The PP-motif in luminal loop 2 of ZnT transporters plays a pivotal role in TNAP activation.

Secretory and membrane-bound zinc-requiring enzymes are thought to be activated by binding zinc in the early secretory pathway. One such enzyme, tissue-non-specific alkaline phosphatase (TNAP), is activated through a two-step mechanism, via protein stabilization and subsequent enzyme activation through metalation, by ZnT5-ZnT6 heterodimers or ZnT7 homodimers. However, little is known about the molecular basis underlying the activation process. In the present study, we found that the di-proline motif (PP-motif) in luminal loop 2 of ZnT5 and ZnT7 is important for TNAP activation. TNAP activity was significantly reduced in cells lacking ZnT5-ZnT6 heterodimers and ZnT7 homodimers [triple knockout (TKO) cells]. The decreased TNAP activity was restored by expressing hZnT5 with hZnT6 or hZnT7, but significantly less so (almost 90% less) by expressing mutants thereof in which the PP-motif was mutated to alanine (PP-AA). In TKO cells, overexpressed hTNAP was not completely activated, and it was converted less efficiently into the holo form by expressing a PP-AA mutant of hZnT5 with hZnT6, whose defects were not restored by zinc supplementation. The zinc transport activity of hZnT7 was not significantly impaired by the PP-AA mutation, indicating that the PP-motif is involved in the TNAP maturation process, although it does not control zinc transport activity. The PP-motif is highly conserved in ZnT5 and ZnT7 orthologues, and its importance for TNAP activation is conserved in the Caenorhabditis elegans hZnT5 orthologue CDF5. These results provide novel molecular insights into the TNAP activation process in the early secretory pathway.

Novel mutations in SLC30A2 involved in the pathogenesis of transient neonatal zinc deficiency.

BACKGROUND: Infants are vulnerable to zinc deficiency. Thus, abnormally low breast milk zinc levels cause transient neonatal zinc deficiency (TNZD) in breast-fed infants. TNZD has been considered to be rare because of a paucity of citations in the published literature. However, recent studies of affected mothers identified four missense mutations in the solute carrier family 30 member 2 gene (SLC30A2), which encodes the zinc transporter, ZnT2. METHODS: Genetic analyses of SLC30A2/ZnT2 in three Japanese mothers secreting low-zinc milk (whose infants developed TNZD) were performed. The effects of identified mutations were examined in a cell-based assay. Furthermore, 31 single-nucleotide polymorphisms (SNPs) in SLC30A2/ZnT2 were evaluated for their potential involvement in low-zinc levels in milk. RESULTS: Each mother had a different novel heterozygous mutation in SLC30A2/ZnT2. One mutation reduced splicing efficiency of the SLC30A2/ZnT2 transcript, and all ZnT2 mutants were defective in zinc transport and were unstable in cells. Moreover, four SNPs caused a significant loss of zinc-transport activity, similar to that in disease-causing ZnT2 mutants. CONCLUSION: Our results indicate that many SLC30A2/ZnT2 mutations cause or potentially cause TNZD. Genetic information concerning TNZD pathogenesis is limited, and our results suggest that the TNZD frequency may be higher than previously thought.

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

The physiological roles of Zn transporter (ZNT) proteins are being increasingly recognized, and three dimensional structures of ZNT bacterial homologs have facilitated our understanding of their biochemical characteristics at the molecular level. However, the biological role of the unique structural features of vertebrate ZNTs, which are absent in their bacterial homologues, is not completely understood. These ZNT sequences include a cytosolic His-rich loop between transmembrane helices IV and V and the cytosolic N-terminus. This study investigated the contribution of these features to zinc transport by ZNT proteins. The importance of the His residues in the cytosolic His-rich loop was investigated using ZNT2 Ala substitution and deletion mutants. The presence of His residues was not essential for zinc transport, even though they possibly participate in modulation of zinc transport activity. Furthermore, we determined the role of the N-terminus by characterizing ZNT2 and ZNT3 domain-swapped and deletion mutants. Unexpectedly, the N-terminus was also not essential for zinc transport by ZNT2 and the domain-swapped ZNT2 mutant, in which the cytosolic His-rich loop was substituted with that of ZNT3. These results provide molecular insights into understanding the roles of the cytosolic parts of ZNT2, ZNT3, and probably other members of their subgroup.

Physiological roles of zinc transporters: molecular and genetic importance in zinc homeostasis.

Zinc (Zn) is an essential trace mineral that regulates the expression and activation of biological molecules such as transcription factors, enzymes, adapters, channels, and growth factors, along with their receptors. Zn deficiency or excessive Zn absorption disrupts Zn homeostasis and affects growth, morphogenesis, and immune response, as well as neurosensory and endocrine functions. Zn levels must be adjusted properly to maintain the cellular processes and biological responses necessary for life. Zn transporters regulate Zn levels by controlling Zn influx and efflux between extracellular and intracellular compartments, thus, modulating the Zn concentration and distribution. Although the physiological functions of the Zn transporters remain to be clarified, there is growing evidence that Zn transporters are related to human diseases, and that Zn transporter-mediated Zn ion acts as a signaling factor, called "Zinc signal". Here we describe critical roles of Zn transporters in the body and their contribution at the molecular, biochemical, and genetic levels, and review recently reported disease-related mutations in the Zn transporter genes.

Overview of Inherited Zinc Deficiency in Infants and Children.

Zinc nutrition is of special practical importance in infants and children. Poor zinc absorption causes zinc deficiency, which leads to a broad range of consequences such as alopecia, diarrhea, skin lesions, taste disorders, loss of appetite, impaired immune function and neuropsychiatric changes and growth retardation, thus potentially threatening life in infants and children. In addition to dietary zinc deficiency, inherited zinc deficiency, which rarely occurs, is found during the infant stage and early childhood. Recent molecular genetic studies have identified responsible genes for two inherited zinc deficiency disorders, acrodermatitis enteropathica (AE) and transient neonatal zinc deficiency (TNZD), clarifying the pathological mechanisms. Both of these zinc deficiencies are caused by mutations of zinc transporters, although the mechanisms are completely different. AE is an autosomal recessive disorder caused by mutations of the ZIP4 gene, consequently resulting in defective absorption of zinc in the small intestine. In contrast, TNZD is a disorder caused by mutations of the ZnT2 gene, which results in low zinc breast milk in the mother, consequently causing zinc deficiency in the breast-fed infant. In both cases, zinc deficiency symptoms are ameliorated by a daily oral zinc supplementation for the patients. Zinc is definitely one of the key factors for the healthy growth of infants and children, and thus zinc nutrition should receive much attention.

Molecular pathogenesis of spondylocheirodysplastic Ehlers-Danlos syndrome caused by mutant ZIP13 proteins.

The zinc transporter protein ZIP13 plays critical roles in bone, tooth, and connective tissue development, and its dysfunction is responsible for the spondylocheirodysplastic form of Ehlers-Danlos syndrome (SCD-EDS, OMIM 612350). Here, we report the molecular pathogenic mechanism of SCD-EDS caused by two different mutant ZIP13 proteins found in human patients: ZIP13(G64D), in which Gly at amino acid position 64 is replaced by Asp, and ZIP13(ΔFLA), which contains a deletion of Phe-Leu-Ala. We demonstrated that both the ZIP13(G64D) and ZIP13(ΔFLA) protein levels are decreased by degradation via the valosin-containing protein (VCP)-linked ubiquitin proteasome pathway. The inhibition of degradation pathways rescued the protein expression levels, resulting in improved intracellular Zn homeostasis. Our findings uncover the pathogenic mechanisms elicited by mutant ZIP13 proteins. Further elucidation of these degradation processes may lead to novel therapeutic targets for SCD-EDS.