title: Molecular Basis of Transient Neonatal Zinc Deficiency: NOVEL ZnT2 MUTATIONS DISRUPTING ZINC BINDING AND PERMEATION.
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.
authors.affiliation: From the Department of Biology, Fred Wyszkowski Cancer Research Laboratory and.
authors.forename: Yarden
authors.initials: Y
authors.lastname: Golan
authors.affiliation: the Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Naoya
authors.initials: N
authors.lastname: Itsumura
authors.affiliation: the Bioinformatics Knowledge Unit, The Lorry I. Lokey Interdisciplinary Center for Life Sciences and Engineering, Technion, Haifa 32000, Israel and.
authors.forename: Fabian
authors.initials: F
authors.lastname: Glaser
authors.affiliation: From the Department of Biology, Fred Wyszkowski Cancer Research Laboratory and.
authors.forename: Bluma
authors.initials: B
authors.lastname: Berman
authors.affiliation: the Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan kambe1@kais.kyoto-u.ac.jp.
authors.forename: Taiho
authors.initials: T
authors.lastname: Kambe
authors.affiliation: From the Department of Biology, Fred Wyszkowski Cancer Research Laboratory and assaraf@tx.technion.ac.il.
authors.forename: Yehuda G
authors.initials: YG
authors.lastname: Assaraf
mesh_terms: D019943:Amino Acid Substitution; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000235:genetics*; D005260:Female; D057895:Haploinsufficiency; D006801:Humans; D007231:Infant, Newborn; D061986:MCF-7 Cells; D009154:Mutation; D020125:Mutation, Missense; D011485:Protein Binding; D015032:Zinc / Q000172:deficiency* / Q000378:metabolism
chemical_list: D027682:Cation Transport Proteins; C486299:SLC30A2 protein, human; D015032:Zinc
keywords: bimolecular fluorescence complementation; dimerization; metal homeostasis; mutant; neonatal zinc deficiency; transport; transport metals; zinc; zinc transport