Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 293, Issue 42, Pages 16206-16216Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA118.005136
Keywords
zinc; transport; monoclonal antibody; inhibitor; pancreatic islet; diabetes; drug discovery; ZnT8; mAb; drug screening; pancreatic beta-cells; HTRF; therapeutic target
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Funding
- National Institutes of Health Shared Instrumentation Grant [S10OD016374]
- National Institutes of Health [S10 OD016315, S10 RR13777001]
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Solute carrier family 30 member 8 (SLC30A8), encoding the pancreatic zinc transporter ZnT8, is a susceptibility gene for type 2 diabetes (T2D). Reducing ZnT8 transport activity or down-regulating its cellular expression is hypothesized to be an antidiabetogenic strategy mimicking the protective effect of SLC30A8 haploinsufficiency in humans. However, research tools to inhibit ZnT8 activity and measure cellular ZnT8 levels are not available. Here, we report the identification of two anti-ZnT8 mAbs applicable to addressing these unmet needs. Both mAbs exhibited subnanomolar affinities for human ZnT8 and were selective against homologous zinc transporters with distinct cross-species reactivities and epitope recognition. We showed that antigen-binding fragments (Fabs) protected ZnT8 from unfolding and inhibited ZnT8-mediated zinc transport in proteoliposomes. Negative-stain EM revealed a ternary binding complex of a ZnT8 monomer and two different Fabs at a 1:1:1 stoichiometry. Moreover, dual bindings of two different mAbs to a single ZnT8 protein multiplied the individual anti-ZnT8 specificities, enabling quantification of cellular ZnT8 levels by homogeneous time-resolved fluorescence (HTRF). Our results demonstrate the utilities of the two generated mAbs as allosteric inhibitors and highly specific biosensors of human ZnT8.
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