a-1 Antitrypsin binds preprohepcidin intracellularly and prohepcidin in the serum Edina Pandur 1 , Judit Nagy 1,2 , Viktor S. Poo ´ r 1,2 ,A ´ kos Sarnyai 2 , Andra ´ s Husza ´ r 1 , Attila Miseta 2 and Katalin Sipos 1 1 Department of Forensic Medicine, University of Pe ´ cs, Hungary 2 Institute of Laboratory Medicine, University of Pe ´ cs, Hungary Hepcidin is the only hormone directly involved in iron regulation. It is synthesized as an 84-amino-acid (AA) preprohormone, and is present in the plasma as a mature 25-AA peptide and as a 60-AA prohormone form. Maturation is facilitated by the serine peptidase furin. The aim of this study was to determine whether prepro- and prohormones show significant interactions with proteins, which may affect the maturation of the hormone in the cell and its cleavage to active hormone in blood. Iron is one of the essential trace elements in living organisms. In vertebrates, the plasma iron level is in the micromolar range and circulating iron is associ- ated predominantly with the transport protein trans- ferrin. The blood iron level and the saturation of transferrin are frequently used indicators of the body Keywords blood serum; cellular transport; hepcidin; iron transport; a-1 antitrypsin Correspondence K. Sipos, Department of Forensic Medicine, University of Pe ´ cs, 12 Szigeti u ´ t, Pe ´ cs H-7624, Hungary Fax: +36 72 536 242 Tel: +36 72 536 230 E-mail: katalin.sipos@aok.pte.hu (Received 13 November 2008, revised 22 January 2009, accepted 26 January 2009) doi:10.1111/j.1742-4658.2009.06937.x Recent discoveries have indicated that the hormone hepcidin plays a major role in the control of iron homeostasis. Hepcidin regulates the iron level in the blood through the interaction with ferroportin, an iron exporter molecule, causing its internalization and degradation. As a result, hepcidin increases cellular iron sequestration, and decreases the iron concentration in the plasma. Only mature hepcidin (result of the cleavage of prohepcidin by furin proteases) has biological activity; however, prohepcidin, the prohormone form, is also present in the plasma. In this study, we aimed to identify new protein–protein interactions of preprohepcidin, prohepcidin and hepcidin using the BacterioMatch two-hybrid system. Screening assays were carried out on a human liver cDNA library. Preprohepcidin screening gave the following results: a-1 antitrypsin, transthyretin and a-1-acid glycoprotein showed strong interactions with preprohepcidin. We further confirmed and examined the a-1 antitrypsin binding in vitro (glutathione S-transferase, pull down, coimmunoprecipitation, MALDI-TOF) and in vivo (ELISA, cross-linking assay). Our results demonstrated that the serine protease inhibitor a-1 antitrypsin binds preprohepcidin within the cell during maturation. Furthermore, a-1 antitrypsin binds prohepcidin significantly in the plasma. This observation may explain the presence of prohormone in the circulation, as well as the post-translational regulation of the mature hormone level in the blood. In addition, the lack of cleavage protection in patients with a-1 antitrypsin deficiency may be the reason for the disturbance in their iron homeostasis. Abbreviations A1AT, a-1 antitrypsin; AA, amino acid; CTCK, carbenicillin–tetracycline–chloramphenicol–kanamycin; CTKXi, chloramphenicol–tetracycline– kanamycin–Gal-X–b-galactosidase inhibitor; DSS, disuccinimidyl suberate; Gal-X, 5-bromo-4-chloroindol-3-yl-b- D-galactoside; GST, glutathione S-transferase; pBT, bait plasmid; pTRG, target plasmid. 2012 FEBS Journal 276 (2009) 2012–2021 ª 2009 The Authors Journal compilation ª 2009 FEBS iron status [1,2]. Both iron deficiency and iron over- load are potentially dangerous conditions, which may cause anaemia, enzyme dysfunctions or degenerative liver, spleen and kidney diseases [3–6]. The most important organs and tissues involved in the regula- tion of iron stores are the liver, placenta, intestine and macrophages [7,8]. Recent findings have indicated that the hormone hepcidin plays a major role in controlling iron homeostasis [9–11]. This peptide is synthesized in the liver as an 84-AA preprohormone [12,13], and is targeted to the secretion pathway by a 24-AA N-terminal targeting sequence. The resulting 60-AA prohepcidin is processed further into a mature C-terminal 25-AA active peptide. The maturation is facilitated by the serine protease furin (Fig. 1) [14,15]. Furin belongs to the prohormone convertase family, which recognizes the consensus sequence R(X ⁄ R ⁄ K) (X ⁄ R ⁄ K)R [16]. Hepcidin regulates the iron level in the blood through its interaction with ferroportin, an iron expor- ter molecule. Ferroportin is expressed in hepatocytes, duodenal enterocytes and macrophages [17]. After binding hepcidin, ferroportin is internalized, phosphor- ylated, ubiquitinated and degraded by hepatocytes and macrophages. [18,19]. The action of hepcidin is differ- ent in intestinal cells: instead of ferroportin degrada- tion, the hormone causes the reduction of DMT1 (divalent metal ion transporter 1) expression [20]. As a result, hepcidin increases cellular iron sequestration in hepatocytes and macrophages, and reduces the iron level in the plasma. The known signals for the induc- tion of hepcidin synthesis are the elevation of the plasma iron level, inflammation and bacterial invasions [21–25]. To date, the only proven interaction of hepcidin is with the iron exporter molecule ferroportin. We were interested in whether we could identify new protein– protein interactions of preprohepcidin, prohepcidin and hepcidin in vivo. For these experiments, we used the BacterioMatch system, a two-hybrid screening assay system developed in bacteria. The most consis- tent and strongest interaction occurred with the serine protease inhibitor a-1 antitrypsin (A1AT). This associ- ation was further tested by both in vivo and in vitro methods to evaluate its significance. Results In vivo interactions of preprohepcidin and hepcidin with hepatocyte proteins The reporter strain of the BacterioMatch two-hybrid system harbours two reporter genes: lacZ and carbeni- cillin resistance genes. These genes are transcribed by RNA polymerase if the bait and target proteins, which are expressed by the bait plasmid (pBT) and target plasmid (pTRG), interact. In the case of transcrip- tional activation, bacterial colonies will be blue on 5-bromo-4-chloroindol-3-yl-b-d-galactoside (Gal-X) indicator plates, and will show a similar growth rate to positive control on carbenicillin–tetracycline–chloram- phenicol–kanamycin (CTCK) plates in the presence of carbenicillin. First, the screening of protein interactions of preprohepcidin as bait and human liver cDNA library as target was carried out on Luria–Bertani (LB) agar plates in the presence of Gal-X. In cases of protein–protein interactions, dark blue colonies appeared, which were restreaked onto plates with 250 or 500 lgÆmL )1 carbenicillin. Plasmids from bacterial colonies growing at high carbenicillin concentration were isolated and cotransformed repeatedly into the reporter strain to confirm the association between proteins. After the second cotransformation, plasmids were isolated and the cDNA insert of pTRG was sequenced. (Screening with the liver cDNA library was repeated: consistently interacting entities were further studied.) The results of the BacterioMatch screening are shown in Table 1. Preprohepcidin exhibited binding to transthyretin (or prealbumin), a serum protein known as a thyroid hormone carrier molecule. We also found the associa- tion of preprohepcidin with a-1 acid protein (orosomu- coid), a major plasma protein with unknown function. The level of this protein is elevated in the blood in the case of inflammation, and it is used as a diagnostic marker in inflammatory diseases (acute phase protein). The strongest association of preprohepcidin proved to be with A1AT, a member of the serine protease inhibitor (serpin) family. A1AT was ‘fished out’ at the screenings more times than any other interacting protein (one-third of all sequenced cDNA clones), indicating Fig. 1. Structure and maturation of preprohepcidin. The first 24 AAs serve as a signal sequence for secretion. To generate the mature 25-AA hepcidin peptide, there is a furin cleavage site in the C-terminal part of prohepcidin. E. Pandur et al. In vivo interactions of preprohepcidin and prohepcidin FEBS Journal 276 (2009) 2012–2021 ª 2009 The Authors Journal compilation ª 2009 FEBS 2013 a consistent and potentially relevant interaction with preprohepcidin. However, a more abundant represen- tation of A1AT clones, when compared with other positive clones, cannot be excluded. The strong binding between preprohepcidin and A1AT was confirmed when the latter was cloned into pTRG, and cotransformed with preprohepcidin expressing pBT into BacterioMatch competent cells. These cells were able to grow on CTCK plates in the presence of 500 lgÆmL )1 carbenicillin con- centration. As furin, a serine protease involved in the maturation of hepcidin, is also inhibited by A1AT, we considered this as a potentially important observation. This cotransformation was repeated with the same protease inhibitor expressed in pTRG, and either the 60-AA prohepcidin (without the targeting sequence) or the 25-AA-containing mature hepcidin cloned into pBT. We detected the growth of the cotransformed BacterioMatch strain on carbenicillin indicator (CTCK) plate in the case of prohepcidin (60 AA), but not with mature hepcidin. We found that the protease inhibitor molecule binds selectively to the preprohor- mone and prohormone, but not to the processed hepci- din or to the targeting sequence of preprohepcidin (84 AA) (Table 2). There were other proteins (cytochrome P450, ATP ⁄ ADP translocase, enoyl-CoA hydratase) which gave weak interactions with preprohepcidin. Alignment of the coding regions of these proteins did not show significant similarities. Nor could we identify common structural domains that may provide further clues to preprohepcidin binding. BacterioMatch screening carried out with the mature 25-AA peptide resulted in significantly fewer positive clones when compared with the screening with the preprohormone. None of these proteins was identical with the screening results of the 84-AA peptide. The only strong and consistent interaction of the mature peptide was with membrane protein CD74. Further experiments are needed to evaluate this finding. In vitro pull-down assay of preprohepcidin and A1AT Both preprohepcidin and A1AT were cloned into inducible plasmids and expressed in bacteria. Preprohepcidin carried a glutathione S-transferase (GST) fusion tag for attachment to an affinity purifica- tion column. This column was used to pull down expressed A1AT from bacterial lysate or human serum. The interaction of A1AT with preprohepcidin was verified by the elution of protein complexes from the column, followed by western blotting developed with anti-A1AT IgG. The in vitro binding of the two molecules appeared to be specific, as GST-carrying affinity columns produced only negligible quantities of A1AT tethering (Fig. 2). Hepcidin expression causes parallel alterations in A1AT mRNA levels Next, we studied the influence of the overexpression or downregulation of preprohepcidin on the A1AT mRNA level. We transfected WRL68 cells with prep- rohepcidin ⁄ pTriex3-Neo plasmid and were able to demonstrate a 470-fold increase in the copy number of preprohepcidin mRNA by real-time quantitative PCR. Using antisense RNA, we reduced the preprohepcidin mRNA level to 63% (Fig. 3A). The same samples were processed for A1AT mRNA level measurement. We found that the A1AT mRNA level increased by more than two-fold when preprohepcidin was overexpres- sed. Even more significantly, the 37% decrease in preprohepcidin expression caused by antisense RNA Table 2. In vivo interactions of a-1 antitrypsin with preprohepcidin, prohepcidin and mature hepcidin. Insert in pBT Insert in pTRG Growth on CTCK plates a Preprohepcidin a-1 Antitrypsin +++ Prohepcidin a-1 Antitrypsin +++ Hepcidin a-1 Antitrypsin ) a Colony growth was classified as follows: ), no growth; +++, strong growth. Table 1. In vivo protein interactions of the 84-AA preprohepcidin using the BacterioMatch two-hybrid system. Target protein Swiss-Prot no. Function Localization Transthyretin P02766 Thyroid hormone binding Secreted to plasma a-1 Acid protein P02763 Acute-phase protein Secreted to plasma a-1 Antitrypsin P01009 Serine protease inhibitor Secreted to plasma Cytochrome P450 P05181 Drug metabolism Membrane protein ATP ⁄ ADP translocase P12235 ATP–ADP exchange Mitochondrion Enoyl-CoA hydratase P30084 Fatty acid oxidation Mitochondrion In vivo interactions of preprohepcidin and prohepcidin E. Pandur et al. 2014 FEBS Journal 276 (2009) 2012–2021 ª 2009 The Authors Journal compilation ª 2009 FEBS coincided with a nearly fourfold reduction of A1AT mRNA. (Fig. 3B) These data suggest a regulatory link between the preprohormone and antiprotease expres- sion, underlining a physiologically important relation- ship between the hormone and A1AT. In vivo cross-linking of preprohepcidin and A1AT in cell culture We wished to confirm whether preprohepcidin interacts with A1AT in vivo within the cells before secretion. For this experiment, we overexpressed prep- rohepcidin in cultured hepatic cells. Huh7 cells were transfected with His-tagged preprohepcidin for 24 h and then treated with cross-linking reagent (disuccin- imidyl suberate, DSS). Preprohepcidin and cross-linked proteins were affinity purified with NiNTA agarose beads which bind His-tag specifically. After washing the beads, protein complexes were eluted with Laemmli buffer and probed with A1AT antibody. The purified His-tagged preprohepcidin gave a clear reaction with anti-A1AT, illustrating effective cross-linking between the two molecules, but there was no signal after control pTriex3-Neo plasmid transfection (Fig. 4). Prohepcidin binds to A1AT in the serum Next, we studied the interaction of prohepcidin and plasma A1AT in the circulation. We carried out ultra- filtration assays with sera collected from presumably healthy volunteers. After measuring the prohepcidin level with ELISA, the serum was filtered through a 30 kDa cut-off membrane and the prohepcidin level was determined in the filtrate (first ultrafiltrate). Prohepcidin itself did not bind to the filter of the Microcon tube, and A1AT did not appear in the serum ultrafiltrate (data not shown). We found that the serum prohepcidin level was 210 lgÆL )1 , whereas the first Fig. 3. Changes in mRNA levels of preprohepcidin and A1AT caused by preprohepcidin overexpression or preprohepcidin silencing with antisense technique in cultured WRL68 cells. mRNA levels were determined by a real-time PCR method, and expression ratios were calculated using b-actin as reference gene. Values represent the mean ± standard error of the mean (SEM) of three independent experiments. (A) Preprohepcidin mRNA levels follow- ing the two different treatments of cell cultures. (B) A1AT mRNA levels displayed parallel changes to the amount of preprohepcidin mRNA. *P < 0.01 versus untreated cells. Fig. 4. Cross-linking of A1AT with preprohepcidin. Cultured Huh7 cells were transfected with His-tagged preprohepcidin-expressing plasmid and then treated with the cross-linker DSS. Protein complexes were purified on NiNTA agarose beads, and western blots were probed with anti-A1AT IgG. (A) Cells were transfected with pTriex3-Neo plasmid. (B) Transfection of cultured cells was carried out with preprohepcidin ⁄ pTriex3-Neo plasmid DNA. AB Fig. 2. Preprohepcidin–A1AT in vitro binding (pull-down) assay. Glutathione–Sepharose 4B beads were employed to purify expressed GST or GST–preprohepcidin fusion protein from cell lysates. Protein complexes were eluted from the beads and western blotting analyses were carried out with anti-A1AT IgG. (A) A1AT expressing BL21 total lysate was used as positive control (a). Interac- tion of A1AT expressing BL21 lysate and GST-coated Glutathione– Sepharose beads served as negative control (b). Pull-down assay with A1AT expressing BL21 cell lysate and GST–preprohepcidin bound to Glutathione–Sepharose beads (c). (B) Interaction of human serum and GST-coated Glutathione–Sepharose beads used as nega- tive control (a). Pull-down assay with human serum and Glutathione– Sepharose beads carrying GST–preprohepcidin (b). E. Pandur et al. In vivo interactions of preprohepcidin and prohepcidin FEBS Journal 276 (2009) 2012–2021 ª 2009 The Authors Journal compilation ª 2009 FEBS 2015 filtrate contained 71.7 lgÆL )1 (34% of the total) (Fig. 5). Although these data prove that normally more than 60% of the total prohepcidin is bound to serum proteins larger than 30 kDa, no evidence could be found that A1AT binds prohepcidin significantly. To demonstrate the capability for binding ‘free’ (filterable) prohepcidin to A1AT, the above experiment was repeated after the addition of 1.5 gÆL )1 A1AT to the first serum ultrafiltrate. The prohepcidin concentra- tion in the second ultrafiltrate was further reduced to 46.6 lgÆL )1 (22% of the total), or to 65% of the first filtrate (Fig. 5). To reveal the specificity of the preceding binding reaction, we performed coimmunoprecipitation assays. We attached A1AT antibody to a column of CNBr- activated Sepharose beads, and incubated this with serum. Sepharose beads were washed and A1AT-asso- ciated proteins were eluted with Laemmli buffer. Next, we probed the eluent with anti-hepcidin IgG. Results of the dot blot displayed strong positive signals, indi- cating that A1AT and prohepcidin associated in vivo in the serum. Ultrafiltrated ‘free’ prohepcidin by itself gave no binding to the activated Sepharose beads (Fig. 6). Similar affinity purification was carried out using the ZipTip method, in which A1AT antibody was attached to the C18 column of ZipTip and incubated with serum, as in the previous experiment. The eluted sample was analysed on a MALDI-TOF mass spectrometer. The spectrum was compared with that obtained in the case of bacterially expressed His-tagged prohepcidin with a molecular weight of 7760.08 Da. In the latter case, two major peaks appeared in the spectrum, at m ⁄ z 1410.96 and 6349.12. The peak at m ⁄ z 1410.96 corresponds to a fragment of 6· His and 5 AA from the C-terminal end of prohepcidin (MCCKTHHHHHH) (Fig. 7A). The affinity-purified prohepcidin from serum gave the same m ⁄ z 6349.14 peak as above, suggesting a similar fragmentation of the prohormone (Fig. 7B). In this experiment, the C-terminal 5-AA (MCCKT) fragment does not appear, as detection was performed between m⁄ z 1000 and 7500 to exclude matrix peaks in the low mass ranges. Not only does this affinity purification assay reveal that A1AT binds prohepcidin, but it also confirms that the whole prohepcidin molecule is involved in the reaction. Discussion Hepcidin is a novel peptide hormone which is synthe- sized by the liver [26]. This hormone, unusually, has two major functions in humans: it regulates iron metabolism of the body and fights against microbial invasions [27–30]. It has been proven that it is produced as an 84-AA preprohepcidin, targeted to the secretory pathway, and cleaved into a 25-AA mature peptide by furin [14]. We used a bacterial two-hybrid assay system, BacterioMatch, to identify interactions of preprohepcidin with human liver-expressed proteins. Our results demonstrate that the serpin peptidase inhibitor A1AT robustly interacts with preprohepcidin, as well as with prohepcidin, but not with mature hepcidin. This finding indicates that A1AT may pro- tect prohepcidin from cleavage by furin, a serine prote- ase, which is responsible for the maturation of the hormone. Indeed, data in the literature show that the inherited mutations of A1AT are associated with increased iron accumulation and liver disease [31]. One of the effects of A1AT modifications is hyperferritina- emia [32]. A possible explanation is that the mutated protease inhibitor does not protect prohepcidin sufficiently. Consequently, more mature hepcidin is produced, which binds to ferroportin, causing Prohepcidin (ng·mL –1 ) Fig. 5. Serum ultrafiltration assay. Human serum with a known A1AT level was centrifuged in a Microcon YM-30 tube. The ultrafiltrate (first ultrafiltrate) was incubated with additional 1.5 gÆ L )1 A1AT and centrifuged again (second ultrafiltrate). Prohepcidin levels of the original serum, first and second ultrafiltrates were deter- mined with the Hepcidin Prohormone ELISA kit. Values are displayed as means ± standard error of the mean (SEM) of three different experiments. *P < 0.01 versus serum; **P < 0.01 versus first ultrafiltrate. A C B Fig. 6. Identification of A1AT–prohepcidin binding with coimmuno- precipitation. Anti-A1AT IgG was coupled to CNBr-activated Sepha- rose 4B beads and utilized for the purification of A1AT-associated protein complexes from serum. Eluted proteins were analysed by dot blotting with the application of anti-hepcidin IgG. (A) Serum ultrafiltrate with ‘free’ (unbound) prohormone was incubated with Sepharose beads in the same way as described above. This was used as a negative control for the experiment. (B) Synthetic mature hepcidin peptide was the positive control for the anti-hepcidin IgG. (C) Coimmunoprecipitation result with human serum. In vivo interactions of preprohepcidin and prohepcidin E. Pandur et al. 2016 FEBS Journal 276 (2009) 2012–2021 ª 2009 The Authors Journal compilation ª 2009 FEBS intracellular degradation of the iron exporter. The result will be iron overload in the reticuloendothelial system and parenchymal tissues, with a simultaneous elevation in the serum ferritin level. The prohepcidin ELISA kit has been tested previ- ously in different diseases, but few correlations have been found between serum prohepcidin levels and clinical laboratory parameters [13,33–36]. The pres- ence of prohepcidin in the circulation is proof in itself that prohepcidin is effectively protected to some extent against proteolytic cleavage. It seems possible that the antibody in the ELISA kit is not always able to react with the prohormone because it is covered by the protease inhibitor. In the case of inflammation, the blood level of the active 25-AA hepcidin increases very rapidly, even before mRNA synthesis is activated [37,38]. Also, in different pathological conditions, the serum contains similar amounts of prohepcidin, but different concentrations of hepcidin [39]. The possible reasons for this are the elevated protease activity and ⁄ or the difference in protection of the prohepcidin molecule by protease inhibitor. It is known that A1AT is increased early in inflammation. Its main function is to inhibit elastase released from granulo- cytes. Consequently, the availability of A1AT for prohepcidin may actually decrease in acute inflamma- tion. Further studies are needed to substantiate this hypothesis. Additional significant interactions of preprohepcidin involve the a-1 acid protein and transthyretin. The former is a major plasma protein, but its physiological functions have not yet been elucidated; the latter is a thyroid-binding transfer protein. The blood level of a-1 acid protein is elevated in different conditions associated with acute and chronic inflammation [40]. Chronic inflammation is frequently associated with tissue iron overload, as well as with anaemia [5,41,42]. Weaker interactions of the preprohormone of unknown relevance were also found with some intra- cellular proteins. Fig. 7. ZipTip affinity purification and mass spectrometric analysis of A1AT-bound serum prohepcidin. ZipTip C18 with bound anti-A1AT was applied to purify the A1AT-coupled prohepcidin from human serum. The eluted samples were analysed using a MALDI-TOF mass spectrom- eter. (A) Bacterially expressed prohepcidin–His fusion protein was used as a prohepcidin standard in the mass spectrometric analysis. The molecular weight of prohepcidin–His was 7760.08 Da. The peaks at m ⁄ z 1410.96 and 6349.12 correspond to two fragments of the prohepcidin– His protein. The peak at m ⁄ z 1410.96 represents the 6· His and 5 AAs of the C-terminal end of prohepcidin (MCCKTHHHHHH). (B) Identification of the affinity-purified prohepcidin from serum. The peak at m ⁄ z 6349.14 demonstrates the same fragmentation of prohepcidin as described above. E. Pandur et al. In vivo interactions of preprohepcidin and prohepcidin FEBS Journal 276 (2009) 2012–2021 ª 2009 The Authors Journal compilation ª 2009 FEBS 2017 None of the proteins which contacted preprohepci- din interacted with the mature form of hepcidin in BacterioMatch screening. This further supports the possibility that these proteins may play a role in the protection of prohepcidin from the serine protease furin. Materials and methods Bacterial two-hybrid system The BacterioMatch Two-Hybrid System Vector Kit (Strata- gene, La Jolla, CA, USA) was used for protein interaction assays. The cDNA coding the 84-AA preprohepcidin was amplified and cloned into pBT of the kit with the restriction enzymes BamHI and XhoI. Tables S1 and S2 show the con- structs and primer sequences used in our experiments. The recombinant pBT was transformed into the Escherichia coli strain JM109, and plasmid was purified with a QIAprep Spin Miniprep Kit (Qiagen Inc., Hilden, Germany) according to the manufacturer’s protocol. The human liver cDNA library cloned into pTRG with the restriction sites EcoRI and XhoI was used for screening. Screening with human liver cDNA library The BacterioMatch Two-Hybrid System Reporter strain cells (Stratagene) were cotransformed via electroporation with 0.5 lg of preprohepcidin expressing pBT and 1 lLof 1 : 10 diluted plasmid cDNA library (already amplified according to the manufacturer’s protocol). The electropo- ration was carried out with a Gene Pulser Xcell Electro- poration System (Bio-Rad, Hercules, CA, USA) using a pre-set bacterial electroporation protocol in 1 mm gap electroporation cuvettes. After electroporation at 1.8 kW, bacteria were immediately resuspended in ice-cold LB medium and grown at 30 °C for 1.5 h with shaking at 350 r.p.m. After incubation, bacteria were pelleted, resuspended in 200 lL of LB medium, plated on LB– chloramphenicol–tetracycline–kanamycin–Gal-X–b-galacto- sidase inhibitor (CTKXi) agar plates and incubated overnight at 30 °C. These indicator plates were supple- mented with chloramphenicol (34 lgÆmL )1 ), tetracycline (15lgÆmL )1 ), kanamycin (50 lgÆmL )1 ), Gal-X (80 lgÆ mL )1 ) and 0.2 mm b-galactosidase inhibitor (i). Plasmids pro- vided in the kit were used as positive control; recombinant pBT cotransformed with positive control pTRG+ was used as negative control. Colonies that appeared blue on Gal-X-containing indica- tor plates were streaked onto LB–CTCK agar plates (containing 250–500 lgÆmL )1 carbenicillin instead of Gal-X) for assay validation. The plates were incubated at 30 °C overnight and the growth rates of the colonies from screening were compared with the growth rates of controls. Protein–protein interaction validation Single colonies were taken from LB–CTCK agar plates and inoculated into 10 mL of LB supplemented with TCK. The cultures were incubated at 30 °C overnight with shaking at 140 r.p.m. Recombinant pBT and cDNA containing pTRG were isolated using a QIAprep Spin Miniprep Kit (Qiagen Inc.) and transformed into BacterioMatch Two-Hybrid System Reporter strain cells. The growth rates of the colo- nies were tested as before, first on LB–CTKXi indicator plates, and then on LB-CTCK plates with 250 lgÆmL )1 carbenicillin. Sequencing of target DNA The cDNAs cloned into pTRG from positive colonies (which were blue on the indicator plate and showed growth on the carbenicillin plate) were amplified by PCR with the following vector-specific primers: 5¢-CAGCCTGAAGTGA AAGAA-3¢ and 5¢-ATTCGTCGCCCGCCATAA-3¢. The PCR products were purified from agarose gel with a QIA- quick Gel Extraction Kit (Qiagen Inc.) and sequenced by the CEQ 8000 Dye Terminator Cycle Sequencing Chemistry Protocol (Beckman Coulter, Inc., Fullerton, CA, USA). The expressed protein was identified with the blastn program (http://www.ncbi.nih.gov/blast/Blast.cgi). Binding of A1AT to preprohepcidin, prohepcidin and mature hepcidin The cDNA of A1AT was cloned into pTRG with the restric- tion sites EcoRI and XhoI, and the coding cDNAs of prep- rohepcidin, prohepcidin and mature hepcidin were cloned into pBT with the restriction enzymes BamHI and XhoI. Recombinant pBT and pTRG were isolated using a QIAprep Spin Miniprep Kit (Qiagen Inc.), and BacterioMatch Two- Hybrid System Reporter strain cells were cotransformed via electroporation as described above. The growth rate of the colonies was tested first on LB-CTKXi indicator plates, and then on LB-CTCK plates with 500 lgÆmL )1 carbenicillin. GST fusion protein binding assay The preprohepcidin coding cDNA was cloned into pGex4T-1 (expression of preprohepcidin was demonstrated by western blot, applying anti-hepcidin IgG; Fig. S1) and A1AT was cloned into pET51b(+). The constructs were then trans- formed into E. coli BL21. GST or GST–preprohepcidin fusion protein was produced in E. coli BL21 after induction with 0.5 m m isopropyl thio- b -d-galactoside for 2 h at 30 °C. Cells were harvested by centrifugation and resuspended into STE (10 mm Tris ⁄ HCl, pH 8, 150 mm NaCl, 1 mm EDTA). The bacteria were lysed by mild sonication at 4 °CinSTE with a final concentration of 1.5% sarcosyl. The supernatant In vivo interactions of preprohepcidin and prohepcidin E. Pandur et al. 2018 FEBS Journal 276 (2009) 2012–2021 ª 2009 The Authors Journal compilation ª 2009 FEBS was gently mixed with STE-washed Glutathione–Sepha- rose 4B beads (Amersham Biosciences, Uppsala, Sweden) at 4 °C for 1 h. GST proteins bound to beads were collected by centrifugation at 3000 g, followed by three successive washes with STE. In vitro protein–protein interaction assay (GST pull-down) was carried out by incubating 30 lL of GST– preprohepcidin and GST beads with an equal volume of A1AT expressing BL21 lysate for 1 h in 5 mL of binding buf- fer (50 mm Tris ⁄ HCl, pH 8, 50 mm NaCl, 10% glycerol, 0.1% Triton X-100). After centrifugation, the beads were washed three times with binding buffer, resuspended in 30 lLof4· Laemmli buffer and centrifuged. The super- natant was loaded onto 8% SDS-PAGE and transferred by electroblotting to nitrocellulose membranes (Hybond C; Amersham Pharmacia Biotech, Uppsala, Sweden). The pro- tein–protein interaction was detected using anti-A1AT IgG (Dako, Glostrup, Denmark). The experiment was repeated with A1AT originating from human serum (400 lL). Real-time PCR WRL68 (HPACC, Salisbury, UK) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supple- mented with 10% fetal bovine serum. Cells grown in six- well dishes were transiently transfected with 1 lg per well of preprohepcidin ⁄ pTriex3-Neo or preprohepcidin anti- sense ⁄ pCDNA3.1 DNA using 2 lL of Transfectin reagent (BioRad, Hercules, CA, USA) for 24 h. Total RNA was isolated from a pellet of transfected cells using the RNeasy Mini Kit (Qiagen Inc.). First-strand cDNA was generated by reverse transcription of 1 lg of total RNA using a Tran- scriptor High Fidelity cDNA Synthesis Kit (Roche Diag- nostics, Meylan, France), according to the manufacturer’s instructions, in a total reaction volume of 20 lL. Reverse and forward oligonucleotide primers, specific to the chosen candidate and housekeeping genes, were designed using primer3 software (http://frodo.wi.mit.edu). The sequences for the reference gene were as follows: b-actin sense, 5¢-AG AAAATCTGGCACCACACC-3¢; antisense, 5¢-GGGGTG TTGAAGGTCTCAAA-3¢; preprohepcidin sense, 5¢-CAG CTGGATGCCCATGTT-3¢; antisense, 5¢-TGCAGCACAT CCCACACT-3¢; A1AT sense, 5¢-CCTATGATGAAGCGT TTAGG-3¢; antisense, 5¢-TATCGTGGGTGAGTTCATT T-3¢. Real-time PCR was performed in a LightCycler 2.0 (Roche Diagnostics) thermal cycler. Each reaction was performed in a 20 lL volume, using the Fast Start DNA MasterPLUS SYBR Green I master mix (Roche Diagnos- tics), with 200 nm final concentrations of each primer. Dissociation curves were generated after each quantitative PCR run to ensure that a single specific product was ampli- fied. Both target and reference genes were amplified with efficiencies near 100% and within 5% of each other. For the relative gene expression analysis, the 2 DDCt (Livak) method was used. The expression level of the gene of interest was compared with the level of b-actin in each sample. These relative expression rates were then compared between the treated and untreated samples. In vivo cross-linking Huh7 (HPACC) cells (10 7 ) were cultured in MEM supple- mented with 10% fetal bovine serum and transiently trans- fected with 30 lg preprohepcidin ⁄ pTriex-Neo3 (insert with C-terminal His-tag) for 24 h with Transfectin reagent (BioRad). In fresh medium, a specific cross-linker (DSS; Sigma-Aldrich Corporation, St Louis, MO, USA) was added to the cells in a 0.2 mm final concentration for 30 min at room temperature. The reaction was stopped with 50 mm Tris ⁄ HCl (pH 7.4). Cells were collected and washed twice with NaCl ⁄ P i and then lysed on ice for 1 h in 150 lL of lysis buffer (50 mm Hepes, pH 7.4, 150 mm NaCl, 1 mm MgCl 2 , 10% glycerol, 0.5% Triton X-100 with protease inhibitors). After incubation, the lysate was clarified with centrifugation and the His-tagged preprohepcidin was affin- ity purified on NiNTA agarose beads (Qiagen Inc.). Bound protein complexes were eluted with 30 lL Laemmli loading buffer, run on 8% SDS-PAGE, blotted onto nitrocellulose membrane and probed with anti-A1AT IgG. Serum ultrafiltration assay A1AT measurements from human serum samples were performed by turbidimetry (Cobas Integra 800 analyzer; Roche Diagnostics). Serum (200 lL) from healthy volunteers with a known A1AT content was centrifuged in a Microcon YM-30 (Millipore Corp., Bedford, MA, USA) filter unit (first ultrafiltrate). A1AT (Sigma-Aldrich Corporation) (150 lg) was added to 100 lL of ultrafiltrate and incubated for 15 min at 30 °C. It was then centrifuged again in a Microcon tube (second ultrafiltrate). The prohepcidin levels of the ori- ginal serum, the first serum ultrafiltrate and the second serum ultrafiltrate were determined with a Hepcidin Prohormone ELISA Kit (DRG International, Mountainside, NJ, USA) according to the manufacturer’s protocol. Coimmunoprecipitation Anti-A1AT IgG was coupled to CNBr-activated Sepha- rose 4B beads (Amersham Biosciences), according to the procedure recommended by the manufacturer. Serum was ultrafiltrated in Microcon YM-30, and 20 lL of the concen- trated serum was incubated with 25 lL of Sepharose beads in incubation buffer (100 mm Tris ⁄ HCl, pH 7.4, 100 mm NaCl, 0.1% Triton X-100, 10% glycerol) for 30 min at room temperature. The beads were washed six times with 1.2 mL of incubation buffer and then eluted with 20 lL4· Laemmli. The total eluted volume was dotted onto nitrocellulose membrane (Hybond C) and probed with anti-hepcidin IgG (Alpha Diagnostic, San Antonio, TX, USA). Synthetic E. Pandur et al. In vivo interactions of preprohepcidin and prohepcidin FEBS Journal 276 (2009) 2012–2021 ª 2009 The Authors Journal compilation ª 2009 FEBS 2019 hepcidin peptide (Sigma-Aldrich Corporation) was used as positive control for the western blot. Serum ultrafiltrate with unbound prohormone served as a negative control for the experiment. Mass spectrometry after ZipTip C18 affinity purification ZipTip (Millipore Corp.) was washed ten times with 20 lL of 50% acetonitrile, 0.1% trifluoroacetic acid and then incubated with 20 lL of anti-A1AT IgG. After incubation, the tip was washed three times with 10 lL of NaCl ⁄ P i (pH 7.4) and blocked with 20 lLof20mgÆmL )1 bovine serum albumin, and washed again with NaCl ⁄ P i . Human serum (15 lL) concentrated with Microcon YM-30 was pipetted up and down for 5 min. The proteins which did not bind to the column were eliminated with two NaCl ⁄ P i and Milli-Q water (produced by Milli-Q Element Ultrapure Water System; Millipore Corp.) washing steps. The elution was carried out with 3 lL of 1% trifluoroacetic acid. The sample was mixed with an equal volume of a-cyano- 4-hydroxycinnamic acid matrix, and 1 lL of the mix was dropped on a Bruker 384 ground steel plate. Mass spectrometric analysis was performed on a Bruker Autoflex II MALDI-TOF-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). The instrument uses a 337 nm pulsed nitrogen laser, model MNL-205MC (LTB Lasertechnik Berlin GmbH, Berlin, Germany). The ions were accelerated under delayed extraction conditions (90 ns) in positive ion mode. Data acquisition was performed in linear detector mode. Bruker flexcontrol 2.4 software was used for control of the instrument and Bruker flexanalysis 2.4 software for spectral evaluation. Acknowledgements We would like to thank Ilona Ga ´ bor, Gergely Montsko ´ and Attila M. Peti for their excellent technical assis- tance. Financial assistance was provided by grants from the Hungarian Fund (OTKA T-048793) to K. S. and Medical Research Council (ETT 401/2006) and National Office for Research and Technology (NKTH MEDIPOLISZ) to A. M. References 1 Ganz T & Nemeth E (2006) Regulation of iron acquisi- tion and iron distribution in mammals. 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Descriptions of constructs used in different experiments. Table S2. Sequences of primers used to generate constructs. This supplementary material can be found in the online version of this article. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. E. Pandur et al. In vivo interactions of preprohepcidin and prohepcidin FEBS Journal 276 (2009) 2012–2021 ª 2009 The Authors Journal compilation ª 2009 FEBS 2021 . a-1 Antitrypsin binds preprohepcidin intracellularly and prohepcidin in the serum Edina Pandur 1 , Judit Nagy 1,2 , Viktor. In vivo interactions of a-1 antitrypsin with preprohepcidin, prohepcidin and mature hepcidin. Insert in pBT Insert in pTRG Growth on CTCK plates a Preprohepcidin