From the § Department of Pediatrics, Harbor-UCLA Medical
Center, and Departments of Medicine and Pathology,
UCLA School of Medicine, Los Angeles, California 90059
Received for publication, September 29, 2000, and in revised form, November 9, 2000
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ABSTRACT |
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Cysteine-rich antimicrobial peptides are abundant
in animal and plant tissues involved in host defense. In insects, most
are synthesized in the fat body, an organ analogous to the liver of vertebrates. From human urine, we characterized a cysteine-rich peptide
with three forms differing by amino-terminal truncation, and we named
it hepcidin (Hepc) because of its origin in the liver and its
antimicrobial properties. Two predominant forms, Hepc20 and Hepc25,
contained 20 and 25 amino acid residues with all 8 cysteines connected
by intramolecular disulfide bonds. Reverse translation and search of
the data bases found homologous liver cDNAs in species from fish to
human and a corresponding human genomic sequence on human chromosome
19. The full cDNA by 5' rapid amplification of cDNA ends was
0.4 kilobase pair, in agreement with hepcidin mRNA size on
Northern blots. The liver was the predominant site of mRNA
expression. The encoded prepropeptide contains 84 amino acids, but only
the 20-25-amino acid processed forms were found in urine. Hepcidins
exhibited antifungal activity against Candida albicans,
Aspergillus fumigatus, and Aspergillus niger and antibacterial activity against Escherichia coli,
Staphylococcus aureus, Staphylococcus
epidermidis, and group B Streptococcus. Hepcidin may
be a vertebrate counterpart of cysteine-rich antimicrobial peptides
produced in the fat body of insects.
Innate immunity relies on a variety of effector mechanisms to
defend against microbial invasion. Among them are the abundant and
widely distributed disulfide-linked cationic antimicrobial peptides
found in both the plant and animal kingdoms. Generally, these peptides
exhibit a broad range of activity against bacteria, fungi, protozoa,
and enveloped viruses. Plants produce many cysteine-rich antimicrobial
peptides including thionins, plant defensins, and the cysteine rich
Ib-AMP 1-4 (1-3). In insects, cysteine-rich antimicrobial peptides
are produced in the fat body (functional homologue of the mammalian
liver) and transcriptionally induced and released into the hemolymph in
response to infection or injury. These include insect defensins,
heliomicin, drosomycin, and thanatin (4-7). Mollusks also produce
cationic and cysteine-rich antimicrobial peptides such as mytilin,
mytimicin, and myticin (8). In mammals, similar antimicrobial peptides
include Like the insect fat body, the vertebrate liver is also centrally
involved in innate immune response to infection. The "acute phase"
response to infection or inflammation is a pattern of increased hepatic
synthesis of many secreted proteins involved in host defense and the
selective suppression of synthesis of other secreted proteins. In
contrast to the abundant fat body-derived antimicrobial peptides of
insects, no vertebrate antimicrobial peptides originating in the liver
have been described to date. In this work, we report the discovery of a
novel hepatic antimicrobial peptide, hepcidin, whose processed form is
found in urine.
Purification from Urine--
Cationic peptides were extracted
from pooled urine of one to five healthy donors using methods that we
described previously for the isolation of human Amino Acid Sequence--
Purified hepcidin was modified by
reduction and carboxymethylation before amino acid sequencing.
Lyophilized peptide was resuspended in reduction and alkylation buffer
(0.5 M Tris buffer, pH 8, 6 M guanidine
hydrochloride, and 20 mM EDTA) to a concentration of 1 mg/ml. Reduction was accomplished by the addition of a 1000-fold molar
excess of dithiothreitol (DTT), and the solution was overlaid with
N2 gas and then incubated at 52 °C for 2.5 h. Fresh
DTT (500-molar excess) was added and incubated for an additional hour
at 52 °C. The solution was cooled to room temperature for 10 min,
and iodoacetamide freshly dissolved in reduction and alkylation buffer
was added to a final concentration of 3-fold molar excess over DTT and
incubated for 10 min in the dark. The reaction was stopped by the
addition of DTT (1500-fold molar excess of the peptide). The peptide
was then purified on a C18 RP-HPLC column equilibrated in
0.1% h-heptafluorobutyric acid (v/v). Peptides were
eluted using linear ACN gradients at a flow rate of 3 ml/min and
monitored by absorbance at 230 and 280 nm as follows: 1% ACN
increment/min for 20 min, followed by a 0.5% increment/min for the
last 60 min. HPLC fractions were lyophilized and resuspended in 5%
acetic acid, and the peak fraction (32-35% ACN) was submitted for
amino acid sequencing by Edman degradation at the UCLA Peptide
Sequencing Facility.
Circular Dichroism Spectroscopy--
CD spectra were recorded
using an AVIV 62DS spectropolarimeter (AVIV Associates, Lakewood, NJ).
Peptide at a concentration of 1 mg/ml was dissolved either in 10 mM NaPO4, pH 7.4, alone or in a 1:1 (v/v)
solution of trifluoroethylene:phosphate buffer. Peptide solutions were
scanned in 0.1 mm light path demountable cells over a 270 nm to 185 nm
wavelength range at 10 nm/min and a sample interval of 0.2 nm.
cDNA and Genomic Cloning--
An initial search of the
protein data base showed no homology of hepcidin to any known protein.
Therefore, the primary amino acid sequence was reverse translated and
searched against the GenBankTM human EST data base using
the BLAST (tblastn) nucleic acid search program (13). A matching
cDNA clone (American Type Culture Collection yb44e08.r1, clone
74054; GenBankTM accession number T48277) was purchased
from American Type Culture Collection (Manassas, VA). The plasmid clone
was transformed and amplified in Bluescript Escherichia coli
(Stratagene, La Jolla, CA) and purified using a Qiagen Plasmid Midi
Prep kit according to the manufacturer's instructions (Valencia, CA).
5' Rapid Amplification of cDNA Ends (RACE)--
The sequence
of the 5' end of the cDNA was determined using Marathon-Ready
cDNA from a liver library (CLONTECH). The
gene-specific primers for hepcidin are antisense (for 5'-RACE)
5'-CCCAAGACCTATGTTCTGG-3' and 5'-TCTGTCTGGCTGTCCCACTGCTGG-3' and sense
(for 3' RACE) 5'-CATGTTCCAGAGGCGAAGG-3'. PCR was carried out according
to the manufacturer's instructions. PCR products were purified by
phenol/chloroform extraction and ethanol precipitation (14). The PCR
product was sequenced using an ABI Prism DNA sequencer (Applied
Biosystems) at the UCLA Sequencing Facility. The Omiga program (Oxford
Molecular Co.) was used to analyze and search for potential consensus
sites within the gene.
Northern Blot Analysis: Human Organ Screen--
The hepcidin
cDNA clone and the gene-specific primers above were used in the PCR
reaction to generate a 173-base pair product from a segment of the
coding region. The PCR product was used to probe
CLONTECH organ blot membranes as described
previously (11). The organ blots contained poly(A) selected human
mRNA from spleen, thymus, appendix, peripheral blood leukocyte,
bone marrow, fetal liver, heart, brain, placenta, lung, adult liver, skeletal muscle, kidney, pancreas, stomach, thyroid, spinal cord, lymph
node, trachea, adrenal gland, prostate, testes, ovary, small intestine,
and colon.
Human Liver--
Frozen normal human liver blocks obtained from
the UCLA Human Tissue Resource Center (Los Angeles, CA) were pulverized
in liquid nitrogen using a mortar and pestle. Approximately 0.1 g of tissue was homogenized in 1 ml of Trizol reagent (Life Technologies, Inc.), and total RNA was extracted according to the manufacturer's instructions. Poly(A) RNA was separated from the total RNA preparation using a Qiagen Oligotex mRNA isolation kit. Purified poly(A) RNA was separated on a 1% agarose gel (Bio-Rad) containing 3.3% formalin in 10 mM NaPO4, transferred onto GeneScreen
Plus (PerkinElmer Life Sciences, Boston, MA), and hybridized with a
radioactive probe as described above.
Antimicrobial Assays--
The 20- and 25-amino acid forms of
hepcidin (Hepc20 and Hepc25), each of which was homogeneous by
electrophoretic and mass spectrum analysis, were quantified by UV
absorbance at 215 and 220 nm using the formula concentration
(mg/ml) = (A215 Germination Assay--
The two human clinical isolates of
filamentous fungi Aspergillus fumigatus and
Aspergillus niger used in this assay were a generous gift
from Dr. Dexter Howard (UCLA Department of Microbiology, UCLA, Los
Angeles, CA). Spores were harvested as described previously (15). The
glycerol stocks of spores from each strain were utilized in a
spectrophotometric germination assay as follows: spores were diluted to
a concentration of 1.25 × 104 /ml in culture
medium (half-strength potato dextrose broth (BBL), 10 µg/ml
tetracycline (Sigma), and 100 µg/ml cefotaxime (Life Technologies,
Inc.). Eighty µl of each spore suspension was placed in sterile
flat-bottomed polystyrene 96-well plates (Costar, Corning, NY), and 20 µl of peptide or peptide diluent (water) was added to yield a final
spore concentration of 104 /ml. The plate was
incubated at 30 °C in a humidified chamber in the dark. Germination
was microscopically monitored for the appearance of hyphae using a
light microscope. After 48 h, the absorbance was measured at 600 nm using a SpectraMAX250 EIA plate reader (Molecular Devices,
Sunnyvale, CA). To test for activity in high salt conditions, the assay
medium was supplemented with 150 mM NaCl.
Fungicidal Assay--
To determine whether the effect of
hepcidin was fungicidal or fungistatic, spores were incubated in the
presence of Hepc20 and Hepc25 at the highest concentrations necessary
to inhibit germination as described above. After 48 h at 30 °C,
the well contents were transferred to sterile microfuge tubes and
centrifuged for 5 min at 2000 × g. The supernatant
containing the peptides was removed, and the spore pellet was
resuspended in 100 µl of fresh media and transferred into a new
96-well plate. The spores were incubated for an additional 48 h at
30 °C and then analyzed for germination as described above. If no
hyphae were observed, then the effect of hepcidin was judged to be fungicidal.
Cytotoxicity Assay--
K562 cells (American Type Culture
Collection leukemic cell line CCL-243) were cultured in RPMI 1640 with
L-glutamine (Life Technologies, Inc.) and 10% fetal calf
serum (Hyclone, Logan, UT) for 48 h and then washed and
resuspended to 106 cells/ml in serum-free RPMI 1640 media.
A volume of 50 µl of cells was aliquoted into sterile flat-bottomed
96-well plates (Nalgene Nunc, Rochester, NY). Various concentrations of
10× peptide stocks were added to the cells and incubated at
37 °C/5% CO2 for 12-16 h. Viability was determined by
trypan blue dye exclusion.
Peptide Synthesis and Refolding--
Peptide synthesis reagents
including Fmoc amino acids (AnaSpec Inc., San Jose, CA) and coupling
solvents (PE Biosystems, Applied Biosystems, Foster City, CA) were used
to synthesize Hepc20 peptide on an ABI 431A peptide synthesizer
(Applied Biosystems) using double coupling for all residues. The scale
of synthesis was 0.25 mmol using a FastMocTM strategy (16)
with PS-PEG resin (PE Perceptive Biosystems, Connecticut Path,
MA). After cleavage, the crude synthetic peptide (sHEP) was resuspended
in reducing buffer (6 M guanidine-HCl, 0.02 M
EDTA, and 0.5 M Tris-HCl, pH 8.07) to a final concentration of 0.5 mg/ml (w/v), homogenized with 20 strokes in a Dounce
homogenizer, and incubated in a 50 °C sonicating water bath for 30 min. The resuspended peptide was reduced by adding DTT to a final
concentration of 0.01 M, overlaying with N2
gas, and then incubating in a 50 °C water bath overnight. The
reduced peptide was loaded onto a C18 Sep-Pak cartridge
(Waters) equilibrated with 0.1% trifluoroacetic acid, desalted with
15% ACN and 0.1% trifluoroacetic acid, and eluted with 40% ACN and
0.1% trifluoroacetic acid. The 40% ACN fraction was lyophilized and
then further purified by RP-HPLC on a Vydac C18 column
using the same gradient program as described above in purification of
the native peptide from urine. The major peak was lyophilized,
resuspended to 0.03 mg/ml with distilled H2O, adjusted to
pH 7.5 with ammonium hydroxide, and then air-oxidized in an open vessel
at room temperature with stirring for 18 h. The refolded peptide
was then purified on a C18 Sep-Pak cartridge followed by
RP-HPLC as described for the reduced peptide.
Purification and Amino Acid Analysis--
Cationic proteins were
extracted from urine using a weak cation exchange resin and then
further purified by RP-HPLC (Fig. 1), and
hepcidin peaks were identified by characteristically migrating bands in
Coomassie Blue-stained acid-urea PAGE. The fractions corresponding to the hepcidin peptides eluted between 24% and 30%
acetonitrile. The identity of each peptide in peaks A and B of Fig. 1 was confirmed by MALDI-TOF-MS and amino acid
sequencing. The peptides were 20 and 25 amino acids long (Hepc20 and
Hepc25, respectively) and differed by amino-terminal truncation but
preserved the cysteine-rich domain (Fig.
2). Mass analysis identified the peptide
in peak C of Fig. 1 as the 22-amino acid form (Hepc22). The
mass data (Table I) indicate that all 8 cysteine residues are connected by disulfide bonds. In urine, Hepc20
and Hepc25 are the major forms, whereas Hepc22 is a minor species.
Collectively, the concentration of hepcidin ranges between 10 and 30 µg/liter (4-12 nM) in urine from normal donors. The
remaining large peaks visible in Fig. 1 had previously been identified
as variably amino-terminal-truncated forms of another antimicrobial
peptide, human CD Spectroscopy--
The CD spectra (Fig.
3) in phosphate-buffered saline solution
(100 mM sodium chloride and 20 mM sodium
phosphate, pH 7.4) and the structure-promoting solvent system
trifluoroethanol-20 mM phosphate buffer, pH 7.4 (trifluoroethylene:buffer, 1:1, v:v), were consistent with a structure
that has a series of Northern Blot Analysis--
Membranes preloaded with human organ
tissue poly(A) selected mRNA were probed with a 173-base pair probe
generated by PCR from the cDNA clone. mRNA was found to be
highly expressed in fetal and adult liver, with a much lower signal
detected in the heart and spinal cord (Fig.
4). A fourth membrane loaded with prostate, testes, ovary, small intestine, and colon was also probed, but no signal was detected (data not shown). A weak mRNA signal corresponding to 2.37 kilobases was seen in fetal and adult liver and
spinal cord but was not further analyzed. Although the peptide was
found in the urine, the mRNA was not detectable in the kidney or
the bladder.
cDNA Cloning--
A search of the protein data base revealed
no sequence homology to any known peptides. Using the primary sequence
of the peptide, the BLAST (tblastn) nucleic acid search program of the
human EST data base identified a cDNA clone that spans the region
of the peptide from Asp40 to Thr84
(I.M.A.G.E. consortium clone 74054; DNA GenBankTM
accession number T48277; EST yb44e08) (17). An identical genomic
sequence match was found on human chromosome 19 (GenBankTM
accession number AD000684; locus CH19R30879). In addition, a search of
EST data bases found matches to cDNA sequences from the liver of
pig, rat, mouse, flounder, and the long-jawed mudsucker (Fig.
5). All of the matches are to peptides
that have yet to be isolated and characterized. The similarity is
strongest in the region that corresponds to the peptides isolated from
urine, namely, residues Arg55 to Thr84.
5' RACE and Gene Analysis--
The PCR products of the 5' RACE
reaction were sequenced and compared with the genomic sequence (Fig.
6). The mRNA, 0.4 kilobase in length
(GenBankTM accession numbers AAG23966 for cDNA
and P81172 for the corresponding hepcidin precursor), matched the
estimated size on the Northern blot. The gene consists of three exons
and two introns, and the third exon encodes the sequence of the
peptides found in urine. The putative unprocessed peptide encoded by
all three exons would be 84 amino acid residues long. A potential
signal-peptide cleavage site between Gly24 and
Ser25 would generate a 60-amino acid propeptide species.
Consensus sites for myristylation sites and phosphorylation sites occur on Gly44 and Thr24, respectively. In addition,
a potential furin cleavage site is found at Arg59 (18).
Since our initial studies, additional human EST sequences have appeared
in the GenBankTM data bases (GenBankTM
accession numbers AI937227, AI829866, and AI797446) with minor sequence
variations in the noncoding regions.
Antimicrobial Activity--
The colony-forming unit assay (19) was
used to determine the antimicrobial activity of purified hepcidin forms
against various microbial strains. In the initial screen, all the
strains were tested against 30 µM Hepc20 and Hepc25
(Hepc22 was not tested because it is present in small concentrations in
the urine). Both peptides were antimicrobial against E. coli
ML35p and, to a lesser extent, against S. epidermidis,
S. aureus, C. albicans, and group B
Streptococcus, but both were inactive against
Pseudomonas aeruginosa (Fig.
7). A dose-response CFU assay performed
against E. coli and S. epidermidis showed that
both hepcidin peptides were microbicidal at the highest concentration
of 30 µM against both strains (data not shown). In this
assay and in the initial screen, Hepc20 was more active than Hepc25;
however, both peptides were inhibited by the addition of 100 mM NaCl to the assay mixture.
Inhibition of Germination--
Spores of two human pathogenic
strains of A. fumigatus and A. niger were
subjected to a germination assay (Fig.
8). Hepc20 was more potent than Hepc25
against both strains. Hepc20 was antifungal against A. niger, where no hyphae were detected after 48 h at 20 µM, but 40 µM was required against the more
resistant strain, A. fumigatus. Hepc25 only retarded spore
germination in A. niger at the highest concentration of 40 µM and was ineffective against A. fumigatus at
the same concentration. Neither peptide was active in the presence of
150 mM NaCl.
Fungicidal Activity--
We assessed fungicidal activity by
following germination after removal of the antifungal peptides and
additional incubation in fresh media. No germination was observed after
48 h. At the highest concentrations, Hepc20 was fungicidal against
both strains, as indicated by the lack of spore germination in fresh
medium. Hepc25 did not exhibit fungicidal activity at any concentration used in this assay.
Cytotoxicity Assay--
To test for cytotoxic effects of hepcidin,
K562 cells were assessed for viability after incubation with Hepc20 and
Hepc25, using trypan blue as an indicator of membrane integrity. At the highest concentration of 30 µM, cells incubated for
15 h in the presence of Hepc20 or Hepc25 were 88% and 74%
viable, respectively. When cells were treated with 30 µM
human Chemical Synthesis of Hepcidin--
Crude synthetic material was
completely reduced with DTT and purified by RP-HPLC. The major HPLC
peaks were analyzed by AU-PAGE and MALDI-TOF-MS. As expected, the
peptide migrated more slowly upon reduction. The gain of 8 Da
corresponding to eight additional hydrogen atoms in MALDI-MS confirmed
that the synthetic peptide was completely reduced. The reduced peptide
was refolded by air oxidation and compared with the native peptide. The
synthetic Hepc20 had identical AU-PAGE migration, eluted
identically in C18 RP-HPLC, and had an identical
electrospray mass and antimicrobial activity against E. coli
in a CFU assay compared with native Hepc20.
We report here the first member of a new vertebrate family of
small antimicrobial peptides that contain 8 cysteine residues and are
active against both bacteria and fungi. Hepcidin mRNA encodes a
larger precursor and is found primarily in the liver, but the peptides
were first discovered in and isolated from urine. The native peptide
was purified from human urine by cation exchange chromatography and
RP-HPLC and characterized by amino acid sequencing, MALDI-TOF-MS, and
CD spectroscopy.
The human hepcidin forms, 2-3 kDa in size, have an overall charge of
+3 at neutral pH and are only 20-25 amino acids in length (8 of these
amino acids are cysteine residues (~30% cysteine)). Mass
spectrometry data confirm all 8 cysteines to be engaged in four
intramolecular disulfide bonds. The symmetric arrangement of pairs of
cysteines around a cationic segment (HRSK) resembles that of
antimicrobial protegrins and tachyplesins, peptides that have a
two-strand The cDNA structure suggests that the peptide is translated as an
84-amino acid prepropeptide that is amino-terminally processed to the
20- to 25-amino acid peptide (Fig. 6). A strong consensus sequence
(score = 16.45 using the PC Gene program) for a signal sequence
cleavage site is located between Gly24 and
Ser25 that would produce a 60-residue propeptide. Another
likely processing site is carboxyl-terminal to Arg59, a
consensus site for the subtilisin/kexin family of mammalian propeptide
processing enzymes (propeptide convertases) with a preference for
cleavage after the paired basic residues Lys-Arg and Arg-Arg. Among
other functions, the propeptide convertases cleave propeptides to
generate one or more bioactive peptides from a single precursor (18).
The abundance of propeptide convertases in the liver may explain our
inability to isolate the larger propeptide from native sources (liver
tissue, bile, and blood serum). The small size of hepcidin, its
conservation between animal species, and its compact folding pattern
may account for our difficulties in producing a useful antibody.
A search of the protein data base revealed no homology to hepcidin in
vertebrates and invertebrates. However, searching the EST data base
revealed liver cDNA homologues in pig, rat, mouse, flounder, and
the long-jawed mudsucker (Fig. 5). All 8 cysteine residues are
conserved with particularly strong sequence similarity in residues
60-84 that encompass the processed forms isolated from the urine. It
is noteworthy that the putative propeptide convertase cleavage sites
are also conserved. In humans, the paired basic residues are Arg-Arg,
whereas the other homologues have the same recognition sequence
or an alternative recognition sequence of Lys-Arg.
It is possible that if the proregion is cleaved from the precursor as a
single peptide, it could also have a biological function. To test this
possibility, the proregion (Ser25-Arg59) was
synthesized, and an antibody reactive with the synthetic peptide was
produced (Research Genetics). Preliminary data utilizing the synthetic
propiece peptide have shown that it exhibits an antibacterial activity
equally potent to that of Hepc20 and Hepc25 (data not shown). However,
a Western blot analysis capable of detecting 500 ng of the synthetic
peptide did not detect the propiece peptide in cationic extracts of
urine, serum, liver extract, and bile (data not shown), making it
unlikely that this form is stable and abundant. Acid liver extracts did
contain a peptide with a mass identical to Hepc25 that eluted at the
same acetonitrile concentration as Hepc25 from urine. While this study
was under review, we noted an interim report that an antimicrobial
peptide identical to Hepc25, LEAP-1, was isolated from human plasma
ultrafiltrate (22). The presence of Hepc25 in ultrafiltrate suggests
that the peptide originating in the liver reaches the kidney in
blood plasma.
Hepcidin may be the functional homologue of the insect defensin-like
peptide drosomycin. Hepcidin is expressed in human liver, whereas
drosomycin is found in the fat body (the liver equivalent) of
Drosophila; both peptides have four disulfide bonds, and
both are potent antifungal peptides. In parallel studies, Pigeon
et al.2 identified
a murine hepcidin as a hepatic mRNA inducible in vivo by
iron overload and lipopolysaccharide. The ability of lipopolysaccharide to increase hepcidin mRNA in vivo and in isolated
hepatocytes is consistent with the proposed role of hepcidin in
inflammation and host defense. Like hepcidin, drosomycin is expressed
constitutively but is also inducible upon microbial challenge. Like
drosomycin, both Hepc20 and Hepc25 forms inhibit spore germination in
the two pathogenic strains of Aspergillus tested. Future
studies will be necessary to determine whether hepcidin, like the
cysteine-rich antimicrobial defensins (23), interacts with specific
cellular receptors and functions also as a signaling molecule.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
- and
-defensins and protegrins (9, 10).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-defensin-1 (11).
Briefly, urine was filtered to remove cells and extracted with the weak
cation exchange matrix CM Macroprep (Bio-Rad, Richmond, CA). Cationic peptides were eluted with 5% acetic acid and further purified by
RP-HPLC1 (Waters Model 626;
Waters, Milford, MA) on a Vydac C18 column (218TP510)
equilibrated in 0.1% trifluoroacetic acid (v/v). Peptides were
eluted with an acetonitrile (ACN) gradient of 4% ACN increment/min for
5 min and washed with 20% ACN for 5 min, followed by a 0.5% ACN
increment/min for the last 30 min (12). HPLC peak fractions were
analyzed by acid urea polyacrylamide gel electrophoresis, and peptide
masses were determined by MALDI-TOF-MS (UCLA Mass Spectrometry Facility
(Los Angeles, CA) or Emory University Microchemical Facility (Atlanta, GA)).
A225) × 0.144, and they were tested for
antimicrobial activity against bacterial strains (E. coli
ML35p, Staphylococcus aureus, Staphylococcus epidermidis, and group B Streptococcus) and yeast
(Candida albicans) in a CFU assay as described previously
(11). Culture densities were measured spectrophotometrically at 620 nm
for bacterial strains and at 450 nm for yeast and then resuspended to a
final concentration of 106 CFU/ml. An
A620 reading of 0.2 corresponds to
107 CFU/ml for the staphylococcal and streptococcal strains
and 5 × 107 CFU/ml for E. coli. An
A450 reading of 1.0 is equivalent to
2.86 × 107 CFU/ml for C. albicans. The
organisms were incubated with various concentrations of peptide at
37 °C with constant shaking for 1, 3, and 24 h. Surviving
microbes were plated in triplicate on trypticase soy broth
plates (Microdiagnostic Products Inc., Lombard, IL) using a spiral
plating system (Spiral Biotech, Bethesda, MD). In some experiments, the
assay medium was supplemented with 100 mM NaCl to mimic
higher salt conditions characteristic of blood plasma.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-defensin-1 (11).
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Fig. 1.
RP-HPLC purification of hepcidin from human
urine. Cationic peptides in urine were purified by RP-HPLC on a
Vydac C18 column using the following linear gradient in
0.1% trifluoroacetic acid: 4% ACN increment/min for 5 min,
20% ACN wash for 5 min, then 0.5% ACN increment/min for the last 30 min. Peak A, Hepc20; peak B, Hepc25; peak
C, Hepc22. Absorbance was monitored at = 215 nm.
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Fig. 2.
Amino acid sequence of hepcidin and proposed
cysteine connectivity. Purified hepcidin was carboxymethylated and
then sequenced by Edman degradation. The three processed forms differ
by amino-terminal truncation as denoted by arrows. The
proposed cysteine linkage pattern is 1-4, 2-8, 3-7, and 5-6, as shown.
The cationic residues are indicated by asterisks
(*).
Characteristics of hepcidin peaks A-C
-turns, loops, and distorted
-sheets.
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Fig. 3.
Circular dichroism spectra of Hepc20.
The CD spectra were recorded in (A) phosphate-buffered
saline solution (100 mM sodium chloride and 20 mM sodium phosphate, pH 7.4) and (B) the
structure-promoting solvent system trifluoroethanol-20 mM
phosphate buffer, pH 7.4 (trifluoroethylene:buffer, 1:1, v:v).
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Fig. 4.
Northern blot analysis of human organ
mRNA. A membrane preloaded with human organ poly(A) selected
mRNA was purchased from CLONTECH and probed
with a radiolabeled probe generated by PCR. A: lane
1, spleen; lane 2, thymus; lane 3, appendix;
lane 4, peripheral blood leukocyte; lane 5, bone
marrow; and lane 6, fetal liver (FL).
B: lane 1, heart (H); lane
2, brain; lane 3, placenta; lane 4, lung;
lane 5, adult liver (AL); lane 6,
skeletal muscle; lane 7, kidney; and lane 8,
pancreas. C: lane 1, stomach; lane 2,
thyroid; lane 3, spinal cord (SC); lane
4, lymph node; lane 5, trachea; and lane 6,
adrenal gland. An RNA marker ladder is shown. Hepc mRNA, 0.4-0.5
kilobase, was detected in fetal liver (FL) and adult
liver (AL), with trace message detected in the heart
(H) and spinal cord (SC).
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Fig. 5.
Comparison of hepcidin sequences from various
species. A BLAST search of GenBankTM EST entries
revealed cDNA homologues of human hepcidin (hHEPC) in
pig (pHEPC), rat (rHEPC), mouse
(mHEPC), flounder (fHEPC), and the long-jawed
mudsucker Gillichthys mirabilis (gHEPC). Putative
peptide sequences were translated from the cDNA sequences isolated
from the liver of each species. The positions of cysteine residues
(outlined in gray boxes) are conserved.
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Fig. 6.
Hepcidin gene and precursor peptide. An
intron-exon diagram and the peptide sequences encoded by the three
exons A, B, and C are shown. The exons encode an 84-amino acid
prepropeptide. Arrows denote three processed forms isolated
from urine. An arrow indicates the putative signal sequence
(SS) cleavage site.
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Fig. 7.
Antimicrobial assays. Various microbial
strains were subjected to a CFU assay using Hepc20 and Hepc25 at 30 µM concentration in 10 mM NaPO4,
pH 7.4, 0.01× trypticase soy broth after a 1-h incubation. The
organisms tested were E. coli ML35p (EC),
P. aeruginosa (PA), S. aureus
(SA), S. epidermidis (SE), group B
Streptococcus (SB), and C. albicans
(CA). The amount of CFU/ml is indicated by the
bars: first gray bar, microbial input
(t = 0); second gray bar, medium only
(t = 1 h); dark gray bar, Hepc25;
black bar, Hepc20.
View larger version (13K):
[in a new window]
Fig. 8.
Fungal germination assay. Graph of a
germination assay is shown. Hepc20 ( ), Hepc25 (
), and synthetic
Hepc20 (
) were tested against the spores of two human
pathogenic strains of A. fumigatus (A) and
A. niger (B). In a 96-well plate, 104
spores were incubated with and without peptide at 30 °C in the dark.
After 48 h, spore germination was monitored at
= 600 nm.
-defensin HNP-1, only 9% viable cells remained at the end of
the incubation period. Thus, hepcidin is not cytotoxic at a
concentration that is ~3000-fold higher than that found in the urine.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-sheet structure stabilized by interstrand disulfide
bonds. Based on this similarity and on preliminary molecular models, we
propose the following cysteine connectivity pattern: 1-4, 2-8, 3-7, and
5-6 (Fig. 2). This arrangement of disulfide bonds exhibits three
possible
-turns and provides a fold with the least steric hindrance
among the side chains. The proposed disulfide connectivity pattern
makes the hepcidin structure similar to the "cystine knot"
class of antimicrobial peptides (20, 21).
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ACKNOWLEDGEMENTS |
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We thank Drs. Christelle Pigeon and Olivier Loreal for several corrections to the manuscript and for sharing their own work with us prior to publication and Dr. Edith Porter for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant HL 46809.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Medicine, CHS 37-055, UCLA School of Medicine, Los Angeles, CA 90095-1690. Tel.: 310-825-6112; Fax: 310-206-8766; E-mail: tganz@mednet.ucla.edu.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M008922200
2 Pigeon, C., Ilyin, G., Courselaud, B., Leroyer, P., Turlin, B., Brissot, P., and Loreal, O. (2001) J. Biol. Chem. 276, 7811-7819
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ABBREVIATIONS |
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The abbreviations used are: RP-HPLC, reverse phase-high performance liquid chromatography; ACN, acetonitrile; CFU, colony-forming unit; DTT, dithiothreitol; Hepc, hepcidin; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time of flight mass spectrometry; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; UCLA, University of California Los Angeles; EST, expressed sequence tag; poly(A), polyadenylated.
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REFERENCES |
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