Báo cáo khoa học: a-1 Antitrypsin binds preprohepcidin intracellularly and prohepcidin in the serum pdf

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:
(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 ( />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 (). 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.
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Supporting information
The following supplementary material is available:
Fig. S1. Bacterial expression of preprohepcidin.
Table S1. 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