We describe the preparation, evaluation, and application of an S100A12 protein-conjugated solid support, hereafter the “A12-resin,” that can remove 99% of Zn(II) from complex biological solutions without significantly perturbing the concentrations of other metal ions. The A12-resin can be applied to selectively deplete Zn(II) from diverse tissue culture media and from other biological fluids, including human serum. To further demonstrate the utility of this approach, we investigated metabolic, transcriptomic, and metallomic responses of HEK293 cells cultured in medium depleted of Zn(II) using S100A12. The resulting data provide insight into how cells respond to acute Zn(II) deficiency. We expect that the A12-resin will facilitate interrogation of disrupted Zn(II) homeostasis in biological settings, uncovering novel roles for Zn(II) in biology.
Sensitive measurements of cellular Zn(II) uptake currently rely on quantitating radioactive emissions from cells treated with 65Zn(II). Here, we describe a straightforward and reliable method employing a stable isotope to sensitively monitor Zn(II) uptake by metazoan cells. First, biological media selectively depleted of natural abundance Zn(II) using A12-resin [Richardson, C. E. R. et al. J. Am. Chem. Soc. 2018, 140, 2413] is restored to physiological levels of Zn(II) by addition of a non-natural Zn(II) isotope distribution comprising 70% 70Zn(II). The resulting 70Zn(II)-enriched media facilitates quantitation of Zn(II) uptake using inductively coupled plasma-mass spectrometry (ICP-MS). This sensitive and reliable assay assesses Zn(II)-uptake kinetics at early timepoints and can be used to delineate how chemical and genetic perturbations influence Zn(II) uptake. Further, the use of ICP-MS in a Zn(II)-uptake assay permits simultaneous measurement of multiple metal ion concentrations. We used this capability to show that, across three cell lines, Zn(II) deficiency enhances selectivity for Zn(II) over Cd(II) uptake.
Zinc deficiency is correlated with many pathologies, but the molecular connection between disease states and insufficient zinc is still poorly understood. To investigate the connection between zinc deficiency and organismal phenotypes, we developed a technique to specifically diminish zinc levels in mammalian cell culture media without perturbing the concentrations of other metal ions. Growth of cells in such depleted medium evokes some of the transcriptional responses previously noted in organismal models of zinc deficiency, which can be restored upon addition of zinc to the original levels. RNASeq and miRNA data similarly support a zinc‐specific response to the depletion. We also report how the zinc transcriptome remodels in the context of zinc limiting growth. This robust and transportable methodology offers investigators a unique tool for studying zinc deficiency in the context of mammalian cell culture.Support or Funding InformationThe work was supported by the US National Institutes of Health and the Mallinckrodt Faculty Scholar Award.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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