A dominant negative heterozygous G87R mutation in the zinc transporter, ZnT-2 (SLC30A2), results in transient neonatal zinc deficiency.
Zinc is an essential mineral, and infants are particularly vulnerable to zinc deficiency as they require large amounts of zinc for their normal growth and development. We have recently described the first loss-of-function mutation (H54R) in the zinc transporter ZnT-2 (SLC30A2) in mothers with infants harboring transient neonatal zinc deficiency (TNZD). Here we identified and characterized a novel heterozygous G87R ZnT-2 mutation in two unrelated Ashkenazi Jewish mothers with infants displaying TNZD. Transient transfection of G87R ZnT-2 resulted in endoplasmic reticulum-Golgi retention, whereas the WT transporter properly localized to intracellular secretory vesicles in HC11 and MCF-7 cells. Consequently, G87R ZnT-2 showed decreased stability compared with WT ZnT-2 as revealed by Western blot analysis. Three-dimensional homology modeling based on the crystal structure of YiiP, a close zinc transporter homologue from Escherichia coli, revealed that the basic arginine residue of the mutant G87R points toward the membrane lipid core, suggesting misfolding and possible loss-of-function. Indeed, functional assays including vesicular zinc accumulation, zinc secretion, and cytoplasmic zinc pool assessment revealed markedly impaired zinc transport in G87R ZnT-2 transfectants. Moreover, co-transfection experiments with both mutant and WT transporters revealed a dominant negative effect of G87R ZnT-2 over the WT ZnT-2; this was associated with mislocalization, decreased stability, and loss of zinc transport activity of the WT ZnT-2 due to homodimerization observed upon immunoprecipitation experiments. These findings establish that inactivating ZnT-2 mutations are an underlying basis of TNZD and provide the first evidence for the dominant inheritance of heterozygous ZnT-2 mutations via negative dominance due to homodimer formation.
The Fred Wyszkowski Cancer Research Laboratory, Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel.
Inbal
I
Lasry
Young Ah
YA
Seo
Hadas
H
Ityel
Nechama
N
Shalva
Ben
B
Pode-Shakked
Fabian
F
Glaser
Bluma
B
Berman
Igor
I
Berezovsky
Alexander
A
Goncearenco
Aharon
A
Klar
Jacob
J
Levy
Yair
Y
Anikster
Shannon L
SL
Kelleher
Yehuda G
YG
Assaraf
D019943:Amino Acid Substitution; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D003593:Cytoplasm; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005260:Female; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D007232:Infant, Newborn, Diseases / Q000235:genetics / Q000378:metabolism; D007599:Judaism; D008297:Male; D026901:Membrane Transport Proteins; D008661:Metabolism, Inborn Errors / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D020125:Mutation, Missense; D017510:Protein Folding; D055503:Protein Multimerization / Q000235:genetics*; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D040681:Structural Homology, Protein; D015032:Zinc / Q000172:deficiency*
D027682:Cation Transport Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C486299:SLC30A2 protein, human; C486459:YiiP protein, E coli; D015032:Zinc