Immunoreactive hephaestin and ferroxidase activity are present in the cytosolic fraction of rat enterocytes

Abstract

Discovered over a decade ago, hephaestin (Heph) has been implicated as a ferroxidase (FOX) vital for intestinal iron absorption. Stringent structural or kinetic data derived from purified, native protein is however lacking, leading to the hypothesis that an alternate, undiscovered form of Heph could exist in mammalian enterocytes. This possibility was tested using laboratory rodent and cell culture models. Cytosolic and membrane fractions were obtained from rat enterocytes and purity of the fractions was assessed. Western blot analyses revealed Heph in cytosol obtained by three different methods, ruling out the possibility of a method-induced artifact being the major contributor to this observation. Absence of two different membrane-proteins, ferroportin 1 and Menke's copper ATPase in cytosol, and the absence of lipids in representative cytosolic samples tested by thin layer chromatography, eliminated significant membrane contamination of cytosol. Further, immunohisto- and immunocyto-chemical analyses identified Heph in rat enterocytes and in two intestinal epithelial cell lines, IEC-6 and Caco-2, intracellularly. Additionally, cytosolic Heph increased upon iron-deprivation but more important, decreased significantly upon copper-deprivation, mimicking the response of membrane-bound Heph. Moreover, FOX activity was present in rat cytosol, and was partly inhibited by anti-Heph antibody. Finally, lack of immunodetectable ceruloplasmin (Cp) by western blot precluded Cp as an underlying cause of this activity. These data demonstrate that rat enterocytes contain a soluble/cytosolic form of Heph possibly contributing to the observed FOX activity.

Fig 1

Presence of immunoreactive Heph in cytosolic and membrane fractions and fraction purity. Panel a western blot of Heph protein in cytosolic and membrane fractions of rat duodenal enterocytes prepared by three different methods. D4 indicates the particular anti-Heph Ab used. Panel b western blot of Atp7a protein in 60 μg protein samples containing various ratios of membrane to cytosolic proteins. 54–10 indicates the particular anti-Atp7a Ab used. Panel c western blot of FPN protein in rat duodenal enterocytes and KNRK (a transformed rat kidney [NRK] cell line) lysate (positive control). Results from two individual rats are shown. Stained blots in panels b and c exemplify proper loading and transfer of proteins. The blank space and black line in panel c indicate removal of unrelated lanes. Panel d thin layer chromatography (TLC) of water, buffers, enterocyte cytosol and membrane fractions, and P-lipid (phospholipid, used as a positive control)

Fig 2

Immunofluorescence analysis of rat duodenum, IEC-6 cells, and Caco-2 cells. Panels a and b immunohistochemical analysis of duodenal sections from an iron-deficient rat (a) with and (b) without the D4 anti-Heph antibody. Panels c and d immunocytochemical analysis of pre-confluent IEC-6 and Caco-2 cells. Data in all panels are representative of three independent experiments performed

Fig 3

Effects of iron- and copper-deprivation on Heph protein expression. Panel a shows denaturing and panel b shows native western blot analysis of Heph in cytosolic and membrane fractions of Ctrl and FeD rat enterocytes. Data in panel a are representative of experiments done with 12 individual rats per group. Panels c and d show denatured western blot analysis of Heph in cytosolic and membrane fractions of Ctrl and CuD rat enterocytes by (c) D4 and (d) GeneTex anti-Heph Abs. Stained blots below show equal loading and transfer of protein samples. In panels a–c data from two rats per group are shown, and in panel d data from three rats per group are shown. Ctrl control, FeD iron-deficient, CuD copper-deficient

Fig 4

FOX activity of rat duodenal enterocyte cytosol and antibody inhibition. Panel a shows the enzyme progress curve of rat cytosol FOX activity by Tf assay. Data represent three individual rats, mean ± SD is shown. Panel b effect of anti-Heph (D4) and anti-Alox15 (against an unrelated protein) Abs on cytosolic FOX activity by Tf assay. n = 2 individual rats, mean is shown

Fig 5

Ceruloplasmin expression in rat duodenal enterocyte fractions and serum. A western blot for Cp is shown above. The stained blot below exemplifies equal loading and transfer of protein samples. Data are representative of two independent experiments that were performed with identical results. MW, molecular weight markers. Memb, membrane