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Experimental Studies |
Antithyroid Peroxidase Antibodies in Hashimoto’s Thyroiditis: Epitope-Mapping Analysis1
Department of Biochemistry, Medical Center of Postgraduate Education (B.C., M.J.-.B.), Marymoncka 99, Warsaw 01 813, Poland; the Department of Medicine, University of Sheffield Clinical Sciences Center, Northern General Hospital (R.S.M., M.S.A., P.F.W., E.H.K., A.P.W.), Sheffield, United Kingdom S5 7AU; and Faculté de Médicin, Laboratoire de Biochimie Endocrinienne et Metabolique (P.C.), Marseille, France
Address all correspondence and requests for reprints to: Prof. A. P. Weetman Department of Medicine, University of Sheffield Clinical Sciences Center, Northern General Hospital, Sheffield, United King-dom S5 7AU.
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Abstract |
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Fab fragments from two patients with
Hashimoto’s thyroiditis and demonstrated the similarity of these Fab
sequences to those published previously, mainly derived from patients
with Graves’ disease. In this paper, we describe epitope mapping of
these Fabs using a previously characterized panel of murine monoclonal
antibody (mAb) and show that the Fabs bind to two neighboring epitopes
on native TPO. Although the epitope-mapping method differs from that
used to characterize previously published TPO-reactive Fab sequences,
it indicates a similarly restricted response to neighboring epitopes in
both Graves’ disease and Hashimoto’s thyroiditis. The epitope mapping
included mAb 47, which binds to a linear TPO peptide of known sequence
in addition to native TPO. Although TPO-reactive Fab did not inhibit
the binding of mAb 47, mAb 47 did inhibit the binding of Fab,
indicating the likely site of the immunodominant region on native TPO.
These results confirm the restricted nature of TPO antibody and further
delineate the immunodominant region of native TPO as defined by the
mAb. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The only comparative study between the two principle mapping methods published to date suggests that the two immunodominant regions on native TPO are not the same. In particular, TPO-reactive Fab bound to TPO did not inhibit binding of monoclonal Ab (mAb) 47 (11). mAb 47 was used for this comparative analysis because it also binds denatured TPO, allowing mapping to the linear epitope C21 (8, 15). In addition, replacement of the C21 epitope or surrounding areas by the homologous region of MPO did not alter the binding of TPO-reactive Fab (16). However, an earlier study, in which a larger portion of TPO, including the C21 sequence, was replaced with MPO sequence did result in inhibition of Fab binding (13). The site of the immunodominant region of native TPO is, therefore, still unknown (5, 12, 16).
We have produced 37 Fab fragments reacting to native human TPO from phage display combinatorial libraries from two Hashimoto’s thyroiditis (HT) patients and shown that the Ig sequence restriction present in anti-TPO Fab from patients with GD is shared with Fab from HT patients (17). In the present study we used a previously characterized panel of murine mAb to human TPO (3) to characterize the epitope reactivity of these Fab. These experiments show that the Fab bind to the two neighboring major conformational domains also recognized by patient sera, and that the patterns of binding correlate with Ig sequence. As part of the mAb 47 epitope has been localized on TPO, this also suggests the likely region on native TPO recognized by these Fab and by Ab reactive to native TPO in general.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The two HT patients from whom cervical lymph node (LN) tissue (n = 2) and thyroid tissue (n = 1) were obtained and were used to derive high titer combinatorial libraries have been previously described (17, 18). Libraries were selected using purified recombinant human TPO between three and five times, phage library DNA from the final screened library was prepared, and Fab solubilization was performed (17, 18). Fab were then screened for reactivity to TPO by enzyme-linked immunosorbent assay (ELISA) using purified human TPO-coated ELISA plates (provided by Cogent Diagnostics, Edinburgh, UK). Analysis of epitope specificity was carried out on Fab prepared in culture medium (17, 18).
Analysis of epitope specificity
Epitope specificity was defined using a panel of 13 murine
mAb (3). Dual binding assays were used to define four antigenic domains
recognized by these mAb: domains A (mAb 2, 9, 47, and 60), B (mAb 15,
18, 59, and 64), C (mAb 24), and D (mAb 1, 30, 40, and 53); domains A
and B are adjacent (3). Two types of competition studies were
performed. Initially, the dilution of Fab corresponding to 50% maximum
binding to immunoaffinity-purified TPO was established in ELISA plates
coated with 100 µL 1 µg/mL TPO prepared as described previously
(3); Fab binding was detected using a 1:10,000 dilution of
immunoaffinity-purified antihuman IgG Fab conjugated to horseradish
peroxidase (Jackson ImmunoResearch, West Grove, PA). Absorbance at 450
nm was read after 10-min incubation at room temperature. Nonspecific
binding of secondary antimouse and antihuman antibodies to TPO-coated
plates did not exceed an OD450 absorbance of 0.1. Fab
binding was then determined in the presence of 1:100 dilutions of mAb
by the addition of mixed mAb and Fab at appropriate dilutions to
TPO-coated wells. In the second study, mAb were used at a dilution
equivalent to 50% maximum binding and Fab was used at a 1:100
dilution, with detection of mAb binding by immunoaffinity-purified
antimouse IgG Fab conjugated to horseradish peroxidase (Jackson
ImmunoResearch). In both cases, 1:100 dilutions of mAb or Fab were
equivalent to 90% or greater of the plateau (maximal) binding. Intra-
and interassay variations ranged from 2–5% and 3.7–10%,
respectively. In the absence of competing Ab, OD450
readings for mAb ranged from 0.30–1.50, and those for Fab ranged from
0.20–1.50; typical values are presented in Table 1
.
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Human leukocyte antigen (HLA)-DR typing
Peripheral blood lymphocytes were prepared from heparinized blood using Ficoll-Hypaque density gradient centrifugation. Genomic DNA was prepared from peripheral blood lymphocytes using a Puregene DNA isolation kit (Gentra Systems, Raleight, NC), and HLA-DR typing was performed as previously described (19).
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Results |
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Cross-reactive inhibition of binding between murine mAb and Fab
was carried out for Fab from the patient HT126 LN library and the
thyroid library that had been panned three times, studying Fab
inhibition of mAb binding. Fab from all four selected libraries were
studied by mAb inhibition of Fab binding. The results of these
experiments are presented in Figs. 1 and 2, respectively. The epitope binding data are summarized
in Table 2.
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; 126B) or domain B mAb (Fig. 1; 126TP15),
inhibition of domain A mAb with some cross-reaction to domain B (Fig. 1; 126A, 126TP6, and 126TP1), and inhibition of domain B mAb with some
cross-reaction to domain A (Fig. 1; 126TP9). No inhibition of binding
was observed for mAb 1, 24, 30, 40, 47, or 53, which all bind other
domains on TPO, with the exception of mAb 47, which binds domain A
(with an approximate affinity of 10 x 10-9 mol/L)
(3). Inhibition of mAb 47 binding was not observed even at very low
dilutions of mAb (1:75,000; data not shown).
Inhibition of Fab binding by mAb followed one of five general patterns:
exclusive inhibition by domain A mAb (Fig. 2; 126TP1) or by domain B
mAb (Fig. 2; 131TP2), inhibition by domain A mAb with some inhibition
by domain B mAb (Fig. 2; 126TP8, 126H, 126TO15, and 131TP14) or
vice versa (Fig. 2; 126TP9 and 126TO3), and inhibition by
both domain A and domain B mAb (Fig. 2; 131TP5). Inhibition of Fab
binding was observed with mAb 1 (affecting 126TP9, -13, -14, and -15;
range, 16.7–23.9%) and mAb 30 (affecting Fab 126TP6, -9 and -13;
range, 11.6–14.7%); these mAb have previously been shown to exhibit
interactions with mAb from domain B (3). mAb 24, 40, and 53 did not
affect Fab binding.
Correlation of Fab binding and sequence data
The two inhibition methods gave similar results (Table 2); in
particular, the assignment of major domain reactivity was identical for
both methods. Patient HT126 Fab, expressing the VO12
light chain in combination with the VH1–3 segment, bound
exclusively or preferentially to domain B [nomenclature of Ruf
et al. (3)] regardless of the DH,
JH, and J used. Patient HT131 Fab,
expressing VO12 (VI) in combination with
the VH3–23 heavy chain, bound equally to domains A and B,
preferentially to domain B, or exclusively to domain B; again, this was
not influenced by the J region used. Patient HT126 Fab,
expressing the VL8 (VI) light chain,
bound exclusively or preferentially to domain A; for all Fab studied,
this light chain was associated with a VH3–21 heavy chain.
Fab 131TP14, with a DP58 VH3 heavy chain and a
VL2 (VIII) light chain, bound
preferentially to domain A. Thus, Fab mapped to domain B were those
containing VO12 light chain in combination with either
VH1 or VH3 heavy chains. The Fab that mapped to
domain A were those containing VL8 (VI)
or VL2 (VIII) light chains in combination
with VH3 heavy chains.
HLA-DR typing
The patients were HLA-DRB610101 homozygous (patient HT126) and HLA-DRB110301 (DR17) and DRB111501–1504 (DR15; patient HT131).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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anti-TPO Fabs from two HT patients, using a previously characterized
panel of 13 murine mAb (3). TPO-reactive Fab sequences have been
published several times previously by two groups studying patients with
GD (4, 5, 20) and HT (21, 22). The Fab used in the present study
constitute a limited number of heavy and light chain gene combinations,
reflecting the restricted nature of the TPO Ab response typical in
individual patients. The differences in sequence between the two
patients may be caused in part by the different HLA-DR types of the
patients. The Fab share considerable similarity in sequence with these
previously published anti-TPO Fab despite the use of LN tissue to
generate two of the three starting libraries and carrying out the
analysis on patients with HT (17). In common with previous TPO-reactive
Fab (11, 20), the Fab described here did not bind TPO in Western blots
(data not shown). Binding inhibition was carried out using two complementary experimental approaches for Fab from two of the panned libraries to assess any inhibition caused by nonspecific interactions and to determine the frequency of discrepancies in the interactions of single mAb and Fab. The mAb have a mixed pattern of reactivity to TPO, with some mAb inhibiting binding of others that do not themselves inhibit the binding of the first mAb (3). There is also a reported degree of cross-competition between mAb from different domains (3). Although the reasons for the discrepancies in interaction are unclear, it may be due in part to differences in mAb affinity for TPO and the exact topography of the binding sites of the interacting mAb and the affinity for the various regions of the binding site (3). In particular, the observed one-way inhibition of Fab by mAb 47 requires further discussion.
From the original reference to the mAb (3), the approximate affinity of mAb 47 for native TPO can be calculated as 10 x 10-9 mol/L, a value consistent with the results of more recently conducted experiments (data not shown). The affinity of mAb 47 for denatured TPO, of which the response to the C21 linear determinant forms a dominant part, is higher than that for native TPO (11). Thus, the affinity of mAb 47 for native TPO may result from a high affinity for only a part of its binding site. The Fabs used in the study have approximate affinities in the range 0.4–10 x 10-9 mol/L and thus have affinities for TPO equal to or higher than that for mAb 47. However, should the C21 linear determinant be accessible in the presence of bound Fab, the higher affinity of mAb 47 for this determinant could result in displacement of Fab. Conversely, the high affinity of mAb 47 for the C21 determinant will prevent displacement of mAb 47 by Fab, thus resulting in the one-way displacement observed. In addition, greater steric hindrance might be caused by mAb 47, an intact IgG, than by the smaller Fab fragments.
The data summarized in Table 2 indicate that the two binding methods
resulted in similar data; the major domain assigned to each Fab was
identical, with discrepancies only noted in assigning minor domain
reactivity. Differences in minor domain reactivity alone were also
noted between Fabs expressing similar heavy and light chains. However,
where this occurred, Fab differed in heavy and/or light chain amino
acid sequences, caused either by differences in junctions introduced
during independent recombination events or by somatic
hypermutation.4 There was no correlation
between the epitope recognized and the affinity of Fab for native TPO
(in the range 0.4–10 x 10-9 mol/L; see Footnote
1).
The best correlation between the domain with which the Fab bind and the
Fab sequence is between Fab containing VO12 light chain
sequences and recognition of the domain designated B by Ruf et
al. (Ref. 3 and this paper) and that designated A by Chazenbalk
et al. (4, 23). Fab analyzed in this study contained the
VO12 light chain sequence with either VH1–3
or VH3–23 heavy chains; VO12-derived
sequences have also been reported in TPO-specific Fab from five of six
AITD patients (5). Taken together, the strong similarity in the
germ-line V regions used and the division of epitope binding patterns
between Fab with or without VO12-derived light chains
are highly suggestive that the Fab described here bind to the same
immunodominant region as that defined previously using TPO-reactive Fab
(4). Additional evidence is provided by the fact that the TPO-reactive
Fab described previously and the murine mAb panel used here inhibit the
binding of the majority of TPO Ab activity to native TPO in patient
sera (3, 4, 5, 24). Using the presence of the VO12 light
chain as an important element of epitope preference, it, therefore,
appears that epitope A of Chazenbalk et al. (4) is the
approximate topological equivalent to epitope B of Ruf et
al. (3), and vice versa.
Given the published reactivity of murine mAb 47 to a linear sequence of TPO (C21) (8, 15), it is further possible to speculate that the immunodominant region of native TPO is close to the mAb 47/C21 sequence. However, there is evidence against this hypothesis. Firstly, preincubation of TPO with TPO-reactive Fab was reported not to inhibit binding of mAb 47 (11). However, we have noted the same phenomenon, with TPO-reactive Fab not inhibiting binding of mAb 47, whereas preincubation with mAb 47 does inhibit binding of TPO-reactive Fab. As discussed above, this may be due to the high affinity of mAb 47 for the linear C21 determinant, the arrangement of the epitopes on the surface of TPO, or the greater steric hindrance caused by an intact IgG molecule. Chazenbalk et al. used mAb 47 at a final dilution of 1:6,000 (11), and the TPO-reactive Fab reported here did not inhibit mAb 47 binding even at a dilution of 1:75,000 (data not shown).
Secondly, in an analysis using replacement of small segments of TPO sequence with MPO sequence, replacement of the C21 sequence itself and of surrounding areas in the native TPO molecule with MPO amino acid sequence were reported not to affect the binding of TPO-reactive Fab (16). However, it is unclear whether perturbation of a linear sequence within a conformational epitope would be expected to abolish binding completely, and the affinity of Fab for the chimeric TPO/MPO sequences was not established (16). If the C21 sequence flanks rather than constitutes a central part of the immunodominant region, replacement of this sequence alone would not be expected to affect Fab binding. In another study, Nishikawa et al. showed that replacement of a larger region containing the C21 sequence (chimera G) prevented binding of Fab, although it was postulated that this may be due to disruption of the native TPO structure rather than removal of elements of the conformational epitope itself (13). It is, therefore, currently unclear whether the chimeric TPO/MPO system can be used to exclude areas of the surface of TPO from constituting the immunodominant region.
Finally, in support of our hypothesis, we have studied the area around the C21 sequence using the crystal structure of human MPO (25) as a guide to the likely structure of TPO. Murine mAb 9, which interacts with the same domain as mAb 47, is reported to bind poorly to TPO in which the tyrosine residues have been iodinated (3), and a similar lack of binding to iodinated TPO was noted in some of the Fab mapped here. This would indicate the presence of a surface tyrosine residue close to the C21 sequence. Using amino acid numbering for the whole MPO and TPO sequences (Refs. 26 and 27, respectively), a tyrosine residue present in the human TPO sequence (Tyr226), but not present in MPO (Arg248) is juxtaposed to the C21 sequence (Lys713-Ser720). Tyr516 and Tyr723 in the human MPO sequence, which would juxtapose and form part of the C21 sequence, respectively, are not present in the human TPO sequence (Phe508 and Phe714, respectively).
It is clear that the TPO-reactive Fab whose mapping is described here bind to a site near the C21 sequence. Because of the similarities in the sequence and epitope pattern between these and previously published Fab sequences, we postulate that the immunodominant regions previously mapped using TPO-reactive Fab and murine mAb are topologically equivalent. It is likely, however, that definitive mapping of the immunodominant domain will require co-crystallization of TPO with Fab (5).
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Acknowledgments |
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Footnotes |
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2 Current address: Division of Molecular and Cellular Immunology,
Department of Clinical Laboratory Sciences, Floor A, West Block,
Queen’s Medical Center, Nottingham, Untied Kingdom NG7 2UH.
3 Sponsored by the Ministry of Education, Pakistan.
4 McIntosh, R. S., M. S. Asghar, E. H. Kemp, et
al., manuscript submitted.
Received December 12, 1996.
Revised April 4, 1997.
Accepted April 30, 1997.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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and IgG
anti-thyroid peroxidase
antibodies from different tissues from patients with Hashimoto’s
thyroiditis. J Endocrinol 148(Suppl):P207.
and IgG
anti-thyroglobulin autoantibodies from a patient with Hashimoto’s
thyroiditis: evidence for in vivo antigen-driven repertoire
selection. J Immunol. 157:927–935.[Abstract]
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