From the Unit 38 of INSERM and the Laboratoire de Biochimie
Endocrinienne et Métabolique, Faculté de
Médecine, 27, boulevard Jean Moulin,
F-13385 Marseille Cedex 5, France
To investigate the B-cell autoimmune epitopes on
human thyroid peroxidase (TPO), we generated proteolytic peptides by
enzymatic hydrolysis of TPO in nondenaturing and nonreducing
conditions. The hydrolysate was chromatographed on a reverse phase
column. We eluted a material immunoreactive with both a TPO monoclonal antibody recognizing a linear epitope (mAb47, amino acid 713-721) and
TPO autoantibodies (aAb) from patients. The aAb immunoreactivity, but
not that of mAb47, was lost after reduction. Western blots after
electrophoresis without reduction showed that the aAb and mAb47 were
immunoreactive with a 66-kDa band and that aAb identified a doublet at
20 kDa. For electrophoresis under reducing conditions, the 66-kDa band
resolved into two peptides of 40 and 26 kDa, whereas the doublet at 20 kDa remained unchanged. None of these reduced peptides was
immunoreactive with aAb, whereas the 40-kDa peptide was immunoreactive
with mAb47. The 40-kDa peptide extends from amino acid 549 to 933 of
TPO, and its last 192 amino acids overlap the autoimmune 20-kDa
peptide. After iodine labeling, the 20-kDa peptide lost its
immunoreactivity. We conclude that the C-terminal end of the
extracellular part of TPO, which includes all the tyrosine residues of
the 20-kDa peptide, contains at least one conformational B-cell epitope
involved in autoimmune thyroid diseases.
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INTRODUCTION |
Thyroperoxidase (TPO)1
is a membrane-bound enzyme that faces the colloid and that acts at the
apical pole of the thyrocytes. TPO catalyzes the iodination of
thyroglobulin and the coupling of some iodotyrosine residues to form
thyroid hormone residues. This catalysis is under thyrotropin control
through its specific receptor (1, 2). Like thyroglobulin and the
thyrotropin receptor, TPO is one of the main autoantigens (aAg) in
autoimmune thyroid disease (AITD). However, the immune response to this
sequestered aAg is not clear (for review, see Ref. 3). After TPO was
identified as microsomal aAg (4, 5), many studies investigated the human immune response to this enzyme. We showed there were two autoimmune domains on the surface of the molecule (6); this was
confirmed by another group (7, 8). Disappointingly, no difference was
observed in autoantibody (aAb) response to TPO for patients with
Graves' disease and Hashimoto's thyroiditis, the two well defined
AITD (9).
One of the major tasks in AITD is to identify the immunodominant B-cell
epitopes of the main aAg. This may help explain the mechanisms causing
immunopathological states and, consequently, may provide targets for
diagnosis and therapeutic strategies. TPO is a valuable model for such
studies, given the preponderance of its corresponding aAb in AITD.
Moreover, TPO is implicated in the physiological function of the
thyroid, and aAb binding to TPO might impair thyroid hormone synthesis
through cytotoxic processes (10), thus leading to hypothyroidism. Some
authors (11-14) but not others (5, 15) claimed that TPO aAb inhibit the catalytic activity of the enzyme at the iodine and the aromatic sites. However, aAb from patients with thyroiditis may block the enzyme
function by binding to epitopes different from the enzymatic sites, as
speculated for bispecific thyroglobulin and TPO aAb (16).
Many attempted to localize and identify the main TPO B-cell
autoepitopes forming the immunodominant regions targeted by the pathologic aAb from patients with AITD. Various linear TPO epitopes were identified through cDNA sublibraries or recombinant bacterial proteins. Others, however, using a eukaryotic expression system to
preserve the three-dimensional structure of the protein, claimed that
TPO B-cell autoepitopes are conformational (for review, see Refs.
17-19). Since molecular biology techniques identify only minimum
peptides not truly representative of the conformational B-cell
epitopes, we mapped the immunodominant region of TPO by an alternative
approach: proteolytic peptides generated by enzymatic hydrolysis of the
native immunopurified human TPO. Through pilot experiments, we selected
endopeptidase Lys-C, which can cleave at 26 lysine residues along the
amino acid sequence of TPO. We found an extracellular conformational
B-cell epitope susceptible to reduction and iodination near the
membrane anchorage and the spanning region of TPO.
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EXPERIMENTAL PROCEDURES |
Murine mAb to TPO and aAb from Patients with AITD--
We used
eight TPO mAb, previously produced and characterized, directed to
epitopes from two immunodominant antigenic regions of TPO (6). The TPO
aAb were immunopurified as described (20) from sera of 40 adult
patients thought to have AITD on the basis of clinical examination and
selected for their high titer in TPO aAb as assessed by Dynotest
(BRAHMS diagnostica, Berlin, Germany). The TPO mAb and aAb were titered
for their TPO reactivity in ELISA and used at saturating dilution in
this work.
Purification and Hydrolysis of TPO--
TPO was immunopurified
from sodium deoxycholate-solubilized microsomes from Graves' thyroid
tissue (4). Native, purified TPO (at a final concentration of 3 mg/ml)
was treated with endoproteinase Lys-C (EC 3.4.21.50 sequencing grade
from Lysobacter enzymogenes (Boehringer Mannheim, Germany)
at an enzyme to substrate ratio of 1:100 (w/w) in 50 mM
NH4HCO3 with 10% acetonitrile for 18 h at
37 °C. Enzymatic digestion was stopped by freeze-drying the hydrolysate.
Reverse Phase HPLC--
The lyophilized hydrolysate of TPO was
restored with ultrapure water containing 0.1% trifluoroacetic acid and
20% acetonitrile (starting buffer). For each run, 250 µg of material
was loaded onto a C-18 reverse phase 3.9 × 150 mm column (Waters,
Millipore, Milford, MA) equilibrated in the starting buffer. Five min
after starting, a linear gradient of 20-90% acetonitrile was applied for 60 min at 0.5 ml/min. The column was then reequilibrated with the
starting buffer for 30 min. Elution of the peptides was monitored by
absorbance reading at 215 nm. Fractions (0.5 ml) were collected in
silicone-coated glass tubes. The fractions from runs corresponding to
the same peak were pooled and freeze-dried.
ELISA--
ELISA was used to detect the immunoreactive peptides
from HPLC fractions recognized by TPO mAb and aAb. Briefly, wells of Immulon II microtiter plates (Dynatech, Chantilly, VA) were filled with
100 µl of HPLC fractions adjusted to 5 µg/ml in PBS, pH 7.3, overnight at 4 °C under humidified atmosphere. The wells were then
washed, overcoated with BSA, washed again, and filled with a saturating
amount of TPO mAb or aAb in PBS, 0.1% Tween-20, 1% BSA. After 2 h at 37 °C, unbound antibodies were removed by extensive washing.
mAb and aAb bindings were detected by an antimouse or antihuman second
antibody labeled with alkaline phosphatase; p-nitrophenyl phosphate was the substrate. Absorbance was read at 405 nm. HPLC fraction 3+4 and native TPO were also tested in ELISA after chemical treatments. For reduction and alkylation, the antigenic material was
coated, the wells were filled with 100 µl of PBS containing 10 mM dithiothreitol and incubated for 15 min at room
temperature. After being washed with PBS, 0.1% Tween-20, the wells
were filled with 100 µl of PBS containing 40 mM
iodoacetamide, incubated for 10 min at room temperature, and washed
again. For iodination, the antigenic material was treated before
coating as for the radiolabeling procedure (see below) but with or
without nonradioactive iodide. After the various treatments, ELISA was
done as above.
SDS-PAGE and Western Blot--
The peptide content of the HPLC
fractions was analyzed by Tricine SDS-PAGE according to Schägger
et al. (21). The lyophilized samples (10 µg/lane) were
restored in 50 mM Tris-HCl, pH 6.8, containing 30%
glycerol, 1% SDS, and 0.02% G-250 Coomasie Brillant Blue, heated for
4 min with 2%
-mercaptoethanol for reducing conditions, and loaded
onto a 16.5% acrylamide, 80 × 100-mm minigel, 0.5 mm thick.
Peptides were stained with the G-250 Coomassie Brillant Blue or
directly electrotransferred onto a 0.2-µm Trans-blot polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA). Western blots were
done by incubating the TPO mAb47 or aAb diluted in PBS, 3% BSA for
2 h at room temperature with constant shaking after saturation of
the membrane with PBS containing 3% non-fat dried milk. The membrane
was then washed three times for 15 min in PBS. The antimouse or
antihuman second antibody labeled with horseradish peroxidase was
incubated for 2 h in PBS, 3% BSA at room temperature under shaking. After additional washes, the blots were developed with 4-chloro 1-naphthol as substrate.
Amino Acid Analysis--
The peptides of interest were
electroeluted from the stained bands in the gel by the Bio-Rad model
422 electroeluter according to the manufacturer instructions. Eluted
peptides were then electrodialyzed against 50 mM
NH4HCO3, 0.001% SDS by the same apparatus.
Next, they were concentrated and dialyzed against methanol and
ultrapure water by a centrifuge concentrator (Amicon, Beverly, MA). The salt-free isolated peptides were placed in PicoTag hydrolysis tubes
(Waters, Millipore) and freeze-dried. The peptides were hydrolyzed in
vapor phase with 6 M HCl, under vacuum, at 110 °C for
24 h. Amino acid compositions were determined from
phenylisothiocyanate-derived amino acids by reverse phase-HPLC
separation (Waters, Millipore). Amino acid sequences were determined by
a computer program (22) that identifies a proteolytic peptide of a
protein through the sequence of the protein and the amino acid
composition of the peptide. We used the TPO sequence reported by
Magnusson et al. (23). We also entered the apparent
molecular weight of the peptides, estimated from the tricine SDS-PAGE,
to calculate the amino acid percentages and to compare them with the
experimental results. The program kept the best fitted peptide on the
basis of a least squares method.
Sequence Determination--
The amino acid sequence of the
20-kDa peptide was determined on material electroeluted from tricine
SDS-PAGE as above and then blotted on a PVDF membrane in a Prospin
cartridge (Applied Biosystems, Foster City, CA). The
NH2-terminal sequence was analyzed in an Applied Biosystem
Procise Sequencer at the Pasteur Institute (Paris, France).
Peptide Labeling and Radioimmunoassay--
Peptides from
HPLC fraction 3+4 were labeled with 125I-Na by the
chloramine-T method. Briefly, 10 µg of material was mixed with 5 µl
of 125I-Na (500 µCi) and 10 µg of chloramine T in 200 mM sodium phosphate buffer pH 7.2. After 1 min, the
reaction was stopped by adding 20 µg of
Na2S2O5. The labeled peptides were
separated by gel filtration through a Superdex 75 column (Pharmacia
Biotech Inc., Uppsala, Sweden) equilibrated with PBS, pH 7.3, containing 0.1% BSA and 0.02% NaN3. The fractions
collected from the column were analyzed for their
125I-peptide content by Tricine SDS-PAGE in nonreducing
condition (5,000 cpm/fraction). The gel was then scanned with a
phosphoimager (FujixBass1000, Japan) equipped with a Tina 2.09 computer
program (Raytest, Courbevoie, France). The column fractions were tested for immunoreactivity by a solid phase radioimmunoassay (6). Briefly,
Startubes (Nunc, Roskilde, Denmark) were coated with purified TPO aAb
or mAb47, overcoated with BSA, and incubated with 100,000 cpm of
125I-peptides. After extensive washing, the radioactive
material bound to the tube was counted.
Protein Assay--
The protein contents of the native TPO
preparation and of the HPLC fractions of hydrolyzed TPO were estimated
by PicoTag amino acid analysis (Waters, Millipore) as above.
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RESULTS |
Proteolytic Peptides from TPO--
To obtain relevant
peptides, we cleaved native TPO with endoproteinase Lys-C. The
hydrolysis products were submitted to reverse phase-HPLC. The elution
profile resolved into seven major peaks and many minor ones, with a
total protein recovery of 50% (Fig. 1).
Peak 0 was free of protein; it was due to an air bubble entrapped during the injection. For convenience, fractions corresponding to peaks
3 and 4 were pooled and named fraction 3+4. Nonhydrolyzed, native TPO
eluted as one major peak corresponding to fraction 7 at the end of the
acetonitrile gradient (data not shown). The peptides in the HPLC
fractions were further analyzed by Tricine SDS-PAGE. In native
conditions, various bands were obtained from fractions 2 to 6 (Fig.
2A). The most heterogeneous
fraction was fraction 3+4, yielding bands from 3.5 to 66 kDa. Fraction
1 showed no band, suggesting that its peptides were less than 3.5 kDa. Fraction 7 and, to a minor extent, fraction 6 showed a major band at
110 kDa, corresponding to poorly hydrolyzed TPO. Submitted to
reduction, most of the bands from the fractions remained unchanged (Fig. 2B). A few were modified, thus yielding bands with
lower molecular mass, e.g. the 66-kDa band in fraction 3+4
yielded a 40- and a 26-kDa band. These modifications resulted from the
presence, in native conditions, of TPO peptides linked by disulfide
bridges.

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Fig. 1.
HPLC elution profile of the proteolytic TPO
peptides. 250 µg of hydrolyzed TPO was loaded onto a C-18
reverse phase column and separated as described under "Experimental
Procedures." The various peaks were monitored by absorbance reading
at 215 nm. The slope indicates the acetonitrile gradient.
The dark areas represent the fractions retained for further
use.
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Fig. 2.
Tricine SDS-PAGE of the HPLC fractions.
The fractions were electrophoresed in native conditions (A)
or after treatment with -mercaptoethanol (B). Each
lane was loaded with 10 µg of protein. After the run, the
gel was stained with G-250 Coomassie Brillant Blue. The numbers of the
HPLC fractions are at the top of the figure. The molecular
masses of peptide standards are on the left. The positions
of bands of interest are shown by arrows.
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Immunoreactivity of TPO mAb and aAb with TPO Peptides--
By
ELISA, we searched for relevant peptides containing TPO epitopes in the
HPLC fractions. Fractions were tested in native conditions with mAb and
aAb directed to potential TPO autoepitopes. Native, nondegraded TPO
served as control to ensure all TPO antibodies were in saturating
conditions (Fig. 3). Fractions 6 and 7 were the most frequently recognized by the panel of antibodies. Among the remaining fractions, fraction 3+4 was the most immunoreactive with
TPO mAb47 and aAb. To identify the immunoreactive peptides, we tested
the HPLC fractions by Western blot with mAb47 and aAb as specific
reagents. From native Tricine SDS-PAGE, mAb47 (Fig. 4A) and aAb (Fig.
4B) both identified a broad band at 66 kDa in fraction 3+4,
and aAb identified a doublet at 20 kDa. Both reagents revealed a band
at 110 kDa in fractions 6 and 7. The mAb47 and aAb immunoreactive band
at 66 kDa shifted to 40 and 26 kDa when fraction 3+4 was separated in
Tricine SDS-PAGE under reducing conditions (see Fig. 2), and only the
40-kDa bands remained reactive with mAb47 (Fig. 4C). In
contrast, these bands and the aAb immunoreactive doublet from fraction
3+4 were no longer revealed by aAb in reducing conditions (Fig.
4D). The 110-kDa band of TPO was revealed by mAb47 in
reduced fractions 6 and 7, whereas only the 110-kDa band of fraction 7 was revealed by aAb. To confirm that recognition of aAb depends on the
antigenic conformation, we tested fraction 3+4 in ELISA after reduction
and alkylation of the coated material. As a control, native TPO was
tested in the same way. After treatment, fraction 3+4 was not
recognized by aAb (Fig. 5A),
whereas the autoreactivity of the native TPO decreased slightly (Fig.
5B). In contrast, mAb47 was slightly more reactive on
treated than untreated materials.

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Fig. 3.
Immunoreactivity of the HPLC fractions with
TPO mAb and aAb. 500 ng of the HPLC fractions was coated and
tested for the presence of TPO autoepitopes by ELISA. We used (i) eight
TPO mAbs cross-reactive with TPO aAb and directed to antigenic domains
A and B of the TPO and (ii) TPO aAb from patients investigated for
AITD. Native, nondegraded TPO was used as control. Background levels
were determined for each test by using noncoated wells; these levels
were subtracted from specific values. Results are expressed as
absorbance reading at 405 nm and represent the mean of triplicate tests
(coefficient of variation less than 10%).
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Fig. 4.
Western blot patterns of TPO mAb47 and aAb
reactivities with the peptides from the HPLC fractions. The TPO
peptides electrophoresed on Tricine SDS-PAGE were electrotransferred
onto a PVDF membrane and tested with TPO mAb47 (A and
C) and aAb (B and D). Western blots
were done with samples used in native conditions (A and
B) or after treatment with -mercaptoethanol (C
and D). The numbers of the HPLC fractions and the molecular
mass standards are the same as in Fig. 2. Arrows indicate
the immunoreactive bands.
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Fig. 5.
Role of the dissulfide bridges in aAb
recognition of TPO epitopes. The ELISA was done with TPO aAb and
mAb47 (as a control) to test HPLC fraction 3+4 (A) and
native TPO (B) with (Treated) or without
(Untreated) reduction and alkylation. The results are
expressed as absorbance reading at 405 nm (duplicate experiments,
mean ± S.D.).
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Identification of the Immunoreactive Peptides--
The 40- and
20-kDa bands reactive with the mAb47 and the aAb, respectively, were
electroeluted from fraction 3+4 after they were run in Tricine SDS-PAGE
under reducing conditions. The amino acid composition was determined
for these peptides, and a computer program localized the best fitted
fragments in the entire TPO amino acid sequence. Table
I shows the experimental and calculated amino acid percentages for the two TPO peptides. The optimized errors
were 1.97 and 2.35% for the 40- and 20-kDa peptides, respectively. The
two peptides were within the 550-923 and 744-922 C-terminal amino
acid sequences of the TPO, respectively (Fig.
6). Taking into account the localization
of the theoretical lysine peptides, we deduced that the 40-kDa peptide
encompassed 12 noncleaved lysine peptides from amino acid 549 to 933 at
the C-terminal end of the TPO and that the last five lysine peptides
from amino acid 742 to 933 overlapped the 20-kDa peptide.

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Fig. 6.
Localization of the TPO peptides in the
entire amino acid sequence of TPO (23). The amino acid sequences
of the 40- and 20-kDa peptides best fitted with the experimental
results are underlined and double underlined,
respectively. The lysine residues (K) and the N-terminal amino acid
numbers of the theoretical peptides are in bold characters.
Arrows show the tyrosine residues (Y) on the 20-kDa peptide.
The mAb47 binding site and the putative membrane spanning region are
boxed. Single amino acid code is used (see Table I).
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Localization of the Autoimmune Epitopes--
The 20-kDa peptide
encompassed an extracellular part of the molecule followed by the
transmembrane region and an intracytoplasmic region. Consequently, the
autoimmune epitopes could be situated inside and/or outside the
thyrocyte. Considering that the 20-kDa peptide contained three tyrosine
residues in the extracellular part of the TPO molecule, we tested the
immunoreactivity of TPO aAb for the peptide after modification of the
tyrosine residues by iodination. After 125I labeling and
gel fitration of the peptides from HPLC fraction 3+4, the 66- and
20-kDa labeled peptides were in fractions 4 and 10, respectively (Fig.
7B). The mAb47 recognized the
66-kDa 125I-labeled peptide. In contrast, TPO aAb
recognized none of the peptides modified by iodination (Fig.
7A). To ascertain that the loss of TPO aAb reactivity to the
iodinated 66- and 20-kDa peptides resulted from the modification of the
tyrosine residues and not the oxidative stress of the chloramine-T
method, we tested by ELISA the TPO aAb and mAb47 immunoreactivity of
the HPLC fraction 3+4 after treatment by the chloramine-T method with
and without iodide. The TPO aAb immunoreactivity for the HPLC fraction
3+4 decreased only when the peptides were iodinated (Fig.
8A). In the absence of iodide,
the oxidative stress of chloramine-T did not abolish the autoimmune
epitopes on the peptides. As expected, the linear epitope recognized by
the mAb47 containing no tyrosine residue was not affected by the
iodination procedure. The treatment of native TPO by the chloramine-T
method, with or without iodide, slightly affected the immunoreactivity
of TPO aAb but not that of mAb47 (Fig. 8B).

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Fig. 7.
Immunoreactivity of 125I-labeled
peptides from HPLC fraction 3+4. A coated-tube radioimmunoassay
was done with TPO aAb and mAb47 to test the radioiodinated peptides
from HPLC fraction 3+4 (A). Results are expressed as
radioiodinated peptide bound to TPO aAb ( ) or mAb47 ( ). The
elution profile of the radioiodinated peptides loaded on the gel
filtration column is also shown ( ). The radiolabeled peptide content
from each fraction was visualized by scanning of the Tricine SDS-PAGE
(B). The molecular masses of peptide standards are on the
left of the figure. The 66- and 20-kDa peptides shown by
arrows are in fractions 4 and 10, respectively.
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Fig. 8.
Role of iodination in aAb recognition of TPO
epitopes. The ELISA was done with TPO aAb and mAb47 (as a control)
to test HPLC fraction 3+4 (A) and native TPO (B)
after treatment by the chloramine-T method with or without iodide. The
results are expressed as percent of immunoreactivity of treated as
compared with nontreated antigenic material (duplicate experiments,
mean ± S.D.).
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DISCUSSION |
We localized a new immunodominant region within amino acids
742-848 at the C-terminal end of the extracellular part of TPO. This
region is deprived of potential sites of glycosylation and contains 11 cysteine residues, some of which form disulfide bridges implicated in
the three-dimensional structure of the evidenced autoepitopes. TPO was
previously used to map the interaction of a panel of 13 mouse mAbs to 4 antigenic regions of TPO; TPO aAb from patients with Graves' or
Hashimoto's disease were directed predominantly against two of the 4 regions (6). Interestingly, all but one of the epitopes in these two
regions were conformational. The linear epitope (recognized by mAb47)
was resistant to denaturation, and further studies localized the
corresponding sequence at amino acids 713-721 (24). Human mAb were
produced to conformational epitopes in two overlapping regions on the
surface of native TPO that were recognized by about 80% of TPO aAb in
individual patient's sera (25). We reported that treatment of TPO by
denaturing agents inactivated most of the aAb reactivity whereas
treatment with a reducing agent completely abolished the autoimmune
recognition (20). We therefore generated peptides from our conventional preparation of native TPO. Enzymatic hydrolysis, peptide separation, and antibody binding were done without heating and reducing agents to
maintain the native conformation of the generated peptides.
The most interesting peptides were recovered from HPLC fraction 3+4,
which eluted with a 41-43% acetonitrile gradient. This percent of
acetonitrile does not change antibody recognition since fraction 7, eluted with 52% acetonitrile, retained almost all the native TPO
autoreactivity in ELISA. The 26-kDa peptide, which is linked to the
40-kDa peptide by one or more disulfide bridges, showed no TPO antibody
reactivity per se and, consequently, was not further
investigated. We chose to localize on the TPO amino acid sequence, the
40- and the 20-kDa peptides, which reacted with the mAb47 and aAb,
respectively. A computer program (22) revealed they were in the
C-terminal end of the known TPO sequence (23). The 40-kDa peptide
extends from amino acid 549 to 933 and overlaps the 20-kDa peptide by
its last 192 amino acids. This overlapping probably explains why these
two peptides displayed similar hydrophobicity and, consequently, eluted
in the same HPLC fraction. The sequence of the 20-kDa peptide was
confirmed by direct sequencing of the five NH2-terminal
amino acids. The 40-kDa sequencing failed to provide reliable
information, but the mAb47 reactivity revealed that the reported mAb47
linear sequence (24) was in the deduced sequence. More precisely, the
autoimmune epitopes was localized in the C-terminal part of the
molecule immediately before the transmembrane region, i.e.
from amino acid 742 to 848. Effectively, the three tyrosine residues on
the 20-kDa peptide were in this part of the molecule, and the
iodination of the peptide abolished the conformational epitopes
recognized by TPO aAb.
Most attempts to identify and locate the B-cell epitopes on TPO were
made on recombinant TPO fragments that obviously did not always adopt
the same structure as their native counterparts in intact TPO (for
review, see Refs. 17-19). Identification of such linear epitopes was
questioned because B-cell epitopes, unlike T-cell epitopes, are usually
conformational i.e. highly dependent on the
three-dimensional structure of the protein (26). Thus, to explain the
autoreactivity of linear epitopes, it was proposed that short peptide
fragments of TPO may be part of larger discontinuous epitopes (27). At
variance with molecular biology studies (28-32), we observed no
autoreactivity in small peptides with low molecular weight.
Autoreactive bands were very scarce, and their immunoreactivity was
very faint despite the large excess of aAb. Hydrolysis at lysine
residues may have damaged some autoepitopes including C2 and C21, as
described by Vassart (28, 29), which are within TPO amino acids
590-622 and 709-721, respectively, and which consequently map in the
40-kDa peptide region (549-933). However, as expected, the mAb47
epitope (713-721), which is virtually identical to the C21 epitope,
was evidenced in the 40-kDa peptide but not in the 20-kDa peptide. This
introduced an additional autoepitope of interest outside the C2 and
C21/mAb47 epitopes at the C-terminal end of the TPO molecule.
A region within amino acids 657-767 harbors a major and frequently
used autoepitope (30). The last 26 amino acids of this region overlap
our 20-kDa peptide. An autoepitope is within residues 873-933 (33),
and there are various autoepitopes along the TPO amino acid sequence,
including the C-terminal amino acids 709-933 (34). All these
autoepitopes, however, are immunoreactive under denaturating and
reducing conditions. On the other hand, limited tryptic digestion, as
part of the purification procedure of TPO, generates various peptidic
fragments (35). One of the trypsin cleavage sites is close to or on the
luminal side of the apical membrane. The possible presence of a major
autoepitope around this cleavage site precludes the use of trypsinized
TPO or truncated recombinant TPO for aAb clinical testing. More
recently, eight TPO mutants whose creation was guided by the crystal
structure of myeloperoxidase, a closely related molecule, were used
(36). These mutations were strategically located to alter the surface of the TPO molecule. Unfortunately, the eight mutageneses did not
affect the aAb recognition of the molecule. The mutation nearest the
C-terminal end of the molecule was within TPO amino acids 722-727,
i.e. exactly 15 amino acids upstream from the 20-kDa peptide
region. By default, this result adds to the validity of our
determination and provides evidence for the involvement of this TPO
region in autoimmune recognition.
A large proportion of the epitopes recognized by aAb require the
correct three-dimensional structure of TPO, but mapping these regions
is a considerable challenge. This is the first report describing a TPO
region that contains at least one conformational epitope recognized by
aAb. Further investigations of this region should determine the aAb
epitopes at the molecular level and evaluate the clinical significance
of the corresponding aAb.
We thank Profs. J.-F. Henry, B. Conte-Devolx,
and Dr. C. De Micco for the thyroid specimens and patients' sera. We
thank Drs. B. Mallet and P.-J. Lejeune for discussions. The Association
pour la Recherche en Biologie Cellulaire is thanked for financial
support.