Proceedings of The Physiological Society
University College London (2011) Proc Physiol Soc 24, C03 and PC03
Indentification and characterisation of ZnT10
H. Bosomworth1,2, G. M. Hann1, D. Ford1, R. A. Valentine1,2
1. Epithelial Research Group and Human Nutrition Research Centre, Newcastle University, Newcastle Upon Tyne, United Kingdom. 2. School of Dental Sciences, Newcastle University, Newcastle Upon Tyne, United Kingdom.
Zn is an important element throughout the body, essential for many proteins and enzymes. Homeostasis of Zn is undertaken by Zn transporters of which there are two families; ZIP and ZnT (Lichten and Cousins, 2009). Little is known about the role and expression pattern of ZnT10, however in silco data predict expression to be restricted to foetal tissue (Seve et al., 2004). We have shown by analysis of a human tissue panel by qPCR a restricted expression profile for ZnT10 in adult human tissue. Expression was identified in the cervix, liver, brain, ovary, testes, small intestine and colon at the mRNA level. Bioinformatic analysis of ZnT10 has confirmed ZnT family topology, with 6 conserved transmembrane spanning domains (Seve et al., 2004, TMPred, TMHMM) and a CDF motif predicted to lie between amino acids 34-50 (ScanProsite). In addition, a cleavage site between amino acid position 29 and 30 is predicted (SignalP). Three N-, one O-glycosylation and fifteen possible phosphorylation sites have been predicted (NetNGlyc, NetOGlyc). Using qPCR we have identified a decrease at the mRNA level of ZnT10 in response to extracellular Zn2+ in Caco-2 cells (0.37 ± 0.05 compared with 9.7 x 10-2 ± 0.03 at 150 µM Zn2+ (n = 3), when expressed as a ratio of ZnT10:GAPDH, p<0.01 by Student’s t test). This response for a ZnT family member is not without precedence; ZnT5 also shows down regulation in response to extracellular Zn. A putative Zn responsive element (ZRE) has been identified in the promoter region of ZnT5 (Coneyworth and Ford, Unpublished data). Deletion of this sequence and mutations of specific base pairs in this element eliminated the response to Zn. We dissected the 5’-UTR of ZnT10 for a putative ZRE consensus sequence. A 3 kb upstream region from the ATG was screened using the nucleotide alignment package, Fuzznuc. This identified a putative ZRE 72 bp upstream of the translational ATG start site. To measure activity, a reporter construct comprising the E-Coli β-galactosidase gene downstream of a 942 bp product containing the putative ZRE was generated. In Caco-2 cells transiently expressing this construct, β-galactosidase activity decreased significantly in media containing 150 µM extracellular Zn2+ when compared with untreated cells expressing the same construct, indicating decreased transcription from the putative promoter (Normalised mean β-galactosidase activity (adjusted by control for each experiment) (U/mg) 1.00 ± 0.05 compared with 0.74 ± 0.07 at 150 µM Zn2+ (n = 14), p < 0.006 by Student’s t-test). The ZRE in this construct has been mutated and will be analysed in this system. In summary we have identified that ZnT10 is differentially expressed in human adult tissues is down regulated at the mRNA level in response to Zn, potentially mediated through transcriptional regulation involving a ZRE in the putative promoter region of ZnT10.
Where applicable, experiments conform with Society ethical requirements