Structure/Function Analysis of human ZnT8 (SLC30A8): A Diabetes Risk Factor and Zinc Transporter

Mark J Daniels; Maciej Jagielnicki; Mark Yeager

doi:10.1016/j.crstbi.2020.06.001

Structure/Function Analysis of human ZnT8 (SLC30A8): A Diabetes Risk Factor and Zinc Transporter

Curr Res Struct Biol. 2020 Jun 27:2:144-155. doi: 10.1016/j.crstbi.2020.06.001. eCollection 2020.

Authors

Mark J Daniels¹, Maciej Jagielnicki^{1

2}, Mark Yeager^{1

3

4}

Affiliations

¹ Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA, 22908, USA.
² Department of Biochemistry, University of Toronto, Toronto, ON, M5G 1M1, Canada.
³ Department of Medicine, Division of Cardiovascular Medicine, University of Virginia Health System, Charlottesville, VA, 22908, USA.
⁴ Center for Membrane and Cell Physiology, University of Virginia School of Medicine, Charlottesville, VA, 22908, USA.

Abstract

The human zinc transporter ZnT8 (SLC30A8) is expressed primarily in pancreatic β-cells and plays a key function in maintaining the concentration of blood glucose through its role in insulin storage, maturation and secretion. ZnT8 is an autoantigen for Type 1 diabetes (T1D) and is associated with Type 2 diabetes (T2D) through its risk allele that encodes a major non-synonymous single nucleotide polymorphism (SNP) at Arg325. Loss of function mutations improve insulin secretion and are protective against diabetes. Despite its role in diabetes and concomitant potential as a drug target, little is known about the structure or mechanism of ZnT8. To this end, we expressed ZnT8 in Pichia pastoris yeast and Sf9 insect cells. Guided by a rational screen of 96 detergents, we developed a method to solubilize and purify recombinant ZnT8. An in vivo transport assay in Pichia and a liposome-based uptake assay for insect-cell derived ZnT8 showed that the protein is functionally active in both systems. No significant difference in activity was observed between full-length ZnT8 (ZnT8A) and the amino-terminally truncated ZnT8B isoform. A fluorescence-based in vitro transport assay using proteoliposomes indicated that human ZnT8 functions as a Zn²⁺/H⁺ antiporter. We also purified E. coli-expressed amino- and carboxy-terminal cytoplasmic domains of ZnT8A. Circular dichroism spectrometry suggested that the amino-terminal domain contains predominantly α-helical structure, and indicated that the carboxy-terminal domain has a mixed α/β structure. Negative-stain electron microscopy and single-particle image analysis yielded a density map of ZnT8B at 20 Å resolution, which revealed that ZnT8 forms a dimer in detergent micelles. Two prominent lobes are ascribed to the transmembrane domains, and the molecular envelope recapitulates that of the bacterial zinc transporter YiiP. These results provide a foundation for higher resolution structural studies and screening experiments to identify compounds that modulate ZnT8 activity.

Keywords: Circular dichroism spectrometry; Diabetes mellitus; Electron microscopy; Membrane protein expression; Zinc transporter.

Abstract

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