From the Ottawa Health Research Institute, Ottawa,
Ontario K1H 8L6, the § Department of Biochemistry,
Microbiology, and Immunology, University of Ottawa, Ottawa,
Institute for Biological Sciences National
Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada, the
§§ Department of Pediatrics, University of
Turku, Turku FIN-20520, Finland, and ¶¶ Nutrition Research
Division, Health Canada, Ottawa, Ontario K1A 0L2, Canada
Received for publication, October 17, 2002
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ABSTRACT |
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The development of autoimmune type 1 diabetes involves complex interactions among several genes and
environmental agents. Human patients with type 1 diabetes show an
unusually high frequency of wheat gluten-sensitive enteropathy; T-cell
response to wheat proteins is increased in some patients, and high
concentrations of wheat antibodies in blood have been reported. In both
major models of spontaneous type 1 diabetes, the BioBreeding (BB) rat and non-obese diabetic mouse, at least half of the cases are
diet-related. In studies of BB rats fed defined semipurified diets,
wheat gluten was the most potent diabetes-inducing protein source. A
major limitation in understanding how wheat or other dietary antigens affect type 1 diabetes has been the difficulty in identifying specific
diabetes-related dietary proteins. To address this issue, we probed a
wheat cDNA expression library with polyclonal IgG antibodies from
diabetic BB rats. Three clones were identified, and the intensity of
antibody binding to one of them, WP5212, was strongly associated with
pancreatic islet inflammation and damage. The WP5212 putative protein
has high amino acid sequence homology with a wheat storage globulin,
Glb1. Serum IgG antibodies from diabetic rats and humans recognized low
molecular mass (33-46 kDa) wheat proteins. Furthermore, antibodies to
Glb1 protein were found in serum from diabetic patients but not in
age-, sex-, and HLA-DQ-matched controls. This study raises the
possibility that in some individuals, type 1 diabetes may be induced by
wheat proteins. Also, it provides a first candidate wheat protein that
is not only antigenic in diabetic rats and human patients but is also closely linked with the autoimmune attack in the pancreas.
Type 1 diabetes is an autoimmune disease that results when a
chronic inflammatory process of unknown origin destroys most of the
insulin-producing The two most studied environmental factors are viruses and diet.
Enteroviruses may be involved (7), but as yet a diabetes-inducing enterovirus has not been identified (8). Epidemiological evidence of
infectious hotspots or traceable routes of infection is lacking (9),
and there are conflicting data with respect to the presence of
candidate viruses in the pancreas or immune cells of diabetic patients
(10-12). The highest incidence of spontaneous diabetes in
BB1 rats and NOD mice occurs
when they are maintained in ultraclean conditions and gnotobiotic
animals still develop diabetes (13). If animals that are maintained in
strict viral antibody-free conditions still develop diabetes then that
leaves diet as the major environmental stimulus.
Although bovine proteins have been a central focus, a recent blinded,
multicenter study demonstrated that a milk-free, wheat-based diet
produced the highest diabetes frequency in diabetes-prone BioBreeding
(BBdp) rats and NOD mice in three widely separate locations (14),
confirming numerous reports that the highest incidence of spontaneous
diabetes occurs in animals fed mixed plant-based diets in which wheat
is the major component (2, 3, 15, 16). Defined diets in which wheat is
the sole protein source are potent inducers of diabetes in BB rats (2,
17). In a different model of diabetes, the NOD mouse, wheat-based diets also resulted in high diabetes frequency (15, 16, 18, 19). In addition,
an unusually high proportion of patients with type 1 diabetes (2-10%)
have wheat gluten-sensitive enteropathy (celiac disease) (20), a rate
that is 10-33 times that in the normal population, and ~1/3 of
diabetes patients have antibodies against the celiac disease
autoantigen, tissue transglutaminase (20, 21). Other reports indicate
that increased peripheral blood T-cell reactivity to wheat gluten was
more frequent in newly diagnosed patients (22) than in controls. These
data are consistent with the involvement of dietary wheat proteins in
diabetes pathogenesis.
Although considered to be a T-cell-mediated disease, studies of the
prediction and pathogenesis of type 1 diabetes in humans rely heavily
on serum autoantibodies as biomarkers of the destructive process. The
humoral immune response to selected autoantigens correlates with
histologic damage in the pancreas of newly diagnosed patients (23).
Indeed, all of the major autoantigens in type 1 diabetes were
identified by virtue of binding by autoantibodies from diabetic
individuals. The 64-kDa autoantigen originally reported in BB rat
and human islets (24, 25) was first discovered using this approach.
This autoantigen was subsequently identified in patients concordant for
both the neurologic disease, Stiff-man syndrome and type 1 diabetes, as
glutamic acid decarboxylase, a major autoantigen in type 1 diabetes
(26). Despite continued progress, the antigens that initiate and
maintain the process leading to In the studies reported here, we used antibodies from rats that
spontaneously develop autoimmune diabetes to identify (i) patterns of
increased binding to low molecular mass wheat proteins as a function of
diabetes risk and age and (ii) individual diabetes-related antigens
from wheat by screening a wheat cDNA expression library. We have
identified a wheat storage protein, Glb1, that is highly antigenic in
diabetic BB rats, the intensity of antibody binding to this protein
correlated with inflammation and damage in the pancreatic islets, and
it was also recognized by IgG antibodies in serum from diabetic
patients but not from controls. This report details studies that
identify a first candidate diabetes-related wheat protein.
Wheat cDNA Library Construction and Probing for Antigenic
Proteins--
Total RNA was isolated (27) from hard red spring wheat,
AC Barrie, provided by Dr. V. Burrows, Eastern Cereal Oilseed Research Centre, of Agriculture and Agri-Food Canada, Ottawa, Canada. Caryopses were harvested at ~10-20 days after pollination, and total RNA was
prepared and sent to Stratagene (La Jolla CA) to construct a ZAP
Express® Custom cDNA library. The cDNA was inserted into the
EcoRI/XhoI cloning site in the amino-terminal
region of the lacZ gene in the ZAP Express vector (Stratagene).
XL1-Blue-MRF' Escherichia coli were infected with 3.5 × 104 plaque-forming units per plate (150 × 15 mm)
of phage from the wheat ZAP Express Custom cDNA library following
the manufacturer's instructions (Stratagene). Protein expression was
induced by the addition of 15 µl of 2 M
isopropyl-1-thio-
The agar plugs were placed in 500 µl of SM buffer (100 mM NaCl, 8 mM
MgSO4·7H2O, 50 mM Tris-HCl, pH
7.5, 0.01% (w/v) gelatin) containing 20 µl of chloroform and stored
at 4 °C. Screening was repeated until the positive phage reached
clonality. Single clone excision was performed to allow in
vivo excision and recircularization of the cloned insert,
according to the manufacturer's instructions (Stratagene). Resistance
to kanamycin indicated the presence of the pBK-CMV phagemid.
Phagemid DNA was prepared for sequencing using a Plasmid Midi Kit
(Qiagen, Mississauga, Ontario, Canada). The cDNA inserts were
sequenced at the University of Ottawa Biotechnology Research Institute
on a 373 Stretch sequencer (Applied Biosystems, Foster City, CA) using
standard T3 forward and T7 reverse primers. For clone WP5212, internal
primers were designed to sequence the full cDNA insert (forward,
5'-ACCACGGGTTCGTCAAGG-3'; reverse, 5'-AACACCTCCTGCACCTCC-3'). Nucleotide and translated BLAST (28) searches of the
GenBankTM 2 and TIGR Wheat
Gene Index3 data bases were
performed for each sequence.
Human Subjects--
Blood samples for serum were obtained from
Finnish children newly diagnosed with type 1 diabetes but not yet
treated with insulin (n = 23; mean age 9.8 ± 3.4 years) and non-diabetic control children (n = 37; mean
age 9.9 ± 3.5 years), matched for age, sex, and HLA-DQ major
histocompatibility complex class II haplotype. Permission for blood
sampling and ethics approval were obtained from the local ethics
committee at the University of Turku.
Animals--
Male and female diabetes-prone BioBreeding (BBdp)
and control BB rats (BBc) were obtained from the Animal Resources
Division of Health Canada (Ottawa). The animals are maintained in
laminar flow protected cages under specific pathogen-free conditions. The mean incidence of diabetes in BBdp rats from this colony fed a
standard cereal-based diet (31) has remained constant over the past 5 years at 65.3 ± 14.9% (mean ± S.D.). This colony is directly descended from the original diabetic rats discovered at
BioBreeding laboratories near Ottawa in 1974 and transferred to Health
Canada in 1977. The colony is not completely inbred but has remained a
closed colony for the past 25 years, and recent genotyping for selected
markers indicates the animals are ~80% identical at the DNA level.
These animals carry the same mutation at the Iddm1/lyp locus
as BB/W rats that is attributable to a frameshift deletion in a novel
member of the immune-associated nucleotide-related gene family, Ian5
(32). BBc rats are derived from an early subline of animals from the
original BB rat colony that does not spontaneously develop diabetes.
Tests in sentinel animals indicate the colony is antibody-free with
respect to Sendai virus, pneumonia virus of mice, rat corona
virus/sialodacryoadenitis virus, Kilham rat virus, Toolan's H-1 virus,
reovirus type 3, and Mycoplasma pulmonis. Animals were
weaned at 23 days of age, caged in banks of 30 wire-bottom cages, and
given free access to food and water. The principles of laboratory
animal care as described by the Canadian Council on Animal Care were followed.
Animals were tested twice weekly for glucose in urine using Testape
(Lily, Toronto, Ontario, Canada) after 60 days of age. Those with a
value greater than 2+ were fasted overnight, and blood glucose in tail
blood was measured the next morning using a glucometer. Diabetes was
diagnosed when fasting blood glucose was >11.1 mmol/liter. Diabetic
animals were killed within 24 h of diagnosis by exsanguination
while under anesthesia with 3% halothane in oxygen.
Insulitis Scores--
All histological analyses were performed
on coded samples. Hematoxylin and eosin-stained sections of pancreas
fixed in Bouin's solution were evaluated at ×100 magnification and
confirmed at ×200 magnification using an Axiolab microscope (Zeiss,
Mississauga, Ontario, Canada). Subjective overall rating of pancreatic
islet inflammation (insulitis (33)) was performed using the following scale: 0, normal islet appearance; 1, infiltration in islet periphery only; 2, infiltration concentrated in islet periphery with infiltration in the islet core; 3, infiltration concentrated in one-third of the
islet core; 4, infiltration concentrated in up to one-half of the islet
core; 5, end stage islets with widespread Diets--
The NTP-2000 diet (Zeigler Bros., Gardners, PA) is an
open formula (the percentage composition is known) and nonpurified diet for rodents developed by the United States National Toxicology Program
of the NIEHS of the National Institutes of Health. NTP-2000 does not
contain any milk protein. This is a mainly plant-based (milk-free) diet
with wheat as the major component (37%), followed by corn, soybean
meal, alfalfa meal, oat hulls, fish meal, and cellulose. The diet
contains ~14.6% protein, 8.2% fat, 9.9% crude fiber, 52%
carbohydrate, 10.7% moisture; the remainder is native and added
micronutrients. The NTP-2000 diet used in these studies was irradiated
and contained low levels of chemical and microbial contaminants (31).
WP semipurified diets were made up of 22.5% wheat gluten (ICN
Biochemicals, Cleveland, OH), 50.2% corn starch, 12.0% sucrose, 5.0%
corn oil, 5.0% fiber (Solka-Floc), 3.5% AIN-76 (or AIN-93G) mineral
mix (ICN), 1.0% AIN-76A (or AIN-93G), vitamin mix (ICN), supplemented
with 0.2% choline bitartrate, 0.02% DL-methionine, 0.5%
L-lysine, and 0.08% L-threonine to compensate
for low sulfur amino acids in wheat proteins. Hydrolyzed casein (HC)
diets contained 51.0% corn starch, 12.0% sucrose, 20.0% casein
hydrolysate (pancreas S enzymatic hydrolysate, Redstar Bioproducts,
Mississauga, Ontario, Canada), 7.0% soybean oil, 5.0% fiber, 3.5%
AIN-76 (or AIN-93G) mineral mix, 1.0% AIN-76A (or AIN-93G) vitamin
mix, 0.2% choline bitartrate, and 0.3% L-cystine. Both
semipurified diets were isocaloric and isonitrogenous.
Probing Wheat Clones for Antibody Reactivity Using Serum from
Individual Rats Fed WP-based Diets--
Serum (diluted 1:200 in
SMP-TBS) from individual diabetic (n = 7), asymptomatic
(no clinical symptoms of diabetes by 150 d; n = 10) BBdp, and BBc (n = 9) rats was used to screen the
wheat clones in the same manner as for the library screening.
Densitometric analysis of regions of interest on nitrocellulose blots
of wheat clones was performed using a Kodak Digital
ScienceTM image station 440CF. The mean intensity/pixel for
each region of interest was tabulated. A clone was randomly chosen from
the library to represent background antibody binding. This clone, WPCON, had an ORF 366 bp long and an expected expression product size
of 121 amino acids (Table I). WPCON shared 91% identity across 326 nucleotides with barley ascorbate peroxidase mRNA (Hordeum vulgare, GenBankTM accession number AF411228.1) and
shared 96% identity across 86 amino acids with the ascorbate
peroxidase protein (H. vulgare, GenBankTM
accession number AAL08496.1).
One-dimensional Western Immunoblotting of Wheat
Proteins--
Proteins were extracted from wheat gluten powder (ICN)
using lysis buffer as described previously (34). Samples were
electrophoresed in 10% SDS-PAGE gels (35), transferred to
nitrocellulose, and blocked with 5% (w/v) SMP-TBS, pH 7.5. Blots were
incubated with sera diluted in SMP-TBS, 1:600. Samples were from
rats at different risk of diabetes and fed WP diet as follows: control
BBc (n = 10), asymptomatic BBdp (no clinical symptoms
of diabetes by 120 days, n = 7), and pre-diabetic BBdp
animals (developed overt diabetes before 120 days, n = 7, or animals with overt diabetes, BBd). Sera from individual patient
(n = 23) and non-diabetic HLA-DQ-matched control
children (n = 37) were diluted 1:50. Following 5 times 5-min washes with TBS containing 1% (v/v) Tween 20, the membrane was
exposed to horseradish peroxidase-conjugated goat anti-rat IgG
(Fc Two-dimensional Western Immunoblotting of Wheat
Proteins--
150 µg of wheat gluten proteins in lysis buffer were
added to the IEF buffer (4% CHAPS, 7 M urea, 2 M thiourea, 40 mM Trizma base, 2 mM
tributylphosphine, and 0.4% Bio-lyte 3/10 (Bio-Rad)) and applied to
rehydrated Ready Strips (Bio-Rad) with an immobilized linear pH
gradient from pH 3 to 10. Wheat proteins were focused at 21 °C for a
total of 100,000 V-h on the Protean IEF cell (Bio-Rad), reduced with 20 mg/ml of dithiothreitol, and alkylated with 25 mg/ml iodoacetamide.
Proteins were separated in the second dimension in 10% SDS-PAGE gels
by electrophoresis at 30 mA for 15 min and 60 mA for 2 h,
transferred to nitrocellulose membrane, and blocked using SMP-TBS
overnight at 4 °C. Serum was pooled from the 23 patients and 37 controls, diluted 1:500 in SMP-TBS buffer, and used to probe
two-dimensional Western blots (1 h at 4 °C). The secondary antibody,
rabbit anti-human total IgG conjugated with horseradish peroxidase
(Dako), was diluted to 1:2000 with SMP-TBS buffer and incubated with
the membranes for 30 min at 4 °C. Antibody binding was visualized
using ECL as recommended by the manufacturer (Amersham Biosciences) and
analyzed using two-dimensional analysis software (PDQuest,
Bio-Rad).
Mass Spectrometry Analysis--
Two-dimensional gels of wheat
gluten proteins were stained with a non-fixing silver stain (36).
Excised gel plugs were digested overnight at 37 °C with 200 ng of
modified sequencing grade trypsin (Promega) in the
ProGestTM automatic digester (Genomic Solutions, Ann Arbor,
MI) as described (36). Rapid capillary LC-MS/MS was performed using a
Waters CapLC liquid chromatograph (Waters, Milford, MA) coupled to a Q-TOF2 mass spectrometer (Micromass, Manchester, UK) with an
electrospray ionization interface. The digested extracts were
redissolved in 5% (v/v) acetonitrile, 0.5% acetic acid, and 10 µl
was loaded onto a 0.3 × 5 mm C18 micro pre-column
cartridge (Dionex/LC-Packings) for each analysis. Rapid peptide elution
was achieved using a linear gradient of 5-60% acetonitrile, 0.2%
formic acid in 6 min (flow rate of 1 µl/min). The mass spectrometer
was operated in data-dependent acquisition mode.
Statistical Analysis--
Comparisons between sample populations
were made using one-way ANOVA and Scheffe's or LSD post hoc
tests (Statistica version 4.5, StatSoft Inc., 1993, Tulsa, OK).
Fisher's Exact test (two-tailed) was used to compare the frequency of
individuals with antibody reactivity to wheat proteins. Pearson
Product-Moment correlation was used to determine r and
p values. Survival analysis using the log Rank test was used
to compare the effect of different diets on diabetes incidence (Statistica).
Wheat Protein Diets Can Modulate Diabetes Outcome--
Animals fed
a non-purified, defined, mainly wheat-based (31), NTP-2000 diet showed
the highest incidence of diabetes (n = 6 experiments,
total of 169 rats, 65.3 ± 14.9%, Fig.
1). When comparing only defined,
isocaloric, and isonitrogenous semi-purified diets with amino acids
from wheat gluten or hydrolyzed casein, there were more cases of
diabetes in BBdp rats fed WP diets (n = 12 experiments,
total of 282 rats, 50.6 ± 11.1%) compared with BBdp rats fed a
protective HC diet (n = 14 experiments, total of 322 rats, 18.8 ± 10.6% Fig. 1; ANOVA/LSD, p < 1 × 10 Three Immunogenic Wheat Clones Isolated from a Wheat cDNA
Expression Library--
A wheat cDNA expression library consisting
of over one million recombinant phage was generated. The primary
screening of the library, using pooled diabetic BB rat serum
(n = 7), yielded 48 positive clones. Eight of these
were found to be true positives and were repeatedly screened until they
reached clonality. The eight clones could be categorized into three
groups based on cDNA insert size (2.1, 1.1, and 0.8 kb). Sequencing
the cDNA inserts confirmed the presence of three distinct sets of
positive clones (Table I). Representative
clones, WP5212, WP12111, and WP23112, from each distinct set were used
for all further analyses.
Nucleotide and translated BLAST searches of GenBankTM2 and
TIGR Wheat Gene Index3 data bases were performed (Table I).
Clone WP5212 contained a 1890-bp open reading frame (ORF), including 95 bp of the lacZ gene. It shared 90% identity across 1387 nucleotides with the Triticum aestivum wheat storage protein
(Glb1) gene (GenBankTM accession number M81719.1). The
expected translated amino acid sequence was 629 amino acids in length
and shared 80% identity across 642 amino acids with the T. aestivum wheat storage protein (GenBankTM accession
number AAA34269.1), Glb1.
WP5212 also shared sequence homologies with the peanut allergen Ara h 1 (GenBankTM accession number P43237; 25% identity across
630 amino acids; Table II), which is
associated with food-induced type 1 hypersensitivity. The
antibody-binding epitopes have been mapped for Ara h 1, and WP5212
shares homology with three of four immunodominant epitopes, as well as
four of five other commonly recognized epitopes (37). WP5212 also had
sequence homology with two other plant allergens, a soybean protein
(Glycine max, GenBankTM accession number
BAB21619) and a dandelion root protein (Taraxacum
officinale, GenBankTM accession number RAP_TAROF). A
BLAST search of the human genome and NCBI data bases retrieved
sequence homologies to tight junction protein 2 (Homo
sapiens, accession number 4759342, and Gallus gallus,
accession number 7512238), similar to tight junction protein ZO-1
(H. sapiens, accession number 17436387) and similar to tight junction protein ZO-2 (H. sapiens, accession number
13639591).
The ORF for cDNA clone WP12111 was 789 bp coding for a putative
product of 262 amino acids (Table I). The nucleotide sequence shared
96% identity across 138 nucleotides with clone CNW03PL453 ITEC CNW
from a wheat powdery mildew-resistant line library (accession number
BE401554) and 63% identity across 144 amino acids with an unknown
Arabidopsis thaliana protein (accession number
AAK25945).
Clone WP23112 had an ORF of 624 bp coding for a putative product of 207 amino acids (Table I). WP23112 shared 96% identity across 542 nucleotides with the BAC clone T16L24 from A. thaliana DNA
chromosome 3 (accession number 6899943) and 62% identity across 150 amino acids with the gene product, a putative A. thaliana protein (accession number CAB75463.1). Clone WP23112 also shared 100%
identity across 511 nucleotides with a clone from a Brevor mature wheat
embryo ABA library (accession number WHE0606).
Diabetic Rats have Increased Frequency and Intensity of Antibody
Reactivity to Glb1--
To determine whether antibody reactivity to
WP5212, WP12111, and WP23112 was related to diabetes risk, the clones
were screened with serum antibodies from individual diabetic
(n = 7), asymptomatic (n = 10), and
control (n = 9) BB rats (Fig.
2, panels A and B). Antibody reactivity was measured by densitometry and is reported as
intensity/pixel. Antibody reactivity to WP5212 in diabetic rats was
significantly higher than in asymptomatic (p = 0.005) and control (p = 10
The frequency of rats with antibodies to wheat proteins was determined
(Fig. 2, panel C). A positive antibody level was defined as
an antibody reactivity value greater than the mean intensity plus 2 S.D. for WPCON screened with control serum. More diabetic (p = 0.009) and asymptomatic (p = 0.02)
rats had antibodies to WP5212 than control rats, but there was no
difference in frequency of antibody reactivity to WP5212 between
diabetic and asymptomatic rats. There was no difference in frequency of
antibody reactivity to WP12111 and WP23112 among the rat groups.
Antibody Reactivity to a Glb1 Protein Correlates with Islet
Inflammation and Damage--
To determine whether antibody reactivity
to the cloned wheat proteins correlated with damage to the target
tissue, the proportion of islets infiltrated with mononuclear cells was
calculated, as well as the mean insulitis score. A relationship with
diabetogenesis was considered to occur when both percent infiltration
(degree of inflammation) and mean insulitis score showed a significant correlation with antibody intensity on the dot blots. Diabetic rats had
significantly fewer islets than both asymptomatic and control rats
(Table III). There was no difference in
total islet number between asymptomatic and control rats. Diabetic rats
had a higher percent of infiltrated islets and mean insulitis score compared with both asymptomatic (p = 0.02 and
p = 0.0001) and control (p = 10 Increased Humoral Immune Reactivity to Low Molecular Mass Wheat
Proteins in Pre-diabetic Rats--
To examine whether differences in
antibody binding to wheat proteins were associated with the development
of disease, Western blots of wheat gluten proteins were probed with
serum obtained prospectively at 50 and 70 days from BB rats at
different risk of developing diabetes. Western blots of wheat proteins
showed antibody reactivity increased with age in BBdp rats (Fig.
4, panel A). At day 50 the
level of antibodies in asymptomatic and pre-diabetic rats was similar.
Compared with animals that remained asymptomatic, higher signal
intensity was detected for wheat proteins around 46 kDa
(p = 0.02, Fig. 4, panel B) in prediabetic
animals at approximately day 70. At necropsy, animals with overt
diabetes had stronger reactivity to 36-kDa wheat proteins compared with
asymptomatic rats (p = 0.006). Blots probed with BBc
rat serum at 1:600 showed low antibody binding to wheat proteins (data
not shown). The frequency of rats reacting to these wheat proteins did
not differ when comparing BBc, BBdp, or overt diabetic animals.
One-dimensional and Two-dimensional Western Blots Show Increased
IgG Binding to Wheat Proteins in Patient Serum; Glb1 Protein Is Bound
by Antibodies from Patients but Not Controls--
One-dimensional
Western blots were used to investigate antibody binding to wheat
proteins (Fig. 5, panel A).
Signal intensity for the 33-kDa proteins was higher in patients than in
controls in 19 of 23 case control comparisons (83%), whereas it was
higher in HLA-DQ-matched non-diabetic children in only 3 of 23 case
controls (p = 0.03). In one comparison, neither patient
nor HLA-DQ-matched control showed antibody binding to the 33-kDa wheat
proteins.
Two-dimensional Western blots of wheat proteins probed with pooled sera
from the same patients showed IgG antibody binding to several wheat
proteins (Fig. 5, panel C). As in the case of diabetic BB
rats, binding of antibodies to wheat proteins was widespread and more
intense compared with controls (Fig. 5). Wheat storage globulin, Glb1,
consists of two subunits with a molecular mass of 49 (pI 6.6)
and 35 kDa (pI 6.9) (38). One of the proteins bound by antibodies from
diabetic children (but not controls) displayed a mass of 50 kDa and pI
of 6.5. When the nature of this protein was determined using LC-MS/MS,
it was found to have peptides homologous to both Glb1 and WP5212. The
expected (theoretical) peptide fragments of Glb1 and WP5212 and the
experimental fragmentation detected by mass spectrometry are shown in
Fig. 6.
When fed to diabetes-prone BB rats, diets in which wheat gluten
was the sole protein source induced nearly three times as many cases of
diabetes as a hydrolyzed casein-based diet (Fig. 1). To analyze as many
potential diabetes-related wheat proteins as possible, we screened more
than one million recombinant phage from a wheat cDNA expression
library with pooled sera from diabetic rats. We isolated eight positive
clones that were shown by nucleotide sequencing to contain three
distinct sets of cDNA inserts. Of three representative clones,
reactivity against WP5212 was strongest. BLAST searches revealed high
similarity at the nucleotide and translated amino acid level with the
wheat storage globulin protein, Glb1. IgG reactivity against Glb1 was
strain-specific, highest in overt diabetic, lower in asymptomatic BB
rats, and lowest in non-diabetes-prone BBc rats.
The autoimmune process involves progressive infiltration into the
Wheat gluten is a large macromolecular complex of polypeptides
consisting mostly (80%) of gliadin and glutenin proteins that remain
after repeated extraction of wheat flour with water, a process that
removes most starch, albumins, and globulins. The endosperm of the
growing wheat seed consists of starch granules embedded in a matrix
composed mostly of storage proteins that provide nourishment and
structure. Following two-dimensional electrophoresis, at least 1,300 endosperm proteins are visible (39). Traditionally, wheat proteins have
been classified according to solubility; the major storage proteins
(~80%) are the gliadins (soluble in aqueous alcohol) and glutenins
(soluble in dilute acid or alkali), whereas albumins (water soluble)
and globulins (salt-soluble) are minor constituents (~20%) (40). The
classification by solubility does not clearly demarcate protein
classes, and several proteins occur in more than one fraction. The
complexity of the endosperm cell proteome, not only with respect to
number but also with respect to size, physicochemical properties, and
function, has made it difficult to identify specific diabetes-related
proteins. Indeed, the wheat genome is estimated to be 16.5 gigabases, more than five times the size of the human genome.
Identifying Glb1, a salt-soluble globulin considered to be absent from
wheat gluten, as a major diabetes-related protein was unexpected.
However, wheat gluten proteins are difficult to separate into distinct
fractions, and globulin proteins can remain trapped in the wheat gluten
matrix (41). In the present study, Glb1 was identified in extracts of
wheat gluten using two-dimensional Western blots and mass spectrometric
analyses (Fig. 5, panel C, and Fig. 6). Thus, there are
several possible interpretations of our findings: (i) Glb1 could be the
main diabetes-related wheat protein; (ii) Glb1 is a normal component of
wheat gluten; (iii) a diabetes-related antigenic structure in Glb1 is
common among other wheat gluten proteins; and (iv) Glb1 is one
diabetes-related protein among several candidates whose antigenicity or
diabetogenicity may differ among wheat-induced diabetes cases.
Considering these possibilities, we interpret our findings as follows.
Glb1 is a normal trace component that becomes trapped in the wheat
gluten protein matrix (41). It contains peptides that are highly
antigenic in diabetes-prone BB rats fed wheat, and this immune
reactivity closely parallels pancreatic damage. There was broad
reactivity to wheat proteins in diabetes-prone BB rats and also in
newly diagnosed, untreated diabetic patients, suggesting that abnormal reactivity to wheat is a common feature in diabetes-susceptible individuals. Our study indicates that of these wheat proteins, Glb1 was
particularly antigenic and is a candidate diabetes-related protein.
Wheat proteins are related through structure and evolution to each
other and also to other groups of seed proteins (42). The 2 S albumins
and the The prospective Western analysis showed a marked humoral response to
certain low molecular mass (36 and 46 kDa) wheat proteins, particularly
in animals that later developed overt diabetes (Fig. 4). These bands
are similar in size to the 35- and 49-kDa subunits of Glb1 (45). Higher
antibody binding to the 33-kDa band was present in 83% of diabetic
children. This indicates a broad response to wheat proteins, one of
which is Glb1. It is not yet clear whether reactivity to Glb1 is a
specific response to a single diabetes-related protein or involves
other wheat proteins. This broad immunoreactivity to wheat might
reflect antigen spreading of the Glb1 shares protein sequence homologies with an important
immunomodulatory food protein, the peanut allergen, Ara h I. This member of the vicilin seed storage family is a major allergen in more
than 90% of peanut-sensitive patients (47). The antibody-binding epitopes of the peanut allergen Ara h 1 have been mapped, and Glb1
shares homology with three of four immunodominant epitopes, and four of
five other commonly recognized epitopes (37). This suggests common
epitopes in both these immunomodulatory food proteins, a point that
will require further analysis.
Globulins are the major protein constituent (90%) of soybean, a
protein source that has been reported to promote the development of
diabetes in BBdp rats, albeit to a lesser extent than the WP diet (2,
33). Furthermore, the Glb1 protein shares sequence homology with a
soybean glycinin protein, suggesting that wheat and soybean might have
common immunogenic and possibly diabetogenic proteins. Further studies
are needed to clarify if these proteins are related to diabetes.
Sequence homologies were also observed between Glb1 and tight junction
protein 2, which is part of a complex of proteins that controls the
permeability of the intestinal epithelium. This is of particular
interest because abnormally increased gut permeability to mannitol, a
marker of paracellular transport, has been reported in pre-diabetic BB
rats (29) and newly diagnosed patients with type 1 diabetes (30).
Cross-reactivity between Glb1 and tight junction proteins might be
expected to damage the gut mucosa of BBdp rats making it more permeable
to dietary antigens, possibly overwhelming the normal oral tolerance
mechanisms, and leading to increased antibody production against
dietary wheat proteins.
Two-dimensional blots also showed higher antibody binding
in diabetic children to several other as yet unidentified wheat proteins (Fig. 5, panels A and C). Glb1 was among
these proteins but absent in the two-dimensional blots probed with
control serum in keeping with the result of the one-dimensional
analysis (Fig. 5, panel A). Our results support the
interpretation that diabetic patients have unique patterns of immune
reactivity, some of which include Glb1. Increased peripheral blood
T-cell reactivity to wheat proteins was seen in 24% of newly diagnosed
patients with type 1 diabetes, compared with only 5% of non-diabetic
controls (22). Taken together, these data are consistent with the
proposition that wheat antigens are the target of inappropriate immune
responses in certain individuals who are genetically susceptible to
develop autoimmune diabetes.
In patients with type 1 diabetes, the presence of autoantibodies to
either glutamic acid decarboxylase or islet antigen-2 has been shown to
be closely correlated with in situ pancreatic islet
inflammation (insulitis) and/or hyperexpression of major histocompatibility complex class I antigens in islets (23). Similarly,
antibodies from BBdp and diabetic rats showed strong reactivity to
the Glb1 protein, and this immunoreactivity correlated closely with the
destructive immune process that targets the pancreatic islet
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DISCUSSION
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-cells in the pancreatic islets of Langerhans. Genetic susceptibility to diabetes is inherited, and there is evidence
that environmental cofactors strongly influence disease expression as
follows: <50% pairwise concordance in identical twins, 3.0% annual
increase in global incidence since 1960 (1), wide geographic variation,
and results from numerous studies in animals showing environmental
factors can modify the development of spontaneous autoimmune diabetes
(2, 3). A major unresolved issue is the identification of the
environmental factors that promote the development of type 1 diabetes
(4). This task has proven difficult because of the multifactorial
nature of the disease (4, 5), difficulty in linking past exposures to
development of diabetes, lack of knowledge of the environmental
antigens, and the large number of predisposing genes in individuals at
risk (6).
-cell destruction remain poorly understood.
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-D-galactopyranoside per 600 µl of
E. coli. Plaque lifts were performed, and the nitrocellulose membranes were screened following the manufacturer's instructions (Stratagene, La Jolla, CA). The primary antibody (diluted 1:200 in skim
milk powder in Tris-buffered saline (SMP-TBS)) consisted of pooled sera
from seven diabetic BB rats fed a wheat protein (WP) diet from weaning.
The BB rat antibodies were pre-absorbed with E. coli phage
lysate. The secondary antibody, alkaline phosphatase-conjugated AffiniPure goat anti-rat IgG, Fc
fragment-specific antibody (Jackson ImmunoResearch, West Grove PA), was diluted 1:5000 in SMP-TBS. Antibody
binding was detected using alkaline phosphatase development solution
(100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl2) containing nitro blue tetrazolium
chloride (0.3 mg/ml) and 5-bromo-4-chloro-3-indolyl phosphate (0.15 mg/ml). Positive clones were detected as dark purple plaques and cored
from the agar.
-cell destruction and/or
core filled with infiltrating mononuclear cells. The mean of 10 islets
per animal was used for an overall insulitis score. Inflammation of the
islets was also measured as the percent of infiltrated islets.
-fragment specific; Cedarlane Laboratories, Hornby, Ontario, Canada) or rabbit anti-human total IgG antibody (Dako), diluted 1:2000
with SMP-TBS. Bands were visualized using ECL according to the
manufacturer's instructions (ECL-Western blotting detection, Amersham
Biosciences) and quantified by densitometry. Digital images of the
Western blot films were acquired using the Kodak Digital
ScienceTM image station 440CF (Rochester, NY) and analyzed
using the Kodak Digital ScienceTM one-dimensional image
software (New Haven, CT).
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5).
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Fig. 1.
Modification of diabetes development in
diabetes-prone BB rats by wheat-based diets. Survival curves and
final diabetes incidence (inset) in BBdp rats fed from
weaning a mixed, cereal-based, mainly wheat-based, NTP-2000 (NTP) diet
(31) or two semipurified, isocaloric, isonitrogenous diets in which the
sole amino acid source was either HC or WP plus supplemental sulfur
amino acids. Animals fed the NTP-2000 diet had the highest incidence,
65.3 ± 14.9% ( 6 versus HC). There were more cases of
diabetes in BBdp rats fed WP diets (n = 12 experiments,
total of 282 rats, 50.6 ± 11.1%) than those fed a protective HC
diet (n = 14 experiments, total of 322 rats, 18.8 ± 10.6%, 14 experiments;
denotes p = 10
5).
Identification and characterization of clones isolated from a wheat
cDNA expression library
Proteins with sequence homologies to Glb1 found by BLAST
(28) searches of the GenBankTM 2
and NCBI human genome data bases
6) rats. Asymptomatic
BBdp rats also had increased antibody reactivity to WP5212 compared
with control rats (p = 0.0004). Diabetic rats had
higher antibody reactivity to WP12111 than asymptomatic rats (p = 0.02). Antibody reactivity to WP23112 did not
differ among the rat groups. Diabetic rats had increased antibody
reactivity to WPCON compared with asymptomatic and control rats.
Antibody reactivity in serum from BB control rats was not different
among any of the proteins analyzed, suggesting that this level
represented nonspecific antibody reactivity.
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Fig. 2.
Examples of plaque lifts of clones screened
with serum from diabetic, asymptomatic, or control rats (panel
A), antibody reactivity to four clones (panel
B), and frequency of antibody reactivity to the wheat
proteins (panel C). Panel A, plaque
lifts of clones WP5212, WP12111, WP23112, and WPCON screened with serum
from five representative diabetic, asymptomatic, or control rats.
Panel B, mean antibody reactivities (intensity/pixel) ± S.D. to the recombinant wheat proteins screened with diabetic (n = 7, cross-hatched bars), asymptomatic (n = 10, hatched
bars), or control (n = 9, open bars) BB rats are shown.
Individual values for diabetic (diamonds), asymptomatic
(squares), or control (circles) rats are shown.
Panel C, the frequency of diabetic (cross-hatched
bars), asymptomatic (hatched bars), and control
(open bars) BB rats with positive antibody reactivity to the
wheat proteins is shown. A positive antibody level was defined as an
antibody reactivity greater than the mean intensity of WPCON screened
with control rat serum plus two S.D. (ANOVA/LSD; indicates
significant difference versus control rats,
p
0.02; * indicates significant versus
asymptomatic rats, p
0.02).
6 and p < 10
7) rats. In
asymptomatic rats, the percent of infiltrated islets was higher, as was
the mean insulitis score compared with control rats (p = 0.0001 and p = 0.002). A positive correlation was
observed between antibody intensity to WP5212 and percent of
infiltrated islets (Fig. 3, panel
A, r = 0.81, p = 10
6) and mean insulitis score (Fig. 3, panel
B, r = 0.78, p = 3 × 10
6). There was no correlation between antibody
reactivity to WP12111 or WP23112 and percent of islets infiltrated and
mean insulitis score (Fig. 3, panels C-F).
Number of islets and inflammation in the pancreas of rats whose serum
was used to screen wheat clones
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Fig. 3.
Antibody reactivity to the Glb1 clone is
strongly associated with pancreatic inflammation or insulitis. The
correlations between the percent of islets infiltrated (left
column) or insulitis score (right column) and antibody
reactivity (mean intensity/pixel) to three recombinant wheat proteins
in diabetic (diamonds), asymptomatic (squares),
or control (circles) rats are shown. The Pearson
Product-Moment correlation r and p values are
indicated.
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Fig. 4.
One-dimensional Western analysis of wheat
proteins probed with serum collected prospectively from BB rats at
different risk of developing diabetes. One-dimensional Western
blots of wheat proteins probed with serum from prediabetic or
asymptomatic BB rats at 50 and 70 days and at necropsy are shown in
A. The mean intensity ± S.D. of each wheat protein
band is shown for the prediabetic period (70 days) or at necropsy for
asymptomatic (open bars) or diabetic (filled
bars) in B; ANOVA/LSD; , p = 0.02;
, p = 0.006.
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Fig. 5.
One-dimensional and two-dimensional Western
analysis of antibody binding to wheat proteins in patients and HLA-DQ
matched controls. Examples of (panel A) one-dimensional
Western blots of wheat proteins probed with serum samples from diabetic
children and control children without diabetes. Panel B, the
mean absorbance ± S.D. of (each) wheat protein band probed with
serum from diabetic children (filled bars) and
HLA-DQ-matched controls (open bars) (ANOVA/LSD; * indicates
p = 0.005). Panel C, two-dimensional Western
blot of wheat proteins probed with pooled serum samples from newly
diagnosed diabetic children (left) or control children
(right). Wheat storage globulin, Glb1, was bound by
antibodies in serum from children with diabetes, but there was no
binding using serum from non-diabetic controls.
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Fig. 6.
Identification of wheat storage globulin,
Glb1, by in-gel tryptic digestion and capillary LC-MS/MS analysis.
Panel A, the MS/MS spectrum of the doubly protonated ion
(MH
DISCUSSION
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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-cell-containing core of the islets by mononuclear cells and
macrophages, a process called insulitis. The severity and prevalence of
insulitis or its sequelae (end stage islets) reflect the extent of
damage in the pancreas. When sera from individual rats at different
risks of developing diabetes were used, IgG reactivity against the Glb1
clone showed a remarkably close correlation with overall islet
infiltration and damage (insulitis rating), as well as inflammation of
individual islets (Fig. 3). The two other positive clones, WP12111 and
WP23112, which shared amino acid homology with unidentified proteins
from A. thaliana showed similar antibody reactivity to the
control WPCON clone (ascorbate peroxidase, H. vulgare), and
there was no correlation between antibody reactivity and islet
inflammation or damage (Fig. 2, panel B, and Fig. 3). These
clones were not investigated further. These results demonstrated not
only a strong immune reaction against the Glb1 protein in wheat-fed,
diabetes-prone BB rats but also a close link with the diabetogenic
process in the target tissue.
-amylase/trypsin inhibitors of cereals are part of the
so-called prolamin superfamily (prolamins, 2 S albumins, and cereal
inhibitors (globulins)) (43). These proteins form part of a fraction
previously termed the chloroform/methanol-soluble fraction, and their
removal from a wheat-based diet inhibited the development
of diabetes in NOD mice (15). There are immunologically relevant
structural similarities among the wheat proteins as shown by
cross-reactivity of monoclonal antibodies between conserved epitopes in
albumins and globulins (44). The present finding suggests that
wheat-induced diabetes in BB rats may result at least in part from a
misdirected immune reaction against non-gluten proteins that are
co-isolated during the preparation of wheat gluten.
-cell reactive process and unique
individual patterns of abnormally high immune reactivity to wheat as
reported in children with celiac disease (46).
-cells
in the pancreas. The close correlation between antibody reactivity to
Glb1 and islet inflammation in BB diabetes-prone and diabetic rats
represents a new association between a previously unidentified wheat
antigen and the target tissue. The fact that higher immunoreactivity to
Glb1 was observed in patients compared with HLA-DQ matched non-diabetic
children raises the possibility that wheat may also be involved in the
pathogenesis of human type 1 diabetes.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. R. Sardana of the Dept. of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Canada, for assistance in the preparation of the wheat cDNA library. We thank Dr. O. Pulido and P. Smyth of the Pathology Section, Toxicology Research Division, Food Directorate, Health Canada, Ottawa, Canada, for assistance with histology. Dr. P. Thibeau and T. L. Tremblay of the Institute for Biological Sciences, National Research Council, Ottawa, Canada, provided assistance with mass spectrometry analysis. We thank J. Souligny and D. Patry of the Animal Resources Division, Health Canada, for animal care, and Dr. G.-S. Wang, M. Green, H. Gruber, H. Cloutier, and P. Jee for technical support. We also thank H. Kolb of the German Diabetes Research Institute, Heinrich Heine University of Düsseldorf, Germany, for advice, discussions, and technical assistance.
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FOOTNOTES |
---|
* This work was supported in part by the Canadian Institutes of Health Research, Juvenile Diabetes Research Foundation, Ontario Research and Development Challenge Fund, Canada Foundation for Innovation, Health Canada, Academy of Finland, and the Sigried Juselius Foundation, Finland.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Both authors contributed equally to this work.
Recipient of an award from the Ontario Graduate Scholarship Program.
** Recipient of a doctoral research award from the Diabetic Children's Foundation and the Canadian Institutes of Health Research.
To whom correspondence should be addressed: Autoimmune
Disease Group/Diabetes, Molecular Medicine, Ottawa Health Research Institute, 501 Smyth Rd., Ottawa, Ontario K1H 8L6, Canada. Tel.: 613-737-8929; Fax: 613-739-6189; E-mail: fscott@ohri.ca.
Published, JBC Papers in Press, October 29, 2002, DOI 10.1074/jbc.M210636200
2 NCBI (2002) website address: www.ncbi.nlm. nih. gov/BLAST/, National Center for Bio/Technology Information, National Library of Medicine, Bethesda, MD.
3 TIGRWheat data base (2001) website address: ww.tigr.org/tdb/tagi/, Institute for Genomic Research, Rockville, MD.
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ABBREVIATIONS |
---|
The abbreviations used are: BB, BioBreeding; NOD, non-obese diabetic; WP, wheat protein; ANOVA, analysis of variance; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; HC, hydrolyzed casein; ORF, open reading frame; LC-MS, liquid chromatography-mass spectrometry; LSD, least significant difference; SMP-TBS, skim milk powder in Tris-buffered saline; NTP, National Toxicology Program.
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