications for our understanding of the mechanism by which progastrin stimulates growth of the colonic mucosa. The inability of the CCK-A and gastrin/CCK-B receptors to recognize progastrin clearly indicates that neither receptor is involved in the hyperplasia (5) or enhanced development of aberrant crypt foci (6) or tumors (7) observed in the colonic mucosa of mice rendered hyperprogastrinemic by expression of a progastrin transgene in the liver. Experiments are underway to determine whether or not other high affinity receptors selective for progastrin are present in the normal colonic mucosa and to define the signaling pathways involved in the proliferative effects. In summary, this paper describes the first synthesis of recombinant human progastrin6 – 80. The observation of progastrindependent proliferation of the mouse colonic cell line YAMC confirms that the recombinant peptide is biologically active and is consistent with the previously reported proliferative effects of endogenous progastrin on the colonic mucosa of transgenic mice (5). The observations that progastrin does not bind to either the CCK-A or the gastrin/CCK-B receptors and that antagonists selective for either the CCK-A or the gastrin/ CCK-B receptor do not affect progastrin-induced proliferation

PROTEIN STRUCTURE AND FOLDING: Biologically Active Recombinant Human Progastrin 6−80Contains a Tightly Bound Calcium Ion Graham S. Baldwin, Frédéric Hollande, Zhiyu Yang, Yulia Karelina, Adrienne Paterson, Rosslyn Strang, Daniel Fourmy, Greg Neumann and Arthur Shulkes

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J. Biol. Chem. 2001, 276:7791-7796. doi: 10.1074/jbc.M009985200 originally published online December 11, 2000