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Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4180-4187
Analysis of Ferritins in Lymphoblastoid Cell Lines and in the Lens of
Subjects With Hereditary Hyperferritinemia-Cataract Syndrome
By
Sonia Levi,
Domenico Girelli,
Federica Perrone,
Marcella Pasti,
Carole Beaumont,
Roberto Corrocher,
Alberto Albertini, and
Paolo Arosio
From Dibit, Institute H. San Raffaele, Milano, Italy; Institute of
Medical Pathology, University of Verona, Verona, Italy; Cattedra di
Chimica, University of Brescia, Brescia, Italy; and INSERM U409,
Faculté Bichat, Paris, France.
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ABSTRACT |
Hereditary hyperferritinemia-cataract syndrome (HHCS) is an
autosomal and dominant disease caused by heterogeneous mutations in the
iron responsive element (IRE) of the 5 untranslated flanking region of ferritin L-chain mRNA, which reduce the binding to the trans
iron regulatory proteins and make L-chain synthesis constitutively upregulated. In the several families identified so far, the serum and
tissue L-ferritin levels are fivefold to 20-fold higher than in
nonaffected control subjects, iron metabolism is apparently normal, and
the only relevant clinical symptom is early onset, bilateral cataract.
Some pathogenetic aspects of HHCS remain obscure, with particular
reference to the isoferritins produced by HHCS cells, as well as the
mechanism of cataract formation. We analyzed lymphoblastoid cell lines
obtained from two nonaffected control subjects and from HHCS patients
carrying the substitution A40G (Paris-1), G41C (Verona-1), and the
deletion of the residues 10-38 (Verona-2) in the IRE structure.
Enzyme-linked immunosorbent assays specific for the H- and L-type
ferritins showed that L-ferritin levels were up to 20-fold higher in
HHCS than in control cells and were not affected by iron
supplementation or chelation. Sequential immunoprecipitation
experiments of metabolically-labeled cells with specific antibodies
indicated that in HHCS cells about half of the L-chain was assembled in
L-chain homopolymers, which did not incorporate iron, and the other
half was assembled in isoferritins with a high proportion of L-chain.
In control cells, all ferritin was assembled in functional
heteropolymers with equivalent proportion of H- and L-chains. Cellular
and ferritin iron uptake was slightly higher in HHCS than control
cells. In addition, we analyzed the lens recovered from cataract
surgery of a HHCS patient. We found it to contain about 10-fold more
L-ferritin than control lens. The ferritin was fully soluble with a low
iron content. It was purified and partially characterized.
Our data indicate that: (1) in HHCS cells a large proportion of
L-ferritin accumulates as nonfunctional L-chain 24 homopolymers; (2)
the concomitant fivefold to 10-fold expansion of ferritin
heteropolymers, with a shift to L-chain-rich isoferritins, does not
have major effects on cellular iron metabolism; (3) L-chain
accumulation occurs also in the lens, where it may induce cataract
formation by altering the delicate equilibrium between other
water-soluble proteins (ie, crystallins) and/or the antioxidant
properties.
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INTRODUCTION |
HUMAN FERRITIN IS encoded by the genes
for the heavy H-chain (182 amino acids) and the light L-chain (174 amino acids) on chromosome 11 and 19, respectively.1
Despite differences in the primary structure (about 50% identity at
the amino acid level), the two chains have remarkably similar
three-dimensional structures and coassemble in different proportions in
the 24-mer protein to originate families of
isoferritins.1,2 Both H- and L-chain mRNAs carry iron
responsive element (IRE) structures close to the 5 termini,
which interact with the trans iron regulatory proteins (IRPs), leading
to an equivalent iron-regulated translational regulation.3,4 The different accumulation of the two chains and messengers in the various cells is mainly attributed to different transcriptional regulations.5,6
The major functional role of ferritin is to store iron into its large
cavity, and consequently several in vitro studies analyzed the
interaction between iron and ferritin.7 Biochemical and crystallographic studies on the recombinant H-chain homopolymers showed
that H-chains contain ferroxidase centers, which promote iron oxidation
and accelerate iron incorporation into the ferritin.8,9 The
L-chains lack this activity, but are richer in carboxyl groups exposed
on the cavity surface, which facilitate iron nucleation and
mineralization.10 The complementary functional
specificities of the two chains make the heteropolymers more efficient
in the in vitro incorporation of iron. For instance, molecules with
30% to 100% H-chains incorporated iron at the same rate, but the ones richer in L-chain were more efficient in iron core
formation.11 The finding that L- is more stable than the
H-ferritin and that it is predominant in tissues with main iron storage
functions suggested that it is designed for long-term iron storage,
whereas the H-chain is probably necessary for an active cellular iron sequestration and detoxification. This hypothesis was confirmed by
recent studies showing that even a minor overexpression of H-chain in
mouse erythroleukemic cell lines induced an iron-deficient phenotype
and repression of globin synthesis.12
Some of us recently described a new disease, the hereditary
hyperferritinemia-cataract syndrome (HHCS), clinically characterized by
the combination of a substantial elevation of serum L-ferritin levels
and early onset bilateral cataract, without other apparent symptoms.13 Typically, hyperferritinemia is not related to
iron overload and persists even if HHCS patients develop iron-deficient anemia after inappropriate phlebotomies.14 After the
descriptions in Italian and French families,13-20 HHCS has
been also found in Germany,21 the United
Kingdom,22 and the United States (D.G., personal
communication, November 1997), suggesting that the disease may be largely distributed in the world and probably not sporadic. This
autosomal and dominant genetic disorder is associated with heterogeneous mutations of the L-chain IRE sequences that
reduce/eliminate binding to IRPs and make L-chain expression
constitutive and noniron regulated. Despite the identification of the
genetic basis, not all is clear about the pathophysiology of HHCS,
which also offers a unique human model to study ferritin biological
roles.13,14 In particular, the structural and functional
properties of the isoferritins produced in HHCS cells, as well as the
mechanism of cataract formation, remain unclear. A genotype-phenotype
correlation has been noted in HHCS, depending on the position of the
different mutations in the IRE stem-loop structure and their effect on
IRP binding affinity. Mutation in the lower stem has been associated with lower serum ferritin and milder, asymptomatic cataract as compared
with mutations in the CAGUGN hexaloop and/or deletion, which
determine ferritin levels greater than 1,000 µg/L and severe cataract.15,19 The parallelism between serum ferritin
levels, tissue ferritin levels, and severity of the cataract suggested that the three are linked and related to the abnormality of ferritin L-chain regulation.15-17 Unexpectedly, the noniron
regulated fivefold to 20-fold increases in L-ferritin levels in serum
and did not have significant effects on body iron metabolism, except
that some subjects were found prone to develop iron
deficiency.14,15 Ferritin is ubiquitous, and all tissues so
far analyzed, red blood cells, peripheral monuclear cells,
and liver were found to accumulate large excesses of ferritin in the
HHCS subjects.15,19 Analysis of lymphoblastoid cell lines
of subjects from the Paris-1 family indicated a large unbalance of
L-chain synthesis over the H-chain.17
To study in more detail the relationship between ferritin synthesis,
cellular iron metabolism, and cataract development, we analyzed
cultured lymphoblastoid cell lines from subjects with three different
HHCS mutations and a specimen from cataract surgery of one HHCS
subject. HHCS lymphoblastoid cells were found to contain a large amount
of ferritin rich in L-chain in the form of nonfunctional L-homopolymers
and functional L-rich heteropolymers. Cellular iron metabolism,
monitored by iron incorporation and IRPs activity, was very similar in
control and HHCS cells. A similar modification of ferritin accumulation
was found in HHCS lens.
The results indicate that the accumulation of L-chain homopolymers and
the shift in isoferritin composition is probably common to most tissues
and has minor consequences on iron metabolism. Cataract formation may
be due to the indirect effects of L-ferritin overexpression on the
solubility of other lens proteins or on antioxidant properties of the
lens.
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MATERIALS AND METHODS |
Reagents.
Recombinant human ferritin H- and L-homopolymers from
Escherichia coli (E coli) are described in Levi
et al8,10 and monoclonal antibodies for H- and L-ferritin
chains in Luzzago et al.23 Radioactive
35S-methionine and 59FeCl3 were
from Amersham (Arlington Heights, IL).
Cell culture.
The Epstein-Barr virus (EBV)-transformed cultured B
lymphoblastoid cell lines were obtained by infection of peripheral
blood mononuclear cells with supernatant from EBV-infected B95-8
Marmoset cells (American Type Culture Collection CRL 1612, Rockville,
MD). They were grown in RPMI 1640 medium (Dulbecco's
modified Eagle's medium; GIBCO, Grand Island, NY)
supplemented with 10% fetal calf serum (FCS, GIBCO), 80 µg/mL gentamycin, 2 mmol/L glutamine, 1% sodium
pyruvate and maintained at a concentration of 106 cell/mL.
Metabolic labeling and immunoprecipitation.
Cells (2 × 106) were grown for 1 hour in
methionine-free MEM medium (GIBCO), 100 mg/mL streptomycin, 100 U/mL
penicillin, 2 mmol/L glutamine, 0.5% FCS (GIBCO), 0.5% bovine serum
albumin, and then labeled for 2 hours with 50 µCi/mL
35S-Met. The cells were collected by centrifugation, washed
with phosphate-buffered saline, and then lysed with 500 µL
of lysis buffer (20 mmol/L Tris-HCl, pH 8.0; 200 mmol/L LiCl; 1 mmol/L EDTA; 0.5% NP-40). Total radioactivity associated with the soluble proteins was determined by trichloroacetic acid (TCA)
precipitation. For immunoprecipitation studies, 2 × 106 cpm of cytosolic lysates were precleared by incubation
with 100 µL of protein A-Sepharose 50% vol/vol (Sigma, St Louis,
MO) for 1 hour at 4°C with gentle shaking. After
centrifugation for 1 minute at 14,000 rpm, the supernatants were added
to 30 µL of protein A-Sepharose preincubated with the antiferritin
H-chain antibody rH02 at a concentration of 3 mg/mL, incubated 1 hour at 4°C, and precipitated as above. The soluble fraction was further added to 30 µL of protein A-Sepharose preincubated with 3 mg/mL of
antiferritin L-chain antibody L03, incubated for 1 hour at 4°C, and
precipitated. The immunobeads were washed, resuspended in sodium
dodecyl sulfate (SDS) buffer, boiled for 10 minutes, and loaded on 15%
SDS-polyacrylamide gel electrophoresis (PAGE). Alternatively, they were
resuspended in SDS loading buffer containing 4 mol/L urea, run on 6%
polyacrylamide SDS-PAGE containing 4 mol/L urea, and subjected to
autoradiography. Under these conditions, ferritin is not
denatured.24 The gels were treated with autoradiography image enhancer (Amplify; Amersham), dried, and exposed. The intensity of ferritin subunit bands was quantified by densitometry (Molecular Dynamics, Sunnyvale, CA).
59Fe cellular uptake.
Cells (106) were grown for 18 hours in RPMI
supplemented with 10 µCi/mL of 59Fe nitrilo triacetate
(59FeNTA) and 200 µmol/L ascorbic acid. The cells were
washed and lysed as above, the homogenates centrifuged at 13,000 rpm
for 5 minutes at 4°C, and the radioactivity associated with soluble and insoluble fraction measured on a  -counter (Packard, Downers Grove, IL). The extracts were analyzed on 6% native-PAGE
and exposed to autoradiography or subjected to the sequential
immunoprecipitation procedure described above and analyzed in 4 mol/L
urea on 6% PAGE under nondenaturing conditions.24
Ferritin and protein quantification in cellular extracts.
Ferritin content of 107 cell extracts was measured by
enzyme-linked immunosorbent assay (ELISA) based on monoclonal
antibodies specific for the H-ferritin (rH02) and the L-ferritin (LF03)
calibrated on the corresponding recombinant homopolymers expressed in
E coli.23,24 Protein content was evaluated by
bicinchoninic acid (BCA) method (Pierce, Rockford,
IL) calibrated on bovine serum albumin. Immunoblotting experiments were performed as described in Santambrogio et
al.11 IRP activity measurements of cell lysates were
determined as in Cairo and Pietrangelo.25
Lens ferritin purification and analysis.
A 25-year-old female HHCS patient belonging to the family
Verona-114 recently underwent cataract surgery by means of
the phacoemulsification technique with posterior chamber intraocular
lens implantation, which is widely used in modern cataract
surgery.26 During the procedure, after opening of the
anterior capsule, the lens is fragmented by ultrasound and then
aspirated in a closed irrigation-aspiration system. The recovered lens
material consisted of a suspension in a buffered saline solution.
Specimens were obtained from the HHCS patient, as well as from several
patients with cataracts unrelated to HHCS, as controls. Soluble
homogenates of the solid phases, which were amorphous and not suitable
for histological examination, were obtained by incubation in 0.2%
NP-40 followed by centrifugation. These samples and the washing
solutions were analyzed for ferritin content with the ELISA assays
described above. Iron content was measured by electrothermal atomic
absorption spectroscopy (EAAS) using a Varian Spectra A
300 (Varian, Sugar Land, TX) equipped with a graphite
tube atomizer (Zeeman effect). Ferritin purification was achieved by
heating the homogenates at 75°C for 10 minutes followed by
anion-exchange column chromatography (20 HQ-Poros). The ferritin
fraction was concentrated on Centricon 30 (Amicon, Danvers,
MA). To test in vitro functionality, the purified protein
(0.5 mg/mL to 0.1 µmol/L) was incubated with 0.1 mmol/L ferrous
ammonium sulfate in 0.1 mol/L HEPES, pH 7.0, for 2 hours at room
temperature, run on 7.5% polyacrylamide nondenaturing gel
electrophoresis, and stained with Prussian blue.10
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RESULTS |
Ferritin in immortalized lymphocytes.
Figure 1 summarizes the substitutions of
human ferritin L-chain IRE sequences, which have been so far associated
with HHCS, including the ones analyzed here. We determined ferritin
content in the lymphoblastoid cells lines from subjects of HHCS
families coded Verona-1,14 Verona-2,15 and
Paris-117 cell homogenates with ELISA assays based on
monoclonal antibodies for the H- and L-ferritin and calibrated on
recombinant human H- and L-homopolymers.23,24 The two
control cells had similar levels of ferritin: about 100 ng of the
L-type and about 190 ng of the H-type per milligram of total soluble
protein. The three HHCS cells had an accumulation of L-ferritin up to
more than 2,000 ng/mg of protein (ie, 13-fold to 28-fold higher than
controls), and a variable decrease in H-ferritin content
(Table 1). In HHCS cells, the L:H ferritin
ratios ranged between 15 and 118:1, whereas in control cells, it was
approximately 0.5:1.
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| Fig 1.
Predicted secondary structure of L-subunit IRE and
mutations so far described in HHCS families. Town names indicate the
different mutations: Paris-114 is the same as
Montpellier-115 and differs from Verona-1,16
Verona-2,17 Pavia-1 and Pavia-2,18 and Paris-2
and Milano-1.20
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To assess the iron-dependent regulation of ferritin expression, the
cells were incubated for 18 hours in the presence of 100 µmol/L of
the chelator desferrioxamine (DFO) or of 100 µmol/L Fe(III) as ferric
ammonium citrate (FAC) (Table 2). In the
control cells, iron supplementation determined an  2-fold increase
of both H- and L-ferritins, and iron chelation a 1.3-fold to 2-fold decrease of the ferritins, as expected. In the Verona-1 cells, H-ferritin expression was similarly modulated by iron, while L-ferritin appeared to be almost totally insensitive to either DFO of FAC treatment (Table 2). Similar results were obtained with Verona-2 and
Paris-1 cells (not shown).
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Table 2.
Ferritin Concentration in Control and Verona-1
Lymphoblastoid Cells With and Without Iron Supplementation or
Chelation
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In HHCS cells, the large excess of L- over the H-ferritin (ratio up to
118:1, to be compared with the 23:1 of molecules with a single H-chain
) suggested that at least part of the L-chain accumulated in
homopolymers, but the proportion of the ferritin L-homopolymers and the
subunit composition of the ferritin heteropolymers remained unknown. To
separate the two ferritin populations, we performed sequential
immunoprecipitation experiments of soluble homogenates from
35S-methionine-labeled cells. The first precipitate,
induced by saturating amounts of monoclonal antibody specific for the
H-chain, was expected to contain all available H-chains and the
associated L-chains, ie, the heteropolymeric fraction of
ferritin.24 The supernatant was further incubated with
saturating amounts of anti-L-ferritin antibody, which precipitate the
possible remaining ferritin L-chains not associated with the H-chain,
ie, L-homopolymers. We found that in metabolically-labeled control
cells the first precipitate contained comparable amounts of H- and
L-chains, plus some nonspecific bands due to the interaction of protein
A with endogenous immunoglobulins of the lymphoblastoid cells, and the
second precipitate did not contain detectable ferritin subunits
(Fig 2A). In contrast, two precipitates
from Verona 1 cell homogenates contained comparable amounts of L-chain,
the difference being a minor proportion of H-chain in the first
precipitate. Similar results were obtained with Verona-2 and Paris-1
cells (not shown). The findings indicate that HHCS cells express two
immunologically and structurally distinct ferritin populations of
similar size: one composed of L-homopolymers and the other one of
heteropolymers with an estimated content of 1-2 H-chains per 24-mer.
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| Fig 2.
Sequential immunoprecipitation experiments of
metabolically labeled lymphoblastoid cells with antibodies specific for
human ferritin H-chain ( H) and L-chain (L). (A) Control cells
(C1) and Verona-1 cells (Ver-1) were metabolically labeled with
35S-methionine, lysed, and 2 × 106 cpm of the
soluble fraction of homogenates precipitated with saturating amounts of
anti-H chain antibody. The soluble fraction was then precipitated again
with saturating amounts of anti-L-chain antibody. The precipitates
were analyzed on SDS-PAGE under denaturing conditions and exposed to
autoradiography. (B) Verona-1 cells were metabolically labeled with
35S-methionine and immunoprecipitated as in (A), the
pellets were resuspended in 4 mol/L urea, and separated on 6%
polyacrylamide gels containing 4 mol/L urea under conditions that
disrupt antibody antigen interactions without affecting ferritin
structure. (C) As in (B), except that cells were metabolically labeled
with 59FeNTA.
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A similar approach was used to analyze the function of the two ferritin
populations: cells were metabolically labeled either with
35S-methionine or 59FeNTA, the homogenates
sequentially immunoprecipitated with anti-H and anti-L ferritin
antibodies, as above, and the precipitates resuspended in 4 mol/L urea
under conditions that disrupt the association with the antibody without
affecting the structural integrity of the highly stable ferritin
molecules.24 The samples were then separated on
nondenaturing PAGE and exposed to autoradiography (Fig 2B and C). The
two precipitates from 35S-methionine-labeled Verona-1
cells showed a similar ferritin content, whereas those from the
corresponding 59Fe-labeled cells differed: the first
precipitate retained the radioactive iron, and the second remained
unlabeled. This result was confirmed on three separate experiments on
Verona-1 cells and twice on Verona-2 and Paris-1 cells. The finding
implies that although the heteropolymeric and homopolymeric ferritin
populations are synthesized in similar amounts, only the heteropolymers
can take up iron.
Cellular iron incorporation.
Cells were incubated for 18 hours with 59FeNTA in the
presence of 200 µmol/L ascorbic acid and, after washing, the
radioactivity associated with cells or with ferritins was determined.
Total cellular iron uptake was slightly higher in the three HHCS cells than in control (Fig 3A), but the
experiments showed some variability. Autoradiography of
59Fe-labeled cellular homogenates separated on
nondenaturing PAGE showed that essentially all protein bound
radioactivity was associated with ferritin and that band intensities
were comparable and slightly stronger in the three HHCS than in the
control cells (Fig 3B). Densitometry of various experiments indicated
some variability (Fig 3C). It should be noted that
59Fe-labeled ferritin in HHCS cells had a slower mobility
than that of control cells (Fig 3B), in agreement with the higher
L-subunit content. Analyses of IRPs activity performed by band shift
assays did not show differences between HHCS and control cells (not
shown). It was concluded that despite the large increase in L-ferritin and the dramatic shift in the isoferritin profile, the cellular iron
metabolism of HHCS cells is apparently similar to that of controls.
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| Fig 3.
59Fe incorporation in lymphobastoid cells and
ferritin. (A) Cells were incubated for 18 hours in RPMI supplemented
with 10 µmol/L 59FeNTA and 200 µmol/L ascorbic acid,
washed, and the radioactivity in the soluble homogenates counted. The
values were normalized on the data of the control cells. Means and
standard deviation (SD) of three independent experiments are
shown. (B) The soluble homogenates from the same number
of cells were subjected to electrophoresis under nondenaturing
conditions and exposed to autoradiography. Ferritin mobility is
indicated by the arrow. (C) Means and SD of densitometometric
quantitation of three independent experiments, as in (B), are
shown. Values normalized on the control cells.
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Ferritin in the lens.
The lens specimens from cataract surgery of an HHCS subject of Verona-1
family were made available to us. The solid samples were too damaged
for histological examination and were homogenized in the presence of
the mild NP-40 detergent. The ferritin concentrations in the soluble
extracts determined with the H- and L-ferritin ELISA assays are
reported in Table
3. In the control, age-matched cataracts, ferritin was present in
detectable amounts (0.01% of total proteins) and the L- was more
abundant than the H-ferritin type (L:H ratio 3-6:1). In HHCS lens,
L-ferritin accounted for a significant proportion of total proteins
( 0.15%), whereas H-ferritin content apparently decreased. Similar
data were obtained in the washing solutions obtained from surgery (not
shown). EAAS analyses detected similar levels of iron in trace amounts
in the homogenates of HHCS and control lens (0.008 µg/mg proteins in HHCS; 0.01 ± 0.002 µg/mg proteins in four controls).
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| Fig 4.
Analysis of the insoluble fraction of lens extracts.
SDS-PAGE of the insoluble fraction of homogenates from lens specimens of a control (C1) and Verona-1 subject (Ver-1). (A) Coomassie blue
stain of the precipitates. (B) Immunoblotting stained with anti-L
ferritin antibody of the two insoluble fractions and a control of
recombinant L ferritin (rLF). The arrows indicate the mobility of H and
L chains.
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| Fig 5.
Electrophoretic analyses of ferritin purified from HHCS
lens. (A) SDS-PAGE of the purified ferritin compared with human H and L
ferritin chains, Coomassie blue stain. (B) Nondenaturing PAGE of the
purified lens ferritin compared with recombinant H and L ferritins,
Coomassie blue stain. (C) The same as in (B), except that ferritins
were preincubated with Fe(II) (1,000 Fe atoms per molecule, pH 7.0) and
the gels stained with Prussian Blue.
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SDS-PAGE analysis of the insoluble fractions of the lens homogenates
showed a major band of size compatible with -crystalline (23 kD). Immunoblotting with anti-L antibodies did not show
the presence of L-chain in the control and HHCS specimens
(Fig 4). Thus, the band was attributed to
-crystalline, and no insoluble ferritin could be detected.
The amount of ferritin in HHCS lens was sufficient to attempt
purification, and heat treatment at 75°C of the homogenate yielded a ferritin preparation 90% pure, as judged by SDS-PAGE analysis. It
contained a single major band comigrating in SDS-PAGE with human
ferritin L-chain (Fig 5A) recognized by anti-L-ferritin antibody (not shown). On nondenaturing PAGE, the protein ran slightly faster than the recombinant L-chain homopolymers (Fig 5B). Similar mobility differences between natural and recombinant ferritins have
already been reported and attributed to the lack of blocking of the
N-terminal amino group in the proteins expressed from E coli.27 The protein could be stained with Prussian Blue
only after in vitro preincubation at pH 7.0 with Fe(II), like the
recombinant L-homopolymers (Fig 5C), indicating an iron-poor ferritin.
EAAS analyses detected only trace amounts of iron in the preparation from HHCS lens, compatible with a ferritin iron content below 20 Fe
atoms per molecule.
| |
DISCUSSION |
Ferritin expression in immortalized lymphocytes.
The point mutations of Verona-1, Paris-1, and Pavia-1, which affect
residues in the loop and bulge of IRE structure involved in IRPs
binding, were shown to abolish the in vitro specific binding to
IRPs.14,17,19 The extensive Verona-2 deletion (Fig 1) could be expected to lead to a more severe phenotype; however, the clinical symptoms of the four families were remarkably similar, with serum ferritin in the range of 1,000 to 2,000 µg/L and early onset of bilateral cataracts.15 Accordingly, we found very similar
L-ferritin levels in Verona-1 and Paris-1 and Verona-2 lymphoblastoid
cells (Table 1). Iron chelation and supplementation treatments
modulated H- and L-ferritin accumulation in the control cells, but had
no effect on L-ferritin accumulation in HHCS cells, a finding
consistent with the observation that even severe iron deprivation did
not modify serum ferritin levels in Verona-1 subjects.14
These data suggest that the activity of the normal IRP-regulated
L-ferritin allele is obscured by the constitutive upregulation of the
abnormal allele. However, an analysis of the de novo synthesis of
ferritin subunits in the same Paris-1 cells, and under the same
conditions of iron supplementation or chelation, showed that L-chain
synthesis is iron modulated, albeit slightly,17 indicating
that the normal L-chain allele is active.
Our data indicated that H-ferritin tended to be lower in HHCS than
control cells possibly caused by L-ferritin overexpression (Table 1).
However, the sandwich-type ELISA assays we used need at least two
separated antibody binding sites for recognition, and the H-ferritin
assay was shown to underestimate the H-chain-poor isoferritins.28 Ferritin heteropolymers in HHCS cells
contain very few H-chains (1 or 2 per molecule) (Fig 2A). Thus, the
decrease of immunologic H-ferritin in HHCS cells may just reflect a
structural shift of isoferritins from H-chain-rich to H-chain-poor
molecules, rather than a decrease in H-chain synthesis/accumulation, in
agreement with data presented by Beaumont et al,17
indicating that H-chain de novo synthesis in control and Paris-1 cells
is comparable.
The cellular and total ferritin capacity to incorporate radioactive
iron (Fig 3) and IRPs activity were comparable in HHCS and control
cells, indicating that the size of the regulatory iron pool and iron
metabolism were not modified by L-chain overexpression. This is in
agreement with the clinical observation that HHCS subjects have no
evident abnormalities in iron metabolism. On the other hand, L-chain
overexpression induced the formation of L-homopolymers up to about 50%
of total ferritin and caused a dramatic shift in the heteropolymer
composition (Fig 2). The strong tendency of ferritin subunits to form
copolymers11 favors L-chains coassembly with H-chains until
these are available and then form L-homopolymers. This implies that for
constant amounts of limiting H-chains, the total amount of isoferritins
containing 5% H-chain (as in HHCS cells) is 10-fold that of
isoferritins with 50% H-chain (as in control cells), in agreement
with the experimental data.
That the L-homopolymers did not take up radioactive iron within the
cells (Fig 2C) was expected because these molecules do not incorporate
iron during expression in E coli and in vitro under conditions
of slow rates of iron autoxidation,6,7 and the natural
iron-poor isoferritins are the richest in L-chains.29 More
unexpected was the finding that the overall activity of heteropolymeric ferritins in HHCS and control cells was comparable, despite the major
differences in the amount and composition. In vitro studies showed that
the ferroxidase activity in the heteropolymers increases almost
linearly with the increase of H-chain in the range of 4% to 30%
to 50% H-chain.11,30 Because this is the range of isoferritin modification observed in the control and HHCS cells, the
total H-chain activity should not be affected by isoferritin reshuffling in the two cell types. In other words, the increased number
of active ferritin molecules appears to be compensated by the lower
specific activity of the single molecules in HHCS cells.
We think that this interpretation of the data has several implications.
First, the overall ferritin iron sequestration activity within the
cells is mainly determined by the amount of available H-chain, rather
than by isoferritin composition, consistent with the finding that the
artificial overexpression of H-chain in mouse erythroleukemic cells
induced an iron-deficient phenotype.12 Second, in vitro
ferritin iron incorporation increases with H-chain content up to 30%
to 50%, then it levels off.11,30 Consequently, an L-chain
overexpression may have little effect in tissues where L-chain is
predominant, whereas in tissues where H-chain is predominant (such as
heart 66%, erythroid cells >70%), it may dilute the H-chains and
determine an increase of ferroxidase activity; this is expected to
decrease the regulatory iron pool and possibly explain the observation
that HHCS subjects are prone to develop iron deficiency. Finally, the
major effect of L-chain overproduction is to expand cellular iron
storage capacity (ie, the number of ferritin molecules capable of
incorporating iron) without major effects on cellular iron metabolism.
This observation goes some way to explain why tissues, in particular
liver, respond to chronic iron overload in a similar way, by increasing
L-chain accumulation with an expansion of total ferritin population and
an isoferritin shift towards L-rich molecules,7 a mechanism
that increases ferritin iron storage capacity without altering
regulatory iron pool. Thus, in vitro and in vivo studies indicate that
the L-chain biological role is truly of iron storage, whereas the
H-chain has more complex functions to regulate iron metabolism and to
store it.
Ferritin in the lens.
The only tissue where L-ferritin overexpression shows clinical
manifestation in HHCS is the lens, which becomes opaque. We had the
opportunity to analyze specimens from surgical cataract treatment of an
HHCS and two control subjects. The procedure involves a harsh treatment
of the samples, which cannot be examined by immunohistochemistry.
Immunologic evaluation of cellular homogenates showed that ferritin is
a significant constituent of non-HHCS lens accounting for about 0.01%
of the total soluble proteins, whereas in HHCS lens, it accounted for
about 0.15%. In controls, the L:H ferritin ratio was 3-6:1 and
increased to 214:1 in HHCS samples. This indicates that in normal lens
ferritin is L-rich and becomes even more so in HHCS. Biochemical
analysis confirmed the immunologic data: ferritin could be purified
from HHCS lens homogenates, but not from control lens and could be
stained with Prussian blue after in vitro iron loading, demonstrating a
functional iron-poor ferritin. It showed an electrophoretic mobility
slightly faster than the control recombinant L-ferritin (Fig 5B and C), a difference fully compatible with previous observations that the
exposed N-terminal amino groups are blocked by acetylation in the
natural human ferritins and not in the recombinant ferritins from E
coli.27 The extra positive charges reduce the
electrophoretic mobility of the recombinant proteins in native PAGE.
The presence of H-chain in the lens preparation was revealed by
sensitive immunoblotting assays (not shown), but not in SDS-PAGE (Fig
5A), a finding consistent with the low proportion (<3%) indicated by
the immunoassays. It should be noted that on SDS-PAGE, the purified
HHCS lens ferritin showed a single peptide corunning with the L-chain,
while the glycosylated G-subunit (23 kD), typical of serum ferritin,
was undetectable even with immunoblotting stains (not shown). These data support the hypothesis that ferritin in HHCS lens is of the intracellular type, made of L-chains with trace amounts of H-chains, like the one of the corresponding lymphoblastoid cells composed of
L-homopolymers and L-rich heteropolymers. This ferritin could not be
stained by Prussian blue, and attempts to evaluate Fe concentration by
atomic absorption indicated an iron loading below 20 Fe atoms per
molecule (in the liver, it is approximately 1,500 Fe atoms per
molecule).
Ferritin in HHCS lens is readily soluble and does not form aggregates
in vitro, suggesting that ferritin precipitation is not the primary
cause of lens opacity. Clearly, the absence of aggregates in vivo can
be ruled out only after in situ staining.
Our data do not provide direct indications on the relationship between
cataract and L-ferritin overexpression, and we may speculate that this
may alter the hydrodynamic equilibrium necessary to maintain lens
transparency by affecting either the solubility of other proteins or
the antioxidant defense of this tissue. It is known that the ocular
lens is exposed to high levels of oxidative stress from oxidants
surrounding the lens and from oxidants generated within the
lens.31 The aqueous humor and the lens contain high levels
of H2O2 and vitamin C, agents that can act as
strong prooxidants in the presence of iron,31 and oxidative
damage appears a primary cause of cataract formation.31 It
was observed that the oxidative damage of lens structural proteins
(crystallines) peptides induced by Fe and H2O2
not only reduced their solubility, but also decreased the chaperone
activity of -crystalline.32,33 An antioxidant role of
ferritin in the lens has been recently proposed by McGahan et
al.34
| |
FOOTNOTES |
Submitted November 11, 1997;
accepted January 21, 1998.
Supported in part by Grant No. E.547 of Telethon-Italy (to D.G.) and by
the CNR (Italy)-INSERM (France) collaboration programme.
Address reprint requests to Sonia Levi, PhD, Dibit, H. San
Raffaele, Via Olgettina 58, 20132 Milano, Italy.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract