Proteomic analysis shows the upregulation of erythrocyte dematin in zinc-restricted human subjects

Abstract

Background: Although the importance of adequate zinc intake has been known for decades, the estimated global prevalence of zinc deficiency remains high. This substantiates the need for a specific and sensitive status assessment tool.

Objective: The objective was to evaluate erythrocyte zinc transporters as candidate molecules with the potential of being a biomarker of dietary zinc status in humans.

Design: A 24-d observational study with acclimation (7 d, 10.4 mg Zn/d), zinc-depletion (10 d, 0.3 mg Zn/d), and zinc-repletion (7 d, 29.5 mg Zn/d) phases was conducted in healthy men (n = 9). Proteomic approaches including Western blot analyses and tandem mass spectrometry were implemented to identify the zinc responsiveness of selected red blood cell membrane proteins.

Results: Zinc transporter 1 (ZnT1) and Zrt/Irt-like proteins ZIP8 and ZIP10 were detected in human erythrocyte membranes. No effects of short-term dietary zinc depletion were observed on the amounts of these proteins. However, changes in a cytoskeletal protein, dematin, by zinc depletion were identified through the nonspecific signals produced by an anti-ZIP8 antibody. This response was further validated by a dematin-specific antibody and with erythrocytes collected from mice fed a zinc-deficient diet.

Conclusions: The presence of ZnT1, ZIP8, and ZIP10 in human red blood cells implicates their role in the regulation of cellular zinc metabolism in the human erythroid system. The zinc responsiveness of membrane dematin suggests its capability to serve as a biomarker for dietary zinc depletion and its involvement in impaired erythroid membrane fragility by zinc restriction. This trial was registered at clinicaltrials.gov as NCT01221129.

Figures

FIGURE 1.

Zinc transporter expression in the plasma membrane of human erythrocytes. The presence of ZnT1(A), ZIP8 (B), and ZIP10 (C) in erythrocyte ghosts was detected by Western blot analysis. The specificity of signals was determined by preincubating each primary antibody with their respective antigenic peptides. D: Glycosylation of ZnT1 was identified by PNGaseF treatment. NS, nonspecific band; PNGaseF, peptide-N-glycosidase F; ZnT1, zinc transporter 1; ZIP8, Zrt/Irt-like protein 8; ZIP10, Zrt/Irt-like protein 10.

FIGURE 2.

Effects of acute dietary zinc depletion on zinc transporter expression in human erythrocytes. A: The amounts of each transporter before (ZnA) and after (ZnD) 10 d of dietary zinc depletion were determined by Western blot analysis by using affinity-purified antibodies. B: Signal intensities were quantified by densitometric analysis, and mean (±SD) values were normalized to baseline values. *Significantly different at P < 0.05 (paired t test, n = 3 subjects in each pooled sample). ZIP8, Zrt/Irt-like protein 8; ZIP10, Zrt/Irt-like protein 10; ZIP8-NS, the nonspecific band detected with the ZIP8 antibody; ZnA, zinc adequate; ZnD, zinc deficient; ZnT1, zinc transporter 1.

FIGURE 3.

Identification of the protein nonspecifically detected by the human ZIP8 antibody. A: Detection of IgG incorporated into the protein sample during immunoprecipitation with the ZIP8 antibody (α-ZIP8) by an anti-IgG secondary antibody (α-IgG). B: Position of the zinc-responsive band nonspecifically produced by α-ZIP8 band on a Western blot. The nonspecific band is indicated by an asterisk. C: Coomassie stain of a counterpart gel of the Western blots shown in panels A and B. The region migrating at the size of the nonspecific protein is indicated by a dashed box and was excised from the stained gel for LC-MS/MS. D: The peptide profile of the isolated proteins determined by LC-MS/MS. Dematin, with an expected molecular weight of 46 kDa, showed the highest normalized spectrum count among the 11 identified proteins. E: Peptide sequences unique for dematin identified by LC-MS/MS are highlighted in gray. HC, heavy chain; IP, immunoprecipitation; LC, light chain; LC-MS/MS, liquid chromatography–tandem mass spectrometry; ZIP8, Zrt/Irt-like protein 8.

FIGURE 4.

Upregulation of erythrocyte membrane dematin by zinc-deprived conditions. A: Response of dematin to 10 d of dietary zinc depletion in humans (n = 3 subjects per pooled sample). Dematin amount was measured by either the signals produced by the nonspecific signals from the ZIP8 antibody or those from a dematin-specific antibody. B: Changes in erythrocyte membrane dematin amounts in mice (n = 3) after being fed a low-zinc diet for 21 d. Signal intensities of Western blots produced by human (C) and mouse (D) samples were quantified by densitometric analysis, and mean (±SD) values were normalized to their respective control values. *,**Significantly different (paired t test and Student's t test for human and mouse samples, respectively): *P < 0.05, **P < 0.005. ZIP8, Zrt/Irt-like protein 8; ZIP8-NS, nonspecific band detected with the ZIP8 antibody; ZnA, zinc adequate; ZnD, zinc depleted.