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INTRODUCTION |
The iron storage protein ferritin has a structure highly conserved
among plants, animals, and bacteria. Ferritin has 24 subunits that are
assembled into a spherical shell characterized by 4-, 3-, and 2-fold
symmetry (432 symmetry). Up to 4500 Fe(III) atoms can be stored as an
inorganic complex in the inner cavity of assembled ferritin, rendering
these atoms nontoxic and biologically available (1). Structural
analyses of vertebrate and bacterial ferritins indicate that each
subunit consists of a four-helix bundle (helices A, B, C, and D) and a
fifth short helix (E helix). The E helix exists around the 4-fold
intersubunit symmetry axes of the protein shell and forms a hydrophobic
pore (1-4). In mammals, two distinct ferritin subunits (H and L) are
found (5). These subunits have 50% identity and similar
three-dimensional structures. The H subunit has ferroxidase activity
(6, 7) and catalyzes oxidation of Fe(II), which is the first step in
iron storage. The L subunit promotes nucleation of the iron core (8).
The synthesis of ferritin in vertebrates is regulated during
translation. It was suggested that both the 5'- and 3'-untranslated
regions of ferritin mRNA contributed to translational control (9).
The iron-responsive element, which was first identified in the 5'-
untranslated region of human ferritin mRNA (10), is highly
conserved among mammals and other vertebrates. Despite the probable
common ancestry of plant and vertebrate ferritins (12), expression of
plant ferritin is regulated primarily at the transcriptional level, in
response to iron administration (11). A sequence with similarity to the iron-responsive element is absent in the 5'-untranslated region of
plant ferritin mRNA; however, Wei and Theil (13) recently suggested
the existence of an "iron regulatory element" in the promoter region of the soybean ferritin gene, which controls the transcription together with a trans-acting factor (13).
Only one polypeptide chain type has been identified as a functional
subunit of plant ferritins (11). Like the mammalian H subunit, with
which it shares about 40% sequence homology, this subunit has
ferroxidase activity (14, 15). During the last decade, evidence for
ferritin multigene families in maize (16), cowpea (17, 18), and soybean
(18) was provided. Cowpea has at least four different ferritin genes,
one that encodes a protein with 97% sequence identity to soybean
ferritin (11, 19). The peptides encoded by the other cowpea genes in
the ferritin family are more divergent. Despite the evidence that
multiple ferritin genes exist in plants, it is not clear whether the
products of these different genes are functionally divergent. This is
in contrast to the situation reported in pigs, frogs and salmon, where
two functional H-type subunits showing different tissue specificity are
found (20-22).
The ferritin subunits of soybean and many other legumes are synthesized
as 32-kDa precursor proteins (23), which contain a unique two-domain
N-terminal sequence (19). These N-terminal domains are not present in
mammalian or other ferritins. The first domain, which consists of
40-50 residues and is known as the "transit peptide"
(TP),1 is presumed to
facilitate transport of the ferritin precursor to plastids (14). The
function of the second domain, which is a part of the mature protein
and is termed extension peptide (EP), is currently unclear. The
ferritin subunit purified from soybean seed is 28 kDa, but it is
apparently converted in significant amounts to a 26.5-kDa subunit (19,
24). In 1990, Ragland et al. (19) suggested that such
truncation occurs by cleavage of the EP during germination or free
radical damage. They considered the 28-kDa subunit to be an
iron-containing form and the 26.5-kDa subunit to be an iron releasing
form (23, 25). This proposal has yet to be unsubstantiated by
experimental evidence.
Here we report evidence that the iron conversion mechanism of soybean
seed ferritin involves two distinct subunits. One of these is novel and
presented here for the first time, while the other is identical to that
previously identified. These data about soybean ferritin maturation
allow us to hypothesize a novel mechanism by which iron is released
from the assembled ferritin molecule.
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EXPERIMENTAL PROCEDURES |
Purification of Native Soybean Ferritin--
500 g of soybean
dry seeds (Glycine max Merrill cv.
Kita-no-shiki) were crushed into flour by a mill. The
soybean seed flour was suspended in 50 mM Tris-HCl buffer
(pH 7.5) containing 1 mM EDTA and 10 mM
2-mercaptoethanol, homogenized, and centrifuged at 10,000 × g for 10 min. The supernatant was fractionated using 20%
saturation of ammonium sulfate. An amber-colored precipitant was
collected by centrifugation and dialyzed against 50 mM
Tris-HCl (pH 7.5) buffer. The dialyzed sample was applied to a
DEAE-Toyopearl (TOSOH, Tokyo, Japan) column previously equilibrated by
50 mM Tris-HCl (pH 7.5) containing 1 mM EDTA.
The column was washed with a buffer containing 0.15 M
sodium chloride, and amber-colored ferritin was eluted. The eluate was
again fractionated with 20% saturated ammonium sulfate. The
supernatant was collected by centrifugation, applied to a
butyl-Toyopearl (TOSOH) column and eluted with a linear gradient of 20 to 0% saturated ammonium sulfate. Fractions containing soybean
ferritin were pooled and dialyzed against 50 mM Tris-HCl
(pH 7.5) containing 1 mM EDTA and applied to Q-Sepharose columns (Amersham Pharmacia Biotech). Proteins were eluted with a
linear gradient of sodium chloride from 0 to 0.7 M.
Fractions containing soybean ferritin were pooled and concentrated and
finally loaded to Superdex 75-pg gel filtration columns (Amersham
Pharmacia Biotech) equilibrated before use in 10 mM
Tris-HCl buffer (pH 7.5) containing 0.15 M NaCl.
Apoferritin was obtained using methods described by Chasteen and Theil
(26). Purified ferritin was dialyzed against 50 mM HEPES/NaOH buffer (pH 7.0) containing 1% thioglycolic acid, followed by successive changes of HEPES buffer with (0.1%) or without
thioglycolic acid. Protein was then dialyzed against HEPES buffer
containing 13 g/liter of Chelex-100 (Bio-Rad) and 0.2 M
NaCl and finally dialyzed against deionized water. The concentration of
purified proteins was determined using a protein assay kit (Bio-Rad)
and densitometer (Amersham Pharmacia Biotech).
Amino Acid Sequence Analysis--
Soybean ferritin subunits were
separated electrophoretically using a 12.5% SDS-polyacrylamide gel
(SDS-PAGE) and transferred to a polyvinylidene difluoride membrane. The
membrane was stained with 0.1% Ponceau S (Sigma) in 2% (v/v) acetic
acid, and two distinctive bands of 28.0 and 26.5 kDa were independently
cut from the membrane. N-terminal amino acid sequence analysis was
performed by automatic Edman degradation using an Applied Biosystems
model 477A pulse-liquid sequencer system.
C-terminal amino acid sequence analysis of the 26.5-kDa subunit was
performed using p-phenylene diisothiocyanate controlled pore
glass (Sigma) (27). Briefly, the 26.5-kDa subunit was digested with
lysyl endopeptidase, and the resulting peptide fragments were
covalently bound to p-phenylene diisothiocyanate controlled pore glass. Only the C-terminal fragment, which contained no lysyl residue, was detached by cleavage of the first residue with
trifluoroacetic acid (27). The sequence of the C-terminal fragment was
determined by automatic Edman degradation, as described above.
Cloning of a Novel Ferritin Gene cDNA from
Soybean--
Primers for amplification of the subunit containing a
novel N-terminal amino acid sequence were designed using the expressed sequence tag sequences (AW185525, AI966037, AW397605, AI443722,
AI900240) of soybean registered in GenBankTM. 5'-Rapid
amplification of cDNA ends (RACE) and 3'-RACE was carried out using
a SMART RACE cDNA Amplification kit (CLONTECH)
according to the manufacturer's instructions; total RNA extracted from
10-day-old seedlings contained developed bifoliates, an epicotyl
and a terminal bud was used as a template. About 10 candidate sequences
of a target sequence coding the novel subunit were determined.
Preparation of Recombinant Soybean Ferritin--
DNA sequences
encoding the mature region of soybean ferritin, previously reported by
Lescure et al. (11), were amplified by polymerase chain
reaction using the primers -TP (5'-GCGCATATGTCAACGGTGCCTCTCAC-3') and C
(5'-GCGGGATCCTAATCAAGAAGTCTTTG-3'). -TP and C contained NdeI
and BamHI restriction sites, respectively. The resulting fragments, which were missing the TP sequence, were ligated to the
NdeI and BamHI sites on the expression vector
pET3a (Novagen) to generate pESF. The expression plasmid pESF was
transformed into the Escherichia coli strain BL21(DE3)pLys.
An E. coli strain harboring the expression plasmid was
cultured in LB medium supplemented with ampicillin (50 µg/ml) at
37 °C. Bacterial growth was monitored with a spectrophotometer at
600 nm. When an absorbance of 0.6 was reached,
isopropyl-
-D-thiogalactopyranoside was added to a final
concentration of 1 mM. The cells were harvested by
centrifugation, and proteins were extracted using BugBuster protein
extraction reagent (Novagen). Recombinant ferritin was purified using
the same methods described above for native ferritin.
Soybean Ferritin Degradation Analysis--
Soluble protein was
extracted from 200 mg of soybean leaves by homogenization with 1 ml of
extraction buffer (50 mM K2PO4, 10 mM 2-mercaptoethanol, 0.1% Triton X-100, 0.1% sarcosine)
and sea sand followed by centrifugation at 12,000 rpm. The supernatant was used for degradation experiments of soybean ferritin. Recombinant ferritin or native soybean ferritin (150 ng each) were added to 20 µl
of the leaf extract and incubated at room temperature for 0, 1, 10, 30, 60, and 120 min, respectively. After SDS-PAGE and electroblotting to a
polyvinylidene difluoride membrane, protein immunodetection was
performed. Antiserum was raised against the soybean ferritin as
previously described (28). Probing of blots with soybean ferritin
antibody was carried out using anti-rabbit IgG sheep immunoglobulin
coupled with biotinylated horseradish peroxidase (Vectastain ABC kit;
Vector Laboratories, Burlingame, CA). Immunostain HRP-1000
(Konica, Tokyo, Japan) was used for visualizing the signal (28,
29).
Iron Incorporation and Iron Release Measurement--
Reactions
examining iron uptake by native soybean and recombinant ferritin were
performed in 100 mM HEPES/NaOH buffer (pH 7.0) with a
Fe2+/ferritin molar ratio of 1000:1 (0.1 mM
ferrous sulfate and 0.1 µM ferritin) at room temperature.
Iron incorporation by each type of ferritin was monitored by measuring
the absorbance at 310 nm (30, 31) using a UV spectrophotometer (U3000;
Hitachi, Tokyo, Japan).
For iron release experiments, native soybean and recombinant
apoferritins (2 µM each) were mineralized using freshly
prepared ferrous sulfate (1 mM) in 0.1 M MOPS
containing 0.2 M NaCl (15, 33) for 2 h at room
temperature and then overnight at 4 °C. The excess iron that was not
incorporated into the ferritin shell was precipitated by the
centrifugation and separated from the mineralization mixture. External
iron was then removed using Econo-Pac 10 DG (Bio-Rad). Iron release was
initiated by the addition of 1 mM sodium ascorbate and 4 mM ferrozine (15); the former was previously reported to
enhance the reductive release of iron from the ferritin shell (15, 34).
Exogenous Fe2+ was measured by the absorbance of the
Fe2+-ferrozine complex at 560 nm using the same
spectrophotometer described above. The data of iron uptake and release
were obtained from three different preparations. The levels of
reductive iron release by the two subunits were compared statistically
using t tests (p = 0.05).
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RESULTS |
Isolation of Soybean Ferritin from Seeds--
Soybean ferritin was
extracted from dry seeds and isolated using the following sequential
chromatographic purification steps: anion exchange, hydrophobicity, and
gel filtration. Soybean ferritin was eluted in a single peak after each
chromatographic step. Purified ferritin subunits were separated as two
peptides (28 and 26.5 kDa) by SDS-polyacrylamide gel electrophoresis
(Fig. 1A). Densitometric analysis indicated that the 28- and 26.5-kDa subunits are present in
purified native ferritin in nearly equivalent amounts (28 versus 26.5 kDa = 1:1.09). Nondenaturing gel
electrophoresis (native PAGE) resolved purified soybean ferritin as a
single complex, estimated to be about 550-560 kDa (Fig.
1B).

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Fig. 1.
SDS-PAGE and native PAGE analysis of purified
soybean ferritin. Samples were added after the final gel
filtration step (see "Experimental Procedures"). A,
purified soybean ferritin was analyzed by SDS-PAGE and stained with
Coomassie Brilliant Blue (lane 1). B,
native PAGE analysis of soybean ferritin. Lane 1,
native soybean ferritin purified from seed; lane
2, recombinant soybean ferritin expressed in E. coli. Lane M, protein markers and their
corresponding molecular masses.
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Amino Acid Sequence Analyses of Ferritin Subunits--
Comparisons
between the N-terminal sequences of the 28-kDa subunit
(Ala-Ser-Asn-Ala-Pro-Ala) and the 26.5-kDa subunit
(Ala-Ser-Thr-Val-Pro-Leu) revealed that the 28-kDa subunit was
different from any previously reported. Further determination for the
N-terminal sequence verified that the 28-kDa subunit was a novel
subunit (Fig. 2). The N-terminal sequence
of the 26.5-kDa subunit was identical to that of the EP region of the
sequence reported by Lescure et al. (11). Our analyses
revealed that the transit peptide is cleaved at the carboxyl side of
the 48th cysteine in the 26.5-kDa subunit, which contrasts with the
previously reported cleavage site at position 49 (alanine) (Fig. 2)
(11, 23).

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Fig. 2.
Deduced amino acid sequences of soybean
ferritin subunits. Top, amino acid sequence of the
novel, H-2 subunit identified during this study. Bottom,
amino acid sequence of the H-1 subunit, which is identical to the
sequence previously reported by Ragland et al.
(19). Conserved residues between the two subunits are shown in
black. Residues in green indicate those that have
been suggested to form the deduced ferroxidase center. The N-terminal
32 residues of the H-2 subunit determined during amino acid sequence
analysis are shown boxed in green. The
peptide fragment containing the C-terminal residue of the cleaved
26.5-kDa form of the H-1 subunit is shown boxed in
red. The cleavage sites of the TPs in both the H-2 and H-1
subunits are indicated by a yellow arrowhead. The
mature regions of both subunits are downstream from here. The cleavage
site for conversion of the H-1 subunit from 28 to 26.5 kDa is indicated
by a green arrowhead.
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To detect the site of cleavage for hypothesized conversion of the
28-kDa subunit to the 26.5-kDa subunit (11, 13), the C-terminal amino
acid sequence of the 26.5-kDa subunit was determined. The sequence of
the peptide fragment containing the C-terminal residue was
Ser-Glu-Tyr-Val-Ala-Gln-Leu-Arg-Arg (Fig. 2), which was the same as
that previously reported (11). The C-terminal residue was determined to
be the 234th arginine, which is situated 17 residues upstream from the
C terminus. These data demonstrated that the 26.5-kDa subunit in the
soybean seed ferritin is generated as a result of proteolytic cleavage
of the C-terminal 16 residues of the previously described subunit.
cDNA Isolation and the Deduced Amino Acid Sequence of a Novel
Ferritin Subunit--
A cDNA encoding a newly identified subunit
(28 kDa) of soybean ferritin was cloned using a polymerase chain
reaction-based strategy. The predicted amino acid sequence was compared
with the previously reported sequence of ferritin subunit (11), and several differences were found (Fig. 2). Based on the conservation of
several residues, thought to comprise the ferroxidase site (6, 7), we
defined the 26.5- and the 28-kDa subunits as H-1 and H-2, respectively.
The mature region of the H-2 subunit, in which the TP is detached from
the precursor, is composed of 209 residues. It is seven residues longer
than the H-1 subunit (202 residues). The amino acid sequence of the
mature region of the H-2 subunit has 82% identity with that of the H-1
subunit. In contrast to a high identity in the mature regions, low
sequence identity between the TP sequences of H-1 and H-2 is found
(41%). In the sequence of the mature region, the deduced helical
regions (A to E helices) (4) are highly conserved between the H-1 and H-2 subunits, while the loop region between the B and C helices and the
EP region are relatively variable. Specifically, the amino acid
sequence identities of the helical, loop, and EP regions are 90, 81, and 63%, respectively (Table I). The H-2
subunit has C-terminal extension consisting of five residues (four of them charged). A comparison between the C-terminal cleavage site of H-1
(
-amino side of position 234) with the putatively homologous position in H-2 (position 235) revealed the presence of an arginine in
H-1 and a leucine in H-2 (Fig. 2). The unsusceptibility of the H-2
subunit to C-terminal cleavage is probably due to the presence of
leucine instead of arginine at this position.
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Table I
The partial amino acid sequence identity between the H-1 and H-2
subunits
The amino acid sequences of both subunits were compared with each other
in eight parts.
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Degradation of the Soybean Ferritin H-1 Subunit--
The full
mature region of the soybean ferritin H-1 subunit was expressed in
E. coli. Native PAGE (Fig. 1B) and gel filtration (data not shown) analysis showed that the recombinant soybean ferritin
subunit assembles into a 24-mer, presumably in a similar fashion to
native ferritin. We abbreviate the recombinant H-1 subunit as
"rH-1" and native soybean ferritin purified from seeds as "seed
H-1/H-2." The rH-1 was composed of only the H-1 subunit in the
original, uncleaved form (28 kDa), while the seed H-1/H-2 included both
the H-2 and cleaved H-1 (26.5 kDa) subunits in nearly equal amounts
(Fig. 1A). The rH-1 was incubated with soybean leaf extract
(Fig. 3). Prior to the incubation with
the soybean leaf extract, the rH-1 subunit remained in its 28-kDa form;
however, it was degraded quickly following the addition of leaf
extract. About half the amount of the rH-1 subunit was degraded to the cleaved form (26.5 kDa) in 10 min, and after 1 h of incubation, the original 28-kDa form had completely disappeared; only a small amount of the cleaved 26.5-kDa form was detected. Subsequently, the
cleaved form degraded completely after 2 h of incubation. In
contrast, the seed H-1/H-2 was still detectable after 2 h of incubation, although the 28-kDa form was not present. Since we used an
antiserum raised against the H-1 subunit, specific detection of the H-2
subunit in seed H-1/H-2 was not expected to occur; this may explain the
absence of a 28-kDa band in Fig. 3.

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Fig. 3.
Degradation of recombinant and native soybean
ferritin. The rH-1 and seed H-1/H-2 were incubated separately in
soybean leaf extract. Ferritin subunits were detected by anti-H-1
subunit antiserum. Lane 1, soybean leaf extract;
lane 2, 150 ng of the rH-1 subunit;
lanes 3-8, 150 ng of rH-1 subunit after
incubation with leaf extract for 0, 1, 10, 30, 60, and 120 min,
respectively; lane 9, 150 ng of seed H-1/H-2;
lanes 10 and 11, 150 ng of seed
H-1/H-2 with leaf extract for 60 and 120 min, respectively.
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Effect of Cleavage on Iron Uptake and Release--
To investigate
the effects of C-terminal cleavage in H-1 on the multimeric complex, we
compared iron uptake and release activities between seed H-1/H-2 and
rH-1. A recombinant form of the cleaved H-1 subunit (whose C terminus
was deleted) did not assemble into a 24-mer (data not shown). Both rH-1
and seed H-1/H-2 showed iron uptake activity (Fig.
4A). Progression plots
indicated that the uptake rate of the seed H-1/H-2 was slower than that
of the rH-1, despite the presence in all of the subunits of ferroxidase
sites. Both the uptake rates were faster than that of Fe(II)
autoxidation (control).

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Fig. 4.
A, progression plots of iron uptake in
soybean ferritin. Experiments were performed in 0.1 M
HEPES-Na, pH 7.0, containing 0.1 µM of each ferritin type
(rH-1 and seed H-1/H-2) and 0.1 mM ferrous sulfate. The
control indicates the rate of Fe(II) autoxidation. B, rates
of iron release from assembled ferritin shells. Seed H-1/H-2 and rH-1
(2 µM) were mineralized in vitro by mixing
with 1 mM ferrous sulfate in 0.1 M MOPS (pH
7.0) and 0.2 M NaCl. Iron release from mineralized
ferritins was initiated by the addition of 1 mM ferrozine
and 4 mM sodium ascorbate. Released iron was detected in
the Fe2+-ferrozine complex form by monitoring the
absorbance at 560 nm. The results were obtained from three
experiments.
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The rate of reductive release of iron from seed H-1/H-2 and rH-1 was
assessed (Fig. 4B). The level of ferrous atoms released from
the ferritin shell was calculated via absorbance measurement of the
Fe(II)-ferrozine complex. At every assessed time, the amounts of the
Fe(II)-ferrozine complex were significantly larger with seed H-1/H-2
than with rH-1. This result suggested that cleavage of C-terminal 16 residues accelerated iron release from the protein shell.
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DISCUSSION |
In this study, we demonstrated that two polypeptide chains with
different sequences are found in soybean ferritin purified from seeds.
Previously, it was hypothesized that only one type of polypeptide
chain, which was in either a cleaved or uncleaved form, was present in
the multimer (11). We found that the two different subunits exist in
nearly equal amounts in the ferritin of soybean seeds. Two kinds of
subunits with different amino acid sequences were also detected
previously in clover seed (35), although one of them existed only at
very low levels, and its amino acid sequence was not unambiguously
identified. From amino acid sequence and cDNA analysis, we
designated the two soybean ferritin subunits as H-1 and H-2. H-1 had a
sequence identical to that previously described (11), and experiments
involving rH-1 and leaf extracts revealed that this 28-kDa subunit
could be readily converted to 26.5-kDa by cleavage of the 16 residues at the C terminus. In the amino acid sequence profiles, no
contaminating peaks were detectable, indicating that the H-1 subunit
was perfectly converted to the cleaved 26.5-kDa form. The H-2 subunit
had a novel amino acid sequence and, unlike H-1, appeared unsusceptible to cleavage, remaining in its 28-kDa mature form. These data are in
conflict with the previous hypothesis that the 26.5-kDa subunit is
generated by cleavage of the N-terminal EP of the 28-kDa subunit and
that the 28- and 26.5-kDa subunits are identical apart from this
cleavage (23).
In degradation experiments using the extract of soybean leaves, the
homo-24-mer of rH-1 was unstable compared with the seed H-1/H-2 (Fig.
3). The instability of the H-1 multimer is probably due to cleavage of
the C-terminal 16 residues in each subunit. In support of this
hypothesis, a mutant recombinant form of H-1, which lacked the
C-terminal 16 residues, could not assemble into a native ferritin-like
complex. Luzzago and Cesareni (37), and Levi et al. (36)
also remarked on the importance of the C-terminal region for shell
stability in human H-chain ferritin. Notably, the H-2 subunit was not
susceptible to cleavage of the C-terminal region and was stable during
incubation with the leaf extraction. These data indicate that the H-2
subunit stabilizes the native ferritin 24-multimer in soybean seeds.
In vertebrate ferritins, the three-dimensional structures of the single
subunit and assembled 24-mer have been analyzed in detail (2, 3, 7).
Ferritin subunits in the spherical 24-mer are related by 432 symmetry.
There are narrow channels around the 3-fold and the 4-fold symmetry
axes (1). Residues around the 3-fold channels are mainly hydrophilic,
and the channels are proposed as the main entrance for iron atoms
(38-40). In contrast, four subunits are tightly packed around the
4-fold channels, with hydrophobic interactions occurring among the
nonpolar residues in the E helices (1-3). Our results indicate that
conversion of the H-1 subunit from 28 to 26.5 kDa is due to the
cleavage of the C-terminal 16 amino acid residues. From amino acid
sequence alignment data and the deduced three-dimensional structure of plant ferritin subunits (41), the cleaved 16 residues correspond to the
E helix of vertebrate ferritin, which is a short helix forming
the narrow channels around the 4-fold intersubunit interaction axes. In
the case of amphibian red cell L-chain ferritin, it was reported that
the diameter of this channel is about 1.5 Å, while that of the 3-fold
channel is about 3.7 Å at its narrowest point (2). Thus, the channel
around the 4-fold axes does not appear to have enough pore size to
allow ions to move freely (2). What might the functional significance
of cleavage of the E-helices in soybean ferritin be? It is likely that
the pore size of the channel around the 4-fold axes would be expanded
drastically. Although half of the subunits (H-2) in soybean ferritin
are not susceptible to proteolysis, the pore generated by cleavage of the H-1 E-helices is expected to be large enough to allow iron atoms to
pass freely. Indeed, the rate of iron release from seed H-1/H-2 was
faster than that from the rH-1 (uncleaved 28-kDa form) (Fig.
4B). These data suggest that the rate of iron release is accelerated by cleavage of the E helix. In contrast to the case of iron
entrance in mammalian ferritin, few studies have been performed on iron
release pathways in either plant or animal ferritin (42). Therefore,
the large pore generated by the cleavage of the E helix provides a
novel hypothesis for iron release from ferritin. Conversely, the iron
uptake activity of seed H-1/H-2 was relatively low compared with the
rH-1, despite the fact that both the H-1 and H-2 subunits possess the
predicted ferroxidase center. The apparent difference in the rate of
iron uptake is probably due to the C-terminal cleavage in H-1, which
facilitates reductive release of incorporated iron from native
ferritin. C-terminal mutation in the human H-chain ferritin has also
been reported to affect iron incorporation and ferroxidase activity
(36).
In plant ferritin, some functional genes have already been identified
(16-18). Here, we have identified a novel soybean ferritin subunit
(H-2), whose maturation process is different from that of the
originally described subunit (H-1). The novel subunit proved to be one
of the major subunits of soybean seed ferritin. The primary structure
of the novel ferritin subunit was closely related to that of the known
(11) subunit (82% identity). However, the difference in the maturation
process of H-2 appears to promote the novel function of "iron
release." The 28-kDa (presumably H-2-like) and partially cleaved
26.5-kDa (presumably H-1-like) subunits are also found in pea and other
legume ferritins (22, 23, 35). Notably, a leucine is found at a similar
position in one of the cowpea ferritin subunits to that of the leucine
at position 235 in H-2 (Fig. 2) (18). Thus, like the case of soybean,
it is possible that the different sized subunits observed in many legume ferritins derive from different genes and play cooperative roles
in the storage and release of iron atoms.
In agreement with the results of Lobréaux and Briat (43),
concentration of ferritin subunits in soybean cotyledon decreased gradually during germination and finally disappeared after about 10 days (data not shown). To date, many studies have examined expression
of plant ferritin in legume plants and maize. While ferritin
accumulates in developing nodules, cotyledons, and embryo axes (43-45)
of soybean and pea, it has not been detected in green leaves in bean
(43). In conflict with the latter result, van der Mark and colleagues
reported that ferritin was detectable in normal green leaves of bean
(46, 47). Interestingly, these authors provided preliminary evidence
for the existence of multiple subunits of ferritin in bean (32). A
consensus on patterns of ferritin expression in plants has not yet been
reached, and we suggest that a contributing factor to this has been the
exclusive study of the H-1 gene and its product during analysis. We are currently analyzing the expression and tissue distribution of the newly
identified H-2 subunit, as well as the H-1 subunit, in order to
elucidate their respective functions and to obtain information on the
significance of the multigene family of soybean ferritin.