Figure 1.
Identification of two missense mutations in the SLC30A2/ZnT2 gene in the mother of a zinc-deficient infant.
(A) Photograph of an affected infant with severe zinc deficiency. The dermatitis was erythematous and erosive, particularly around the infant's mouth. (B) Predicted topology of hZnT2 indicating the positions of the W152R and S296L substitutions found in this study. (C) Electropherograms showing SLC30A2/ZnT2 mutations in the affected mother. W152R and S296L mutations were found at exons 4 and 7 on different alleles.
Figure 2.
Sequence alignment among hZnT2, hZnT3 and hZnT8.
The positions of tryptophan (corresponding to W152 in hZnT2) and serine (S296) residues (indicated by black arrowheads) identified in the affected mother with low milk zinc are completely conserved in hZnT3 and semi-conserved in hZnT8. The positions of histidine (corresponding to H54 in hZnT2) and glycine (G87) residues that have been identified are also indicated by gray arrowheads. Identical amino acids are indicated by *. The putative transmembrane regions, which are predicted by SOSUI (http://bp.nuap.nagoya-u.ac.jp/sosui/) using hZnT2 sequence, are shaded in gray.
Figure 3.
No mutations were found in and around the promoter region of the SLC30A2/ZnT2 gene.
Alignment of the sequences of the mother with low milk zinc (“mother”) and the human genomic sequence deposited in the GenBank database (“human”). To show high homology in this region among mammals, where the MRE is completely conserved, the sequences are also aligned with those of rats and mice deposited in the GenBank database (corresponding regions from −110 to +22 of mouse Znt2 are shown. The transcription start site is indicated by gray shading). Identical nucleotides are indicated by * and the MRE sequence is indicated with bold letters.
Figure 4.
W152R hZnT2 loses the ability to transport zinc, but the S296L hZnT2 does not.
(A) The carboxyl-terminal epitope tags do not interfere with hZnT2 expression. Untagged and HA- or FLAG-tagged hZnT2 were stably expressed in ZnT1−/−MT−/−ZnT4−/− cells. Immunoblotting was performed using anti-hZnT2 antibody. (B) Confirmation of stable expression of the WT hZnT2-HA, W152R, S296L and other mutants of hZnT2-HA in ZnT1−/−MT−/−ZnT4−/− cells. Immunoblotting was performed using an anti-HA antibody. In both (A) and (B), 20 µg of total cellular protein was loaded onto each lane, and the same membrane was used for detection of both hZnT2 and tubulin. Tubulin is shown as a loading control. (C) Effects of zinc on MT-luciferase reporter gene expression in ZnT1−/−MT−/−ZnT4−/− cells stably expressing WT hZnT2-HA, W152R or S296L mutant hZnT2-HA. Relative activity of Luc is shown (the luciferase activity of ZnT1−/−MT−/−ZnT4−/− cells cultured without ZnSO4 is defined as 1). Each value is the mean ± SD of triplicate experiments. * denotes a significant difference of relative activity of Luc between the cells expressing WT and W152R mutant hZnT2 (P<0.05).
Table 1.
Evaluation of zinc transport activities of hZnT2 proteins using ZnT1−/−MT−/−ZnT4−/− cells.
Figure 5.
W152R hZnT2 mutant is not dominant negative because it fails to form functional dimers.
Tagged-hZnT2 WT or mutants were immunoprecipitated (IP) with antibodies against either the FLAG or HA epitopes. The immunoprecipitates were analyzed by immunoblotting using antibodies against the FLAG or HA tags. To estimate the amount of tagged hZnT2 WT and mutant proteins, 10% of each aliquot was subjected to immunoblot analysis (input panels). The IP experiments were performed four times, which gave the same results. The panels show the representative results.
Figure 6.
S296L mutation causes hZnT2 destabilized.
(A) The expression level of the hZnT2 protein at each time point. The ZnT1−/−MT−/−ZnT4−/− cells expressing WT hZnT2 or S296L mutant were treated with CHX and collected periodically over 4 h. Immunoblot analysis was performed to monitor hZnT2 levels (left panel). The band intensities of hZnT2 protein (○, WT; •, S296L mutant) are shown as the percentage of the intensity at 0 h (T0) after normalized by that of tubulin at each time (right panel). * and ** denote a significant difference between expression levels of the WT and S296L mutant hZnT2 at each time point (* P<0.05, ** P<0.01) (B) Lysosome inhibitor bafilomycin A1 and proteasome inhibitor MG132 block the degradation of S296L hZnT2 mutant. Immunoblot analysis (left panel) and the band intensities of hZnT2 protein (○, MG132; Δ, bafilomycin A1; • no inhibitor, right panel) are shown. In the right panels of both (A) and (B), each value is the mean ± SD of triplicate experiments. The same membrane was used for detection of both hZnT2 and tubulin. Tubulin is shown as a loading control. * and ** denote significant differences between expression levels in the absence and presence of bafilomycin A1 or MG132 at each time point (* P<0.05, ** P<0.01).
Figure 7.
Protein stability of W152R, H54R and G87R hZnT2 mutants.
The expression levels of the WT (○) and W152R (•) mutant hZnT2 proteins (A), the WT (○) and H54R (•) mutant hZnT2 proteins (B), and the WT (○) and G87R (•) mutant hZnT2 proteins (C) at each time point. Immunoblot analysis was performed to monitor hZnT2 levels (left panel), as described in Figure 6. In the right panels of (A) – (C), each value is the mean ± SD of triplicate experiments. The same membrane was used for detection of both hZnT2 and tubulin. Tubulin is shown as a loading control. * and ** denote a significant difference between expression levels of the WT and W152R, H54R or G87R mutant hZnT2 at each time point (* P<0.05, ** P<0.01).