Ferritin from the spleen of the Antarctic teleost
Trematomus bernacchii
is an M-type homopolymer
Guiseppina Mignogna
1
, Roberta Chiaraluce
1
, Valerio Consalvi
1
, Stefano Cavallo
1
, Simonetta Stefanini
1
and Emilia Chiancone
1,2
1
Department of Biochemical Sciences and
2
CNR, Center of Molecular Biology, Department of Biochemical Sciences ‘A. Rossi Fanelli’,
University of Rome ‘La Sapienza’, Italy
Ferritin from the spleen of the Antarctic teleost Trematomus
bernacchii is composed of a single subunit that contains both
the ferroxidase center residues, typical of mammalian
H c hains, and the carboxylate residues forming the micelle
nucleation site, typical of mammalian L chains. C omparison
of the amino-acid sequence w ith those available from lower
vertebrates indicates that T. bernacchii ferritin can be
classified a s an M-type homopolymer. Interestingly, the
T. bern acchii ferritin chain shows 85.7% identity with a cold-
inducible ferritin chain of the rainbow trout Salmo gairdneri.
The s tructural a nd functional properties i ndicate that cold

480 k Da), the cavity of which can accommodate up to 4500
iron atoms as an inorganic micellar core [4].
Mammalian ferritins are h eteropolymers of two genetic-
ally distinct subunits, L and H, of s imilar sequence,
molecular mass (19–21 kDa, respectively) and with the
same four-helix-bundle tertiary conformation. The ferritin
subunits are expressed in different proportions in various
cells and tissues [5]. Thus, L-rich copolymers predominate in
spleen and liver, which have an iron-storage function,
whereas H-rich f erritins are found in other tissues such as
heart and kidney, which do not [6]. Accordingly, the H
and L subunits have distinct and c omplementary functions.
The H chains contain in the four-helix bundle a dinuclear
ferroxidase center, which promotes the oxidation of Fe
2+
in
the p resence of molecular oxygen [7]. The i ron ligands are
highly conserved and are provided by residues E27, E61,
E62, H65, E107 and Q141 [7]. The L chains lack such a
center, but contain specific carboxylic groups (E57, E60,
and E64 using the H-chain numbering) facin g the inner
surface of the apoferritin shell, that provide efficient
nucleation sites for iron accumulation [8].
Ferritins f rom l ower vertebrates have re ceived r elatively
little attention. In amphibians, specifically in bullfrog
tadpole erythrocytes, the occurrence of three distinct ferritin
cDNAs and their cell-specific expression has been described.
The corresponding subunits were named H (heavy),
M (middle) and L (light) as they show distinct mobilities
in denaturing gels [9]. With respect to t he sequence elements

rainbow trout cells (Salmo gairdneri) revealed that the
transcription and accumulation of the m RNA correspond-
ing to three ferritin H isoforms H1, H 2 and H3 is enhanced
[11]. In turn, the induction of ferritin H expression during
cold acclimation may suggest that this ferritin is particularly
apt to function at low temperatures.
This study was undertaken to characterize ferritin from
an Antarctic fish and thereby establish whether cold
adaptation affects t he structural–functional p roperties of
this protein. Ferritin extracted from the spleen of the
Antarctic teleost Trematomus bernacchii, which lives at a
constant temperature of )1.9 °C, was chosen. To our
knowledge only s pleen ferritin from another A ntarctic
teleost, Gymnodraco acuticeps, has been partially character-
ized; it is an H -type homopolymer, as i ndicated by the
N-terminal amino-acid sequence, that is able to accumulate
iron as an L-rich mammalian ferritin m olecule [12].
The results show that native T. bernacchii ferritin i s a
homopolymer with a h igh iron content ( 2500 iron atoms
per molecule) and a high ferroxidase activity. The amino-
acid seq uence o f the constitutive subunit shows a high
similarity to one o f the cold-inducible chains of S. gairdneri
ferritin; like this chain, it contains the functional residues
characteristic of both mammalian L and H chains. The
molecular adaptation essential to function at low tempera-
ture is not accompanied by a significant modification o f the
protein stability to chemical and physical denaturants with
respect to the mesophilic proteins.
MATERIALS AND METHODS
Enzymes a nd chemicals were purchased from the f ollowing

M
Tris/HCl, pH 7 .5, containing 2 m
M
EDTA, 4
M
guanidi-
nium chloride and 12 lmol dith iothreitol, and incubated for
3hat55°C. Thereafter, 4-vinylpyridine (90 lmol, 10 lL)
was added, and, after 10 min incubation, the protein was
desalted by HPLC using a guard c artridge (C
8
,
4.6 m m · 30 mm). A n aliquot (0.5 mg) of t he denaturated
pyridylethylated protein was dissolved in 0.2 m L 80% ( v/v)
trifluoroacetic a cid, incu bated i n the dark with 4 mg CNBr
for 24 h at room temperature, and lyophilized. A second
aliquot of protein (0.5 mg) was suspended in 0.5 mL 10 m
M
Tris/HCl, p H 7.5, c ontaining 10% acetonitrile, and incuba-
ted at 37 °C overnight after the addition of 4 lgAsp-N
endoproteinase. A third aliquot (0.3 mg) w as dissolved in
0.2 m L 5% (v/v) formic acid, and incubat ed with 6 lg
pepsin at 25 °C for 5 min. The peptide mixtures obtain ed
after enzymatic digestions were purified immediately after
the incubation with proteases, w ithout lyophiliz ation.
The peptide mixtures were p urified by H PLC using a
Beckman S ystem Gold chromatographer on a macroporous
reversed-phase column (C8208TP52; 4.6 mm · 250 mm;
5 lm Vydac; Esperia, CA, USA). T hey were eluted with a
linear gradient from 0 to 35% acetonitrile in 0.2% (v/v)

benzoic acid d issolved in water. The mixture of peptide and
matrix was placed on the MALDI stainless-steel plate and
allowed to dry spontaneously. Ions were generated by
irradiating the sample area with a nitrogen laser at a
wavelength of 337 nm. Calibrations were carried out using a
mixture o f angiotensin I (1297.51 MH
+
), adrenocortico-
tropic hormone ACTH (clip 1–17) (2094.46 MH
+
), ACTH
(clip 18–39) (2466.72 MH
+
), ACTH (clip 7–38) (3660.19
MH
+
) and bovine insulin (5734.59 MH
+
) (Sequazime
TM
Peptide Mass Standards kit; Applied B iosystems).
Mass analysis of the N-terminal-blocked peptide
An aliquot (10 lg) of pyridylethylated protein was dissolved
in 50 lL50m
M
NH
4
HCO
3
, pH 8.5, and incubated a t

assess iron incorporation, at the end o f the reaction the
samples were analyzed by nondenaturing gel electrophoresis
(staining with Prussian blue for i ron and Coomassie blue for
protein) and by sedimentation velocity in a B eckman
Optima XL-A analytical ultracentrifuge at 49 000 g and
10 °C. The s edimentation coefficients w ere reduced to s
20, w
by standard procedures.
Analysis of the state of association
The state of association was analysed by size- exclusion
chromatography experiments at 20 °C on a Superose 12
column (Pharmac ia) eluted w ith 20 m
M
sodium phosphate,
pH 7.0, containing 0.15
M
NaCl at a flow rate of
0.5 m LÆmin
)1
controlled b y a Dionex gradient pump. After
24 h incubation at pH 1.5–4.0, the samples were diluted
20-fold i nto the column injection loop. The Superose
column was calibrated with horse spleen apoferritin
(440 kDa, elution volume V
e
¼ 7.8 mL), rabbit muscle
aldolase (161 kDa, elution volume V
e
¼ 9.2 mL), horse
liver alcohol dehydrogenase (80 kDa, elution v olume V

24 h incubation at 20 °C, the samples were analyzed by CD,
fluorescence spectroscopy, and size exclusion chromato-
graphy.
Spectroscopic methods
Intrinsic fluorescence emission and light-scattering measure-
ments were carried out with an LS50B PerkinElmer
spectrofluorimeter using a 1-cm pathlength quartz cuvette.
Intrinsic fluorescence emission spectra were recorded at
300–400 nm (1 nm sampling interval) with the excitation
wavelength set at 295 nm. Light scattering was measured
with both excitation and emission wavelength set at 480 nm.
CD spectra were recorded on a J asco J-720 spectropolari-
meter. Far-UV (190–250 nm) and near- UV CD (250–
310 n m) measurements were performed in a 0.1-cm and
1.0-cm pathlength quartz c uvette, respectively. The r esults
are expressed as mean residue ellipticity ([Q]) assuming a
mean residue weight of 110 per amino-acid residue. A ll the
spectroscopic measurements were performed at 20 °C.
Thermal denaturation
For thermal scans, the protein samples (0.06 mgÆmL
)1
)in
20 m
M
sodium phosphate at pH 7.0 and in 40 m
M
glycine/
HCl at pH 4 .0 were heated from 10 to 95 °Cand
subsequently cooled to 10 °C with a heating/cooling rate
of 1 degreeÆmin

128–158 (Fig. 2). This four-helix pat tern is analogous to the
four-helix-bundle characteristic of mammalian ferritin [ 4].
A search i n the SwissProt-TrEMBLE Database with the
T. bern acchii ferritin as a p robe re trieved many ferritin
sequences. The alignment, obtained using the program
1602 G. Mignogna et al.(Eur. J. Biochem. 269) Ó FEBS 2002
CLUSTALW
, is r eported in F ig. 2 where, for the sake of
simplicity, only the human L and H chains are shown to
represent m ammalian ferritins. T he percentage identity
among the various sequences ranges from 87.5 to 59, the
latter value pertaining to human L chains. The most similar
to the T. bernacchii constitutive chain are the H2 chain from
S. ga irdneri (87.5%) and the M chains from S. salar
(86.9%) and Gillichthys m irabilis (78.7%). The percentage
identity for the H c hains from S. salar and
Oncorhynchus nerka is significantly lower (70.5 and 70.4,
respectively). Despite the paucity of available sequence data,
it appears that T. bern acchii ferritin can be classified as an
M homopolymer and that the H2 chain from S. gairdneri
should be likewise considered an M chain.
The amino-acid residues of functional relevance in
mammalian L and H chains are a ll conserved in the
T. bernacchii spleen ferritin chain. More specifically, E27,
E61, E62, H65, E107 and Q141, correspond to amino acids
characteristic of the H-chain ferroxidase site, while E57,
D60 and E64 correspond to sites of iron nucleation in
L c hains. This characteristic, first described for the poly-
peptide chains of bullfrog f erritin [9], is common to fish
ferritins on the basis of the available sequences.

¼ 715 s) when the
temperature is decreased from 20 °Cto4°C. The human
recombinant H homopolymer shows a similar decrease in
the catalytic activity (t
1/2
¼ 360s)at4°C. The effect of
temperature on t he half-time of t he iron-oxidation reaction,
measured between 4 °Cand50°C, was a nalysed using
the Arrhenius equation. The a ctivation energy, E
a
,of
T. bernacchii apoferritin i s 74.9 k JÆmol
)1
, a value only
slightly lower than that measured for the recombinant
H protein (80.8 k JÆmol
)1
).
All t he added iron is incorporated inside the apoferritin
shell as i ndicated by native g el electrophoresis and s edimen-
tation velocity experiments. The reconstitution products
obtained o n i ncubation of apoferritin with 2500 iron a toms
Fig. 1. Complete amino-acid sequence of T. bernacchii ferritin. The
extent of the various fragments used t o r eco nstruct the sequence i s
shown. B, CNBr peptides; A, Asp-N peptides; P, peptic peptides.
Ac-M, acetylmethionine.
Fig. 2. Amino-acid sequence comparison among T. be rnacchii ferritin
and M, H a nd L chain of ferritins. The alignment was obtained using
ClustalW. TbS_M,MchainfromT. bernacchii spleen; SgG_H2 ,H2
chain f rom S. gairdneri gonadal fibroblast (TrE MBL accession num-

range 3.0–1.5 at 20 °C for 24 h, a time established to be
sufficient to reach equilibrium. T. bernacchii apoferritin
maintains its quaternary assembly when incubated at
pH 3.0 and at pH 2.5, as indicated by the corresponding
elution volumes from a Superose 12 column, w hich are
decreased only slightly (V
e
¼ 7.7 mL) compared with that
of the native protein at pH 7.0 (V
e
¼ 7.8 mL). On
incubation at pH 3.0, the s econdary structure of native
apoferritin i s a lmost completely preserved, a s indicated by
the far-UV C D s pectrum (Fig. 4A). Likewise, the near-UV
CD spectrum resembles that measured at pH 7.0 with
minor differences (Fig. 4B). Consistently with the modest
changes observed i n t he near-UV and far-UV CD spectra
compared with the protein at pH 7.0, the fluorescence
emission of apoferritin at pH 3.0 is decreased by only 20%,
and is not red-shifted relative to the protein at pH 7.0,
which shows a k
max
¼ 333 nm on excitation at 295 nm
(Fig. 4 C).
Incubation of T. be rnacchii apoferritin at pH 2.5 (3.2 m
M
HCl) does not induce any change in the Superose 12 elution
profile, but alters significantly the protein spectral proper-
ties. The near-UV CD s pectrum displays a consistent
decrease in all t he aromatic residue contributions. Interest-

sodium phosphate, –––), pH 3.0 (1.0 m
M
HCl, – Æ –), pH 2.0 (10.0 m
M
HCl, —–), pH 2.5 (3.2 m
M
HCl, –
ÆÆ
–), pH 2.0 pCl 1.5
(31.6 m
M
NaCl – ) –), and pH 1 .5 (31.6 m
M
HCl, ÆÆÆÆÆÆ).
1604 G. Mignogna et al.(Eur. J. Biochem. 269) Ó FEBS 2002
ing of the maximum emission intensity and a red shift of the
k
max
to 345 nm compa red with the protein at pH 7.0.
Incubation of T. bernacchii apoferritin at p H 2.0 (10 m
M
HCl) and 1.5 (31.6 m
M
HCl) results in the disassembly o f
the quaternary structure, a s indicated by the shift of the size-
exclusion chromatography elution volume from 7.8 mL
(pH 7 .0) to 11.4 mL. The de polymerization of T. bernacchii
apoferritin incubated at pH 2.0 and 1.5 is paralleled by a
significant loss in secondary structure as indicated by the
far-UV CD spectra. The spectra are characterized by a

The temperature-induced far-UV CD changes in
T. bernacchii apoferritin w ere mon itored continuously at
222 nm at two pH values, 7.0 and 4.0. The observed
transitions were irreversible, and the spectra measured at
the end of the cooling phase were different f rom those of
the native a poprotein. The m idpoints of t he tran sitions at
pH 7.0 a nd p H 4. 0 correspond to 82 and 7 4 °C,
respectively (data not shown). These values are closer
to those m easured in mammalian H-type ferritins (77
and 67 °C)thantothosemeasuredinL-richapoferritin
(93 and 90 °C) [21].
DISCUSSION
The p resent characterization of ferritin f rom t he Antarctic
fish T. bernacchii describes for the first time the s tructural
and functional properties of a homopolymer constructed
from an unusual s ubunit, the M chain, which i s capable of
carrying out both t he iron-oxidation and the i ron-mineral-
ization process. In the mammalian proteins, these two
reactions are c arried out by two distinct chains. The stability
of the T. bernacchii homopolymer does not differ signifi-
cantly from that of mesophilic ferritins, indicating that c old
acclimation does not significantly affect the quaternary
construction.
The a mino-acid s equence of the T. bernacchii polypeptide
chain shows the presence of b oth the amino-acid residues at
the ferroxidase center of the m ammalian H chains and t he
carboxylate g roups, w hich promote iron incorporation and
mineralization in the mammalian L chains [4]. In accord-
ance with the sequence d ata, the T. be rnacchii ferritin
homopolymer is able to both oxidize and accumulate iron

pH. The slight decrease in intrinsic fluorescence is
probably due to dynamic quenching caused by minor
tertiary structure perturbations leading to an increased
mobility of the W93 s ide chain. At pH 2 .5, the quaternary
assembly is not altered, as indicated by size exclusion
chromatography. However, changes in protein tertiary
structure occur, as indicated by the near-UV CD and
fluorescence spectra (Fig. 4B,C). The ellipticity attribut-
able to tryptophan and tyrosine residues d ecreases; in
accordance with these findings, the fluorescence intensity
decreases and the e mission wavelength shows a modest
shift towards the red, indicating further exposure of the
W93 residue to solvent. Interestingly, the CD band a t
262 nm attr ibutable to phenylalanine residues c hanges
sign, possibly b ecause of t he presence of several pheny-
lalanines a t o r n ear t he subunit contact areas. Collectively
these changes point to a quaternary construction with
increased local flexibility at t he interfaces. A t pH 2 .5,
most of the protein secondary-structure elements are
present a s indicated by t he far-UV CD spectrum
(Fig. 4 A), w hich shows only a modest blue shift of the
zero intercept and a s mall decrease in the overall ellipticity
with respect to the p rotein at pH 7.0 and 3.0. Such
secondary-structure elements may provide t he residual
tertiary cont acts necessary to maintain the quaternary
structure of the protein. Below pH 2 .5, where the protein
dissociates, t he depolymerization process is accompanied
by the almost complete loss of secondary structure. This is
indicated by t he significant decrease in dichroic activity in
the far-UV, and by the progressive exposure of W93 to

S. gaird neri supports the c ontention [11] that t he expression
of such protein s plays a significant role in cold acclimation.
ACKNOWLEDGEMENT
This research was supported by the Italian N ational Programme for
Antarctic Research (PNRA).
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