From the CNRS Unité Mixte de Recherche
(UMR) 5094, Faculté de Pharmacie, 15 avenue Charles
Flahault, B. P. 14491, Montpellier 34093 Cedex 5, ¶ CNRS-Institut National de la Recherche Agronomique UMR 5087,
Station de Recherche de Pathologie Comparée, St.
Christol-lez-Alès 30380, and ** INSERM U397, Institut
Fédératif de Recherche Louis Bugnard, Centre Hospitalier
Universitaire Rangueil, Toulouse 31403 Cedex 4, France
Received for publication, November 22, 2002, and in revised form, December 23, 2002
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ABSTRACT |
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The discontinuous immunodominant region
(IDR) recognized by autoantibodies directed against the thyroperoxidase
(TPO) molecule, a major autoantigen in autoimmune thyroid diseases, has
not yet been completely localized. By using peptide phage-displayed
technology, we identified three critical motifs,
LXPEXD, QSYP, and EX(E/D)PPV, within selected mimotopes which interacted with the human recombinant anti-TPO autoantibody (aAb) T13, derived from an antibody
phage-displayed library obtained from thyroid-infiltrating TPO-selected
B cells of Graves' disease patients. Mimotope sequence alignment on
the TPO molecule, together with the binding analysis of the T13 aAb on
TPO mutants expressed by Chinese hamster ovary cells, demonstrated that
regions 353-363, 377-386, and 713-720 from the myeloperoxidase-like domain and region 766-775 from the complement control protein-like domain are a part of the IDR recognized by the recombinant aAb T13.
Furthermore, we demonstrated that these regions were involved in the
binding to TPO of sera containing TPO-specific autoantibodies from
patients suffering from Hashimoto's and Graves' autoimmune diseases.
Identification of the IDR could lead to improved diagnosis of thyroid
autoimmune diseases by engineering "mini-TPO" as a target
autoantigen or designing therapeutic peptides able to block undesired
autoimmune responses.
Human thyroid peroxidase
(TPO),1 described previously
as the "thyroid microsomal antigen" (1), is a membrane-bound enzyme expressed at the apical pole of thyrocytes (2). TPO generates the
functional form of thyroglobulin by iodination and coupling of tyrosine
residues (3). During autoimmune thyroid diseases (AITD), TPO represents
a major target for the immune system (4, 5), leading to high titer
TPO-specific autoantibodies (aAbs) in the sera of patients suffering
from Hashimoto's thyroiditis and Graves' disease. Besides their role
as efficient and early diagnostic markers of AITD, TPO-specific aAbs
also act as effector molecules either through modulating antigen
presentation to T cells or by mediating thyroid destruction after
complement activation or antibody-dependent cell
cytotoxicity (6-12). Alignment studies and structural homologies have
shown that TPO is formed by three distinct domains: a myeloperoxidase
(MPO)-like, a complement control protein (CCP)-like, and an EGF-like
domain, from the N- to the C-terminal extremities (13). Although the
structure of each domain has been elucidated in part by
three-dimensional modeling (13-16), the full three-dimensional
structure of TPO remains unknown, even though low resolution crystals
have been obtained (17, 18). The flexibility observed for the hinge
regions probably make difficult the exact positioning of each domain in
relation to the others (15).
These observations denote a highly complex structure of TPO, thus
explaining the reason why TPO aAbs from patients' sera preferentially recognize discontinuous epitopes on TPO (19-22). Different approaches have been used to determine the epitopic regions recognized by anti-TPO
aAbs from patients' sera as well as critical amino acids involved in
these interactions: (i) competition for TPO binding between mouse mAbs
or rabbit polyclonal antisera and human anti-TPO aAbs (13, 23-25),
(ii) analysis of human anti-TPO aAb binding to recombinant and/or
truncated antigen (15, 16, 26-30), (iii) generation of TPO fragments
by enzymatic hydrolysis following by analysis of their binding to TPO
aAbs (31, 32), and (iv) eukaryotic cell expression of chimeric MPO-TPO
proteins obtained by "guided" mutagenesis and binding analysis to
TPO aAbs (33-35). These approaches have demonstrated a restricted
response of TPO-specific aAbs, which mainly recognize a discontinuous
immunodominant region (IDR). By competition between human anti-TPO from
patients' sera and a pool of murine anti-TPO mAbs, the IDR was divided
into two overlapping domains named A and B (25). Using monoclonal human anti-TPO antibody fragments (Fabs) as competitor, Chazenbalk and co-workers (23) defined the A and B domains but inverted the nomenclature. This second nomenclature, defined by the human Fabs, will
be used here. The IDR was only partially characterized, however, with
no clear direct relation between the identified regions. We hypothesize
that most of the techniques currently in use for mapping mAb linear
epitopes are inadequate for precisely defining a discontinuous epitope
on molecules like TPO, which have a highly organized architecture. To
overcome this problem, we decided to combine two technological advances
already used to delineate highly structured epitopes (36-39). First,
antibody-specific peptides were selected for their ability to mimic
natural epitopes by screening phage-displayed peptides libraries.
Second, the sequence alignments of such selected mimotopes on the
primary sequence of the antigen together with knowledge of the
three-dimensional structure of the antigen allowed the identification
of a potential conformational epitope.
We applied this strategy to the identification of the TPO
immunodominant epitope involved in thyroid autoimmunity by using the
human IDR-specific recombinant IgG1 aAb T13 derived from
antibody phage-displayed libraries obtained from B cells extracted from thyroid tissue of Graves' disease patients (40, 41). Four distinct
regions, belonging to the MPO-like and CCP-like domains of TPO,
delimited the discontinuous IDR, recognized by the human recombinant
aAb T13 and identified by sera from patients suffering from
Hashimoto's thyroiditis or Graves' disease.
Patient Sera--
Sera from five patients with Graves' disease,
five with Hashimoto's thyroiditis, and four healthy donors were
obtained from Dr. L. Baldet and Dr. A. M. Puech (Guy de Chauliac
Hospital, Montpellier, France). The anti-TPO aAb titers were
determined by radioimmunoassay using the TPO-AB-CT Kit (Cis bio, Gif
sur Yvette, France). The patients' sera were characterized further for
the presence of anti-TSH receptor aAbs by a radio receptor assay using
the TRAK human kit (BRAHMS, Hennigsdorf, Germany) and for
triiodothyronine, thyroxine, and thyroid-stimulating hormone levels
(Table I). As controls, sera from eight
patients suffering from other autoimmune affections, two with type 1 diabetes, four with human systemic lupus erythematosus, one with
vascularitis, and one with rheumatoid arthritis were tested.
Materials--
The human TPO (hTPO), purified (greater than 95%
pure) from thyroid glands, was obtained from HyTest Ltd. (Turku,
Finland). The plasmid encoding full-length hTPO was kindly provided by
Dr. B. Rapoport (Cedars-Sinai Medical Center, Los Angeles, CA).
Anti-TPO mAb 47 was provided by Dr. J. Ruf (INSERM Unit 555, Marseille, France) (42). Anti-TPO mAb 6F5 and rabbit polyclonal anti-TPO were
purchased from HyTest Ltd., and Abcys S.A. (Paris, France), respectively.
Cloning, Expression, and Purification of Human aAb T13--
The
recombinant human anti-TPO scFv T13 was selected from an antibody
phage-displayed library obtained from B cells extracted from thyroid
tissue of Graves' disease patients (40). The T13 aAb was expressed as
an entire IgG1 antibody by using the baculovirus/insect cell system as described (41, 43, 44). IgG1 T13 was
purified on a protein G affinity column and the concentration
determined by the A280 nm,
E0.1% of 1.40.
Purity, Specificity, and Kinetic Parameters of aAb T13--
The
purity was analyzed on 8% SDS-PAGE under nonreducing conditions, then
Coomassie Brilliant Blue stained or transferred to a polyvinylidene
difluoride membrane (Novex, San Diego), and revealed by a
peroxidase-conjugated anti-human IgG Fc-specific Ab (Sigma) diluted
1:1,000, using the ECL detection system (Amersham Biosciences). Then,
the aAb specificity was determined by ELISA. The microtiter plate wells
were coated with 9 nM (1 µg/ml) TPO in 100 mM
NaHCO3, pH 9, overnight at 4 °C. Plates were washed with
0.05% Tween 20 in PBS (PBS-T), pH 7.3, and blocked with 2% powdered
milk in PBS-T for 1 h at 37 °C. After washing, aAb T13 or
purified human IgG1 (Sigma) as control was incubated with
1% powdered milk in PBS-T (incubation buffer) for 1.5 h at
37 °C. After washing, an alkaline phosphatase-conjugated anti-human
IgG Ab (Sigma, diluted 1:1,000) in the incubation buffer was added, and
the plates were incubated for 1 h at 37 °C. After three
washings, the reactivity was revealed with 1 mg/ml of 4-nitrophenyl
phosphate in 0.5 M diethanolamine/HCl, pH 9.6, for 1 h
at 37 °C. Kinetic parameters of aAb T13 were obtained by real time
analysis as described previously (41). The aAb T13, expressed as scFv
or IgG1, inhibits between 7.3 and 100% of the binding to TPO
of serum samples from patients with AITD that were tested (40, 41).
Panning and Screening of Phage-displayed Peptides--
Four
phage-displayed libraries (LX-8/f88.4, X15/f88.4,
X8CX8/f88.4, and X30/f88.4) in
which peptides were displayed at the N terminus of the pVIII coat
protein on the surface of the filamentous bacteriophage M13, were
obtained from Dr. J. Scott (Simon Fraser University, Burnaby, Canada)
(45). Phages specific for aAb T13 were selected by biopanning as
described previously (46). Antibody-bound phages were eluted (i) by 3 ml of acid elution (0.1 M glycine-HCl (pH 2.2), 1 mg/ml
bovine serum albumin) for 30 min at room temperature and further
neutralization by adding 300 µl of 2 M Tris/HCl, pH 9.2;
(ii) by competition with a 180 nM (20 µg/ml) TPO solution in 5 ml of NaCl/Tris (50 mM Tris, 150 mM NaCl,
0.05% Tween 20, pH 7.5) for 2 h at 37 °C. Phages were
amplified after each round of panning using an exponentially growing
culture of Escherichia coli K91 cells, and T13-specific
phages were cloned and characterized by ELISA as described (46). Clones
giving an absorbance more than twice the background level were selected.
Each selected and purified clone was checked further by ELISA for its
ability to be inhibited by soluble TPO at a concentration of 90 nM (10 µg/ml). Briefly, wells from microtiter plates were coated with 13 nM aAb T13 in 100 mM
NaHCO3, pH 9, overnight at 4 °C. Plates were washed with
PBS-T, and then 2% powdered milk in PBS-T was added for 1 h at
37 °C. After saturation and four washings, a 1:10 dilution of
purified phages from each clone was coincubated with soluble TPO for
1.5 h at 37 °C. After washing, a horseradish
peroxidase-conjugated anti-M13 (Amersham Biosciences; diluted 1:5,000
with 1% powdered milk in PBS-T) was added for 1 h at 37 °C.
Three washings were performed, and residual bound phages were revealed
with 4 mg/ml 2-phenylenediamine solution containing 0.03% (v/v)
hydrogen peroxide in 0.1 M citrate buffer, pH 5.0. After 20 min, the reaction was stopped by adding 50 µl of 2 M
H2SO4 to each well, and the resulting
absorbance was measured at 490 nm. Selected phages, whose binding to
aAb T13 was inhibited by more than 15%, were sequenced for peptide
sequence identification. As a control, the effect of a solution
containing 90 nM soluble thyroglobulin (60 µg/ml) on the
binding of these selected phages to aAb T13 was tested.
Mimotope Synthesis, Alanine Scanning, and Immunoassay on
Cellulose Membrane-bound Peptides--
The general protocol of Spot
parallel peptide synthesis has been described previously (47). 12 pentadecapeptides corresponding to the selected T13-specific mimotopes
and their 15 alanine analogs were synthesized by the Spot method. The
membrane-bound peptides were probed by coincubation of a 0.1 µM solution of aAb T13 with peroxidase-conjugated
anti-human Fc specific antibody (Sigma) diluted 1:1,000 for 1.5 h at 37 °C. After three washings, the complex was revealed
using the ECL detection system on a sensitive film, and the intensities
of the spots were evaluated with ScionImage software. The membrane was
reused after a regeneration cycle.
Sequence Alignments and Molecular Modeling--
Each amino acid
sequence corresponding to an immunoreactive mimotope was aligned with
the other selected peptides or with the hTPO sequence using the
multiple sequence alignment program Multalin (48). The sequence
homologies between T13-specific mimotopes and hTPO were located on a
three-dimensional model of TPO, using the atomic coordinates kindly
provided by Hobby et al. (13).
Directed Mutagenesis and Stable Expression of Wild-type and
Mutated TPO cDNA--
A KpnI restriction site was added
by PCR just after the stop codon of the full-length hTPO cDNA.
Wild-type TPO cDNA was cloned using the HindIII and
KpnI sites into the pcDNA5/FRT expression vector from
the Flp-In system (Invitrogen). The mutants were constructed by overlap
extension PCR as described previously (49). The mutation replacements
were performed as follows. The
353HARLRDSGRAY363 sequence from wt TPO was
replaced by sequence 353NQAFQANGAAL363 in the
TPO353-363 mutant, wt TPO
(377PEPGIPGETR386) by TPO377-386
(377LLTNRLGRIG386), wt TPO
(506ASFQEHPDL514) by TPO506-514
(506NRYLPMEPN514), wt TPO
(713KFPEDFES720) by TPO713-720
(713SYARLAVN720), wt TPO
(737PQDD740) by TPO737-740
(737ALAA740), and wt TPO
(766YSCRHGYELQ775) by TPO766-775
(766LTCEGGFRIS775). All sequences were verified
by the dideoxynucleotide termination method (50). The Flp-In system was
used to generate isogenic stable mammalian cell lines expressing wt and
mutated TPO. CHO cells (ATCC CCL61) were routinely grown in Dulbecco's
modified Eagle's medium/nutrient mixture F-12 containing 10% fetal
calf serum. Stable transfectants were obtained as recommended by the manufacturer. Briefly, the CHO host cell line was constructed by
transfection with 0.5 µg of the pFRT/lacZeo vector and
selection with growth medium containing 500 µg/ml ZeocinTM (Cayla,
Toulouse, France). ZeocinTM-resistant clones were screened to identify
those containing a single integrated FRT site and expressing a high Membrane Protein Extraction--
Stably transfected CHO cells
were washed three times with PBS and scraped at 4 °C. Membrane
protein extraction was performed as follows. After centrifugation at
1,000 rpm for 5 min at 4 °C, membrane proteins were solubilized by
adding 500 µl of lysis buffer (50 mM Tris/HCl, pH 7.3, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1%
Triton X-100, and a protease inhibitor mixture tablet/10 ml of lysis
buffer (Roche Molecular Biochemicals)) and incubated for 30 min on ice.
After centrifugation at 13,000 rpm for 30 min at 4 °C, the
supernatants containing membrane proteins were recovered and conserved
at Western Blotting Analysis and Competition
Experiments--
Approximately 20 µg of membrane proteins was mixed
with protein buffer (0.2 mM Tris/HCl, pH 6.8, 50%
glycerol, 1% SDS, and 0.1% bromphenol blue) for native conditions,
supplemented with 10% dithiothreitol, 10% 2-mercaptoethanol, and
boiled 5 min for denaturing conditions. Each sample was loaded into
individual wells and electrophoresed through a 9% SDS-PAGE. Proteins
were transferred to nitrocellulose membranes, which were then saturated with 5% powdered milk in PBS-T for 30 min at room temperature. The
first antibodies, diluted with the incubation buffer, were applied
overnight at 4 °C. After three washings with PBS-T for 10 min at
room temperature, the membranes were incubated with horseradish
peroxidase-conjugated secondary antibodies diluted in incubation buffer
for 1 h at 37 °C. After three washings, the signal was detected
with chemiluminescence ECL on a sensitive film.
Competitive ELISA experiments were performed as follows. Briefly, the
wells were coated with 9 nM (1 µg/ml) TPO in 100 mM NaHCO3, pH 9, overnight at 4 °C. Then
they were washed and blocked with 2% bovine serum albumin in PBS-T
(saturation buffer) for 1 h at 37 °C. After three washings, 3 nM mAb T13 (at a dilution giving an
A490 of 1.5) was incubated with or without
decreasing concentrations of inhibitor in saturation buffer for 1.5 h at 37 °C under mild shaking. The wells were washed, and
horseradish peroxidase-conjugated anti-human IgG antibody diluted
1:2,000 in saturation buffer was added for 1 h at 37 °C. Three
washings were performed, and then aAb T13 bound to TPO was detected by adding 4 mg/ml 2-phenylenediamine solution containing 0.03% (v/v) hydrogen peroxide in 0.1 M citrate buffer, pH 5.0. The
reaction was stopped with 2 M
H2SO4, and the resulting absorbance was
measured at 490 nm.
ELISA with mAbs and Human Sera--
Wells were coated with 20 µg/ml membrane proteins in PBS overnight at 4 °C. The plates were
washed with PBS-T and blocked with the saturation buffer for 1 h
at 37 °C. After three washings, mAb or serum was incubated in the
saturation buffer for 1.5 h at 37 °C. The plates were washed,
and horseradish peroxidase-conjugated anti-human IgG or anti-mouse
antibody (diluted 1:2,000 in saturation buffer) was added for 1 h
at 37 °C. Three washings were performed, and the reactivity was
revealed as described above.
Characterization of aAb T13 Expressed as Whole IgG1 in
Insect Cells--
The anti-TPO aAb T13 was first produced in the form
of a human scFv (40). For the present study, we produced the entire human IgG1 T13 by using the baculovirus/insect cell system
and purified it by protein G affinity chromatography. The purity was analyzed on 10% SDS-PAGE stained with Coomassie Brilliant Blue under
nonreducing conditions (Fig.
1A, left panel). We
visualized a 150 kDa band corresponding to human IgG1 T13,
and the purity was estimated to be superior to 95%. The identity of
the band was confirmed by Western blotting, under nonreducing
conditions, with a peroxidase-conjugated anti-human IgG Fc-specific Ab
(Fig. 1A, right panel). Approximately 5 mg of
purified aAb T13 was obtained per liter of Sf9 insect cell
culture supernatant. The binding specificity of aAb T13 to hTPO was
determined by ELISA. As shown in Fig. 1B, aAb T13
specifically bound hTPO in a dose-dependent manner, whereas
polyclonal human IgG1 as control did not. Moreover, no
binding was observed for aAb T13 to thyroglobulin (data not shown).
The kinetic parameters of aAb T13 were assessed by real
time analysis, using a BIACORE 2000 as described previously (41), by
injecting three concentrations of purified hTPO on aAb T13 or
irrelevant Ab (Table II). We found that
aAb T13 showed a high affinity for hTPO (KD = 0.9 nM, average of three experiments) with a low
dissociation rate (kd = 1.6 10 Selection of T13-binding Mimotopes by Phage Display
Technology--
We have demonstrated previously that aAb T13 strongly
inhibits the TPO binding of aAb in the sera of patients suffering from AITD (40, 41). Moreover, we have shown that aAb T13 does not recognize
a linear epitope,2 thus
suggesting the conformational nature of this dominant cognate epitope.
To address this question, biopannings were performed with a mix of four
phage libraries expressing peptides with 8, 15, 17, or 30 amino acids
at their surface, to obtain specific mimotopes selected by human aAb
T13, which mimic the conformational epitope. 100 phages were eluted
with a pH 2.2 buffer after three rounds of panning on aAb
T13 and another 100 phages after four rounds of panning for the elution
by competition. All 200 phages were isolated and then purified. As
shown in Table III, 12 T13-specific mimotopes, 6 obtained by acid elution and 6 by competition, were further selected according to (i) their binding level on aAb T13 by
ELISA and (ii) their ability to compete with human soluble TPO as
inhibitor for aAb T13 binding; only mimotopes with at least 15%
inhibition are shown. This inhibition ranging from 19 and 55% for the
selected mimotopes. The binding of these mimotopes to the aAb T13 was
not inhibited by thyroglobulin, used as a control protein (data not
shown). Only peptides derived from the X15/f88.4 library
were obtained, suggesting that the conformation of 15-mer peptides
leads to better recognition by aAb T13. On the other hand, similar
experiments performed with another human anti-TPO aAb permitted the
selection of peptides from X15/f88.4,
X8CX8/f88.4, and X30/f88.4
libraries.3 Except for
mimotope RLAPEPDDPITPMTK (Table III), each peptide sequence was
obtained individually.
Analysis of T13-specific Mimotopes by Sequence Alignment and Spot
Technology--
T13-specific mimotopes were aligned with each other
and grouped into three families according to their sequence homology
(Table IV). Families 1, 2, and 3 contain
mimotopes bearing LXPEXD,
(S/T)X2QSYPXP, and
EX2PPVX6L homologous
motifs, respectively (where X represents any amino acid).
However, peptides KNSRQSYPEPAPVYH, QLSPESDYDDHGMRY, and SQSYPEPARGSVPMP
showed similarities found in both families 1 and 2. More generally, the
mimotopes often presented a proline-rich sequence representing between
6.6 and 27% of their residues. Acidic amino acids were highly
represented in the sequences of Family 1 (between 6.6 and 33% of the
residues) and to a lesser extend in Family 3 (between 13.3 and 20% of
their residues).
To determine critical amino acids implicated in the recognition of each
peptide by aAb T13, we synthesized on a cellulose membrane, by the Spot
method, each mimotope followed by an alanine scanning of their amino
acid sequences. Most of the 12 mimotopes reacted strongly with the T13
aAb (Fig. 2A). Mimotope 8, showing no reactivity with aAb T13, and mimotope 3, demonstrating a
cross-reaction with the secondary Ab (Fig. 2A), were
eliminated from further alanine scanning studies. As exemplified in
Fig. 2B, the systematic alanine replacement for each
mimotope revealed critical residues for T13 binding and identifies
three different critical motifs, LXPEXD, QSYP,
and EX(E/D)PPV for Families 1, 2, and 3 respectively; these
motifs correlated with those demonstrated by sequence homology. Moreover, the alanine scanning patterns clarified the ambiguity for the
peptide assignment indicated above, i.e. peptides 7 and 12, KNSRQSYPEPAPVYH and SQSYPEPARGSVPMP, were definitively assigned to
Family 2, whereas peptide 10, QLSPESDYDDHGMRY, belongs to Family 1 (Table IV).
Localization of Putative T13 Autoantibody Interaction Sites on
Human TPO--
Amino acid sequences from T13-specific mimotopes were
aligned on the primary sequence of hTPO to identify the regions that contain sequence homology on TPO. First, we performed the alignment of
critical motifs, identified previously by the Spot technology (Fig.
2B), with the primary sequence of hTPO. Then, the other amino acids for each peptide were aligned according to the best homology with the primary sequence of hTPO. As shown in Fig.
3, this procedure identified four
different regions (residues 356-382, 713-720, 737-747, and 766-775)
which demonstrated the best matching with mimotopes and their critical
motifs. Interestingly, two regions bear the amino acids
Lys713 and Tyr772, described previously as
critical for immunodominance (15, 32). Analysis of the
three-dimensional model of hTPO showed that these regions are located
at the surface of the globular structure (Fig.
4A) and could form an
immunodominant binding surface (IBS) between the MPO- and CCP-like
domains because of the flexibility of the hinge regions (Fig. 4,
A and B). Because these locations correspond to
the putative T13 aAb interaction sites on TPO, we produced six TPO
mutants by overlap extension PCR (Table
V) according to sequence alignment. The
three-dimensional model revealed that region 356-382 presented two
exposed loops around the putative interaction site; thus two distinct
TPO mutants, each showing a mutated region on each distinct loop
(TPO353-363 and TPO377-386) were constructed.
As control, a TPO mutant, bearing a mutated region (residues 506-514)
not identified by mimotope alignment but located close to the IBS (Fig.
4, A and B), was also generated. All the
mutations were performed according to two rules: (i) when different,
the amino acids were replaced by those present in the primary sequence
of the human MPO for the mutants TPO353-363,
TPO377-386, TPO506-514, and
TPO713-720 or the primary sequence of the CCP domain
of the factor H protein for the mutant TPO766-775; and
(ii) when similar, the residues were present in the TPO/MPO and
TPO/factor H sequences, amino acids were replaced by uncharged residues
(alanine or leucine). For example the amino acid Arg355 was
replaced by Ala355 in the mutated protein
(TPO353-363). For the mutant TPO737-740,
located in the fringe of the MPO and CCP modules, we used only alanine
or leucine substitutions. Finally, the Cys768 residue was
not changed because it could contribute to the overall folding of the
TPO molecule by formation of a disulfide bridge in the CCP-like
domain.
Expression and Analysis of Wild-type and Mutated TPO--
To
demonstrate that the mutated regions are a part of the IDR, the wt and
the chimeric proteins were expressed in eukaryotic cells by using the
new Flp-InTM expression system able to produce a similar level of
different proteins of interest. By immunofluorescence, we observed a
defect in the transport of the mutant TPO766-775 to the
plasma membrane (data not shown). A similar observation was described
recently by Umeki et al. (51), who showed that when the
Gly771 is replaced by Arg771 in the TPO genes
of patients, this results in a localization defect (accumulation of
mutated TPO in the endoplasmic reticulum) and causes congenital
hypothyroidism. To overcome this problem, we decided to extract all
membrane proteins from wt and stably transfected CHO and to study them
by Western blotting and ELISA. All antibodies tested by Western
blotting were reactive, under nonreducing conditions, with a protein of
~110 kDa, corresponding to the hTPO (Fig.
5A). The polyclonal rabbit
antiserum was able to recognize all TPO mutants whatever the
conditions. The mutations did not globally affect the expression level
for most of the chimeric proteins, even though the expression of mutant
TPO737-740 was lower than that of the five others. This
lower expression was confirmed with all mAb tested. As expected, mAb
47, which recognizes a linear determinant (713-721) (42), did not bind to the mutant TPO713-720 but reacted with all of the other
mutants under native or denatured conditions. Surprisingly, mAb 6F5, as
well as aAb T13, was not reactive under denaturing conditions with wt
or mutated TPO (Fig. 5A). Furthermore, only the binding of
mAb 6F5 to the mutant TPO766-775 was eliminated under
native conditions. These results demonstrate that mAbs 6F5 and T13 are
directed against two different conformational epitopes that overlap in
region 766-775. We conclude from these observations that (i) the
folding of the TPO mutants is homologous to that of the wt TPO and (ii)
in the mutated regions, amino acids 766-775 compose only a part of the
conformational epitope recognized by mAb 6F5. Finally, the reactivities
of aAb T13 to native proteins are different depending on the mutants.
Strong signals were observed with wt TPO and mutant
TPO506-514, whereas binding of aAb T13 to mutants
TPO353-363, TPO377-514, and
TPO766-775 was markedly decreased or totally abrogated for
mutant TPO713-720. To confirm that the epitope of aAb T13
is in both the MPO- and the CCP-like domains, mAbs 47 and 6F5 were used
in competition with aAb T13 for the binding to TPO in an ELISA. As
shown in Fig. 5B, the murine mAbs competed in a
dose-dependent manner with aAb T13 and partially inhibited
the binding of aAb T13, an inhibition of 45 and 35% for mAb 47 and
6F5, respectively, with a molar excess of 100-fold for the inhibitor.
No inhibition was observed with a murine mAb as control. Nevertheless,
we could not exclude a steric hindrance by using the competition
method, therefore we studied the interaction of mAb 6F5 and aAb T13
with membrane proteins from wt and mutated TPO by ELISA (Fig.
6A). Whereas mAb 6F5
recognized similarly the wt and mutant TPO (except for mutant
TPO766-775), aAb T13 showed a significant and specific
binding both to wt TPO and, as expected, to TPO506-514.
However, the other mutations significantly reduced the T13 aAb binding
on TPO. Mutations 353-363, 377-386, and 766-775 partially affected
aAb T13 recognition of TPO, whereas amino acid replacements in 713-720
totally abolished the binding of aAb T13 to TPO. These results
reinforce the previous observations obtained by Western blotting (Fig.
5A).
Analysis of TPO Mutant Recognition by Patient Serum--
To place
this study in a pathological context, the binding of anti-TPO aAbs from
sera of patients suffering from AITD, other autoimmune affections, and
healthy donors was investigated by ELISA as performed above with mAb
6F5 and aAb T13 (Fig. 6B). Whereas the sera from healthy
donors or patients suffering from autoimmune pathologies (type 1 diabetes, systemic lupus erythematosus, rheumatoid arthritis, or
vascularitis) did not react with any clone (data not shown), all sera
from patients with Hashimoto's thyroiditis or Graves' disease
specifically bound to wt TPO in a dose-dependent fashion.
Control mutations in positions 506-514 did not globally affect the
binding of patient serum, whereas mutations in other positions
decreased the binding to TPO. Interestingly, these mutants could be
classified into two groups, which weakly (TPO377-386) or
strongly (TPO353-363, TPO713-720,
TPO766-775) affected the binding of the anti-TPO aAbs. In
contrast to competition studies between anti-TPO mAb and polyclonal
patients' sera described previously by our group and others (13, 14,
25, 40, 41), where the results could be biased by steric hindrance, the
effects of each mutation on the binding of all polyclonal anti-TPO sera studied here resulted in the same pattern for the particular mutation. Similar results were obtained by McLachlan and Rapoport's group (30).
The three-dimensional structure of the hTPO molecule has not been
solved by crystallographic studies, even if low resolution diffracting
crystals have been obtained (17, 18). However, analysis of the
three-dimensional structure of hTPO as assessed by homology modeling
denotes a highly complex structure comprising three distinct modules,
MPO-, CCP-, and EGF-like domains (13). This highly organized
architecture of the TPO autoantigen is a plausible explanation for the
anti-TPO aAb reactivity, which is directed mainly to a discontinuous
IDR on the native structure of their autoantigen (19, 52). Previous
investigations aimed at defining the IDR used rabbit polyclonal
antisera directed against TPO polypeptide fragments (13, 24) or murine
mAbs (25) in competition with human aAbs. These studies, even if they
lead to a better knowledge of the IDR, used Abs produced by
animal immunization which do not exactly reflect the hTPO-specific aAb repertoire in AITD. Others studies (33-35), which used eukaryotic cells transfected with MPO-TPO chimeras, did not lead to the structural identification of the IDR, probably because of the low antigen expression of the transfected clones and/or high heterogeneity of TPO
expression by the clones. More recently, Guo et al. (30) clearly excluded the EGF-like domain as being a part of the IDR and
localized this region in the junction between MPO- and CCP-like domains
but with a much larger extension in the MPO module. Although these
results are crucial to localize globally the IDR, up to now few data
have been reported on the identification of restricted regions on these
domains recognized by human aAbs and on the location of the IDR in the
three-dimensional structure of hTPO.
We hypothesize that phage-displayed peptide technology followed by
sequence alignment is presently the most powerful strategy to position
discontinuous epitopes on a highly complex structure like hTPO (38, 39,
53). In the present study, we have applied this technology to the well
characterized human anti-TPO aAb T13 in an attempt to identify peptides
able to mimic its discontinuous and immunodominant epitope. After
sequence alignments between critical peptide motifs and the primary
sequence of hTPO, we clearly demonstrated by directed mutagenesis on
hTPO and generation (with the new Flp-In system) of isogenic stable
mammalian cells expressing the same level of wt and mutated TPO that
four restricted regions (regions 353-363, 377-386, and 713-720 in
the MPO-like domain and region 766-775 in the CCP-like domain of TPO)
delineated the IBS on the three-dimensional structure of hTPO.
Moreover, by comparison with wt and mutant TPO, aAb T13 and patients'
sera showed different binding levels, depending on the mutated region.
Whereas mutations in region 377-386 only weakly affected the binding
of human aAbs, mutations in regions 353-363, 713-720, and 766-775
strongly reduced their recognition (Fig. 6, A and
B).
These data emphasize the discontinuous nature of the IDR and provide
new insights into the MPO- and CCP-like positioning. Indeed, the
CCP-like module needs to be close to the MPO-like domain to form the
putative interaction surface with the human recombinant aAb T13. As
shown in Fig. 4B, regions 377-386, 713-720, and 766-775
(constituting the extremities of the IBS) delineate an autoantigenic
surface. The antigen/antibody interfacial areas identified for other
discontinuous epitopes by x-ray crystallographic analyses and NMR
studies have been reported to be between 650 and 1,000 Å2
(54, 55). Thus, to form an IBS compatible with these areas, the
distance between the regions 377-386, 713-720, and 766-775 must be
close enough to each other. The structural folding of the three domains
constituting the hTPO appears to be homologous with known proteins such
as MPO, the CCP module of the factor H, and EGF (13), but no
information is presently available concerning the exact folding of one
domain in relation to the others. Estienne et al. (15)
underlined the high flexibility of the hinge region separating the CCP-
and EGF-like domains. We hypothesize a similar flexibility between the
MPO- and CCP-like domains to form the IBS. Furthermore, we propose two
possibilities to explain this phenomenon: either the MPO-like and
CCP-like domains interact directly by interactions such as hydrogen
bonds or salt links or the flexibility of the hinge region results in
constant rearrangement among the three modules, and the binding of an
aAb (like T13) on at least two domains "freezes" the structure into
a stable conformation. If the latter alternative is true, this could
explain why it is so difficult to obtain high resolution diffracting
crystals of the TPO ectodomain (17, 18). Co-crystallization of TPO with
a specific Fab stabilizing the structure should solve this problem definitively.
Using the phage-displayed peptide technology, we generated peptides
recognized by aAb T13 and identified critical motifs
(LXPEXD, QSYP, and EX(E/D)PPV)
involved in its interaction with these mimotopes. Controversial results
have assigned the mAb 47/C21 epitope (713-721) sometimes inside the
IDR (25, 32, 56) and sometimes outside this region (23, 35, 57).
McLachlan and Rapoport's group (32) deduced from their studies using
recombinant antibody TR1.9 that TPO residue Lys713, which
is within the human aAb IDR, lies at the fringe of the mouse mAb 47/C21
epitope (32), as is also suggested for the 713-720 region identified
by our investigation. Interestingly, other residues from this region
such as Pro715, Glu716, and Asp717
align with the motif PEXD identified in T13-specific
mimotopes from Family 1, suggesting that they could contribute to the
aAb binding to TPO. Furthermore, the Tyr772 residue,
recently identified by the Ruf and Carayon's group (15) as critical
for immunodominance, is involved in the region 766-775 we
characterized as belonging to the IDR. The QSYP motif, identified as
critical for aAb T13 binding to mimotopes from Family 2, comprises three residues also found in the 766-775 region of the CCP-like domain. We hypothesize that the QSYP motif mimics a motif composed of
residues Tyr772 close to Ser667, and
Gln775 in the three-dimensional TPO structure. These
residues could be contact amino acids within the aAb immunodominant
epitope. Of interest, the other strongly contributing region, 353-363, also shows residues (Ser359, Tyr363) aligned
with the mimotope motif of Family 2 (QSYP) identified by aAb T13. These
observations should give us some leads to carry out rational
mutagenesis experiments on hTPO and precisely identify the amino acids
involved in the IDR, even if we cannot formally exclude that one of
these regions contains amino acids essential for the folding of the IDR
and is not directly involved in the interaction with aAbs.
Another previously described region, called C2 (16, 58), has been
restricted to region 599-617 on TPO and has been defined as one major
autoantigenic determinant by inhibition of human anti-TPO aAbs with
rabbit polyclonal antisera directed against this peptide (13).
Inspection of the three-dimensional TPO model shows that this epitope
is located on the opposite side of the MPO-like domain with regard to
the IBS we describe here. This observation could be explained by the
fact that overlapping antigenic domains have been characterized on the
TPO molecule. Particularly, two overlapping immunodominant domains,
named A and B (23, 25), have been described based on the competition
between polyclonal anti-TPO from patients and human or murine Abs. It
is important to note that the A domain defined by the murine mAbs (25)
corresponds to the B domain defined by the human Fabs (23) and vice
versa. As we used a human anti-TPO aAb in our experiments as did
McLachlan and Rapoport's group (23), we chose to use the nomenclature of the IDR/A and B defined by this group. Consequently, the
Lys713 residue is part of the epitope recognized by the
IDR/B-specific antibody TR1.9 (32), and region 713-720 belongs to the
immunodominant epitope mapped by aAb T13. Furthermore, mAb 47, which
recognizes the IDR/B, competes in part with the human aAb T13 for
binding to the TPO molecule (Fig. 5B). In addition,
non-IGKV1-39 aAbs such as IGLV1-40 aAb T13 have been described to
interact with TPO IDR/B, whereas antibodies that use the IGKV1-39 germ
line light chain gene have been assigned to IDR/A recognition (23). Taken together, we assume that the human recombinant aAb T13 recognizes the IDR/B on the TPO molecule, even if we cannot formally excluded cross-reaction with the A domain caused by overlapping between IDR/A
and B.
In conclusion, by combining phage-displayed peptide technology with
mimotope sequence alignment on the TPO molecule, we have precisely
identified three regions (353-363, 377-386, and 713-720) belonging
to the MPO-like domain and one (766-775) located on the CCP-like
domain as being a part of the IDR. We have also localized on the
three-dimensional model of hTPO the IBS, which comprises these four
regions that are crucial for the binding of human anti-TPO aAbs. This
IBS is recognized (i) by the human recombinant aAb T13 obtained with
antibody phage-displayed library constructed using TPO-purified B cells
from Graves' disease patients (40, 41) and (ii) by anti-TPO aAbs from
sera of patients suffering from Hashimoto's and Graves' thyroid
diseases. Moreover, our demonstration was obtained by using the
full-length TPO autoantigen expressed by eukaryotic cells. Because
TPO-specific aAbs from patients' sera are directed against this IDR,
such a finding would certainly improve our understanding of TPO
recognition by the immune system during AITD. Furthermore, the
synthesis of a constrained "mini-TPO," which mimics the IDR we
identified, could be of great diagnostic value to detect TPO-specific
aAbs. From a therapeutic point of view, the findings presented here
should help us to design rational bioactive peptides able to block
antigen presentation by B cell membrane-bound TPO-specific aAbs (6, 7),
leading to the autoimmune T cell response in AITD and to block the
cytotoxic activity of aAbs from patients suffering from AITD, both of
these mechanisms resulting in thyroid destruction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Description of sera from patients suffering from autoimmune thyroid
diseases
-galactosidase level. A single clone was selected and amplified for
further studies. The generation of wt or mutated TPO was obtained by
cotransfection, into the selected ZeocinTM-resistant CHO host cell
line, of the hygromycin-resistant pcDNA5/FRT plasmids bearing each
form of TPO and the pOG44 plasmid which constitutively expresses the
Flp recombinase. Two days later, the cells were fed with growth medium
supplemented with 300 µg/ml hygromycin B (Invitrogen). The clones of
interest were selected for hygromycin resistance, ZeocinTM
sensitivity, and lack of
-galactosidase activity.
80 °C. The protein concentrations were evaluated by the BCA
protein assay reagent (Pierce).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (16K):
[in a new window]
Fig. 1.
Purity and specificity of aAb T13.
Purity and specificity were studied as described under "Experimental
Procedures." A, each sample (2 µg of proteins) was
charged on two 8% polyacrylamide-SDS gels. The first was stained with
Coomassie Brilliant Blue R-250 (lanes 1 and 2)
and the second analyzed by Western blotting (lanes 3 and
4). Lanes 1 and 3 show the baculovirus
supernatant before purification. Lanes 2 and 4 show the aAb T13 after purification (black arrow). The
molecular masses (MW) in kDa are shown on the
left of the figure. B, the specificity of aAb T13
was analyzed by ELISA. The binding of aAb T13 (open circles)
and human polyclonal IgG1 as control (closed
triangles) on TPO is shown.
4
s
1, average of three experiments), whereas hTPO did not
bind to the irrelevant Ab.
Kinetic parameters of aAb T13 as assessed by real time analysis
Peptide sequences selected by phage-displayed peptide library
Classification of mimotopes by sequence homology
View larger version (30K):
[in a new window]
Fig. 2.
Analysis of T13-specific mimotopes by the
Spot technology. A, immunoreactivity of peptides with
aAb T13 was studied by the Spot technology. aAb T13 reactivity
complexed with the secondary antibody and the secondary antibody
reactivity alone are shown on the right (see "Experimental
Procedures"). B, identification by alanine scanning of
critical residues of mimotopes. A series of alanine analogs of each
peptide sequences found previously to be reactive with aAb T13 was
prepared and tested by the Spot method. Replaced amino acid
indicates which residue was replaced by Ala (or Gly when the natural
residue is Ala). T13-specific reactivity on the Spot membrane is
represented at the top of each histogram, and the percent of
binding of aAb T13 to each analog compared with the wt sequence is
shown by the histograms. Only one representative mimotope for each
family is exemplified. Underlined amino acids represent the
critical residues observed for several peptides stemming from each
family.
View larger version (36K):
[in a new window]
Fig. 3.
Sequence alignment between mimotopes and hTPO
sequences. The alignments were obtained as described under
"Experimental Procedures." The critical motifs of T13-specific
mimotopes are underlined. Identical residues between
mimotopes and the primary sequence of hTPO are represented in
bold letters.
View larger version (29K):
[in a new window]
Fig. 4.
Location of the mutated regions on a
three-dimensional ribbon diagram of the structure of hTPO.
A, ribbon diagram of the structure of hTPO showing the MPO-,
CCP-, and EGF-like domains, reproduced with permission (13). This
representation corresponds to the juxtaposition of the
three-dimensional model of each domain. The mutations produced by
"guided" mutagenesis are represented, positions 353-363 in
yellow, 377-386 in red, 506-514 in
green, 713-720 in blue, 737-740 in
pink, and 766-775 in orange. The flexibility of
the hinge regions is represented by a white arrow.
B, ribbon diagram representing the possible folding of the
CCP-like domain on the MPO-like domain (to simplify the figure, the
EGF-like domain is not represented). A white arrow indicates
the plausible movement of the CCP domain to form the IBS virtually
represented by three yellow dotted lines. The junction
between MPO- and CCP-like domains is shown by white dotted
lines. The distance (in Å) between the regions 377-387 and
713-720 is shown. The model was adjusted by using Swiss-PDB viewer
3.7b2 freeware available at www.expasy.ch/spdbv.
Comparison of the amino acid sequences between wild-type and mutated
TPO
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[in a new window]
Fig. 5.
Western blotting and ELISA evidence that aAb
T13 recognizes both MPO- and CCP-like domain. A,
Western blots with native or denatured membrane proteins were performed
as indicated under "Experimental Procedures." mAb 47 (10 µg/ml),
mAb 6F5 (10 µg/ml), aAb T13 (10 µg/ml), and rabbit polyclonal
antiserum (1:300) were used and are identified on the right
of the figure. TPO-specific bands are shown by black arrows,
and the molecular masses in kDa are given on the left.
B, competitive ELISA experiments were performed with mAb 47, mAb 6F5, and murine mAb 2C2 against digoxigenin (control) as competitor
for the binding of aAb T13 to TPO (see "Experimental
Procedures").
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[in a new window]
Fig. 6.
The binding of anti-TPO mAb or patients'
sera to membrane proteins is affected by TPO mutations. The
effects of TPO mutations on the binding of anti-TPO mAb or patients'
sera binding were evaluated by ELISA. A, the results
obtained with aAb T13 or mAb 6F5 are shown. B, patients'
sera with Hashimoto's thyroiditis (patient 1 ( ), patient 2 (
),
patient 3 (
), patient 4 (
), and patient 5 (*)) and Graves'
disease (patient 6 (
), patient 7 (
), patient 8 (
), patient 9 (
), and patient 10 (x)) were tested for their ability to bind
to the membrane proteins extracted from wt or stably transfected CHO
(see "Experimental Procedures"). The results are given in
absorbance after subtraction of the background values corresponding to
the binding of sera to membrane proteins from nontransfected CHO and
are expressed as the mean ± S.D. of duplicate values.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Dr. S. L. Salhi for carefully reading the manuscript. We also thank Drs. J. Ruf and P. Carayon for providing the mAb 47; Drs. B. J. Sutton, P. Hobby, and J. P. Banga for permission to use the three-dimensional model of TPO; and Drs. L. Baldet and A. M. Puech for providing patient sera. We acknowledge Dr. N. Chapal for performing scFv T13 characterization, S. Bonniol and Dr. D. Laune for assistance in the cloning of aAb T13, and A. Ozil and M. Ozil for technical assistance with baculovirus expression. We are also grateful to B. Nguyen for expert technical assistance and S. Villard for the Spot synthesis.
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FOOTNOTES |
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* 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.
§ Recipient of a fellowship from the CNRS and the Région Languedoc-Roussillon. To whom correspondence should be addressed. Tel.: 33-467-548-609; Fax: 33-467-548-610; E-mail: damienbresson@yahoo.fr.
Supported by the Agence National de Recherche contre le Sida.
Supported by the Association pour la Recherche contre le Cancer.
Published, JBC Papers in Press, December 24, 2002, DOI 10.1074/jbc.M211930200
2 D. Bresson, M. Cerutti, G. Devauchelle, M. Pugnière, F. Roquet, C. Bès, C. Bossard, T. Chardès, and S. Péraldi-Roux, unpublished data.
3 D. Bresson, M. Cerutti, G. Devauchelle, S. Bonniol, D. Laune, T. Chardès, and S. Péraldi-Roux, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: TPO, thyroid peroxidase; aAb, autoantibody; AITD, autoimmune thyroid disease(s); CCP, complement control protein; CHO, Chinese hamster ovary; EGF, epidermal growth factor; ELISA, enzyme-linked immunosorbent assay; hTPO, human TPO; IBS, immunodominant binding surface; IDR, immunodominant region; mAb, monoclonal antibody; MPO, myeloperoxidase; PBS, phosphate-buffered saline; wt, wild-type.
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1. | Kaufman, K. D., Rapoport, B., Seto, P., Chazenbalk, G. D., and Magnusson, R. P. (1989) J. Clin. Invest. 84, 394-403[Medline] [Order article via Infotrieve] |
2. | Zimmer, K. P., Scheumann, G. F., Bramswig, J., Bocker, W., Harms, E., and Schmid, K. W. (1997) Histochem. Cell Biol. 107, 115-120[CrossRef][Medline] [Order article via Infotrieve] |
3. | Taurog, A., Dorris, M. L., and Doerge, D. R. (1996) Arch. Biochem. Biophys. 330, 24-32[CrossRef][Medline] [Order article via Infotrieve] |
4. | McLachlan, S. M., and Rapoport, B. (1992) Endocr. Rev. 13, 192-206[Medline] [Order article via Infotrieve] |
5. | McLachlan, S. M., and Rapoport, B. (2000) Int. Rev. Immunol. 19, 587-618[Medline] [Order article via Infotrieve] |
6. | Guo, J., Quaratino, S., Jaume, J. C., Costante, G., Londei, M., McLachlan, S. M., and Rapoport, B. (1996) J. Immunol. Methods 195, 81-92[CrossRef][Medline] [Order article via Infotrieve] |
7. | Guo, J., Wang, Y., Rapoport, B., and McLachlan, S. M. (2000) Clin. Exp. Immunol. 119, 38-46[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Guo, J.,
Jaume, J. C.,
Rapoport, B.,
and McLachlan, S. M.
(1997)
J. Clin. Endocrinol. Metab.
82,
925-931 |
9. | Metcalfe, R. A., Oh, Y. S., Stroud, C., Arnold, K., and Weetman, A. P. (1997) Autoimmunity 25, 65-72[Medline] [Order article via Infotrieve] |
10. | Parkes, A. B., Othman, S., Hall, R., John, R., Richards, C. J., and Lazarus, J. H. (1994) J. Clin. Endocrinol. Metab. 79, 395-400[Abstract] |
11. | Rodien, P., Madec, A. M., Ruf, J., Rajas, F., Bornet, H., Carayon, P., and Orgiazzi, J. (1996) J. Clin. Endocrinol. Metab. 81, 2595-2600[Abstract] |
12. | Chiovato, L., Bassi, P., Santini, F., Mammoli, C., Lapi, P., Carayon, P., and Pinchera, A. (1993) J. Clin. Endocrinol. Metab. 77, 1700-1705[Abstract] |
13. |
Hobby, P.,
Gardas, A.,
Radomski, R.,
McGregor, A. M.,
Banga, J. P.,
and Sutton, B. J.
(2000)
Endocrinology
141,
2018-2026 |
14. |
Estienne, V.,
Blanchet, C.,
Niccoli-Sire, P.,
Duthoit, C.,
Durand-Gorde, J. M.,
Geourjon, C.,
Baty, D.,
Carayon, P.,
and Ruf, J.
(1999)
J. Biol. Chem.
274,
35313-35317 |
15. |
Estienne, V.,
Duthoit, C.,
Blanchin, S.,
Montserret, R.,
Durand-Gorde, J. M.,
Chartier, M.,
Baty, D.,
Carayon, P.,
and Ruf, J.
(2002)
Int. Immunol.
14,
359-366 |
16. |
Arscott, P. L.,
Koenig, R. J.,
Kaplan, M. M.,
Glick, G. D.,
and Baker, J. R.
(1996)
J. Biol. Chem.
271,
4966-4973 |
17. | Gardas, A., Sohi, M. K., Sutton, B. J., McGregor, A. M., and Banga, J. P. (1997) Biochem. Biophys. Res. Commun. 234, 366-370[CrossRef][Medline] [Order article via Infotrieve] |
18. | Hendry, E., Taylor, G., Ziemnicka, K., Grennan Jones, F., Furmaniak, J., and Rees Smith, B. (1999) J. Endocrinol. 160, R13-R15[Abstract] |
19. | Portolano, S., Chazenbalk, G. D., Seto, P., Hutchison, J. S., Rapoport, B., and McLachlan, S. M. (1992) J. Clin. Invest. 90, 720-726[Medline] [Order article via Infotrieve] |
20. | Finke, R., Seto, P., and Rapoport, B. (1990) J. Clin. Endocrinol. Metab. 71, 53-59[Abstract] |
21. | Frorath, B., Abney, C. C., Scanarini, M., Berthold, H., Hunt, N., and Northemann, W. (1992) J. Biochem. (Tokyo) 111, 633-637[Abstract] |
22. | Banga, J. P., Barnett, P. S., Ewins, D. L., Page, M. J., and McGregor, A. M. (1990) Autoimmunity 6, 257-268[Medline] [Order article via Infotrieve] |
23. | Chazenbalk, G. D., Portolano, S., Russo, D., Hutchison, J. S., Rapoport, B., and McLachlan, S. M. (1993) J. Clin. Invest. 92, 62-74[Medline] [Order article via Infotrieve] |
24. | Gardas, A., Watson, P. F., Hobby, P., Smith, A., Weetman, A. P., Sutton, B. J., and Banga, J. P. (2000) Redox Rep. 5, 237-241[CrossRef][Medline] [Order article via Infotrieve] |
25. | Ruf, J., Toubert, M. E., Czarnocka, B., Durand-Gorde, J. M., Ferrand, M., and Carayon, P. (1989) Endocrinology 125, 1211-1218[Abstract] |
26. | Zanelli, E., Henry, M., and Malthiery, Y. (1992) Clin. Exp. Immunol. 87, 80-86[Medline] [Order article via Infotrieve] |
27. | Zanelli, E., Henry, M., and Malthiery, Y. (1993) Cell. Mol. Biol. (Noisy-le-grand) 39, 491-501 |
28. | Grennan Jones, F., Ziemnicka, K., Sanders, J., Wolstenholme, A., Fiera, R., Furmaniak, J., and Rees Smith, B. (1999) Autoimmunity 30, 157-169[Medline] [Order article via Infotrieve] |
29. | Ewins, D. L., Barnett, P. S., Tomlinson, R. W., McGregor, A. M., and Banga, J. P. (1992) Autoimmunity 11, 141-149[Medline] [Order article via Infotrieve] |
30. |
Guo, J.,
McLachlan, S. M.,
and Rapoport, B.
(2002)
J. Biol. Chem.
277,
40189-40195 |
31. |
Estienne, V.,
Duthoit, C.,
Vinet, L.,
Durand-Gorde, J. M.,
Carayon, P.,
and Ruf, J.
(1998)
J. Biol. Chem.
273,
8056-8062 |
32. |
Guo, J.,
Yan, X. M.,
McLachlan, S. M.,
and Rapoport, B.
(2001)
J. Immunol.
166,
1327-1333 |
33. | Nishikawa, T., Nagayama, Y., Seto, P., and Rapoport, B. (1993) Endocrinology 133, 2496-2501[Abstract] |
34. | Nishikawa, T., Rapoport, B., and McLachlan, S. M. (1994) J. Clin. Endocrinol. Metab. 79, 1648-1654[Abstract] |
35. | Nishikawa, T., Rapoport, B., and McLachlan, S. M. (1996) Endocrinology 137, 1000-1006[Abstract] |
36. |
Ganglberger, E.,
Grunberger, K.,
Sponer, B.,
Radauer, C.,
Breiteneder, H.,
Boltz-Nitulescu, G.,
Scheiner, O.,
and Jensen-Jarolim, E.
(2000)
FASEB J.
14,
2177-2184 |
37. |
Hong, S. S.,
Karayan, L.,
Tournier, J.,
Curiel, D. T.,
and Boulanger, P. A.
(1997)
EMBO J.
16,
2294-2306 |
38. |
del Rincon, I.,
Zeidel, M.,
Rey, E.,
Harley, J. B.,
James, J. A.,
Fischbach, M.,
and Sanz, I.
(2000)
J. Immunol.
165,
7011-7016 |
39. |
Myers, M. A.,
Davies, J. M.,
Tong, J. C.,
Whisstock, J.,
Scealy, M.,
Mackay, I. R.,
and Rowley, M. J.
(2000)
J. Immunol.
165,
3830-3838 |
40. |
Chapal, N.,
Chardès, T.,
Bresson, D.,
Pugnière, M.,
Mani, J. C.,
Pau, B.,
Bouanani, M.,
and Peraldi-Roux, S.
(2001)
Endocrinology
142,
4740-4750 |
41. | Bresson, D., Chardès, T., Chapal, N., Bès, C., Cerutti, M., Devauchelle, G., Bouanani, M., Mani, J. C., and Peraldi-Roux, S. (2001) Hum. Antibodies 10, 109-118[Medline] [Order article via Infotrieve] |
42. | Finke, R., Seto, P., Ruf, J., Carayon, P., and Rapoport, B. (1991) J. Clin. Endocrinol. Metab. 73, 919-921[Abstract] |
43. | Poul, M. A., Cerutti, M., Chaabihi, H., Devauchelle, G., Kaczorek, M., and Lefranc, M. P. (1995) Immunotechnology 1, 189-196[CrossRef][Medline] [Order article via Infotrieve] |
44. | Poul, M. A., Cerutti, M., Chaabihi, H., Ticchioni, M., Deramoudt, F. X., Bernard, A., Devauchelle, G., Kaczorek, M., and Lefranc, M. P. (1995) Eur. J. Immunol. 25, 2005-2009[Medline] [Order article via Infotrieve] |
45. | Bonnycastle, L. L., Mehroke, J. S., Rashed, M., Gong, X., and Scott, J. K. (1996) J. Mol. Biol. 258, 747-762[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Ferrieres, G.,
Villard, S.,
Pugnière, M.,
Mani, J. C.,
Navarro-Teulon, I.,
Rharbaoui, F.,
Laune, D.,
Loret, E.,
Pau, B.,
and Granier, C.
(2000)
Eur. J. Biochem.
267,
1819-1829 |
47. |
Laune, D.,
Molina, F.,
Ferrieres, G.,
Mani, J. C.,
Cohen, P.,
Simon, D.,
Bernardi, T.,
Piechaczyk, M.,
Pau, B.,
and Granier, C.
(1997)
J. Biol. Chem.
272,
30937-30944 |
48. | Corpet, F. (1988) Nucleic Acids Res. 16, 10881-10890[Abstract] |
49. | Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 51-59[CrossRef][Medline] [Order article via Infotrieve] |
50. | Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467[Abstract] |
51. | Umeki, K., Kotani, T., Kawano, J. I., Suganuma, T., Yamamoto, I., Aratake, Y., Furujo, M., and Ichiba, Y. (2002) Eur. J. Endocrinol. 146, 491-498[Medline] [Order article via Infotrieve] |
52. |
Rapoport, B.,
and McLachlan, S. M.
(2001)
J. Clin. Invest.
108,
1253-1259 |
53. | Lang, S., Xu, J., Stuart, F., Thomas, R. M., Vrijbloed, J. W., and Robinson, J. A. (2000) Biochemistry 39, 15674-15685[CrossRef][Medline] [Order article via Infotrieve] |
54. |
Davies, D. R.,
and Cohen, G. H.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
7-12 |
55. |
Spiegel, P. C., Jr.,
Jacquemin, M.,
Saint-Remy, J. M.,
Stoddard, B. L.,
and Pratt, K. P.
(2001)
Blood
98,
13-19 |
56. |
Czarnocka, B.,
Janota-Bzowski, M.,
McIntosh, R. S.,
Asghar, M. S.,
Watson, P. F.,
Kemp, E. H.,
Carayon, P.,
and Weetman, A. P.
(1997)
J. Clin. Endocrinol. Metab.
82,
2639-2644 |
57. | Chazenbalk, G. D., Costante, G., Portolano, S., McLachlan, S. M., and Rapoport, B. (1993) J. Clin. Endocrinol. Metab. 77, 1715-1718[Abstract] |
58. | Libert, F., Ludgate, M., Dinsart, C., and Vassart, G. (1991) J. Clin. Endocrinol. Metab. 73, 857-1860[Abstract] |