Characterization of ZnT8 Monoclonal Antibodies

1. Characterization of ZnT8 Monoclonal Antibodies Christina Miller, Suparna Sarkar, Catherine Lee, Jay Walters, Janet Wenzlau, Julie Wu, Howard Davidson, John HuttonBarbara Davis Center for Diabetes, University of Colorado School of Medicine, Aurora, CO Results Introduction Insulin ZnT8 Phogrin TGN38 Merge 17H2 Type 1 Diabetes (T1D) is a multifactorial disease characterized by an autoimmune attack on pancreatic β cells in the islets of Langerhans (1). Previous studies have identified in humans with T1D autoantibodies reactive to β cell antigens: glutamate decarboxylase (GAD), protein tyrosine phosphatase (IA2), insulin, islet cytoplasm (IC), and most recently zinc transporter 8 (ZnT8) (2).The presence of these autoantibodies serve as a predictor of diabetes and the associated antigens are potential targets for therapy. The ZnT8 autoantibody is detected in 60-80% of individuals with new-onset T1D (3). The function of ZnT8 in pancreatic β cells is to transport zinc into insulin granules in order to stabilize insulin for storage. Insulin is stored as a solid hexamer bound to two Zn+2. Since zinc is also released with insulin upon granule secretion, it is likely that it is used for paracrine/autocrine communication with nearby pancreatic cells (4). Therefore, ZnT8 plays a crucial role in β cell function through zinc homeostasis. However, there is still little known about the autoantigen. Developing and optimizing monoclonal antibodies for ZnT8 could be very useful for purifying the protein in order to study ZnT8’s characteristics. A B Merge ZnT8 Insulin Glucagon Merge ZnT8 Insulin Glucagon 10D7 17H2 17H2 7G4 10D7 10D7 4D2 7G4 7G4 Figure 2. Immunocytochemistry of ZnT8 antibodies and cell compartment markers on INS-1 cells: INS-1 cells were treated with 5.6 mM glucose. Primary antibodies were ZnT8 monoclonal antibodies (17H2, 7G4, 10D7, and 4D2), insulin, phogrin (IA2), and TGN38. Pseudocolors were used for the merge images. Marker = 7µm. 4D2 4D2 Figure 3. Western Blot of ZnT8 DNA constructs transfected in COS-7 cells, INS-1 and MIN-6 cells: Lane 1) human ZnT8 W, 2) human ZnT8 R, 3) mouse ZnT8, 4) rat ZnT8, 5) MIN-6 cells, 6) INS-1 cells and 7) plasmid vector only. The immunoblot was performed with ZnT8 mouse monoclonal antibodies (17H2, 10D7, 7G4, and 4D2). Thick bands in lane 2 and 3 of 10D7 and 7G4 western blots (25 - 37 kDa) represent the ZnT8 protein. kDa C Figure 1 A, B. Immunohistochemistry of ZnT8 antibodies on mouse pancreatic islets: Wild type mouse pancreatic islets (A) and knock out mouse pancreatic islets (B) were stained with primary antibodies: ZnT8 (17H2, 10D7, 7G4, and 4D2), insulin, and glucagon. Marker = 15 µm. Figure 1 C. Co-localization of insulin and ZnT8 in mouse islet cells. Magnified images (40x oil) were taken of wild type mouse islets stained with primary 17H2 mouse antibody for ZnT8, as well as insulin and glucagon. Marker = 7µm. Merge Glucagon ZnT8 Insulin Objectives 17H2 Merge TGN38 Insulin ZnT8 • Overall goal: to further characterize mouse ZnT8 monoclonal antibodies that previously were found to be specific for ZnT8 in both mouse and human pancreatic islet tissue (5). Specific goals of this project were to: • compare the reactivity of the monoclonal antibodies to ZnT8 in wild type mouse and knock out mouse pancreatic tissue • determine if of the antibodies are specific to rat ZnT8 in insulinoma cells • identify any co-localization of ZnT8 with other subcelluar compartment markers • characterize the ZnT8 antibodies’ specificity in western blots 2.2mM Conclusions 5.6mM • Monoclonal antibodies showed a reactivity for ZnT8 in wild type mouse islets. As expect, no fluorescent signal was present in knock out mouse islets, confirming previous research that the antibodies are specific to mouse ZnT8 (5). • 17H2 and 7G4 antibodies had greater fluorescent signal intensity in mouse islets, suggesting that they may be more appropriate for use in further immunohistochemistry. • Monoclonal antibodies may be reactive to rat ZnT8 in INS-1 cell cultures. ZnT8 was ubiquitous within INS-1 cells and co-localization with other cellular markers was unclear. • Only 10D7 and 7G4antibodies were specific for human and mouse ZnT8 in a western blot. However, they were only specific to human ZnT8 DNA constructs (R) and MIN-6 cell ZnT8. • INS-1 cells treated with either 2.2 mM, 5.6 mM, and 20.0 mM glucose showed no difference in ZnT8 or insulin fluorescent signal intensity. 20mM Methods Figure 4. Signal intensity in 2.2 mM, 5.6 mM, and 20.0 mM glucose in INS-1 cells. INS-1 cells incubated in either 2.2 mM, 5.6 mM, or 20.0 mM glucose and stained with primary antibodies: ZnT8 (17H2), insulin, phogrin, and TGN38. Images taken with 3I Marianas inverted spinning disk microscope (University of Colorado School of Medicine, Aurora, CO). Marker = 7µm. Immunohistochemistry: pancreas tissue of mice (both wild type and knock out for SLC8A30)were incubated in 1°ZnT8 mouse monoclonal antibodies (17H2, 10D2, 7G4, and 4D2), insulin (guinea pig), and glucagon (rabbit). Tissues were then incubated in 2°antibodies tagged with fluorescent dyes and imaged at 20x magnification. Cell Cultures: INS-1 cells were starved in no glucose media for 30 minutes and then treated with 2.2 mM glucose, 5.6 mM, or 20.0 mM glucose for 2 hours. Some cell cultures were fixed with paraformaldehyde (4%) for staining, and others were harvested for western blotting. MIN-6 and COS-7 cellswere grown with normal glucose media. Immunocytochemistry: fixed INS-1 cells were incubated in 1°ZnT8 mouse monoclonal antibodies (17H2, 10D2, 7G4, and 4D2) and subcellular marker antibodies: insulin (guinea pig), phogrin (rabbit) and TGN38 (sheep). Cells were then incubated in 2°antibodies tagged with fluorescent dyes and imaged at 60x oil magnification. Transfection: COS-7 cells were transfected with DNA constructs of mouse, rat, and human (R and W) ZnT8. Western Blot: protein from transfected COS-7 cells, INS-1 cells (only 5.6 mM glucose) and MIN-6 cells was loaded into an SDS-page gel electrophoresis. The gel was transferred to a PVDF membrane and immunoblot with the 1°ZnT8 mouse monoclonal antibodies (17H2, 10D7, 7G4, 4D2). The membrane was incubated in a 2°antibody and exposed on x-ray film. Acknowledgements Gillespie K. (2006). Canadian Medical Association Journal. 175(2): 165-170. Wenzlau JM et al. (2008). Immunology of Diabetes V. 1150: 256-259 Wenzlau JM et al. (2007). PNAS. 104(43): 17040-17045 Chimienti et al. (2005) BioMetals. 18:313-317. Gitomer et al. (2007). [poster] Optimizing antibodies to ZnT8 immunohistochemistry. This project was funded by the Department of Health and Human Services, Public Heath Services Grant number: 3 T32 DK 63687- 9 S1.