Citrullination of glucokinase is linked to autoimmune diabetes

Inflammation, including reactive oxygen species and inflammatory cytokines in tissues amplify various post-translational modifications of self-proteins. A number of post-translational modifications have been identified as autoimmune biomarkers in the initiation and progression of Type 1 diabetes. Here we show the citrullination of pancreatic glucokinase as a result of inflammation, triggering autoimmunity and affecting glucokinase biological functions. Glucokinase is expressed in hepatocytes to regulate glycogen synthesis, and in pancreatic beta cells as a glucose sensor to initiate glycolysis and insulin signaling. We identify autoantibodies and autoreactive CD4+ T cells to glucokinase epitopes in the circulation of Type 1 diabetes patients and NOD mice. Finally, citrullination alters glucokinase biologic activity and suppresses glucose-stimulated insulin secretion. Our study define glucokinase as a Type 1 diabetes biomarker, providing new insights of how inflammation drives post-translational modifications to create both neoautoantigens and affect beta cell metabolism.


Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used 2a-2c in the separate spreadsheets of "Source Data-File 1" file. The representative MS/MS spectra with annotation of the neutral loss of isocyanic acid for all citrullinated sites identified in PAD-treated rhGK are listed in the separate spreadsheets of "Source Data-File 2" file. N= minimum of 3 biological replicates and between 3 and 6 technical replicates were used to generate data. Sample size was designed on the basis of trial experiments or the results from the first iteration of each experiment and were increased to achieve statistical significance if necessary.
No data was excluded. All attempts at 3 or more replication were successfully reproducible.
Randomization was not applicable in this study. Sex and age matched animals were used for experimental and control groups. We chose patient groups and/or samples with confirmed diagnostic criteria and clinical criteria for type 1 diabetes or from normal healthy individuals. 1) For murine subjects, blinding is not relevant since the genetics were identical for all processed murine subjects in the same experiment. 2) For human subjects, all transportation and experiments were done by investigators blinded to the group allocation.
INS-1 cells were karyotyped and confirmed for phenotype in our laboratory. INS-1E cell lines was karyotyped by the contributor and phenotype confirmed on a regular basis by evaluating glucose induced-insulin secretion.
No wild animals were used.
No field-collected samples were used.
All animal studies were performed in accordance with the guidelines of the Yale University Institutional Animal Care and Use Committee.
The relevant population characteristics are available within the "Methods" and/or its "supplementary information", supplementary Table 4 and 5.
For serum and peripheral blood samples: Subjects for this study are recruited through the BRI Diabetes and Control Registry modules of the Immune Mediated Disease Registry and Repository. New potential participants may contact BRI unsolicited through the BRI Diabetes Research Line (1-800-888-4187), BRI website, or email (diabetes@benaroyaresearch.org). Subjects

March 2021
Ethics oversight Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation Instrument Software Cell population abundance Gating strategy are also recruited via physician referral, through community diabetes events (i.e. camps, ADA/JDRF events and walks, and patient forums), letters and advertisements. They may also be recruited through collaborating institutions with appropriate IRB oversight. We have not identified any specific biases in our population based on this broad recruitment strategy, but any such biases that may exist are unlikely to impact this cross-sectional retrospective study.
For serum and peripheral blood samples: This study was reviewed and approved by the Benaroya Research Institute Institutional Review Board, with annual continuing review.
For human islets: This work is designated as "Not human subjects research" by the Yale Institutional Review Board since it did not involve data obtained through intervention or interaction with the individual and did not contain identifiable private information (per 45 CFR 46.102) For INS-1 cells: Cells were seeded in 6-well plates (1,000,000 cells per condition) cultured with or without recombinant mouse IFN (1000 units/mL; R&D Systems) and recombinant human IL-1 (50 units/mL; R&D Systems) for 48 hours. After trypsinization, cells were fixed by 95% methanol and 4% paraformaldehyde for 30 min on ice. Cells were stained with anti-peptidyl-citrulline, clone F95 (Millipore), and anti-mouse Alexa Fluor 488 (Life Technology) for intracellular citrullination level and/or with antiglucokinase (Proteintech) and anti-rabbit Alexa Fluor 647 (Life Technology) for glucokinase expression in staining buffer (PBS containing 1% BSA and 0.1% Tween-20).

For human T cells:
The sample preparation for each experiment setting are available within the "Methods" and its "supplementary information", supplementary Figure 3, 4 and 5.
For human beta cells: Single-cell suspensions were prepared from pancreatic islets by trypsin dissociation and then stained with FluoZin-3 (Invitrogen) and TMRE (Life Technologies) for cell sorting. For human beta cells, TMRE+ and zinc+ was used for sorting with a FACSAria II and the percentage was around 30-60% of total pancreatic islet cells.
For INS-1 cells: Cell were first gated by FSC/SSC to exclude dead cells and debris and then positive and negative threshold were set using an isotype Ig control with the same fluorophore.
For human T cells: Cells were first gated on FSC-A/SSC-A to determine bulk lymphoctye population. Second, cells were passed to a FSC-W/FSC-H gate to exclude FSC-doublet cells. Third, cells were passed to a SSC-W/SSC-H gate to exclude SSC-doublet cells. Cells were then gated on Viability/CD4. Viability-CD4+ cells were passed on and used to determine individual's memory (CCR7/CD45RA) and chemokine (CXCR3/CCR4/CCR6) expression, which was used later when applied to individual person's tetramer+ cells.