Báo cáo khoa học: Definition of the residues required for the interaction between glycine-extended gastrin and transferrin in vitro pptx

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Definition of the residues required for the interactionbetween glycine-extended gastrin and transferrin in vitroSuzana Kovac1, Audrey Ferrand1, Jean-Pierre Este`ve2, Anne B. Mason3and Graham S. Baldwin11 Department of Surgery, University of Melbourne, Austin Health, Victoria, Australia2 INSERM U.858, Plateforme d’interaction mole´culaire, Institut Louis Bugnard, Toulouse, France3 College of Medicine, Department of Biochemistry, University of Vermont, Burlington, VT, USAIntroductionIron plays a central role in cellular processes because ofits ability to accept or donate electrons readily, and tocycle between ferric (Fe3+) and ferrous (Fe2+) forms.Iron is essential for DNA synthesis, respiration andKeywordsferric; gastrin; iron; transferrinCorrespondenceG. S. Baldwin, University of MelbourneDepartment of Surgery, Austin Health,Studley Road, Heidelberg, Victoria 3084,AustraliaFax: +61 3 9458 1650Tel: +61 3 9496 5592E-mail: grahamsb@unimelb.edu.au(Received 2 March 2009, revised 27 May2009, accepted 30 June 2009)doi:10.1111/j.1742-4658.2009.07186.xTransferrin is the main iron transport protein found in the circulation, andthe level of transferrin saturation in the blood is an important indicator ofiron status. The peptides amidated gastrin(17) (Gamide) and glycine-extended gastrin(17) (Ggly) are well known for their roles in controllingacid secretion and as growth factors in the gastrointestinal tract. Severallines of evidence, including the facts that transferrin binds gastrin, thatgastrins bind ferric ions, and that the level of expression of gastrins posi-tively correlates with transferrin saturation, suggest the possible involve-ment of the transferrin–gastrin interaction in iron homeostasis. In thepresent work, the interaction between gastrins and transferrin has beencharacterized by surface plasmon resonance and covalent crosslinking.First, an interaction between iron-free apo-transferrin and Gamide or Gglywas observed. The fact that no interaction was observed in the presence ofthe chelator EDTA suggested that the gastrin–ferric ion complex was theinteracting species. Moreover, removal of ferric ions with EDTA reducedthe stability of the complex between apo-transferrin and gastrins, and nointeraction was observed between Gamide or Ggly and diferric transferrin.Second, some or all of glutamates at positions 8–10 of the Ggly molecule,together with the C-terminal domain, were necessary for the interactionwith apo-transferrin. Third, monoferric transferrin mutants incapable ofbinding iron in either the N-terminal or C-terminal lobe still bound Ggly.These findings are consistent with the hypothesis that gastrin peptides bindto nonligand residues within the open cleft in each lobe of transferrin andare involved in iron loading of transferrin in vivo.Structured digital abstractlMINT-7212832, MINT-7212849: Apo-transferrin (uniprotkb:P02787) and Gamide (uni-protkb:P01350) bind (MI:0407)bysurface plasmon resonance (MI:0107)lMINT-7212881, MINT-7212909: Ggly (uniprotkb:P01350) and Apo-transferrin (uni-protkb:P02787) bind (MI:0407)bycross-linking studies (MI:0030)lMINT-7212864: Apo-transferrin (uniprotkb:P02787) and Ggly (uniprotkb:P01350) bind(MI:0407)bycompetition binding (MI:0405)AbbreviationsApoTf, apo-transferrin; Gamide, amidated gastrin(17); Ggly, glycine-extended gastrin(17); HoloTf, holo-transferrin; RU, resonance units; SEM,standard error of the mean.4866 FEBS Journal 276 (2009) 4866–4874 ª 2009 The Authors Journal compilation ª 2009 FEBSmetabolic processes as a key component ofcytochromes, oxygen-binding molecules such ashemoglobin and myoglobin, and iron–sulfur clusters inmany enzymes. Because of its crucial biological func-tions, iron must be readily available throughout thebody.Transferrin is the main iron transport protein in thecirculation. The biological importance of transferrin isshown by the fact that hypotransferrinemic hpx mice [1]die from severe anemia within 14 days post partum [2].Transferrin is able to bind two ferric ions with veryhigh affinity, and can then donate iron to cells through-out the body via transferrin receptor-1. The crystalstructure of the single transferrin polypeptide chain(consisting of 680–690 amino acids) has been deter-mined in both diferric [3] and iron-free [apo-transferrin(ApoTf)] forms [4]. The chain is folded into two lobes,the N-lobe and C-lobe, derived from the N-terminaland C-terminal halves of the protein, respectively. Thetwo lobes share 60% homology, and are presumed tohave arisen by gene duplication and fusion [5]. Eachlobe is folded into two subdomains, which cometogether to form a cleft that provides a binding site forone ferric ion [6]. In vitro studies have shown that thetwo lobes are kinetically and thermodynamically dis-tinct, and that cooperativity between the lobes isrequired for iron release [7,8]. Transferrin adopts a‘closed’ (holo) conformation when iron enters the cleft,and an ‘open’ (apo) conformation when iron isreleased. In healthy humans, although the concentra-tion of transferrin in the serum is 25–50 mm, onlyapproximately 30% is saturated with iron. The propor-tions of the four possible forms are as follows: 27% dif-erric; 23% monoferric N-lobe; 11% monoferric C-lobe;and 39% ApoTf [9]. Transferrin saturation is an impor-tant indicator of iron status, as it modulates the con-centration of hepcidin, the peptide responsible forregulation of iron release from cells that store iron.The gastrointestinal peptide hormone gastrin [amidat-ed gastrin(17), Gamide] is well known as a stimulant ofgastric acid secretion, and as a growth factor for the gas-tric mucosa [10]. More recently, nonamidated precursorforms, such as progastrin and glycine-extended gas-trin(17) (Ggly), have also been shown to stimulate pro-liferation and migration of cell lines derived from avariety of gastrointestinal tumors, although, in contrastto stimulation of growth by Gamide, that by Ggly invivo is restricted to the colorectal mucosa [10]. Fluores-cence quenching data have revealed the presence of twoferric ion-binding sites in both Ggly and Gamide, with aKdof 0.6 lm in aqueous solution [11]. Glu7 serves as aligand for one ferric ion, and Glu8 and Glu9 bind a sec-ond ferric ion, in both Ggly [12] and Gamide [13].Although both Ggly and Gamide bind iron, only in thecase of Ggly is biological activity dependent on ferricion binding [12]; Gamide is fully active in the absence ofmetal ions [13].Evidence for a connection between gastrins and ironhomeostasis was first provided in a search for gastrin-binding proteins in porcine gastric mucosa [14]. Aninteraction between Gamide and transferrin was identi-fied by covalent crosslinking assays [14], and subse-quently a more detailed ultracentrifugal study revealedthat, at pH 7.4, ApoTf bound two molecules of gastrinwith a Kdof 6.4 lm [15]. Importantly, no significantbinding of Gamide to diferric transferrin was detected.The observations that circulating gastrin concentra-tions are increased in the iron-loading disorder hemo-chromatosis [16], and that circulating Gamideconcentrations are correlated with transferrin satura-tion in both mice and humans [17], suggest that theinteraction between gastrins and transferrin may beimportant in the regulation of iron homeostasis. Inde-pendent evidence for a connection between gastrinsand iron status has been provided by a microarraycomparison of gene expression profiles in the stomachsof gastrin-deficient and wild-type mice. The concentra-tion of gastric hepcidin mRNA in gastrin-deficientmice was only 40% of that in wild-type mice, andGamide infusion restored the hepcidin mRNA concen-tration to 130% of the wild-type value [18].The biochemical basis of the gastrin–transferrin inter-action is still unknown. Knowledge of the regions oftransferrin required for the binding of gastrin, and ofthe regions in gastrin required for the interaction withtransferrin, is obviously essential to a full understandingof the interaction. The independent involvement of iron[17] and nonamidated gastrins such as Ggly [10] in thedevelopment of colorectal cancer make it particularlyimportant to establish whether or not Ggly also inter-acts with transferrin. Here, surface plasmon resonanceand covalent crosslinking have been used to explorewhether Ggly interacts with transferrin in vitro,toinvestigate whether iron is required for the Ggly–trans-ferrin interaction, to define the domains ⁄ residues ofGgly involved in the interaction (using Ggly mutants),and, finally, to determine the regions of transferrinrequired for the interaction with gastrins.ResultsBoth Gamide and Ggly interact with ApoTf butnot holo-transferrin (HoloTf)An interaction between immobilized Gamide or Gglypeptides and ApoTf was clearly observed using surfaceS. Kovac et al. The interaction between Ggly and transferrinFEBS Journal 276 (2009) 4866–4874 ª 2009 The Authors Journal compilation ª 2009 FEBS 4867plasmon resonance (Fig. 1A), whereas no binding wasfound for HoloTf (Fig. 1B). The apparent rateconstants for association (ka) and dissociation (kd)were as follows: for Gamide, ka= 5.94 · 105m)1Æs)1,and kd= 8.06 · 10)4s)1, and for Ggly, ka= 5.20 ·105m)1Æs)1, and kd= 1.06 · 10)3s)1. The data areconsistent with the hypothesis that gastrins bind withinthe iron-binding cleft, which needs to be in the open(apo) conformation for the association betweengastrins and transferrin to occur.Covalent crosslinking experiments confirmed thatGgly interacts with ApoTf but not with HoloTf (Fig. 1C). Thus, two different approaches demonstrate thattransferrin must be in the open (iron-free) conforma-tion to be able to interact with Ggly, as was previouslyfound for Gamide [14,15]. To measure the affinity ofApoTf for Ggly, a titration curve was constructedusing unlabeled Ggly (Fig. 1D). The IC50for bindingof Ggly to ApoTf was found to be 39 ± 1 lm.Importance of ferric ions for the gastrin–ApoTfinteractionAs both Gamide and Ggly bind two ferric ions [11],the iron chelator EDTA was coinjected with ApoTfinto the BIAcore channel to determine whether the fer-ric ions were required for the interaction between gast-rins and ApoTf. In the presence of EDTA, nointeraction between ApoTf and either Gamide or Gglywas observed (Fig. 2A). Therefore, ferric ions must bepresent for formation of the complex between ApoTfand Ggly or Gamide.The effect of ferric ions on the stability of the gas-trin–ApoTf complex was then investigated. After for-mation of the gastrin–ApoTf complex, EDTA wasinjected into the BIAcore to chelate any available iron.As soon as the EDTA was injected, the associationbetween gastrins and ApoTf was disrupted, indicatingthat ferric ions were essential for the stability of thegastrin–ApoTf complex (Fig. 2B).–20 0 20 40 60 80 –100 0 100 200 300 400 500 Time (s) Response differential (RU) HoloTf Gamide Ggly–20 0 20 40 60 80 0 100 200 300 400 500 Time (s) –100 ApoTf Gamide GglyResponse differential (RU) ApoTf Total protein Crosslinked proteinHoloTf [Ggly] µM 0 25 50 75 100 0 25 50 75 100 Ggly concentration [log M] –12.0 –5.5 –5.0 –4.5 –4.0 –3.5 Relative density (%)0 20 40 60 80 100 120 A B C D Fig. 1. Both Gamide and Ggly interact with ApoTf but not HoloTf. (A) Following injection of ApoTf (10 lgÆmL)1) into the BIAcore channel, aninteraction was observed with both Gamide (red line) and Ggly (blue line) by surface plasmon resonance. After removal of ApoTf from therunning buffer (thick arrow), the interaction between Ggly ⁄ Gamide and ApoTf gradually declined. (B) Upon injection of HoloTf (10 lgÆmL)1)into the BIAcore channel, no interaction was observed with Gamide (red line) or Ggly (blue line). (C) The interaction between Ggly and ApoTfwas also detected using covalent crosslinking. [125I]Ggly(2–17) was prereacted with the bivalent crosslinker disuccinimidyl suberate beforebeing mixed with ApoTf in 50 mM Hepes buffer (pH 7.6) in the absence or presence of increasing concentrations of unlabeled Ggly. TheApoTf–Ggly complex was separated from the unreacted Ggly by SDS ⁄ PAGE, and the extent of incorporation of radioactivity was determinedby phosphoimager and densitometric analysis. Unlabeled Ggly inhibited the interaction in a dose-dependent manner. Lack of interactionbetween Ggly and HoloTf was also confirmed. (D) The IC50for binding of Ggly to ApoTf was found to be 39 ± 1 lM by curve-fitting, with anintercept of 92.3%. Data points are means ± SEM, where n =3.The interaction between Ggly and transferrin S. Kovac et al.4868 FEBS Journal 276 (2009) 4866–4874 ª 2009 The Authors Journal compilation ª 2009 FEBSCharacterization of Ggly domains involved in theinteraction with ApoTfWe have previously demonstrated that Glu7 acts as aligand for the first ferric ion, and that Glu8 and Glu9act as ligands for the second ferric ion, in the gastrin–ferric ion complex for both Ggly [12] and Gamide [13].To characterize the involvement of the glutamates inthe interaction of the peptide with ApoTf, Gglymutants in which alanine was substituted for glutamateat positions 7 and 8–10 (E7A and E8–10A, respec-tively) were used (Table 1). As the residual crosslinkingof ApoTf to125I-labeled Ggly(2–17) in the presence of100 lm unlabeled Ggly was less than 35% of the valuein its absence, Ggly mutants were also tested at thisconcentration. Mutant E7A significantly competedwith radiolabeled Ggly for the binding to ApoTf(66.5% relative density; P < 0.001), although theextent of competition was significantly less than withthe parental Ggly peptide (Fig. 3A). The triple mutant,E8–10A, did not compete with Ggly for ApoTf bind-ing. Thus, the lack of interaction between ApoTf andthe E8–10A peptide suggests that either some or all ofGlu8, Glu9 and Glu10 are involved in the interactionwith ApoTf. Alternatively, these results could indicatethat the ferric ion bound to Glu8 and Glu9 itself bindsto transferrin.To determine whether the N-terminus or C-terminusof Ggly is also required for the interaction betweenGgly and ApoTf, short N-terminal and C-terminalfragments of Ggly with or without the polyglutamateregion (Table 1) were included as unlabeled competi-tors in the crosslinking experiments (Fig. 3B).Although the peptide Ggly(1–11) did not interact withApoTf, the fragment Ggly(5–18), which contains boththe glutamate region and the C-terminal portion, inter-acted with ApoTf with similar potency (30.5% relativedensity, P < 0.05) to the parental Ggly peptide(36.6% relative density, P < 0.05). However, the pep-tide Ggly(12–18), with the C-terminal portion alone(i.e. lacking the pentaglutamate sequence), did notinteract with ApoTf. Thus, neither the pentaglutamatesequence nor the C-terminal portion is alone sufficientfor interaction with ApoTf to occur.Mutation of the N-terminal or C-terminaliron-binding sites of transferrin does notprevent interaction with GglyN-lobe and C-lobe transferrin mutants were used toinvestigate the effect of loss of either iron-binding siteon the affinity of transferrin for Ggly (Fig. 4). Thetransferrin mutants contained mutations that com-pletely disrupted iron binding to either the N-lobe(Mono C, Y95F ⁄ Y188F) or the C-lobe (Mono N,Y426F ⁄ Y517F), and hence each bound only one ferricion [19]. The affinity of full-length recombinant ApoTffor Ggly (31 ± 1 lm) (Fig. 4A) was nearly identical tothe affinity of commercially available ApoTf(39 ± 1 lm) (Fig. 1C). Although the two transferrinmutants (Mono N and Mono C) each bound Ggly,and the intensity of the radioactive crosslinked bandwas not significantly different in either case from thatTable 1. Gastrin peptides used for the crosslinking studies. Thepentaglutamate sequence of gastrins is shown in bold. Amino acidsthat differ from the naturally occurring sequence are underlined.Peptide Amino acid sequence1 6 10 18Gamide ZGPWLEEEEEAYGWMDFNH2Ggly ZGPWLEEEEEAYGWMDFGOHGgly(1–11) ZGPWLEEEEEAOHGgly(12–18) YGWMDFGOHGgly(5–18) LEEEEEAYGWMDFGOHGglyE7A ZGPWLEAEEEAYGWMDFGOHGglyE8–10A ZGPWLEEAAAAYGWMDFGOH–100 0 100 200 300 400 500 600 700 800Time (s) –40 –20 0 –200 ApoTf + EDTA –200 –100 0 100 200 300 400 500 600 700 800 EDTA Time (s) –40 –20 0 20 40 60 80 ApoTf Response differential (RU) Response differential (RU) Gamide Gamide GglyGglyA B Fig. 2. Ferric ions are important for both the formation and stabilityof the gastrin–ApoTf complex. (A) Injection of the iron chelatorETDA (3 mM) into the BIAcore channel at the same time as ApoTfprevented the association between the ApoTf and either Ggly (blueline) or Gamide (red line). (B) Following injection of ApoTf into theBIAcore channel, a complex was formed between ApoTf and Ggly(blue line) or Gamide (red line). After addition of the iron chelatorEDTA to the flow buffer, the gastrin–ApoTf complexes dissociated.S. Kovac et al. The interaction between Ggly and transferrinFEBS Journal 276 (2009) 4866–4874 ª 2009 The Authors Journal compilation ª 2009 FEBS 4869observed for ApoTf, the affinity in each case was lowerthan the affinity of wild-type ApoTf for Ggly (Fig. 4B,C). The IC50values for the interaction between Gglyand the Mono N and Mono C transferrins were96 ± 1 lm and 64 ± 1 lm, respectively.DiscussionThe in vitro formation of a complex between Gamideand ApoTf was first demonstrated over 20 years ago[14,15]. Although evidence was obtained for acomplex between two molecules of Gamide andApoTf, no association was observed between Gamideand iron-loaded transferrin (HoloTf). Our observationthat the iron saturation of serum transferrin was cor-related with circulating Gamide concentrations inboth mice and humans strongly suggested that theinteraction between Gamide and transferrin is physio-logically relevant. Thus, serum transferrin saturationwas reduced in gastrin-deficient mice at 4 weeks, andwas increased in hypergastrinemic cholecystokinin 2receptor-deficient mice at 4 weeks. Similarly, inpatients with multiple endocrine neoplasia type 1,approximately 40% of whom develop hypergastrin-emia, there was a significant correlation betweenserum transferrin saturation and serum Gamide con-centrations [17]. On the basis of these data, we sug-gested a mechanism, based on the well-known factthat efficient loading of ApoTf requires an anion(such as bicarbonate) or an anionic chelator (such asnitrilotriacetate), to explain the correlation betweencirculating Gamide concentrations and serum trans-ferrin saturation. The model proposed that, followingexport of ferrous ions from the enterocyte by ferro-portin and their oxidation to ferric ions by hephaes-tin, circulating Gamide or Ggly might act aschaperones for the uptake of ferric ions by ApoTf.The failure to detect significant binding of Gamide todiferric transferrin [14,15] suggested that Gamidedissociates after iron transfer has occurred, and henceRelative density (%)020406080100120140160180**–Total proteinCrosslinked proteinApo-Tf incubated with:–GglyGgly E7AGgly E8–10AGglyGgly E7AGgly E8–10ATotal proteinCrosslinked proteinApo-Tf incubated with:–GglyGgly 1–11Ggly 5–18Ggly 12–18G-glyGgly 1–11Ggly 5–18Ggly 12–18Relative density (%)050100150200**–**ABFig. 3. Both Glu8–Glu10 and the C-terminal portion of the Gglypeptide are important for the interaction between Ggly and ApoTf.(A) Binding of Glu fi Ala mutants of Ggly to ApoTf was assessedby competition with radiolabeled Ggly(2–17) in a covalent crosslink-ing assay. A representative analysis of the interaction betweenApoTf and Ggly glutamate mutants (100 lM) by SDS ⁄ PAGE isshown, followed by densitometric quantification of the data.Mutant E7A (coarse-hatched bar) significantly competed with radio-labeled Ggly(2–17) for binding to ApoTf [66.5% of control (gray bar)with no unlabeled peptide; ***P < 0.001], although with reducedpotency as compared with the parental Ggly peptide (fine hatchedbar). The triple mutant E8–10A (cross-hatched bar) did not competewith Ggly for ApoTf binding. (B) Short N-terminal and C-terminalfragments of Ggly with or without the polyglutamate region wereused to determine whether the N-terminus or C-terminus of Ggly isrequired for the interaction between Ggly and ApoTf. A typical anal-ysis of the interaction between ApoTf and Ggly fragments (100 lM)by SDS ⁄ PAGE is shown, followed by densitometric quantificationof the data. Ggly(1–11) (medium-hatched bar) did not interact withApoTf, whereas Ggly(5–18) (coarse-hatched bar), which containsboth the glutamate region and the C-terminal portion, interactedwith ApoTf with greater potency [30% of control (gray bar) with nounlabeled peptide, *P < 0.05] than the parental Ggly peptide (fine-hatched bar). Peptide Ggly(12–18) (cross-hatched bar), which lacksthe polyglutamate region, did not interact with ApoTf. Data aremeans ± SEM, where n =3.The interaction between Ggly and transferrin S. Kovac et al.4870 FEBS Journal 276 (2009) 4866–4874 ª 2009 The Authors Journal compilation ª 2009 FEBSplays a catalytic role consistent with the difference inthe circulating concentrations of Gamide and trans-ferrin. In the present study, we explored further theinteraction between Gamide and transferrin, andcharacterized the interaction between Ggly andtransferrin for the first time. Using two differentin vitro techniques, namely surface plasmon resonanceand covalent crosslinking, we observed that Ggly,like Gamide, only interacts with ApoTf (Fig. 1). Onthe basis of the facts that the signals observed oninteraction of Gamide and Ggly with ApoTf in thesurface plasmon resonance study were of similarmagnitude, and that Gamide and Ggly differ by asingle amino acid, it is very likely that two moleculesof Ggly will also bind to one molecule of ApoTf.Ggly has previously been reported to bind two ferricions, the first via Glu7, and the second via Glu8 andGlu9 [12]. In order to determine whether both of theseiron-binding sites are involved in the interaction withtransferrin, we used Ggly mutants in which the gluta-mates had been mutated to alanines (Table 1, Fig. 3).Analysis of the Ggly mutants revealed that the GglyE7A peptide still bound to ApoTf. Therefore, neitherGlu7 nor the first ferric ion is directly involved in theinteraction with ApoTf. Additionally, the first ferricion is unlikely to be transferred to ApoTf. The secondferric ion-binding site is formed by Glu8 and Glu9[12]. The observation that the Ggly E8–10A peptide nolonger bound to ApoTf in the crosslinking assays sug-gests either that binding to transferrin occurs throughone or more of Glu8, Glu9 and Glu10, or that thebinding of the second ferric ion to Glu8 and Glu9 iscrucial in the recognition of Ggly. Clearly, in the lattercase, the second ferric ion is likely to be involved inloading ApoTf.The role of the N-terminus and C-terminus of Gglyin the interaction with transferrin was investigated byApo-transferrin Mono N 0 20 40 60 80 100 120 140 160 180 200 220 –12.0 –6.0 –5.5 –5.0 –4.5 –4.0 –3.5 –12.0 –6.0 –5.5 –5.0 –4.5 –4.0 –3.5 Ggly concentration [log M] Ggly concentration [log M] Ggly concentration [log M] –12.0 –6.0 –5.5 –5.0 –4.5 –4.0 –3.5 Relative density (%)0 20 40 60 80 100 120 140 160 180 200 220 Relative density (%)Relative density (%)0 20 40 60 80 100 120 140 160 180 200 220 Mono C W T W T + G g l y M o n o N M o n o N + G g l y M o n o C MonoC + G g l y Relative density (%) 0 20 40 60 80 100 120 140 160 A B C D Fig. 4. Both the N-terminal and C-terminal lobes of transferrin caninteract with Ggly. (A) ApoTf and ApoTf mutants were crosslinkedto radiolabeled Ggly(2–17) in the presence or absence of 100 lMunlabelled Ggly, and the samples were separated by SDS ⁄ PAGE toremove the unbound radiolabel. The extent of crosslinking was notsignificantly different between recombinant wild-type ApoTf (WT),ApoTf that only binds iron in the N-lobe (Mono N), and ApoTf thatonly binds iron in the C-lobe (Mono C). Data are the means ± SEMfrom three independent experiments. (B) The interaction betweenGgly and recombinant wild-type ApoTf. The amount of radioactivityassociated with transferrin in the presence of increasing concentra-tions of unlabeled Ggly was determined by densitometric scanning,and was expressed as a percentage relative to sample with nounlabeled Ggly. The line of best fit was drawn with an IC50of31 ± 1 lM and an intercept of 101%. (C) The interaction betweenGgly and ApoTf that only binds iron in the N-lobe (Mono N). Theline of best fit was drawn with an IC50of 96 ± 1 lM and an inter-cept of 115%. (D) The interaction between Ggly and ApoTf thatonly binds iron in the C-lobe (Mono C). The line of best fit wasdrawn with an IC50of 64 ± 1 lM and an intercept of 134%.S. Kovac et al. The interaction between Ggly and transferrinFEBS Journal 276 (2009) 4866–4874 ª 2009 The Authors Journal compilation ª 2009 FEBS 4871crosslinking experiments (Fig. 3), using the Ggly frag-ments listed in Table 1. The fact that Ggly(1–11) didnot significantly inhibit the interaction of [125I]Gglywith transferrin suggested that the N-terminal domainof Ggly is not involved in the association with trans-ferrin. However, the observations that Ggly(5–18) wasas effective as Ggly as a competitor and that Ggly(12–18) was ineffective indicated that both the C-terminusof Ggly and the pentaglutamate sequence are criticalto the interaction with ApoTf. Thus, one or more ofthe seven C-terminal amino acids of Ggly is necessaryfor the formation of the complex.As it is well established that each lobe of transferrinbinds one ferric ion, the crosslinking analysis wasextended to transferrin mutants in which the iron-binding tyrosines in either the N-lobe or C-lobe hadbeen replaced by phenylalanines. This experimentallowed determination of whether or not the iron-binding residues in either lobe were required for theinteraction with Ggly. The affinity of Ggly for eachof the two authentic monoferric transferrins was simi-lar and only slightly weaker than the affinity forrecombinant wild-type ApoTf (which is capable ofbinding iron in both lobes) (Fig. 4). The simplestexplanation for this result is that there is no directinvolvement of the iron-binding residues in either lobein the interaction with Ggly. However, as each mole-cule of ApoTf binds two molecules of gastrin (pre-sumably with one molecule of gastrin bound to eachlobe), the possibility remained that mutation of theiron-binding residues did affect gastrin binding, andthat the observed binding was to the unmutated lobe.The observation that the extent of crosslinking wasthe same for Mono N and Mono C transferrin as forwild-type ApoTf (Fig. 4A) strongly suggests that bothmutant transferrins still bound two molecules of gas-trin, and hence that the first explanation was correct.Further studies showing the binding of gastrin to atransferrin with the iron-binding residues in bothlobes mutated, or to the individually expressed N-lobeor C-lobe with and without the iron-binding residuesmutated, would conclusively disprove the secondexplanation.Our data also provide some information on themechanisms of iron transfer from gastrin to transfer-rin. The fact that no interaction was observed betweenApoTf and either Gamide or Ggly in the presence ofEDTA (Fig. 2A) shows that gastrin peptides must bindferric ions in order to interact with ApoTf. Further-more, the preformed complex between ApoTf andeither Gamide or Ggly dissociates immediately uponaddition of EDTA (Fig. 2B). One attractive possibilityis that this dissociation is triggered by the transfer of aferric ion from one of the relatively low-affinity bind-ing sites on gastrin to one of the relatively high-affinitybinding sites on transferrin, as our data clearly indicatethat HoloTf does not bind gastrins (Fig. 1C). As dis-cussed above, the study with Ggly mutants supportsthe second iron-binding site on gastrin as the morelikely iron donor.In conclusion, the current work provides a muchbetter understanding of the complex formed betweengastrin peptides and ApoTf. Taken together, the dataare consistent with our hypothesis [17] that gastrinpeptides catalyze the loading of iron onto transferrin,and hence gastrins should be considered as part of therapidly expanding network of molecules that play arole in iron homeostasis. Moreover, the demonstrationof an interaction between Ggly and transferrin suggeststhat the stimulatory effects of Ggly and iron on thedevelopment of colorectal carcinoma may be linked,perhaps through a Ggly-dependent increase in transfer-rin saturation with a concomitant increase in the avail-ability of iron to the tumor cells.Experimental proceduresPeptidesGgly(2–17) was obtained from Mimotopes, and all othergastrin peptides and fragments (Table 1) were from AuspepPty. Ltd (Melbourne, Australia). All Ggly peptides wereused at 100 l m and were made up in dimethylsulfoxide.ApoTf was from Sigma–Aldrich (St Louis, MO, USA). Themutant Mono C transferrin, with the mutationsY95F ⁄ Y188F, the mutant Mono N transferrin, with themutations Y426F ⁄ Y517F, and full-length recombinanthuman transferrin were prepared as described previously[19].Iron removal from transferrinsPrior to crosslinking or surface plasmon resonance analy-sis, iron was removed from the transferrin mutants usinga previously reported procedure [20]. Briefly, solutions ofMono C and Mono N transferrin were placed in Centr-icon 10 microconcentrators (Millipore, North Ryde,Australia), together with 2 mL of buffer containing 0.5 msodium acetate (pH 4.9), 1 mm EDTA, and 1 mm nitrilo-triacetic acid. Sample volumes were reduced to 100 lLbycentrifugation at 5110 g for 2 h, during which periodthe characteristic salmon-pink color of iron-loaded trans-ferrin disappeared. The samples were subsequently washedonce with 2 mL of 100 mm KCl, once with 2 mL of100 mm sodium perchlorate, three times with 2 mL of100 mm KCl, and five times with 2 mL of 100 mmNH4HCO3.The interaction between Ggly and transferrin S. Kovac et al.4872 FEBS Journal 276 (2009) 4866–4874 ª 2009 The Authors Journal compilation ª 2009 FEBSLabeling of peptides with I125Ggly(2–17) (2 mgÆmL)1) was iodinated using the iodogenmethod, and the mono-iodinated peptide was separatedfrom di-iodinated and unlabeled peptide by RP-HPLC aspreviously described [14].CrosslinkingThe radiolabeled Ggly(2–17) was reacted with the bivalentcrosslinker disuccinimidyl suberate (0.6 mm), via the singleN-terminal amino group, in 50 mm Hepes buffer (pH 7.6)for 15 min at 4 °C. ApoTf (113 lgÆmL)1) was mixed withunlabeled Ggly, and the crosslinked125I-labeled Ggly(2–17)was added. In order to find the regions of Ggly necessaryfor transferrin interaction, Ggly mutants with alaninessubstituted for glutamates or short Ggly fragments wereused in the crosslinking experiments instead of the unla-beled Ggly. The reaction was stopped by addition ofreduced 2 · SDS loading dye, and the samples were boiledfor 5 min at 100 °C.SDS/PAGEThe ApoTf–Ggly complex (2 lg of protein) was separatedfrom unreacted Ggly by SDS ⁄ PAGE. Subsequently, the gelwas stained with Coomassie blue and destained overnightwith a solution containing 7% acetic acid, 5% methanol, and2% glycerol. The extent of incorporation of radioactivitywas determined by phosphoimager (FujiBAS 1800 II; Fuji-film, Melbourne, Australia) and densitometric analysis usingmultigauge software (Fujifilm). A reduction in intensity ofthe radioactive signal indicated binding of the unlabeledpeptide to ApoTf. Data are expressed as a percentage of thedensity observed with ApoTf and125I-labeled Ggly(2–17)only, after correction for variation in protein loading.Surface plasmon resonanceThe kinetics of transferrin binding to immobilized Gamideand Ggly were measured with a BIAcore 3000 biosensorinstrument (BIAcore, Uppsala, Sweden). Binding of trans-ferrin to immobilized peptides was measured in resonanceunits (RU) (1000 RU = 1 ng of protein bound per mm2offlow cell surface). The running buffer was Hanks’ balancedsalt buffer with no added iron salts, and the same bufferwas used for diluting samples before injection. Syntheticbiotinylated Gamide (biotin-QGPWLEEEEEAYGWMDFa-mide) and Ggly (biotin-QGPWLEEEEEAYGWMDFG)peptides were immobilized onto streptavidin-coated carbo-xymethylated dextran chips. To measure binding interac-tions, the transferrins, at a concentration of 10 lgÆmL)1,were passed over the immobilized peptides at a flow rate of20 lLÆmin)1at 25 °C. After each binding assay, flow cellswere regenerated by short pulses of 5 lL of 0.01% SDS.Statistical analysisStatistics were analyzed by Student’s t-test using the pro-gram sigmastat (Jandel Scientific, San Rafael, CA, USA).Values of the IC50were determined by fitting crosslinkingdata to the equation for one-site competitionf = min. + (max. – min.) ⁄ [1 + 10^ (x – logIC50)]and dose–inhibition curves were plotted using sigmaplot(Jandel Scientific). Data are presented as mean ± standarderror of the mean (SEM) from three separate experiments.AcknowledgementsThis work was supported by grant 5 RO1 GM065926from the National Institutes of Health (to G. Bald-win), grants 400062 (to G. Baldwin) and 566555 (to G.Baldwin) from the National Health and MedicalResearch Council of Australia, grant R01 (DK 21739)from the United States Public Health Service (to A. B.Mason), and grant CT8917 from Medical Researchand Technology in Victoria which is managed by ANZTrustees (to A. Ferrand).References1 Huggenvik JI, Craven CM, Idzerda RL, Bernstein S,Kaplan J & McKnight GS (1989) A splicing defect inthe mouse transferrin gene leads to congenital atransfer-rinemia. Blood 74, 482–486.2 Andrews NC (2000) Iron homeostasis: insights fromgenetics and animal models. Nat Rev Genet 1, 208–217.3 Bailey S, Evans RW, Garratt RC, Gorinsky B, HasnainS, Horsburgh C, Jhoti H, Lindley PF, Mydin A, SarraR et al. (1988) Molecular structure of serum transferrinat 3.3-A resolution. Biochemistry 27, 5804–5812.4 Wally J, Halbrooks PJ, Vonrhein C, Rould MA, EverseSJ, Mason AB & Buchanan SK (2006) The crystalstructure of iron-free human serum transferrin providesinsight into inter-lobe communication and receptorbinding. J Biol Chem 281, 24934–24944.5 Park I, Schaeffer E, Sidoli A, Baralle FE, Cohen GN& Zakin MM (1985) Organization of the humantransferrin gene: direct evidence that it originated bygene duplication. Proc Natl Acad Sci USA 82, 3149–3153.6 Baker HM, He QY, Briggs SK, Mason AB & BakerEN (2003) Structural and functional consequences ofbinding site mutations in transferrin: crystal structuresof the Asp63Glu and Arg124Ala mutants of theN-lobe of human transferrin. Biochemistry 42,7084–7089.7 Bali PW & Harris WR (1989) Cooperativity andheterogeneity between the two binding sites of diferrictransferrin during iron removal by pyrophosphate.J Am Chem Soc 111, 4457–4461.S. Kovac et al. The interaction between Ggly and transferrinFEBS Journal 276 (2009) 4866–4874 ª 2009 The Authors Journal compilation ª 2009 FEBS 48738 Chasteen ND, Grady JK, Woodworth RC & MasonAB (1994) Salt effects on the physical properties of thetransferrins. Adv Exp Med Biol 357, 45–52.9 Williams J & Moreton K (1980) The distribution ofiron between the metal-binding sites of transferrinhuman serum. Biochem J 185, 483–488.10 Aly A, Shulkes A & Baldwin GS (2004) Gastrins, chole-cystokinins and gastrointestinal cancer. Biochim BiophysActa 1704, 1–10.11 Baldwin GS, Curtain CC & Sawyer WH (2001)Selective, high-affinity binding of ferric ions byglycine-extended gastrin(17). Biochemistry 40,10741–10746.12 Pannequin J, Barnham KJ, Hollande F, Shulkes A,Norton RS & Baldwin GS (2002) Ferric ions areessential for the biological activity of the hormoneglycine-extended gastrin. J Biol Chem 277,48602–48609.13 Pannequin J, Tantiongco JP, Kovac S, Shulkes A &Baldwin GS (2004) Divergent roles for ferric ions in thebiological activity of amidated and non-amidatedgastrins. J Endocrinol 181, 315–325.14 Baldwin GS, Chandler R & Weinstock J (1986) Bindingof gastrin to gastric transferrin. FEBS Lett 205,147–149.15 Longano SC, Knesel J, Howlett GJ & Baldwin GS(1988) Interaction of gastrin with transferrin: effects offerric ions. Arch Biochem Biophys 263, 410–417.16 Smith KA, Kovac S, Anderson GJ, Shulkes A & Bald-win GS (2006) Circulating gastrin is increased in hemo-chromatosis. FEBS Lett 580, 6195–6198.17 Kovac S, Smith K, Anderson GJ, Burgess JR, ShulkesA & Baldwin GS (2008) Interrelationships betweencirculating gastrin and iron status in mice andhumans. Am J Physiol Gastrointest Liver Physiol 295,G855–G861.18 Friis-Hansen L, Rieneck K, Nilsson HO, Wadstrom T& Rehfeld JF (2006) Gastric inflammation, metaplasia,and tumor development in gastrin-deficient mice. Gas-troenterology 131, 246–258.19 Mason AB, Halbrooks PJ, James NG, Connolly SA,Larouche JR, Smith VC, MacGillivray RT & ChasteenND (2005) Mutational analysis of C-lobe ligands ofhuman serum transferrin: insights into the mechanismof iron release. Biochemistry 44, 8013–8021.20 He QY, Mason AB, Woodworth RC, Tam BM, Wads-worth T & MacGillivray RT (1997) Effects of muta-tions of aspartic acid 63 on the metal-binding propertiesof the recombinant N-lobe of human serum transferrin.Biochemistry 36, 5522–5528.The interaction between Ggly and transferrin S. Kovac et al.4874 FEBS Journal 276 (2009) 4866–4874 ª 2009 The Authors Journal compilation ª 2009 FEBS . Knowledge of the regions of transferrin required for the binding of gastrin, and of the regions in gastrin required for the interaction with transferrin, is. Definition of the residues required for the interaction between glycine-extended gastrin and transferrin in vitro Suzana Kovac1, Audrey Ferrand1,
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