Biochem. J. (2010) 427, 313–321 (Printed in Great Britain)
313
doi:10.1042/BJ20100015
A novel EP-involved pathway for iron release from soya bean seed ferritin
Xiaoping FU*, Jianjun DENG*, Haixia YANG*, Taro MASUDA†, Fumiyuki GOTO‡, Toshihiro YOSHIHARA‡ and Guanghua ZHAO*1
*CAU and ACC Joint Laboratory of Space Food, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China, †Laboratory of Food Quality
Design and Development, Division of Agronomy and Horticultural Science, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan, and ‡Biotechnology
Sector, Environmental Science Research Laboratory, Central Research Institute of Electric Power Industry, 1646 Abiko, Chiba 270-1194, Japan
Iron in phytoferritin from legume seeds is required for seedling
germination and early growth. However, the mechanism by which
phytoferritin regulates its iron complement to these physiological
processes remains unknown. In the present study, protein degradation is found to occur in purified SSF (soya bean seed ferritin)
(consisting of H-1 and H-2 subunits) during storage, consistent
with previous results that such degradation also occurs during
seedling germination. In contrast, no degradation is observed with
animal ferritin under identical conditions, suggesting that SSF
autodegradation might be due to the EP (extension peptide) on
the exterior surface of the protein, a specific domain found only in
phytoferritin. Indeed, EP-deleted SSF becomes stable, confirming
the above hypothesis. Further support comes from a protease
activity assay showing that EP-1 (corresponding to the EP of
the H-1 subunit) exhibits significant serine protease-like activity,
whereas the activity of EP-2 (corresponding to the EP of the H-2
subunit) is much weaker. Consistent with the observation above,
rH-1 (recombinant H-1 ferritin) is prone to degradation, whereas
its analogue, rH-2, becomes very stable under identical conditions.
This demonstrates that SSF degradation mainly originates from
the serine protease-like activity of EP-1. Associated with EP
degradation is a considerable increase in the rate of iron release
from SSF induced by ascorbate in the amyloplast (pH range, 5.8–
6.1). Thus phytoferritin may have facilitated the evolution of the
specific domain to control its iron complement in response to cell
iron need in the seedling stage.
INTRODUCTION
26.5 kDa (H-1) subunit and a 28.0 kDa (H-2) subunit, which share
∼ 80 % amino acid sequence identity [8,9]. The two subunits are
encoded by two distinct genes, SferH-1 (GenBank® accession
number M64337) and SferH-2 (GenBank® accession number
AB062754) respectively [8], and are synthesized as a precursor
(32 kDa) that contains a TP (transit peptide) and a following EP
(extension peptide) at its N-terminal. The TP is responsible for the
precursor targeting plastids [10]. Upon transport to the plastids,
the TP is cleaved from the subunit precursor, producing the
mature subunit which assembles in a 24-mer ferritin within
the plastids [11]. Thus in mature phytoferritin, 24 EP domains per
molecule represent the major structural difference between animal
and plant ferritin. In PSF, each EP domain is composed of 24
amino acid residues, 11 of which form a specific α-helix, termed
the P-helix, flanked by proline residues (X and L) [6]. Differing
from the TP in function, it was recently discovered that the EP
serves as a second binding and ferroxidase centre contributing to
iron core mineralization at high ferritin iron loadings (> 48 iron/
protein shell) [12], indicative of the role of the EP in iron oxidative
deposition in phytoferritin. Although Arabidopsis ferritin is
considered crucial to protecting cells against oxidative damage
rather than for iron storage [13], in legume seeds, the majority
of total iron is stored in ferritin in the amyloplast; such storage
is known to meet plant demand during seedling germination and
growth [1,14]. Therefore elucidating whether or not the EP plays
a role in phytoferritin iron release in the seedling stage is the focus
of the present study.
In the present study, protein degradation was found to occur
upon SSF (soya bean seed ferritin) standing at 4 ◦C, an observation
consistent with a previous report where PSF showed the same
Ferritins are a class of multimeric iron storage and detoxification
proteins. The importance of their dual function is underscored
by their ubiquitous distribution throughout all organisms, with
the exception of fungi. The ferritin complex has 24 subunits
assembled into a spherical shell characterized by a 432-point
symmetry. Up to 4500 Fe3+ atoms can be stored either as the
crystalline mineral ferrihydrite or as amorphous hydrous ferric
oxyphosphate in the inner cavity of the assembled ferritin shell
[1–3]. Structural analyses of vertebrate, plant and bacterial
ferritins indicate that each subunit consists of a four-helix bundle
(helices A, B, C and D) and a fifth short helix (helix E) [1,4,5].
In mammals, two distinct ferritin subunits (H and L) are found
with similar three-dimensional structures. The H subunit has
ferroxidase centres responsible for fast Fe2+ oxidation. In contrast,
L subunits lack ferroxidase centres, and thus do not exhibit fast
Fe2+ oxidation kinetics, but facilitate nucleation of the mineral
core [5].
Amino acids involved in the definition of the ferroxidase centre
are strictly conserved in all plant ferritins except for PSF (pea seed
ferritin), where a histidine residue is replaced with a glutamic acid
residue at position 62 of the amino acid sequence in the ferroxidase
centre [6,7]. However, plant and animal ferritins are remarkably
different in their cytological localization. In contrast with animal
ferritin existing in the cell cytoplasm, plants store iron within
ferritin mainly in plastids, such as the amyloplast in seeds [3].
Unlike animal ferritin, in which two types of subunits (H and L)
occur, only the H-type subunit has been described in phytoferritin.
Ferritin from dried soya bean seed consists of two subunits, a
Key words: autodegradation, extension peptide, iron release,
phytoferritin, serine protease-like activity.
Abbreviations used: AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; AMC, 7-amino-4-methylcoumarin; Boc, t-butoxycarbonyl; EP, extension peptide;
MALDI–TOF-MS, matrix-assisted laser-desorption ionization–time-of-flight MS; MCA, (7-methoxycoumarin-4-yl)acetyl; PSF, pea seed ferritin; rH-1 (rH-2),
recombinant H-1 (H-2) ferritin; SSF, soya bean seed ferritin; TP, transit peptide; WT, wild-type.
1
To whom correspondence should be addressed (email [email protected]).
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314
X. Fu and others
protein degradation taking place during seed germination [7,14].
Further studies revealed that the EP of SSF has serine proteaselike activity, which causes its degradation, probably because of
its exposure to the exterior surface of the protein. Interestingly,
removal of the EP from WT (wild-type) SSF considerably
facilitates iron release from ferritin with the presence of ascorbate
in the pH range 5.8–6.1, demonstrating that the EP is closely
associated with iron release from phytoferritin.
EXPERIMENTAL
Reagents
All chemicals used were of reagent grade or purer. Boc-GlnAla-Arg-MCA [where Boc is t-butoxycarbonyl and MCA is
(7-methoxycoumarin-4-yl)acetyl] and N-succinyl-Ala-Phe-LysMCA were purchased from Sigma–Aldrich. PMSF, AEBSF [4-(2aminoethyl)benzenesulfonyl fluoride], antipain, EDTA, pepstatin,
benzamidine and leupeptin were from Amresco.
Determination of EP protease-like activity
Potential serine protease activity was determined as previously
described [21,22] with slight modifications. Enzyme assays
using peptide–MCA substrates (Boc-Gln-Ala-Arg-MCA and Nsuccinyl-Ala-Phe-Lys-MCA) were performed by fluorometric
determination of liberated AMC (7-amino-4-methylcoumarin).
Briefly, 3.94 ml of 50 mM Tris/HCl buffer solution (pH 8.0)
containing 100 mM NaCl and 10 mM CaCl2 was added to 40 μl
of peptide MCA substrate dissolved in DMSO (10 mM), followed
by mixing with 20 μl of EP into a fluorescence cuvette at 25 ◦C.
The AMC liberation by enzymatic hydrolysis was monitored with
a Cary Eclipse spectrofluorimeter (Varian) at 25 ◦C for 120 s.
Fluorescence was measured using an excitation wavelength of
380 nm and an emission wavelength of 460 nm [22]. Control
samples were created under the same conditions, except that the
above protein solution was replaced with either 20 μl of BSA
(40 μg) or 20 μl of Alcalase (1000-fold dilution of Alcalase 2.4L).
Reactions between the EP and PMSF
Preparation of WT SSF, recombinant SSF and EP-deleted SSF
SSF was purified [12,15] and rH-1 (recombinant H-1) prepared
[8,16] as described previously. The expression plasmid for rH-2
(recombinant H-2) was constructed by inserting the H-2 cDNA
into the NcoI/BamHI site of pET21d using a PCR-based method.
Constructs were then introduced into the Escherichia coli strain
BL21 (DE3). Positive transformants of each construct were grown
at 37 ◦C on an LB (Luria–Bertani) medium supplemented with
50 mg/l carbenicillin, and protein expression was induced
with 100 μM IPTG (isopropyl β-D-thiogalactoside) when cell
density reached an A600 of 0.6. Both rH-1 and rH-2 were
purified using the same methods described above for native SSF.
Apoferritin was prepared as described previously [17,18]. The
concentrations of all ferritin types were determined according
to the Lowry method with BSA as a standard. SDS/PAGE was
performed under reducing conditions using 15 % mini-slab gels
according to a previously reported method [19]. Preparation and
identification of SSF with the EP deleted was carried out as
reported recently [12].
CD and fluorescence spectroscopy
CD spectra were recorded on a PiStar-180 spectrometer (Applied
Photophysics) at 25 ◦C under a constant flow of nitrogen gas.
Typically, a cell with a 0.1-cm pathlength was used for spectral
measurements between 190 and 260 nm. The spectra represent
an average of 6–10 scans. CD intensities reported in the figures
are expressed in ε (M−1 · cm−1 ). Fluorescence spectra were
measured using a Cary Eclipse spectrofluorimeter (Varian) at
25 ◦C with 280 nm as an excitation wavelength. The spectral
resolution was 1.0 nm.
Effect of protease inhibitors on SSF degradation
Different kinds of protease inhibitors were added to the
SSF solution to obtain the final concentration (5 mM PMSF,
5 mM EDTA, 20 μM pepstatin, 2 mM benzamidine and 20 μM
leupeptin). The effective inhibiting concentrations of the protease
inhibitors were used as described previously [20,21]. After
incubation of the mixture at 4 ◦C for 40–50 days, samples were
applied to SDS/PAGE. A control test was performed without the
addition of inhibitors.
c The Authors Journal compilation c 2010 Biochemical Society
A 1 μl aliquot of 140 mM PMSF was added to 100 μl of EP-1
(140 μM) in 5 mM Mops at pH 7.0, and the resulting solution
was incubated overnight at 4 ◦C. Subsequently, the solution was
mixed with 0.1 % TFA (trifluoroacetic acid) and 10 mg/ml CHCA
(α-cyano-4-hydroxy cinnamic acid) prior to MALDI–TOF-MS
(matrix-assisted laser-desorption ionization–time-of-flight MS)
analyses.
The degraded products of the ∼ 24.5 and 26.5 kDa SSF subunits
were separated electrophoretically using SDS/PAGE (15 % gels).
Gel spots were prepared as described previously [9]. All mass
spectra of MALDI–TOF-MS were obtained on a Bruker Ultraflex
III TOF/TOF (Bruker Daltonik) in positive-ion mode at an
accelerating voltage of 20 kV with a nitrogen laser (337 nm)
[12].
Kinetics of iron release from holoferritin
Iron release from SSF was investigated with a stopped-flow
instrument (a Hi-Tech SFA-20M apparatus) in conjunction
with a Cary 50 spectrophotometer (Varian) using the assay
procedure described previously [23]. All concentrations stated
were final after mixing the two reagents. The mixing dead
time was determined to be 6.8 +
− 0.5 ms using the DICP
(dichloroindophenol) and ascorbic acid test reaction [24]. The
development of [Fe(ferrozine)3 ]2+ was measured by recording the
increase in absorbance at 562 nm, and the iron released estimated
using ε562 = 27 900 M−1 · cm−1 [25]. The initial rate (ν 0 ) of iron
release was measured as described previously [26].
Preparation and pH measurement of amyloplasts from soya bean
seed cotyledons
Amyloplasts were prepared according to previously reported
methods, with slight modifications [27,28]. Soya bean seeds were
imbibed in ddH2 O (double-distilled H2 O) at room temperature
(25 ◦C) for 12 h in the dark and sown in a petri dish. After
germination at 12, 48 and 72 h respectively, amyloplasts were
isolated from cotyledons, which were chopped with a razor blade
and immediately frozen with liquid nitrogen. The cotyledon slices
were then homogenized with 10-fold ultrapure water in a Waring
Blendor. This homogenate was quickly filtered through a double
layer of nylon mesh (300 mesh). The filtrate was centrifuged at
500 g for 10 min at 4 ◦C. The pellets were washed 6–8 times with
EP autodegradation facilitates iron release
Figure 2
Figure 1
SDS/PAGE analysis of SSF degradation
The purified SSF was incubated in buffer B at 4 ◦C at different time intervals. Lanes 0–5
correspond to incubation times of 0, 10, 20, 30, 40 and 50 days respectively. Lane M, protein
markers and their corresponding molecular masses.
315
Native PAGE and SDS/PAGE analyses of SSF with the EP deleted
(A) Native PAGE at pH 8.3. (B) SDS/PAGE at pH 8.3. Lane 1, SSF with the EP deleted (molecular
mass 440 kDa); lane 2, wild-type SSF (molecular mass 560 kDa); and lane M, protein markers
and their corresponding molecular masses.
ultrapure water and were centrifuged under the same conditions
as above until the intact amyloplast was examined by light
microscopy (Supplementary Figure S6B). The amyloplast pellets
were crushed using an oscillator for 10 min. After centrifugation
at 13 200 g for 20 min at 4 ◦C, the pH value of the supernatant was
determined with a microelectrode (MI 402; Microelectrodes).
RESULTS
Autodegradation of SSF
SSF was purified to homogeneity with an apparent molecular
mass estimated to be approx. 560 kDa by native PAGE
(Supplementary Figure S1A at http://www.BiochemJ.org/bj/427/
bj4270313add.htm). Furthermore, SDS/PAGE analysis indicated
that the ferrritin complex contained two kinds of subunits (28.0
and 26.5 kDa) (Supplementary Figure S1B) present in purified
native ferritin in an approximately 1:1 ratio, as reported previously
[8]. To determine whether SSF was stable, the protein was allowed
to stand at 4 ◦C for 10–50 days. Since SSF exhibits good solubility
at pH 8.0, 50 mM phosphate buffer containing 150 mM NaCl
(buffer B) at pH 8.0 was used as a sample buffer. As shown in
Figure 1, two ferritin subunits began to degrade into ∼ 24.5 kDa
polypeptide(s) after 10 days of incubation. As the incubation time
increased from 10 to 50 days, degradation significantly increased.
Similar results were obtained with other buffers, such as Mops
or Mes in the 6.0–7.5 pH range (results not shown), and protein
samples from three different protein preparations/purifications,
suggesting that the observed degradation is not sample/buffer/pHdependent. In contrast, such protein degradation was not observed
in animal ferritins such as HoSF (horse spleen ferritin) and HuHF
(human H-chain ferritin) under the same experimental conditions
(results not shown).
No autodegradation with EP-deleted SSF
The EP represents the major structural difference between
plant and animal ferritins [1,8,12], raising a question as to
whether the EP is involved in SSF autodegradation. To answer
this question, EP-deleted SSF was prepared by incubating
WT SSF with a commercially available protease, Alcalase
2.4L, at 60 ◦C for 5 min. The newly prepared holoSSF has a
Figure 3 Amino acid sequence of (A) WT SSF and (B) 15 N-terminal
sequence residues of two subunits of SSF whose EP has been deleted by
Alcalase 2.4L
The EP is indicated in boldface.
molecular mass of ∼ 440 kDa (Figure 2A, lane 1) and contains
the same amount of iron within the interior core as WT
holoSSF (results not shown). SDS/PAGE analysis indicated that
the 28.0 and 26.5 kDa subunits were completely hydrolysed
to two small subunits with molecular masses of 23.5 and
21.0 kDa respectively (Figure 2B, lane 1). Subsequently, the
first 15 amino acid residues of the N-terminal of the two new
subunits were determined (and are listed in Figure 3B), further
confirming removal of the EP. Moreover, both WT and EPdeleted SSF had nearly the same CD (Supplementary Figure
S2A at http://www.BiochemJ.org/bj/427/bj4270313add.htm) and
fluorescence spectra (Supplementary Figure S2B), indicating that
the structure of EP-deleted SSF is similar to that of WT SSF.
To compare the stability of the two proteins, experiments were
conducted wherein both WT and EP-deleted SSF were allowed to
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316
X. Fu and others
S4 (at http://www.BiochemJ.org/bj/427/bj4270313add.htm), SSF
degradation was partially inactivated by 0.5 mM PMSF. In
contrast, when the PMSF concentration reached 1 mM, a nearly
complete inhibition was obtained. Therefore 1 mM PMSF was
used during the isolation and purification of SSF.
Serine protease-like activity of the EP domain of SSF
Figure 4 Comparison of the stability of WT SSF and SSF with the EP deleted
by SDS/PAGE analysis
The samples were incubated in buffer B at 4 ◦C for 60 days. Lane 1, holoSSF; lane 2, apoSSF;
lane 3, holoSSF with the EP deleted; lane 4, apoSSF with the EP deleted; and lane M, protein
markers and their corresponding molecular masses.
stand at 4 ◦C over a period of 60 days. As observed above, protein
degradation occurred pronouncedly in both WT holoSSF and
apoSSF (Figure 4, lanes 1 and 2), whereas EP-deleted holoSSF
and apoSSF were relatively stable (Figure 4, lanes 3 and 4).
This points to a functional role for the EP that is associated with
protein degradation through a certain way (see below). Even upon
standing for 60 days or longer, no subunits with a molecular mass
less than 23.5 and 21.0 kDa were generated (results not shown),
indicating that the EP itself is susceptible to degradation. In
addition, WT apoSSF was also unstable, indicating that iron was
not involved in such degradation, or at least was not a main factor.
Effect of different kinds of protease inhibitors on SSF degradation
The above observation raises the possibility that the EP has
protease-like activity. To confirm this, SSF was mixed with
different kinds of protease inhibitors followed by standing at 4 ◦C
for 40 days. Final concentrations of these inhibitors were used as
described previously [20,21]. As shown in Figure 5(A), holoSSF
degradation was nearly completely inhibited by a serine protease
inhibitor, PMSF, whereas EDTA (a metalloprotease inhibitor),
pepstatin (an aspartic protease inhibitor) and leupeptin (a serine
protease and cysteine protease inhibitor) hardly had any inhibitory
effect on the degradation. A trypsin-like serine protease inhibitor,
benzamidine, did not display an inhibitory effect. Moreover, the
combination of PMSF with EDTA or other inhibitors did not
exhibit an enhanced inhibitory effect. As expected, other serine
protease inhibitors, such as AEBSF and antipain, also exhibited
nearly identical effects with PMSF (Supplementary Figure S3
at http://www.BiochemJ.org/bj/427/bj4270313add.htm). Thus it
can be concluded from these results that the activity of serine
proteases is responsible for the observed degradation of the
phytoferritin EP. Nearly the same results were obtained after
incubation of these protease inhibitors with SSF for 50 days at
4 ◦C (Figure 5B), further supporting this conclusion.
To determine what concentration of PMSF is effective to
prevent SSF degradation, a series of PMSF solutions with different
concentrations were tested. As shown in Supplementary Figure
c The Authors Journal compilation c 2010 Biochemical Society
To seek direct evidence of where the serine protease-like activity
originates from, both EP-1 with the sequence ASTVPLTGVIFEPFEEVKKSELAVPT (corresponding to the EP from the H-1
subunit) and EP-2 with the sequence ASNAPAPLAGVIFEPFQELKKDYLAVPI (corresponding to the EP from the H-2
subunit) were synthesized and their hydrolysing activity was
measured with Alcalase (1000-fold dilution of Alcalase 2.4L)
and BSA as control samples. As expected, BSA has almost
no activity against the two peptides, whereas Alcalase had the
strongest activity among all tested samples (Figure 6). Similar
to BSA, EP-2 had a much weaker activity compared with the
analogue EP-1, which showed good activity against Boc-GlnAla-Arg-MCA (Figure 6A) and N-succinyl-Ala-Phe-Lys-MCA
(Figure 6B), indicating that EP-1 is mainly responsible for the
EP hydrolysing activity. Furthermore, it was observed that
the combination of EP-1 and EP-2 exhibited marginally stronger
activity than EP-1 alone. Similar to other serine proteases, EP-1
also revealed a stronger activity against Boc-Gln-Ala-Arg-MCA
than against N-succinyl-Ala-Phe-Lys-MCA [21]. Although the
relative catalytic activity of EP-1 is profoundly stronger than that
of EP-2, its absolute catalytic activity is still very low, excluding
the characterization of enzymology for EP-1.
To gain insight into the mechanism by which PMSF prevents
SSF degradation, the interaction of EP-1 with PMSF was studied
by MALDI–TOF-MS. The MS profile of the intact EP-1 showed
a single peak at m/z 2790.120 Da (Figure 7A). This indicates
a singly charged peptide monomer [M+H]+ of the peptide,
which is in good agreement with the predicted molecular mass
of 2790.240 Da. This peak rose to 2810.474 Da upon treatment
with PMSF (Figure 7B). The difference in molecular mass
between treated EP and untreated EP is 20.354 Da, which is
approximately equal to the mass of one molecule of hydrogen
fluoride (20.006 Da), which is derived from PMSF hydrolysis
[29]. Therefore the observed inhibition of EP degradation by
PMSF might be derived from the combination of EP-1 and
hydrogen fluoride. This result provided a good explanation for
why PMSF can completely inhibit SSF degradation.
Comparison of stability of rH-1 and rH-2 SSF
To seek further evidence of the protease activity of EP-1, the
stabilities of rH-2 and rH-1 were compared by SDS/PAGE
analysis. Similar to WT SSF, rH-1 also began to degrade
into smaller polypeptides after 20 days of incubation at 25 ◦C
(Figure 8B), whereas rH-2 was very stable (Figure 8A), with no
degradation under the same conditions. This confirms the above
conclusion that EP-1, but not EP-2, has the serine protease-like
activity responsible for the observed SSF autodegradation during
storage. Consistent with the present observations, MALDI–TOFMS results showed that the peptide mass fingerprint of the
degraded product of SSF with a molecular mass of ∼ 24.5 kDa
(Figure 1) corresponded significantly with that of the 26.5 kDa
SSF subunit (Supplementary Figure S5 at http://www.BiochemJ.
org/bj/427/bj4270313add.htm), as suggested by its high probability-based Mowse score (131). This, once again, demonstrates
that the H-1 subunit is prone to degradation.
EP autodegradation facilitates iron release
Figure 5
317
Effect of different kinds of protease inhibitors on SSF degradation
(A) The purified SSF was incubated in buffer B at 4 ◦C for 40 days; (B) the sample was incubated in buffer B at 4 ◦C for 50 days. Lane 1, 5 mM EDTA; lane 2, 5 mM PMSF; lane 3, 20 μM pepstatin;
lane 4, 2 mM benzamidine; lane 5, 20 μM leupeptin; lane 6, 5 mM EDTA and 5 mM PMSF; lane 7, 5 mM EDTA and 20 μM pepstatin; lane 8, 5 mM EDTA, 5 mM PMSF and 20 μM pepstatin; lane 9,
control (without protease inhibitors); and lane M, protein markers and their corresponding molecular masses.
Comparison of the rate of iron release from WT and EP-deleted
holoSSF
To shed light on the physiological function of EP autodegradation,
the rate of iron release from WT holoSSF induced by ascorbic
acid was compared with that from EP-deleted holoSSF. To
determine what pH values should be used for the iron release
from SSF, the pH of amyloplasts isolated from SSF during
seedling germination and growth was directly measured as
described previously [27,28]. It was found that the amyloplast
pH was in the range 5.8–6.1 (Supplementary Figure S6 at
http://www.BiochemJ.org/bj/427/bj4270313add.htm) at different
germination periods. Therefore the level of ferrous atoms released
from the ferritin shell was monitored at pH 6.5 by the formation
of the Fe2+ –ferrozine complex [23]. Generally, iron release
from EP-deleted holoSSF was much faster than that from WT
holoSSF at all pH values tested (Table 1). For example, at
pH 6.0, the initial rate of iron release from EP-deleted holoSSF
(2.36 +
− 0.15 μM/s) is 12-fold larger than that from EP-deleted
holoSSF (0.194 +
− 0.013 μM/s) (Table 1 and Figure 9). These
results indicate that EP removal from SSF favours iron release
from the protein shell to a large extent. The initial rate of iron
release from both WT and EP-deleted SSF at pH 5.8 is smaller
than that at pH 6.0, which may be due to larger protein aggregation
occurring at pH 5.8 compared with at pH 6.0 (results not shown).
DISCUSSION
Phytoferritin is unique among all known ferritins in that it contains
a specific EP domain at its N-terminal sequence that could impart
special properties to the protein [1,6,10]. Although it has been
almost 20 years since EP was first reported [10], its biological role
remains unknown. Until recently, the EP was reported to serve as
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318
X. Fu and others
Figure 7 MS profiles of EP-1 (A) and the EP treated with PMSF
(PMSF/EP = 10:1) (B) acquired by MALDI–TOF-MS
Figure 6 Relative enzyme activities of EP-2, EP-1, and EP-2/EP-1 (1:1)
towards two peptide–MCA substrates, Boc-Gln-Ala-Arg-MCA (A) and N succinyl-Ala-Phe-Lys-MCA (B)
BSA and Alcalase (1000-fold dilution of Alcalase 2.4L) were used as control samples.
Conditions: 0.7 μM EP-2, EP-1 or EP-2/EP-1 (1:1), 0.1 mM Boc-Gln-Ala-Arg-MCA or
N -succinyl-Ala-Phe-Lys-MCA, in 50 mM Tris/HCl solution (pH 8.0) containing 100 mM NaCl
and 10 mM CaCl2 . Values are expressed as means +
− S.D. (n = 3).
Table 1 Initial rate of iron release from WT holoSSF and holoSSF with the
EP deleted, induced by ascorbate (1 mM) at different pH values
Values are means +
− S.D. (n = 3).
Initial rate of iron release (μM/s)
Samples
pH 5.8
pH 6.0
pH 6.5
WT holoSSF
EP-deleted holoSSF
0.0569 +
− 0.0045
0.608 +
− 0.063
0.194 +
− 0.013
2.36 +
− 0.15
0.145 +
− 0.023
1.787 +
− 0.112
a second centre responsible for iron binding and oxidation, at high
Fe2+ flux into phytoferritin, through a novel pathway by which
the iron is ultimately deposited in the mineral core [12]. However,
there is little information available on its function during seedling
germination and growth.
The present study demonstrates that WT SSF degradation
mainly occurs due to the serine protease-like activity of
c The Authors Journal compilation c 2010 Biochemical Society
EP-1 on the outer surface of protein, and that such degradation
facilitates iron release from the protein. Initially, WT holoSSF
degradation took place during storage (Figure 1). Consistent with
this observation, previous reports showed that degraded holoSSF
can be directly separated during seed germination, and the amount
of degraded holoPSF rose with increasing soaking time [7,14].
The same phenomenon was also observed previously by another
group, showing that a 22 kDa subunit was obtained after the soya
bean seeds had been soaked long enough to induce germination
[30]. Thus phytoferritin degradation seems to constantly take
place in vitro and in vivo.
More importantly, the reason why SSF autodegradation occurs
during seed germination or storage in vitro was addressed in
the present study. Previous studies found that the production
of hydroxyl radical (HO䊉 ) during iron release from holoPSF
in the presence of ascorbate can be completely inhibited by
iron chelators such as o-phenanthroline and desferrioxamine B.
Based on this observation, it was proposed that holoPSF
degradation is due to damage by the iron-induced hydroxyl
radical [14,15]. However, the fact that protein degradation
comes from pure SSF alone (Figure 1), and that the addition
of either o-phenanthroline or desferrioxamine B to WT SSF
has no effect on such degradation (Supplementary Figure S7 at
http://www.BiochemJ.org/bj/427/bj4270313add.htm), has raised
uncertainties concerning this proposal. If the observed protein
EP autodegradation facilitates iron release
Figure 8
319
Comparison of the stability of WT SSF, rH-2 and rH-1 by SDS/PAGE analysis
(A) The samples were incubated in buffer B at 25 ◦C for 20 days. Lane 1, WT SSF; lane 2, rH-2; and lane M, protein markers and their corresponding molecular masses. (B) Lane 1, rH-1 incubated
in buffer B at 25 ◦C for 1 day; lane 2, rH-1 incubated in buffer B at 25 ◦C for 20 days; and lane M, protein markers and their corresponding molecular masses.
Figure 9 Comparison of iron release from WT holoSSF and holoSSF with
the EP deleted in the presence of ascorbic acid at pH 6.0
Conditions: 0.2 μM holoSSF or the holoSSF with the EP deleted, 1 mM ascorbic acid, 100 μM
ferrozine, in 100 mM Mes and 100 mM NaCl (pH 6.0, 25 ◦C).
degradation is attributed to the iron-induced HO䊉 , it is not
understood why no degradation occurs with EP-deleted holoSSF
(Figure 4, lane 3) containing the same amount of iron within a
protein shell as WT holoSSF (results not shown). The finding that
protein degradation occurs with WT apoSSF (Figure 4, lane 2)
precludes the possibility that protein degradation stems
from iron-induced HO䊉 . Thus the protein shell, not iron,
is responsible for protein degradation, specifically holoSSF
autodegradation. As expected, upon EP removal from holoSSF,
the resultant protein (consisting of 23.5 and 21.0 kDa subunits
with a molecular mass of ∼ 440 kDa) becomes stable with no
degradation under the same experimental conditions (Figure 4,
lanes 3 and 4), demonstrating that SSF autodegradation
actually corresponds to EP degradation. Thus the EP is a
protease-susceptible sequence, possibly because of its exposure
on the outer surface of the protein as predicated by the
three-dimensional model [6]. However, except for the EP,
the other part of SSF seems to be resistant to proteolysis, as
suggested by no further degradation in SSF (Figure 1). Consistent
with the present observation, it was found that subtilisin can only
catalyse a slight hydrolysis of SSF, independent of reaction time
[30].
As a typical inhibitor of serine proteases, PMSF is capable
of preventing EP degradation, whereas other kinds of protease
inhibitors such as EDTA, pepstatin, benzamidine and leupeptin
are not (Figure 5). This suggests that EP degradation originates
from its serine protease-like activity. Upon treatment of SSF or
EP-1 with PMSF, hydroxy groups located on the side chain of
serine residues in the EP were deactivated by its integration with
hydrogen fluoride (Figures 5 and 7), which was generated from
PMSF hydrolysis due to its instability in aqueous solutions at
pH ∼ 7.0 [29]. This eliminates the serine protease-like activity
of the EP, ultimately inhibiting EP degradation. Agreeing with
this idea, two other serine protease inhibitors, containing AEBSF
and antipain, also have such an ability (Supplementary Figure
S3). Direct evidence of this view comes from protease activity
measurements showing that either EP-1 or EP-1 plus EP-2
exhibit significant hydrolysing activity against two peptide–MCA
substrates, Boc-Gln-Ala-Arg-MCA (Figure 6A) and N-succinylAla-Phe-Lys-MCA (Figure 6B). In comparison, hydrolysing
activity of the EP-2 alone is much weaker. Thus EP-1 is mainly
responsible for SSF degradation. The large difference between
the hydrolysing activity of EP-1 and EP-2 may reside in their
somewhat dissimilar amino acid residues. EP-1 contains two serine residues at positions 2 and 20, whereas EP-2 has only one
serine residue at position 2 (Figure 3A). The above conclusion
was further confirmed by a comparison of rH-1 and rH-2 stability,
showing that rH-2 is a stable protein molecule whereas its
analogue, rH-1, is susceptible to degradation (Figure 8). These
results clearly demonstrate that WT SSF autodegradation during
storage mainly stemmed from the serine protease-like activity of
EP-1. Consistent with the present observation, previous studies
showed that the H-2 subunit was more resistant to proteolysis
compared with the H-1 subunit [8]. Further support comes from
a recent report showing that recombinant His6 –H-1 ferritin (in
which each subunit contains six histidine residues at its N-terminal
for convenient purification) is prone to degradation, whereas its
analogue, recombinant His6 –H-2 ferritin, is stable [31].
What advantage might EP autodegradation render to phytoferritin during seedling germination and growth? The answer to
this question may lie in the experimental results of iron reductive
release from WT and EP-deleted holoSSF (Figure 9 and Table 1).
c The Authors Journal compilation c 2010 Biochemical Society
320
X. Fu and others
The rate of iron release from EP-deleted holoSSF is pronouncedly
larger than that from WT holoSSF at different pH values (5.8, 6.0
and 6.5) (Table 1). Thus EP deletion facilitates iron release from
ferritin. In support of this conclusion, it has been established that
the majority of iron in soya bean seed was stored within ferritin
[3,32] to meet the demand of various key physiological processes,
such as electron transfer and DNA synthesis in the seedling stage
[1,3]. It was visualized that two-thirds of iron in soya bean seeds
move from bean cotyledons to the seedling axis during the first
week of germination [33]. Thus fast iron release from ferritin
might be beneficial to these processes. Alternatively, phytoferritin
is not only an iron depot, but a phosphorus storage molecule with
a iron/phosphorus ratio of <3:1 (results not shown), in accordance
with previous measurements [1,11]. Accompanied by iron release,
phosphate ions were therefore also liberated more quickly from
EP-deleted holoSSF than its analogue, again favouring the above
physiological processes [14].
The crystal structure analysis of the soya bean ferritin indicates
that the EP stabilizes the protein shell by maintaining the
conformation of the C-helix [4]. Therefore it is likely that removal
of the EP domain destabilizes the outer case of the ferritin
structure, leading to the facile release of iron and phosphorus.
This is reminiscent of the mechanism of iron release from animal
ferritin which is usually a cytosolic molecule. Many studies
have shown that cytosolic ferritin gains entry into lysosomes by
autophagy, and that ferritin degradation within lysosomes is
responsible for iron release [34,35]. Thus there is something
in common between the mechanism of iron release from plant
and animal ferritins, namely, their degradation occurs prior to
iron exit from protein. However, phytoferritin degradation is
slightly partial degradation. As a result, the degraded phytoferritin
might retain an intact protein shell, as suggested by the result
showing that it contains the same amount of iron within a
protein shell as WT holoSSF (results not shown). Thus reductants,
such as ascorbate, are still required for reducing Fe3+ caged in
phytoferritin, and resultant ferrous ions diffuse out of ferritin shell
plant ferritin through 3- or 4-fold channels [1–3]. In contrast,
complete degradation of animal ferritin by hydrolases existing
in the lysosomes results in iron release from protein without
participation of the reductants [34,35].
In conclusion, the present study demonstrates that the EP
on SSF possesses a serine protease-like activity previously
unrecognized, thus resulting in its autodegradation. Associated
with the degradation is faster iron release from ferritin to meet the
requirement of seedling growth, representing a novel pathway for
how phytoferritin controls its iron complement through a specific
domain in phytoferritin, the EP. Alternatively, since SSF is compartmentalized in amyloplasts, one of the most vital and yet leastunderstood organelles in seedling germination and early growth
[28], the detailed knowledge of ferritin shown in the present study
is beneficial to understanding the function of this organelle.
AUTHOR CONTRIBUTION
Xiaoping Fu performed the biochemical analysis, mass spectra and contributed to
manuscript writing. Jianjun Deng contributed to prepration of amyloplasts and ferritin,
and writing. Haixia Yang performed CD and fluorescence spectroscopy. Taro Masuda.
Fumiyuki Goto and Toshihiro Yoshihara contributed to preparation of rH-1 and rH-2.
Guanghua Zhao contributed to experimental design, ideas and manuscript writing.
FUNDING
This work was supported by the National Natural Science Foundation of China [grant
number 30972045]; the Chinese Universities Scientific Fund [grant number 2009-3-10];
and the China High-Tech (863) project [grant number 2007AA10Z333].
c The Authors Journal compilation c 2010 Biochemical Society
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Received 4 January 2010/8 February 2010; accepted 10 February 2010
Published as BJ Immediate Publication 10 February 2010, doi:10.1042/BJ20100015
c The Authors Journal compilation c 2010 Biochemical Society
Biochem. J. (2010) 427, 313–321 (Printed in Great Britain)
doi:10.1042/BJ20100015
SUPPLEMENTARY ONLINE DATA
A novel EP-involved pathway for iron release from soya bean seed ferritin
Xiaoping FU*, Jianjun DENG*, Haixia YANG*, Taro MASUDA†, Fumiyuki GOTO‡, Toshihiro YOSHIHARA‡ and Guanghua ZHAO*1
*CAU and ACC Joint Laboratory of Space Food, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China, †Laboratory of Food Quality
Design and Development, Division of Agronomy and Horticultural Science, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan, and ‡Biotechnology
Sector, Environmental Science Research Laboratory, Central Research Institute of Electric Power Industry, 1646 Abiko, Abiko, Chiba 270-1194, Japan
Figure S1
Native PAGE (A) and SDS/PAGE (B) analysis of purified SSF
Lane 1, crude extract; lane 2, sample fraction only from the DEAE-Sepharose Fast Flow column;
lane 3, purified SSF; and lane M, protein markers and their corresponding molecular masses.
Figure S2 CD spectra (A) and fluorescence spectra (B) of wild-type apoSSF
and EP-deleted apoSSF
(A) WT apoSSF or EP-deleted apoSSF at 0.2 μM, in 0.8 mM Mops solution (pH 7.0).
(B) WT apoSSF or EP-deleted apoSSF at 1 μM, in 5 mM Mops solution (pH 7.0) under
280 nm excitation.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2010 Biochemical Society
X. Fu and others
Figure S3 SDS/PAGE analysis of effect of AEBSF (A) and antipain (B) on
WT holoSSF degradation
The purified SSF was incubated at 37 ◦C for 20 days. Lane 1, control (without proteinase
inhibitors); lane 2, 1 mM AEBSF or 0.1 mM antipain; and lane M, protein markers and their
corresponding molecular masses.
Figure S4 SDS/PAGE analysis of effect of PMSF of different concentrations
on SSF degradation
(A) The purified SSF was incubated at 37 ◦C for 10 days. (B) The sample was incubated at 37 ◦C
for 30 days. Lane 1, 5 mM PMSF; lane 2, 4 mM PMSF; lane 3, 3 mM PMSF; lane 4, 2 mM
PMSF; lane 5, 1 mM PMSF; lane 6, 0.5 mM PMSF; lane 7, control (without PMSF); and lane
M, protein markers and their corresponding molecular masses.
c The Authors Journal compilation c 2010 Biochemical Society
EP autodegradation facilitates iron release
Figure S5 Tryptic peptide mass fingerprint of the degraded product of
SSF subunits (∼ 24.5 kDa) (A) and the 26.5 kDa subunit (B) gel bands from
SDS/PAGE acquired by MALDI–TOF-MS.
Figure S6 Characterization of the amyloplast from soya bean seed
cotyledons
(A) The pH value of the amyloplast from soya bean seed cotyledons as a function of germination
time. (B) Typical light micrograph of an intact amyloplast isolated from soya bean seed cotyledons
12 h after germination in ddH2 O.
c The Authors Journal compilation c 2010 Biochemical Society
X. Fu and others
Figure S7 SDS/PAGE analysis of the effect of o -phenanthroline and
desferrioxamine B on SSF degradation
The purified SSF was incubated at 4 ◦C for 30 days. Lane 1, control (without o -phenanthroline
or desferrioxamine B); lane 2, 0.2 % o -phenanthroline; lane 3, 0.2 % desferrioxamine B; and
lane M, protein markers and their corresponding molecular masses.
Received 4 January 2010/8 February 2010; accepted 10 February 2010
Published as BJ Immediate Publication 10 February 2010, doi:10.1042/BJ20100015
c The Authors Journal compilation c 2010 Biochemical Society