Nuclear ferritin: a ferritoid-ferritin complex in corneal epithelial cells

Abstract

Purpose: Ferritin is an iron storage protein that is generally cytoplasmic. However, in embryonic avian corneal epithelial (CE) cells, the authors previously observed that the ferritin was predominantly nuclear. They also obtained evidence that this ferritin protects DNA from oxidative damage by UV light and hydrogen peroxide and that the nuclear localization involves a tissue-specific nuclear transporter, termed ferritoid. In the present investigation, the authors have determined additional properties of the nuclear ferritoid-ferritin complexes.

Methods: For biochemical characterization, a combination of molecular sieve chromatography, immunoblotting, and nuclear-cytoplasmic fractionation was used; DNA binding was analyzed by electrophoretic mobility shift assay.

Results: The CE nuclear ferritin complex has characteristics that differentiate it from a "typical" cytoplasmic ferritin, including the presence of ferritin and ferritoid subunits; a molecular weight of approximately 260 kDa, which is approximately half that of cytoplasmic ferritin; its iron content, which is below our limits of detection; and its ability to bind to DNA.

Conclusions: Within CE cell nuclei, ferritin and ferritoid are coassembled into stable complex(es) present in embryonic and adult corneas. Thus, ferritoid not only serves transiently as a nuclear transporter for ferritin, it remains as a component of a unique ferritoid-ferritin nuclear complex.

Figure 1

Gel filtration chromatography of proteins extracted with HEPES buffer from E15 embryonic CE tissue. (A) Calibration curve showing the molecular weight makers in kilodaltons, and the corresponding numbers of the fractions collected and subsequently analyzed for ferritoid (FTD) and ferritin (FTN) by denaturing SDS-PAGE, followed by Western blot analysis. (B) Western blot analysis of the fractions probed for FTD and FTN after denaturing SDS-PAGE. (C) Immunoprecipitation of the fractions with anti–FTN antibody followed by Western blot analysis for FTD.

Figure 2

(A) Schematic diagram of the cornea depicting the central area (gray) in which the ferritin (FTN) and ferritoid (FTD) of CE cells have a predominantly nuclear localization and the peripheral ring (black) in which FTN and FTD are cytoplasmic, as shown in the accompanying immunoconfocal micrographs for FTD and FTN, and for DNA (by Hoechst staining). The area of the ring is approximately 40% of the central region [π(r² − r − r/7)² versus π(r − r/7)²]. Also shown are Western blot analyses for cytoplasmic (cyto) and nuclear (nucl) fractions using antibodies against FTD, FTN, and histone 3 (His3). (B, C) Subcellular localization of the FTD-FTN complexes. Analyses of the nuclear (B) and cytoplasmic (C) extracts of cultured CE cells, separated by gel filtration chromatography, followed by immunoprecipitation for FTN (IP/FTN) and Western blot for FTD (WB/FTD). The fraction number and molecular weight (MW) of each marker is shown at the bottom.

Figure 3

(A) Denaturing SDSPAGE of the heat-enriched ferritoidferritin (FTD-FTN) fraction of a CE tissue extract, stained with Coomassie. Left: molecular weights. Also shown are Western blots (WB) for FTN and FTD. (B) Gel filtration chromatography of the heat-enriched material, followed by SDS-PAGE visualized by silver staining (upper, FTD and FTN). Lower: Western blot detection of FTD in the material immunoprecipitated with the anti–FTN antibody. Positions of molecular weight markers and fraction numbers are shown at the bottom. Also shown are the results of iron detection (± signs) in the fractions. (C) Electrophoretic mobility shift assay of column fractions 3, 8, 14, and 16. Double asterisks: shift in DNA mobility indicative of protein binding. Arrow: free probe.

Figure 4

(A) Expression and assembly of ferritin (FTN) and ferritoid (FTD) in CE from E8 and E11 and in the adult tissue (analyzed by quantitative RT-PCR and normalized to β-actin). (B) Coimmunoprecipitation analysis of protein fractions collected by gel filtration chromatography of the HEPES extracts from adult chicken CE. Western blot detection of FTD (WB/FTD) in the material immunoprecipitation with the anti–FTN antibody (IP/FTN). The fraction numbers and corresponding molecular weight (MW) markers are shown at the bottom.

Figure 5

Proposed models of dodecameric nuclear complexes composed of homodimeric subunits of ferritin and ferritoid in ratios of 2:1 (280 kDa), 1:1 (300 kDa), and 1:2 (320 kDa).