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Binding of pepsinogen to the 78 kDa gastrin binding protein
Biochimica et Biophysica Acta 1428 (1999) 21^28 www.elsevier.com/locate/bba
Binding of pepsinogen to the 78 kDa gastrin binding protein Kristy A. Rorison a , Gregory M. Neumann b , Graham S. Baldwin a
a;
*
University Department of Surgery, Austin Campus, A p RMC, Studley Road, Heidelberg, Melbourne, Victoria 3084, Australia b Department of Biochemistry, LaTrobe University, Melbourne, Victoria, Australia Received 5 January 1999; received in revised form 11 March 1999; accepted 24 March 1999
Abstract An endogenous ligand of the 78 kDa gastrin-binding protein (GBP) has been purified from detergent extracts of porcine gastric mucosal membranes by ion exchange chromatography and preparative gel electrophoresis. The ligand bound to the GBP with high affinity (mean IC50 value of 0.31 þ 0.09 Wg/ml, or 8 nM), as assessed by inhibition of cross-linking of iodinated gastrin2;17 to the GBP. Both the N- and C-terminal halves of the GBP, which had been expressed individually as glutathioneS-transferase fusion proteins in Escherichia coli, and purified on glutathione^agarose beads, bound the ligand. Two peptides derived from the ligand were purified by reversed-phase high-performance liquid chromatography (HPLC), and characterised by mass spectrometry and Edman sequencing. The peptides were 97% and 100% identical, respectively, to amino acids 119^157 and 199^219 of porcine pepsinogen A. Commercial samples of pepsinogen also bound to the GBP, with a mean IC50 value of 3.9 þ 1.2 Wg/ml (100 nM). We conclude that the ligand is closely related, but not identical, to pepsinogen A. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Gastrin binding protein; Pepsinogen
1. Introduction The puri¢cation of a 78 kDa gastrin-binding protein (GBP) from detergent extracts of porcine gastric mucosal membranes has been described previously [1,2]. Cloning of the cDNA encoding the GBP [3] revealed that its amino acid sequence was closely related to the sequences of a family of proteins involved in the oxidation of long-chain fatty acids [4].
Abbreviations: CCK, cholecystokinin; GBP, gastrin-binding protein ; GST, glutathione-S-transferase; IC50 , concentration required for 50% inhibition; IPTG, isopropylthiogalactoside * Corresponding author. Fax: +61-3-9458-1650; E-mail: [email protected]
The closest relative was the K-subunit of a mitochondrial trifunctional protein which catalysed long-chain enoyl CoA hydratase and 3-hydroxyacyl CoA dehydrogenase activity [5,6]. The subsequent demonstration of long-chain enoyl CoA hydratase (G.S. Baldwin, unpublished data) and 3-hydroxyacyl CoA dehydrogenase activity [7] associated with the GBP established that it was also related functionally to the K-subunit of the mitochondrial trifunctional protein. The critical metabolic importance of the trifunctional protein has recently been demonstrated by the description of mutations in the human K-subunit [8]. A¡ected children present with a spectrum of clinical features, including hypoketotic hypoglycaemia, muscle weakness, cardiomyopathy and microvesicular fat deposition in the liver, and failure to modify fat intake may lead to coma and death [9].
0304-4165 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 9 9 ) 0 0 0 4 2 - 2
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Several lines of evidence suggest that the GBP may play a role in the proliferation of human colorectal cancer cells. Firstly the anti-proliferative e¡ects of gastrin receptor antagonists on human colorectal carcinoma cell lines [10] appear to be mediated via the GBP [11]. Secondly evidence has been presented that binding of non-steroidal anti-in£ammatory drugs to the GBP may contribute to the inhibitory e¡ects of the drugs on the proliferation of colorectal carcinoma cell lines in vitro, and the development of colorectal carcinoma in vivo [7,12]. Finally the gastrin/CCK-C receptor [11], a low-a¤nity binding site for gastrin on the surface of gastric [13] and colorectal [10] carcinoma cell lines, appears to be a membrane-bound form of the GBP. During the puri¢cation of the GBP no cross-linking of iodinated gastrin was observed until samples had been passed through DEAE^Sepharose [2]. We therefore postulated that in cell extracts the gastrin binding site was occupied by an endogenous ligand, which had a greater a¤nity for DEAE^Sepharose than for the GBP, and which consequently was removed by passage through the anion-exchange resin. In the present paper we describe the puri¢cation of the endogenous ligand, and its characterisation as a novel relative of pepsinogen.
2.3. Puri¢cation of the endogenous GBP ligand
2. Materials and methods
The GBP was partially puri¢ed from detergent extracts of porcine gastric mucosal membranes by sequential chromatography on concanavalin-A^ and DEAE^Sepharose [1,2]. Cross-linking of [125 I](Nle15 )gastrin2;17 to the GBP in the presence of increasing concentrations of the endogenous ligand or of pepsinogen was measured as described previously [1,11]. Brie£y, [125 I](Nle15 )-gastrin2;17 (0.4 nM) was reacted with 0.2 mM disuccinimidylsuberate in 50 mM Na Hepes (pH 7.6) for 15 min at 0³C. 25-Wl aliquots (10 fmol, approx. 30 000 cpm) were added to 25-Wl aliquots of the GBP which had been preincubated for 15 min at 0³C in the presence of increasing concentrations of the endogenous ligand or of pepsinogen in the same bu¡er containing 0.1% Triton X-100. After 20 min at 0³C, the reaction was stopped by addition of 50 Wl Laemmli loading bu¡er, and the samples were treated for 5 min at 95³C and electrophoresed on sodium dodecyl sulfate^10% polyacrylamide gels. After staining with Coomassie blue and
2.1. Materials (Nle15 )-gastrin2;17 and gastrin17 were from Research Plus (Bayonne, NJ, USA). Porcine pepsinogen and pepsin were from Sigma (St. Louis, MO, USA). 2.2. Iodinations (Nle15 )-gastrin2;17 was iodinated by the iodogen method, and puri¢ed by chromatography on a C18 SepPak (Waters Associates, Milford, MA, USA) as described previously [14]. The DEAE^Sepharose 500 mM NaCl eluate was also iodinated by the iodogen method, but the reaction was stopped by the addition of sodium metabisul¢te to 1 mg/ml, and labelled proteins were separated from unincorporated iodide by passage through a 10 ml column of Sephadex G25 Fine (Pharmacia, Uppsala, Sweden).
The endogenous GBP ligand was partially puri¢ed from Triton X-100 extracts of porcine gastric mucosal membranes by chromatography on concanavalin-A^Sepharose as described previously [1,2]. The ligand was separated from the GBP by chromatography of the concanavalin-A^Sepharose eluate on DEAE^Sepharose. 10 ml DEAE^Sepharose was equilibrated with 20 ml 50 mM Na Hepes (pH 7.6), containing 1 mM EDTA, 1 mM PMSF and 0.1% Triton X-100 (bu¡er A), and transferred to a 50-ml tube containing the concanavalin-A^Sepharose eluate which had been made 2 mM in EDTA. The tube was rotated end over end for 60 min at 4³C and centrifuged at 1000Ug for 5 min. The supernatant containing the GBP was decanted, and the DEAE^ Sepharose was washed with 20 ml bu¡er A, followed by 20 ml bu¡er A containing 150 mM NaCl. The endogenous GBP ligand was then eluted with 20 ml bu¡er A containing 500 mM NaCl, and concentrated with an Amicon ultra¢ltration cell (Model 8050, Amicon Corp., Danvers, MA, USA). Protein concentrations were measured by the Bradford assay [15]. 2.4. Cross-linking of iodinated gastrin to the GBP
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drying, the radioactivity associated with the GBP was detected and quantitated with a Phosphorimager (Molecular Dynamics, Sunnyvale, CA, USA). Estimates of IC50 values and of the levels of cross-linking in the absence of inhibitor were obtained with the program SIGMASTAT (Jandel Scienti¢c, San Rafael, CA, USA) by nonlinear regression to the equation y a= 1 x=b, where y is the amount of label incorporated expressed as a percentage of the value a observed in the absence of the endogenous ligand or of pepsinogen, x is the concentration of the endogenous ligand or of pepsinogen, and b is the IC50 value. 2.5. Preparation of GST fusion proteins The N- and C-terminal halves of the GBP were expressed independently in Escherichia coli as glutathione-S-transferase fusion proteins as described previously [16]. Brie£y, E. coli strain NM522 was transformed with the appropriate pGEX plasmids [17] and grown overnight at 37³C with shaking in LB medium containing 100 Wg/ml of ampicillin. The overnight culture (15 ml) was used to inoculate the same medium (135 ml). When an absorbance at 600 nm of 0.8 was reached the culture was induced with 0.1 mM IPTG for 2 h. The cells were harvested by centrifugation at 4000 rpm for 10 min. The cell pellet was washed in cold STE bu¡er (10 mM Tris^ HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA), and lysozyme was added to a ¢nal concentration of 100 Wg/ml. After incubation on ice for 15 min, DTT was added to 5 mM and proteins were solubilised with 1.5% Sarkosyl [18]. After vortexing for 15 s, cells were sonicated for 20 s (power level 4, duty cycle 50%) in a Model 250 Soni¢er (Branson Sonic Power Co., Danbury, CT, USA). The lysate was clari¢ed by centrifugation, and the supernatant was made 2% in Triton X-100. After vortexing for 10 s, washed glutathione^agarose beads (2 ml, 50% v/v suspension in PBS) were added and the suspension was gently mixed by rotation at 4³C for 1 h. The beads with GST-fusion proteins attached were then washed twice with ice-cold PBS, and stored at 370³C in 2 ml storage bu¡er (50 mM Na Hepes (pH 7.4), 150 mM NaCl, 5 mM DTT, 10% v/v glycerol).
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2.6. Binding of the endogenous ligand to the GBP-fusion proteins GST-fusion proteins bound to glutathione^agarose beads (50 Wl) were washed twice with 1 ml 50 mM Na Hepes (pH 7.6), 0.1% Triton X-100, 1 mM dithiothreitol and 0.02% NaN3 (bu¡er B), and incubated for 2 h at 4³C with 250 000 cpm iodinated endogenous ligand (DEAE^Sepharose 500 mM NaCl eluate). All subsequent steps were at 4³C. The beads were then washed twice with 1 ml bu¡er B, and resuspended in 50 Wl loading bu¡er [19] containing 50 mM dithiothreitol, boiled for 5 min, and centrifuged at 13 000Ug for 1 min. Aliquots (10 Wl) of the supernatant were then electrophoresed on a 10% SDS^polyacrylamide gel [19], and proteins were detected by staining with Coomassie blue. 2.7. Preparation and reversed-phase HPLC of tryptic digests The endogenous GBP ligand was precipitated with methanol, reduced and carboxymethylated by treatment with iodoacetic acid in the presence of 8 M urea, and digested with trypsin, as previously described [2]. Tryptic digests of reduced and carboxymethylated proteins were acidi¢ed to 0.5% TFA, ¢ltered (0.2 Wm PVDF ¢lter) and subjected to reversedphase HPLC on a C18 microbore column (Vydac 218TP51; 1U250 mm; 5 Wm particle size; 30 nm pore size) attached to a Hewlett^Packard HP-1090 liquid chromatograph. The column was protected by a 0.2-Wm pre¢lter and a C18 guard column (Alltech macrosphere 300-C18-5u). After column equilibration in 0.1% TFA at a £ow rate of 40 Wl/min, 250-Wl samples were loaded via a 500-Wl loop, followed by a 30-min column wash (0.1% TFA), after which the loop was switched out of the £ow line. Peptides were then eluted from the column at the same £ow rate, using linear gradients of CH3 CN/ 0.1%TFA (0^35% CH3 CN over 100 min, followed by 35^70% CH3 CN over a further 20 min). 20^100Wl fractions were manually collected while monitoring UV absorbance at 215 nm. 2.8. Electrospray ionisation mass spectrometry Samples (2^4 Wl) of reversed-phase HPLC frac-
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tions were analysed directly by electrospray ionisation mass spectrometry using a Perkin^Elmer Sciex API-300 triple quadrupole mass spectrometer ¢tted with a micro-ionspray ion source. Samples were infused through 20 Wm i.d. fused silica tubing using a 10-Wl Hamilton gas-tight syringe driven by a Harvard syringe pump at a £ow rate of 0.2 Wl/min. Signalaveraged spectra were obtained from 50^100 scans over 5^10 min using a scan range of m/z 100^2500 u and a constant peak width (at half height) of 0.6 u. Prior to sample analysis, peak widths were adjusted and the mass scale calibrated to an accuracy of 0.01% using singly-charged poly(propyleneglycol) reference ions. 2.9. 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 PTH-amino acid standards prior to each sequencing run. 3. Results and discussion 3.1. Puri¢cation of the endogenous GBP ligand The ¢rst evidence for the presence of an endogenous ligand of the 78 kDa GBP was our previous report that no binding of iodinated gastrin to the GBP was observed prior to passage of the GBP through DEAE^Sepharose [2]. We now report that material which elutes from the DEAE^Sepharose column with 500 mM NaCl is able to inhibit by over 90% the cross-linking of iodinated gastrin to the GBP (Fig. 1). Signi¢cantly less competition is observed with the same volumes of the 75 mM NaCl or 1 M NaOH eluates (31% and 30%, respectively). The inhibitory material could be precipitated by addition of methanol to 80%, followed by storage at 370³C (data not shown), and was therefore likely to be a protein. In order to determine the size of the inhibitory material, the 500 mM NaCl eluate from the DEAE^Sepharose column was radioactively labelled with 125 I by the iodogen method. After removal of unincorporated iodide by passage through a Sepha-
Fig. 1. Binding of the endogenous GBP ligand to DEAE^ Sepharose. The GBP was puri¢ed from extracts of porcine gastric mucosal membranes [1,2], and identi¢ed by covalent crosslinking to [125 I](Nle15 )-gastrin2;17 as described in Section 2 (tracks 1^4). No cross-linking to the GBP was observed in the eluate from the concanavalin-A^Sepharose column (track 1), or in the 75 mM or 500 mM NaCl eluates from the DEAE^Sepharose column (tracks 3 and 4, respectively), but a radioactive band with an apparent molecular mass of 80 kDa was apparent in the percolate from the DEAE^Sepharose column (track 2). Subtraction of the mass of [125 I](Nle15 )-gastrin2;17 (2 kDa) yielded an apparent molecular mass of 78 kDa for the GBP. The ability of the salt eluates from the DEAE^Sepharose column to inhibit covalent cross-linking of [125 I](Nle15 )-gastrin2;17 to the GBP in the percolate from the DEAE^Sepharose column was also investigated (tracks 5^11). Cross-linking to the GBP could be blocked by prior incubation of the percolate with the 500 mM NaCl eluate from the DEAE^Sepharose column (track 7), or with 12 WM unlabelled gastrin17 (track 11). Quantitation by phosphorimager indicated that the residual cross-linking in the presence of the 500 mM NaCl eluate was less than 10% of the value observed in the presence of 500 mM NaCl alone (track 8). In contrast the residual cross-linking observed following incubation of the percolate with the 75 mM NaCl eluate (track 5), or the 1 M NaOH eluate (track 9), was 69% and 70%, respectively, of the values observed in the presence of the corresponding bu¡ers alone (tracks 6 and 10). The sizes of molecular mass markers are given in kDa.
dex G-25 column, the radioactively labelled material was incubated with glutathione^agarose beads to which had been attached fusion proteins containing glutathione-S-transferase (GST) linked to either the N-terminal enoyl CoA hydratase or C-terminal 3-hydroxyacyl CoA dehydrogenase domains of the GBP. After thorough washing, bound radioactivity was released by heating in Laemmli loading bu¡er and analysed by electrophoresis on polyacrylamide gels (Fig. 2). Autoradiography revealed that a major doublet at 45 kDa, and a minor band at 40 kDa, were present in the eluates from both the N-terminal hydratase or C-terminal dehydrogenase domains of the GBP. No radioactivity was present in control eluates from glutathione^agarose beads alone, or from glu-
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tathione^agarose beads to which had been attached GST itself. We conclude that proteins of 40 and 45 kDa bind to both the N-terminal hydratase and Cterminal dehydrogenase domains of the GBP. The presence of binding sites for the endogenous ligand on both domains of the GBP may at ¢rst sight be surprising. However, the GBP catalyses two consecutive reactions in the oxidation of long-chain fatty acids, and the product of the enoyl CoA hydratase reaction is the substrate of the 3-hydroxyacyl CoA dehydrogenase reaction. Consequently the hydratase and dehydrogenase active sites must share some similar features. Furthermore, binding of gastrin to both N-terminal hydratase and C-terminal dehydrogenase domains of the GBP has been demonstrated directly with GST-fusion proteins [16], and the hydratase and dehydrogenase active sites are involved, since substrates of the hydratase reaction and products of the dehydrogenase reaction compete with gastrin for binding to the intact GBP [11]. The fact that the endogenous ligand also competes with gastrin for binding to the GBP suggests that the endogenous ligand also recognises the structural features common to both hydratase and dehydrogenase active sites. The endogenous ligand was further puri¢ed by elution from DEAE^Sepharose with a NaCl gradient. Inhibitory fractions were pooled, concentrated, and reduced and carboxymethylated. The component proteins were then separated by preparative polyacrylamide gel electrophoresis. Three major Coomassie blue-staining bands, with apparent molecular masses of 53 kDa, 50 kDa and 48 kDa, were detected
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Fig. 2. Characterisation of the endogenous GBP ligand. Proteins in the 500 mM NaCl eluate from the DEAE^Sepharose column were iodinated by the iodogen method, and separated from unincorporated iodide, as described in Section 2. The radiolabelled proteins were then incubated with glutathione^agarose beads (B), or with glutathione^agarose beads to which had been bound fusion proteins between glutathione-S-transferase and the N- and C-terminal halves of the GBP (N and C, respectively), or glutathione-S-transferase alone (G). After thorough washing the bound proteins, including the N- and C-terminal GBP fusion proteins and glutathione-S-transferase, were removed by heating at 95³C in loading bu¡er, and separated by polyacrylamide gel electrophoresis. Bound radiolabelled proteins were detected by autoradiography (A); unlabelled proteins were detected by staining with Coomassie blue (B). The sizes of molecular mass markers are given in kDa, and the positions of the origin (O) and dye front (DF) are indicated by arrows.
Table 1 Comparison of peptides isolated from the endogenous ligand with pepsinogen sequences Peptide T7/59 Pepsinogen T7/50 Pepsinogen
Residues
119^157 199^219
Molecular mass (Da) Obs.
Pred.
3970.5 þ 0.4
3970.3 4076.5 2148.6 2148.6
2148.4 þ 0.3
Sequence GGTGSMTGILGYDTVQVGGISDTNQIFGLSETEPGSFLY(T)YGTGSMTGILGYDTVQVGGISDTNQIFGLSETEPGSFLY(Y) LSSNDDSGSVVLLGGIDSSYY(Y)LSSNDDSGSVVLLGGIDSSYY(T)
The indicated peptides were puri¢ed from tryptic digests of reduced and carboxymethylated samples of the endogenous ligand by microbore reversed-phase HPLC as described in Section 2. Peptide sequences were determined by amino acid sequencing and are compared with the amino acid sequence of porcine pepsinogen translated from the cDNA sequence reported by Tsukagoshi et al. [20]. Observed molecular masses (Obs.) were determined by electrospray ionisation mass spectrometry with the uncertainties indicated; predicted molecular masses (Pred.) were calculated from the indicated sequences. The amino acids adjacent in the pepsinogen sequence to the puri¢ed peptides are indicated in parentheses.
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in the range 40^60 kDa (data not shown). All three proteins were isolated and concentrated by electroelution, and digested with trypsin in the presence of 0.5 M urea. The resultant peptides from each protein were separated by microbore reversed-phase HPLC, but because of shortage of material in the digest from the 48 kDa protein, subsequent analysis concentrated on peptides derived from the 53 and 50 kDa proteins. 3.2. Identi¢cation of the GBP ligand as a relative of pepsinogen A Two major peaks from the peptide map of the 53 kDa protein were characterised by electrospray ionisation mass spectrometry, and their sequences determined by Edman degradation. The properties of both peptides were very similar to fragments of porcine pepsinogen A (Table 1). Thus peptide T7/50 had the predicted molecular mass and sequence for amino acids 199^219 of porcine pepsinogen A. Peptide T7/59 had the predicted sequence for amino acids 119^157 of porcine pepsinogen A, except that tyrosine 119 was replaced by glycine. The substitution was con¢rmed by the molecular mass of the peptide, as determined by mass spectrometry (Table 1). A peak with the same retention time as peptide T7/59, and an almost identical molecular mass of 3970.8 þ 0.5, was also observed in the peptides derived from the 50 kDa protein. We therefore concluded that the 53 kDa and 50 kDa proteins were both related to pepsinogen A, and that the 50 kDa and 48 kDa proteins were likely to be degradation products of the 53 kDa protein. 3.3. Structure, function and location of pepsinogen A Pepsinogen A is a member of a family of aspartic acid proteinase precursors [21]. Family members share the ability to hydrolyse proteins at acid pH, but can be separated immunologically and by amino acid sequence into four major groups, pepsinogen A, pepsinogen C, cathepsin D and cathepsin E. The apparent molecular mass of pepsinogen A determined by polyacrylamide gel electrophoresis is 43 kDa, but a substantial increase, similar in magnitude to that observed with the endogenous ligand, is observed following reduction and carboxymethylation
Fig. 3. Commercial pepsinogen and pepsin bind to the GBP. Cross-linking of [125 I](Nle15 )-gastrin2;17 to the GBP with disuccinimidylsuberate was measured in the presence of increasing concentrations of pepsinogen (circles) or the concentrated 500 mM NaCl eluate from the DEAE^Sepharose column (squares) as previously described [1,2]. Reaction products were separated by electrophoresis on duplicate SDS^polyacrylamide gels and the radioactivity associated with the GBP was detected, quanti¢ed with a phosphorimager, and expressed as a percentage ( þ S.E.M.) of the value observed in the absence of any competitor. Estimates of IC50 values, and of the levels of [125 I](Nle15 )gastrin2;17 bound in the absence of competitor (pepsinogen: 1.11 Wg/ml, 98%; DEAE 500 mM eluate: 0.43 Wg/ml, 125%), were obtained by non-linear regression with the program SIGMASTAT as described in Section 2, and were used to calculate the lines of best ¢t shown in the ¢gure. The IC50 values were then combined with the values obtained in at least two other experiments to obtain the mean values presented in the text. The concentration of pepsinogen in the concentrated 500 mM NaCl eluate from the DEAE^Sepharose column was estimated by measuring the proportion of pepsinogen by densitometric scanning of the Coomassie blue-stained sample after electrophoresis on SDS^polyacrylamide gels, and the total concentration of protein by the Bradford assay [15] with a pepsinogen standard. Pepsinogen concentrations greater than 1 Wg/ml could not be achieved in the DEAE eluate titration because of limited amounts of material.
(data not shown). At least ¢ve minor variants of pepsinogen A have been distinguished on the basis of electrophoretic di¡erences [21]. Additional work will be required to determine whether the observed amino acid substitution in the puri¢ed peptides is a re£ection of the selective binding of one of the minor variants to the GBP, or merely the result of allelic variation. Pepsinogen A is synthesised in the chief cells of the gastric mucosa, and stored in cytoplasmic granules
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[22]. Following release into the acid lumen of the stomach, pepsinogen is activated autocatalytically into the aspartic proteinase pepsin. A statistical survey of bond cleavage by pepsin in proteins revealed a preference for hydrophobic amino acids on either side of the scissile bond, with some speci¢city for leucine, phenylalanine, tryptophan and glutamic acid on the N-terminal side, and for tryptophan, tyrosine, isoleucine and phenylalanine on the C-terminal side [23]. The speci¢city of the four cleavages required to generate peptides T7/50 and T7/59 is not inconsistent with the speci¢city of pepsin since, with the exception of the N-terminus of peptide T7/ 59, all had occurred C-terminal to a tyrosine residue. One possible explanation is that the 53 kDa pepsinogen-like protein had refolded after reduction and carboxymethylation in 8 M urea, and that the fragments were generated by auto-catalytic degradation. None of the cleavages appeared to be catalysed by trypsin and, with the bene¢t of hindsight, trypsin was not the best protease to choose, since the lysine and arginine residues in pepsinogen are clustered in the N-terminal 36, and C-terminal 20, amino acids. Because the intervening 314 amino acids are devoid of lysine and arginine residues, the majority of pepsinogen will not be digested by trypsin. 3.4. Binding of pepsinogen to the GBP In order to investigate further the relationship between the endogenous ligand and pepsinogen, we next tested the ability of commercial pepsinogen to compete with gastrin for binding to the GBP. Increasing concentrations of commercial pepsinogen inhibited the cross-linking of iodinated gastrin to the GBP in a dose-dependent manner, with a mean IC50 value ( þ S.E.M.) of 3.9 þ 1.2 Wg/ml (or 98 nM assuming a molecular mass of 39 627 [24]) (Fig. 3). The IC50 value for commercial pepsin was very similar (data not shown). In contrast to the intermediate a¤nity of commercial pepsinogen for the GBP, the pepsinogen in the DEAE 500 mM NaCl eluate bound to the GBP with high a¤nity. The mean IC50 value ( þ S.E.M.) of 0.31 þ 0.09 Wg/ml (7.8 nM) for the pepsinogen in the DEAE 500 mM NaCl eluate was 12-fold lower than the values for commercial pepsinogen and pepsin, and this di¡erence was statistically signi¢cant (P = 0.011, Student's t-test). The
27
only structural di¡erence observed to date between the endogenous ligand and pepsinogen is the substitution of tyrosine 119 by glycine. However, over 80% of the sequence of the endogenous ligand remains to be established, and it is premature to speculate on whether the di¡erence in apparent IC50 value between the endogenous ligand and pepsinogen is caused by a di¡erence in amino acid sequence, or by post-translational modi¢cation. Examination of the subcellular location of the GBP and pepsinogen suggests that complex formation may well occur in vivo. The majority of pepsinogen in the gastric chief cell is found in cytoplasmic granules [22]. Recent data indicate that the GBP may be a sub-population of the trifunctional protein, which is targeted to the membranes of the endoplasmic reticulum rather than the mitochondrion by splicing of an alternative signal peptide (M. Katinka, personal communication). One plausible explanation for the observed interaction is that the GBP is in some way involved in granule formation. However, the possibility should be borne in mind that the observed interaction between the two proteins may be an artefact which occurs only after homogenisation of the gastric mucosa. Formation of the complex between the GBP and pepsinogen may in principle be modulated by gastrin. Binding of the pepsinogen A-like molecule to the GBP is reversible, since the complex can be disrupted simply by passage through DEAE^Sepharose (Fig. 1). Similarly, binding of gastrin2;17 to the GBP can be reversed by addition of either the pepsinogen A-like molecule or commercial pepsinogen A (Fig. 3). The IC50 values for binding of the pepsinogen A-like molecule or gastrin17 to the GBP are both in the nM^WM range (7.8 nM and 230 nM [11], respectively). Hence, the occupancy of the gastrin binding site on the GBP may vary depending on the relative concentrations of the pepsinogen A-like molecule and gastrin17 in the local microenvironment. In summary, the endogenous ligand of the GBP has been identi¢ed as a pepsinogen-like protein, on the basis of molecular mass and internal amino acid sequence. The high a¤nity interaction between the pepsinogen-like protein and the GBP has been con¢rmed with commercial samples of pepsinogen, although the interaction in this case is 12-fold weaker. Future work will aim to de¢ne the biological sig-
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ni¢cance of the interaction between pepsinogen and the GBP. Acknowledgements We gratefully acknowledge the assistance of Rosemary Condron with the sequencing, of Bronwyn Roberts with the HPLC, and of Nicos Nicola with stimulating discussions. This work was supported in part by NH and MRC Grant number 960182.
References [1] G.S. Baldwin, R. Chandler, D.B. Scanlon, J. Weinstock, J. Biol. Chem. 261 (1986) 12252^12257. [2] G.S. Baldwin, R. Chandler, B. Grego, M.R. Rubira, K.L. Seet, J. Weinstock, Int. J. Biochem. 26 (1994) 529^538. [3] T. Mantamadiotis, P. Sobieszczuk, J. Weinstock, G.S. Baldwin, Biochim. Biophys. Acta 1170 (1993) 211^215. [4] G.S. Baldwin, Comp. Biochem. Physiol. 104B (1993) 55^61. [5] Y. Uchida, K. Izai, T. Orii, T. Hashimoto, J. Biol. Chem. 267 (1992) 1034^1041. [6] T. Kamijo, T. Aoyama, A. Komiyama, T. Hashimoto, Biochem. Biophys. Res. Commun. 199 (1994) 818^825. [7] G.S. Baldwin, V.J. Murphy, Z. Yang, T. Hashimoto, J. Pharmacol. Exp. Ther. 286 (1998) 1110^1114.
[8] L. Ijlst, R.J.A. Wanders, S. Ushikubo, T. Kamijo, T. Hashimoto, Biochim. Biophys. Acta 1215 (1994) 347^350. [9] A.C. Sewell, S.W. Bender, S. Wirth, H. Munterfering, L. Ijlst, R.J.A. Wanders, Eur. J. Pediatr. 153 (1995) 745^750. [10] N.M. Hoosein, P.A. Kiener, R.C. Curry, L.C. Rovati, D.K. McGilbra, M.G. Brattain, Cancer Res. 48 (1988) 7179^7183. [11] G.S. Baldwin, Proc. Natl. Acad. Sci. USA 91 (1994) 7593^ 7597. [12] Z. Yang, F. Hollande, G.S. Baldwin, Cancer Lett. 124 (1998) 187^191. [13] J. Weinstock, G.S. Baldwin, Cancer Res. 48 (1988) 932^937. [14] L. Seet, L. Fabri, E.C. Nice, G.S. Baldwin, Biomed. Chromatogr. 2 (1988) 159^163. [15] S.M. Read, D.H. Northcote, Anal. Biochem. 116 (1981) 53^ 64. [16] V.J. Murphy, T. Mantamadiotis, G.S. Baldwin, Int. J. Biochem. Cell Biol. 28 (1996) 1233^1240. [17] D.B. Smith, K.S. Johnson, Gene 67 (1988) 31^40. [18] J.V. Frangioni, B.G. Neel, Anal. Biochem. 210 (1993) 179^ 187. [19] J. King, U.K. Laemmli, J. Mol. Biol. 62 (1971) 465^473. [20] T. Tsukagoshi, Y. Ando, Y. Tomita, R. Uchida, T. Takemura, T. Sasaki, H. Yamagata, S. Udaka, Y. Ichihara, K. Takahashi, Gene 65 (1988) 285^292. [21] I.M. Samlo¡, Gastroenterology 96 (1989) 586^595. [22] I.M. Samlo¡, Gastroenterology 61 (1971) 185^188. [23] V.K. Antonov, Adv. Exp. Med. Biol. 95 (1977) 179^198. [24] X.-L. Lin, R.N.S. Wong, J. Tang, J. Biol. Chem. 264 (1989) 4482^4489.
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Binding of pepsinogen to the 78 kDa gastrin binding protein Kristy A. Rorison a , Gregory M. Neumann b , Graham S. Baldwin a
a;
*
University Department of Surgery, Austin Campus, A p RMC, Studley Road, Heidelberg, Melbourne, Victoria 3084, Australia b Department of Biochemistry, LaTrobe University, Melbourne, Victoria, Australia Received 5 January 1999; received in revised form 11 March 1999; accepted 24 March 1999
Abstract An endogenous ligand of the 78 kDa gastrin-binding protein (GBP) has been purified from detergent extracts of porcine gastric mucosal membranes by ion exchange chromatography and preparative gel electrophoresis. The ligand bound to the GBP with high affinity (mean IC50 value of 0.31 þ 0.09 Wg/ml, or 8 nM), as assessed by inhibition of cross-linking of iodinated gastrin2;17 to the GBP. Both the N- and C-terminal halves of the GBP, which had been expressed individually as glutathioneS-transferase fusion proteins in Escherichia coli, and purified on glutathione^agarose beads, bound the ligand. Two peptides derived from the ligand were purified by reversed-phase high-performance liquid chromatography (HPLC), and characterised by mass spectrometry and Edman sequencing. The peptides were 97% and 100% identical, respectively, to amino acids 119^157 and 199^219 of porcine pepsinogen A. Commercial samples of pepsinogen also bound to the GBP, with a mean IC50 value of 3.9 þ 1.2 Wg/ml (100 nM). We conclude that the ligand is closely related, but not identical, to pepsinogen A. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Gastrin binding protein; Pepsinogen
1. Introduction The puri¢cation of a 78 kDa gastrin-binding protein (GBP) from detergent extracts of porcine gastric mucosal membranes has been described previously [1,2]. Cloning of the cDNA encoding the GBP [3] revealed that its amino acid sequence was closely related to the sequences of a family of proteins involved in the oxidation of long-chain fatty acids [4].
Abbreviations: CCK, cholecystokinin; GBP, gastrin-binding protein ; GST, glutathione-S-transferase; IC50 , concentration required for 50% inhibition; IPTG, isopropylthiogalactoside * Corresponding author. Fax: +61-3-9458-1650; E-mail: [email protected]
The closest relative was the K-subunit of a mitochondrial trifunctional protein which catalysed long-chain enoyl CoA hydratase and 3-hydroxyacyl CoA dehydrogenase activity [5,6]. The subsequent demonstration of long-chain enoyl CoA hydratase (G.S. Baldwin, unpublished data) and 3-hydroxyacyl CoA dehydrogenase activity [7] associated with the GBP established that it was also related functionally to the K-subunit of the mitochondrial trifunctional protein. The critical metabolic importance of the trifunctional protein has recently been demonstrated by the description of mutations in the human K-subunit [8]. A¡ected children present with a spectrum of clinical features, including hypoketotic hypoglycaemia, muscle weakness, cardiomyopathy and microvesicular fat deposition in the liver, and failure to modify fat intake may lead to coma and death [9].
0304-4165 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 9 9 ) 0 0 0 4 2 - 2
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Several lines of evidence suggest that the GBP may play a role in the proliferation of human colorectal cancer cells. Firstly the anti-proliferative e¡ects of gastrin receptor antagonists on human colorectal carcinoma cell lines [10] appear to be mediated via the GBP [11]. Secondly evidence has been presented that binding of non-steroidal anti-in£ammatory drugs to the GBP may contribute to the inhibitory e¡ects of the drugs on the proliferation of colorectal carcinoma cell lines in vitro, and the development of colorectal carcinoma in vivo [7,12]. Finally the gastrin/CCK-C receptor [11], a low-a¤nity binding site for gastrin on the surface of gastric [13] and colorectal [10] carcinoma cell lines, appears to be a membrane-bound form of the GBP. During the puri¢cation of the GBP no cross-linking of iodinated gastrin was observed until samples had been passed through DEAE^Sepharose [2]. We therefore postulated that in cell extracts the gastrin binding site was occupied by an endogenous ligand, which had a greater a¤nity for DEAE^Sepharose than for the GBP, and which consequently was removed by passage through the anion-exchange resin. In the present paper we describe the puri¢cation of the endogenous ligand, and its characterisation as a novel relative of pepsinogen.
2.3. Puri¢cation of the endogenous GBP ligand
2. Materials and methods
The GBP was partially puri¢ed from detergent extracts of porcine gastric mucosal membranes by sequential chromatography on concanavalin-A^ and DEAE^Sepharose [1,2]. Cross-linking of [125 I](Nle15 )gastrin2;17 to the GBP in the presence of increasing concentrations of the endogenous ligand or of pepsinogen was measured as described previously [1,11]. Brie£y, [125 I](Nle15 )-gastrin2;17 (0.4 nM) was reacted with 0.2 mM disuccinimidylsuberate in 50 mM Na Hepes (pH 7.6) for 15 min at 0³C. 25-Wl aliquots (10 fmol, approx. 30 000 cpm) were added to 25-Wl aliquots of the GBP which had been preincubated for 15 min at 0³C in the presence of increasing concentrations of the endogenous ligand or of pepsinogen in the same bu¡er containing 0.1% Triton X-100. After 20 min at 0³C, the reaction was stopped by addition of 50 Wl Laemmli loading bu¡er, and the samples were treated for 5 min at 95³C and electrophoresed on sodium dodecyl sulfate^10% polyacrylamide gels. After staining with Coomassie blue and
2.1. Materials (Nle15 )-gastrin2;17 and gastrin17 were from Research Plus (Bayonne, NJ, USA). Porcine pepsinogen and pepsin were from Sigma (St. Louis, MO, USA). 2.2. Iodinations (Nle15 )-gastrin2;17 was iodinated by the iodogen method, and puri¢ed by chromatography on a C18 SepPak (Waters Associates, Milford, MA, USA) as described previously [14]. The DEAE^Sepharose 500 mM NaCl eluate was also iodinated by the iodogen method, but the reaction was stopped by the addition of sodium metabisul¢te to 1 mg/ml, and labelled proteins were separated from unincorporated iodide by passage through a 10 ml column of Sephadex G25 Fine (Pharmacia, Uppsala, Sweden).
The endogenous GBP ligand was partially puri¢ed from Triton X-100 extracts of porcine gastric mucosal membranes by chromatography on concanavalin-A^Sepharose as described previously [1,2]. The ligand was separated from the GBP by chromatography of the concanavalin-A^Sepharose eluate on DEAE^Sepharose. 10 ml DEAE^Sepharose was equilibrated with 20 ml 50 mM Na Hepes (pH 7.6), containing 1 mM EDTA, 1 mM PMSF and 0.1% Triton X-100 (bu¡er A), and transferred to a 50-ml tube containing the concanavalin-A^Sepharose eluate which had been made 2 mM in EDTA. The tube was rotated end over end for 60 min at 4³C and centrifuged at 1000Ug for 5 min. The supernatant containing the GBP was decanted, and the DEAE^ Sepharose was washed with 20 ml bu¡er A, followed by 20 ml bu¡er A containing 150 mM NaCl. The endogenous GBP ligand was then eluted with 20 ml bu¡er A containing 500 mM NaCl, and concentrated with an Amicon ultra¢ltration cell (Model 8050, Amicon Corp., Danvers, MA, USA). Protein concentrations were measured by the Bradford assay [15]. 2.4. Cross-linking of iodinated gastrin to the GBP
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drying, the radioactivity associated with the GBP was detected and quantitated with a Phosphorimager (Molecular Dynamics, Sunnyvale, CA, USA). Estimates of IC50 values and of the levels of cross-linking in the absence of inhibitor were obtained with the program SIGMASTAT (Jandel Scienti¢c, San Rafael, CA, USA) by nonlinear regression to the equation y a= 1 x=b, where y is the amount of label incorporated expressed as a percentage of the value a observed in the absence of the endogenous ligand or of pepsinogen, x is the concentration of the endogenous ligand or of pepsinogen, and b is the IC50 value. 2.5. Preparation of GST fusion proteins The N- and C-terminal halves of the GBP were expressed independently in Escherichia coli as glutathione-S-transferase fusion proteins as described previously [16]. Brie£y, E. coli strain NM522 was transformed with the appropriate pGEX plasmids [17] and grown overnight at 37³C with shaking in LB medium containing 100 Wg/ml of ampicillin. The overnight culture (15 ml) was used to inoculate the same medium (135 ml). When an absorbance at 600 nm of 0.8 was reached the culture was induced with 0.1 mM IPTG for 2 h. The cells were harvested by centrifugation at 4000 rpm for 10 min. The cell pellet was washed in cold STE bu¡er (10 mM Tris^ HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA), and lysozyme was added to a ¢nal concentration of 100 Wg/ml. After incubation on ice for 15 min, DTT was added to 5 mM and proteins were solubilised with 1.5% Sarkosyl [18]. After vortexing for 15 s, cells were sonicated for 20 s (power level 4, duty cycle 50%) in a Model 250 Soni¢er (Branson Sonic Power Co., Danbury, CT, USA). The lysate was clari¢ed by centrifugation, and the supernatant was made 2% in Triton X-100. After vortexing for 10 s, washed glutathione^agarose beads (2 ml, 50% v/v suspension in PBS) were added and the suspension was gently mixed by rotation at 4³C for 1 h. The beads with GST-fusion proteins attached were then washed twice with ice-cold PBS, and stored at 370³C in 2 ml storage bu¡er (50 mM Na Hepes (pH 7.4), 150 mM NaCl, 5 mM DTT, 10% v/v glycerol).
23
2.6. Binding of the endogenous ligand to the GBP-fusion proteins GST-fusion proteins bound to glutathione^agarose beads (50 Wl) were washed twice with 1 ml 50 mM Na Hepes (pH 7.6), 0.1% Triton X-100, 1 mM dithiothreitol and 0.02% NaN3 (bu¡er B), and incubated for 2 h at 4³C with 250 000 cpm iodinated endogenous ligand (DEAE^Sepharose 500 mM NaCl eluate). All subsequent steps were at 4³C. The beads were then washed twice with 1 ml bu¡er B, and resuspended in 50 Wl loading bu¡er [19] containing 50 mM dithiothreitol, boiled for 5 min, and centrifuged at 13 000Ug for 1 min. Aliquots (10 Wl) of the supernatant were then electrophoresed on a 10% SDS^polyacrylamide gel [19], and proteins were detected by staining with Coomassie blue. 2.7. Preparation and reversed-phase HPLC of tryptic digests The endogenous GBP ligand was precipitated with methanol, reduced and carboxymethylated by treatment with iodoacetic acid in the presence of 8 M urea, and digested with trypsin, as previously described [2]. Tryptic digests of reduced and carboxymethylated proteins were acidi¢ed to 0.5% TFA, ¢ltered (0.2 Wm PVDF ¢lter) and subjected to reversedphase HPLC on a C18 microbore column (Vydac 218TP51; 1U250 mm; 5 Wm particle size; 30 nm pore size) attached to a Hewlett^Packard HP-1090 liquid chromatograph. The column was protected by a 0.2-Wm pre¢lter and a C18 guard column (Alltech macrosphere 300-C18-5u). After column equilibration in 0.1% TFA at a £ow rate of 40 Wl/min, 250-Wl samples were loaded via a 500-Wl loop, followed by a 30-min column wash (0.1% TFA), after which the loop was switched out of the £ow line. Peptides were then eluted from the column at the same £ow rate, using linear gradients of CH3 CN/ 0.1%TFA (0^35% CH3 CN over 100 min, followed by 35^70% CH3 CN over a further 20 min). 20^100Wl fractions were manually collected while monitoring UV absorbance at 215 nm. 2.8. Electrospray ionisation mass spectrometry Samples (2^4 Wl) of reversed-phase HPLC frac-
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tions were analysed directly by electrospray ionisation mass spectrometry using a Perkin^Elmer Sciex API-300 triple quadrupole mass spectrometer ¢tted with a micro-ionspray ion source. Samples were infused through 20 Wm i.d. fused silica tubing using a 10-Wl Hamilton gas-tight syringe driven by a Harvard syringe pump at a £ow rate of 0.2 Wl/min. Signalaveraged spectra were obtained from 50^100 scans over 5^10 min using a scan range of m/z 100^2500 u and a constant peak width (at half height) of 0.6 u. Prior to sample analysis, peak widths were adjusted and the mass scale calibrated to an accuracy of 0.01% using singly-charged poly(propyleneglycol) reference ions. 2.9. 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 PTH-amino acid standards prior to each sequencing run. 3. Results and discussion 3.1. Puri¢cation of the endogenous GBP ligand The ¢rst evidence for the presence of an endogenous ligand of the 78 kDa GBP was our previous report that no binding of iodinated gastrin to the GBP was observed prior to passage of the GBP through DEAE^Sepharose [2]. We now report that material which elutes from the DEAE^Sepharose column with 500 mM NaCl is able to inhibit by over 90% the cross-linking of iodinated gastrin to the GBP (Fig. 1). Signi¢cantly less competition is observed with the same volumes of the 75 mM NaCl or 1 M NaOH eluates (31% and 30%, respectively). The inhibitory material could be precipitated by addition of methanol to 80%, followed by storage at 370³C (data not shown), and was therefore likely to be a protein. In order to determine the size of the inhibitory material, the 500 mM NaCl eluate from the DEAE^Sepharose column was radioactively labelled with 125 I by the iodogen method. After removal of unincorporated iodide by passage through a Sepha-
Fig. 1. Binding of the endogenous GBP ligand to DEAE^ Sepharose. The GBP was puri¢ed from extracts of porcine gastric mucosal membranes [1,2], and identi¢ed by covalent crosslinking to [125 I](Nle15 )-gastrin2;17 as described in Section 2 (tracks 1^4). No cross-linking to the GBP was observed in the eluate from the concanavalin-A^Sepharose column (track 1), or in the 75 mM or 500 mM NaCl eluates from the DEAE^Sepharose column (tracks 3 and 4, respectively), but a radioactive band with an apparent molecular mass of 80 kDa was apparent in the percolate from the DEAE^Sepharose column (track 2). Subtraction of the mass of [125 I](Nle15 )-gastrin2;17 (2 kDa) yielded an apparent molecular mass of 78 kDa for the GBP. The ability of the salt eluates from the DEAE^Sepharose column to inhibit covalent cross-linking of [125 I](Nle15 )-gastrin2;17 to the GBP in the percolate from the DEAE^Sepharose column was also investigated (tracks 5^11). Cross-linking to the GBP could be blocked by prior incubation of the percolate with the 500 mM NaCl eluate from the DEAE^Sepharose column (track 7), or with 12 WM unlabelled gastrin17 (track 11). Quantitation by phosphorimager indicated that the residual cross-linking in the presence of the 500 mM NaCl eluate was less than 10% of the value observed in the presence of 500 mM NaCl alone (track 8). In contrast the residual cross-linking observed following incubation of the percolate with the 75 mM NaCl eluate (track 5), or the 1 M NaOH eluate (track 9), was 69% and 70%, respectively, of the values observed in the presence of the corresponding bu¡ers alone (tracks 6 and 10). The sizes of molecular mass markers are given in kDa.
dex G-25 column, the radioactively labelled material was incubated with glutathione^agarose beads to which had been attached fusion proteins containing glutathione-S-transferase (GST) linked to either the N-terminal enoyl CoA hydratase or C-terminal 3-hydroxyacyl CoA dehydrogenase domains of the GBP. After thorough washing, bound radioactivity was released by heating in Laemmli loading bu¡er and analysed by electrophoresis on polyacrylamide gels (Fig. 2). Autoradiography revealed that a major doublet at 45 kDa, and a minor band at 40 kDa, were present in the eluates from both the N-terminal hydratase or C-terminal dehydrogenase domains of the GBP. No radioactivity was present in control eluates from glutathione^agarose beads alone, or from glu-
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tathione^agarose beads to which had been attached GST itself. We conclude that proteins of 40 and 45 kDa bind to both the N-terminal hydratase and Cterminal dehydrogenase domains of the GBP. The presence of binding sites for the endogenous ligand on both domains of the GBP may at ¢rst sight be surprising. However, the GBP catalyses two consecutive reactions in the oxidation of long-chain fatty acids, and the product of the enoyl CoA hydratase reaction is the substrate of the 3-hydroxyacyl CoA dehydrogenase reaction. Consequently the hydratase and dehydrogenase active sites must share some similar features. Furthermore, binding of gastrin to both N-terminal hydratase and C-terminal dehydrogenase domains of the GBP has been demonstrated directly with GST-fusion proteins [16], and the hydratase and dehydrogenase active sites are involved, since substrates of the hydratase reaction and products of the dehydrogenase reaction compete with gastrin for binding to the intact GBP [11]. The fact that the endogenous ligand also competes with gastrin for binding to the GBP suggests that the endogenous ligand also recognises the structural features common to both hydratase and dehydrogenase active sites. The endogenous ligand was further puri¢ed by elution from DEAE^Sepharose with a NaCl gradient. Inhibitory fractions were pooled, concentrated, and reduced and carboxymethylated. The component proteins were then separated by preparative polyacrylamide gel electrophoresis. Three major Coomassie blue-staining bands, with apparent molecular masses of 53 kDa, 50 kDa and 48 kDa, were detected
25
Fig. 2. Characterisation of the endogenous GBP ligand. Proteins in the 500 mM NaCl eluate from the DEAE^Sepharose column were iodinated by the iodogen method, and separated from unincorporated iodide, as described in Section 2. The radiolabelled proteins were then incubated with glutathione^agarose beads (B), or with glutathione^agarose beads to which had been bound fusion proteins between glutathione-S-transferase and the N- and C-terminal halves of the GBP (N and C, respectively), or glutathione-S-transferase alone (G). After thorough washing the bound proteins, including the N- and C-terminal GBP fusion proteins and glutathione-S-transferase, were removed by heating at 95³C in loading bu¡er, and separated by polyacrylamide gel electrophoresis. Bound radiolabelled proteins were detected by autoradiography (A); unlabelled proteins were detected by staining with Coomassie blue (B). The sizes of molecular mass markers are given in kDa, and the positions of the origin (O) and dye front (DF) are indicated by arrows.
Table 1 Comparison of peptides isolated from the endogenous ligand with pepsinogen sequences Peptide T7/59 Pepsinogen T7/50 Pepsinogen
Residues
119^157 199^219
Molecular mass (Da) Obs.
Pred.
3970.5 þ 0.4
3970.3 4076.5 2148.6 2148.6
2148.4 þ 0.3
Sequence GGTGSMTGILGYDTVQVGGISDTNQIFGLSETEPGSFLY(T)YGTGSMTGILGYDTVQVGGISDTNQIFGLSETEPGSFLY(Y) LSSNDDSGSVVLLGGIDSSYY(Y)LSSNDDSGSVVLLGGIDSSYY(T)
The indicated peptides were puri¢ed from tryptic digests of reduced and carboxymethylated samples of the endogenous ligand by microbore reversed-phase HPLC as described in Section 2. Peptide sequences were determined by amino acid sequencing and are compared with the amino acid sequence of porcine pepsinogen translated from the cDNA sequence reported by Tsukagoshi et al. [20]. Observed molecular masses (Obs.) were determined by electrospray ionisation mass spectrometry with the uncertainties indicated; predicted molecular masses (Pred.) were calculated from the indicated sequences. The amino acids adjacent in the pepsinogen sequence to the puri¢ed peptides are indicated in parentheses.
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in the range 40^60 kDa (data not shown). All three proteins were isolated and concentrated by electroelution, and digested with trypsin in the presence of 0.5 M urea. The resultant peptides from each protein were separated by microbore reversed-phase HPLC, but because of shortage of material in the digest from the 48 kDa protein, subsequent analysis concentrated on peptides derived from the 53 and 50 kDa proteins. 3.2. Identi¢cation of the GBP ligand as a relative of pepsinogen A Two major peaks from the peptide map of the 53 kDa protein were characterised by electrospray ionisation mass spectrometry, and their sequences determined by Edman degradation. The properties of both peptides were very similar to fragments of porcine pepsinogen A (Table 1). Thus peptide T7/50 had the predicted molecular mass and sequence for amino acids 199^219 of porcine pepsinogen A. Peptide T7/59 had the predicted sequence for amino acids 119^157 of porcine pepsinogen A, except that tyrosine 119 was replaced by glycine. The substitution was con¢rmed by the molecular mass of the peptide, as determined by mass spectrometry (Table 1). A peak with the same retention time as peptide T7/59, and an almost identical molecular mass of 3970.8 þ 0.5, was also observed in the peptides derived from the 50 kDa protein. We therefore concluded that the 53 kDa and 50 kDa proteins were both related to pepsinogen A, and that the 50 kDa and 48 kDa proteins were likely to be degradation products of the 53 kDa protein. 3.3. Structure, function and location of pepsinogen A Pepsinogen A is a member of a family of aspartic acid proteinase precursors [21]. Family members share the ability to hydrolyse proteins at acid pH, but can be separated immunologically and by amino acid sequence into four major groups, pepsinogen A, pepsinogen C, cathepsin D and cathepsin E. The apparent molecular mass of pepsinogen A determined by polyacrylamide gel electrophoresis is 43 kDa, but a substantial increase, similar in magnitude to that observed with the endogenous ligand, is observed following reduction and carboxymethylation
Fig. 3. Commercial pepsinogen and pepsin bind to the GBP. Cross-linking of [125 I](Nle15 )-gastrin2;17 to the GBP with disuccinimidylsuberate was measured in the presence of increasing concentrations of pepsinogen (circles) or the concentrated 500 mM NaCl eluate from the DEAE^Sepharose column (squares) as previously described [1,2]. Reaction products were separated by electrophoresis on duplicate SDS^polyacrylamide gels and the radioactivity associated with the GBP was detected, quanti¢ed with a phosphorimager, and expressed as a percentage ( þ S.E.M.) of the value observed in the absence of any competitor. Estimates of IC50 values, and of the levels of [125 I](Nle15 )gastrin2;17 bound in the absence of competitor (pepsinogen: 1.11 Wg/ml, 98%; DEAE 500 mM eluate: 0.43 Wg/ml, 125%), were obtained by non-linear regression with the program SIGMASTAT as described in Section 2, and were used to calculate the lines of best ¢t shown in the ¢gure. The IC50 values were then combined with the values obtained in at least two other experiments to obtain the mean values presented in the text. The concentration of pepsinogen in the concentrated 500 mM NaCl eluate from the DEAE^Sepharose column was estimated by measuring the proportion of pepsinogen by densitometric scanning of the Coomassie blue-stained sample after electrophoresis on SDS^polyacrylamide gels, and the total concentration of protein by the Bradford assay [15] with a pepsinogen standard. Pepsinogen concentrations greater than 1 Wg/ml could not be achieved in the DEAE eluate titration because of limited amounts of material.
(data not shown). At least ¢ve minor variants of pepsinogen A have been distinguished on the basis of electrophoretic di¡erences [21]. Additional work will be required to determine whether the observed amino acid substitution in the puri¢ed peptides is a re£ection of the selective binding of one of the minor variants to the GBP, or merely the result of allelic variation. Pepsinogen A is synthesised in the chief cells of the gastric mucosa, and stored in cytoplasmic granules
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[22]. Following release into the acid lumen of the stomach, pepsinogen is activated autocatalytically into the aspartic proteinase pepsin. A statistical survey of bond cleavage by pepsin in proteins revealed a preference for hydrophobic amino acids on either side of the scissile bond, with some speci¢city for leucine, phenylalanine, tryptophan and glutamic acid on the N-terminal side, and for tryptophan, tyrosine, isoleucine and phenylalanine on the C-terminal side [23]. The speci¢city of the four cleavages required to generate peptides T7/50 and T7/59 is not inconsistent with the speci¢city of pepsin since, with the exception of the N-terminus of peptide T7/ 59, all had occurred C-terminal to a tyrosine residue. One possible explanation is that the 53 kDa pepsinogen-like protein had refolded after reduction and carboxymethylation in 8 M urea, and that the fragments were generated by auto-catalytic degradation. None of the cleavages appeared to be catalysed by trypsin and, with the bene¢t of hindsight, trypsin was not the best protease to choose, since the lysine and arginine residues in pepsinogen are clustered in the N-terminal 36, and C-terminal 20, amino acids. Because the intervening 314 amino acids are devoid of lysine and arginine residues, the majority of pepsinogen will not be digested by trypsin. 3.4. Binding of pepsinogen to the GBP In order to investigate further the relationship between the endogenous ligand and pepsinogen, we next tested the ability of commercial pepsinogen to compete with gastrin for binding to the GBP. Increasing concentrations of commercial pepsinogen inhibited the cross-linking of iodinated gastrin to the GBP in a dose-dependent manner, with a mean IC50 value ( þ S.E.M.) of 3.9 þ 1.2 Wg/ml (or 98 nM assuming a molecular mass of 39 627 [24]) (Fig. 3). The IC50 value for commercial pepsin was very similar (data not shown). In contrast to the intermediate a¤nity of commercial pepsinogen for the GBP, the pepsinogen in the DEAE 500 mM NaCl eluate bound to the GBP with high a¤nity. The mean IC50 value ( þ S.E.M.) of 0.31 þ 0.09 Wg/ml (7.8 nM) for the pepsinogen in the DEAE 500 mM NaCl eluate was 12-fold lower than the values for commercial pepsinogen and pepsin, and this di¡erence was statistically signi¢cant (P = 0.011, Student's t-test). The
27
only structural di¡erence observed to date between the endogenous ligand and pepsinogen is the substitution of tyrosine 119 by glycine. However, over 80% of the sequence of the endogenous ligand remains to be established, and it is premature to speculate on whether the di¡erence in apparent IC50 value between the endogenous ligand and pepsinogen is caused by a di¡erence in amino acid sequence, or by post-translational modi¢cation. Examination of the subcellular location of the GBP and pepsinogen suggests that complex formation may well occur in vivo. The majority of pepsinogen in the gastric chief cell is found in cytoplasmic granules [22]. Recent data indicate that the GBP may be a sub-population of the trifunctional protein, which is targeted to the membranes of the endoplasmic reticulum rather than the mitochondrion by splicing of an alternative signal peptide (M. Katinka, personal communication). One plausible explanation for the observed interaction is that the GBP is in some way involved in granule formation. However, the possibility should be borne in mind that the observed interaction between the two proteins may be an artefact which occurs only after homogenisation of the gastric mucosa. Formation of the complex between the GBP and pepsinogen may in principle be modulated by gastrin. Binding of the pepsinogen A-like molecule to the GBP is reversible, since the complex can be disrupted simply by passage through DEAE^Sepharose (Fig. 1). Similarly, binding of gastrin2;17 to the GBP can be reversed by addition of either the pepsinogen A-like molecule or commercial pepsinogen A (Fig. 3). The IC50 values for binding of the pepsinogen A-like molecule or gastrin17 to the GBP are both in the nM^WM range (7.8 nM and 230 nM [11], respectively). Hence, the occupancy of the gastrin binding site on the GBP may vary depending on the relative concentrations of the pepsinogen A-like molecule and gastrin17 in the local microenvironment. In summary, the endogenous ligand of the GBP has been identi¢ed as a pepsinogen-like protein, on the basis of molecular mass and internal amino acid sequence. The high a¤nity interaction between the pepsinogen-like protein and the GBP has been con¢rmed with commercial samples of pepsinogen, although the interaction in this case is 12-fold weaker. Future work will aim to de¢ne the biological sig-
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ni¢cance of the interaction between pepsinogen and the GBP. Acknowledgements We gratefully acknowledge the assistance of Rosemary Condron with the sequencing, of Bronwyn Roberts with the HPLC, and of Nicos Nicola with stimulating discussions. This work was supported in part by NH and MRC Grant number 960182.
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