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Dieter Rondas,1 Inne Crèvecoeur,1 Wannes D’Hertog,1 Gabriela
Bomfim Ferreira,1 An Staes,2,3
Abhishek D. Garg,4 Decio L. Eizirik,5 Patrizia Agostinis,4 Kris
Gevaert,2,3 Lut Overbergh,1 andChantal Mathieu1
Citrullinated Glucose-Regulated Protein 78 Is anAutoantigen in
Type 1 DiabetesDiabetes 2015;64:573–586 | DOI:
10.2337/db14-0621
Posttranslational modifications of self-proteins play a
sub-stantial role in the initiation or propagation of the
autoim-mune attack in several autoimmune diseases, but
theircontribution to type 1 diabetes is only recently emerging.
Inthe current study, we demonstrate that inflammatory
stress,induced by the cytokines interleukin-1b and
interferon-g,leads to citrullination of GRP78 in b-cells. This is
coupledwith translocation of this endoplasmic reticulum chaperoneto
the b-cell plasma membrane and subsequent secretion.Importantly,
expression and activity of peptidylargininedeiminase 2, one of the
five enzymes responsible for citrul-lination and a candidate gene
for type 1 diabetes in mice, isincreased in islets from
diabetes-prone nonobese diabetic(NOD) mice. Finally, (pre)diabetic
NOD mice have autoanti-bodies and effector T cells that react
against citrullinatedGRP78, indicating that inflammation-induced
citrullinationof GRP78 in b-cells generates a novel autoantigen in
type 1diabetes, opening new avenues for biomarker developmentand
therapeutic intervention.
Type 1 diabetes is an autoimmune endocrine disease inwhich loss
of central and peripheral tolerance toward b-cellantigens is
proposed as the underlying mechanism. How-ever, b-cells themselves
also contribute to trigger and/orpropagate the autoimmune attack,
leading to a dialoguewith immune infiltrating cells that may
amplify local in-flammation (insulitis) in genetically predisposed
individuals(1). Insulin (or proinsulin) is probably the primary
autoan-tigen in type 1 diabetes (2), but antigen spreading occurs
as
the autoimmune assault progresses, with autoantibodiesappearing
against several non–b-cell-specific autoantigens,such as GAD65 (3),
islet antigen 2 (IA2) (4), heat shockprotein 60 (HSP60) (5), and
chromogranin A (ChgA) (6).
During insulitis, local production of inflammatory media-tors,
such as the cytokines interleukin (IL)-1b and interferon-g(IFNg),
triggers b-cell oxidative and endoplasmic reticulum(ER) stress.
These, and other signals, may lead to alternativesplicing and
misfolding of b-cell proteins as well as posttrans-lational
modifications (PTMs) (7–9). In other autoimmunediseases, like
rheumatoid arthritis (RA), multiple sclerosis,and celiac disease,
such posttranslationally modified proteinsbehave as autoantigens
(10,11), but their relevance in type 1diabetes is only starting to
be explored (12–15).
Building on our previous observation that the ERchaperone 78 kDa
glucose-regulated protein (GRP78; alsonamed binding immunoglobulin
protein [BiP]) is post-translationally modified in cytokine-exposed
insulin-producing INS-1E cells (9), we now identify this
modificationas citrullination and show that
inflammation-inducedcitrullinated GRP78 is an autoantigen in type 1
diabetes.These findings suggest a novel role for GRP78 beyond
itswell-known function in the ER, leading to the loss oftolerance
to b-cells in type 1 diabetes.
RESEARCH DESIGN AND METHODS
Western blotting, mass spectrometry, GRP78 cloning,expression,
purification, and in vitro citrullination areavailable in the
Supplementary Data.
1Laboratory for Clinical and Experimental Endocrinology, KU
Leuven, Leuven,Belgium2Department of Medical Protein Research, VIB,
Ghent, Belgium3Department of Biochemistry, Ghent University, Ghent,
Belgium4Laboratory for Cell Death Research and Therapy, KU Leuven,
Leuven, Belgium5Laboratory of Experimental Medicine and Université
Libre de Bruxelles Center forDiabetes Research, Medical Faculty,
Université Libre de Bruxelles, Brussels, Belgium
Corresponding author: Lut Overbergh,
[email protected].
Received 18 April 2014 and accepted 28 August 2014.
This article contains Supplementary Data online at
http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0621/-/DC1.
D.R. and I.C. contributed equally to this study.
L.O. and C.M. contributed equally to this study.
© 2015 by the American Diabetes Association. Readers may use
this article aslong as the work is properly cited, the use is
educational and not for profit, andthe work is not altered.
Diabetes Volume 64, February 2015 573
IMMUNOLOGYAND
TRANSPLANTATIO
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Reagents and AntibodiesPrimary antibodies were as follows: mouse
anti-poly(ADP-ribose) monoclonal antibody (mAb) (Enzo Life
Sciences,Antwerp, Belgium), rabbit anti-GRP78 and
anti-CHOPpolyclonal antibody (pAb) (Santa Cruz Biotechnology,Santa
Cruz, CA), rabbit anti-eIF2a pAb, anti–p-eIF2a(Ser51) pAb,
anti-PERK mAb and anti–p-PERK(Thr980)mAb (Cell Signaling, Beverly,
MA), and mouse anti-actinmAb (Sigma-Aldrich, Diegem, Belgium) for
Western blot-ting; rabbit anti–b-catenin mAb (Cell Signaling
Technol-ogy) and mouse anti-GRP78 pAb (Abcam, Cambridge,U.K.) for
immunocytochemistry. Anti–cit(510)-GRP78was raised in rabbits
against the following peptides: C-aminohexanoic
acid-IDVNGIL[citrulline]VTAEDKG-amideand
acetyl-IDVNGIL[citrulline]VTAEDKG-aminohexanoicacid-C-amide,
through five subsequent injections (21stCentury Biochemicals).
Specificity of the antibody wasconfirmed by dot blot against the
citrullinated peptideand its native counterpart. Secondary
antibodies were asfollows: donkey anti-rabbit horseradish
peroxidase, don-key anti-mouse Alexa Fluor 488, and donkey
anti-rabbitAlexa Fluor 555 (Invitrogen, Merelbeke, Belgium).
Oval-bumin and rabbit peptidylarginine deiminase (PAD) en-zyme were
from Sigma-Aldrich.
Cell Lines and Culture ConditionsRat INS-1E cells, a gift from
Prof. Wollheim (CMU,Geneva, Switzerland), were cultured as
previously de-scribed (9). INS-1E cells were exposed to
recombinantrat IFNg (500 units/mL; R&D Systems),
recombinanthuman IL-1b (10 units/mL; R&D Systems),
thapsigargin(Tg) (15 and 50 nmol/L; Sigma-Aldrich), tunicamycin(Tn)
(2 and 5 mg/mL; Sigma-Aldrich), high glucose(HG) (25 mmol/L;
Sigma-Aldrich), and palmitate (Pa)(0.5 mmol/L; Sigma-Aldrich).
Mouse MIN6 cells, a giftfrom Dr. Miyazaki (Osaka University, Osaka,
Japan),were cultured in DMEM (Invitrogen) containing 15%(volume for
volume) FCS, 100 units/mL penicillin, 100mg/mL streptomycin, and 70
mmol/L b-mercaptoethanol.MIN6 cells were exposed to recombinant
mouse IFNg (500units/mL; R&D Systems) and human recombinant
IL-1b(10 units/mL).
Apoptosis MeasurementsThe percentage of living and apoptotic
cells was assessedas previously described (9).
MiceC57Bl/6 mice were obtained from Harlan Laboratories(Horst,
the Netherlands) and nonobese resistant (NOR)mice from The Jackson
Laboratory (Bar Harbor, ME).Nonobese diabetic (NOD) mice have been
inbred in ouranimal facility since 1989 and are kept under
semi-barrier conditions. For all experiments, a mix of male
andfemale mice was used. All animal manipulations werein compliance
with the principles of laboratory care andapproved by the
Institutional Animal Ethics Committee ofKU Leuven.
Islet Isolation and CulturePancreatic islets were isolated from
3- or 10-week-oldC57Bl/6, NOD, and NOR mice. Islet isolation and
culturewere performed as previously described (16). C57Bl/6islets
were exposed to recombinant mouse IFNg (1,000units/mL) and
recombinant human IL-1b (50 units/mL).
ImmunofluorescenceImmunofluorescence on isolated islets was
performed aspreviously described (17). Fixed INS-1E cells or
sectionedislets were incubated with primary antibody in 1% BSAfor 1
h, followed by four washes in PBS before incubationwith the
secondary antibody in 1% BSA for another hour.Nuclei were detected
with DNA-binding dye DRAQ5TM(Biostatus Ltd., Leicestershire, U.K.).
Specificity was con-firmed by including negative controls with
secondary anti-bodies alone. INS-1E samples were observed under a
ZeissLSM 510 microscope using a Plan-Neofluar 403/1.3 oilDIC lens.
Images were acquired and processed using LSM510 software (Carl
Zeiss AG, Jena, Germany). Mouse isletsections were observed under a
Nikon Eclipse Ti micro-scope using a Plan-Fluor 403/0.75 DIC lens,
and imageswere acquired and processed using Nis-Elements Viewer4.20
software (Nikon Instruments Inc.).
Cell Surface BiotinylationINS-1E or MIN6 cells were incubated
with the indicatedstressors for 12–15 h and then treated as
previously de-scribed (18).
GRP78 ELISATo determine GRP78 concentration in conditioned
mediafrom INS-1E cells, the GRP78 ELISA kit (Enzo LifeSciences,
Antwerp, Belgium) was used, according to themanufacturer’s
protocol.
Two-Dimensional Gel Electrophoresis AnalysisTwo-dimensional gel
electrophoresis (2D-GE) analysis wasperformed as previously
described (9).
Quantitative RT-PCRQuantitative RT-PCR was performed as
previously de-scribed (19).
Measurement of PAD ActivityTo determine PAD activity levels in
islets and pancreatafrom C57Bl/6, NOR, and NOD mice, the
antibody-basedassay for PAD activity (ABAP) (ModiQuest Research,
Oss,the Netherlands) was used, according to the manufac-turer’s
protocol.
Autoantibody ELISA Against Native or CitrullinatedGRP78Serum
autoantibodies against two native and citrullinatedGRP78 peptides
(amino acids 500–519 TFEIDVNGILRVTAEDKGTG and amino acids 295–314
AKRALSSQHQARIEIESFYE) were determined by ELISA as previously
de-scribed (20). For the citrullinated forms, arg510 or arg306were
replaced by citrulline (synthesized by PolyPeptide Lab-oratories,
Strasbourg, France).
574 GRP78 Is an Autoantigen in Type 1 Diabetes Diabetes Volume
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IFNg MeasurementSplenocytes from 10-week-old and new-onset
diabeticNOD mice and age-matched C57Bl/6 and NOR mice werecultured
in flat-bottom 96-well plates (1 3 106 cells/well)in the absence or
presence of the indicated stimuli. IFNglevels were measured in cell
culture supernatant after48 h of culture, using Meso Scale
Discovery technology(Rockville, MD), according to the
manufacturer’s protocol.
StatisticsStatistical analyses of data were performed using
GraphPadPrism 6 (GraphPad Software, San Diego, CA). Data
areexpressed as means6 SEM and were analyzed by a Kruskal-Wallis
test followed by a Dunn multiple comparisons test,unless stated
otherwise in the figure legend. P values ,0.05were considered
significant.
RESULTS
GRP78 Is Translocated to the b-Cell Surface UponCytokine
ExposureWe investigated cytokine-mediated regulation of GRP78at
both transcriptional and translational levels after 12–15 hexposure
and at cytokine concentrations that inducedonly minor apoptosis
(Fig. 1A). No significant increasein GRP78 mRNA (Fig. 1B) and total
protein expression(Fig. 1C, center panel, and Fig. 1D) were
observed ascompared with control INS-1E cells. However, a
detailedanalysis of the plasma membrane fraction revealed a
lim-ited presence of GRP78 at the cell surface under
basalconditions, which was increased upon IL-1b+IFNg expo-sure, but
not upon single cytokine exposure (Fig. 1C, bot-tom panel, and Fig.
1E). This was confirmed in MIN6 cellsafter 12 h treatment (Fig.
1F), a preapoptotic time point(Fig. 1G). In parallel with the
increased plasma membranetranslocation of GRP78, we also observed
an increase inGRP78 secretion upon cytokine exposure of INS-1E
cells(Fig. 1H).
These observations were confirmed by immunocyto-chemistry
showing increased GRP78 staining on theplasma membrane of
nonpermeabilized cytokine-exposedINS-1E cells (Fig. 2A), and a
clear increase in colocaliza-tion between GRP78 and the plasma
membrane markerb-catenin in cytokine-exposed INS-1E cells (Fig.
2B). Im-portantly, a similar membrane translocation was observedin
cytokine-exposed C57Bl/6 mouse islets (Fig. 2C), againat an early
and preapoptotic time point (Fig. 1I). Thus,surface expression of
GRP78 upon cytokine exposure isnot solely taking place in clonal
b-cell models but also inprimary b-cells, and is not a consequence
of nonspecificchanges associated with cell death, increasing the
rele-vance of these findings.
GRP78 Is Translocated to the Plasma Membrane UponChemical ER
Stress, but Not Upon Metabolic StressCytokine exposure of INS-1E
cells and primary rat, mouse,and human b-cells is known to induce
ER stress–dependentapoptosis (21–25). We further evaluated the
contribution
of ER stress to the observed cytokine-induced GRP78 mem-brane
translocation by investigating the effect of the chem-ical ER
stressors Tg and Tn. Cytokines (12–15 h) induceda clear activation
of the PERK-eIF2a-CHOP pathway, at thedose tested (Supplementary
Fig. 1). For Tg, there wasa dose-response effect both in terms of
apoptosis (Fig.3A) and ER stress induction (Fig. 3B and
SupplementaryFig. 1), with minor ER stress at 15 nmol/L but a clear
in-duction of the PERK-eIF2a-CHOP branch at 50 nmol/L Tg,toward
levels similar to those observed upon cytokine ex-posure. Of note,
Xbp1 splicing was induced by Tg at 50nmol/L, which was not the case
upon cytokine exposure(Supplementary Fig. 1B). INS-1E cells were
more resistantto Tn, with less apoptosis (Fig. 3A) and an
intermediateactivation of PERK-eIF2a-CHOP and Xbp1 splicing, both
at2 and 5 mg/mL (Supplementary Fig. 1), as compared withthe higher
doses of Tg and cytokines. As illustrated in Fig.3C–E, 15 nmol/L Tg
and 2 mg/mL Tn did not induce mem-brane translocation of GRP78.
Importantly, the higherdose of 50 nmol/L Tg led to a clear membrane
transloca-tion, which was paralleled by more marked expression ofER
stress markers (see above). A similar effect was ob-served with 5
mg/mL Tn, although less pronounced.
Finally, upon metabolic stress (Pa [0.5 mmol/L] or
thecombination of HG [25 mmol/L] + Pa), most of the ERstress
markers were increased, although the increase inCHOP mRNA and
protein was less marked than thatobserved with chemical ER
stressors or cytokines (Sup-plementary Fig. 1). On the other hand,
the total andmembrane-associated GRP78 protein levels remained
un-altered (Fig. 3F–H). Taken together, these findings indi-cate
that GRP78 membrane translocation occurs uponinflammation- and
severe chemical–induced ER stress,but not upon metabolic
stress.
GRP78 Is Posttranslationally Modified in Cytokine-Exposed
b-CellsBesides the above described membrane translocation ofGRP78,
we observed extensive PTM of GRP78 uponcytokine exposure of INS-1E
cells (Fig. 4A and previouslydescribed [9]) and C57Bl/6 mouse
islets (Fig. 4B). Thiswas also the case, although to a lesser
degree, for IL-1bor IFNg exposure alone (Fig. 4A). Of particular
interest,2D-GE analysis of the plasma membrane fraction of con-trol
and cytokine-exposed INS-1E (Fig. 4C) and MIN6cells (Fig. 4D) not
only demonstrated the presence ofthe three different
cytokine-responsive GRP78 isoforms,but also showed a
cytokine-mediated upregulation of nu-merous acidic GRP78 isoforms
as compared with controlcells. On the other hand, metabolic- and
chemical-induced(both at low and high concentrations) ER stress did
notinduce detectable PTMs of GRP78 (Fig. 4E), confirmingthe
specific effects of proinflammatory cytokines.
GRP78 Is Citrullinated in Cytokine-Exposed INS-1ECellsIn order
to identify the nature and site of cytokine-induced PTMs in GRP78,
we subjected the three different
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GRP78 isoforms observed in INS-1E cells to massspectrometry (MS)
analysis. Sequence coverage rangedfrom 62.54 to 74.46% (n = 2).
When comparing theresulting peptide profiles of isoform 1 (I1)
versus isoform2 (I2) and I1 versus isoform 3 (I3), one specific
peptide,VTAEDKGTGNK (AA511–521), was identified exclusivelyin I1.
This was confirmed by a quantitative differentialanalysis using
trypsin digestion in combination with differ-ential N-butyrylation
and endoproteinase Lys-C (endo Lys-C)
digestion combined with differential N-propionylation(Fig. 5A
and B). As the peptide upstream of VTAEDKGTGNKcould not be
identified using both methods, we hy-pothesized the presence of a
PTM in this region, possi-bly on Arg510, which would prevent
tryptic digestion inI2 and I3, thereby rendering the resulting
peptide(AA493–521) too long for retrieval and detection byMS. Of
note, also a second peptide of interest wasfound, which was
identified almost exclusively in I2 and
Figure 1—Cytokine exposure induces membrane translocation of
GRP78 in insulin-secreting INS-1E and MIN6 cells. A: Apoptosis
levelsin INS-1E cells exposed for 12–15 h (white bars) or 24 h
(black bars) to IL-1b (10 units/mL) and/or IFNg (500 units/mL) (n =
3–8independent experiments, each biological replicate is the mean
of two technical duplicates). B: GRP78 mRNA expression in
INS-1Ecells treated for 12–15 h as described above (n = 7, each
biological replicate is the mean of two technical duplicates). C:
Total andplasma membrane-associated (PM) GRP78 protein expression
in INS-1E cells exposed to the indicated stressors. A
representativeWestern blot from four independent experiments is
shown. D and E: The relative intensities of the different protein
bands were quantifiedby densitometry and expressed as a ratio (n =
4). F: Total and PM GRP78 protein levels in control and
cytokine-exposed MIN6 cells. Arepresentative Western blot from two
independent experiments is shown. G: Apoptosis levels in MIN6 cells
exposed for 12 h (white bars)and 24 h (black bars) to IL-1b and
IFNg (n = 5–8 independent experiments, each biological replicate is
the mean of two technicalduplicates). H: GRP78 protein
concentration in the culture medium of control and cytokine-exposed
INS-1E cells. Data are expressed asmeans 6 SEM and were analyzed by
a two-tailed paired Student t test (n = 10 independent
experiments). I: Apoptosis levels in C57Bl/6mouse islets exposed
for 24 h (white bars) and 72 h (black bars) to IL-1b (50 units/mL)
and IFNg (1,000 units/mL) (n = 3–5 independentexperiments, each
biological replicate is the mean of two technical duplicates). *P
< 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001vs.
respective control (Ctrl).
576 GRP78 Is an Autoantigen in Type 1 Diabetes Diabetes Volume
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Figure 2—Microscopic imaging of cytokine-induced GRP78 membrane
translocation in INS-1E cells and mouse islets of Langerhans.
A:Unpermeabilized control and cytokine-exposed INS-1E cells (15 h)
were stained for GRP78 (green), and the nuclei (blue) were stained
withHoechst 33342. Images shown are representative for two
independent experiments. Bar, 10 mm. Control and cytokine-exposed
INS-1Ecells (15 h) (B) and intact mouse islets (24 h) (C ) were
stained for GRP78 (green) and b-catenin (red). Nuclei (blue) were
stained with Hoechst33342. Images shown are representative for
three independent experiments. Scale bars, 20 mm.
diabetes.diabetesjournals.org Rondas and Associates 577
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I3, whereas hardly in I1 (AA307–324) (Fig. 5A and B).Since this
would point to the presence of a PTM in thebasic but not acidic
forms, we did not further analyze therelevance of this peptide.
Based on these findings, we then investigated thenature of PTM
present in the identified region. We didnot succeed in retrieving
by MS the longer, Arg510-containing peptide (AA493–521) or a spiked
tryptic pep-tide with heavy label (AA493–521) used as internal
control.This is probably caused by insolubility and inability
toanalyze on a C18 column and forced us to use insteadspecific
enzymatic and antibody-based assays. We focusedon three potential
PTMs consistent with an acidic shift in
isoelectric point without change in molecular weight
anddescribed to occur in GRP78 in other cell types, namelyADP
ribosylation, phosphorylation, and citrullination(26–29). Both ADP
ribosylation and citrullination occuron Arg residues, whereas
phosphorylation may occur onThr 518.
We initially investigated ADP ribosylation and phos-phorylation
of GRP78, but the absence of a positive signalin 2D Western blots
of cytokine-exposed INS-1E cells withanti–poly(ADP-ribose) (Fig.
6A) and the absence of GRP78staining by Pro-Q Diamond in control-
and cytokine-exposed INS-1E cells (Fig. 6B), as well as persistence
of themodified isoforms upon treatment with calf intestinal
Figure 3—Chemical ER stress, but not metabolic stress, induces
membrane translocation of GRP78 in insulin-secreting INS-1E cells.
A:Apoptosis levels in INS-1E cells exposed for 12–15 h (white bars)
or 24 h (black bars) to Tg (15 or 50 nmol/L) or Tn (2 or 5 mg/mL)
or HG (25mmol/L), Pa (0.5 mmol/L), or the combination (HG+Pa) (n =
5–9 independent experiments, each biological replicate is the mean
of twotechnical duplicates). B: GRP78 mRNA expression in INS-1E
cells treated for 12–15 h as described above (n = 4–10, each
biologicalreplicate is the mean of two technical duplicates). C and
F: Total and plasma membrane-associated (PM) GRP78 protein
expression in INS-1E cells exposed to the indicated stressors. A
representative Western blot from four independent experiments is
shown. D, E, G, and H: Therelative intensities of the different
protein bands were quantified by densitometry and expressed as a
ratio (n = 4–7). *P < 0.05, **P < 0.01,***P < 0.001, and
****P < 0.0001 vs. respective control (Ctrl).
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Figure 4—Cytokine exposure induces PTM of GRP78 in INS-1E cells
and in intact mouse islets. Representative images from 2D
differencegel electrophoresis analysis (selected region of 24 cm,
pH 4–7, 12.5% SDS-PAGE) of GRP78 in control and cytokine-exposed
INS-1E cells(A) and intact mouse islets (B) with corresponding
three-dimensional view and Graph view of the DeCyder analysis. For
each of the threeisoforms (I1, I2, and I3), the fold increase or
decrease is shown, and statistical analysis was performed using a
two-tailed, unpaired Studentt test (n = 4 independent experiments;
*P < 0.05 and **P < 0.01 vs. control). Representative images
of 2D-GE analysis (selected region of24 cm, pH 4–7, 12.5% SDS-PAGE)
of intracellular and membrane-associated GRP78 in control and
cytokine-exposed INS-1E cells (onerepresentative experiment out of
five independent experiments is shown) (C ) and MIN6 cells (one
representative experiment out of twoindependent experiments is
shown) (D). E: Representative 2D-GE analysis (selected region of 24
cm, pH 4–7, 12.5% SDS-PAGE) withcorresponding three-dimensional
view of GRP78 in total lysates from control and differentially
exposed (as indicated) INS-1E cells (onerepresentative experiment
out of three independent experiments is shown).
diabetes.diabetesjournals.org Rondas and Associates 579
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alkaline phosphatase (Fig. 6C) or l-phosphatase (data notshown),
argued against both modifications.
To verify the implication of citrullination, an antibodythat
specifically recognizes citrullinated GRP78 at Arg510(selection
based on the obtained MS data, see Fig. 5) wasraised in rabbits. 2D
Western blots from control andcytokine-exposed INS-1E cells with
anti–cit510-GRP78clearly indicated that the cytokine-induced acidic
isoformsof GRP78 correspond to citrullinated GRP78 at
residueArg510, whereas no reactivity was observed against themost
basic, nonmodified GRP78 isoform 1 (Fig. 6D).
Padi2 Expression and Activity Are Upregulated inNOD MiceNext, we
investigated the potential role of citrullinationin the
diabetes-prone NOD mouse. Elevated Padi2 mRNAexpression was
observed in islets of 3- and 10-week-oldprediabetic NOD mice as
compared with islets fromage-matched C57Bl/6 and NOR control mice
(Fig. 7Aand B). No differences between the strains were ob-served
for Padi1, Padi3, Padi4, and Padi6 expression,
which were either low or undetectable. Further analysisin other
tissues revealed an overall very low expressionof Padi2 in the
immune-related tissues thymus, lymphnodes, and spleen. Except for
kidney, no elevated levelsof Padi2 were observed in the other
tissues analyzed inNOD as compared with C57Bl/6 mice. In addition,
NORmice showed even lower/undetectable Padi2 expressionin most of
the tissues analyzed (Fig. 7C). Furthermore,elevated Padi2 mRNA
expression in NOD islets corre-sponded to higher PAD activity in
total pancreases(Fig. 7D and E) and islets (Fig. 7F and G) of 3-
and 10-week-old NOD mice, compared with both C57Bl/6 andNOR mice,
indicating a marked increase of PAD activityin islets of NOD mice
immediately before and duringinsulitis. Of note, 3-week-old NOD
mice did not showany sign of inflammation in the islets, as
measured by IL-1b (Fig. 7H) and IFNg (Fig. 7I) mRNA expression. In
10-week-old NOD mice, on the other hand, clear signs ofimmune
infiltration were observed, as evidenced by highexpression of IFNg
and IL-1b mRNA, confirming previ-ous findings from our group
(30).
Figure 5—Quantitative mass spectrometric analysis of the three
GRP78 isoforms I1, I2, and I3 using trypsin digestion combined
withdifferential N-butyrylation and endo Lys-C digestion combined
with differential N-propionylation. For each dataset, the ratios
(light [I1]/heavy [I2 or I3]) were converted to their log2 value,
in order to render a normal distribution. In the volcano plots, the
size of the fold change iscompared with the statistical
significance level. Comparison between the most basic isoform of
GRP78 (I1) and the first more acidic isoform(I2) (A) and between I1
and the second more acidic GRP78 isoform (I3) (B) with a sequence
coverage of 77.67 and 71.70%, respectively(494 and 456 out of 654
amino acids of the GRP78_RAT sequence, respectively, with omission
of the 18–amino acid signal peptide). Theidentified tryptic
peptides are marked in red, the identified endo Lys-C peptides in
bold, and the signal peptides in blue. The two peptideswith the
highest z score and fold change are depicted on the volcano plot
and underlined in the sequence.
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NOD Mice Have Circulating Autoantibodies andAutoreactive T Cells
Against Citrullinated GRP78To evaluate whether citrullinated GRP78
contributes to theautoimmune response in NOD mice, serum samples
fromprediabetic and new-onset diabetic NOD mice and age-matched
C57Bl/6 and NOR mice were analyzed for the pres-ence of
autoantibodies against the native and citrullinatedpeptide
containing the epitope of interest (p500–519).Serum levels of
anti-GRP78 antibodies recognizing thiscitrullinated epitope were
significantly higher in new-onsetdiabetic NOD mice as compared with
age-matched C57Bl/6or NOR mice (Fig. 8A, right panel). Furthermore,
in diabeticNOD mice, significant higher serum antibody levels to
thecitrullinated peptide were found compared with those ofthe
native peptide. No such differences were observed inthe case of
another irrelevant (citrullinated) GRP78 peptide(p295–314) tested
(Supplementary Fig. 2). These findingsprovide evidence that this
specific citrullinated epitope isimportant for autoantibody
generation during type 1 diabetesdevelopment in NOD mice.
Next, to determine if NOD mice have autoreactiveT cells against
native or citrullinated GRP78, freshly isolatedsplenocytes from
prediabetic and new-onset diabetic NODmice and age-matched C57Bl/6
and NOR mice werestimulated with various concentrations of native
and in
vitro citrullinated recombinant mouse GRP78 protein.Secretion of
IFNg was used as a measure of effector T-cellactivation. Whereas
little to no effector T-cell activation wasobserved in the three
different strains when culturingsplenocytes with different
concentrations of nativeGRP78 (Fig. 8B, red line and top right
graph), a clear,dose-responsive increase in IFNg secretion was
observedwhen splenocytes from prediabetic and diabetic NOD micewere
cultured in the presence of citrullinated GRP78 (Fig.8B, blue line
and bottom right graph). C57Bl/6 splenocyteswere unresponsive to
citrullinated GRP78, whereas a minorIFNg response was detected in
NOR splenocytes. Absenceof an IFNg response against both the PAD
enzyme aloneand the control protein ovalbumin, either native or in
vitrocitrullinated (at 0.1, 1, and 5 mg/mL) (data not
shown),suggests that the observed autoreactive T-cell response
isspecifically generated against citrullinated GRP78.
DISCUSSION
The role of posttranslationally modified proteins is
wellestablished in several human autoimmune diseases, andevidence
for similar phenomena in the development oftype 1 diabetes is
accumulating (12–14). Most impor-tantly for this study, McGinty et
al. (15) recently demon-strated the relevance of citrullination in
patients with
Figure 6—GRP78 is citrullinated upon cytokine exposure of INS-1E
cells. A: Representative 2D Western blot (11 cm, pH 4–7,
4–12.5%SDS-PAGE) of cytokine-exposed INS-1E lysate detected with
anti–poly(ADP-ribose) antibody (top panel) followed by anti-GRP78
(bottompanel) (one representative experiment out of three
independent experiments is shown). B: 2D-GE gel of cytokine-exposed
INS-1E cells (24cm, pH 4–7, 12.5% SDS-PAGE) stained using Sypro
Ruby to visualize all proteins (top panel) and Pro-Q Diamond dye to
detect phos-phoproteins (bottom panel) (one representative
experiment out of two independent experiments is shown). C: 2D-GE
analysis of GRP78 incontrol and cytokine-exposed INS-1E cells
(selected region of pH 4–7, 12.5% SDS-PAGE is shown), treated or
not with calf intestinalalkaline phosphatase (CIAP) (one
representative experiment out of two independent experiments is
shown). D: 2D-GE analysis of citrullinatedGRP78 in control and
cytokine-exposed INS-1E cells with anti–cit510-GRP78 (top panels)
and total GRP78 Ab (bottom panels) (onerepresentative experiment
out of seven independent experiments is shown).
diabetes.diabetesjournals.org Rondas and Associates 581
http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0621/-/DC1
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type 1 diabetes, by showing an increased response
tocitrullinated GAD65 peptides. We show that the ER chap-erone
GRP78 is citrullinated specifically upon exposure ofb-cells to
inflammatory stress. This is paralleled by trans-location of GRP78
to the b-cell plasma membrane andeventually its secretion. Under
these circumstances, a spe-cific cross-talk between the b-cell and
the immune systemis initiated, resulting in the generation of
autoantibodiesand induction of T-cell autoreactivity against
citrullinatedGRP78 (Fig. 8C). Importantly, we also observed a
markedupregulation of Padi2 in islets of NOD mice, providinga
strong argument for PADI2 being the diabetes suscep-tibility gene
in the recently identified Idd25 locus onmouse chromosome 4 (31)
and adding to a potentialrole for citrullination in type 1 diabetes
(15).
In previous studies, we have shown that GRP78
isposttranslationally modified in INS-1E cells exposed tocytokines
(9), a PTM that we now identified as citrullina-tion. Cytokines
contribute to b-cell dysfunction and deathat least in part through
inducing ER stress (23–25). How-ever, citrullination of GRP78 is
not induced by chemicalER stressors (Tg or Tn) or by metabolic
stress via expo-sure to HG and/or Pa, suggesting that
cytokine-inducedGRP78 citrullination occurs through a mechanism
inde-pendent of its ER stress–inducing capacities. Preliminarydata
suggest that this citrullination is not mediated by
direct upregulation of the PADI2 enzyme by cytokines(data not
shown) but needs to be the consequence ofincreased PAD activity in
b-cells exposed to cytokines.Since PAD activity is highly Ca2+
dependent, changes in Ca2+
fluxes induced in cytokine-exposed b-cells might play a role.The
present findings, together with the recent report
onposttranslationally modified GAD65 (15), add type 1 diabetesto
the list of autoimmune diseases involving citrullination,i.e., RA,
multiple sclerosis, and systemic erhythematosus (32).This suggests
that citrullination is not a specific disease-related event but
rather an inflammation-dependent pro-cess occurring preferentially
in autoimmune target tissues.The role of citrullination in the
induction of auto-antigenicity has been best described in RA, with
severalcitrullinated autoantigens, including GRP78, already
iden-tified (20,33). These citrullinated peptide epitopes are
bet-ter accommodated in the HLA pocket of HLA-DR4–typeindividuals,
determining the strength of the immune re-sponse to citrullinated
peptides and providing a molecularbasis for the genetic
predisposition of HLA-DR4 individualsto RA (34). This mechanism has
recently also been de-scribed in type 1 diabetes (15), where
HLA-DR4 is an im-portant risk allele for the disease (35).
In addition to citrullination, cytokine-induced trans-location
of GRP78 to the b-cell plasma membrane may bea crucial step for
GRP78 to become an autoantigen. This
Figure 7—NOD islets have high Padi2 mRNA expression and PAD
activity. A and B: Padi1, 2, 3, 4, and 6 mRNA expression in islets
ofLangerhans of respectively 3- and 10-week-old C57Bl/6 (black
bars), NOR (gray bars), and NOD (white bars) mice (n = 5–8). C:
Padi2mRNAlevels in different tissues of 3-week-old C57Bl/6 (black
bars), NOR (gray bars), and NOD (white bars) mice (n = 5–10, each
sample consistsof tissue isolated from a single mouse). D and E:
Pancreatic PAD activity in respectively 3- and 10-week-old C57Bl/6
(black bars), NOR(gray bars), and NOD (white bars) mice. Data are
expressed as means 6 SEM and were analyzed by a one-way ANOVA
followed bya Bonferroni multiple comparisons test (n = 4–9). F and
G: Islet PAD activity in respectively 3- and 10-week-old C57Bl/6
(black bars), NOR(gray bars), and NOD (white bars) mice (n = 4).
All replicates refer to biological replicates with samples (islets
or pancreas) obtained fromindividual mice. The PAD activity
experiments were performed at least three times. ND, not
detectable. IL-1b (H) and IFNg (I) mRNA levels in3- and 10-week-old
C57Bl/6 (black bars), NOR (gray bars), and NOD (white bars) mice (n
= 5–8, each sample consists of islets isolated froma single mouse).
*P < 0.05; **P < 0.01; ***P < 0.001; ****P <
0.0001.
582 GRP78 Is an Autoantigen in Type 1 Diabetes Diabetes Volume
64, February 2015
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Figure 8—NOD mice have circulating autoantibodies and
autoreactive T cells against citrullinated GRP78. A: Comparison
between theserum levels of antinative and anticitrullinated peptide
(AA500–519) antibodies in prediabetic and diabetic NOD (NOD DM)
mice and age-matched C57Bl/6 and NOR mice grouped according to age.
Each dot indicates the value of a single mouse. Four independent
experimentswere performed, each containing samples of all
experimental groups. B: IFNg response of splenocytes from
prediabetic (n = 9, light graybars) and diabetic NOD (n = 11, white
bars) mice and age-matched C57Bl/6 (n = 8, black bars) and NOR (n =
11, dark gray bars) micestimulated with various concentrations of
native (red) and citrullinated (blue) recombinant mouse GRP78. Four
independent experimentswere performed, with two to three animals
per group per experiment. *P < 0.05; **P < 0.01; ***P <
0.001; ****P < 0.0001. C: Proposedmodel for the role of b-cell
citrullinated GRP78 in autoantibody generation and T-cell
activation. Exposure of b-cells to cytokines inducescitrullination,
membrane translocation, and secretion of GRP78. Citrullinated
membrane–associated or secreted GRP78 is taken up andprocessed by B
cells and antigen-presenting cells (APCs), resulting in the
generation of specific anticitrullinated GRP78 autoantibodies
andrelease of IFNg by activated effector T cells, respectively.
diabetes.diabetesjournals.org Rondas and Associates 583
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translocation is shown to be an early event in response
toinflammation, suggesting that it is an active process atleast in
part independent from “protein leakage” by apo-ptotic b-cells.
GRP78 membrane translocation, but notcitrullination, was found to
be ER stress dependent andparalleled to the increased CHOP
expression. Membrane-associated GRP78 has been described in
different tumor celltypes (36–38) as well as in exocrine pancreatic
cells (39) andproliferating endothelial and monocytic cells
(40,41). Inthese models, membrane-associated GRP78 acts, amongother
functions, as a cell surface signaling receptor for dif-ferent
ligands such as activated a2-macroglobulin (42,43)and coxsackie A9
virus (44,45) and is found associatedwith the major
histocompatibility complex class I (MHC-I)(44). Of interest,
changes in the topography of membrane-associated GRP78, caused by
PTMs, may convert GRP78into a receptor with autoantigenic
properties. This hasbeen observed in cancer cells where GRP78 is
modified byO-linked glycosylation (38), leading to the generation
ofGRP78 autoantibodies. The present observations that in-flammation
induces extensive GRP78 citrullination, trans-location to the
plasma membrane, and secretion underscoreits putative function as
an autoantigen in type 1 diabetes.Furthermore, based on the
proposed transmembranemodel for GRP78 (46), the reactive p500–519
epitope,against which GRP78 autoantibodies are generated inNOD
mice, is located in the extracellular domain, thusbeing exposed to
infiltrating immune cells.
The possible role for citrullinated GRP78 as an auto-antigen in
type 1 diabetes is supported by the presentobservations showing the
generation of autoantibodies aswell as CD4+ T-cell autoreactivity
against citrullinatedGRP78 in NOD mice. No T-cell reactivity is
observed inC57Bl/6 mice, whereas NOR mice show a minor T-cell
re-sponse. This supports the idea that inflammation is neces-sary
to initiate this process, as low levels of IFNg and IL-1bare
detected in islets of 10-week-old NOR mice, a phenom-enon referred
to as protracted, noninvasive insulitis (47). Amarked upregulation
of PAD2, a key enzyme for proteincitrullination, is observed
exclusively in islets from NODmice, as compared with NOR and
C57Bl/6 islets. PAD ac-tivity is further increased in NOD islets
with increasing ageand insulitis, suggesting that inflammation
plays a role forthis phenomenon, perhaps by increasing cytosolic
Ca2+
concentrations due to cytokine-mediated calcium depletionfrom
the ER (24). Interestingly, expression of Padi2 wasvery low in NOD
thymus and not different from C57Bl/6 andNOR thymus. This may
explain the escape of citrullinatedGRP78 from thymic tolerization
in developing thymo-cytes, thereby clarifying why citrullinated
GRP78 can berecognized as a b-cell–specific autoantigen whereas
nativeGRP78 is ubiquitously expressed. A similar mechanismhas been
proposed for chromogranin A, where exposureof a naturally occurring
cleavage product (peptide WE14)to transglutaminase, expressed in
b-cells, forms high- andlow-molecular weight aggregates, thus
rendering the pep-tide highly antigenic in NOD mice (13).
Whether these observed processes are also applicableto other ER
chaperones or heat shock proteins, such asHSP60, requires further
investigation. It will be of utmostimportance to determine whether
similar processesare involved in human type 1 diabetes. The
knowledgethat inflammation-mediated b-cell stress is taking placein
human type 1 diabetes (48) and the high overlapbetween autoantigens
identified to date in NOD miceand type 1 diabetes patients (49) are
in support of thishypothesis.
In conclusion, local inflammation in the islets
inducescitrullination of GRP78 in the stressed b-cells,
turningcitrullinated GRP78 into an autoantigen. This modifiedGRP78
is recognized by both B and T cells, thus propa-gating and
amplifying the ongoing autoimmune attackagainst the b-cells. Our
findings support and providemechanistic evidence for the concept
that inflammation-induced b-cell stress initiates a specific
communicationbetween the b-cell and the immune system, which
willaggravate and accelerate the development of type 1 dia-betes. A
genetic predisposition for increased citrullinationin the islets,
as shown here for NOD mice, is expected tofurther exacerbate this
process. This proposed mechanism(Fig. 8C), implicating
tissue-specific and inflammation-induced protein modification, cell
surface translocation,and secretion, would also explain why
different tissue-specific autoimmune diseases can have similar
autoantigens.
Acknowledgments. The technical assistance of Frea Coun,
MartineGilis, Jos Laureys, Willem Van Den Berghe, Wim Werckx, and
Farah-DeborahLok (Laboratory of Clinical and Experimental
Endocrinology, KU Leuven) is greatlyappreciated. The authors thank
Katleen Lemaire (Gene Expression Group, KULeuven) and Monique
Beullens (Laboratory for Biosignaling and Therapeutics, KULeuven)
for advice on GRP78 cloning in pET vector. The authors thank the
CellImaging Core (KU Leuven) for providing technical assistance
with confocalmicroscopy.Funding. This work was supported by
Juvenile Diabetes Research FoundationInternational (17-2012-129 and
17-2013-515), the European Community’s Sev-enth Framework Programme
NAIMIT (Natural Immunomodulators as NovelImmunotherapies for Type 1
Diabetes) under grant agreement 241447, theKU Leuven
(Geconcerteerde Onderzoeksactie GOA 12/24 and an F+ fellowshipfor
D.R.), and the Flemish Research Foundation (G.0619.12, a
postdoctoral fel-lowship for G.B.F. and W.D. and a clinical
research fellowship for C.M.).Duality of Interest. No potential
conflicts of interest relevant to this articleare to be
reported.Author Contributions. D.R. and I.C. designed and performed
research,analyzed data, and wrote the manuscript. W.D., G.B.F.,
A.S., and A.D.G. per-formed research. D.L.E. analyzed data and
wrote and edited the manuscript. P.A.and K.G. edited the
manuscript. L.O. and C.M. designed research and wrote andedited the
manuscript. L.O. and C.M. are the guarantors of this work and,
assuch, had full access to all the data in the study and take
responsibility for theintegrity of the data and the accuracy of the
data analysis.
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