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Endocrinology Vol. 141, No. 7 2518-2525
Copyright © 2000 by The Endocrine Society

ARTICLES

Production of Immunoreactive Thyroglobulin C-Terminal Fragments during Thyroid Hormone Synthesis

Christine Duthoit, Valérie Estienne, Frédéric Delom, Josée-Martine Durand-Gorde, Bernard Mallet, Pierre Carayon and Jean Ruf

Unit 38 of the French Institute of Health and Medical Research, Faculté de Médecine Timone, Université de la Méditerranée, Marseille, France

Address all correspondence and requests for reprints to: Dr. Jean Ruf, U38 INSERM, Faculté de Médecine Timone, 27 boulevard Jean Moulin, F-13385 Marseille Cedex 5, France. E-mail: jean.ruf{at}medecine.univ-mrs.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Here, we studied the fragmentation of the prothyroid hormone, thyroglobulin (Tg), which occurs during thyroid hormone synthesis, a process which involves iodide, thyroperoxidase, and the H2O2-generating system, consisting of glucose and glucose oxidase. Various peptides were found to be immunoreactive to autoantibodies to Tg from patients and monoclonal antibodies directed against the immunodominant region of Tg. The smallest peptide (40 kDa) bore thyroid hormones and was identified at the C-terminal end of the Tg molecule, which shows homologies with acetylcholinesterase. Similar peptides were obtained by performing metal-mediated oxidation of Tg via a Fenton reaction. It was concluded that the oxidative stress induced during hormone synthesis generates free radicals, which, in turn, cleave Tg into immunoreactive peptides.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROGLOBULIN (Tg), a large glycoprotein with a molecular mass of 660 kDa, is the precursor of the thyroid hormones T3 and tetraiodothyronine or T4. These two hormones result from the iodination and coupling of a few specific tyrosine residues within the Tg. Iodide is trapped by the thyroid, which serves mainly to concentrate iodine from the serum and return it to the circulation in the form of T3 and T4. Iodination and coupling are carried out by a specific enzyme, thyroperoxidase (TPO). Iodination also requires a H2O2-generating system which, like TPO, is located at the apical surface of the thyroid cell. Iodide migrates from the basal membrane to the apical microvilli, where it undergoes an oxidative process linking it to tyrosine residues within the Tg. The synthesis of T4 may involve the oxidation of DIT into a radical and interactions between two molecules of DIT radicals via a quinol ether intermediate. Likewise, T3 synthesis results from the interaction of one molecule of DIT with one molecule of MIT. Hormonal secretion into the circulation requires the endocytosis of Tg and its transport to a lysosomal compartment, where the hormone is subsequently released as the result of enzymatic hydrolysis (for a general review, see Ref. 1).

On the other hand, Tg and TPO are major autoantigens (aAg) involved in autoimmune thyroid disease. Circulating autoantibodies (aAb) to these aAg have been detected in the sera of patients. The heterogeneity of Tg and TPO aAb is restricted to specific areas of the two molecules, called immunodominant regions (2). However, the reasons for this restriction are unknown and probably relate to the initial perturbed state in the course of the disease. The specific B-cell response to Tg and TPO in autoimmune thyroid disease is generally thought to be an aAg-driven process that is T cell dependent, but the initial event responsible for breaking the T cell tolerance to these aAg has not yet been definitely established. It is possible that protein fragmentation before lysosomal enzymatic digestion may suitably expose cryptic epitopes for the pathogenic autoimmune process to be initiated (3, 4, 5). Numerous changes in the structure of proteins have been observed in the presence of the free radicals produced by Fenton chemistry with specific metals such as iron or copper (6, 7, 8, 9, 10, 11, 12). Interestingly, the synthesis of thyroid hormones involves an oxidation process, resulting in the production of free radicals (13, 14, 15, 16).

Here we report that, during hormone synthesis, a Tg fragmentation process occurs that mimics metal-catalyzed oxidation and generates various immunoreactive fragments, the smallest of which was found to be identical to the C-terminal end of Tg-bearing thyroid hormones.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of Tg and TPO
Native, poorly iodinated human Tg (3.1 iodine atoms per molecule) was purified from colloid goiter by performing slice extraction, sodium phosphate precipitation, and gel filtration on Bio-Gel A-5m (Bio-Rad Laboratories, Inc. Richmond, CA), as previously described (17). Human TPO was immunopurified by performing affinity chromatography (18) from thyroid patients undergoing partial thyroidectomy for Graves’ disease.

Hormone synthesis protocol
Tg (1 µM) dissolved in 0.05 M Tris-HCl buffer, pH 7.2, was incubated at 37 C with 20 µM KI in the presence of TPO (50 µg/ml) and the following H2O2-generating system: glucose (1 mg/ml) and glucose oxidase (2.5 µg/ml) (Roche Molecular Biochemicals, Mannheim, Germany). After a 30-min incubation period, the reaction was stopped by performing a freeze-drying step. This protocol was called Ox1 and corresponded to standard hormone synthesis conditions (19). Protocols called Ox2 to Ox5 were performed on similar lines, using 2-fold to 5-fold doses of KI, TPO, and H2O2 (glucose and glucose-oxidase), in comparison with those used in the Ox1 protocol. The Ox4 protocol was selected for further use.

Metal-catalyzed oxidation reaction
Tg (1 µM) dissolved in 0.05 M Tris-HCl buffer, pH 7.2, was incubated at 37 C in the presence of 100 µM metal salt (MgCl2, FeSO4, FeCl3, CuSO4, or CuCl) and 1 mM H2O2 or 15 mM ascorbate. After incubation periods of 30 min to 2 h, the reactions were stopped by performing a freeze-drying step. In experiments performed in the presence of 1 mM D-penicillamine or 1 mM EDTA, the chelator was added just before adding CuSO4 and H2O2. Competition experiments were performed by adding a 3-fold excess (300 µM) of ZnCl2 for 5 min before adding CuSO4 and H2O2. All the solutions described were freshly prepared.

Monoclonal antibodies (mAb)
We used a panel of ten Tg mAb directed against various antigenic determinants from six antigenic regions of the Tg molecule. Accordingly, the Tg mAb fell into six (I–VI) clusters of reactivity (20).

Patients’ sera
Positive Tg aAb titer sera from six adult patients with various autoimmune thyroid diseases were selected, based on the LUMItest anti-Tg (BRAHMS Diagnostica, Berlin, Germany). They were pooled, aliquoted, and stored at -20 C for further use.

SDS-PAGE and Western blotting
The peptides generated from Tg during hormone synthesis were analyzed by SDS-PAGE. The lyophilized samples (5 µg Tg/lane) were restored in 10 µl of 65 mM Tris-HCl buffer, pH 6.8, containing 2% SDS, 10% glycerol, and 0.025% Bromophenol blue, heated for 5-min with or without 2% ß-mercaptoethanol, to obtain either reducing or native conditions, and loaded onto a 10% acrylamide, 80 x 100 mm, 0.5-mm thick minigel. Peptides were directly electrotransferred onto a 0.45-µm Immobilon-P polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). Western blot experiments were carried out by incubating the blotted membrane with Tg mAb (50 µg) or human sera diluted 10-fold in 5 ml PBS, pH 7.3 containing 3% BSA, for 3 h at room temperature, with constant shaking after saturation of the membrane with PBS containing 5% nonfat dried milk. The membrane was then washed three times for 15-min in PBS. The second, antimouse or antihuman antibody labeled with horseradish peroxidase (Sigma, St. Louis, MO), was incubated for 2 h at room temperature, under shaking. After several additional washes, the blots were developed with 4-chloro 1-naphtol as the substrate.

Peptide labeling and TLC
Ox4 protocol was performed as above on Tg (33 µg) using trace amounts (800 µCi) of 125I-Na. The reaction was stopped by adding 20 µg Na2S2O5. The labeled peptides were separated by performing gel filtration through a Bio-Gel A-1.5m column (Bio-Rad Laboratories, Inc.) equilibrated with PBS, pH 7.3, containing 0.1% BSA and 0.02% NaN3. The fractions collected from the column were analyzed to determine their 125I-peptide content, by SDS-PAGE, under nonreducing conditions (5,000 cpm/fraction). The gel was then scanned with a phosphoimager apparatus (FujixBass1000, Fuji, Japan) equipped with a Tina 2.09 computer program (Raytest, Courbevoie, France). Before TLC, 5,000 cpm of 125I fractions to be analyzed were enzymatically digested by 20% (wt/wt) type XXI protease (Roche Molecular Biochemicals) for 48 h at 37 C, and then by 20% (wt/wt) leucine amino peptidase (Roche Molecular Biochemicals) for 24 h at 37 C. After being lyophilized, the fractions were restored by placing them in a 65% n-butanol, 20% acetic acid, 15% H2O chromatography buffer and washed twice by centrifuging them; the precipitates were discarded, and the supernatants were kept. Five microliters (1,000 cpm) of supernatant from selected fractions found to contain the labeled 40-kDa peptide were then loaded onto a silica-coated glass plate (Merck & Co., Inc., Darmstadt, Germany) and left to migrate with the chromatography buffer. After being dried, the plates were scanned with a phosphoimager apparatus as above.

Iodoamino acid analysis
Iodoamino acid analysis was carried out after digestion of Tg, with type XXI protease and leucine aminopeptidase, as described above, by performing reverse-phase HPLC, according to Baudry et al. (21).

Continuous elution electrophoresis
The peptide was separated with the Model 491 Prep Cell from Bio-Rad Laboratories, Inc. The procedure consisted of separating the proteins from the complex mixture, by SDS-PAGE, prepared in a 28-mm diameter gel tube. Briefly, 2 mg Tg were subjected to the Ox4 protocol, freeze-dried overnight, and restored with 2 ml of 65 mM Tris buffer (pH 6.8) containing 2% SDS, 10% glycerol, and 0.025% Bromophenol blue. This mixture was loaded onto a 10% acrylamide gel, and the separation was run with a Laemmli buffer, according to the manufacturer’s instructions. Fractions from the elution flow were collected, and the absorbance was continuously measured at 280 nm. The eluted 40-kDa peptide was then concentrated on a Centricon 10 (Amicon, Beverly, MA) and quantified by performing a microBCA assay (Pierce Chemical Co., Rockford, IL).

Sequencing of the 40-kDa peptide
The 40-kDa peptide was electrophoresed again by SDS-PAGE (minigel) under reducing conditions (2% ß-mercaptoethanol), and then it was electrotransferred onto a ProBlott membrane (PE Applied Biosystems, Foster City, CA). After coloring it with amidoblack, the 40-kDa band was cut, and this material was sent to the protein-sequencing facility at the Pasteur Institute (Paris, France), where the N-terminal amino acid sequencing of the peptide was carried out.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Fragmentation of Tg during hormone synthesis
Tg iodination and coupling were performed in vitro by TPO using iodide and the glucose-glucose oxidase system to produce H2O2. Tg fragments were revealed on Western blots using Tg mAb 11. As shown in Fig. 1Go, increasing TPO, KI, and H2O2 at a constant amount of Tg resulted in a parallel increase in Tg fragmentation and led to the production of various immunoreactive peptides, the smallest of which had a molecular mass of 40 kDa. Maximum Tg fragmentation was reached with a 4-fold increase in the initial dose of KI, TPO, and H2O2-generating system previously established for standard hormone synthesis protocols (19). Further increasing the dose of these reagents (Ox5) did not improve the Tg cleavages level.



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  		Figure 1. During in vitro hormone synthesis, Tg fragments were produced and revealed on Western blots by Tg mAb 11 after a SDS-PAGE separation step performed under nonreducing conditions. From left to right: calibration standards, nontreated Tg (5 µg), and Tg (5 µg) iodinated with gradually increasing amounts of iodine, TPO, and H2O2-generating system. Lanes 1–5 correspond to Tg treated by performing Ox1 to Ox5 protocols, as described in Materials and Methods. The lane containing maximum fragmentation (lane 4) is boxed. The molecular weights (MW) of the standards are indicated on the left. The arrow indicates the position of the 40-kDa peptide.

 
To confirm that the occurrence of the 40-kDa peptide paralleled the thyroid hormone synthesis, we analyzed the iodoamino acid contents of the Tg molecule before and after performing the various protocols. The results, given in Table 1Go, confirm that Tg was iodinated, because MIT and DIT were produced, and that Tg was also coupled, because T3 and T4 were produced. The total iodide gradually increased from the Ox1 to Ox5 protocol. On the other hand, the level of thyroid hormone synthesis paralleled the level of Tg fragmentation, showing the highest levels with the Ox4 protocol. Accordingly, the Ox4 protocol, which yielded both maximum thyroid hormone production and Tg fragmentation, was selected for further use. We also focused, in particular, on the production of the 40-kDa peptide, which we took to reflect the occurrence of an efficient Tg cleavage process.


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  		Table 1. Iodoamino acid composition of the Tg preparations

 
Tg is fragmented by an oxidative cleavage process
Substantial evidence has, by now, accumulated that free radicals are produced during thyroid hormone synthesis (13, 14, 15, 16). Because free radicals can induce oxidative damage, including protein fragmentation (6, 7, 8), we used the metal-catalyzed Fenton reaction to generate them and addressed the question as to whether the 40-kDa Tg peptide results from free radical-mediated Tg fragmentation. Various metallic cations were mixed with Tg in the presence of H2O2. When incubated for 30 min in the absence of added metal, Tg underwent incomplete fragmentation, yielding only large fragments (Fig. 2Go), as observed in the presence of Mg2+, a metal that is not involved in Fenton chemistry. On the other hand, Cu, whatever its valency, was able to efficiently cleave Tg and produce the 40-kDa peptide (Fig. 2Go), but it failed to produce the 40-kDa peptide under H2O2-free conditions (data not shown). Interestingly, using Tg mAb 11 in Western blot experiments, some of the Tg peptides obtained with this Cu/H2O2 protocol were found to be the same as those obtained with the Ox4 protocol. Surprisingly, adding Fe did not induce the 40-kDa peptide in the same way as with Cu. Because the prooxidative rate of iron is relatively slow (9) and it increases with ascorbate (6, 10, 11, 12), we extended the incubation time from 30 min to 2 h with FeCl3 plus 15 mM ascorbate. Under these conditions, the fragmentation process occurred, yielding various immunoreactive fragments, some of which were found to be similar to those obtained with the Cu/H2O2 protocol, especially the 40-kDa peptide (Fig. 3Go). Ferric ions are able to act in two catalytic ways with ascorbate: 1) O2 oxidates ascorbate, yielding H2O2; and 2) hydroxyl radicals are produced from H2O2 in a Fenton reaction (22). Similar results have been previously obtained using ascorbate on catalase (11, 12). In short, we observed that the oxidative cleavage of Tg was absolutely dependent on the metals mediating the Fenton chemistry (Fe, Cu).



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  		Figure 2. Metal-mediated oxidative fragmentation of Tg. Tg (1 µM) was mixed with various metals (100 µM) in the presence of H2O2 (1 mM). Tg peptides were detected on Western blots, as in Fig. 1Go. The molecular weight standards on the left are the same as those in Fig. 1Go. The arrow indicates the position of the 40-kDa peptide.

 


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  		Figure 3. Metal-mediated inhibition of oxidative fragmentation of Tg. Tg (1 µM) was mixed with 100 µM CuSO4 and 1 mM H2O2 (left panel) and with 100 µM FeSO4 and 15 mM ascorbate (right panel). The inhibitory effects of 1 mM D-penicillamine, 1 mM EDTA, and 300 µM ZnCl2 on metal-mediated Tg fragmentation (lanes + D-Pen, EDTA, and Zn, respectively) were detected by adding the chelators or the inhibitor before the metal and H2O2 or ascorbate. Tg peptides were detected on Western blots, as in Fig. 1Go. The molecular weight standards on the left are the same as those in Fig. 1Go. The arrow indicates the position of the 40-kDa peptide.

 
The effects of metal chelators on Tg fragmentation were therefore assessed to check whether the oxidative reaction yielding the 40-kDa peptide occurred at metal-binding sites on the target molecule. The production of the 40-kDa peptide by the Cu2+/H2O2 protocol was inhibited by D-penicillamine and by EDTA (Fig. 3Go); whereas the Fe3+/Asc-induced fragmentation was potentiated by D-penicillamine and, to a much lesser extent, by EDTA. Because D-penicillamine is a broader-specificity copper chelator, it is not possible to discuss its specific effects in the presence of Fe. On the other hand, the surprising result obtained with EDTA might be attributable to its enhancing influence on the rate of ascorbate autoxidation in the presence of iron (11). It emerges from these experiments that Cu and Fe behaved differently with chelators in mediating the oxidative Tg cleavages. Previous studies have also shown that the effects of chelators on the metal-catalyzed oxidation process depend both on the metal ion and the substrate involved; the multiple effects of chelators on the oxidation of lipids might be attributable to their influence on the redox-potential and on the accessibility of the metal-chelator complex to hydroperoxide (23). We further established that Zn, a metal that potentially competes with Cu or Fe for the same protein binding sites but is unable to mediate Fenton reactions (6, 8), did not affect the ability of Cu and Fe to cleave Tg (Fig. 3Go). The latter data suggested that metal binding to Tg was not required for the generation of the 40-kDa peptide to occur.

Mapping of epitopes located in the various fragments
The immunoreactivity of the Ox4 fragments separated by SDS-PAGE under nonreducing conditions was tested in Western blotting experiments using a panel of previously characterized Tg mAb. As shown in Fig. 4Go, all the mAb recognized high molecular weight Tg fragments, whereas only mAb 5, 6, 8, 10, and 11 also bound to the 40-kDa peptide and other Tg fragments produced using the Ox4 protocol. All these mAb belong to cluster I, except mAb 5 from cluster IV, which partly overlaps with cluster I. These mAb were recently found to cross-react with cloned human Tg Fab from a patient with Hashimoto’s thyroiditis; this finding confirmed that they were directed against an immunodominant region on the surface of the Tg molecule (24).



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  		Figure 4. Epitope mapping of the Tg fragments. Tg was fragmented by performing the Ox4 protocol, and the Tg peptides were detected on Ox4-treated and nontreated Tg by performing Western blotting with a panel of 10 Tg mAb. The Ox4 Tg peptides were recognized by Tg mAb from cluster I (i.e. mAb 6, 8, 10, and 11) and by mAb 5 from cluster IV, which partially overlaps with cluster I. The molecular weight standards on the left are the same as in Fig. 1Go. The arrow indicates the position of the 40-kDa peptide. Tg mAb nomenclature from (20 ): mAb 1, J7C9.3; mAb 2, J8B6.12; mAb 3, J8B89.5; mAb 5, J7C44.6; mAb 6, J7C73.7; mAb 7, J7C76.20; mAb 8, J8A53.9; mAb 9, J8B45.5; mAb 10, J8A32.13; mAb 11, J7B49.15.

 
To further confirm whether the 40-kDa peptide contained a B-cell autoepitope, Western blotting experiments were performed using a pool of patients’ Tg aAb-positive sera. The 40-kDa peptide and the other immunoreactive Tg peptides obtained with the Ox4 protocol showed up again (Fig. 5Go). The specificity of the Tg aAb binding for all the Tg peptides was confirmed by the loss of the Tg aAb reactivity, observed after previous absorption of the pooled sera with Tg but not TPO. Most of the B-cell epitopes targeted by these patient’s aAb are generally found to be conformational (25). We noted that all the Tg mAb and aAb reactivities were, in fact, abolished when Western blotting was performed under reducing conditions (data not shown).



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  		Figure 5. Immunoreactivity of the Tg peptides with pooled human patients’ sera. Tg was fragmented by performing the Ox4 protocol, and the Tg peptides were detected on Ox4-treated and nontreated Tg, by performing Western blotting with Tg aAb from pooled patients’ sera (left panel). Western blotting were also performed with pooled sera preincubated with a saturating amount of Tg and TPO (middle and right panels, respectively). The molecular weight standards on the left are the same as those in Fig. 1Go. The arrow indicates the position of the 40-kDa peptide.

 
Physiological relevance of the 40-kDa peptide
Because the 40-kDa peptide was produced during excess thyroid hormone synthesis, we investigated whether it was present in untreated Tg, which would mean that it is produced in vivo. Increasing the amount of loaded Tg in Western blotting experiments led to the occurrence of the 40-kDa peptide and other fragments with higher molecular weights, which were similar to those obtained with the Ox4 protocol (Fig. 6Go). Similar Tg fragments were detected in Tg preparations from two colloid goiters and from normal peritumoral thyroid tissue (data not shown).



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  		Figure 6. The Tg peptides were already present in the initial Tg preparation. Increasing the amount of nontreated Tg loaded onto the gel, before performing SDS-PAGE, from 5 to 20 µg, led to the occurrence of similar Tg peptides on the Western blots to those shown in Fig. 1Go, which was obtained upon loading 5 µg of Ox4-treated Tg. The molecular weight standards on the left are the same as those in Fig. 1Go. The arrow indicates the position of the 40-kDa peptide.

 
Thyroid hormone synthesis involves a coupling reaction in Tg, either between two DIT residues or between one MIT and one DIT residue, resulting in the synthesis of T4 and T3, respectively. The iodotyrosine residue responsible for the phenolic outer ring is known as the donor, whereas the DIT residue responsible for the inner ring is known as the acceptor, or the hormonogenic site (1). We therefore tested the 40-kDa peptide to establish whether it contained a hormonogenic site, by performing the Ox4 protocol using 125I as a tracer. Gel-filtration chromatography was carried out to separate the peptides. The fractions were then analyzed by SDS-PAGE, and some of them were found to contain a labeled 40-kDa peptide (Fig. 7AGo). When analyzed by TLC, four of these fractions containing increasing amounts of the radioactive 40-kDa peptide were also found to contain increasing amounts of thyroid hormones (Fig. 7BGo), which proves that T3 and/or T4 was formed at the hormonogenic site in the 40-kDa peptide during the Ox4 protocol.



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  		Figure 7. Thyroid hormones were formed on the 40-kDa peptide during the Ox4 protocol. Tg was fragmented by performing the Ox4 protocol using radiolabeled iodine. After gel-filtration chromatography, the labeled 40-kDa peptide was detected in fractions 49–55 by performing SDS-PAGE and scanning the radioactivity (A). Four of these fractions were analyzed by performing TLC to determine their iodoaminoacid contents (B). From fractions 48–55, note the progressive decrease in Tg and the parallel increase in both the 40-kDa peptide and the T3 + T4 content.

 
Localization of the 40-kDa peptide
Four main hormonogenic sites have been defined, so far (26). These were aligned with Tyr residue at positions 5, 1290, 2553, and 2746. Tyr 5, in the N-terminal domain of the molecule, is the main hormonogenic site, accounting for about 40% of the Tg hormone. Tyr 2553, at the C-terminal part, is the next most important site, containing about 25% of the hormone. Because mAb 2,3,7,9 which were previously found to be directed toward various epitopes in the N-terminal domain of Tg (27), did not bind to either the 40-kDa peptide or any other Ox4 peptides (see Fig. 4Go), it seemed likely that the hormonogenic site of the 40-kDa peptide might be located at the C-terminus of the molecule. To ascertain whether this was actually the case, the purified 40-kDa peptide from the Ox4 protocol was subjected to N-terminal amino acid sequencing. The six amino acid residues from the N terminus (namely: Ser, Gln, Leu, Phe, Arg, and Arg) perfectly matched all the residues in the entire molecule at positions 2384–2389. This sequence was present only at the C-terminal end of the Tg molecule (28) in the acetylcholinesterase (AChE)-like module (Fig. 8Go). In view of its estimated molecular mass of 40 kDa, it seemed likely that the peptide might extend to the carboxyl terminus of the molecule. The deduced Tg peptide was 365 amino acids long, and its calculated molecular mass was 40.4 kDa. It contained four Cys residues at conserved positions in the AChE molecule (29), a N-glycosylation site at position Asn 2562, and (more importantly) two hormonogenic sites at Tyr 2553 and Tyr 2746.



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  		Figure 8. The C-terminal amino acid sequence of Tg. Sequence homologies with AChE from amino acid 2191–2715 have been underlined. The N-terminal amino acid sequence of the purified 40-kDa peptide is given in gray. The deduced sequence of the entire peptide from amino acid 2384–2748 is boxed. This region contains four cysteine residues (black dots), one N-glycosylation site (asterisk), and two hormonogenic sites at tyrosine 2553 and 2746 (boxed Y).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The presence of immunoreactive Tg fragments, produced during hormone synthesis, was detected in the present study by performing native SDS-PAGE and Western blot experiments. Because Tg is known to be a thyroid aAg in which aAb can be measured in the sera of patients with autoimmune thyroid disease, we addressed the question as to the pathological significance of this fragmentation process in the B-cell autoimmune response. The immunoreactive peptide production was found to occur during the iodination and coupling of tyrosine residues, because we observed that, with the Ox1–4 protocols, both the immunoreactive peptide production and the level of hormone synthesis gradually increased. The fact that the T3 and T4 synthesis was lower under Ox5 conditions than with Ox4 is difficult to explain. However, partial deiodination of Tg during iodotyrosine coupling has been described (30, 31), and the oxidation of insoluble Tg has been found to result in iodide release (32).

Based on the results of electron-spin resonance analysis, the idea has been put forward that free radicals might be generated in the Tg iodination reaction (13). In addition, substantial evidence exists that the iodotyrosine coupling reaction may involve a radical mechanism (14, 15, 16). The putative involvement of free radicals in Tg cleavage was suggested by the fact that similar peptides, especially the 40-kDa peptide, were obtained when Fenton reactions were performed with Tg. The binding of the metal to a specific site on the Tg molecule seemed unnecessary for the oxidative fragmentation to occur. However, metal ions led to the generation of radical species, which resulted in selective cleavages occurring at special sites within the Tg. The reasons for this specificity are not entirely clear, but they may include the following: first, specific amino acid residues may be most susceptible to oxidation (8); and second, it may depend on the accessibility of some sites to the attacking species, the stability of the radicals generated on the protein, and the occurrence of radical transfer reactions (22). Tg cleavages may be conformation-dependent, because it has been established that nonlenticular proteins, such as Tg, are far more susceptible to fragmentation by copper and hydrogen peroxide than lenticular proteins ({alpha}-, ß-, and {gamma}-crystallins), the conformation of which gives a limited accessibility to fragmentation (33).

The 40-kDa peptide was recognized by all the Tg mAb previously found to be directed to the immunodominant region of the Tg molecule (24), and therefore it contains B-cell epitopes. This antigenic region seems to be human-specific, given that all the Tg epitopes recognized by these mAb were not found to be present in canine and porcine Tg (20). As was to be expected, the 40-kDa peptide was also recognized by sera from patients with autoimmune thyroid disease. Like most of the other B-cell epitopes, the epitopes present on this peptide were found to be susceptible to reducing conditions; and hence, they are conformational. The 40-kDa peptide had the lowest molecular weight of those peptides identified in the present Western blotting experiments. Because its immunoreactivity was maintained with the Tg mAb and the patients’ sera, we concluded that it originated from the other, larger, immunoreactive peptides that were simultaneously observed.

The 40-kDa peptide was found to bear T4- and T3-forming sites at Tyr 2553 and 2746, respectively. It was located at the C-terminal part of Tg from residue 2384 to residue 2748. Strikingly, this peptide was mapped in three fifths of the homologous AChE domain of Tg at position 2191–2715. This Tg domain contains amino acids 28% identical to those present in AChE from Torpedo californica (29) and includes 524 residues, involving more than 90% of the AChE molecule. Furthermore, the hydropathy profiles of Tg and AChE are all conserved, and the six cysteine residues are located at homologous positions (34, 35), which suggests that the two proteins may adopt similar folding patterns in their respective three-dimensional structures. However, the hormonogenic tyrosine residue of Tg at position 2553 is not conserved in AChE, nor is the active site of AChE conserved in Tg. The conservation of the overall structure, along with the differences between the functional residues, suggests that the region surrounding the hormone forming site is specifically involved in the B-cell response in autoimmune thyroid disease. It is worth noting that the C-terminal part of Tg has been found to be involved in the induction of experimental autoimmune thyroiditis (36, 37, 38, 39, 40, 41, 42). Some authors have previously described a critical region located around Tyr 2553, the existence of which further suggests that there may be a link between hormone synthesis and autoimmunity (39, 41).

There exists substantial evidence that a process of protein fragmentation, occurring before the processing in antigen-presenting cells, may protect cryptic epitopes from further enzymatic cleavage, thus making it possible for them to interact with MHC molecules (4). On the other hand, assuming that the proteolytic activities involved in antigen processing have little sequence specificity, local structural instabilities within the aAg might provide the basis for selective peptide presentation and, hence, for immunodominance (5). It has been speculated that the continuous oxidative stress exerted in the thyroid gland during TSH-stimulated or basal H2O2 production means that the gland must have a strong antioxidative defense system to protect it from oxidative damage. Accordingly, an increase in the free radical production, or conversely, a drop in the antioxidative defenses attributable, for instance, to a selenium deficiency (42), might increase the production and, thus, the capture of Tg fragments by the thyrocytes. This might, in turn, increase the level of delivery into the processing compartment of cryptic epitopes to a level above a critical threshold level required for T cell activation. Further studies on the T cells epitopes and on the protein structure and dynamics involved should help to establish how strongly local instabilities may control antigen processing and presentation.

It was recently established that the C-terminal part of rat Tg contains a heparin-binding consensus sequence (SRRLKRP) that is involved in megalin binding (43). A related sequence is present in the 40-kDa human Tg peptide, from amino acids 2473–2480 (ARALKRS). In view of its presence on the apical surface of thyroid cells, megalin, a member of the low-density lipoprotein (LDL) receptor family (44), has been thought to act as a receptor in Tg endocytosis (45). Interestingly, it was also recently reported that, as observed in the case of Tg in this study, LDL fragmentation was more efficiently mediated by Cu than by Fe (46). The similarity between the behavior of Tg and LDL in the Fenton chemistry suggests that these two molecules may have similar structural characteristics and, hence, that they may have similar functions in the endocytosis pathway. The 40-kDa peptide and its higher-molecular-weight counterparts were found to exist in trace amounts in native Tg preparations and to bear thyroid hormones, which suggests that they are produced in vivo during hormone synthesis. It is conceivable that, under unique internal and/or external circumstances, endocytosis signals may direct preprocessed Tg peptides toward antigen processing by thyrocytes, leading to a restricted, B-cell autoimmune response to Tg.


   Acknowledgments
 
We thank Dr. O. Chabaud and Dr. A. Giraud for their critical reading of this manuscript.

Received January 14, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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