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The role of cysteine residues in the oxidation of ferritin
Free Radical Biology & Medicine, Vol. 33, No. 3, pp. 399 – 408, 2002 Copyright © 2002 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/02/$–see front matter
PII S0891-5849(02)00915-2
Original Contribution THE ROLE OF CYSTEINE RESIDUES IN THE OXIDATION OF FERRITIN KEVIN D. WELCH,* CHRISTOPHER A. REILLY,†
and
STEVEN D. AUST*
*Biotechnology Center, Utah State University, Logan, UT, USA; and †Department of Pharmacology and Toxicology, Center for Human Toxicology, University of Utah, Salt Lake City, UT, USA (Received 28 February 2002; Revised 30 April 2002; Accepted 9 May 2002)
Abstract—We have shown that ferritin is oxidized during iron loading using its own ferroxidase activity and that this oxidation results in its aggregation (Welch et al., Free Radic. Biol. Med. 31:999 –1006; 2001). In this study we determined the role of cysteine residues in the oxidation of ferritin. Loading iron into recombinant human ferritin by its own ferroxidase activity decreased its conjugation by a cysteine specific spin label, indicating that cysteine residues were altered during iron loading. Using LC/MS, we demonstrated that tryptic peptides of ferritin that contained cysteine residues were susceptible to modification as a result of iron loading. To assess the role of cysteine residues in the oxidation of ferritin, we used site-directed mutagenesis to engineer variants of human ferritin H chain homomers where the cysteines were substituted with other amino acids. The cysteine at position 90, which is located at the end of the BC-loop, appeared to be critical for the formation of ferritin aggregates during iron loading. We also provide evidence that dityrosine moieties are formed during iron loading into ferritin by its own ferroxidase activity and that the dityrosine formation is dependent upon the oxidation of cysteine residues, especially cysteine 90. In conclusion, cysteine residues play an integral role in the oxidation of ferritin and are essential for the formation of ferritin aggregates. © 2002 Elsevier Science Inc. Keywords—Iron, Ferritin, Protein oxidation, Cysteine oxidation, Dityrosine, Free radicals
INTRODUCTION
ferritin via this mechanism could damage the ferritin and any other biomolecules in close proximity. We [11,12] and others [13] have shown that the •OH is indeed produced when iron is loaded into ferritin via its ferroxidase activity. Additionally, we demonstrated that the production of the hydroxyl radical resulted in damage to the ferritin [11,12]. Oxidative damage to ferritin was prevented when iron loading was performed in the presence of an organic buffer, e.g., HEPES. It was postulated that the protective effect of HEPES was the result of its ability to scavenge •OH, thus inhibiting the oxidation of ferritin by •OH. However, the addition of catalase to the reaction mixture (not containing HEPES) did not “protect” ferritin from damage [11]. These data suggest that the oxidation of the ferritin was either a site-specific process or that it was initiated by an oxidant not requiring H2O2 for its formation. Interestingly, the aggregation of ferritin appeared to be a result of cysteine oxidation [11]. This is quite probable because the cysteine residue is one of the more susceptible amino acids to metal-catalyzed oxidation [14 –16], and can subsequently form disulfide cross-links.
Ferritin is an ubiquitous intracellular iron storage protein, wherein up to 2500 atoms of ferric iron can be stored as a ferric oxyhydroxy phosphate complex [1– 4]. Ferritin is composed of 24 subunits of H and L type, which assemble to form a protein shell with a hollow interior of approximately 90 Å. In order for iron to be incorporated into the ferritin core it must be presented as ferrous iron and subsequently oxidized. In general, it is believed that iron is loaded into ferritin by itself, dependent upon a “ferroxidase activity” of the H subunits of ferritin [2,5– 8]. However, the stoichiometry of iron oxidation in this system is 2:1, i.e., two moles of iron oxidized per mole of molecular oxygen reduced, indicating the generation of H2O2 [9,10]. Hydrogen peroxide in the presence of ferrous iron can result in the formation of a strong oxidant such as the hydroxyl radical (•OH), a nonspecific oxidant of biomolecules. Therefore, loading iron into Address correspondence to: Dr. Steven D. Aust, Biotechnology Center, Utah State University, Logan, UT 84322-4705, USA; Tel: (435) 797-2730; Fax: (435) 797-2755; E-Mail: [email protected]. 399
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K. D. WELCH et al. Table 1. Primers for Site-Directed Mutagenesis of Human H Chain Ferritin DNAa
Forward primer (5⬘-3⬘)
Restriction site
CAGGATATCAAGAAACCAGACCGGGATGACTGGG GGGCTGAATGCAATGGAGGCTGCATTACATTTGG CAAAAATGACCCCCATTTGGCCGACTTCATTGAGACAC
BseGI TseI CfrI
Primer ID rHuHFt C90R rHuHFt C102A rHuHFt C130A a
Target codons are underlined and sequence changes bolded.
The H subunit of human ferritin contains three cysteine residues, at positions 90, 102, and 130. Analysis of the predicted 3D structure of the H chain of ferritin demonstrated that cysteine 90, located on the BC-loop, faced the exterior, solvent-exposed surface of the protein. In ferritin from other species cysteine 90 is absent, e.g., rat ferritin contains an arginine at position 90. Interestingly, rat ferritin appears to be less susceptible to aggregation during iron loading versus human ferritin (unpublished observations). In almost all species cysteine 102 and 130 are highly conserved in the H chain as well as cysteine 130 in the L chain. This study was performed to further our understanding of the process of oxidation and subsequent aggregation that occurs when ferritin is loaded with iron via its own ferroxidase activity. Specifically, we wanted to determine the role of the cysteine residues in the oxidation of ferritin. To accomplish this we performed site-directed mutagenesis to engineer ferritin variants where the cysteine residues were substituted with other amino acids. We produced C90R, C102A, C130A, C90R/C102A, and C90R/C102A/C130A ferritin variants. We analyzed all of these variants, in addition to wild-type recombinant human ferritin H chain homomer (wt) ferritin, for protein oxidation and subsequent aggregation. Analysis of the tryptic digests of ferritins by liquid chromatographymass spectrometry (LC/MS) indicated that peptides containing cysteine residues were modified during iron loading into ferritin via its own ferroxidase activity. We also provide data demonstrating that ferritin variants with the C90R substitution were significantly less susceptible to aggregate formation resulting from oxidation during iron loading. MATERIALS AND METHODS
Chemicals Ammonium bicarbonate was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Sucrose and ferrous ammonium sulfate were purchased from Mallinckrodt (Paris, KY, USA). 4-Maleimido-TEMPO and sodium chloride (99.999% pure) were purchased from Aldrich Chemical Company (Milwaukee, WI, USA). N-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid
(HEPES) was purchased from Boehringer Mannheim (Indianapolis, IN, USA). Isopropyl alcohol was purchased from Fisher Scientific (Fair Lawn, NJ, USA). Reagents were prepared using water purified by reverse osmosis with subsequent passage through a NANOpure Infinity Ultrapure Water System (Barnstead/Thermolyne Corporation, Dubuque, IA, USA). Proteins Trypsin was purchased from Sigma Chemical Company. Recombinant human ferritin H chain homomer was produced and purified as described by Van Eden and Aust [17]. Site-directed mutagenesis Gene sequence and 3D crystal structure models for human ferritin were obtained from Genebank (gi:182504) and the Protein Data Bank (2fha), respectively. Rasmol v2.5 (Glaxo Research and Development, Greenford, Middlesex, UK) was used to model protein structure. Mutagenesis was conducted using the QuickChange Site-Directed Mutagenesis protocol from Stratagene (La Jolla, CA, USA). Mutagenesis primers were designed using the program Primer Generator [18] to include unique restriction sites for screening the products of mutagenesis. Macromolecular Resources (Department of Biochemistry, Colorado State University, Ft. Collins, CO, USA) synthesized the primers for site-directed mutagenesis (Table 1). Mutagenesis with each primer set was conducted by PCR using a Gene Amp System 9600 Thermal Cycler (Perkin-Elmer, Emeryville, CA, USA) according to the QuickChange Site-Directed Mutagenesis protocol. The PCR product was transformed into Escherichia coli strain XL1-Blue (Stratagene), plated on LB/Ampicillin agar plates and incubated at 37°C for 20 h. Resistant colonies were selected and cultured in 3 ml of LB/Ampicilin medium for 16 h at 37°C. Plasmid DNA was isolated from the cell pellet derived from 1 ml of the E. coli cultures by a “microprep” protocol [19]. The purified plasmid DNA was subjected to PCR using primers for the 5⬘ and 3⬘ end of the ferritin H chain gene. The PCR product was then digested with the appropriate
Ferritin oxidation
restriction enzyme to test for the incorporation of the mutation in the gene sequence. DNA containing the C90R mutation was used to create a double mutant C90R/C102A and a triple mutant C90R/C10A/C130A using the appropriate mutagenesis primer sets. The modified human ferritin DNA sequences were confirmed by sequencing using an ABI Prism 377 DNA sequencer (PE/Applied Biosystems, Foster City, CA, USA) by the Biotechnology Center Service Laboratory (Utah State University, Logan, UT, USA). The various expression plasmids containing the mutated DNA for H chain human ferritin were transformed into E. coli strain B834 (DE3) pLysS Novagen, Inc. (Madison, WI, USA) for expression of the human ferritin variant proteins. Loading iron into ferritin Loading iron into ferritin was performed as described previously [20]. Briefly, ferritin was incubated with ferrous ammonium sulfate (pH adjusted to ⬃7.5) in 50 mM NaCl, pH 7.0, at 37°C. For specific concentrations refer to appropriate figure legends. Sucrose density-gradient centrifugation Iron loading mixtures were placed on top of 10 ml continuous sucrose gradients (1–25% w/v). The samples were centrifuged in a Beckman SW41Ti swinging bucket rotor at 30,000 rpm (⬃110,000 ⫻ g) for 2.5 h. Aliquots (1 ml) were removed from the top of the gradients by pipette and analyzed for protein by the Bradford Assay. Native PAGE analysis Aliquots were removed directly from iron loading reactions and subjected to native PAGE using a 7.5% resolving gel with a 4% stacking gel. The samples were electrophoresed for approximately 2.5 h and subsequently stained for protein with Coomassie Blue or iron using Prussian Blue. Prussian Blue staining was performed by incubating gels in a solution of 0.1% (w/v) potassium ferrocyanide in 1 N HCl at 25°C for ⬃15 min. Ferritin iron content was qualitatively assessed after the gels were washed with water. Spin labeling of ferritin 4-Maleimido-TEMPO was used to label the cysteine residues of recombinant human H chain ferritin. Briefly, 7.2 mM spin label was incubated with 5 M ferritin in 50 mM HEPES, pH 7.0, at 25°C in the dark for 2 h. Excess spin label was removed by chromatography over a Econo Pac 10DG desalting column (BioRad Laboratories, Her-
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cules, CA, USA). ESR spectra of spin labeled ferritin were recorded at room temperature using a Bruker ECS 106 spectrometer operating at 9.8 GHz with a 50 kHz modulation frequency. Tryptic digestion of ferritin Upon completion of iron loading, isopropyl alcohol and ammonium bicarbonate, pH 8.4, were added to make final concentrations of 40% (v/v) and 10% (v/v) respectively, in 1 ml final volume. The samples were then incubated at 100°C for 5 min. The samples were allowed to cool to 25°C before the addition of trypsin (0.2 mg). The samples were then incubated at 37°C for 24 h. Liquid chromatography-mass spectrometry of ferritin peptides Analysis of ferritin peptides produced from tryptic digestion of ferritin was achieved by high performance liquid chromatography and electrospray-ionization mass spectrometry (ESI LC/MS) using a ThermoQuest/Finnigan TSQ7000 mass spectrometer (ThermoQuest Instruments, San Jose, CA, USA) interfaced with a HewlettPackard series 1100 HPLC system (Agilent Technologies, Palo Alto, CA, USA). Samples of peptides were diluted to a concentration of 1 g/l in 90%, aqueous formic acid (0.1% v/v):10% methanol, placed into an autosampler vial maintained at 4°C, and 10 l were injected onto the HPLC column. Chromatographic separation of the peptides was achieved using a Vydac Protein and Peptides (150 ⫻ 2.5mm, 5 m particle size) C18 reversed-phase HPLC column (Grace Vydac, Hesperia, CA, USA) and a gradient of aqueous formic acid (0.1% v/v) and methanol containing 0.1% (v/v) formic acid. The column was equilibrated at a flow rate of 0.25 ml/min with a mobile phase consisting of 95% 0.1% (v/v) formic acid and 5% methanol/formic acid at 40°C. The mobile phase was maintained at this composition for 5 min and then the methanol was increased linearly to 95% at 80 min. The concentration of methanol was maintained at 95% for 10 min and then returned to its initial concentration. The HPLC effluent was diverted into the mass spectrometer after 5 min and remained diverted into the mass spectrometer for the duration the assay. The mass spectrometer was equipped with an electrospray-ionization source and operated in scan mode for the detection of protonated ions having a mass to charge ratio of 200 –2000 using a scan time of 2 s. The mass spectrometer was calibrated and its settings optimized using apo-myoglobin and the synthetic peptide MRFA, as directed by the instrument manufacturer. All other
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Fig. 1. The effect of iron loading on ESR spin labeling of cysteine residues of ferritin. Reaction mixtures contained 5 M ferritin conjugated with 4-maleimido TEMPO. (A) Apo-wt ferritin, (B) wt ferritin loaded with 500 molar equivalents Fe(II). Instrumental conditions: microwave power, 50 mW; modulation amplitude, 1.01 G; time constant, 40.96 ms; sweep time, 41.96 s; 8 scans.
instrument parameters were set as follows: ESI spray voltage, 4.5 kV; capillary temperature, 250°C; auxiliary gas flow, 10 units; and sheath gas 50 psi. Qualitative analysis of the data was performed using the Xcalibur software package (version 1.1) (ThermoQuest). Identification of the peptides was achieved by comparing the calculated mass and the observed mass spectrum to theoretical M⫹H ions for the predicted tryptic peptides. Data for the theoretical peptide masses and relative retention properties were generated by virtual proteolysis using PepCut (ThermoQuest). Fluorescence spectroscopy Fluorescence spectroscopy was used to monitor for the appearance of dityrosine fluorescence (excitation at 325 nm and emission at 405 nm) of ferritin. Ferritin (0.25 M) was incubated with or without 250 molar equivalents Fe(II):ferritin of ferrous ammonium sulfate for 30 min at 25°C. Spectra were obtained using a Shimadzu RF-1501 spectrofluorophotometer. The fluorescence intensity is reported as relative intensity, i.e., the raw data obtained. RESULTS
We used the ESR technique of spin labeling to determine the effect of iron loading into ferritin on cysteine
residues. The spin label 4-maleimido-TEMPO has been reported to be selective for cysteine residues [21]. Incubation of the spin label with loaded wt ferritin produced an ESR spectrum with ⬃23% lower signal intensity than the spectrum for wt apo-ferritin (Fig. 1), indicating that cysteine residues had been altered during iron loading. A variety of peptides were produced by tryptic digestion, most of which were readily identified by comparing the calculated peptide mass and observed mass spectrum to the theoretical data. On average, the calculated mass was within 1.5 a.m.u. of the expected mass. Approximately 80% of the ferritin protein was represented by the LC/MS data. The identity of the peptides was also confirmed by MS/MS. In general, we found that the loading of ferritin with iron significantly altered the detection of various peptides, however, peptides containing cysteine residues were particularly susceptible to modification. A selected ion chromatogram showing the effects of iron loading in the detection of cysteine containing peptides by LC/MS is represented in Fig. 2. The identities of the peptides in Fig. 2 are presented in Table 2. M⫹H ions specific to each tryptic peptide listed in Table 2 were chosen for representation in the chromatogram based on its relative intensity in the mass spectrum as well as its prevalence in other spectra. In some instances the M⫹H ion used for the selected ion chromatogram was not the most abundant ion for the peptide, however, identical diagnostic M⫹H ions were used to generate the selected
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Fig. 2. LC/MS analysis of ferritin loaded with iron. Representative selected ion mass chromatograms from analysis of 10 g tryptic peptides generated from proteolysis of apo- and loaded ferritin. The peak identities are presented in Table 2. Ferritin (2 mg) was incubated with and without ferrous ammonium sulfate in 50 mM NaCl, pH 7.0, at 37°C (200 l final volume). Fe(II) was added in four increments of 500 molar equivalents of Fe(II):ferritin every 20 min.
ion chromatograms presented in Fig. 2. Selection of less intense ions to represent the peptide was done so that peaks having a much lower intensity were adequately represented in the chromatogram. Within each chromatogram, the peaks were normalized using the largest peak observed in the sample. Yet, as a result of these procedures, the relative size of each peak may not be a true representation of its actual abundance in the sample. However, the complete absence of a peak in the chro-
matogram signifies the loss of the peptide in the sample, presumably due to oxidative damage and a concomitant change in mass and/or retention time. In general, decreases in the detection of cysteinecontaining peptides increased with the degree of iron loading. Using LC/MS we observed a selective, however not limited to, loss of detection of peptides that contained cysteine residues (Fig. 3). Smaller peptides without cysteines, but represented in larger damaged peptides, were
Table 2. Peak Identities from LC/MS Chromatograms of Tryptic Digested Ferritin
Peak #
Peptide sequence
Retention time (min)
I II III IV V VI VII VIII IX X *XI *XII *XIII *XIV
LQNQR LATDK LMK LMKLQNQRGGR LMKLQNQR NVNQSLLELHK IFLQDIK GGRIFLQDIK LATDKNDPHLCDFIETHYLNEQVK LQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEK NVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVK IFLQDIKKPDCDDWESGLNAMECALHLEK KPDCDDWESGLNAMECALHLEKNVNQSLLELHK IFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHK
7.5 9.5 10.5 22.2 23.7 39.8 44.1 48.4 49.4 49.4 54.8 60.3 63.6 64.3
* A significant amount of peptide was detected in loaded C90R/C102A/C30A variant.
Theoretical mass
Experimental mass
657.7 546.6 390.5 1300.5 1030.3 1294.5 876.1 1146.4 2844.1 4272.8 4120.6 3362.8 3781.2 4639.3
657.7 ⫾ 0.0 546.3 ⫾ 0.0 390.2 ⫾ 0.0 1301.6 ⫾ 1.4 1030.3 ⫾ 0.0 1294.0 ⫾ 0.1 875.6 ⫾ 0.1 1146.5 ⫾ 0.6 2843.9 ⫾ 0.4 4275.0 ⫾ 0.7 4120.7 ⫾ 0.3 3361 ⫾ 1.3 3779 ⫾ 1.2 4638 ⫾ 1.3
Effects of iron loading Peptide Peptide Peptide Peptide Peptide Peptide Peptide Peptide Peptide Peptide Peptide Peptide Peptide Peptide
detected detected detected detected detected detected detected detected not detected not detected not detected not detected not detected not detected
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Fig. 3. Sequence alignment of peptides produced from tryptic digestion of apo-ferritin and loaded ferritin. Tryptic peptides were generated by incubating 2 mg of ferritin with 0.2 mg trypsin for 24 h at 37°C in 100 mM ammonium bicarbonate, pH 8.4, containing 40% isopropanol. Analysis and identification of the peptides were achieved by ESI-LC/MS. Identification of the peptides was achieved by comparison of the experimentally observed mass spectrum with theoretical mass spectra generated by virtual digestion of ferritin. Amino acid residues in bold represent consensus/overlapping portions of peptides that were modified (not detectable) in samples prepared from loaded ferritin.
not affected. The loss of cysteine-containing peptides may be related to the ease of oxidation of cysteine residues as well as their location on the peptide. Additionally, other “hot spots” were also observed, which could possibly be attributed to oxidation of histidine and tryptophan residues, which will be addressed in further studies. Wild-type ferritin and C90R, C102A, C130A, C90R/ C102A, and C90R/C102A/C130A variants were loaded with iron and subjected to native-PAGE (Fig. 4). One gel (A) was stained for protein and another (B) for iron. After loading, a significant amount of ferritin migrates as aggregates near the top of the gel [11]. The change of a
cysteine for an arginine altered the overall charge of the ferritin, making it more basic, hence the C90R ferritin variants migrated less than wild-type ferritin. Additionally, the ferritin variants with a C90R substitution migrated as a distinct band, especially the C90R/C102A and C90R/C102A/C130A variants. The majority of the protein for these variants migrated as expected with less formation of aggregates. The majority of the wt ferritin remained at the top of the gel, suggesting that aggregates had formed. The variants C102A and C130A also remained at or near the top of the gel, but in addition migrated as a smear, suggesting [11] that these proteins had also been oxidized and subsequently aggregated.
Fig. 4. Native-PAGE of ferritins loaded with iron. (A) Each well contained approximately 10 g of ferritin. The ferritins were visualized by Coomassie Blue staining. (B) Each well contained approximately 20 g of ferritin. The iron core of the ferritins was visualized by Prussian Blue staining. Lanes (1) contained C90R variant, (2) C102A variant, (3) C130A variant, (4) C90R/C102A variant, (5) C90R/C102A/C130A variant, (6) wt ferritin. Each ferritin was incubated with 1000 molar equivalents of ferrous ammonium sulfate in 50 mM NaCl, pH 7.0 at 37°C.
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Table 3. Changes in Fluorescence of Dityrosine of Recombinant Human Ferritins as a Result of Iron Loading Relative fluorescence intensitya Sample Wild type C90R C102A C130A C90R/C102A C90R/C102A/C130A
Apo-ferritin
Loaded ferritin
8.3 ⫾ 1.1 4.4 ⫾ 0.3 4.9 ⫾ 0.3 5.2 ⫾ 0.6 5.0 ⫾ 0.8 4.1 ⫾ 0.2
90 ⫾ 13 16.2 ⫾ 0.5 40 ⫾ 3 56 ⫾ 4 15.6 ⫾ 1.5 12.1 ⫾ 1.0
a The data are means of triplicate assays with standard deviations. Fluorescence intensity is reported as relative intensity, as described under Materials and Methods.
Fig. 5. Sucrose-density gradient sedimentation patterns of ferritins loaded with iron. (●) wt ferritin, (■) C90R variant, (Œ) C102A variant, () C130A variant, (⽧) C90R/C102A variant, and (E) C90R/C102A/ C130A variant. Ferritin (4 M) was incubated with ferrous ammonium sulfate (pH adjusted to ⬃7.5) in 50 mM NaCl, pH 7.0, at 37°C. Four additions of iron were added in increments of 250 molar equivalents Fe(II):ferritin at 20 min intervals.
Although the Prussian Blue stain of a native gel is only a qualitative measurement it is quite obvious that the variants with the C90R substitution loaded a significant amount of iron and that the protein which migrated as a distinct band towards the bottom of the gel contained a large amount of the iron. Interestingly the wt ferritin that migrated as a distinct band towards the bottom of the gel had very little iron in it, which suggests that the protein that migrated as a distinct band did so because it had not been loaded with iron and therefore was not damaged. The variants C102A and C130A loaded a significant amount of iron, however, the bands were smeared, which indicates heterogeneity in the mass to charge ratio, indicating that the protein had been oxidized. To further determine the role of each cysteine residue in the aggregation of ferritin as a result of iron loading we loaded ferritins with iron and separated them by sucrose-density gradient centrifugation. Loading 1000 atoms of ferrous ammonium sulfate per wt ferritin in 50 mM NaCl resulted in ⬃50% of the protein pelleting at the bottom of the centrifuge tube (Fig. 5). This pelleted protein has been attributed to damaged and consequently aggregated ferritin [11]. The ferritin variants C102A and C130A also had a large percentage of the protein aggregate and thus pellet at the bottom of the centrifuge tube. Interestingly, insignificant amounts of the ferritin vari-
ants with the C90R mutation pelleted at the bottom of the gradients. These data suggest that the cysteine at position 90 was primarily responsible for the aggregation of the ferritin. The formation of dityrosine moieties has been attributed to protein oxidation [22–24]. We have shown previously that the loading of iron into ferritin via its own ferroxidase activity resulted in an increase in dityrosine, as determined by fluorescence spectroscopy [11]. In this study we compared the amount of dityrosine formed during iron loading of wt ferritin with that of the cysteine variants. Interestingly, C90R variants were less prone to dityrosine formation (Table 3). DISCUSSION
The results presented in this study further demonstrate that loading iron into ferritin via its own ferroxidase activity resulted in the oxidation and subsequently aggregation of ferritin. It appeared that cysteine 90 was a key determinant of protein aggregation. Cysteine 90 has been shown to be located at the end of the BC loop of the ferritin H subunit (Fig. 6). The location of this cysteine residue may make it more accessible to metal-catalyzed oxidation as well as more accessible to other ferritin molecules to form aggregates. Interestingly, the signal intensity of the ferritin:spin label adduct decreased ⬃23% due to iron loading. A decrease in signal intensity of the ferritin:spin label adduct upon iron loading indicated that iron loading altered the cysteines such that they could not be conjugated by the spin label, possibly suggesting that they had been oxidized. Mass spectrometry has become a very powerful analytical technique for monitoring protein modification [25–29]. By combining HPLC to separate the proteolytic fragments of the trypsin-digested protein and the detection of peptides by mass spectrometry we were able to identify peptides that were modified during iron loading and localized the sites of damage to specific areas of
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Fig. 6. Ribbon diagram depicting the structure of the H subunit of human ferritin. The three cysteine residues are highlighted and labeled accordingly.
ferritin. In general, we observed a selective loss of peptides that contained cysteines. Interestingly, a comparison of the ion chromatograms of apo- and loaded C90R/ C102A/C130A variant, indicated that detection of the cysteine-containing peptides was not lost (data not shown). These data indicate that the cysteine residues are indeed “hot spots” for the oxidation of ferritin during iron loading by its own ferroxidase activity, and that without the cysteine residues the peptides were not modified. Additionally, upon iron loading we detected several peptides that were not observed in control samples. This is consistent with the fact that the oxidation of protein causes structural rearrangements that result in increased accessibility to proteolysis [30,31]. Therefore, loading iron into ferritin altered the 3-D conformation or tertiary structure of the protein such that new sites were accessible to trypsin. Many of the new recognition sites were close to cysteine residues. This suggests that the cysteine residues were oxidized and consequently the tertiary structure of the protein in the vicinity of the cysteine residues was altered. A number of studies [30, 32–35] have shown that oxidation of proteins does indeed alter the tertiary structure of the protein. We showed previously that using sucrose densitygradient centrifugation to separate ferritins loaded with iron we can get an approximation of how much ferritin aggregated [11]. We demonstrated that any ferritin that sediments completely through a 1–25% sucrose gradient
is damaged and/or aggregated protein. In this study we discovered that the cysteine at position 90 was essential in the aggregation of ferritin. Loading iron into ferritins that contained cysteine 90 resulted in a significant amount of aggregation, whereas any ferritin variants with the C90R mutation had very little aggregation. Additionally, the C102A change decreased the amount of ferritin that aggregated either as an individual change or in combination with C90R. This suggests that cysteine 102 also plays some role in the aggregation of ferritin. Variants with both C90A and C102R mutations exhibited significantly less aggregation as a result of iron loading. It appears from Fig. 5 that the ferritin variants containing the C90R substitution did not load much iron, however, from the Prussian blue stained gel (Fig. 4) it is apparent that they did indeed load iron. Additionally, we compared the ferroxidase activity of all the ferritin variants to that of wt ferritin, and determined they were all equal (data not shown). Therefore, the decrease in aggregation of the C90R-containing variants cannot be attributed to less iron being oxidized and/or loaded. Our first hypothesis was that the aggregation of the ferritin was a result of disulfide formation. However, the data obtained by LC/MS analysis provided no evidence for a M⫹M-2 peptide, i.e., a peptide indicative of two small peptides linked by a disulfide bridge. However, disulfide bonds are not the only covalent bonds formed during aggregation of protein. It has been demonstrated that protein aggregation can be a result of intermolecular dityrosine formation [22–24,30,32,36]. When ferritin is loaded with iron by its own ferroxidase activity there is an increase in dityrosine formation (Table 3). Interestingly, when the cysteine at position 90 was changed to an arginine, there was only a 3-fold increase in dityrosine formation versus the almost 11-fold increase in dityrosine in wt ferritin as a result of iron loading. The C102A variant had less dityrosine formation than both C130A variants and wt ferritin. It is possible that the cysteines of H chain ferritin are a “hot spot” for metalcatalyzed oxidation, especially cysteine 90; however, they are not the only amino acids oxidized. Other researchers have suggested that cysteine residues can be oxidized and then through intraprotein electron transfer catalyze the oxidation of other amino acids [37,38]. Thus the cysteine is the first, or one of the first residues oxidized, however, intraprotein electron transfer results in the oxidation of other residues, e.g., tyrosines. We are currently investigating the role of tyrosine residues in the aggregation of ferritin. We feel that the data presented in this study further demonstrates that iron is not loaded into ferritin via its own ferroxidase activity in vivo. If iron were loaded via this mechanism, the ferritin would be damaged. Recently it has been shown in cell culture experiments, that when
Ferritin oxidation
iron was continually supplied to the cells, the half-life of ferritin increased [39]. Oxidative damage to proteins is a marker for degradation [35,40,41], therefore, if ferritin were damaged during iron loading the half-life would decrease, not increase. One possibility is that during various conditions of iron overload, iron can be loaded into ferritin via its own ferroxidase activity and consequently the ferritin may be damaged. There has been considerable research done to determine the relationship of ferritin and hemosiderin [42– 44]. Recently researchers have suggested that hemosiderin is a degradation product of ferritin [45– 47]. It has been shown under iron overload and also copperdeficient conditions that there is an increase in hemosiderin formation in animals [48]. We propose that under physiological conditions iron is loaded into ferritin with the aid of another enzyme [49]. However, under various conditions of iron overload or a lack of the necessary enzyme, iron could be loaded into ferritin via its own ferroxidase activity and consequently the ferritin would be damaged and subsequently degraded to hemosiderin. We are currently testing this hypothesis. In conclusion, iron loading into ferritin via its own ferroxidase activity resulted in damage to the ferritin. The cysteine residues of ferritin appeared to be very susceptible to oxidation and appear to be involved in the oxidation and aggregation of the ferritin. Cysteine 90, located on the BC loop, appeared to be a key residue for aggregate formation. Even though ferritin variants that contain the C90R substitution do not aggregate, the ferritins were still damaged. Additionally, the cysteine residues may not be solely responsible for the aggregation but may work in concert with tyrosine residues, and aggregation could be a result of intermolecular dityrosine formation. Acknowledgement — The authors would like to thank Terri Maughan for her secretarial assistance in the preparation of the manuscript.
REFERENCES [1] Theil, E. C. Ferritin: structure, gene regulation, and cellular function in animals, plants, and microorganisms. Annu. Rev. Biochem. 56:289 –315; 1987. [2] Harrison, P. M.; Arosio, P. The ferritins: molecular properties, iron storage function and cellular regulation. Biochim. Biophys. Acta 1275:161–203; 1996. [3] Granick, S.; Hahn, P. Speed of uptake of iron by the liver and its conversion to ferritin iron. J. Biol. Chem. 155:661– 669; 1944. [4] deSilva, D.; Guo, J.-H.; Aust, S. D. Relationship between iron and phosphate in mammalian ferritins. Arch. Biochem. Biophys. 303: 451– 455; 1993. [5] Theil, E. C. The ferritin family of iron storage proteins. In: Meister, A., ed. Advances in enzymology and related areas of molecular biology (Vol. 63). New York: John Wiley & Sons, Inc.; 1990:421– 449. [6] Bakker, G. R.; Boyer, R. F. Iron incorporation into apoferritin. The role of apoferritin as a ferroxidase. J. Biol. Chem. 261: 13182–13185; 1986.
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[7] Lawson, D. M.; Treffry, A.; Artymiuk, P. J.; Harrison, P. M.; Yewdall, S. J.; Luzzago, A.; Cesareni, G.; Levi, S.; Arosio, P. Identification of the ferroxidase centre in ferritin. FEBS Lett. 254:207–210; 1989. [8] Yang, X.; Chasteen, N. D. Ferroxidase activity of ferritin: effects of pH, buffer and Fe(II) and Fe(III) concentrations on Fe(II) autoxidation and ferroxidase. Biochem. J. 338:615– 618; 1999. [9] Xu, B.; Chasteen, N. D. Iron autoxidation chemistry in ferritin. Increasing Fe/O2 stoichiometry during core formation. J. Biol. Chem. 266:19905–19970; 1991. [10] Yang, X.; Chen-Barrett, Y.; Arosio, P.; Chasteen, N. D. Reaction paths of iron oxidation and hydrolysis in horse spleen and recombinant human ferritins. Biochemistry 37:9743–9750; 1999. [11] Welch, K. D.; Van Eden, M. E.; Aust, S. D. Modification of ferritin during iron loading. Free Radic. Biol. Med. 31:999 –1006; 2001. [12] Van Eden, M. D.; Aust, S. D. The consequences of hydroxyl radical formation on the stoichiometry and kinetics of ferrous iron oxidation by human apoferritin. Free Radic. Biol. Med. 31:1007– 1017; 2001. [13] Grady, J. K.; Chen, Y.; Chasteen, N. D.; Harris, D. C. Hydroxyl radical production during oxidative deposition of iron in ferritin. J. Biol. Chem. 264:20224 –20229; 1989. [14] Stadtman, E. R. Metal ion-catalyzed oxidation of proteins: biochemical mechanism and biological consequences. Free Radic. Biol. Med. 9:315–325; 1990. [15] Stadtman, E. R. Oxidation of free amino acids and amino acid residues in proteins by radiolysis and by metal-catalyzed reactions. Annu. Rev. Biochem. 62:797– 821; 1993. [16] Kang, J. H.; Kim, K. S.; Choi, S. Y.; Kwon, H. Y.; Won, M. H. Oxidative modification of human ceruloplamsin by peroxyl radicals. Biochem. Biophys. Acta 1568:30 –36; 2001. [17] Van Eden, M. E.; Aust, S. D. Intact human ceruloplasmin is required for the incorporation of iron into human ferritin. Arch. Biochem. Biophys. 381:119 –126; 2000. [18] Turchin, A.; Lawler, J. F. The primer generator: a program that facilitates the selection of oligonucleotides for site-directed mutagenesis. Biotechniques 26:672– 676; 1999. [19] Shepard, A. R.; Rae, J. L. “Microprep” method for rapidly isolating plasmid DNAs for restriction enzyme analysis. Biotechniques 26:868 – 870; 1999. [20] deSilva, D.; Miller, D. M.; Reif, D. W.; Aust, S. D. In vitro loading of apoferritin. Arch. Biochem. Biophys. 293:409 – 415; 1992. [21] Singh, R. J.; Feix, J. B.; Metiaourab, H. S.; Hogg, N.; Kalyanaraman, B. Spin-labeling study of the oxidative damage to lowdensity lipoprotein. Arch. Biochem. Biophys. 320:155–161; 1995. [22] Audette, M.; Blouquit, Y.; Houee-Levin, C. Oxidative dimerization of proteins: role of tyrosine accessibility. Arch. Biochem. Biophys. 376:217–220; 2000. [23] Giulivi, C.; Davies, K. J. A. Dityrosine: a marker for oxidatively modified proteins and selective proteolysis. Methods Enzymol. 233:363–371; 1994. [24] Dalle-Donne, I.; Rossi, R.; Giustarini, D.; Gagliano, N.; Lusini, L.; Milzani, A.; DiSimplicio, P.; Columbo, R. Actin carbonylation: from a simple marker of protein oxidation to relevant signs of severe functional impairment. Free Radic. Biol. Med. 31:1075– 1083; 2001. [25] Kiselar, J. G.; Malekinla, S. D.; Sullivan, M.; Downard, K. M.; Chance, M. R. Hydroxyl radical probe of protein surfaces using synchrotron x-ray radiolysis and mass spectrometry. Int. J. Radiat. Biol. 78:101–114; 2002. [26] Keough, T.; Lacey, M. P.; Strife, R. J. Atmospheric pressure matrix-assisted laser desorption/ionization ion trap mass spectrometry of sulfonic acid derivatized tryptic peptides. Rapid Commun. Mass Spectrom. 15:2227–2239; 2001. [27] Mason, D. E.; Liebler, D. C. Characterization of benzoquinone peptide adducts by electrospray mass spectrometry. Chem. Res. Toxicol. 13:976 –982; 2000. [28] Jones, J. A.; Liebler, D. C. Tandem MS analysis of model peptide
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K. D. WELCH et al. adducts from reactive metabolites of the hepatotoxin 1,1-dichloroethylene. Chem. Res. Toxicol. 13:1302–1312; 2000. Fu, X.; Mueller, D. M.; Heinecke, J. W. Generation of intramolecular and intermolecular sulfenamides, sulfinamides, and sulfonamides by hypochlorous acid: a potential pathway for oxidative cross-linking of low-density lipoprotein by myeloperoxidase. Biochemistry 41:1293–1301; 2002. Davies, K. J. A.; Delsignore, M. E. Protein damage and degradation by oxygen radicals. (III) Modification of secondary and tertiary structure. J. Biol. Chem. 262:9908 –9913; 1987. Rivett, A. J. Preferential degradation of the oxidatively modified form of glutamine synthetase by intracellular mammalian proteases. J. Biol. Chem. 260:300 –305; 1985. Davies, K. J. A. Protein damage and degradation by oxygen radicals. (I) General aspects. J. Biol. Chem. 202:9895–9901; 1987. Davies, K. J. A.; Delsignore, M. E.; Lin, S. W. Protein damage and degradation by oxygen radicals. (II) Modification of amino acids. J. Biol. Chem. 262:9902–9907; 1987. Davies, K. J. A.; Lin, S. W.; Pacifici, R. E. Protein damage and degradation by oxygen radicals. (IV) Degradation of denatured protein. J. Biol. Chem. 262:9914 –9970; 1987. Grune, T.; Reinheckel, T.; Davies, K. J. A. Degradation of oxidized proteins in mammalian cells. FASEB J. 11:526 –534; 1997. Ogino, T.; Okada, S. Oxidative damage of bovine serum albumin and other enzyme proteins by iron-chelate complexes. Biochim. Biophys. Acta 1245:359 –365; 1995. Davies, M. J.; Gilbert, B. C.; Haywood, K. M. Radical-induced damage to bovine serum albumin: role of the cysteine residue. Free Radic. Res. Commun. 18:353–367; 1993. Anderson, R. F.; Hille, R.; Patel, K. B. Inactivation of xanthine oxidase by oxidative radical attack. Int. J. Radiat. Biol. 68:535– 541; 1995. Trufy, J.; Malpe, R.; Linder, M. C. Iron prevents ferritin turnover in hepatic cells. J. Biol. Chem. 276:48775– 48780; 2001. Nagy, I.; Floyd, R. A. Hydroxyl free radical reactions with amino acids and proteins studied by electron spin resonance spectroscopy and spin-trapping. Biochim. Biophys. Acta 790:238 –250; 1984.
[41] Rivett, A. J.; Levine, R. L. Metal-catalyzed oxidation of Escherichia coli glutamine synthetase: correlation of structural and functional changes. Arch. Biochem. Biophys. 278:26 –34; 1990. [42] Ward, R. J.; Ramsey, M.; Dichson, D. P. E.; Hunt, C.; Douglass, T.; Mann, S.; Aouad, F.; Peters, T. J.; Crichton, R. R. Further characterization of forms of hemosiderin in iron-overloaded tissues. Eur. J. Biochem. 225:187–194; 1994. [43] Mann, S.; Wade, V. J.; Dichson, D. P. E.; Reid, N. M. K.; Ward, R. J.; O’Connell, M. J.; Peters, T. J. Structural specificity of haemosiderin iron cores in iron-overload diseases. FEBS Lett. 234:69 –72; 1988. [44] Weir, M. P.; Gibson, J. F.; Peters, T. J. Biochemical studies on the isolation and characterization of human spleen haemosiderin. Biochem. J. 223:31–38; 1984. [45] O’Connell, M. J.; Baum, H.; Peters, T. J. Haemosiderin-like properties of free-radical-modified ferritin. Biochem. J. 240:297– 300; 1986. [46] O’Connell, M. J.; Peters, T. J. Ferritin and haemosiderin in free radical generation; lipid peroxidation and protein damage. Chem. Phys. Lipids 45:241–249; 1987. [47] Iancu, T. C. Ferritin and hemosiderin in pathological tissues. Electron Microsc. Rev. 5:209 –229; 1992. [48] Seo, H.; Xie, B.; Wan, S.; Yoshihawa, H.; Oyamuda, T.; Yoshikawa, T. Ultrastructure of hepatocytes in copper-deficient Sika deer (Cervus nippon Temminck). J. Comp. Path. 114:283–290; 1996. [49] Reilly, C. A.; Aust, S. D. Iron loading into ferritin by an intracellular ferroxidase. Arch. Biochem. Biophys. 359:69 –76; 1998.
ABBREVIATIONS
H2O2— hydrogen peroxide • OH— hydroxyl radical LC/MS—liquid chromatography/mass spectrometry MS/MS—tandem mass spectrometry ESR— electron spin resonance
PII S0891-5849(02)00915-2
Original Contribution THE ROLE OF CYSTEINE RESIDUES IN THE OXIDATION OF FERRITIN KEVIN D. WELCH,* CHRISTOPHER A. REILLY,†
and
STEVEN D. AUST*
*Biotechnology Center, Utah State University, Logan, UT, USA; and †Department of Pharmacology and Toxicology, Center for Human Toxicology, University of Utah, Salt Lake City, UT, USA (Received 28 February 2002; Revised 30 April 2002; Accepted 9 May 2002)
Abstract—We have shown that ferritin is oxidized during iron loading using its own ferroxidase activity and that this oxidation results in its aggregation (Welch et al., Free Radic. Biol. Med. 31:999 –1006; 2001). In this study we determined the role of cysteine residues in the oxidation of ferritin. Loading iron into recombinant human ferritin by its own ferroxidase activity decreased its conjugation by a cysteine specific spin label, indicating that cysteine residues were altered during iron loading. Using LC/MS, we demonstrated that tryptic peptides of ferritin that contained cysteine residues were susceptible to modification as a result of iron loading. To assess the role of cysteine residues in the oxidation of ferritin, we used site-directed mutagenesis to engineer variants of human ferritin H chain homomers where the cysteines were substituted with other amino acids. The cysteine at position 90, which is located at the end of the BC-loop, appeared to be critical for the formation of ferritin aggregates during iron loading. We also provide evidence that dityrosine moieties are formed during iron loading into ferritin by its own ferroxidase activity and that the dityrosine formation is dependent upon the oxidation of cysteine residues, especially cysteine 90. In conclusion, cysteine residues play an integral role in the oxidation of ferritin and are essential for the formation of ferritin aggregates. © 2002 Elsevier Science Inc. Keywords—Iron, Ferritin, Protein oxidation, Cysteine oxidation, Dityrosine, Free radicals
INTRODUCTION
ferritin via this mechanism could damage the ferritin and any other biomolecules in close proximity. We [11,12] and others [13] have shown that the •OH is indeed produced when iron is loaded into ferritin via its ferroxidase activity. Additionally, we demonstrated that the production of the hydroxyl radical resulted in damage to the ferritin [11,12]. Oxidative damage to ferritin was prevented when iron loading was performed in the presence of an organic buffer, e.g., HEPES. It was postulated that the protective effect of HEPES was the result of its ability to scavenge •OH, thus inhibiting the oxidation of ferritin by •OH. However, the addition of catalase to the reaction mixture (not containing HEPES) did not “protect” ferritin from damage [11]. These data suggest that the oxidation of the ferritin was either a site-specific process or that it was initiated by an oxidant not requiring H2O2 for its formation. Interestingly, the aggregation of ferritin appeared to be a result of cysteine oxidation [11]. This is quite probable because the cysteine residue is one of the more susceptible amino acids to metal-catalyzed oxidation [14 –16], and can subsequently form disulfide cross-links.
Ferritin is an ubiquitous intracellular iron storage protein, wherein up to 2500 atoms of ferric iron can be stored as a ferric oxyhydroxy phosphate complex [1– 4]. Ferritin is composed of 24 subunits of H and L type, which assemble to form a protein shell with a hollow interior of approximately 90 Å. In order for iron to be incorporated into the ferritin core it must be presented as ferrous iron and subsequently oxidized. In general, it is believed that iron is loaded into ferritin by itself, dependent upon a “ferroxidase activity” of the H subunits of ferritin [2,5– 8]. However, the stoichiometry of iron oxidation in this system is 2:1, i.e., two moles of iron oxidized per mole of molecular oxygen reduced, indicating the generation of H2O2 [9,10]. Hydrogen peroxide in the presence of ferrous iron can result in the formation of a strong oxidant such as the hydroxyl radical (•OH), a nonspecific oxidant of biomolecules. Therefore, loading iron into Address correspondence to: Dr. Steven D. Aust, Biotechnology Center, Utah State University, Logan, UT 84322-4705, USA; Tel: (435) 797-2730; Fax: (435) 797-2755; E-Mail: [email protected]. 399
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K. D. WELCH et al. Table 1. Primers for Site-Directed Mutagenesis of Human H Chain Ferritin DNAa
Forward primer (5⬘-3⬘)
Restriction site
CAGGATATCAAGAAACCAGACCGGGATGACTGGG GGGCTGAATGCAATGGAGGCTGCATTACATTTGG CAAAAATGACCCCCATTTGGCCGACTTCATTGAGACAC
BseGI TseI CfrI
Primer ID rHuHFt C90R rHuHFt C102A rHuHFt C130A a
Target codons are underlined and sequence changes bolded.
The H subunit of human ferritin contains three cysteine residues, at positions 90, 102, and 130. Analysis of the predicted 3D structure of the H chain of ferritin demonstrated that cysteine 90, located on the BC-loop, faced the exterior, solvent-exposed surface of the protein. In ferritin from other species cysteine 90 is absent, e.g., rat ferritin contains an arginine at position 90. Interestingly, rat ferritin appears to be less susceptible to aggregation during iron loading versus human ferritin (unpublished observations). In almost all species cysteine 102 and 130 are highly conserved in the H chain as well as cysteine 130 in the L chain. This study was performed to further our understanding of the process of oxidation and subsequent aggregation that occurs when ferritin is loaded with iron via its own ferroxidase activity. Specifically, we wanted to determine the role of the cysteine residues in the oxidation of ferritin. To accomplish this we performed site-directed mutagenesis to engineer ferritin variants where the cysteine residues were substituted with other amino acids. We produced C90R, C102A, C130A, C90R/C102A, and C90R/C102A/C130A ferritin variants. We analyzed all of these variants, in addition to wild-type recombinant human ferritin H chain homomer (wt) ferritin, for protein oxidation and subsequent aggregation. Analysis of the tryptic digests of ferritins by liquid chromatographymass spectrometry (LC/MS) indicated that peptides containing cysteine residues were modified during iron loading into ferritin via its own ferroxidase activity. We also provide data demonstrating that ferritin variants with the C90R substitution were significantly less susceptible to aggregate formation resulting from oxidation during iron loading. MATERIALS AND METHODS
Chemicals Ammonium bicarbonate was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Sucrose and ferrous ammonium sulfate were purchased from Mallinckrodt (Paris, KY, USA). 4-Maleimido-TEMPO and sodium chloride (99.999% pure) were purchased from Aldrich Chemical Company (Milwaukee, WI, USA). N-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid
(HEPES) was purchased from Boehringer Mannheim (Indianapolis, IN, USA). Isopropyl alcohol was purchased from Fisher Scientific (Fair Lawn, NJ, USA). Reagents were prepared using water purified by reverse osmosis with subsequent passage through a NANOpure Infinity Ultrapure Water System (Barnstead/Thermolyne Corporation, Dubuque, IA, USA). Proteins Trypsin was purchased from Sigma Chemical Company. Recombinant human ferritin H chain homomer was produced and purified as described by Van Eden and Aust [17]. Site-directed mutagenesis Gene sequence and 3D crystal structure models for human ferritin were obtained from Genebank (gi:182504) and the Protein Data Bank (2fha), respectively. Rasmol v2.5 (Glaxo Research and Development, Greenford, Middlesex, UK) was used to model protein structure. Mutagenesis was conducted using the QuickChange Site-Directed Mutagenesis protocol from Stratagene (La Jolla, CA, USA). Mutagenesis primers were designed using the program Primer Generator [18] to include unique restriction sites for screening the products of mutagenesis. Macromolecular Resources (Department of Biochemistry, Colorado State University, Ft. Collins, CO, USA) synthesized the primers for site-directed mutagenesis (Table 1). Mutagenesis with each primer set was conducted by PCR using a Gene Amp System 9600 Thermal Cycler (Perkin-Elmer, Emeryville, CA, USA) according to the QuickChange Site-Directed Mutagenesis protocol. The PCR product was transformed into Escherichia coli strain XL1-Blue (Stratagene), plated on LB/Ampicillin agar plates and incubated at 37°C for 20 h. Resistant colonies were selected and cultured in 3 ml of LB/Ampicilin medium for 16 h at 37°C. Plasmid DNA was isolated from the cell pellet derived from 1 ml of the E. coli cultures by a “microprep” protocol [19]. The purified plasmid DNA was subjected to PCR using primers for the 5⬘ and 3⬘ end of the ferritin H chain gene. The PCR product was then digested with the appropriate
Ferritin oxidation
restriction enzyme to test for the incorporation of the mutation in the gene sequence. DNA containing the C90R mutation was used to create a double mutant C90R/C102A and a triple mutant C90R/C10A/C130A using the appropriate mutagenesis primer sets. The modified human ferritin DNA sequences were confirmed by sequencing using an ABI Prism 377 DNA sequencer (PE/Applied Biosystems, Foster City, CA, USA) by the Biotechnology Center Service Laboratory (Utah State University, Logan, UT, USA). The various expression plasmids containing the mutated DNA for H chain human ferritin were transformed into E. coli strain B834 (DE3) pLysS Novagen, Inc. (Madison, WI, USA) for expression of the human ferritin variant proteins. Loading iron into ferritin Loading iron into ferritin was performed as described previously [20]. Briefly, ferritin was incubated with ferrous ammonium sulfate (pH adjusted to ⬃7.5) in 50 mM NaCl, pH 7.0, at 37°C. For specific concentrations refer to appropriate figure legends. Sucrose density-gradient centrifugation Iron loading mixtures were placed on top of 10 ml continuous sucrose gradients (1–25% w/v). The samples were centrifuged in a Beckman SW41Ti swinging bucket rotor at 30,000 rpm (⬃110,000 ⫻ g) for 2.5 h. Aliquots (1 ml) were removed from the top of the gradients by pipette and analyzed for protein by the Bradford Assay. Native PAGE analysis Aliquots were removed directly from iron loading reactions and subjected to native PAGE using a 7.5% resolving gel with a 4% stacking gel. The samples were electrophoresed for approximately 2.5 h and subsequently stained for protein with Coomassie Blue or iron using Prussian Blue. Prussian Blue staining was performed by incubating gels in a solution of 0.1% (w/v) potassium ferrocyanide in 1 N HCl at 25°C for ⬃15 min. Ferritin iron content was qualitatively assessed after the gels were washed with water. Spin labeling of ferritin 4-Maleimido-TEMPO was used to label the cysteine residues of recombinant human H chain ferritin. Briefly, 7.2 mM spin label was incubated with 5 M ferritin in 50 mM HEPES, pH 7.0, at 25°C in the dark for 2 h. Excess spin label was removed by chromatography over a Econo Pac 10DG desalting column (BioRad Laboratories, Her-
401
cules, CA, USA). ESR spectra of spin labeled ferritin were recorded at room temperature using a Bruker ECS 106 spectrometer operating at 9.8 GHz with a 50 kHz modulation frequency. Tryptic digestion of ferritin Upon completion of iron loading, isopropyl alcohol and ammonium bicarbonate, pH 8.4, were added to make final concentrations of 40% (v/v) and 10% (v/v) respectively, in 1 ml final volume. The samples were then incubated at 100°C for 5 min. The samples were allowed to cool to 25°C before the addition of trypsin (0.2 mg). The samples were then incubated at 37°C for 24 h. Liquid chromatography-mass spectrometry of ferritin peptides Analysis of ferritin peptides produced from tryptic digestion of ferritin was achieved by high performance liquid chromatography and electrospray-ionization mass spectrometry (ESI LC/MS) using a ThermoQuest/Finnigan TSQ7000 mass spectrometer (ThermoQuest Instruments, San Jose, CA, USA) interfaced with a HewlettPackard series 1100 HPLC system (Agilent Technologies, Palo Alto, CA, USA). Samples of peptides were diluted to a concentration of 1 g/l in 90%, aqueous formic acid (0.1% v/v):10% methanol, placed into an autosampler vial maintained at 4°C, and 10 l were injected onto the HPLC column. Chromatographic separation of the peptides was achieved using a Vydac Protein and Peptides (150 ⫻ 2.5mm, 5 m particle size) C18 reversed-phase HPLC column (Grace Vydac, Hesperia, CA, USA) and a gradient of aqueous formic acid (0.1% v/v) and methanol containing 0.1% (v/v) formic acid. The column was equilibrated at a flow rate of 0.25 ml/min with a mobile phase consisting of 95% 0.1% (v/v) formic acid and 5% methanol/formic acid at 40°C. The mobile phase was maintained at this composition for 5 min and then the methanol was increased linearly to 95% at 80 min. The concentration of methanol was maintained at 95% for 10 min and then returned to its initial concentration. The HPLC effluent was diverted into the mass spectrometer after 5 min and remained diverted into the mass spectrometer for the duration the assay. The mass spectrometer was equipped with an electrospray-ionization source and operated in scan mode for the detection of protonated ions having a mass to charge ratio of 200 –2000 using a scan time of 2 s. The mass spectrometer was calibrated and its settings optimized using apo-myoglobin and the synthetic peptide MRFA, as directed by the instrument manufacturer. All other
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Fig. 1. The effect of iron loading on ESR spin labeling of cysteine residues of ferritin. Reaction mixtures contained 5 M ferritin conjugated with 4-maleimido TEMPO. (A) Apo-wt ferritin, (B) wt ferritin loaded with 500 molar equivalents Fe(II). Instrumental conditions: microwave power, 50 mW; modulation amplitude, 1.01 G; time constant, 40.96 ms; sweep time, 41.96 s; 8 scans.
instrument parameters were set as follows: ESI spray voltage, 4.5 kV; capillary temperature, 250°C; auxiliary gas flow, 10 units; and sheath gas 50 psi. Qualitative analysis of the data was performed using the Xcalibur software package (version 1.1) (ThermoQuest). Identification of the peptides was achieved by comparing the calculated mass and the observed mass spectrum to theoretical M⫹H ions for the predicted tryptic peptides. Data for the theoretical peptide masses and relative retention properties were generated by virtual proteolysis using PepCut (ThermoQuest). Fluorescence spectroscopy Fluorescence spectroscopy was used to monitor for the appearance of dityrosine fluorescence (excitation at 325 nm and emission at 405 nm) of ferritin. Ferritin (0.25 M) was incubated with or without 250 molar equivalents Fe(II):ferritin of ferrous ammonium sulfate for 30 min at 25°C. Spectra were obtained using a Shimadzu RF-1501 spectrofluorophotometer. The fluorescence intensity is reported as relative intensity, i.e., the raw data obtained. RESULTS
We used the ESR technique of spin labeling to determine the effect of iron loading into ferritin on cysteine
residues. The spin label 4-maleimido-TEMPO has been reported to be selective for cysteine residues [21]. Incubation of the spin label with loaded wt ferritin produced an ESR spectrum with ⬃23% lower signal intensity than the spectrum for wt apo-ferritin (Fig. 1), indicating that cysteine residues had been altered during iron loading. A variety of peptides were produced by tryptic digestion, most of which were readily identified by comparing the calculated peptide mass and observed mass spectrum to the theoretical data. On average, the calculated mass was within 1.5 a.m.u. of the expected mass. Approximately 80% of the ferritin protein was represented by the LC/MS data. The identity of the peptides was also confirmed by MS/MS. In general, we found that the loading of ferritin with iron significantly altered the detection of various peptides, however, peptides containing cysteine residues were particularly susceptible to modification. A selected ion chromatogram showing the effects of iron loading in the detection of cysteine containing peptides by LC/MS is represented in Fig. 2. The identities of the peptides in Fig. 2 are presented in Table 2. M⫹H ions specific to each tryptic peptide listed in Table 2 were chosen for representation in the chromatogram based on its relative intensity in the mass spectrum as well as its prevalence in other spectra. In some instances the M⫹H ion used for the selected ion chromatogram was not the most abundant ion for the peptide, however, identical diagnostic M⫹H ions were used to generate the selected
Ferritin oxidation
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Fig. 2. LC/MS analysis of ferritin loaded with iron. Representative selected ion mass chromatograms from analysis of 10 g tryptic peptides generated from proteolysis of apo- and loaded ferritin. The peak identities are presented in Table 2. Ferritin (2 mg) was incubated with and without ferrous ammonium sulfate in 50 mM NaCl, pH 7.0, at 37°C (200 l final volume). Fe(II) was added in four increments of 500 molar equivalents of Fe(II):ferritin every 20 min.
ion chromatograms presented in Fig. 2. Selection of less intense ions to represent the peptide was done so that peaks having a much lower intensity were adequately represented in the chromatogram. Within each chromatogram, the peaks were normalized using the largest peak observed in the sample. Yet, as a result of these procedures, the relative size of each peak may not be a true representation of its actual abundance in the sample. However, the complete absence of a peak in the chro-
matogram signifies the loss of the peptide in the sample, presumably due to oxidative damage and a concomitant change in mass and/or retention time. In general, decreases in the detection of cysteinecontaining peptides increased with the degree of iron loading. Using LC/MS we observed a selective, however not limited to, loss of detection of peptides that contained cysteine residues (Fig. 3). Smaller peptides without cysteines, but represented in larger damaged peptides, were
Table 2. Peak Identities from LC/MS Chromatograms of Tryptic Digested Ferritin
Peak #
Peptide sequence
Retention time (min)
I II III IV V VI VII VIII IX X *XI *XII *XIII *XIV
LQNQR LATDK LMK LMKLQNQRGGR LMKLQNQR NVNQSLLELHK IFLQDIK GGRIFLQDIK LATDKNDPHLCDFIETHYLNEQVK LQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEK NVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVK IFLQDIKKPDCDDWESGLNAMECALHLEK KPDCDDWESGLNAMECALHLEKNVNQSLLELHK IFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHK
7.5 9.5 10.5 22.2 23.7 39.8 44.1 48.4 49.4 49.4 54.8 60.3 63.6 64.3
* A significant amount of peptide was detected in loaded C90R/C102A/C30A variant.
Theoretical mass
Experimental mass
657.7 546.6 390.5 1300.5 1030.3 1294.5 876.1 1146.4 2844.1 4272.8 4120.6 3362.8 3781.2 4639.3
657.7 ⫾ 0.0 546.3 ⫾ 0.0 390.2 ⫾ 0.0 1301.6 ⫾ 1.4 1030.3 ⫾ 0.0 1294.0 ⫾ 0.1 875.6 ⫾ 0.1 1146.5 ⫾ 0.6 2843.9 ⫾ 0.4 4275.0 ⫾ 0.7 4120.7 ⫾ 0.3 3361 ⫾ 1.3 3779 ⫾ 1.2 4638 ⫾ 1.3
Effects of iron loading Peptide Peptide Peptide Peptide Peptide Peptide Peptide Peptide Peptide Peptide Peptide Peptide Peptide Peptide
detected detected detected detected detected detected detected detected not detected not detected not detected not detected not detected not detected
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Fig. 3. Sequence alignment of peptides produced from tryptic digestion of apo-ferritin and loaded ferritin. Tryptic peptides were generated by incubating 2 mg of ferritin with 0.2 mg trypsin for 24 h at 37°C in 100 mM ammonium bicarbonate, pH 8.4, containing 40% isopropanol. Analysis and identification of the peptides were achieved by ESI-LC/MS. Identification of the peptides was achieved by comparison of the experimentally observed mass spectrum with theoretical mass spectra generated by virtual digestion of ferritin. Amino acid residues in bold represent consensus/overlapping portions of peptides that were modified (not detectable) in samples prepared from loaded ferritin.
not affected. The loss of cysteine-containing peptides may be related to the ease of oxidation of cysteine residues as well as their location on the peptide. Additionally, other “hot spots” were also observed, which could possibly be attributed to oxidation of histidine and tryptophan residues, which will be addressed in further studies. Wild-type ferritin and C90R, C102A, C130A, C90R/ C102A, and C90R/C102A/C130A variants were loaded with iron and subjected to native-PAGE (Fig. 4). One gel (A) was stained for protein and another (B) for iron. After loading, a significant amount of ferritin migrates as aggregates near the top of the gel [11]. The change of a
cysteine for an arginine altered the overall charge of the ferritin, making it more basic, hence the C90R ferritin variants migrated less than wild-type ferritin. Additionally, the ferritin variants with a C90R substitution migrated as a distinct band, especially the C90R/C102A and C90R/C102A/C130A variants. The majority of the protein for these variants migrated as expected with less formation of aggregates. The majority of the wt ferritin remained at the top of the gel, suggesting that aggregates had formed. The variants C102A and C130A also remained at or near the top of the gel, but in addition migrated as a smear, suggesting [11] that these proteins had also been oxidized and subsequently aggregated.
Fig. 4. Native-PAGE of ferritins loaded with iron. (A) Each well contained approximately 10 g of ferritin. The ferritins were visualized by Coomassie Blue staining. (B) Each well contained approximately 20 g of ferritin. The iron core of the ferritins was visualized by Prussian Blue staining. Lanes (1) contained C90R variant, (2) C102A variant, (3) C130A variant, (4) C90R/C102A variant, (5) C90R/C102A/C130A variant, (6) wt ferritin. Each ferritin was incubated with 1000 molar equivalents of ferrous ammonium sulfate in 50 mM NaCl, pH 7.0 at 37°C.
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Table 3. Changes in Fluorescence of Dityrosine of Recombinant Human Ferritins as a Result of Iron Loading Relative fluorescence intensitya Sample Wild type C90R C102A C130A C90R/C102A C90R/C102A/C130A
Apo-ferritin
Loaded ferritin
8.3 ⫾ 1.1 4.4 ⫾ 0.3 4.9 ⫾ 0.3 5.2 ⫾ 0.6 5.0 ⫾ 0.8 4.1 ⫾ 0.2
90 ⫾ 13 16.2 ⫾ 0.5 40 ⫾ 3 56 ⫾ 4 15.6 ⫾ 1.5 12.1 ⫾ 1.0
a The data are means of triplicate assays with standard deviations. Fluorescence intensity is reported as relative intensity, as described under Materials and Methods.
Fig. 5. Sucrose-density gradient sedimentation patterns of ferritins loaded with iron. (●) wt ferritin, (■) C90R variant, (Œ) C102A variant, () C130A variant, (⽧) C90R/C102A variant, and (E) C90R/C102A/ C130A variant. Ferritin (4 M) was incubated with ferrous ammonium sulfate (pH adjusted to ⬃7.5) in 50 mM NaCl, pH 7.0, at 37°C. Four additions of iron were added in increments of 250 molar equivalents Fe(II):ferritin at 20 min intervals.
Although the Prussian Blue stain of a native gel is only a qualitative measurement it is quite obvious that the variants with the C90R substitution loaded a significant amount of iron and that the protein which migrated as a distinct band towards the bottom of the gel contained a large amount of the iron. Interestingly the wt ferritin that migrated as a distinct band towards the bottom of the gel had very little iron in it, which suggests that the protein that migrated as a distinct band did so because it had not been loaded with iron and therefore was not damaged. The variants C102A and C130A loaded a significant amount of iron, however, the bands were smeared, which indicates heterogeneity in the mass to charge ratio, indicating that the protein had been oxidized. To further determine the role of each cysteine residue in the aggregation of ferritin as a result of iron loading we loaded ferritins with iron and separated them by sucrose-density gradient centrifugation. Loading 1000 atoms of ferrous ammonium sulfate per wt ferritin in 50 mM NaCl resulted in ⬃50% of the protein pelleting at the bottom of the centrifuge tube (Fig. 5). This pelleted protein has been attributed to damaged and consequently aggregated ferritin [11]. The ferritin variants C102A and C130A also had a large percentage of the protein aggregate and thus pellet at the bottom of the centrifuge tube. Interestingly, insignificant amounts of the ferritin vari-
ants with the C90R mutation pelleted at the bottom of the gradients. These data suggest that the cysteine at position 90 was primarily responsible for the aggregation of the ferritin. The formation of dityrosine moieties has been attributed to protein oxidation [22–24]. We have shown previously that the loading of iron into ferritin via its own ferroxidase activity resulted in an increase in dityrosine, as determined by fluorescence spectroscopy [11]. In this study we compared the amount of dityrosine formed during iron loading of wt ferritin with that of the cysteine variants. Interestingly, C90R variants were less prone to dityrosine formation (Table 3). DISCUSSION
The results presented in this study further demonstrate that loading iron into ferritin via its own ferroxidase activity resulted in the oxidation and subsequently aggregation of ferritin. It appeared that cysteine 90 was a key determinant of protein aggregation. Cysteine 90 has been shown to be located at the end of the BC loop of the ferritin H subunit (Fig. 6). The location of this cysteine residue may make it more accessible to metal-catalyzed oxidation as well as more accessible to other ferritin molecules to form aggregates. Interestingly, the signal intensity of the ferritin:spin label adduct decreased ⬃23% due to iron loading. A decrease in signal intensity of the ferritin:spin label adduct upon iron loading indicated that iron loading altered the cysteines such that they could not be conjugated by the spin label, possibly suggesting that they had been oxidized. Mass spectrometry has become a very powerful analytical technique for monitoring protein modification [25–29]. By combining HPLC to separate the proteolytic fragments of the trypsin-digested protein and the detection of peptides by mass spectrometry we were able to identify peptides that were modified during iron loading and localized the sites of damage to specific areas of
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Fig. 6. Ribbon diagram depicting the structure of the H subunit of human ferritin. The three cysteine residues are highlighted and labeled accordingly.
ferritin. In general, we observed a selective loss of peptides that contained cysteines. Interestingly, a comparison of the ion chromatograms of apo- and loaded C90R/ C102A/C130A variant, indicated that detection of the cysteine-containing peptides was not lost (data not shown). These data indicate that the cysteine residues are indeed “hot spots” for the oxidation of ferritin during iron loading by its own ferroxidase activity, and that without the cysteine residues the peptides were not modified. Additionally, upon iron loading we detected several peptides that were not observed in control samples. This is consistent with the fact that the oxidation of protein causes structural rearrangements that result in increased accessibility to proteolysis [30,31]. Therefore, loading iron into ferritin altered the 3-D conformation or tertiary structure of the protein such that new sites were accessible to trypsin. Many of the new recognition sites were close to cysteine residues. This suggests that the cysteine residues were oxidized and consequently the tertiary structure of the protein in the vicinity of the cysteine residues was altered. A number of studies [30, 32–35] have shown that oxidation of proteins does indeed alter the tertiary structure of the protein. We showed previously that using sucrose densitygradient centrifugation to separate ferritins loaded with iron we can get an approximation of how much ferritin aggregated [11]. We demonstrated that any ferritin that sediments completely through a 1–25% sucrose gradient
is damaged and/or aggregated protein. In this study we discovered that the cysteine at position 90 was essential in the aggregation of ferritin. Loading iron into ferritins that contained cysteine 90 resulted in a significant amount of aggregation, whereas any ferritin variants with the C90R mutation had very little aggregation. Additionally, the C102A change decreased the amount of ferritin that aggregated either as an individual change or in combination with C90R. This suggests that cysteine 102 also plays some role in the aggregation of ferritin. Variants with both C90A and C102R mutations exhibited significantly less aggregation as a result of iron loading. It appears from Fig. 5 that the ferritin variants containing the C90R substitution did not load much iron, however, from the Prussian blue stained gel (Fig. 4) it is apparent that they did indeed load iron. Additionally, we compared the ferroxidase activity of all the ferritin variants to that of wt ferritin, and determined they were all equal (data not shown). Therefore, the decrease in aggregation of the C90R-containing variants cannot be attributed to less iron being oxidized and/or loaded. Our first hypothesis was that the aggregation of the ferritin was a result of disulfide formation. However, the data obtained by LC/MS analysis provided no evidence for a M⫹M-2 peptide, i.e., a peptide indicative of two small peptides linked by a disulfide bridge. However, disulfide bonds are not the only covalent bonds formed during aggregation of protein. It has been demonstrated that protein aggregation can be a result of intermolecular dityrosine formation [22–24,30,32,36]. When ferritin is loaded with iron by its own ferroxidase activity there is an increase in dityrosine formation (Table 3). Interestingly, when the cysteine at position 90 was changed to an arginine, there was only a 3-fold increase in dityrosine formation versus the almost 11-fold increase in dityrosine in wt ferritin as a result of iron loading. The C102A variant had less dityrosine formation than both C130A variants and wt ferritin. It is possible that the cysteines of H chain ferritin are a “hot spot” for metalcatalyzed oxidation, especially cysteine 90; however, they are not the only amino acids oxidized. Other researchers have suggested that cysteine residues can be oxidized and then through intraprotein electron transfer catalyze the oxidation of other amino acids [37,38]. Thus the cysteine is the first, or one of the first residues oxidized, however, intraprotein electron transfer results in the oxidation of other residues, e.g., tyrosines. We are currently investigating the role of tyrosine residues in the aggregation of ferritin. We feel that the data presented in this study further demonstrates that iron is not loaded into ferritin via its own ferroxidase activity in vivo. If iron were loaded via this mechanism, the ferritin would be damaged. Recently it has been shown in cell culture experiments, that when
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iron was continually supplied to the cells, the half-life of ferritin increased [39]. Oxidative damage to proteins is a marker for degradation [35,40,41], therefore, if ferritin were damaged during iron loading the half-life would decrease, not increase. One possibility is that during various conditions of iron overload, iron can be loaded into ferritin via its own ferroxidase activity and consequently the ferritin may be damaged. There has been considerable research done to determine the relationship of ferritin and hemosiderin [42– 44]. Recently researchers have suggested that hemosiderin is a degradation product of ferritin [45– 47]. It has been shown under iron overload and also copperdeficient conditions that there is an increase in hemosiderin formation in animals [48]. We propose that under physiological conditions iron is loaded into ferritin with the aid of another enzyme [49]. However, under various conditions of iron overload or a lack of the necessary enzyme, iron could be loaded into ferritin via its own ferroxidase activity and consequently the ferritin would be damaged and subsequently degraded to hemosiderin. We are currently testing this hypothesis. In conclusion, iron loading into ferritin via its own ferroxidase activity resulted in damage to the ferritin. The cysteine residues of ferritin appeared to be very susceptible to oxidation and appear to be involved in the oxidation and aggregation of the ferritin. Cysteine 90, located on the BC loop, appeared to be a key residue for aggregate formation. Even though ferritin variants that contain the C90R substitution do not aggregate, the ferritins were still damaged. Additionally, the cysteine residues may not be solely responsible for the aggregation but may work in concert with tyrosine residues, and aggregation could be a result of intermolecular dityrosine formation. Acknowledgement — The authors would like to thank Terri Maughan for her secretarial assistance in the preparation of the manuscript.
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ABBREVIATIONS
H2O2— hydrogen peroxide • OH— hydroxyl radical LC/MS—liquid chromatography/mass spectrometry MS/MS—tandem mass spectrometry ESR— electron spin resonance