The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 2 643-648
Copyright © 1999 by The Endocrine Society
Species-Specific Autoantibodies in Type 1 Diabetes1
C. S. Hampe,
E. Örtqvist,
O. Rolandsson,
M. Landin-Olsson,
C. Törn,
Å. Ågren,
B. Persson,
D. B. Schranz2 and
Å. Lernmark
Department of Medicine, University of Washington (C.S.H., D.B.S.,
A.L.), Seattle, Washington 98195; and the Department of Medicine,
University Hospital (M.L.-O., C.T.), Lund; the Department of Family
Medicine, Umea University (O.R., A.A.), Umea; and the Department of
Woman and Child Health, Karolinska Institute (E.O., B.P.), Stockholm,
Sweden
Address all correspondence and requests for reprints to: Dr. Christiane S. Hampe, Department of Medicine, Box 357710, University of Washington, Seattle, Washington 98195. E-mail: champe{at}u
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Abstract
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GAD65 autoantibodies (GAD65Ab) are important markers for type 1
(insulin-dependent) diabetes mellitus. Although most patients have
GAD65Ab at the time of clinical diagnosis, there are also
GAD65Ab-positive individuals in the population at low risk of
developing type 1 diabetes. The aim of this study was to test the
hypothesis that the GAD65Ab reactivity to GAD65 cloned from human,
mouse, and rat in newly diagnosed type 1 diabetic patients differ from
antibody-positive healthy individuals. Sera from 254 new-onset 0- to
34-yr-old type 1 diabetic patients and 270 controls were assayed for
their reactivity to human, mouse, and rat GAD65. Among the type 1
diabetic patients there was a significant better binding of human GAD65
compared to either mouse (P = 0.03) or rat GAD65
(P = 0.0005). The preference for human GAD65
increased with increasing age at onset (P =
0.0002). This differentiation was not observed in 88 GAD65Ab-positive
control subjects. Our data indicate that recognition of epitopes by
GAD65Ab in type 1 diabetes is different from that in nontype 1
diabetes, GAD65Ab-positive individuals.
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Introduction
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INSULIN-DEPENDENT (type 1) diabetes
mellitus is a chronic autoimmune disease. It is characterized by
lymphocytic infiltration of the islet of Langerhans (1) associated with
a gradual and specific destruction of pancreatic ß-cells (2). This
process can last several years and eventually results in complete
ß-cell destruction, hyperglycemia, and life-long insulin dependency.
Most type 1 diabetic patients have circulating autoantibodies directed
to islet cell autoantigens (3, 4). The main autoantigens identified are
insulin (5, 6), the Mr 65,000 isoform of glutamic acid
decarboxylase (GAD65) (5, 7), and tyrosine phosphatase IA-2 (8). These
autoantibodies are often detected long before the clinical onset of
type 1 diabetes and may therefore predict disease (5, 8, 9). In
particular, the presence of all three autoantibodies predicts type 1
diabetes among first degree relatives (8, 9). GAD65 autoantibodies
(GAD65Ab) tend to be the first to appear several years before the onset
of disease (5, 10), with a diagnostic sensitivity of 75–85% (5, 11)
and a diagnostic specificity of 98–99% (10, 12). GAD65Ab are found in
only 1–2% of healthy subjects (13) and may therefore mark that the
type 1 diabetes process is present. GAD65Ab do not bind denatured
GAD65, GAD65 protein fragments, or synthetic peptides (14, 15), which
implies that they bind protein conformation-dependent epitopes. The
identification of type 1 diabetes-specific GAD65 epitopes will be of
major importance for the value of GAD65Ab as a major predictive marker.
GAD65Ab appear to be predominately directed toward epitopes located at
the middle (amino acids 240–435 = epitope 1 or E1) (14, 15) and
the carboxyl-terminal (amino acids 451–570 = epitope 2 or E2)
regions of GAD65 (14, 15). These epitopes were identified by the use of
GAD65/GAD67 chimeric proteins or GAD65, modified by site-directed
mutagenesis. The use of these molecules may, however, introduce
conformational changes and thus eliminate certain epitopes. GAD65 is a
highly conserved protein. It has been isolated and characterized in
several mammals (human, murine, and rat) (16, 17, 18). To our knowledge,
naturally occurring variants of GAD65 have not been used to determine
the presence of epitope-specific anti-GAD65 Ig. In this study we have
taken advantage of the limited sequence difference, previously viewed
as insignificant for the binding by type 1 diabetes-associated
anti-GAD65 IgG (19), among human, rat, and murine GAD65 to determine
their ability to bind autoantibodies in new-onset 0- to 35-yr-old type
1 diabetic patients and in antibody-positive nontype 1 diabetes control
subjects.
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Subjects and Methods
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Human sera
Three groups of type 1 diabetes sera were used in this study
(Table 1
). The first group represents 10
children who were diagnosed with diabetes at age 7–12 yr (median, 10
yr) and subjected to plasmapheresis (20). These samples have been used
in all Immunology of Diabetes Workshops to standardize islet cell
autoantibodies (ICA) (21) and GAD65Ab (22). One sample from this
set of 10 samples is serving as the worldwide standard for expression
of ICA levels in JDF units (21) and of GAD65Ab as a GAD65 antibody
index (7). The second group consists of 2- to 18-yr-old newly diagnosed
patients (n = 126) with type 1 diabetes. All of these patients
were from the St. Gorans Children Hospital (Stockholm, Sweden) and
represent 90% of all children diagnosed at this clinic during
1993–1995. The third group consists of randomly selected (n =
118), 15- to 35-yr-old newly diagnosed Swedish insulin-dependent
patients. The subjects were registered between 1992–1993 in the
Diabetes Incidence Study in Sweden.
Four groups of sera were used as controls (Table 1
). The first group
(n = 132) consists of randomly selected, 15- to 35-yr-old Swedish
individuals (mean age, 26 yr). The second group (n = 50) consists
of 2- to 18-yr-old healthy Swedish individuals. The third group
consisted of type 2 diabetic patients (n = 58) who were also part
of the Diabetes Incidence Study in Sweden study. The fourth group
(n = 30) consists of healthy controls with a GAD65Ab index above
the cut-off detected in a population-based screening of 2276 adults
(mean age, 47 yr). Of these 30 GAD65Ab-positive individuals, 83%
(n = 25) were normoglycemic, and 13% (n = 5) had impaired
glucose tolerance. These individuals participated in The
Västerbotten Intervention Program, Sweden. All serum samples were
kept frozen at -80 C as small aliquots for 1–2 yr before analysis.
The study was approved by the ethics committee of the Karolinska
Institute (Stockholm, Sweden) and Umea University (Umea, Sweden). All
individuals gave their informed consent to participate in the
study.
Construction of murine and rat GAD65 and of chimeric
molecules
Full-length murine and rat GAD65 complementary DNA (cDNA; both
were provided by Drs. Daniel Kaufman and Alan Tobin, respectively,
University of California, Los Angeles, CA) were inserted into the
vector pcDNAII (Invitrogen Corp., San Diego, CA) and coded
pcKoM215 and pcKoR91, respectively. An additional 14 bp containing the
Kozak sequence GGATCCAATTCACC were inserted directly 5' of the coding
sequences. The chimerical molecule consisting of the amino-terminal
amino acids (aa) of human GAD65 (aa 1–83)/GAD67 (aa 89–593) was
constructed as described previously (23). The amino-terminal portion of
human GAD65 (aa 1–83) was substituted by the amino-terminal portion of
rat GAD65, using a native PstI site in both cDNA clones.
GAD65 antibody (GAD65Ab) RIA with recombinant human, murine, and
rat GAD65
Recombinant [35S]GAD65 antigens were produced in
an in vitro coupled transcription/translation system with
SP6 ribonucleic acid polymerase and nuclease-treated rabbit
reticulocyte lysate (Promega Corp., Madison, WI) as
described previously (24). The in vitro translated
[35S]GAD65 was kept at -70 C and used within 2 weeks in
RIAs.
Equal amounts of GAD65 in all three preparations were verified by
densitometric analysis of SDS-PAGE. GAD65Ab were determined by a
previously described RIA (7, 24). Human serum samples were tested at a
final serum dilution of 1:25 unless indicated otherwise. Recombinant
human GAD65 (rhGAD65) expressed in and purified from a baculovirus
system (BioSyn, Stockholm, Sweden) was used in the GAD65Ab competitive
RIA. The RIA was performed as described above, but in the presence of
the indicated concentrations of rhGAD65. The intraassay average
coefficient of variations was 5.2; the highest value was 20, and the
lowest value was 0.1.
Statistical analysis
Antibody levels were expressed as relative indexes using one
positive and two negative standard sera, as previously described (7, 24): GAD65Ab index = (cpm of tested sample - average cpm of
two negative standards)/(cpm of positive standard - average cpm
of two negative standards). Antibody-positive and -negative samples
were included in every assay to correct for interassay variation. The
upper limit (index of 0.07) of the normal range was established as the
99th percentile of the levels of 182 healthy control subjects. The
Juvenile Diabetes Foundation ICA standard (25), which is also GAD65Ab
positive, as verified by immunoprecipitation (26), was used as the
GAD65 antibody-positive standard. A randomly selected control serum
from a healthy volunteer was used as the negative standard. All samples
were tested in duplicate, and the coefficient of variations was
determined for each sample. Differences in binding to the three GAD65
antigens were evaluated using the nonparametric Mann-Whitney U test.
P < 0.05 was considered statistically significant.
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Results
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GAD65 from human, mouse, and rat
GAD65 has been isolated and sequenced from several mammalian
species including human, mouse, and rat (16, 17, 18). The sequence
comparison in Fig. 1 shows that human
GAD65 differs from rat at 22 residues and from mouse GAD65 at 24
residues. The majority of the amino acid substitutions are located at
the first 100 amino acids [54% (7) human/rat and 62% (19)
human/mouse]. The substitutions are radical in 83% (10 of 12 for
human/rat) or 86% (13 of 15 for human/mouse), such as proline to
serine. The mouse GAD65 differs from rat at 8 residues.
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Figure 1. Alignment of human, rat, and mouse GAD65
(pex9, pKoR91, and pKoM215, respectively). The respective amino acid
substitutions are indicated.
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Immunoreactivity of the three GAD65 species to standard ICA
sera
The immunoreactivity of the different GAD65 species was first
tested in RIAs using the ICA standard sera (Fig. 2). The sera were tested at different
dilutions (1:25–1:1000) with all three radiolabeled antigens. Sera
from two healthy individuals were used as negative controls. GAD65
antibodies were detected in eight of these sera (Table 2). All of the GAD65Ab-positive sera
recognized all three GAD65 species. Five of them (sera 4, 5, 6, and 8)
clearly differentiated between human and rodent GAD65. All of them
immunoprecipitated higher amounts of human than of rodent GAD65. The
difference in antigen precipitation was observed at all serum dilutions
except the very high dilutions, where the frequency of GAD65Ab
decreased and gradually overlapped with the range of
immunoprecipitation of the healthy control sera. Serum 2 differentiated
between human and rodent GAD65 to some extent, whereas sera 1 and 7 did
not distinguish among the three antigens. Sera 9 and 10 were both
negative for GAD65Ab. Serum 9 is included in the figure as an example
of a negative serum.
Displacement of human, mouse, and rat GAD65 by rhGAD65
Sera 7 and 8 were analyzed at a dilution of 1:50 for displacement
with unlabeled rhGAD65 (Fig. 3). Serum 7
did not show significant differences in the binding of the three GAD65
species (Fig. 2), and the anti-GAD65 IgG was displaced equally well
from all three antigens with increasing concentrations of rhGAD65 (Fig. 3). Serum 8 displayed significant differences in the binding of the
three GAD65 species (Fig. 2) and showed different displacement curves
(Fig. 3). Although human GAD65 was displaced completely already at 60
ng/mL rhGAD65, both rodent antigens were displaced to a lesser extent
at this concentration. Mouse GAD65 was displaced completely by 100
ng/mL rhGAD65, whereas rat GAD65 was still not displaced entirely at
this concentration.
GAD65Ab reactivity to human, murine, and rat GAD65 in two groups of
type 1 diabetic patients, GAD65Ab-positive type 2 diabetic patients,
and healthy controls
Sera from two groups of type 1 diabetic patients were tested for
their immunoreactivity with the three antigens. The patients had
developed diabetes either at 2–18 yr of age or at 15–34 yr of age.
Both groups (244 patients) showed a clear preference in binding human
GAD65 compared to either mouse (P = 0.03) or rat GAD65
(P = 0.002; Fig. 4). In
the patients who developed type 1 diabetes at 15–34 yr of age, both
preferences were more pronounced (P = 0.03 and
P = 0.0005) than in the group of patients who developed
the disease at a younger age (2–18 yr; both P = 0.05).
There were no significant differences between binding to rat or mouse
GAD65 in either patient group (P = 0.07). The two
healthy control groups (total n = 182) matching the two above
patient groups showed no binding to any of the antigens above the
cut-off value. In analyzing the third control group (n = 58)
consisting of GAD65Ab-positive type 2 diabetic patients and the fourth
group (n = 30) consisting of healthy individuals with GAD65Ab, we
made the surprising observation that there was no preference in binding
between human and either rat or mouse GAD65 (P = 0.38;
Fig. 5).
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Figure 4. Difference between GAD65Ab index of human
GAD65 and 1) mouse GAD65 in 2- to 18-yr-old type 1 diabetic patients,
2) rat GAD65 in 2- to 18-yr-old type 1 diabetic patients, and 3) mouse
GAD65 in 15- to 34-yr-old type 1 diabetic patients, and 4) rat GAD65 in
15- to 34-yr-old type 1 diabetic patients.
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Figure 5. Difference between GAD65Ab index of human
GAD65 and 1) mouse GAD65 in healthy GAD65Ab-positive individuals, 2)
rat GAD65 in healthy GAD65Ab-positive individuals, 3) mouse GAD65 in
GAD65Ab-positive type 2 diabetic patients, and 4) rat GAD65 in
GAD65Ab-positive type 2 diabetic patients.
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Differentiation between human and rat GAD65 is age
dependent
The above observation of age-dependent preference for human over
rodent GAD65 was further examined by plotting the differences in GAD
indexes (GAD index human - GAD index rat) to the age at
diagnosis. The data (Fig. 6) demonstrate
that the difference between human and rat GAD65Ab indexes increases
with increasing age at diagnosis (P = 0.0002).
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Figure 6. Plot of differences between the human and
rat GAD65Ab indexes and age at onset. The dotted line
indicates the division of the two age groups tested.
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Type 1 diabetic patients do not recognize epitopes in the
N-terminus of GAD65
We next studied binding of the GAD65Ab of type 1 diabetic patients
to the N-terminus of the GAD65 molecule. Therefore, we employed a
chimeric molecule constructed by substituting the first 83 amino acids
of rat GAD67 with the respective sequence of human GAD65 only. Thirty
of 202 (15%) sera from type 1 diabetic patients bound to the
chimera, whereas in type 2 diabetic patients and healthy
GAD65Ab-positive individuals, 9 of 29 (33%) and 18 of 39 (46%) sera
bound, respectively (P = 0.024 and 0.0036,
respectively). The majority of the binding was due to cross-reactivity
with GAD65, as only 7 of 155 (4%) sera from the type 1 diabetic
patients bound to the chimera and not to GAD67 (data not shown),
whereas 4 of 23 (17%) sera from the type 2 diabetic patients and 6 of
28 (21%) sera from the GAD65Ab-positive individuals bound only to the
chimera and not to GAD67 (P = 0.01 and 0.001,
respectively), indicating that these sera recognize epitopes in the
N-terminus of the molecule. The data confirm that the N-terminal end of
GAD65 does not have an important epitope for antibody binding in type 1
diabetic patients.
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Discussion
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The identification of disease-specific epitopes is of major
importance for the prediction of type 1 diabetes. In most studies only
the presence or absence of GAD65Ab is analyzed (12, 27). Several
studies of new-onset patients and controls have attempted to define
diagnostic sensitivity and specificity (9, 11, 13, 23). The cut-off for
positivity has been arbitrarily estimated by receiver operating
characteristics analysis (28), percentiles, or mean + 3
SD (13). However, less attention has been paid to levels of
GAD65Ab as a potential factor for diabetes risk (3, 8).
Conformation-specific GAD65Ab to predict type 1 diabetes have been used
only in one study (23). We here tested the hypothesis that IgG
anti-GAD65 in type 1 diabetic patients are species specific. Our
GAD65Ab RIA with protein A detects primarily IgG (except IgG3) and not
antibodies of other isotypes. Furthermore, GAD65-specific IgM was not
detected in the sera tested here (Schranz, D. B., personal
communication). The use of GAD65 cloned from human, mouse, and rat
guarantees preservation of conformational intact GAD65. Our major
findings in testing 254 type 1 diabetic sera are that GAD65Ab showed
significantly preferred binding to human GAD65 compared to both rodent
GAD65 species. This observation may seem obvious, but it has been
claimed that GAD65Ab in type 1 diabetic patients do not distinguish
GAD65 species differences (19). The preference for human GAD65 is
independent of the GAD65Ab index and seems to be age related, as it was
more prominent in the patient group who developed type 1 diabetes
at an older age (18–35 yr) than in those who developed the disease at
a younger age (2–18 yr). It is also noted that 8% (11 of 126) of the
young type 1 diabetic patients showed significant preference for rodent
GAD65. Studies to determine whether this pattern changes over time or
is stable are currently being conducted.
GAD65Ab are important markers in the prediction of type 1 diabetes. The
GAD65Ab-specific epitopes are believed to be conformational (7, 15). Previous studies with chimeric GAD65/67 have failed to identify
epitopes located at the N-terminus (23). These results were confirmed
in this study (data not shown). The N-terminal portion of the molecule
carries most of the amino acid substitutions among the three species.
The remaining molecule is 98% identical between both rodent and human.
Four of the amino acid substitutions observed in the three antigens
involve proline (amino acid positions 19, 62, 63, and 83). These
proline substitutions may have a major influence on conformation and
explain the differences in binding. The conformational differences in
rodent GAD65 compared to human GAD65 could hinder the accessibility of
antibodies to bind to one or both epitopes. The epitopes in type 1
diabetic patients’ sera and sera from healthy controls are identical,
but patients’ sera bind significantly better to the C-terminal epitope
(23). We speculate that the molecular folding typical for rodent GAD65
may hinder antibody binding to the C-terminal epitope. Studies
involving different chimeric molecules are currently underway in our
laboratory.
Our study shows that sera from type 1 diabetic patients can
differentiate between GAD65 species. Only patients’ GAD65Ab
differentiate between species, whereas GAD65Ab found in
GAD65Ab-positive healthy individuals (n = 30) and type 2 diabetic
patients (n = 58) do not. Therefore, GAD65Ab in these two control
groups are more alike than those found in type 1 diabetic patients.
This indicates that GAD65Ab-positive type 1 diabetic patients recognize
different epitopes from those recognized in these two GAD65Ab-positive
control groups. Although GAD65Ab of both healthy GAD65Ab-positive
individuals and type 2 diabetic patients show a broad immune response
to GAD65, as shown by equal binding of all three isoforms, GAD65Ab in
newly diagnosed type 1 diabetic patients represent a more specific
subgroup of antibodies, as they preferentially recognize human GAD65.
Epitope analysis as a measure of type 1 diabetes prediction may
therefore be critical and useful. The comparison of GAD65Ab binding to
human and rodent GAD65 may increase the predictive value for type 1
diabetes and broaden our understanding of the underlying autoimmune
process.
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Acknowledgments
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We thank Terri Daniels for excellent technical assistance. The
samples from the 15- to 34-yr-old new-onset patients were randomly
selected from the Diabetes Incidence Study in Sweden, a
population-based investigation coordinated by Jan Östman, Hans J.
Arnqvist, Göran Blohmè, Folke Lithner, Bengt Littorin,
Lennarth Nystràm, Gàran Sundkvist, and Lars
Wibell.
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Footnotes
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1 This work was supported by the Juvenile Diabetes Foundation
International and the NIH (Grants DK-42654, DK-26190, and
DK-53004). 
2 Juvenile Diabetes Foundation International Research Fellow.
Received September 9, 1998.
Revised November 4, 1998.
Accepted November 10, 1998.
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Top
Abstract
Introduction
), mouse (
), and rat (
) GAD65 to standard sera was measured
in RIA. The GAD65Ab index for sera 1–3 is given on the
left of panel 1; the GAD65Ab index scale for sera
4–9 is given on the left of panel 4. Dilution curves of
eight GAD65Ab-positive standard sera (