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The Randall Centre and Guy’s, King’s and St. Thomas’ School of Medicine (A.M.M., J.P.B.), King’s College London, London, United Kingdom SE1 1UL; and Medical Centre of Postgraduate Education (A.G.) and Department of Medical Informatics, Warsaw University Medical School (R.R.), 99 01-813 Warsaw, Poland
Address all correspondence and requests for reprints to: Dr. B. J. Sutton, The Randall Centre, King’s College London, New Hunt’s House, Guy’s Campus, London Bridge, London, United Kingdom SE1 1UL. E-mail: brian.sutton{at}kcl.ac.uk
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TPO belongs to the family of mammalian peroxidases that include myeloperoxidase (MPO), eosinophil peroxidase, lactoperoxidase, and salivary peroxidase (19, 20, 21). The crystal structure of only MPO is known, and it reveals a disulfide-linked dimer (22, 23). We have generated crystals of TPO, but they were unsuitable for x-ray diffraction analysis (24). More recently, diffracting crystals of TPO have been described, but they do not diffract to sufficiently high resolution for a structure determination (25). The degree of sequence similarity between TPO and MPO, however, is sufficient for homology modeling.
We report here a model of the extracellular domains of TPO, from which we have selected peptides and generated antisera to accessible surface features of the molecule. Binding studies with these antipeptide antibodies and inhibition of patients’ autoantibodies identify a major autoreactive epitope in TPO. Furthermore, competition with murine mabs relates this epitope to the A and B determinants identified previously; thus, the three-dimensional structure and location of an immunodominant epitope of TPO is now defined.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-Cß bonds of the
overlapping residues at the splice points aligned well, and if main
chain 
and 
angles were acceptable; minimal steric interaction
with adjacent SCRs was the final criterion for selection; 5) side-chain
coordinates for the TPO sequence, where different from the template,
were generated using a rotamer library (within HOMOLOGY); 6) splice
points between SCRs and VRs were refined, placing a torsion force to
constrain the main chain 
angle to 180° during local energy
minimization (splice repair subroutine of HOMOLOGY/DISCOVER); 7) energy
minimization of the entire structure of each domain was performed (100
cycles of steepest descent followed by 900 cycles of conjugate
gradient); and 8) structures were assessed using PROCHECK (28) to
monitor stereochemistry. Solvent accessibilities of peptide segments
within the model were calculated using NACCESS (S. J. Hubbard,
Department of Biomolecular Sciences, University of Manchester).
Purification of human TPO (hTPO)
Thyroid microsomes were prepared from pooled Graves’ thyroid
tissue and human TPO (hTPO), purified as previously described (29).
Antibodies
Serum from patients with thyroid autoimmune disease was obtained
with permission from the Warsaw out-patient endocrine clinic. Diagnosis
was made by standard clinical and biochemical criteria, identifying
sera from patients with Graves’ disease (n = 45) and lymphocytic
hypothyroid disease (n = 20). Pooled sera from normal healthy
individuals (n = 20) were used a control. Autoantibody to TPO was
measured by enzyme-linked immunosorbent assay (ELISA), standardized to
the WHO/Medical Research Council International Standard 66/387 (29).
Murine mabs to TPO, in the form of ascites of mabs 2, 9, 47, 15, 18,
and 64, were provided by Dr. J. Ruf (13).
Rabbit antibody to synthetic peptides of TPO
Peptides were synthesized by F-moc chemistry with C-terminal
amides and purified by HPLC in trifluoroacetic acid/water and
lyophilized. A cysteine residue was added to the N- or C-terminus of
each peptide for coupling to carrier protein. All peptides were checked
for purity by mass spectrometry. Peptides were conjugated to
maleimide-activated keyhole limpet hemocyanin (1 mg peptide/1 mg
keyhole limpet hemocyanin) and further purified by chromatography on
Ultrogel AcA22 chromatography in PBS as described previously (29) for
peptides 11–13. Either one or two New Zealand White rabbits per
peptide were injected according to the schedule described previously
(29).
RIA
TPO was iodinated by the Iodogen method as previously described
(30). Briefly, to an Eppendorf tube
(Sigma-Aldrich, Poznan, Poland) coated with 25 µg
Iodogen/tube, 10 µl containing 100 µCi
[125I]Na (Swierk, Warsaw, Poland), and
100 µg hTPO in PBS (100 µl) were added and incubated for 10 min.
The contents were transferred to another tube containing 100 µl
PBS/BSA and applied to a HPLC column (Superose 6 HR 10/30,
Pharmacia Biotech, Uppsala, Sweden) and run in 0.1
M Tris-HCl buffer, pH 8.5, containing 0.1% deoxycholate
and 0.5 M NaCl. Fractions containing protein and
radioactivity were assayed for reactivity with rabbit anti-hTPO
antibodies and pooled.
Antisera were diluted from 1:100 to 1:5,000 in PBS, pH 8.0, containing
0.5% Triton X-100 and 3% polyethylene glycol 6000. Labeled TPO
(25,000 cpm) was added to each tube, mixed, and incubated overnight at
8 C. Then, 100 µl goat antirabbit serum were added and mixed, and
after 15 min the precipitated complexes were microfuged at 10,000
gav for 10 min. The supernatant was
carefully discarded, and the radioactivity of the pellet was measured
in a 
-counter (LKB Wallac, Inc., Turku,
Finland). The amount of labeled TPO precipitated by rabbit
polyclonal anti-hTPO antibody at 1:100 dilution was taken as 100%.
When human sera were tested, the precipitating antibody was rabbit
antihuman, instead of goat antirabbit serum. In all experiments the
control was preimmune rabbit or human serum negative for TPO
antibodies.
Inhibition of autoantibody binding to TPO and ELISA
Microtiter plates (Nunc, Copenhagen, Denmark) were coated with
purified hTPO (0.5 µg/ml), and 100 µl of 100-fold diluted rabbit
antipeptide antiserum were added and incubated for 1 h at room
temperature. After washing, patients’ sera that had been diluted to
give 10 IU/ml TPO autoantibodies were added to the wells and incubated
for 1 h at room temperature. After washing three times in PBS
Tween (saline with 0.1% Tween-20), horseradish
peroxidase-conjugated rabbit antihuman IgG (diluted 1:2000) was added
and incubated for 1 h, followed by three washes with PBS
Tween. The plates were developed with
tetramethylbenzidine solution, and optical density was measured
at 450 nm. Preimmune rabbit serum was used for controls, and wells
without addition of human serum were considered blank. Inhibition of
autoantibody binding was calculated in comparison to preimmune serum
according to the formula: (A - B/T - B) x 100, where
B is the OD of the background, T is the OD of preimmune serum, and A is
the OD of antipeptide antiserum. Inhibition of binding of murine mabs
to TPO was measured in the same way, but using rabbit antimouse
IgG-horseradish peroxidase conjugate (diluted 1:2000). All ELISAs were
performed as described previously (29).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Within this domain (overall sequence
identity, 46%; overall sequence similarity, 71% between TPO and MPO)
there are several extensive structurally conserved regions (SCRs;
boxed regions in Fig. 1). These regions include conservation
of the 13 cysteine residues that in MPO form 6 intramolecular disulfide
bridges and 1 intermolecular bridge between subunits across the dimer
interface. There is 1 additional cysteine residue in TPO (residue
146) in a 3-residue insertion located at the surface; this may bridge
to a conserved cysteine (residue 133) in the N-terminal segment
(residue 1–141) that is not present in mature MPO. All of the VRs
(Fig. 1) correspond to surface loops. The first of the 2 large VRs
(249–256, SKAAFGGG) corresponds to the segment that in MPO is cleaved
from the mature protein to generate the 2 polypeptide chains, and the
second is an insertion of 10 residues (299–308: GDQGALFGNL) in a
surface location close to the dimer interface in the MPO structure.
Model structures for these 2 loops were generated by conformational
loop search (GENLOOPS) and are the only 2 regions where the modeled
backbone conformation must be considered tentative. With the exception
of a 3-residue insertion (376–378), the other VRs involve only single
residue insertions or deletions, and all are readily accommodated
within the structure. The extensive SCRs and the surface location of
all VRs are illustrated in the model of TPO shown in Fig. 2A. [PGH2
synthase-1 has sequence homology with MPO and a similar overall fold
(31), but the mode of dimerization is quite different, and the long VRs
of TPO have no counterparts in PGS to assist modeling.]
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-helical regions
separated by turns, suggestive of a folded -helical bundle motif, a
result confirmed by fold recognition analysis (using the UCLA-DOE Fold
Recognition server).
To the C-terminal side of the MPO-like domain, between this domain and
the predicted transmembrane region (Fig. 1
), lies a sequence (residues
739–841) that corresponds precisely to the products of exons 13 and 14
(20). Comparison of this region with the SCOP database indicates a
highly significant match with the 15th domain of complement factor H
over the entire sequence corresponding to exon 13 (residues 739–795;
Fig. 1; 21% identity, 48% similarity between the two sequences). This
domain of factor H is a typical complement control protein (CCP) module
(32), and the alignment of four of the five cysteine residues in the
exon 13 sequence of TPO with the only four cysteine residues in the CCP
domain of factor H (where they form two disulfide bridges) strongly
implicates this fold in TPO. Only one deletion of a five-residue loop
from the factor H structure and a single residue insertion are required
to model the main chain conformation of this domain (Figs. 1 and 2B).
A strikingly similar match was obtained for the entire sequence
corresponding to exon 14 (residues 796–841), which aligns with several
epidermal growth factor (EGF)-like domains, as noted previously (20).
The highest score was with the EGF-like domain of fibrillin (33) (36%
identity, 62% similarity between the two sequences). Again, all six
cysteine residues of the exon 14 sequence (Fig. 1) could be aligned
with the six cysteine residues of the EGF-like domain (where they
formed three disulfide bridges), strongly implicating this fold in TPO.
Deletion of only one extended loop region of the EGF-like structure was
required to provide a model for the main chain conformation of this
domain (Figs. 1 and 2B).
Predicted quaternary structure of TPO
If the TPO model is superimposed upon the MPO dimer, there are no
steric clashes between TPO subunits, and most of the residues at the
dimer interface of MPO are either identical, including the bridging
cysteine residue 296, or conservatively substituted in TPO. The
10-residue loop insertion in TPO (residues 299–308; Figs. 1 and 2A)
has been modeled in such a way so as not to interfere with
dimerization, but could conceivably adopt a conformation that would
clash with the second subunit. Similarly, the location of the
N-terminal domain (residues 1–141) is not known and might prevent
dimerization, but could equally well be accommodated within a dimeric
model of TPO, for which there is experimental evidence (34). (Note
the location of the N- terminus of the MPO-like domain indicated in
Fig. 2B.)
The relative disposition of the three modeled domains (Fig. 2B) cannot
be predicted, but is constrained by the fact that there are only five
residues between the C-terminal end of the MPO-like domain fold and the
start of the CCP domain, and there is no linker sequence between the
CCP and EGF-like domains, which must therefore be closely packed
together. Seven residues connect the C-terminal end of the EGF-like
domain to the predicted transmembrane region. Finally, four potential
sites for N-linked glycosylation are found in the
extracellular sequence of TPO, one in the structurally undefined
N-terminal region and three within the MPO-like domain, all of which
lie at the surface of the TPO model.
Location of peptide epitopes
The peptides used in this study, which all correspond to sequences
within the MPO-like domain, are listed in Table 1, together with the accessibility of
each to solvent, as calculated from the model structure. The total
solvent accessible surface area for each peptide is quoted together
with the fraction (percentage) that this represents of the theoretical
maximum surface area for that particular sequence as an isolated
peptide in an extended conformation. The location of the surface
accessible peptides may be seen in Fig. 2B, in which the domain is
viewed in virtually the same orientation as in Fig. 2A, i.e.
from the side opposite that of the active site. Peptide 6 consists of a
surface loop with little or no secondary structure and is furthest from
the putative dimer interface (Fig. 2A). Peptide 14 is highly exposed
and consists of ß-strand/turn/-helix/turn, overlapping with
peptide 15, -helix/turn/-helix. Peptide 16 is a very exposed
surface loop with turns, and lies close to the putative dimer
interface. Peptide 17 is an extended surface peptide in the monomeric
domain model, but would form part of the interface in a TPO dimer
(compare accessibility values for monomer and dimer in Table 1). A
number of buried peptides were selected as controls (not shown in Fig. 2B); peptide 11 is totally buried, whereas peptides 1, 12, and 13
include a few exposed residues. All of the peptides, both exposed and
buried, correspond to SCRs in TPO (Fig. 1) and are therefore modeled
with confidence.
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). Binding to TPO was also evaluated by
RIA. Antibodies to peptide 14 from two different immunized rabbits
precipitated the highest amounts of labeled TPO (60–75% of the total
labeled TPO at dilutions between 1:100 to 1:1000; Table 2), and
antibodies to peptide 6 gave moderate precipitation of TPO (6–22%),
but antibodies to peptides 15, 16, and 17 showed negligible
precipitation. As RIA involves protein modification (tyrosine
iodination), inhibition ELISA with native soluble TPO was also
performed. These data show that all of the antipeptide 14 antibodies
react with native TPO (complete inhibition; Fig. 3), which correlates well with the fact
that peptide 14 exhibits the greatest accessible surface area in the
structure (Table 1). The other antipeptide antisera (peptides 6, 15,
16, and 17) do not show complete inhibition by native TPO, but this is
consistent with their lower accessibility values. Antibodies to three
buried sequences in the TPO model (peptides 1, 11, and 13) showed
negligible binding to native TPO as expected, although antisera to a
fourth buried peptide sequence (peptide 12) showed moderate reactivity
with native TPO (Fig. 3) and precipitation of labeled TPO in RIA (Table 2), presumably due to reactivity with the few residues that are
accessible.
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). The remaining antipeptide antibodies
(Table 1) did not show any inhibition of binding of any of the murine
mabs (data not shown).
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). The mean level of inhibition with
antipeptide 14 antibodies was 41% (SD = 16%),
whereas the mean level for all other antipeptide antisera was 5.8%
(SD = 4.4%).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the MPO-like domain, all of the insertions and deletions in the TPO sequence were found to occur at the surface of the molecule, and with only two exceptions (the longer loops 249–256 and 299–308) could be modeled with considerable confidence. Biochemical studies indicate that membrane-bound TPO in thyroid cells exists as a disulfide-linked dimer (34), and conservation of many of the residues at the putative dimer interface, including the essential cysteine residue that in MPO covalently links the two subunits, supports a dimeric structure. Dimerization of the MPO-like domain at the membrane surface could be accommodated by flexibility at the link region between this domain and the CCP module. Similarity between the enzyme mechanisms of TPO and MPO is suggested by conservation of the critical residues surrounding the heme group, including the proximal and distal histidines (residues 494 and 239) and an adjacent arginine (residue 396) (22, 23, 38). Residues that bind a Ca2+ ion in the MPO structure (Asp240, Thr321, Asp325, and Ser327) are also conserved, implying that TPO also binds Ca2+.
Based upon this model, peptides corresponding to accessible surface features were synthesized, and antisera were raised in rabbits to probe for autoreactive epitopes, using buried peptides as controls. ELISA measurements showed that the antipeptide antisera cross-reacted well with TPO, but this was the case for both the surface peptides and the buried peptides, indicating that denaturation of the TPO may have occurred upon coating of the plates (39). RIA measurements showed minimal reactivity with radiolabeled TPO for three of the four antisera directed to buried peptides, but three of the five antisera to accessible peptides did not react either, perhaps because labeling affects the conformation of TPO. Inhibition ELISAs were therefore carried out with native TPO in solution, and these data showed that all the antisera to peptides predicted to be accessible do indeed react with native TPO; furthermore, there is an approximate correlation between the degree of inhibition (i.e. extent of reactivity with native TPO) and the accessibility of the peptides predicted from the model. Antipeptide 14 antisera are clearly the most reactive, and it is this peptide that in the model is the most accessible and protrusive of the peptides tested.
The relationship between the epitopes recognized by these antipeptide
antisera and the previously identified immunodominant determinants A
and B was investigated by competition with the murine mabs originally
used to define them. The antipeptide 14 antisera inhibited TPO binding
for all three mabs associated with determinant B, whereas none of the
other antisera inhibited any of the mabs associated with either
determinant. Thus, peptide 14 is either close to or part of one of the
dominant epitopes on TPO, and the fact that the antipeptide 14 antisera
inhibited all three mabs that define the B determinant suggests that it
may be a central feature of this epitope. Its prominent, exposed
location distant from both the membrane, putative dimer interface and
active site is shown in Fig. 2B (inset).
The relevance of the epitope defined by peptide 14 to the human autoantibody response was then investigated, with the striking result that antipeptide 14 antisera inhibited to some degree all human patient sera tested, whereas none of the other antipeptide antisera tested showed any inhibition above that of control serum. It is known that 80–90% of autoantibodies from patients with thyroid disease recognize the A and B determinants (13), and that the fraction directed to each varies among patients’ sera (15, 40, 41). Our results confirm that determinant B is indeed a dominant epitope and that peptide 14 represents a critical structural feature, the location of which is now mapped onto the structure of TPO.
An earlier study (12) identified autoantigenic determinants within a recombinant fragment of TPO comprising amino acids 589–633, which includes the sequence of peptide 14 (residues 599–617). However, antibodies to determinants within this region recognized only denatured TPO, indicating that these determinants were not accessible in the native molecule, in contrast to our findings that antibodies to peptide 14 not only inhibit patients’ autoantibodies but also bind to native TPO. Furthermore, the highly accessible structure adopted by residues 599–617 in native TPO is quite different from the ß-turn structure predicted for the isolated peptide of residues 592–613 by Arscott and colleagues (12). In another study (42), a peptide fragment of residues 742–848 displayed reactivity with patients’ sera, and the conformational nature of these epitopes is consistent with the fact that the peptide corresponds almost exactly to the CCP and EGF-like domains together.
It is striking that peptide 14, although a linear sequence, defines, by
virtue of its conformation in the folded structure, a surface
accessible epitope of about the size recognized by an antibody. This
may in part account for the high level of reactivity of antipeptide 14
antibodies with native TPO, and their efficacy in inhibiting the
binding of patients’ sera, although clearly antibodies to peptide 14
would be expected to block access to immediately adjacent segments
should they also be important determinants. Nevertheless, peptide 14
appears to be a critical feature of the B determinant, the full extent
of which can now be mapped, for example by single site mutagenesis. The
negative results with antisera to peptides 15–17, however, do define
the limits on one side. A clue to the location of the A determinant is
provided by the fact that the only murine mab from the panel to be
mapped to a sequence is mab 47, which binds to both denatured and
native TPO (41) and recognizes residues 713–721 (8). In the model, as
shown in Fig. 2B, this peptide is highly accessible and extended,
consistent with this cross-reactivity. As mab 47 only weakly inhibits
human autoantibody binding (13), the full extent of the A determinant
must also now be mapped onto the structure of TPO.
This study has defined for the first time the structure and location within the TPO molecule of a major part of the B determinant and confirmed that this structure is indeed a dominant epitope in the human autoantibody response in thyroid disease. Knowledge of the structures of such epitopes may provide clues to the origin of this focused response, and the identity of agents that, perhaps through molecular mimicry (43), may trigger autoreactivity.
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Acknowledgments |
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Footnotes |
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2 Both authors contributed equally to this work.
Received November 17, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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antithyroid peroxidase antibodies in Hashimoto’s thyroiditis: epitope
mapping analysis. J Clin Endocrinol Metab 82:2639–2644 anti-thyroid
peroxidase antibodies from different tissues from two patients with
Hashimoto’s thyroiditis. J Clin Endocrinol Metab 82:3818–3825,6-diphenylglycoluril. Biochem Biophys Res
Commun 80:849–857[CrossRef][Medline]
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