Molecular Basis of Transient Neonatal Zinc Deficiency

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.In the past 50 years, the key role of zinc in human health was established (1). Zinc is crucial for central physiological processes including DNA and protein synthesis, enzyme activity, intracellular signaling, immune function, fertility, as well as growth and development (1-3). Zinc homeostasis is primarily regulated by two zinc transporter families as well as by zincbinding proteins. The SLC39A gene family encodes for ZIP1-14 transporters, which are responsible for zinc uptake into the cytosol from the extracellular fluid or from intracellular vesicles (4). In contrast, the SLC30A gene family encodes for the transporters ZnT1-10, which are responsible for zinc secretion from the cytosol to intracellular organelles or through the plasma membrane outside the cells (2, 4).The recent position of the Academy of Nutrition and Dietetics (5) is that breastfeeding in the first 6 months of life can provide adequate micronutrients and macronutrients for optimal growth and development. This statement is correct for the vast majority of infants around the world. However, recent reports indicate that some exclusively breastfed infants suffer from severe zinc deficiency due to lack of zinc in the breast milk they consume (6 -11). These cases were referred to as transient neonatal zinc deficiency (TNZD), 4 as the symptoms can be treated with zinc supplementation to the diet of the infant (12, 13). In contrast to TNZD, which appe...

A genetic variant in SLC30A2 causes breast dysfunction during lactation by inducing ER stress, oxidative stress and epithelial barrier defects

“…One would predict that because there is substantially greater S 288 retained within the ER, then the rate of Zn accumulation should also be greater. As this was not the case, it suggests that the S 288 variant may have reduced Zn transporting activity, similar to our previous observations of several other ZnT2 mutants (K 66 N, Q 71 H, D 103 E, and T 312 K) 1 , and also to that observed by Golan and colleagues (G 280 R, E 355 Q and T 312 M) 53 . Further studies are required to define the precise mechanism(s) through which the S 288 substitution confers defects in Zn transporting activity.…”

Evaluation of the roles of the cytosolic N-terminus and His-rich loop of ZNT proteins using ZNT2 and ZNT3 chimeric mutants

“…Another important aspect of this study was the characterization of ZNT2 SNP mutants, as even heterozygous ZNT2 mutations may reduce zinc levels in the breast milk of affected mothers, leading to TNZD in exclusively breastfed infants 24 , 25 , 42 , 52 , 55 – 58 . TNZD-associated zinc deficiency is effectively counteracted by zinc supplements 12 , 13 and accumulating genetic information associated with TNZD pathogenesis assists in supporting normal growth and development of breastfed infants 13 , 58 .…”

“…YiiP forms homodimers with six transmembrane (TM) helices and functions as a proton-zinc exchanger. Most ZNTs form similar homodimers with six TM helices for transporting zinc across biological membranes 21 – 25 and functioning as proton-zinc exchangers 26 , 27 . However, some ZNTs, including ZNT5 and ZNT6, also form heterodimers 22 , 23 , 28 , 29 , and ZNT5 forms 15 TM helices.…”

Demonstrating aspects of multiscale modeling by studying the permeation pathway of the human ZnT2 zinc transporter

“…In the past decade, the human zinc transporter 2 (ZnT2/SLC30A2) was found to be the predominant transporter mediating the translocation of zinc into breast milk in lactating mammary epithelial cells [ 11 ], involved in clinical cases of transient neonatal zinc deficiency (TNZD) [ 11 – 19 ]. Specifically, mothers harboring loss of function mutations in ZnT2 produce breast milk containing very low zinc levels; consequently, their exclusively breastfed infants suffer from severe zinc deficiency.…”