Molecular Basis of Transient Neonatal Zinc Deficiency: NOVEL ZnT2 MUTATIONS DISRUPTING ZINC BINDING AND PERMEATION

Yarden Golan,Naoya Itsumura,Fabian Glaser,Bluma Berman,Taiho Kambe,Yehuda G Assaraf

doi:10.1074/jbc.m116.732693

Abstract

A gradually increasing number of transient neonatal zinc deficiency (TNZD) cases was recently reported, all of which were associated with inactivating ZnT2 mutations. Here we characterized the impact of three novel heterozygous ZnT2 mutations G280R, T312M, and E355Q, which cause TNZD in exclusively breastfed infants of Japanese mothers. We used the bimolecular fluorescence complementation (BiFC) assay to provide direct visual evidence for the in situ dimerization of these ZnT2 mutants, and to explore their subcellular localization. Moreover, using three complementary functional assays, zinc accumulation using BiFC-Zinquin and Zinpyr-1 fluorescence as well as zinc toxicity assay, we determined the impact of these ZnT2 mutations on vesicular zinc accumulation. Although all three mutants formed homodimers with the wild type (WT) ZnT2 and retained substantial vesicular localization, as well as vesicular zinc accumulation, they had no dominant-negative effect over the WT ZnT2. Furthermore, using advanced bioinformatics, structural modeling, and site-directed mutagenesis we found that these mutations localized at key residues, which play an important physiological role in zinc coordination (G280R and E355Q) and zinc permeation (T312M). Collectively, our findings establish that some heterozygous loss of function ZnT2 mutations disrupt zinc binding and zinc permeation, thereby suggesting a haploinsufficiency state for the unaffected WT ZnT2 allele in TNZD pathogenesis. These results highlight the burning need for the development of a suitable genetic screen for the early diagnosis of TNZD to prevent morbidity.

Highlights

A gradually increasing number of transient neonatal zinc deficiency (TNZD) cases was recently reported, all of which were associated with inactivating ZnT2 mutations
The TNZD Mutations G280R, T312M, and E355Q Retain ZnT2 Homodimerization—We first assessed the ability of the ZnT2 mutants to form homodimers using the bimolecular fluorescence complementation (BiFC) assay and flow cytometric analysis
These mutations, including G280R, T312M, and E355Q, mapped to the C terminus of ZnT2 (Fig. 6) in contrast to the G87R and W152R mutations that were found to be located in TM domains (7, 8), whereas H54R localized at the N terminus (6)

Summary

Introduction

A gradually increasing number of transient neonatal zinc deficiency (TNZD) cases was recently reported, all of which were associated with inactivating ZnT2 mutations. In contrast to TNZD, which appears only in exclusively breastfed infants, and will not emerge after weaning, acrodermatitis enteropathica (OMIM number 201100), an autosomal recessive disease (12, 14), presents after weaning, as a result of loss of function mutations in the SLC39A4 gene (ZIP4). These inactivating mutations impair intestinal zinc absorption via the ZIP4 transporter, thereby leading to severe zinc deficiency after weaning. The SLC30A2 gene encodes for the zinc transporter ZnT2, which is responsible for zinc accumulation in intracellular vesicles, in lactating epithelial mammary gland cells Through exocytosis, these zinc-loaded vesicles secrete zinc into breast milk (13). Excluding one case of compound ZnT2 mutations (7), all TNZD cases reported to date were found in mothers that were heterozygous for mutations in the SLC30A2 gene

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Results

Conclusion