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. 2021 Jan-Jun:296:100269.
doi: 10.1016/j.jbc.2021.100269. Epub 2021 Jan 8.

Zinc transporter mutations linked to acrodermatitis enteropathica disrupt function and cause mistrafficking

Eziz Kuliyev  1 , Chi Zhang  2 , Dexin Sui  2 , Jian Hu  3
Affiliations

Zinc transporter mutations linked to acrodermatitis enteropathica disrupt function and cause mistrafficking

Eziz Kuliyev et al. J Biol Chem. 2021 Jan-Jun.

Abstract

ZIP4 is a representative member of the Zrt-/Irt-like protein (ZIP) transporter family and responsible for zinc uptake from diet. Loss-of-function mutations of human ZIP4 (hZIP4) drastically reduce zinc absorption, causing a life-threatening autosomal recessive disorder, acrodermatitis enteropathica (AE). These mutations occur not only in the conserved transmembrane zinc transport machinery, but also in the extracellular domain (ECD) of hZIP4, which is only present in a fraction of mammalian ZIPs. How these AE-causing ECD mutations lead to ZIP4 malfunction has not be fully clarified. In this work, we characterized all seven confirmed AE-causing missense mutations in hZIP4-ECD and found that the variants exhibited completely abolished zinc transport activity in a cell-based transport assay. Although the variants were able to be expressed in HEK293T cells, they failed to traffic to the cell surface and were largely retained in the ER with immature glycosylation. When the corresponding mutations were introduced in the ECD of ZIP4 from Pteropus Alecto, a close homolog of hZIP4, the variants exhibited structural defects or reduced thermal stability, which likely accounts for intracellular mistrafficking of the AE-associated variants and as such a total loss of zinc uptake activity. This work provides a molecular pathogenic mechanism for AE.

Keywords: ZIP4; acrodermatitis enteropathica; disease-causing mutation; extracellular domain; misfolding; mistrafficking; zinc transporter.

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Conflict of interest statement

Conflict of interest The authors declare no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
Mapping of the AE-causing mutations and the SNPs on the structural model of hZIP4-ECD dimer. Seven residues subjected to AE-causing mutations (highlighted in boxes) are in yellow and stick mode with four in the HRD domain, two in the PCD domain, and one (P200L) in the linker connecting the two subdomains, whereas the residues at which SNPs occur are in red. Additional three SNPs not shown in the model are either in the highly disordered his-rich loop (R251W) or in the signal peptide (V2A and E10A).
Figure 2
Figure 2
Expression and functional characterization of hZIP4 and the variants.A, cell-based zinc transport assay of the AE-associated variants with 10 μM Zn2+ in the medium. The data points of one representative experiment out of 2–6 independent experiments are shown as black dots, and the means and the standard deviations are indicated by bars. B, total expression hZIP4 and the variants detected by western blot using anti-HA antibody. β-actin was used as loading control. C, processing of hZIP4 by PNGase F and Endo H glycosidases. D, zinc transport assay of the N261Q variant. Data are expressed as average ± standard deviation (n = 3).
Figure 3
Figure 3
Cell surface expression of hZIP4 and the AE-associated variants.A, surface-bound anti-HA antibody for detection of surface expression of hZIP4-HA and the variants. β-actin was used as the loading control. The result of one representative experiment out of three independent experiments is shown. B, detection of expression of hZIP4 and its variants. FITC-labeled anti-HA antibody was used to stain HA tagged hZIP4 in nonpermeabilized cells (surface expression, upper row) or permeabilized cells (total expression, lower row). The scale bar = 10 μm.
Figure 4
Figure 4
Colocalization of hZIP4 with the ER marker calreticulin. Representative confocal images (100×) of HEK293T cells transiently expressing hZIP4-HA or the variants are shown. hZIP4 and the variants were detected with an anti-HA tag antibody and an Alexa-568 goat anti-mouse antibody (red). Calreticulin was detected with an anti-calreticulin antibody and an Alexa-488 anti-rabbit antibody (green). Scale bar = 5 μm. Several dual-color images (n = 4–7) were taken and subjected to colocalization analysis using Image J equipped with the JACoP plugins. PCC was calculated for each image and data are expressed as average ± standard deviation for each construct. Statistical analysis was conducted to examine the significant difference of PCC between hZIP4 and the variants. ∗∗p < 0.01.
Figure 5
Figure 5
Purification of pZIP4-ECD and the variants.A, size-exclusion chromatography. The elution volumes of the protein standards are indicated by arrows, from which the apparent molecular weights of the purified proteins were estimated. B, dynamic light scattering of the wild-type protein and the Q299H variant. Data are expressed as average ± standard deviation (n = 2–3). ∗p < 0.05.
Figure 6
Figure 6
CD spectra of pZIP4-ECD and the variants. For each protein, representative spectrum out of three repeats is shown. The α-helical content (α %) was estimated using the K2D3 server. 222/208 indicates the ratio of θ222/θ208. The SDS-PAGEs of purified protein are shown in the insets. The results of 3–5 technical repeats from one experiment are expressed as average ± standard deviation. For the wild-type pZIP4-ECD and the P193L variant, three independent batches of purified proteins were analyzed, from which the α-helical contents were determined to be 66 ± 0% and 65 ± 1%, and the 222/208 ratios are 1.031 ± 0.009 and 0.976 ± 0.016, respectively. ∗p < 0.05.
Figure 7
Figure 7
Thermal stability of the wild-type pZIP4-ECD and the P193L variant.A, CD spectra of wild-type pZIP4-ECD at different temperatures. At 95 °C, the α-helical content was estimated to be 20%. B, comparison of the native and the refolded pZIP4-ECD. Left: size-exclusion chromatography. Peaks a and b are dimer and monomer, respectively, of which SDS-PAGE is shown in the inset. Right: CD spectra with indicated α-helix contents and θ222/θ208 ratios. C, heat denaturation curves of the wild-type pZIP4-ECD and the P193L variant. The error bars indicate standard deviations of three technical repeats in one experiment. The shifted curve of the P193L variant relative to the wild-type protein was consistently observed in three independent experiments. D, the interaction of P193 with W262 (PDB ID: 4X82). The two monomers are colored in green and cyan, respectively. The bridging region is indicated by the dashed oval.
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