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. 2012 Feb 28;109(9):3564-9.
doi: 10.1073/pnas.1120833109. Epub 2012 Feb 13.

Discovery of a cytosolic/soluble ferroxidase in rodent enterocytes

Perungavur N Ranganathan  1 , Yan Lu, Brie K Fuqua, James F Collins
Affiliations

Discovery of a cytosolic/soluble ferroxidase in rodent enterocytes

Perungavur N Ranganathan et al. Proc Natl Acad Sci U S A. .

Abstract

Hephaestin (Heph), a membrane-bound multicopper ferroxidase (FOX) expressed in duodenal enterocytes, is required for optimal iron absorption. However, sex-linked anemia (sla) mice harboring a 194-amino acid deletion in the Heph protein are able to absorb dietary iron despite reduced expression and mislocalization of the mutant protein. Thus Heph may not be essential, and mice are able to compensate for the loss of its activity VSports手机版. The current studies were undertaken to search for undiscovered FOXs in rodent enterocytes. An experimental approach was developed to investigate intestinal FOXs in which separate membrane and cytosolic fractions were prepared and FOX activity was measured by a spectrophotometric transferrin-coupled assay. Unexpectedly, FOX activity was noted in membrane and cytosolic fractions of rat enterocytes. Different experimental approaches demonstrated that cytosolic FOX activity was not caused by contamination with membrane Heph or a method-induced artifact. Cytosolic FOX activity was abolished by SDS and heat (78 °C), suggesting protein-mediated iron oxidation, and was also sensitive to Triton X-100. Furthermore, cytosolic FOX activity increased ∼30% in iron-deficient rats (compared with controls) but was unchanged in copper-deficient rats (in contrast to the reported dramatic reduction of Heph expression and activity during copper deficiency). Additional studies done in sla, Heph-knockout, and ceruloplasmin-knockout mice proved that cytosolic FOX activity could not be fully explained by Heph or ceruloplasmin. Therefore rodent enterocytes contain a previously undescribed soluble cytosolic FOX that may function in transepithelial iron transport and complement membrane-bound Heph. .

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"V体育ios版" Conflict of interest statement

The authors declare no conflict of interest.

Figures (V体育ios版)

Fig. 1.
Fig. 1.
FOX activity in rat enterocyte fractions. Relative catalytic rate (dA460/dt) is shown at the 1-, 2-, and 3-min time points. (A and B) Mean ± SD of data from 12 individual rats in three separate experiments. **P < 0.01; *P < 0.05. Ctrl, control rats; FeD, iron-deficient rats.
Fig. 2.
Fig. 2.
Enterocyte purity and alternative methods. (A) LDH activity assays. Catalytic rate at 75 s is shown reflecting disappearance of the substrate pyruvate. Lysate and cytosol are samples purified from rat enterocytes (n = 3). Purified rabbit muscle LDH (0.05 U, 0.1 U, and 0.2 U) was used as a positive control. **P < 0.01 compared with lysate. (B) Western blot of four rat cytosol and four membrane samples reacted with anti-ZnT1 antibody. (Lower row) Ponceau S-stained blot showing comparable loading and transfer of proteins. The black line indicates where unrelated lanes were removed from the image. (C and D) Progress curves from Tf-coupled FOX assay of enterocyte cytosolic and membrane fractions prepared using alternative methods. Each symbol is the mean of two individual rats. In A, C, and D, blanks are reaction mixes devoid of enzyme source (i.e., protein sample).
Fig. 3.
Fig. 3.
Chemical properties of cyto-FOX. (A) Effect of protein denaturants on enzyme activity. Data shown are reaction rates at 30 s from four rats per treatment. (−), no treatment. ***P < 0.0001 compared to (−). (B) Effect of 1.5% Triton X-100 (TX) on cyto-FOX activity. Progress curves are shown for blank and cytosol samples with and without the addition of detergent. n = 4 individual rats. *P < 0.05. In A and B, data are shown as mean ± SD.
Fig. 4.
Fig. 4.
Effect of copper deficiency on cyto-FOX activity. (A and B) (Upper) Western blot of Cp protein in rat serum (A) and Western blot of Atp7a protein in duodenal enterocyte samples derived from experimental rats (B). 54–10 indicates the particular antiserum used. (Lower) Ponceau S-stained blots exemplify equal loading and transfer of protein. (C) Cyto-FOX activity assays in samples derived from experimental rats. The enzymatic rate (dA460/dt) is shown at various time points. Data are shown as mean ± SD. n = 5. Ctrl, control rats; CuD, copper-deficient rats.
Fig. 5.
Fig. 5.
Heph and Cp protein expression and FOX activity in mutant mouse models. WT mice, sla (Heph sla/y), and Cp-KO (Cp−/−) mice were studied. (A and B) (Upper) Western blot for cytosolic Heph in three WT (+/y) and three mutant (sla/y) mice (A) and Western blot for Cp in three WT, three sla/y, and two Cp−/− mice (B). MW, molecular weight markers. (Lower) Ponceau S-stained blots exemplify equal loading and transfer of protein. Black lines indicate where unrelated lanes were removed from the images. (C and D) FOX activity assays are shown as mean ± SD. Enzymatic rate (dA460/dt) is shown at various time points. n = 3 WT, 6 sla/y, and 3 Cp−/− mice.
Fig. 6.
Fig. 6.
FOX activity in enterocyte cytosol from Heph-KO mice. FOX activity was measured using two spectrophotometric methods, the initial velocity Tf assay (A) and the end-point ferrozine (Fz) assay (B). The enzymatic rate (dA/dt) is shown at different time points for the Tf assay. A570 progress curves are shown for the ferrozine assay. Note that the ferrozine assay is a substrate disappearance assay in which lower numbers indicate higher enzyme activity. No statistical differences were noted between groups in either assay. n = 4 for WT and 4 for Heph -/y mice.
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References

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