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 acclimation and functional a daptation t o l ow temperatures are a chieved w ithout significant modification o f t he protein stability. In fact, the stability of T. bernacchii ferritintode- 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 kJÆmol )1 , a value slightly lower than t hat measured for t he human recombinant H ferritin (80.8 kJÆmol )1 ). Keywords: amino-acid sequence; cold adaptation; iron incorporation; stability; Trematomus bernachii Antarctic fish ferritin. Several m olecular adaptation mechanisms have been devel- oped by living organisms under extreme environmental conditions [1]. In many cases, cold adaptation is achieved by modification o f t he structural and functional properties of proteins [2]. It follows that the correlation between the physicochemical properties o f proteins a nd 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 c haracterized by high ther mal and chemical s tability i n 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 ÔfreeÕ iron. The extremely stable quaternary structure of the ferritin molecule is highly conserved. It consists of a hollow 2 4-mer p rotein shell, apoferritin (molecular mass 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 of functional importance, th e 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 f erroxidase center and, in addition, two out of the t hree carboxylic residues typical of mammalian L chains (E57 and E64, E60 is replaced by a h istidine). 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 i n position 5 7 i s present only in the M chains a nd is substituted by an asparagine in the H chains [8]. It is noteworthy t hat S. salar ferritin d isplays a d ifferent pattern Correspondence to E. Chiancone, CNR Center of Molecular Biology, Department of Biochemical Sciences ÔA. Rossi FanelliÕ, University of Rome ÔLa 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 2 8 September 200 1, revised 3 0 November 2001, accepted 3 January 2002) Eur. J. Biochem. 269, 1600–1606 (2002) Ó FEBS 2002 of subunit expression relative to mammalian f erritins. Thus, H c hains predominate in spleen and liver at variance with thepresenceofL chainsinthesameorgansofmammals[6]. Interestingly, a study of cold-inducible gene expression of 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 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 of T. bernacchii ferritin Specimens of T. bernacchii were sam pled f rom T erra Nova Bay Station, Ross S ea; the spleens were i mmediately 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 0.5 M acetate buffer, pH 4.8, containing 1% (w/v) s odium dithionite and subsequent chelation of Fe 2+ with 2,2 ¢-bipyridyl. Th e concentration of apoferritin was determined from the A 280 using an absorption coefficient (e 1%,1 cm ¼ 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 0.5 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) trifluoroacetic acid at a flow rate of 1.0 mLÆmin )1 .Elution of the p eptides w as monitored using a diode array detector (Beckman model 168) at 220 and 280 n m. The amino-acid sequence of peptide samples was deter- mined b y a utomated Edman degradation using an Applied Biosystems model 476A sequencer. S amples (0.1–0.5 nmol) were loaded on to poly(vinylidene difluoride) membranes (ProBlott; App lied Biosystems), coated with 2 lL polyb rene (100 mg ÆmL )1 ; 5 0% methanol), and run with a B lott 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 a fter 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 p eptides 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 t he m atrix solution: 30% aqueous solution of acetonitrile and 0.1% trifluoroacetic acid saturated with a-cyano-4-hydroxycinnamic a cid ( CHCA) or 2 ,5-dihydroxy- 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 Ó FEBS 2002 Homopolymeric M-type T. bernacchii ferritin (Eur. J. Biochem. 269) 1601 37 °C overnight after addition of 1 lg trypsin. T he peptide mixture w as 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 m ultiple s equence a lignments, and prediction of s econ - dary structures were carried out with the programs from EXPASY (Expert P rotein Analysis System) proteomics s erver of the Swiss Institute of Bioinformatics (SIB). Iron incorporation experiments Iron incorporation experiments were performed by addition of a f reshly prepared anaerobic solution o f ferrous amm o- nium sulfate to an air-equilibrated solution o f apoferritin. T. bern acchii and h uman recombinant a poferritins were used in parallel experiments. Human recombinant H homopolymer (100% H subunit) was overexpressed in Escherichia c oli and purified essentially as described by Levi et al . [15]. The kinetics of i ron oxidation and u ptake w ere followed at the desired tem perature measuring the absorbance o f the ferric o xide hydrate micelle at 310 nm u sing the absorption coefficient of ferritin iron, e 1%,1 cm ¼ 450 [16]. As a control, therateofFe 2+ autoxidation was m easured i n p arallel. To 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 e ¼ 9.8 m L), BSA (66 kDa, elution volume V e ¼ 10.0 mL), ovalbumin (45 kDa, elution volume V e ¼ 10 .3 mL), and cytochrome c (12 k Da, elution volume V e ¼ 12.4 mL). pH-dependence experiments T. bern acchii ferri tin (0.03–1.2 m gÆmL )1 ) w as incubated for 24 h at 20 °CatpH1.5(31.6m M HCl), pH 2.0 (10.0 m M HCl), pH 2.5 (3.2 m M HCl), pH 3.0 (1.0 m M HCl) and pH 2.0 in the presence of 31.6 m M NaCl (pCl 1.5). T he pH of the s olutions was measured with a n I nLab 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 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 )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 m onit- 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. RESULTS Determination of amino-acid sequence The complete sequence o f the single subunit t hat gives rise to T. bernacchii ferritin is reported in Fig. 1. The subunit contains 176 amino-acid residues. T he seq uence was deduced after the isolatio n and identification of an almost complete set of CNBr peptide s, which were ordered w ith the help of overlapping peptides produced by Asp-N a nd pepsin cleavage. The sequence of each peptide was c onfirmed by MS analysis. The automated Edman degradation o f the native protein was unsuccessful for the possible p resence o f a blocked N-terminus. MALDI-TOF MS an alysis of a tryptic digest of the protein indicated for the N-terminal peptide MDSQVR a value of m/z 777, which points t o the presence of acetylme thionine. This r esidue 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 ahelices in the regions corresponding to positions 8–40, 45–75, 94–120 and 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. A further distinctive property of t he fish H chains known to date relative to those o f mammals appears to be the lack of the four-amino-acid extension at the N-terminus. An exception is t he H c hain 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. bern acchii apoferritin was studied at different temperatures in 50 m M 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 w as examined. As shown i n Fig. 3, at 20 °Cthetime course of Fe 2+ oxidation by T. bernacchii apoferritin is characterized by a half-time of about 120 s, which is h igher than that measured under s imilar conditions fo r the human H homopolymer (t 1/2 ¼ 55 s) and significantly lower than that of the L -rich apoferritin of horse spleen (t 1/2 ¼ 60 0 s) [19]. The iron-oxidation capacity is maintained by T. bernacchii apoferritin at low temperature; the rate of the reaction is r educed sixfold (t 1/2 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- ber: P79822); SaL_M,MchainfromS. salar liver (SwissProt acces- sion number: P49947); GmL_M,MchainfromG. mirabilis liver (TrEMBL accession number: Q9DFP0); SaL_H,Hchainfrom S. salar liver (SwissProt accession number: P49946); OnB_H,Hchain from O. nerka brain (TrEMBL accession number: Q98TT0); HuL_H, H ch ain from human liver (SwissProt accession number: P02794); HuL_L, L cha in from h uman 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).Yellowboxes indicate the a helix (A, B, C, D and E) identified in the crystallographic structure of h uman H c hain. The human H chain n umbering has been adopted. Ó FEBS 2002 Homopolymeric M-type T. bernacchii ferritin (Eur. J. Biochem. 269) 1603 per m olecule 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 thosemeasuredforthenativeprotein. Structure of T. bernacchii ferritin as a function of pH The e ffect of low p H o n t he association state of the protein was investigated to compare the stability of T. bernacchii ferritin with that o f L-type a nd 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, 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- ingly, the 262 nm phenylalanine band is of opposite sign t o the protein a t pH 7.0 (Fig. 4B). The f ar-UV CD spectrum of T. bernacchii apofer ritin 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 c oncomitantly 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 apof erritin (– ) –20°C, – ÆÆ –4°C); human recomb inant H homopolymer (–20 °C, ÆÆÆÆ 4 °C). Prot ein concentration: 0.2 l M .Buffer:50m M Mops/NaOH, pH 6.5. Inset: effect of temperature on t 1/2 value in T. bernac chii (d) and human recombinant H homopolymer (j) (Arrhenius plot). Fig. 4. Effect of p H on the spectral properties of T. bernacchii ferritin. (A) Far-UV CD (0.1 cm quartz cuvette) a nd (C) fluorescence (295 nm excitation wavelength) spectra were recorded at 0.05 mgÆmL )1 protein concentration. (B) Near-UV CD spectra were recorded in a 1-cm quartz cuvette at 1.20 mgÆmL )1 protein concentration. All the s pectra were recorded at 20 °C a fter 24 h incubation of the p ro tein 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, – 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 significant decrease in ellipticity relative to pH 3.0, a blue shift of the z ero intercept, and a change in the ratio between the 208 and the 222 nm ba nds (Fig. 4A). In particular, the molar ellipticity ratio ([Q 222 ]/[Q 208 ]) shifts from 1.46 at pH 7.0 to 0.84 a t 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 k max value of 351–352 nm, accompanied b y a notable quenching of the intensity (Fig. 4 C). T he 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 k max value to 349 nm, possibly caused by charge s hielding, which leads to a decrease in the repulsive e ffect. 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 o f the protein. Thus, the elution p rofiles from a Superose 12 column of native ferritin at pH 7.0 and of the protein incubated f or 24 h a t pH 3 .0–1.5 are comparable t o those of the apoprotein (data not shown). Thermal denaturation 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 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 therefo re 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 i n cold-adapted proteins [ 22]. However, t he paucity of available M -type chain sequences does not warrant such an analysis. I n this connection, it is of interest that the t hermal stability o f the apoferritin m olecule, a property exploited during the purification process, is very similar in T. bernacchii ferritin and in the m esophilic 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 t han to the L-type one [21]. This behavior may be attributed at least in part to the absence o f the salt bridge formed within the four-helix bundle of the L c hains between K62 and E107 and thought to play a special role in conferring thermal stability [20]; in the T. bernacchii M c hain, just as in the human H chains, a glutamate replaces lysine in position 62. The stability of T. be rnacchii ferritin at acid pH values likewise r esembles that of mesophilic mammalian ferritins, because the polymeric assembly is maintained at pH 2.5 [20]. At p H 3.0, t he tertiary structure o f T. bernacchii ferritin is essentially unchanged with r espect 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 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 Ó FEBS 2002 Homopolymeric M-type T. bernacchii ferritin (Eur. J. Biochem. 269) 1605 solvent 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 i n accord- ance with the presence in T. bernacchii ferritin of the ferroxidase center residues t ypical 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 E a 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]. T his finding suggests an i ncrease i n local flexibility in relevant positions of the structure. On the basis of s tructural and o r f unctional 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 a ssembled from only one subunit in line with previous preliminary observations [12]. The M-type chain in T. bernacchii ferritin carries the a mino acids that confer on the homopolymer the capacity to carry out efficiently the two processes that lead to incorporation o f iron in the apoferritin shell, namely iron oxidatio n and nucleation of the iron core. The high s equence s imilarity between T. bern acchii ferritin and the cold-inducible H2 chain of S. gaird neri supports the c ontention [11] that t he expression of such protein s plays a significant role in cold acclimation. 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