Identification of zyklopen, a new member of the vertebrate multicopper ferroxidase family, and characterization in rodents and human cells

Abstract

We previously detected a membrane-bound, copper-containing oxidase that may be involved in iron efflux in BeWo cells, a human placental cell line. We have now identified a gene encoding a predicted multicopper ferroxidase (MCF) with a putative C-terminal membrane-spanning sequence and high sequence identity to hephaestin (Heph) and ceruloplasmin (Cp), the other known vertebrate MCF. Molecular modeling revealed conservation of all type I, II, and III copper-binding sites as well as a putative iron-binding site. Protein expression was observed in multiple diverse mouse tissues, including placenta and mammary gland, and the expression pattern was distinct from that of Cp and Heph. The protein possessed ferroxidase activity, and protein levels decreased in cellular copper deficiency. Knockdown with small interfering RNA in BeWo cells indicates that this gene represents the previously detected oxidase. We propose calling this new member of the MCF family "zyklopen."

FIGURE 1

Molecular modeling of Zp. (A) The superimposition of modeled human Zp on Cp structure. The figures were generated using a modified version of Molscript (43) and subsequently rendered in Raster3D version 2.0 (44). (B) The ribbon diagram of Zp shown with top view, bottom view (C), and side view (D). The residues are colored blue to green for domains 1 and 2 (residues 1–370), yellow to red for domains 3 and 4 (residues 371–720), and lilac to gray for domains 5 and 6 (residues 721–1067). The copper and oxygen atoms are shown in blue and red, respectively.

FIGURE 2

Expression of Zp in rodent tissues (A,B) and human cell lines (C). (A) Expression of Zp mRNA in E18 mouse placenta, liver, heart, kidney, E7 placenta, E18 embryo, and enterocytes. GAPDH expression was used as a loading control. (B) Immunoblot with Zp-specific IgG of rat placenta, lactating mouse mammary tissue, mouse E18 embryo, and (C). mouse serum and enterocytes, and human BeWo, MCF7, T47D, and MCF10AT cell line extracts. (D) Immunoblot with Cp- and Heph-specific IgG, respectively, of mouse serum and enterocytes; 50 μg total protein/lane.

FIGURE 3

Immunostaining of Zp in adult (A) and embryonic (B) mouse tissues. (A) Immunohistochemical localization of Zp in adult mouse brain, kidney, testes, and retina at 10 wk of age. No staining is detected in the preimmune control sera (top row). In the brain, Zp was detected in the choroid plexus, the dentate gyrus, and the CA1 region of the hippocampus (arrow). Zp was localized in cross-sectioned tubules in the kidney medulla and medullary rays (arrow). Zp was highly expressed in the mature spermatozoa of the testes and in the interstitial spaces between the tubules where the endocrine Leydig cells are located (arrow). In the retina, Zp was detected in the retinal pigment epithelium (RPE) and ganglion cell layer (GCL) (arrows). (B) Immunohistochemical localization of Zp in embryonic (E17.5) mouse brain, bladder, eye, and brown fat. No staining was detected using the preimmune control sera (top row). Zp expression was high in the choroid plexus of the brain and in the urinary epithelium of the bladder. Zp was also expressed in the E17.5 retina and brown fat. All images were taken at 20× magnification.

FIGURE 4

pPD oxidase (A) and ferroxidase (B) activity of Zp in the BeWo human placental cell line. (A) In-gel pPD oxidase activity was measured in cell extracts (60, 90, 120, and 150 μg total protein/lane) separated under nondenaturing conditions by native gel electrophoresis. Purified human Cp (5–20 μg/lane) was used as a control. (B) In-gel ferroxidase activity of the same extracts in A. (C) Immunoblot of the same extracts in A with Zp-specific IgG.

FIGURE 5

pPD oxidase activity in BeWo cells after siRNA knockdown of Zp. (A) Immunoblot with Zp- and GAPDH-specific IgG of extracts (100 μg total protein/lane) of nontransfected BeWo cells (Ctrl) and BeWo cells transfected with set 1, set 2, and set 3 siRNA targeting Zp. Purified human Cp was used as a control (20 μg/lane). (B) In-gel pPD activity assay of the same samples (100 μg total protein/lane).

FIGURE 6

Regulation of Zp by copper in BeWo cells. (A) BeWo cells were made copper-deficient by incubation with increasing concentrations of BCS, and cell extracts (100 μg total protein/lane) were then immunoblotted with Zp-specific and GAPDH-specific IgG followed by development with chemiluminescence and measurement of band intensity by densitometry. The ratio of each Zp band density to the density of the 0 μmol/L BCS control were multiplied by 100, averaged, and then plotted versus BCS concentration (r² = 0.926; P ≤ 0.0005). Values are means ± SD of results from 3 independent experiments. (B) SOD1 activity as measured in the same cell lysates as in A and plotted versus BCS concentration (r² = 0.982; P ≤ 0.0001). Values are means ± SD, n = 2 independent experiments.