Characterization of ZnT8 Monoclonal Antibodies
Christina Miller, Suparna Sarkar, Catherine Lee, Jay Walters, Janet Wenzlau, Julie Wu, Howard Davidson, John Hutton
Barbara Davis Center for Diabetes, University of Colorado School of Medicine, Aurora, CO
Results
Introduction
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 newonset 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.
Objectives
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:
•
A
ZnT8
Insulin
Glucagon
Merge
B
ZnT8
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
Glucagon
ZnT8
Merge
17H2
17H2
17H2
10D7
10D7
10D7
7G4
7G4
7G4
4D2
4D2
C
Insulin
Phogrin
TGN38
Merge
4D2
ZnT8
Insulin
Glucagon
Merge
17H2
Insulin
Methods
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.
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
compare the reactivity of the monoclonal antibodies to ZnT8 in wild
type mouse and knock out mouse pancreatic tissue
•
Insulin
TGN38
ZnT8
Merge
2.2mM
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.
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.
Conclusions
 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 7G4 antibodies 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.
5.6mM
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 cells were 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
20mM
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.
1.
2.
3.
4.
5.
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.