Rossi Fanelli’, University of Rome ‘La Sapienza’, Italy Ferritin from the spleen of the Antarctic teleost Trematomus bernacchiiis composed of a single subunit that contains both the ferr
Trang 1Ferritin from the spleen of the Antarctic teleost
Guiseppina Mignogna1, Roberta Chiaraluce1, Valerio Consalvi1, Stefano Cavallo1, Simonetta Stefanini1 and Emilia Chiancone1,2
1
Department of Biochemical Sciences and2CNR, 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
bernacchiiis composed of a single subunit that contains both
the ferroxidase center residues, typical of mammalian
H chains, and the carboxylate residues forming the micelle
nucleation site, typical of mammalian L chains Comparison
of the amino-acid sequence with those available from lower
vertebrates indicates that T bernacchii ferritin can be
classified as an M-type homopolymer Interestingly, the
T bernacchiiferritin chain shows 85.7% identity with a
cold-inducible ferritin chain of the rainbow trout Salmo gairdneri
The structural and functional properties indicate that cold
acclimation and functional adaptation to low temperatures
are achieved without significant modification of the protein stability In fact, the stability of T bernacchii ferritin to de-naturation induced by acid or temperature closely resembles that of mesophilic mammalian ferritins Moreover iron is taken up efficiently and the activation energy of the reaction
is 74.9 kJỈmol)1, a value slightly lower than that measured for the human recombinant H ferritin (80.8 kJỈmol)1) Keywords: amino-acid sequence; cold adaptation; iron incorporation; stability; Trematomus bernachii Antarctic fish ferritin
Several molecular adaptation mechanisms have been
devel-oped by living organisms under extreme environmental
conditions [1] In many cases, cold adaptation is achieved by
modification of the structural and functional properties of
proteins [2] It follows that the correlation between the
physicochemical properties of proteins and cold acclimation
is particularly attractive for molecules that are highly
thermostable [3] This is the case for ferritin, the ubiquitous
iron-storage protein, which is characterized by high thermal
and chemical stability in all mesophilic species [4] Ferritins
are able to sequester and store iron in a soluble and available
form thereby protecting the organism against the toxic effect
of ƠfreeÕ iron The extremely stable quaternary structure of
the ferritin molecule is highly conserved It consists of a
hollow 24-mer protein shell, apoferritin (molecular mass
480 kDa), the cavity of which can accommodate up to 4500
iron atoms as an inorganic micellar core [4]
Mammalian ferritins are heteropolymers of two
genetic-ally distinct subunits, L and H, of similar 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 ferritins are found in other tissues such as
heart and kidney, which do not [6] Accordingly, the H and L subunits have distinct and complementary functions The H chains contain in the four-helix bundle a dinuclear ferroxidase center, which promotes the oxidation of Fe2+in the presence of molecular oxygen [7] The iron 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) facing the inner surface of the apoferritin shell, that provide efficient nucleation sites for iron accumulation [8]
Ferritins from lower vertebrates have received relatively 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 the sequence elements
of functional importance, the L chain contains the three negatively charged residues (E57, E60, and E64) responsible for iron nucleation and mineralization in the mammalian protein The H and M chains, although differing in sequence and molecular mass, contain all the ligands of the ferroxidase center and, in addition, two out of the three carboxylic residues typical of mammalian L chains (E57 and E64, E60 is replaced by a histidine)
In fish ferritins, evidence for two subunits was obtained by screening of a liver cDNA library in the Atlantic salmon Salmo salar[10] As in tadpole ferritin, the H and M subunits contain both the ligands typical of the H chain ferroxidase center and the canonical L chain carboxylate residues in positions 60 and 64 The canonical L chain glutamate residue in position 57 is present only in the M chains and is substituted by an asparagine in the H chains [8] It is noteworthy that S salar ferritin displays a different pattern
Correspondence to E Chiancone, CNR Center of Molecular Biology,
Department of Biochemical Sciences ƠA Rossi FanelliÕ, University of
Rome ƠLa SapienzaÕ, P.le A Moro, 5, 00185 Roma, Italy.
Fax: + 39 06 4440062, Tel.: + 39 06 49910761,
E-mail: emilia.chiancone@uniroma1.it
Abbreviation: CHCA, a-cyano-4-hydroxycinnamic acid.
(Received 28 September 2001, revised 30 November 2001, accepted
3 January 2002)
Trang 2of subunit expression relative to mammalian ferritins Thus,
H chains predominate in spleen and liver at variance with
the presence of L chains in the same organs of mammals [6]
Interestingly, a study of cold-inducible gene expression of
rainbow trout cells (Salmo gairdneri) revealed that the
transcription and accumulation of the mRNA
correspond-ing to three ferritin H isoforms H1, H2 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 the structural–functional properties 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 spleen ferritin from another Antarctic
teleost, Gymnodraco acuticeps, has been partially
character-ized; it is an H-type homopolymer, as indicated by the
N-terminal amino-acid sequence, that is able to accumulate
iron as an L-rich mammalian ferritin molecule [12]
The results show that native T bernacchii ferritin is a
homopolymer with a high iron content ( 2500 iron atoms
per molecule) and a high ferroxidase activity The
amino-acid sequence of the constitutive subunit shows a high
similarity to one of 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 of the
protein stability to chemical and physical denaturants with
respect to the mesophilic proteins
M A T E R I A L S A N D M E T H O D S
Enzymes and chemicals were purchased from the following
suppliers: Asp-N endoproteinase and trypsin from Roche
Diagnostics Corporation; pepsin and 4-vinylpyridine from
Sigma; CNBr from Fluka; guanidinium chloride
(recrystal-lized from methanol) from Merck; the liquid
chromatogra-phy solvents, HPLC-grade, from Carlo Erba Reagenti;
sequence-grade chemicals from Applied Biosystems
Purification and characterization ofT bernacchii ferritin
Specimens of T bernacchii were sampled from Terra Nova
Bay Station, Ross Sea; the spleens were immediately
removed and frozen at )80 °C until use Spleen ferritin
was purified following the procedure described previously
[12] Iron was removed from the native protein, which
contains about 2500 iron atoms per molecule, by incubation
for 24 h in 0.5M acetate buffer, pH 4.8, containing 1%
(w/v) sodium dithionite and subsequent chelation of Fe2+
with 2,2¢-bipyridyl The concentration of apoferritin was
determined from the A280using an absorption coefficient
(e1%,1 cm¼ 6.5) calculated as described by Gill & Von
Hippel [13]
Analysis of amino-acid sequence
The protein sample (1.5 mg) was suspended in 0.5 mL 0.5M
Tris/HCl, pH 7.5, containing 2 mM EDTA, 4M
guanidi-nium chloride and 12 lmol dithiothreitol, and incubated for
3 h at 55°C Thereafter, 4-vinylpyridine (90 lmol, 10 lL) was added, and, after 10 min incubation, the protein was desalted by HPLC using a guard cartridge (C8, 4.6 mm· 30 mm) An aliquot (0.5 mg) of the denaturated pyridylethylated protein was dissolved in 0.2 mL 80% (v/v) trifluoroacetic acid, incubated in 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 mM Tris/HCl, pH 7.5, containing 10% acetonitrile, and incuba-ted at 37°C overnight after the addition of 4 lg Asp-N endoproteinase A third aliquot (0.3 mg) was dissolved in 0.2 mL 5% (v/v) formic acid, and incubated with 6 lg pepsin at 25°C for 5 min The peptide mixtures obtained after enzymatic digestions were purified immediately after the incubation with proteases, without lyophilization The peptide mixtures were purified by HPLC using a Beckman System Gold chromatographer on a macroporous reversed-phase column (C8208TP52; 4.6 mm· 250 mm;
5 lm Vydac; Esperia, CA, USA) They were eluted with a linear gradient from 0 to 35% acetonitrile in 0.2% (v/v) trifluoroacetic acid at a flow rate of 1.0 mLÆmin)1 Elution
of the peptides was monitored using a diode array detector (Beckman model 168) at 220 and 280 nm
The amino-acid sequence of peptide samples was deter-mined by automated Edman degradation using an Applied Biosystems model 476A sequencer Samples (0.1–0.5 nmol) were loaded on to poly(vinylidene difluoride) membranes (ProBlott; Applied Biosystems), coated with 2 lL polybrene (100 mgÆmL)1; 50% methanol), and run with a Blott cartridge using an optimized gas-phase fast program N-Terminal sequence analysis of the protein was per-formed on samples (5 lg) electrotransferred on ProBlott membranes after SDS/PAGE [14], using a liquid-phase fast program
Peptides were numbered retrospectively according to their location in the sequence, starting from the N-terminus CNBr peptides were designated with B, Asp-N peptides with A, and peptic peptides with P
MS analysis Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) measurements were performed using a Voyager-DE (Applied Biosystems) mass spectrometer Solutions (1 lL) containing peptides (1–5 pmol) were mixed with 1 lL of the matrix solution: 30% aqueous solution of acetonitrile and 0.1% trifluoroacetic acid saturated with a-cyano-4-hydroxycinnamic acid (CHCA) or 2,5-dihydroxy-benzoic acid dissolved 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 of 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+) (SequazimeTM Peptide Mass Standards kit; Applied Biosystems)
Mass analysis of the N-terminal-blocked peptide
An aliquot (10 lg) of pyridylethylated protein was dissolved
in 50 lL 50 mM NHHCO, pH 8.5, and incubated at
Trang 337°C overnight after addition of 1 lg trypsin The peptide
mixture was desalted using the ZipTipC18 (Millipore) and
then mixed with the matrix solution
(a-cyano-4-hydroxy-cinnamic acid) for MALDI-TOF MS analysis
Structure comparison
A search of the SwissProt-TrEMBLE database, pairwise
and multiple sequence alignments, and prediction of
secon-dary structures were carried out with the programs from
EXPASY(Expert Protein Analysis System) proteomics server
of the Swiss Institute of Bioinformatics (SIB)
Iron incorporation experiments
Iron incorporation experiments were performed by addition
of a freshly prepared anaerobic solution of ferrous
ammo-nium sulfate to an air-equilibrated solution of apoferritin
T bernacchii and human recombinant apoferritins were
used in parallel experiments Human recombinant
H homopolymer (100% H subunit) was overexpressed in
Escherichia coliand purified essentially as described by Levi
et al [15]
The kinetics of iron oxidation and uptake were followed
at the desired temperature measuring the absorbance of the
ferric oxide hydrate micelle at 310 nm using the absorption
coefficient of ferritin iron, e1%,1 cm¼ 450 [16] As a control,
the rate of Fe2+autoxidation was measured in parallel To
assess iron incorporation, at the end of the reaction the
samples were analyzed by nondenaturing gel electrophoresis
(staining with Prussian blue for iron and Coomassie blue for
protein) and by sedimentation velocity in a Beckman
Optima XL-A analytical ultracentrifuge at 49 000 g and
10°C The sedimentation coefficients were reduced to s20, 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 (Pharmacia) eluted with 20 mMsodium phosphate,
pH 7.0, containing 0.15M NaCl at a flow rate of
0.5 mLÆmin)1controlled by a Dionex gradient pump After
24 h incubation at pH 1.5–4.0, the samples were diluted
20-fold into the column injection loop The Superose
column was calibrated with horse spleen apoferritin
(440 kDa, elution volume Ve¼ 7.8 mL), rabbit muscle
aldolase (161 kDa, elution volume Ve¼ 9.2 mL), horse
liver alcohol dehydrogenase (80 kDa, elution volume Ve¼
9.8 mL), BSA (66 kDa, elution volume Ve¼ 10.0 mL),
ovalbumin (45 kDa, elution volume Ve¼ 10.3 mL), and
cytochrome c (12 kDa, elution volume Ve¼ 12.4 mL)
pH-dependence experiments
T bernacchiiferritin (0.03–1.2 mgÆmL)1) was incubated for
24 h at 20°C at pH 1.5 (31.6 mMHCl), pH 2.0 (10.0 mM
HCl), pH 2.5 (3.2 mM HCl), pH 3.0 (1.0 mM HCl) and
pH 2.0 in the presence of 31.6 mMNaCl (pCl 1.5) The pH
of the solutions was measured with an InLab 422 electrode
(Mettler–Toledo AG) connected to a Corning P507 ion
meter before and after the addition of the protein After
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 Jasco J-720 spectropolari-meter Far-UV (190–250 nm) and near-UV CD (250–
310 nm) measurements were performed in a 0.1-cm and 1.0-cm pathlength quartz cuvette, respectively The results are expressed as mean residue ellipticity ([Q]) assuming a mean residue weight of 110 per amino-acid residue All the spectroscopic measurements were performed at 20°C Thermal denaturation
For thermal scans, the protein samples (0.06 mgÆmL)1) in
20 mMsodium phosphate at pH 7.0 and in 40 mMglycine/ HCl at pH 4.0 were heated from 10 to 95°C and subsequently cooled to 10°C with a heating/cooling rate
of 1 degreeÆmin)1 controlled by a Jasco programmable Peltier element Far-UV CD spectra were recorded every 5
or 2.5°C, and the dichroic activity at 222 nm was monit-ored continuously every 0.5°C with 4 s averaging time All the spectra were corrected for the solvent contribution at the different temperatures and pH values examined The melting temperatures were determined by taking the first derivative of the ellipticity signal at 222 nm with respect to temperature
R E S U L T S
Determination of amino-acid sequence The complete sequence of the single subunit that gives rise
to T bernacchii ferritin is reported in Fig 1 The subunit contains 176 amino-acid residues The sequence was deduced after the isolation and identification of an almost complete set of CNBr peptides, which were ordered with the help of overlapping peptides produced by Asp-N and pepsin cleavage The sequence of each peptide was confirmed by
MS analysis The automated Edman degradation of the native protein was unsuccessful for the possible presence of
a blocked N-terminus MALDI-TOF MS analysis of a tryptic digest of the protein indicated for the N-terminal peptide MDSQVR a value of m/z 777, which points to the presence of acetylmethionine This residue is commonly found in the N-terminus of eukaryotic proteins together with N-acetyl Ala, Ser, Gly and Thr [17]
Secondary-structure prediction, performed as described
by Rost [18] shows the presence of four a helices in the regions corresponding to positions 8–40, 45–75, 94–120 and 128–158 (Fig 2) This four-helix pattern is analogous to the four-helix-bundle characteristic of mammalian ferritin [4]
A search in the SwissProt-TrEMBLE Database with the
T bernacchii ferritin as a probe retrieved many ferritin sequences The alignment, obtained using the program
Trang 4CLUSTALW, is reported in Fig 2 where, for the sake of
simplicity, only the human L and H chains are shown to
represent mammalian ferritins The 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 gairdneri (87.5%) and the M chains from S salar
(86.9%) and Gillichthys mirabilis (78.7%) The percentage
identity for the H chains 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 bernacchii 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 all conserved in the
T bernacchiispleen 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 chains This characteristic, first described for the
poly-peptide chains of bullfrog ferritin [9], is common to fish
ferritins on the basis of the available sequences
A further distinctive property of the fish H chains known
to date relative to those of mammals appears to be the lack
of the four-amino-acid extension at the N-terminus
An exception is the H chain of G acuticeps spleen ferritin,
the N-terminal amino-acid sequence of which,
TTASTSQVRQNYHQDSE, shows the typical
four-ami-no-acid extension of mammalian ferritin H chains [12]
Iron incorporation
Iron uptake by T bernacchii apoferritin was studied at
different temperatures in 50 mM Mops/NaOH buffer at
pH 6.5 after the aerobic addition of 500 iron atoms per
molecule In parallel, the recombinant human H
homopoly-mer was examined As shown in Fig 3, at 20°C the time
course of Fe2+ oxidation by T bernacchii apoferritin is
characterized by a half-time of about 120 s, which is higher
than that measured under similar conditions for the human
H homopolymer (t1/2¼ 55 s) and significantly lower than
that of the L-rich apoferritin of horse spleen (t1/2¼ 600 s)
[19] The iron-oxidation capacity is maintained by
T bernacchii apoferritin at low temperature; the rate of the reaction is reduced sixfold (t1/2¼ 715 s) when the temperature is decreased from 20°C to 4 °C The human recombinant H homopolymer shows a similar decrease in the catalytic activity (t1/2¼ 360 s) at 4 °C The effect of temperature on the half-time of the iron-oxidation reaction, measured between 4°C and 50 °C, was analysed using the Arrhenius equation The activation energy, Ea, of
T bernacchii apoferritin is 74.9 kJÆmol)1, a value only slightly lower than that measured for the recombinant
H protein (80.8 kJÆmol)1)
All the added iron is incorporated inside the apoferritin shell as indicated by native gel electrophoresis and sedimen-tation velocity experiments The reconstitution products obtained on incubation of apoferritin with 2500 iron atoms
Fig 1 Complete amino-acid sequence of T bernacchii ferritin The
extent of the various fragments used to reconstruct the sequence is
shown B, CNBr peptides; A, Asp-N peptides; P, peptic peptides.
Ac-M, acetylmethionine.
Fig 2 Amino-acid sequence comparison among T bernacchii ferritin and M, H and L chain of ferritins The alignment was obtained using ClustalW TbS_M, M chain from T bernacchii spleen; SgG_H2, H2 chain from S gairdneri gonadal fibroblast (TrEMBL accession num-ber: P79822); SaL_M, M chain from S salar liver (SwissProt acces-sion number: P49947); GmL_M, M chain from G mirabilis liver (TrEMBL accession number: Q9DFP0); SaL_H, H chain from
S salar liver (SwissProt accession number: P49946); OnB_H, H chain from O nerka brain (TrEMBL accession number: Q98TT0); HuL_H,
H chain from human liver (SwissProt accession number: P02794); HuL_L, L chain from human liver (SwissProt accession number: P02792) Residues conserved in all sequences are in boldface type Amino acids that constitute the H-chain ferroxidase center are in blue; those forming the L chain iron micelle nucleation site are in red Green arrows indicate the four predicted a helices (A, B, C, D) Yellow boxes indicate the a helix (A, B, C, D and E) identified in the crystallographic structure of human H chain The human H chain numbering has been adopted.
Trang 5per molecule in Mops/NaOH buffer, pH 6.5, sediment as a
heterogeneous peak with an average sedimentation
coeffi-cient of about 43 S The distribution of iron micelles and the
value of the sedimentation coefficient are very similar to
those measured for the native protein
Structure ofT bernacchii ferritin as a function of pH The effect of low pH on the association state of the protein was investigated to compare the stability of T bernacchii ferritin with that of L-type and H-type mammalian ferritins, which are known to dissociate at pH 2.5 and 2.8–3.0, respectively [20]
The stability of T bernacchii ferritin at acid pH values was studied after incubation of the apoprotein in the pH 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, which are decreased only slightly (Ve¼ 7.7 mL) compared with that
of the native protein at pH 7.0 (Ve¼ 7.8 mL) On incubation at pH 3.0, the secondary structure of native apoferritin is almost completely preserved, as indicated by the far-UV CD spectrum (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 in the 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 kmax¼ 333 nm on excitation at 295 nm (Fig 4C)
Incubation of T bernacchii apoferritin at pH 2.5 (3.2 mM HCl) does not induce any change in the Superose 12 elution profile, but alters significantly the protein spectral proper-ties The near-UV CD spectrum displays a consistent decrease in all the aromatic residue contributions Interest-ingly, the 262 nm phenylalanine band is of opposite sign to the protein at pH 7.0 (Fig 4B) The far-UV CD spectrum
of T bernacchii apoferritin at pH 2.5 shows a modest blue shift of the zero intercept and an overall decrease in the ellipticity relative to the protein at pH 7.0 (Fig 4A) The fluorescence spectrum concomitantly shows a 47%
quench-Fig 3 Progress curves of iron oxidation uptake by T bernacchii and
human recombinant H apoferritins on addition of 500 Fe atoms/molecule
as ferrous ammonium sulfate at 20 and 4 °C T bernacchii apoferritin
(– ) – 20 °C, – Æ Æ – 4 °C); human recombinant H homopolymer
(–20 °C, ÆÆÆÆ 4 °C) Protein concentration: 0.2 l M Buffer: 50 m M
Mops/NaOH, pH 6.5 Inset: effect of temperature on t 1/2 value in
T bernacchii (d) and human recombinant H homopolymer (j)
(Arrhenius plot).
Fig 4 Effect of pH on the spectral properties of T bernacchii ferritin (A) Far-UV CD (0.1 cm quartz cuvette) and (C) fluorescence (295 nm excitation wavelength) spectra were recorded at 0.05 mgÆmL)1protein concentration (B) Near-UV CD spectra were recorded in a 1-cm quartz cuvette at 1.20 mgÆmL)1protein concentration All the spectra were recorded at 20 °C after 24 h incubation of the protein at pH 7.0 (20 m M 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, – Æ Æ –), pH 2.0 pCl 1.5 (31.6 m M NaCl – ) –), and pH 1.5 (31.6 m M HCl, ÆÆÆÆÆÆ).
Trang 6ing of the maximum emission intensity and a red shift of the
kmaxto 345 nm compared with the protein at pH 7.0
Incubation of T bernacchii apoferritin at pH 2.0 (10 mM
HCl) and 1.5 (31.6 mMHCl) results in the disassembly of
the quaternary structure, as indicated by the shift of the
size-exclusion chromatography elution volume from 7.8 mL
(pH 7.0) to 11.4 mL The depolymerization 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
significant decrease in ellipticity relative to pH 3.0, a blue
shift of the zero intercept, and a change in the ratio between
the 208 and the 222 nm bands (Fig 4A) In particular, the
molar ellipticity ratio ([Q222]/[Q208]) shifts from 1.46 at
pH 7.0 to 0.84 at pH 2.0 A weakening of the protein
tertiary contacts at pH 2.0 and 1.5 is indicated by the
intrinsic fluorescence spectra, which display a red shift of the
emission maximum to a kmax value of 351–352 nm,
accompanied by a notable quenching of the intensity
(Fig 4C) The addition of chloride to the protein at
pH 2.0 did not affect significantly the protein spectral
properties except for a blue shift of the kmax value to
349 nm, possibly caused by charge shielding, which leads to
a decrease in the repulsive effect
A set of experiments was performed on native ferritin
containing about 2500 Fe atoms per polymer The presence
of an iron core does not influence the acid-induced
dissociation of the protein Thus, the elution profiles from
a Superose 12 column of native ferritin at pH 7.0 and of the
protein incubated for 24 h at pH 3.0–1.5 are comparable to
those of the apoprotein (data not shown)
Thermal denaturation
The temperature-induced far-UV CD changes in
T bernacchii apoferritin were monitored 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 from those of
the native apoprotein The midpoints of the transitions at
pH 7.0 and pH 4.0 correspond to 82 and 74°C,
respectively (data not shown) These values are closer
to those measured in mammalian H-type ferritins (77
and 67°C) than to those measured in L-rich apoferritin
(93 and 90°C) [21]
D I S C U S S I O N
The present characterization of ferritin from the Antarctic
fish T bernacchii describes for the first time the structural
and functional properties of a homopolymer constructed
from an unusual subunit, the M chain, which is capable of
carrying out both the iron-oxidation and the
iron-mineral-ization process In the mammalian proteins, these two
reactions are carried out by two distinct chains The stability
of the T bernacchii homopolymer does not differ
signifi-cantly from that of mesophilic ferritins, indicating that cold
acclimation does not significantly affect the quaternary
construction
The amino-acid sequence of the T bernacchii polypeptide
chain shows the presence of both the amino-acid residues at
the ferroxidase center of the mammalian H chains and the
carboxylate groups, which promote iron incorporation and
mineralization in the mammalian L chains [4] In accord-ance with the sequence data, the T bernacchii ferritin homopolymer is able to both oxidize and accumulate iron efficiently (Fig 3) Ferritin from the spleen of another Antarctic teleost, G acuticeps, likewise is a homopolymer that is rich in iron [12] It appears therefore that Antarctic fish ferritins do not require heteropolymeric assemblies to take up iron efficiently because of the structural character-istics of the constituent polypeptide chain
A previous comparison of amino-acid sequences from homologous proteins from mesophiles and psychrophiles established that a number of specific amino-acid substitu-tions occur in cold-adapted proteins [22] However, the paucity of available M-type chain sequences does not warrant such an analysis In this connection, it is of interest that the thermal stability of the apoferritin molecule, a property exploited during the purification process, is very similar in T bernacchii ferritin and in the mesophilic proteins Thus, in thermal denaturation experiments at
pH 7.0 and 4.0, T bernacchii ferritin has melting points that are closer to those of the recombinant human H-type protein than to the L-type one [21] This behavior may be attributed at least in part to the absence of the salt bridge formed within the four-helix bundle of the L chains between K62 and E107 and thought to play a special role in conferring thermal stability [20]; in the T bernacchii
M chain, just as in the human H chains, a glutamate replaces lysine in position 62
The stability of T bernacchii ferritin at acid pH values likewise resembles that of mesophilic mammalian ferritins, because the polymeric assembly is maintained at pH 2.5 [20] At pH 3.0, the tertiary structure of T bernacchii ferritin is essentially unchanged with respect to neutral
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 side 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 decreases; in accordance with these findings, the fluorescence intensity decreases and the emission wavelength shows a modest shift towards the red, indicating further exposure of the W93 residue to solvent Interestingly, the CD band at
262 nm attributable to phenylalanine residues changes sign, possibly because of the presence of several pheny-lalanines at or near the subunit contact areas Collectively these changes point to a quaternary construction with increased local flexibility at the interfaces At pH 2.5, most of the protein secondary-structure elements are present as indicated by the far-UV CD spectrum (Fig 4A), which shows only a modest blue shift of the zero intercept and a small decrease in the overall ellipticity with respect to the protein at pH 7.0 and 3.0 Such secondary-structure elements may provide the residual tertiary contacts necessary to maintain the quaternary structure of the protein Below pH 2.5, where the protein dissociates, the depolymerization process is accompanied
by the almost complete loss of secondary structure This is indicated by the significant decrease in dichroic activity in the far-UV, and by the progressive exposure of W93 to
Trang 7solvent shown by the red shift of the maximum emission
fluorescence wavelength relative to the protein at pH 3.0
Analysis of the iron-incorporation process is in
accord-ance with the presence in T bernacchii ferritin of the
ferroxidase center residues typical of mammalian H-type
ferritin Thus, the reaction rate is comparable in the two
proteins over the whole temperature range explored
(Fig 3) The lower Ea value observed in T bernacchii
compared with the H homopolymer is consistent with
reports on other cold-adapted proteins; it may be considered
as a common mechanism of adaptation at low temperatures
[23] This finding suggests an increase in local flexibility in
relevant positions of the structure On the basis of structural
and or functional data, increased local or global flexibility of
cold-adapted proteins is often, but not always, implicated in
cold adaptation [24,25] It follows that a combination of
different strategies is adopted by organisms to survive at low
temperatures [24]
In conclusion, this study shows that ferritins from
Antarctic fish can be assembled from only one subunit in
line with previous preliminary observations [12] The M-type
chain in T bernacchii ferritin carries the amino acids that
confer on the homopolymer the capacity to carry out
efficiently the two processes that lead to incorporation of iron
in the apoferritin shell, namely iron oxidation and nucleation
of the iron core The high sequence similarity between
T bernacchii ferritin and the cold-inducible H2 chain of
S gairdnerisupports the contention [11] that the expression
of such proteins plays a significant role in cold acclimation
A C K N O W L E D G E M E N T
This research was supported by the Italian National Programme for
Antarctic Research (PNRA).
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