From the University Department of Surgery, Austin
Hospital, Heidelberg, Victoria 3084, the
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 ¶ Faculté de Pharmacie, Université de
Montpellier, Montpellier, and the ** INSERM U 151, CHU Rangeuil,
Toulouse, France
Received for publication, November 2, 2000
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ABSTRACT |
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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.
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 N-terminal
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 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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.
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 progastrin-derived 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 baculovirus-infected 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.
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 µM,
K2 = 0.29 µM, and
K3 = 7.1 µM. 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.
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EXPERIMENTAL PROCEDURES |
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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 µg/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 GST-progastrin 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 µg/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 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 µl/min), previously calibrated to an accuracy of ±0.01% using singly charged poly(propylene glycol) reference ions. Samples (2-4 µl) 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 µM 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 µM 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 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 µg/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 µM 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 µl of Dulbecco's modified Eagle's medium containing
125I-CCK8 (50,000 cpm, 14.5 fmol; Amersham
Pharmacia Biotech), 150 µM phenylmethylsulfonyl fluoride,
0.05% bacitracin, and 0.1% BSA. Cells were then washed twice with PBS
and lysed with 300 µl 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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RESULTS |
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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).
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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 progastrin
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.
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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
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 µM EDTA (Fig. 4) or 100 µM 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 µM EDTA (data
not shown).
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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 µM (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).
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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 125I-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 discrepancy may be due in part to the presence of more
than one class of binding sites (22).
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DISCUSSION |
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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 glutathione-agarose-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 cleavage 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 µM, K2 = 0.29 µM, and K3 = 7.1 µM. 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 C-terminal 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 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 µM (Fig. 5B). Thirdly previous studies have not detected high affinity binding sites for 125I-gastrin17 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 125I-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
progastrin-dependent 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 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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. The 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: 613-9458-1650; E-mail: g.baldwin@surgeryaustin.unimelb.edu.au.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M009985200
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ABBREVIATIONS |
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The abbreviations used are: CCK, cholecystokinin; GST, glutathione S-transferase; HPLC, high pressure liquid chromatography; PBS, phosphate-buffered saline.
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