We were interested in whether we could identify new protein– protein interactions of preprohepcidin, prohepcidin and hepcidin in vivo.. First, the screening of protein interactions of pr
Trang 1prohepcidin in the serum
Edina Pandur1, Judit Nagy1,2, Viktor S Poo´r1,2, A´ kos Sarnyai2
, Andra´s Husza´r1, Attila Miseta2 and Katalin Sipos1
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.
Trang 2iron 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 elevainduc-tion 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.
Trang 3a 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)1carbenicillin
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 prohorpreprohor-mone, but not to the processed
hepci-din or to the targeting sequence of preprohepcihepci-din
(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
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.
Trang 4coincided 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.
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).
Trang 5filtrate 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
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 gÆL)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.
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.
Trang 6intracellular 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.
Trang 7None of the proteins which contacted
preprohepci-din interacted with the mature form of hepcipreprohepci-din 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 lL of
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
resuspended in 200 lL of LB medium, plated on LB–
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
Gal-X) for assay validation The plates were incubated at
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
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
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
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
Cells were harvested by centrifugation and resuspended into
with a final concentration of 1.5% sarcosyl The supernatant
Trang 8was gently mixed with STE-washed
Glutathione–Sepha-rose 4B beads (Amersham Biosciences, Uppsala, Sweden) at
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
0.1% Triton X-100) After centrifugation, the beads were
washed three times with binding buffer, resuspended in
30 lL of 4· 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 six-well
(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
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
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
supple-mented with 10% fetal bovine serum and transiently
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
of lysis buffer (50 mm Hepes, pH 7.4, 150 mm NaCl, 1 mm
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
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
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 lL 4· 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
Trang 9hepcidin 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,
serum (15 lL) concentrated with Microcon YM-30 was
pipetted up and down for 5 min The proteins which did
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
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
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