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Iron and Phosphate Content of Rat Ferritin Heteropolymers
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 357, No. 2, September 15, pp. 293–298, 1998 Article No. BB980847
Iron and Phosphate Content of Rat Ferritin Heteropolymers Shu-Hui Juan and Steven D. Aust1 Biotechnology Center, Utah State University, Logan, Utah 84322-4705
Received May 18, 1998, and in revised form July 6, 1998
An attempt was made to relate the iron and phosphate content of ferritin to its subunit composition. Ferritins from various tissues were separated according to their subunit composition by anion exchange chromatography and according to their iron content by density-gradient centrifugation. Iron and phosphate contents were not related to subunit composition. Recombinant rat liver ferritin heteropolymers of different subunit composition (1, 4, 6, 10, 15, and 17 H chains per 24 mer) were maximally loaded with iron, using ceruloplasmin and phosphate. All loaded approximately the same amount of iron and phosphate (2250 and 380 atoms, respectively). The iron and phosphate content of all ferritin, including the maximally loaded recombinant ferritin heteropolymers, fit an equation we previously reported: [Fe] 5 4404 2 5.61 [Pi] (D. deSilva et al., 1993, Arch. Biochem. Biophys. 303, 451– 455). These results suggest that the amount of iron and apparently the space within the core of ferritin were not related to different subunit composition. © 1998 Academic Press
Ferritin is an intracellular iron storage protein that plays a key role in the regulation of iron homeostasis. Ferritin sequesters iron in a nontoxic form and serves as a reservoir that may provide iron for iron–sulfur proteins, heme protein, and other iron-containing or -requiring enzymes (1, 2). The highly reactive nature of iron in an aerobic environment may result in the generation of partially reduced toxic species of oxygen. Ferritin seems to be distributed ubiquitously in all living organisms to prevent toxicity resulting from iron. Ferritin is composed of 24 subunits of varying proportion of H and L chains. The ratio of H to L chains varies depending on the species and tissue of origin (3). 1 To whom correspondence should be addressed. Fax: (435) 7972755. E-mail: [email protected].
0003-9861/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
There is considerable (55% identical) homology between the two chains although homopolymers do not seem to exist. The role of the H chain seems to be fairly well defined. It seems to be involved in iron loading in two different loading systems. We have shown that the H chain is required for loading iron by ceruloplasmin (4, 5). The iron loading channel was identified in the four a-helix bundle of the H chain by site-directed mutagenesis (6). The channel seems blocked in the L chain by a salt bridge between Lys 62 and Glu 107 in the four a-helix bundle. Opening this channel in the L chain by site-directed mutagenesis resulted in a recombinant L chain homopolymer that could be loaded with iron while closing this channel in the H chain (E62K and H65G) resulted in a recombinant H chain homopolymer that could not be loaded by ceruloplasmin (6). Other investigators have proposed a ferroxidase center located in the four a-helix bundle of ferritin H chain for loading iron into ferritin by itself (7). Mammalian ferritin heteropolymers with different subunit composition and different iron contents have been found in various tissues (3). However, the relationship between subunit composition and iron content of ferritin is still disputed. Wagstaff et al. (8) found that ferritin isolated from human spleen, liver, and heart, consisting largely of L chain, contained the least amount of iron, whereas ferritin with more H chain contained more iron. Similarly, this phenomenon was also observed in horse liver and spleen ferritin (9). In contrast, Bomford et al. (10) showed that there is a preferential synthesis of rat liver L chain after iron administration, presumably to provide ferritin with more L chain for storage of the iron. They proposed that storage of iron is favored because ferritin rich in L chain is less susceptible to intracellular proteolysis and survives longer than ferritin rich in H chain (10, 11). Guanidium hydrochloride was employed by Santambrogio et al. (12) to disassemble recombinant human ferritin homopolymers and subsequently reassembled ferritin heteropolymers by dialysis with different percentages of H and L chain. They found (13, 14) that 293
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iron contents were proportional to the number of L chain in ferritin when the proteins were incubated with ferrous sulfate in Good’s buffer, the system used by most investigators to load iron into ferritin. The L chain has also been proposed to contribute significantly more iron nucleation sites within the core than does the H chain (glu 53, 56, 57, 60, and 62 versus glu 61, 64, and 67) (15). However, we could not show any difference in the amount of iron or phosphate loaded into a mutant H chain homopolymer with various numbers of proposed nucleation sites (16). The composition of the iron core of ferritin has been given as (FeOOH)8(FeO:OPO3H2) (17). The significance of phosphate in the ferritin iron core is not well understood. Reconstitution of ferritin with iron by ceruloplasmin followed by prolonged incubation with phosphate produced cores similar to native ferritin in terms of iron to phosphate ratios (18). In addition, the amount of phosphate in fractionated mammalian ferritin from horse spleen, rat liver, and bovine liver was inversely proportional to the content of iron with a linear correlation; [Fe] 5 4404 2 5.61 [Pi] (18). The present study was designed to investigate the relationship between the subunit content of ferritin and the iron (and phosphate) content. Ferritins can be separated according to iron content by density-gradient centrifugation (10) and according to the content of L to H chain by anion exchange chromatography (5). The maximum amounts of iron and phosphate which could be loaded into ferritin were utilized to estimate the core size in recombinant rat liver ferritin heteropolymers containing different ratios of H to L chain. MATERIALS AND METHODS Chemicals. Rat liver, spleen, and heart were obtained from PelFreez Biologicals (Rogers, AR). Other chemicals were obtained from Sigma (St. Louis, MO) except that L-histidine was purchased from Eastman Kodak (Rochester, NY). The materials and methods to construct a baculovirus transfer vector containing both H and L chain ferritin genes were described previously (5, 16). All enzymes utilized for DNA manipulations were from United States Biochemicals (Cleveland, OH) and Boehringer Mannheim Biochemicals (Indianapolis, IN) and used according to manufacturers’ instructions. Baculovirus transfer vector pAcUW51, linearized baculovirus BaculoGold, and a culture of Spodoptera frugiperda (Sf-21) were purchased from PharMingen (San Diego, CA). Serum-free insect cell medium EX-CELL 401 was obtained from JRH Biochemicals (Lenexa, KS). Expression of recombinant ferritin heteropolymers. Recombinant pAcUW51 (2.0 mg) containing both H and L chain genes was cotransfected into S. frugiperda Sf-21 cells using linearized viral DNA BaculoGold (1.0 mg) in serum-free medium EX-CELL 401 and lipofectin (Gibco BRL, Gaithersburg, MD), as described in detail by O’Reilly et al. (20). Recombinant ferritin heteropolymers were produced as described previously (5) except that the multiplicity of infection was increased to 30 to give different ratios of H to L chains in the heteropolymers. Purification of ferritins. Native rat ferritins, isolated from rat liver, spleen, and heart, were purified by the method described by
Thomas et al. (21), except that heat treatment to remove other proteins was omitted, a Bio-Gel (Bio-Rad Laboratories, Hercules, CA) column was used to replace the Sephadex G-200 column, and chromatography on a Sepharose CL-6B column (Pharmacia Biotech, Piscataway, NJ) on a Pharmacia Biotech FPLC system was added. The purification of recombinant ferritin was similar to native ferritin, except that the final step of ultracentrifugation to pellet the ferritin was omitted since recombinant ferritin expressed in the baculovirus expression system contained no iron, as reported previously (5). Separation of ferritins by iron contents. Density-gradient centrifugation was employed to separate purified native rat liver ferritins with different iron contents. Two milligrams of purified native rat liver ferritin, in 0.5 ml, was loaded on the top of a glycerol gradient, 1.5 ml each of 60, 50, 40, 30, 20, and 10 % of glycerol, and subjected to ultracentrifugation at 100,000g overnight. Aliquots (1 ml each) were collected sequentially from top to bottom and the concentration of ferritin in each fraction was quantitated using the bicinchoninic acid kit (22). Separation of ferritins by subunit composition. The ferritins were chromatographed over a DEAE–Sepharose fast flow column (Pharmacia Biotech, Piscataway, NJ). Ferritins containing different ratios of H to L chains were separated using 10 mls each of 50, 75, 100, 125, 150, and 175 mM NaCl in 50 mM Tris buffer. The concentration of ferritin in each fraction was quantitated using the bicinchoninic acid kit (Pierce, Rockford, IL) using bovine serum albumin as a standard (22). Loading of iron and phosphate into recombinant rat liver ferritins. Iron was incorporated into ferritin using ceruloplasmin as described by deSilva et al. (23). Rat ceruloplasmin was purified as outlined by Ryan et al. (24). Histidine and ferrous chloride at the molar ratio of 5:1 (550:110 uM) were dissolved in argon-purged 50 mM NaCl, pH 7.0. Increments of histidine-ferrous iron (500 atoms of iron per ferritin) were added to a 1:1 molar ratio of ferritin and ceruloplasmin (0.22 uM) in Chelex-treated 50 mM NaCl, pH 7.0, at 37°C. The rate of iron incorporation into the ferritin heteropolymers by ceruloplasmin was continuously monitored as absorbance at 380 nm (E380 5 1.03 mM21 cm21). When iron incorporation ceased, ferrozine was added to chelate any remaining ferrous iron in solution and solutions were passed through a Chelex-100 column (Sigma) to remove remaining iron or nonincorporated iron. Phosphate was incorporated into the iron-loaded ferritin heteropolymers by incubation in 10 mM potassium phosphate (pH 7.0) at 4°C for 24 h. Samples were passed through an Econo-Pac 10 DG desalting column (Bio-Rad Laboratories) to remove any nonincorporated phosphate. Analysis of subunit composition, iron and phosphate contents in ferritins. Equal amount of ferritins (7 mg) from various preparations, as described above, were analyzed on a denaturing 12% Ready Gel (Bio-Rad Laboratories) using a Bio-Rad Mini-Protein II electrophoresis cell to determine the ratios of H to L chain. The gels were stained with Coomassie brilliant blue and scanned (ISCO Model UA-5, Instrumentation Specialties Co.) to integrate the areas of protein bands for H and L chains. The amount of iron and phosphate in the various ferritins were analyzed using total iron (25) and phosphate assays (26).
RESULTS
Subunit composition and iron and phosphate content of rat liver, spleen and heart ferritins. The ratios of H to L chain in ferritin, determined by SDS–PAGE (12%) (Fig. 1), were approximately 9.0 6 0.5 to 15.0 6 0.5, 11.8 6 0.6 to 13.2 6 0.6, and 15.3 6 0.5 to 8.7 6 0.5 (mean 6 standard deviation for three separate preparations) for native rat liver, spleen, and heart ferritins,
IRON AND PHOSPHATE CONTENT OF FERRITINS
FIG. 1. SDS–PAGE of H and L chain of native rat ferritins isolated from liver, spleen, and heart. Approximately 8 mg of the purified ferritins were resolved on SDS–PAGE (12%) and stained for protein with Coomassie blue. They represent rat liver, spleen, and heart ferritins from left to right in the gel, respectively.
respectively. The amounts of iron were 1850 6 30, 2350 6 70, and 1830 6 60 and the amounts of phosphate were 460 6 30, 380 6 20, and 450 6 40 for rat liver, spleen, and heart ferritins, respectively. Subunit composition and iron and phosphate content of density-gradient fractionated native rat liver ferritin. Density-gradient centrifugation of native rat liver ferritin separated ferritins with different iron contents. Ferritin-containing fractions were collected from the density gradient, with ferritin containing more iron found closer to the bottom of the tube. The amount of phosphate was inversely proportional to the amount of iron in ferritin when the amount of iron was more than 1000 atoms of iron per ferritin (Fig. 2). The inset in Fig. 2 shows the SDS–PAGE (12%) of the ferritins fractionated through the glycerol gradient. The ratios of H to L chain in ferritin all averaged approximately 9 to 15. They were similar to the composition of unfractionated rat liver ferritin, as shown in Fig. 1, although they were fractionated according to their iron contents. Subunit composition and iron and phosphate content of native rat spleen ferritins fractionated by anion exchange chromatography. Rat spleen ferritin was fractionated by anion exchange chromatography. The fractions eluted with increasing concentration of NaCl were resolved by SDS–PAGE (12%) and determined to consist of 2.0 6 0.5, 4.0 6 0.8, 6.2 6 0.5, 7.8 6 1.0, 15.0 6 1.4, and 18.1 6 1.2 H chain per ferritin (Fig. 3A). These fractions were also subjected to a native– PAGE (7.5%) (Fig. 3B) and they exhibited electrophoretic mobility proportional to the number of H chain, since H chain is more acidic than L chain (27). Rat liver ferritin was also fractionated using anion exchange chromatography; however, the range of subunit composition was smaller than spleen ferritin. Therefore, only results obtained for rat spleen ferritin are reported. These rat spleen ferritins fractionated by anion exchange chromatography were found to contain similar amounts of iron and phosphate, despite having different ratios of H to L chain (Table I). Subunit composition analysis and incorporation of iron and phosphate into recombinant ferritin heteropolymers. We previously reported (5) that recombinant ferritins containing one or two H chains per 24 subunits were obtained when a multiplicity of infection
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of 10 was used to infect insect cells. By increasing the multiplicity of infection to 30, recombinant ferritin heteropolymers with higher ratios of ferritin H to L chain were obtained. They were separated by anion exchange chromatography and resolved by SDS–PAGE (12%) (data not shown). These were scanned and the composition of H and L chain in ferritin was quantitated using gel densitometry (Fig. 4). These heteropolymers were loaded to the same extent by ceruloplasmin, approximately 2250 atoms of iron per ferritin, as shown in Table II. The amounts of phosphate incorporated following the maximal amount of iron loading by ceruloplasmin were also similar among the recombinant ferritin heteropolymers with different ratios of ferritin H to L chains (Table II).
FIG. 2. The amount of iron and phosphate in ferritins separated by density-gradient centrifugation. The amounts of iron (F) and phosphate (■) in each ferritin-containing fraction, separated by densitygradient centrifugation were determined as described under Materials and Methods. The results are representative of at least three individual measurements. The inset is a typical SDS–PAGE used to determine the ratio of H to L chain in ferritins through the glycerol gradient. The sample from left to right in the gel corresponds to each increasing fraction number displayed in the x-axis. Approximately 8 mg of the gradient-fractionated ferritins were resolved on an SDS– PAGE (12%) and stained for protein with Coomassie blue.
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FIG. 3. SDS–PAGE (A) and native-PAGE (B) of native rat spleen ferritins fractionated by anionic exchange chromatography. The separation of heteropolymers of native rat spleen ferritin was described under Materials and Methods. (A) Approximately 8 mg of the gradient-fractionated ferritins were resolved by SDS–PAGE (12%) and stained for protein with Coomassie blue. (B) Approximately 10 mg of the gradient-fractionated ferritins were resolved by native–PAGE (7.5%) and stained for protein with Coomassie blue. In each case, the lanes from 1 to 6 are the samples eluted in 50, 75, 100, 125, 150, and 175 mM NaCl in 50 mM Tris buffer, respectively.
DISCUSSION
We reported earlier (18) that the iron and phosphate content of ferritin had a linear inverse correlation when the iron content in ferritin was more than 1000 atoms per ferritin. Interestingly, in the present study we found that this relationship was consistent for rat ferritins isolated from different tissues, for ferritins fractionated according to either iron content or subunit composition, and for recombinant rat liver ferritin heteropolymers with vastly different number of H chains (1 to 17) loaded by ceruloplasmin followed by the incubation with phosphate. This may indicate that the size of the iron core in ferritin with different ratios of H to L chain were all similar, estimated by their maximum iron and phosphate content. With respect to the effect of ferritin subunit composition on its iron content, we found that native ferritins
FIG. 4. Ratios of H to L chain in recombinant rat liver ferritin heteropolymers. The number of H chain (F) and L chain (■) subunits in ferritin was quantitated using gel densitometry. The results are the averages of at least three individual measurements.
isolated from liver and heart averaging 15 L and 9 L chains, respectively, contained similar amount of iron: 1850 6 30 and 1830 6 60 atoms. The amounts of iron and phosphate were also not different in fractionated native rat spleen ferritins, although they too were made up of different subunit composition. In addition, native rat liver ferritin fractionated according to iron content using density-gradient centrifugation had similar subunit composition, suggesting that there is no correlation between iron content and subunit composition. Finally, recombinant ferritin with 23 L chains was loaded to a similar extent as a recombinant ferritin with 7 L chains. All of these results consistently showed that the amount of iron was independent of the ratios of ferritin H to L chain, different from the results published by other laboratories (10 –12). Our results obtained with recombinant rat liver heteropolymers were different from the results reported
TABLE I
Iron and Phosphate Content of Native Rat Spleen Ferritin Fractionated by Anion Exchange Chromatography Content of H and L Chains Atoms per ferritin
H2L22
H4L20
H6L18
H8L16
H15L9
H18L6
Iron Phosphate
2380 6 60 350 6 30
2330 6 50 350 6 40
2350 6 70 360 6 30
2370 6 70 340 6 60
2340 6 80 360 6 30
2330 6 50 340 6 50
Note. The heteropolymers of native rat spleen ferritin were separated by anion exchange chromatography and their subunit composition was quantitated as described under Materials and Methods. The amounts of iron and phosphate were determined using total iron (25) and phosphate (26) assays.
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IRON AND PHOSPHATE CONTENT OF FERRITINS TABLE II
Iron and Phosphate Content of Recombinant Rat Liver Ferritin Reconstituted by Ceruloplasmin and Incubation with Phosphate Buffer Content of H and L Chains Atoms per ferritin
H1L23
H4L20
H6L18
H10L14
H15L9
H17L7
Iron Phosphate
2280 6 50 390 6 30
2200 6 70 380 6 30
2220 6 70 380 6 40
2230 6 80 370 6 40
2250 6 70 360 6 50
2230 6 90 380 6 40
Note. Recombinant ferritin heteropolymers were separated by anion exchange chromatography and the subunit composition was quantitated as described under Materials and Methods. The ferritins were then loaded maximally with iron using ceruloplasmin and then incubated in phosphate buffer (18). The ferritins were then chromatographed and the amounts of iron and phosphate were determined using total iron (25) and phosphate (26) assays.
by Santambrogio et al. (12). They solubilized recombinant human ferritin homopolymers in guanidium hydrochloride and reassembled heteropolymers by dialysis. Their homopolymers were expressed in Escherichia coli. They loaded their heteropolymer using ferrous sulfate in Mes buffer, assuming that ferritin has its own ferroxidase activity. We do not believe that this ferritin loading system is physiological as oxygen radicals are produced which can damage the ferritin (23). We use the ferroxidase activity of ceruloplasmin to load iron into ferritin. We have demonstrated an association between the H chain of ferritin and ceruloplasmin that results in efficient loading of iron into ferritin with excellent stoichiometry (i.e., 4FeII 1 O2 3 4FeIII 1 2 H2O) and no damage to the ferritin (23, 28). In addition, we produce our recombinant ferritins (using rat liver genes rather than human) using an insect cell– baculovirus expression system (4, 5). We believe that recombinant ferritins produced by E. coli may be oxidatively damaged (to be published). These different methods may be responsible for the different results reported here and by Levi et al. (13). They observed a 3to 4-fold increase in iron incorporation in ferritin with increasing ratio of L to H chain (70 – 82% L chain). They concluded that ferritins with greater amount of H chain incorporated less of the iron presented (1000 atoms per ferritin) because of precipitation of the ferritin with more H chain. We did not observe any precipitation of ferritin during loading with ceruloplasmin. In addition, we found that ferritins loaded with ceruloplasmin behave similarly to native ferritins with respect to the stability of the iron core (28). Although it was shown that rat liver L chain was preferentially induced to accommodate more iron following iron administration (10, 11), the mechanism for iron storage in ferritin upon the acute increase in the intracellular iron level might be different from normal physiological conditions. For example, it was reported that acute iron administration increased the conversion of ferritin to hemosiderin (15, 29). Additionally,
some confounded results were reported that ferritins isolated from humans and horses having more L chain were found to contain less iron (8, 9, 30), opposite from the results using rat ferritin, in which lower iron content was found in ferritin with less L chain (15, 29). This may address the complicated nature of this issue in that the correlation between ferritin subunit composition and its iron content may vary with ferritin isolated from different species, iron status of animals, or apoferritin reconstituted either by ceruloplasmin or Good’s type buffer. We showed previously (16) that recombinant ferritin H chain homopolymers with additional putative nucleation sites were more stable before phosphate incorporation, however, they loaded the same amount of iron. Therefore, L chain ferritin may be involved in the stability of the iron core in ferritin, as it contains two more putative nucleation sites than H chain ferritin. We also found that recombinant L chain ferritin homopolymers with no iron was more susceptible to proteolytic degradation and aggregation (unpublished work). The proteolytic cleavage product we observed seemed similar to hemosiderin identified by Andrews et al. (31) as a proteolytic product of the L chain. Therefore, we would suggest that the L chain of ferritin may be somehow involved in ferritin turnover, however, the exact role of the L chain of ferritin remains to be determined. ACKNOWLEDGMENTS We gratefully acknowledge the technical assistance of Randy Booth and Ryan Berger. We also thank T. Maughan for her secretarial assistance in the preparation the manuscript. The work was supported by NIH Grant ES05056.
REFERENCES 1. Crichton, R. R. (1990) Adv. Protein Chem. 40, 281–363. 2. Crichton, R. R., and Ward, R. J. (1992) Biochemistry 3, 11255– 11264.
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3. Arosio, P., Adelman, T. G., and Drysdale, J. W. (1978) J. Biol. Chem 253, 4451– 4458. 4. Guo, J.-H., Abedi, M., and Aust, S. D. (1996) Arch. Biochem. Biophys 335, 197–204. 5. Juan, S.-H., Guo, J.-H., and Aust, S. D. (1997) Arch. Biochem. Biophys 341, 280 –286. 6. Guo, J.-H., Juan, S.-H., and Aust, S. D. (1998) Arch. Biochem. Biophys 352, 71–77. 7. Lawson, D. M., Artymiuk, P. J., Yewdall, S. J., Smith, J. M., Livinstone, J., Treffry, A., Luzzago, A., and Arosio, P. (1991) Nature 349, 541–544. 8. Wagstaff, M., Worwood, M., and Jacobs, A. (1978) Biochem. J. 173, 969 –977. 9. Bomford, A., Berger, M., Lis, Y., and Williams, R. (1978) Biochem. Biophys. Res. Commun. 83, 334 –341. 10. Bomford, A., Conlon-Hollingshead, C., and Munro, H. N. (1981) J. Biol. Chem. 356, 948 –955. 11. Kohgo, Y., Yokota, M., and Drysdale, J. W. (1980) J. Biol. Chem. 255, 5195–5200. 12. Santambrogio, P., Levi, S., and Arosio, P. (1993) J. Biol. Chem 268, 12744 –12748. 13. Levi, S., Santambrogio, P., Cozzi, A., Rovida, E., Corsi, B., Tamborini, E., Spada, S., Albertini, A., and Arosio, P. (1994) J. Mol. Biol. 238, 649 – 654. 14. Wade, V. J., Levi, S., Arosio, P., Treffry, A., Harrison, P. M., and Mann, S. (1991) J. Mol. Biol. 221, 1443–1452. 15. Levi, S., Santambrogio. P., Cozzi, A., Rovida, E., Albertini, A., Yewdall, S. J., Harrison, P. M., and Arosio, P. (1992) Biochem. J. 288, 591–596. 16. Juan, S.-H., and Aust, S. D. (1998) Arch. Biochem. Biophys 350, 259 –265.
17. Michaelis, L., Coryell, C. D., and Granick, S. (1943) J. Biol. Chem. 148, 463– 480. 18. deSilva, D., Guo, J,-H., and Aust, S. D. (1993) Arch. Biochem. Biophys 303, 451– 455. 19. Murray, M. T., White, K., and Munro, H. N. (1987) Proc. Natl. Acad. Sci. USA 84, 7438 –7442. 20. O’Reilly, D. R., Miller, L. K., and Luckow, V. A. (1992) Baculovirus Expression Vectors; A Laboratory Manual, 1st Ed., Freeman, New York. 21. Thomas, C. E., Morehouse, L. A., and Aust, S. D. (1985) J. Biol. Chem. 260, 3275–3280. 22. Bradford, M. M. (1976) Anal. Biochem 72, 248 –254. 23. deSilva, D., Miller, D. M., Reif, D. W., and Aust, S. D. (1992) Arch. Biochem. Biophys. 293, 409 – 415. 24. Ryan, T. P., Grover, T. A., and Aust, S. D. (1992) Arch. Biochem. Biophys. 293, 1– 8. 25. Brumby, D. E, and Massey, V. (1967) in Methods in Enzymology (Fleischer, S., and Packer, L., Eds.), Vol. 52, pp. 302–310, Academic Press, New York. 26. Bartlett, G. R. (1959) J. Biol. Chem. 234, 466 – 468. 27. Drysdale, J. W (1977) in Ciba Foundation Symposium 51, Iron Metabolism, pp.41– 67. Elsevier-North-Holland, Amsterdam. 28. deSilva, D., and Aust, S. D. (1992) Arch. Biochem. Biophys. 298, 259 –264. 29. Drysdale, J. W., and Munro, H. N (1966) J. Biol. Chem. 241, 3630 –3637. 30. Russell, S. M., and Harrison, P. M. (1978) Biochem. J. 175, 91–104. 31. Andrews, S. C., Treffry, A., and Harrison, P. M. (1987) Biochem. J. 245, 447– 453.
Vol. 357, No. 2, September 15, pp. 293–298, 1998 Article No. BB980847
Iron and Phosphate Content of Rat Ferritin Heteropolymers Shu-Hui Juan and Steven D. Aust1 Biotechnology Center, Utah State University, Logan, Utah 84322-4705
Received May 18, 1998, and in revised form July 6, 1998
An attempt was made to relate the iron and phosphate content of ferritin to its subunit composition. Ferritins from various tissues were separated according to their subunit composition by anion exchange chromatography and according to their iron content by density-gradient centrifugation. Iron and phosphate contents were not related to subunit composition. Recombinant rat liver ferritin heteropolymers of different subunit composition (1, 4, 6, 10, 15, and 17 H chains per 24 mer) were maximally loaded with iron, using ceruloplasmin and phosphate. All loaded approximately the same amount of iron and phosphate (2250 and 380 atoms, respectively). The iron and phosphate content of all ferritin, including the maximally loaded recombinant ferritin heteropolymers, fit an equation we previously reported: [Fe] 5 4404 2 5.61 [Pi] (D. deSilva et al., 1993, Arch. Biochem. Biophys. 303, 451– 455). These results suggest that the amount of iron and apparently the space within the core of ferritin were not related to different subunit composition. © 1998 Academic Press
Ferritin is an intracellular iron storage protein that plays a key role in the regulation of iron homeostasis. Ferritin sequesters iron in a nontoxic form and serves as a reservoir that may provide iron for iron–sulfur proteins, heme protein, and other iron-containing or -requiring enzymes (1, 2). The highly reactive nature of iron in an aerobic environment may result in the generation of partially reduced toxic species of oxygen. Ferritin seems to be distributed ubiquitously in all living organisms to prevent toxicity resulting from iron. Ferritin is composed of 24 subunits of varying proportion of H and L chains. The ratio of H to L chains varies depending on the species and tissue of origin (3). 1 To whom correspondence should be addressed. Fax: (435) 7972755. E-mail: [email protected].
0003-9861/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
There is considerable (55% identical) homology between the two chains although homopolymers do not seem to exist. The role of the H chain seems to be fairly well defined. It seems to be involved in iron loading in two different loading systems. We have shown that the H chain is required for loading iron by ceruloplasmin (4, 5). The iron loading channel was identified in the four a-helix bundle of the H chain by site-directed mutagenesis (6). The channel seems blocked in the L chain by a salt bridge between Lys 62 and Glu 107 in the four a-helix bundle. Opening this channel in the L chain by site-directed mutagenesis resulted in a recombinant L chain homopolymer that could be loaded with iron while closing this channel in the H chain (E62K and H65G) resulted in a recombinant H chain homopolymer that could not be loaded by ceruloplasmin (6). Other investigators have proposed a ferroxidase center located in the four a-helix bundle of ferritin H chain for loading iron into ferritin by itself (7). Mammalian ferritin heteropolymers with different subunit composition and different iron contents have been found in various tissues (3). However, the relationship between subunit composition and iron content of ferritin is still disputed. Wagstaff et al. (8) found that ferritin isolated from human spleen, liver, and heart, consisting largely of L chain, contained the least amount of iron, whereas ferritin with more H chain contained more iron. Similarly, this phenomenon was also observed in horse liver and spleen ferritin (9). In contrast, Bomford et al. (10) showed that there is a preferential synthesis of rat liver L chain after iron administration, presumably to provide ferritin with more L chain for storage of the iron. They proposed that storage of iron is favored because ferritin rich in L chain is less susceptible to intracellular proteolysis and survives longer than ferritin rich in H chain (10, 11). Guanidium hydrochloride was employed by Santambrogio et al. (12) to disassemble recombinant human ferritin homopolymers and subsequently reassembled ferritin heteropolymers by dialysis with different percentages of H and L chain. They found (13, 14) that 293
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iron contents were proportional to the number of L chain in ferritin when the proteins were incubated with ferrous sulfate in Good’s buffer, the system used by most investigators to load iron into ferritin. The L chain has also been proposed to contribute significantly more iron nucleation sites within the core than does the H chain (glu 53, 56, 57, 60, and 62 versus glu 61, 64, and 67) (15). However, we could not show any difference in the amount of iron or phosphate loaded into a mutant H chain homopolymer with various numbers of proposed nucleation sites (16). The composition of the iron core of ferritin has been given as (FeOOH)8(FeO:OPO3H2) (17). The significance of phosphate in the ferritin iron core is not well understood. Reconstitution of ferritin with iron by ceruloplasmin followed by prolonged incubation with phosphate produced cores similar to native ferritin in terms of iron to phosphate ratios (18). In addition, the amount of phosphate in fractionated mammalian ferritin from horse spleen, rat liver, and bovine liver was inversely proportional to the content of iron with a linear correlation; [Fe] 5 4404 2 5.61 [Pi] (18). The present study was designed to investigate the relationship between the subunit content of ferritin and the iron (and phosphate) content. Ferritins can be separated according to iron content by density-gradient centrifugation (10) and according to the content of L to H chain by anion exchange chromatography (5). The maximum amounts of iron and phosphate which could be loaded into ferritin were utilized to estimate the core size in recombinant rat liver ferritin heteropolymers containing different ratios of H to L chain. MATERIALS AND METHODS Chemicals. Rat liver, spleen, and heart were obtained from PelFreez Biologicals (Rogers, AR). Other chemicals were obtained from Sigma (St. Louis, MO) except that L-histidine was purchased from Eastman Kodak (Rochester, NY). The materials and methods to construct a baculovirus transfer vector containing both H and L chain ferritin genes were described previously (5, 16). All enzymes utilized for DNA manipulations were from United States Biochemicals (Cleveland, OH) and Boehringer Mannheim Biochemicals (Indianapolis, IN) and used according to manufacturers’ instructions. Baculovirus transfer vector pAcUW51, linearized baculovirus BaculoGold, and a culture of Spodoptera frugiperda (Sf-21) were purchased from PharMingen (San Diego, CA). Serum-free insect cell medium EX-CELL 401 was obtained from JRH Biochemicals (Lenexa, KS). Expression of recombinant ferritin heteropolymers. Recombinant pAcUW51 (2.0 mg) containing both H and L chain genes was cotransfected into S. frugiperda Sf-21 cells using linearized viral DNA BaculoGold (1.0 mg) in serum-free medium EX-CELL 401 and lipofectin (Gibco BRL, Gaithersburg, MD), as described in detail by O’Reilly et al. (20). Recombinant ferritin heteropolymers were produced as described previously (5) except that the multiplicity of infection was increased to 30 to give different ratios of H to L chains in the heteropolymers. Purification of ferritins. Native rat ferritins, isolated from rat liver, spleen, and heart, were purified by the method described by
Thomas et al. (21), except that heat treatment to remove other proteins was omitted, a Bio-Gel (Bio-Rad Laboratories, Hercules, CA) column was used to replace the Sephadex G-200 column, and chromatography on a Sepharose CL-6B column (Pharmacia Biotech, Piscataway, NJ) on a Pharmacia Biotech FPLC system was added. The purification of recombinant ferritin was similar to native ferritin, except that the final step of ultracentrifugation to pellet the ferritin was omitted since recombinant ferritin expressed in the baculovirus expression system contained no iron, as reported previously (5). Separation of ferritins by iron contents. Density-gradient centrifugation was employed to separate purified native rat liver ferritins with different iron contents. Two milligrams of purified native rat liver ferritin, in 0.5 ml, was loaded on the top of a glycerol gradient, 1.5 ml each of 60, 50, 40, 30, 20, and 10 % of glycerol, and subjected to ultracentrifugation at 100,000g overnight. Aliquots (1 ml each) were collected sequentially from top to bottom and the concentration of ferritin in each fraction was quantitated using the bicinchoninic acid kit (22). Separation of ferritins by subunit composition. The ferritins were chromatographed over a DEAE–Sepharose fast flow column (Pharmacia Biotech, Piscataway, NJ). Ferritins containing different ratios of H to L chains were separated using 10 mls each of 50, 75, 100, 125, 150, and 175 mM NaCl in 50 mM Tris buffer. The concentration of ferritin in each fraction was quantitated using the bicinchoninic acid kit (Pierce, Rockford, IL) using bovine serum albumin as a standard (22). Loading of iron and phosphate into recombinant rat liver ferritins. Iron was incorporated into ferritin using ceruloplasmin as described by deSilva et al. (23). Rat ceruloplasmin was purified as outlined by Ryan et al. (24). Histidine and ferrous chloride at the molar ratio of 5:1 (550:110 uM) were dissolved in argon-purged 50 mM NaCl, pH 7.0. Increments of histidine-ferrous iron (500 atoms of iron per ferritin) were added to a 1:1 molar ratio of ferritin and ceruloplasmin (0.22 uM) in Chelex-treated 50 mM NaCl, pH 7.0, at 37°C. The rate of iron incorporation into the ferritin heteropolymers by ceruloplasmin was continuously monitored as absorbance at 380 nm (E380 5 1.03 mM21 cm21). When iron incorporation ceased, ferrozine was added to chelate any remaining ferrous iron in solution and solutions were passed through a Chelex-100 column (Sigma) to remove remaining iron or nonincorporated iron. Phosphate was incorporated into the iron-loaded ferritin heteropolymers by incubation in 10 mM potassium phosphate (pH 7.0) at 4°C for 24 h. Samples were passed through an Econo-Pac 10 DG desalting column (Bio-Rad Laboratories) to remove any nonincorporated phosphate. Analysis of subunit composition, iron and phosphate contents in ferritins. Equal amount of ferritins (7 mg) from various preparations, as described above, were analyzed on a denaturing 12% Ready Gel (Bio-Rad Laboratories) using a Bio-Rad Mini-Protein II electrophoresis cell to determine the ratios of H to L chain. The gels were stained with Coomassie brilliant blue and scanned (ISCO Model UA-5, Instrumentation Specialties Co.) to integrate the areas of protein bands for H and L chains. The amount of iron and phosphate in the various ferritins were analyzed using total iron (25) and phosphate assays (26).
RESULTS
Subunit composition and iron and phosphate content of rat liver, spleen and heart ferritins. The ratios of H to L chain in ferritin, determined by SDS–PAGE (12%) (Fig. 1), were approximately 9.0 6 0.5 to 15.0 6 0.5, 11.8 6 0.6 to 13.2 6 0.6, and 15.3 6 0.5 to 8.7 6 0.5 (mean 6 standard deviation for three separate preparations) for native rat liver, spleen, and heart ferritins,
IRON AND PHOSPHATE CONTENT OF FERRITINS
FIG. 1. SDS–PAGE of H and L chain of native rat ferritins isolated from liver, spleen, and heart. Approximately 8 mg of the purified ferritins were resolved on SDS–PAGE (12%) and stained for protein with Coomassie blue. They represent rat liver, spleen, and heart ferritins from left to right in the gel, respectively.
respectively. The amounts of iron were 1850 6 30, 2350 6 70, and 1830 6 60 and the amounts of phosphate were 460 6 30, 380 6 20, and 450 6 40 for rat liver, spleen, and heart ferritins, respectively. Subunit composition and iron and phosphate content of density-gradient fractionated native rat liver ferritin. Density-gradient centrifugation of native rat liver ferritin separated ferritins with different iron contents. Ferritin-containing fractions were collected from the density gradient, with ferritin containing more iron found closer to the bottom of the tube. The amount of phosphate was inversely proportional to the amount of iron in ferritin when the amount of iron was more than 1000 atoms of iron per ferritin (Fig. 2). The inset in Fig. 2 shows the SDS–PAGE (12%) of the ferritins fractionated through the glycerol gradient. The ratios of H to L chain in ferritin all averaged approximately 9 to 15. They were similar to the composition of unfractionated rat liver ferritin, as shown in Fig. 1, although they were fractionated according to their iron contents. Subunit composition and iron and phosphate content of native rat spleen ferritins fractionated by anion exchange chromatography. Rat spleen ferritin was fractionated by anion exchange chromatography. The fractions eluted with increasing concentration of NaCl were resolved by SDS–PAGE (12%) and determined to consist of 2.0 6 0.5, 4.0 6 0.8, 6.2 6 0.5, 7.8 6 1.0, 15.0 6 1.4, and 18.1 6 1.2 H chain per ferritin (Fig. 3A). These fractions were also subjected to a native– PAGE (7.5%) (Fig. 3B) and they exhibited electrophoretic mobility proportional to the number of H chain, since H chain is more acidic than L chain (27). Rat liver ferritin was also fractionated using anion exchange chromatography; however, the range of subunit composition was smaller than spleen ferritin. Therefore, only results obtained for rat spleen ferritin are reported. These rat spleen ferritins fractionated by anion exchange chromatography were found to contain similar amounts of iron and phosphate, despite having different ratios of H to L chain (Table I). Subunit composition analysis and incorporation of iron and phosphate into recombinant ferritin heteropolymers. We previously reported (5) that recombinant ferritins containing one or two H chains per 24 subunits were obtained when a multiplicity of infection
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of 10 was used to infect insect cells. By increasing the multiplicity of infection to 30, recombinant ferritin heteropolymers with higher ratios of ferritin H to L chain were obtained. They were separated by anion exchange chromatography and resolved by SDS–PAGE (12%) (data not shown). These were scanned and the composition of H and L chain in ferritin was quantitated using gel densitometry (Fig. 4). These heteropolymers were loaded to the same extent by ceruloplasmin, approximately 2250 atoms of iron per ferritin, as shown in Table II. The amounts of phosphate incorporated following the maximal amount of iron loading by ceruloplasmin were also similar among the recombinant ferritin heteropolymers with different ratios of ferritin H to L chains (Table II).
FIG. 2. The amount of iron and phosphate in ferritins separated by density-gradient centrifugation. The amounts of iron (F) and phosphate (■) in each ferritin-containing fraction, separated by densitygradient centrifugation were determined as described under Materials and Methods. The results are representative of at least three individual measurements. The inset is a typical SDS–PAGE used to determine the ratio of H to L chain in ferritins through the glycerol gradient. The sample from left to right in the gel corresponds to each increasing fraction number displayed in the x-axis. Approximately 8 mg of the gradient-fractionated ferritins were resolved on an SDS– PAGE (12%) and stained for protein with Coomassie blue.
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FIG. 3. SDS–PAGE (A) and native-PAGE (B) of native rat spleen ferritins fractionated by anionic exchange chromatography. The separation of heteropolymers of native rat spleen ferritin was described under Materials and Methods. (A) Approximately 8 mg of the gradient-fractionated ferritins were resolved by SDS–PAGE (12%) and stained for protein with Coomassie blue. (B) Approximately 10 mg of the gradient-fractionated ferritins were resolved by native–PAGE (7.5%) and stained for protein with Coomassie blue. In each case, the lanes from 1 to 6 are the samples eluted in 50, 75, 100, 125, 150, and 175 mM NaCl in 50 mM Tris buffer, respectively.
DISCUSSION
We reported earlier (18) that the iron and phosphate content of ferritin had a linear inverse correlation when the iron content in ferritin was more than 1000 atoms per ferritin. Interestingly, in the present study we found that this relationship was consistent for rat ferritins isolated from different tissues, for ferritins fractionated according to either iron content or subunit composition, and for recombinant rat liver ferritin heteropolymers with vastly different number of H chains (1 to 17) loaded by ceruloplasmin followed by the incubation with phosphate. This may indicate that the size of the iron core in ferritin with different ratios of H to L chain were all similar, estimated by their maximum iron and phosphate content. With respect to the effect of ferritin subunit composition on its iron content, we found that native ferritins
FIG. 4. Ratios of H to L chain in recombinant rat liver ferritin heteropolymers. The number of H chain (F) and L chain (■) subunits in ferritin was quantitated using gel densitometry. The results are the averages of at least three individual measurements.
isolated from liver and heart averaging 15 L and 9 L chains, respectively, contained similar amount of iron: 1850 6 30 and 1830 6 60 atoms. The amounts of iron and phosphate were also not different in fractionated native rat spleen ferritins, although they too were made up of different subunit composition. In addition, native rat liver ferritin fractionated according to iron content using density-gradient centrifugation had similar subunit composition, suggesting that there is no correlation between iron content and subunit composition. Finally, recombinant ferritin with 23 L chains was loaded to a similar extent as a recombinant ferritin with 7 L chains. All of these results consistently showed that the amount of iron was independent of the ratios of ferritin H to L chain, different from the results published by other laboratories (10 –12). Our results obtained with recombinant rat liver heteropolymers were different from the results reported
TABLE I
Iron and Phosphate Content of Native Rat Spleen Ferritin Fractionated by Anion Exchange Chromatography Content of H and L Chains Atoms per ferritin
H2L22
H4L20
H6L18
H8L16
H15L9
H18L6
Iron Phosphate
2380 6 60 350 6 30
2330 6 50 350 6 40
2350 6 70 360 6 30
2370 6 70 340 6 60
2340 6 80 360 6 30
2330 6 50 340 6 50
Note. The heteropolymers of native rat spleen ferritin were separated by anion exchange chromatography and their subunit composition was quantitated as described under Materials and Methods. The amounts of iron and phosphate were determined using total iron (25) and phosphate (26) assays.
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IRON AND PHOSPHATE CONTENT OF FERRITINS TABLE II
Iron and Phosphate Content of Recombinant Rat Liver Ferritin Reconstituted by Ceruloplasmin and Incubation with Phosphate Buffer Content of H and L Chains Atoms per ferritin
H1L23
H4L20
H6L18
H10L14
H15L9
H17L7
Iron Phosphate
2280 6 50 390 6 30
2200 6 70 380 6 30
2220 6 70 380 6 40
2230 6 80 370 6 40
2250 6 70 360 6 50
2230 6 90 380 6 40
Note. Recombinant ferritin heteropolymers were separated by anion exchange chromatography and the subunit composition was quantitated as described under Materials and Methods. The ferritins were then loaded maximally with iron using ceruloplasmin and then incubated in phosphate buffer (18). The ferritins were then chromatographed and the amounts of iron and phosphate were determined using total iron (25) and phosphate (26) assays.
by Santambrogio et al. (12). They solubilized recombinant human ferritin homopolymers in guanidium hydrochloride and reassembled heteropolymers by dialysis. Their homopolymers were expressed in Escherichia coli. They loaded their heteropolymer using ferrous sulfate in Mes buffer, assuming that ferritin has its own ferroxidase activity. We do not believe that this ferritin loading system is physiological as oxygen radicals are produced which can damage the ferritin (23). We use the ferroxidase activity of ceruloplasmin to load iron into ferritin. We have demonstrated an association between the H chain of ferritin and ceruloplasmin that results in efficient loading of iron into ferritin with excellent stoichiometry (i.e., 4FeII 1 O2 3 4FeIII 1 2 H2O) and no damage to the ferritin (23, 28). In addition, we produce our recombinant ferritins (using rat liver genes rather than human) using an insect cell– baculovirus expression system (4, 5). We believe that recombinant ferritins produced by E. coli may be oxidatively damaged (to be published). These different methods may be responsible for the different results reported here and by Levi et al. (13). They observed a 3to 4-fold increase in iron incorporation in ferritin with increasing ratio of L to H chain (70 – 82% L chain). They concluded that ferritins with greater amount of H chain incorporated less of the iron presented (1000 atoms per ferritin) because of precipitation of the ferritin with more H chain. We did not observe any precipitation of ferritin during loading with ceruloplasmin. In addition, we found that ferritins loaded with ceruloplasmin behave similarly to native ferritins with respect to the stability of the iron core (28). Although it was shown that rat liver L chain was preferentially induced to accommodate more iron following iron administration (10, 11), the mechanism for iron storage in ferritin upon the acute increase in the intracellular iron level might be different from normal physiological conditions. For example, it was reported that acute iron administration increased the conversion of ferritin to hemosiderin (15, 29). Additionally,
some confounded results were reported that ferritins isolated from humans and horses having more L chain were found to contain less iron (8, 9, 30), opposite from the results using rat ferritin, in which lower iron content was found in ferritin with less L chain (15, 29). This may address the complicated nature of this issue in that the correlation between ferritin subunit composition and its iron content may vary with ferritin isolated from different species, iron status of animals, or apoferritin reconstituted either by ceruloplasmin or Good’s type buffer. We showed previously (16) that recombinant ferritin H chain homopolymers with additional putative nucleation sites were more stable before phosphate incorporation, however, they loaded the same amount of iron. Therefore, L chain ferritin may be involved in the stability of the iron core in ferritin, as it contains two more putative nucleation sites than H chain ferritin. We also found that recombinant L chain ferritin homopolymers with no iron was more susceptible to proteolytic degradation and aggregation (unpublished work). The proteolytic cleavage product we observed seemed similar to hemosiderin identified by Andrews et al. (31) as a proteolytic product of the L chain. Therefore, we would suggest that the L chain of ferritin may be somehow involved in ferritin turnover, however, the exact role of the L chain of ferritin remains to be determined. ACKNOWLEDGMENTS We gratefully acknowledge the technical assistance of Randy Booth and Ryan Berger. We also thank T. Maughan for her secretarial assistance in the preparation the manuscript. The work was supported by NIH Grant ES05056.
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