A Method for Selective Depletion of Zn(II) Ions from Complex Biological Media and Evaluation of Cellular Consequences of Zn(II) Deficiency

Abstract

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.

Figure 1

(a/b) Chelex^® resin nonspecifically depletes cations from media (n=4, ±SEM). (c) TPEN treatment of alkaline phosphatase secreted from transfected HEK293T cells diminishes the activity of the enzyme (n=3, ±SEM)

Figure 2

(a) ICP-MS analysis indicating that Freestyle™ medium is depleted of Zn(II) by direct addition of S100A12 followed by removal of Zn(II)-bound S100A12 with a 10-kDa molecular weight cutoff filter (n=3, ±SEM). (b) S100A12 or S100A12 ΔHis₃Asp can be conjugated to agarose to afford two distinct resins. (c) A12-resin specifically depletes Zn(II) from DMEM/FBS, whereas the S100A12Δ His₃Asp (control) resin does not affect the metal ion concentrations of the medium (n=3, ±SEM). (d) A12-resin depletes Zn(II) from an array of diverse cell culture medium formulations (n=4, ±SEM). (e) A12-resin selectively depletes human serum of zinc. (f) Regeneration of the A12-resin in 1.0 M acetic acid (pH=3.8) enables multiple cycles of resin reuse (n=4, ±SEM).

Figure 3

(a) HEK293 Freestyle cells cultured in a Zn(II)-depleted medium exhibit a loss of metabolic activity, as assessed by CellTiter-Glo^® assay over 4 d of culture (n=3, ±SEM). (b) HEK293T cells cultured in a Zn(II)-depleted DMEM/FBS exhibit a loss of metabolic activity, as assessed by a resazurin assay over 4 d of culture (n=3, ± SEM). (c) Heat map from RNA Seq of HEK-293 Freestyle cells cultured in untreated, depleted, and repleted medium reveals the specificity of S100A12 medium depletion for Zn(II). (d) Total cellular Zn(II) content of HEK293T cultured in Zn(II)-depleted medium is diminished relative to cells cultured in Zn(II)-adequate medium (n≥6, ±SEM). (e) Culture of HEK293T cells in Zn(II) depleted medium does not affect intracellular free Zn(II) concentration (n=6, ±SEM).