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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 11 3752-3757
Copyright © 1997 by The Endocrine Society

Original Studies

Human Thyroid Peroxidase (TPO) Isoforms, TPO-1 and TPO-2: Analysis of Protein Expression in Graves’ Thyroid Tissue1

A. Gardas, A. Lewartowska, B. J. Sutton, Z. Pasieka, A. M. McGregor and J. P. Banga

Medical Centre of Postgraduate Education (A.G., A.L.), Marymoncka 99, 01–813 Warsaw; Institute of Endocrinology (Z.P.), Lodz, Poland; and The Randall Institute (B.J.S.) and Department of Medicine (A.M.McG., J.P.B.), King’s College London, London, United Kingdom

Address all correspondence and requests for reprints to: A. Gardas, Medical Center of Postgraduate Education, Clinical Biochemistry Department, Marymoncka 99, Warsaw 01–813, Poland.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Thyroid peroxidase (TPO) is the key enzyme involved in the biosynthesis of thyroid hormones and is an important autoantigen in autoimmune thyroid disease. Different messenger RNA species coding for TPO are present in thyroid tissue, including the species coding for a 933-amino acid protein (termed TPO-1) and a second in which exon 10 is deleted and which is 57 residues shorter (termed TPO-2). However, it is not known whether the smaller, TPO-2 isoform is expressed as a protein in thyroid cells. In SDS-PAGE under reducing conditions, TPO appears in the thyroid microsome and purified protein preparations as a closely migrating double band of approximately 105 (larger form) and 100 kilodaltons (smaller form).

We investigated the presence of the isoform TPO-2 polypeptide in Graves’ thyroid tissue using rabbit antisera to three different synthetic peptides from exon 10 (specific for TPO-1) and a polyclonal rabbit and monoclonal anti-TPO antibody (both of which are specific for the two forms of TPO). The larger and smaller forms of TPO were purified by electroelution after gel electrophoresis of highly purified natural TPO from Graves’ thyroid microsomes. Both of the purified forms of TPO react with all three anti-exon 10 peptide antibodies, the polyclonal anti-TPO and the monoclonal antibody anti-TPO. This shows that both forms of TPO contain exon 10-encoded polypeptide of TPO-1. Interestingly, the proportion of the larger and smaller forms of TPO varied in different Graves’ thyroid microsome preparations. To investigate the presence of the smaller TPO-2 isoform in the purified natural TPO preparation, affinity depletion of TPO-1 using the anti-exon 10 peptide antibodies was carried out. The binding of anti-exon 10 peptide antibodies to the immunodepleted TPO-1 fraction was considerably diminished in comparison to binding of polyclonal anti-TPO, suggesting the presence of small amounts (<10%) of TPO-2 expressed as a protein in thyroid cells.

Our results extend previous observations by showing that the alternatively spliced form of TPO, in which exon 10 is excised, is expressed at low levels in Graves’ thyroid tissue. Furthermore, we confirm that both the larger and smaller forms of TPO observed on gel electrophoresis contain TPO-1, suggesting that the difference is caused by posttranslational modifications. The presence of small amounts of TPO-2 in Graves’ thyroid glands argues for its role in thyroid function, which remains to be clarified.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID peroxidase (TPO) is a major enzyme in the biosynthesis of thyroid hormone. It is an integral apical membrane glycoprotein of thyroid follicular cells that contains heme at its active site. TPO has catalytic activity for two substrates and is responsible for both the iodination and the coupling of tyrosine residues in thyroglobulin, which generates the thyroid hormones T3 and T4 (1). TPO is also the major target of the autoimmune response in autoimmune thyroid disease, in which high levels of antibodies to TPO are present, which are useful in the diagnosis of the disease (2).

Cloning of the human TPO complementary DNA revealed that it corresponds to a protein of 933 amino acids, with a large extracellular region of 845 residues, a short membrane-spanning region, and a cytoplasmic tail of 60 residues (3, 4, 5). The gene also revealed a hitherto unknown second form of TPO in which 57 amino acids (residues 533–589) coded by exon 10 are spliced out, yielding a smaller form of TPO of 876 residues (3, 6, 7). These two forms are known as TPO-1 (the larger) and TPO-2 (the smaller) form, respectively. Varying levels of messenger RNA (mRNA) for both forms are present in Graves’ and normal thyroid tissue (3, 8). In addition, other TPO mRNA species have been described in human thyroid tissues that also arise by alternative splicing (9, 10). Protein analysis of TPO, either by immunoblotting with specific antibodies or protein staining of purified TPO in SDS-PAGE under reducing conditions, has shown that TPO exists as a closely migrating double band of approximately 105 and 100 kilodaltons (kDa) (11, 12, 13, 14), although the reason for this has not been clear. However, expression of TPO-1 cDNA (both full-length or the extracellular, soluble region) in eukaryotic cells such as Chinese hamster ovary (CHO) cells (15, 16) or in insect cells (17–21 and Banga and Gardas, unpublished observations) also leads to the presence of a double TPO band, indicating that the difference arises as a result of differential glycosylation or proteolytic degradation.

Much interest has focused on TPO-2, because deletion of 57 amino acids could result in considerable changes in protein conformation and in an enzyme with altered kinetics and functional properties. In addition, TPO-2 could play a role in thyroid autoimmunity by the presence of unique autoreactive determinants. Interestingly, structural prediction studies have indicated that an essential, highly conserved distal histidine residue at position 486 is deleted in TPO-2, which could lead to an inactive enzyme (22, 23, 24). In contrast to TPO-1, attempts to express TPO-2 as a recombinant protein in CHO cells have been thwarted by the lack of detectable expression (16) or low levels of expression (25), making it difficult to conclusively ascertain whether TPO-2 is enzymatically active. Although TPO-1 expressed as recombinant protein (as the membrane-anchored or soluble-secreted form) in insect cells lacks enzymatic activity (17–19 and Banga and Gardas, unpublished observations), this can be partially or fully restored by the addition of precursors of heme biosynthesis such as hemin or {delta}-aminolevulinic acid respectively (20, 21). However, in a recent report on expression of TPO-2 in insect cells, such additives essential for heme biosynthesis were not added, resulting in an enzymatically inactive protein (26) and thus failing to resolve the issue of the functional activity of TPO-2.

In other studies using antisera specific for the polypeptide corresponding to exon 10, it was shown that both the upper and lower bands of TPO in thyroid microsome preparations from normal and Graves’ thyroid tissues contain the larger, TPO-1 isoform (27). We have extended these studies further by showing that TPO-2 is expressed in thyroid cells, albeit at low levels in comparison to the expression of the larger TPO-1 isoform.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Purification of TPO from Graves’ thyroid glands

Human thyroid tissue was obtained from Graves’ disease patients undergoing thyroidectomy. Thyroid microsomes were prepared by homogenization and differential centrifugation (28). In the routine preparation of TPO, thyroid tissue was pooled from 10 different glands for microsome preparation; in other preparations, microsomes were prepared separately from tissue derived from 8 different glands. Microsomes from the latter glands were prepared immediately after fresh thyroid tissue was received from the operating theater on ice, and the microsomes aliquoted and stored at -70 C. TPO was purified from sodium dexycholate solubilized thyroid microsomes by monoclonal antibody (mAb) affinity chromatography with mAb A4 (28). Purified TPO was stored in aliquots in 50 mM Tris-HCL, 0.15 M NaCl, pH 8.5, containing 0.1% sodium dexycholate at -70 C.

Rabbit antibody to synthetic peptides of TPO

The following peptides were synthesized by fluoren-9-ylmethoxycarbonyl chemistry (Fmoc) chemistry by the Multiple Sclerosis Society synthetic peptide laboratory, Oxford Polytechnic (Oxford, UK). A cysteine residue was added either at the N- or C-terminus of each peptide to facilitate coupling to carrier protein (see below), and all peptides were synthesized as carboxyl terminal amides. The sequence of the peptides, derived from the exon 10 region of TPO, were as follows: peptide 1, NH2-CDLASINLQRGRDHG-CONH2; peptide 2, NH2-LQVQDQLMNEELTERC-CONH2; and peptide 3, NH2-LSNSSTLDLASINLC-CONH2.

Peptides were conjugated to maleimide-activated keyhole limpet hemocyanin (KLH) (5 mg peptide/mg KLH), according to the manufacturers instructions (Boeringer Mannheim, Indianapolis, IN). The conjugated peptides were further purified by chromatography on Ultrogel AcA22 (LKB, Uppsala, Sweden) chromatography in PBS. Two New Zealand White rabbits were injected with each peptide using 1.0 mg peptide-KLH conjugate in 2 mL complete Freund’s adjuvant into several intramuscular sites. This was followed by three subsequent injections with the same dose every 2 weeks in incomplete Freund’s adjuvant. Two weeks after the last injection, serum was collected and tested for the presence of antibody to the respective peptide and to native TPO by enzyme-linked immunosorbent assay (ELISA).

(ELISA)

Microtiter plates (Maxisorb 96 well; Nunc, Denmark) were coated with 100 µL of a 2-µg/mL peptide or purified human TPO (0.5 µg/mL) in carbonate-bicarbonate buffer (pH 9.6) overnight at 4 C and then washed three times with PBST (PBS plus 0.1% vol/vol Tween 20). The plates were blocked in 0.5% (wt/vol) BSA in PBS for 1 h at room temperature and washed with PBS. The 105- and 100-kDa bands of TPO purified by electroelution from preparative gels were coated at different concentrations up to 1 µg/mL into individual wells of the microtiter plates, as described above. Serial dilutions of preimmune (control) and immune sera (1:100–1:500,000) diluted in PBST were applied (100 µL) to wells of peptide or TPO-coated plates and incubated for 1 h at room temperature. After washing three times in PBST, 100 µL 1:2000 diluted horseradish peroxidase conjugated antirabbit IgG (DAKO, Copenhagen, Denmark) was added to the wells and incubated as above. After washing, 100 µL solution of tetra-methyl-benzidine (0.1 mg/mL) in citric-phosphate buffer, pH 4.0 was added. After 15 min incubation, 50 µL 1 M sulphuric acid was added, and the optical density at 450 nm was measured. In the ELISA experiment, the electroeluted upper and lower fractions were coated on microtiter plates, and antisera applied at the the following dilution: all antipeptide antisera were used at 1:50000 dilution, and rabbit anti-whole human TPO serum were used at 1:30000.

Gel electrophoresis, immunoblotting, and electroelution

Thyroid microsomes were analyzed by SDS-PAGE under reducing conditions and immunoblotted onto Hybond C membrane (Amersham Life Sciences, Arlington Heights, IL) and blocked overnight in 0.5% BSA in saline, pH 7.4, containing 0.05% Tween 20 (PBST) (28). mAb A4 ascites (1:10000) or rabbit antipeptide antiserum (1:10000) diluted in 0.5% BSA in PBST was used for detection of TPO in the blots, and after washing three times, horseradish peroxidase conjugated antimouse or antirabbit (1:2000) diluted in the above buffer was used; after washing, bound antibody was detected by using 4-chloro-1-naphtol (Sigma Chemical Co., St. Louis, MO). Densitometry on blots was carried out on Ultrascan TM XL (Pharmacia Biotech, Piscataway, NJ).

For electroelution, purified TPO (500 µg) was electrophoresed in 10% acrylamide, 1.5-mm preparative gels under reducing conditions at 50 V for 12 h. Following electrophoresis, the gels were immersed overnight in 250 mM KCl to precipitate the SDS-bound proteins. Under these conditions, the doublet band of TPO was clearly visible. Horizontal strips of the gel containing the upper and lower bands of TPO were carefully excised. TPO was recovered from the gel slices using an electroelutor (Bio-Rad Labs., Hercules, CA) at 10 mA for 3 h. The eluted fractions were concentrated using Centricon columns (Amicon, W. R. Grace, Beverly, MA). SDS in the concentrated samples was reduced by washing the Centricon tube four times with PBS containing 0.05% Zwitergent 3–10 (Calbiochem, La Jolla, CA).

Immunoprecipitation experiments

IgG from immune sera were purified by diethylaminoethyl-blue (Bio-Rad) according to the manufacturer’s instructions. IgG fraction of antisera against antipeptide 2 (0.5 mg) and affinity-purified human TPO (0.1 mg) in saline buffer containing 0.5% Zwitergent 3–08 (Calbiochem) was incubated overnight at 4 C to allow immune complex formation. In control experiments, IgG from preimmune serum was used. This was followed by adding 200 µL protein A agarose gel (Pharmacia), incubating for 2 h at 4 C, and then briefly microfuged; to the supernatant a second portion (200 µL) of protein A agarose was added and incubated for 2 h as above. During both incubations, the tubes were mixed on a rotary shaker. The supernatant obtained after the second incubation with protein A agarose was coated on half of a 96-well microtiter plate; the remaining half of the same plate was coated with the affinity-purified TPO preparation used above for the immunoprecipitation experiment. Assessment of binding of antipeptide antibodies and rabbit polyclonal antihuman TPO antibodies to coated TPO before and after immunoprecipitation was measured by ELISA, as described above. The same sera dilutions were used as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Rabbit antipeptide antisera

Two rabbits were immmunized with each peptide in adjuvant and, as assessed by ELISA, all rabbits produced high-titer antiserum to the immunizing peptide. The binding of the sera to the peptides was highly specific, because there was no binding with the irrelevant peptide; in addition, all antipeptide sera also showed binding to native, purified TPO (not shown). The preimmune serum did not show any binding to any of the peptides or to purified TPO (not shown). All antipeptide sera reacted with TPO by immunoblotting using thyroid microsomes or purified TPO as starting material (not shown).

Purification of 105- and 100-kDa bands of TPO

Purification of the closely migrating bands of TPO, using affinity-purified TPO as starting material, was achieved by preparative gel electrophoresis and electroelution. Examination of the electroeluted TPO by re-electrophoresis and immunoblotting with the three antipeptide antisera and mAb A4 showed that both the 105- and 100-kDa bands of TPO reacted with all antibodies (Fig. 1Go). The two electroeluted bands of TPO were also examined for binding of antibodies to TPO in ELISA. Both the 105- and 100-kDa bands of TPO bound comparable levels of the antipeptide antibody or mAb A4 (Fig. 2Go). The results of immunoblotting and ELISA using purified 105- and 100-kDa bands of TPO show that TPO-1, detected by the three anti-TPO peptide antibodies, was also present in the lower 100-kDa bands, in addition to being present in the expected 105-kDa band; furthermore, both the bands bound similar amounts of the antipeptide antibody and mouse mAb to TPO, and the binding was no different from binding to affinity-purified TPO used for purification of the upper and lower bands.



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  		Figure 1. Immunoblotting on upper and lower isoforms of human TPO rerun on an SDS-PAGE. Two isoforms were isolated from antibody affinity-purified TPO by electroelution following separation in SDS-PAGE, as described in Materials and Methods. 1, Rabbit antipeptide 1 antibody; 2, rabbit antipeptide 2 antibody; 3, rabbit antipeptide 3 antibody; 4, murine mAb to TPO (mAb A4).

 


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  		Figure 2. Binding of antibodies to upper and lower isoforms of TPO by ELISA. Two isoforms were separated as described in legend to Fig. 1Go. Concentration of purified TPO isoform used for coating microtiter ELISA plates is shown on horizontal axis, and optical density reading is shown on vertical axis. •, Antipeptide 1 antibody binding to upper band TPO isoform; {circ}, antipeptide 1 antibody binding to lower band TPO isoform; {blacksquare}, mAb A4 binding to upper band TPO isoform; {square}, mAb A4 binding to lower band TPO isoform.

 
Examination of TPO doublet in different thyroid glands

Thyroid microsomes freshly prepared from eight different thyroid glands were examined for the 105- and 100-kDa bands of TPO by immunoblotting. Immunoblotting with mAb A4 showed that both of the closely migrating bands of TPO were clearly detectable in all the microsome preparations, but their relative amounts varied (Fig. 3Go and Table 1Go). Thus, some microsome preparations contained lower levels of the 100-kDa band (Fig. 3Go, lanes 1, 2, 3, 6, and 8), whereas other microsome preparations contained higher levels of the 100-kDa band (Fig. 3Go, lanes 4 and 7 and Table 1Go).



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  		Figure 3. Immunoblotting on thyroid microsome preparations from eight individual Graves’ thyroid tissue with murine mAb A4. All Graves’ thyroid microsomes (lanes 1–4 and 6–9) were prepared immediately after thyroidectomy, and 20 µg total protein loaded per lane. For comparison, affinity-purified TPO (5 µg protein) prepared from other pooled Graves’ glands is also shown (lane 5).

 

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  		Table 1. Proportion of upper and lower bands in eight individual human microsomal preparations measured by densitometry of a immunoblot with mAb to TPO (mAbA4)

 
Immunoprecipitation of TPO-1 by antipeptide antibodies

Immune complexes of TPO-1 with antipeptide 2 rabbit IgG were precipitated by protein A Sepharose. Binding of the three antipeptide sera and rabbit antihuman TPO serum to supernatant left after TPO-1 precipitation with rabbit antipeptide antibodies, was compared with antibody binding to TPO before immunoprecipitation (Fig. 4Go). The results indicate that the affinity-purified TPO preparation was depleted of TPO-1 by immunoprecipitation, and that the TPO-2 content in supernatant left after immunoprecipitation was relatively increased, to the extent that differences in antibody binding were clearly visible (Fig. 4Go). The experiment was repeated three times with two different TPO preparations, and the variation between assays was 6–10%.



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  		Figure 4. Binding of antibodies to upper and lower isoforms of TPO by ELISA following immunodepletion of TPO-1 with rabbit antipeptide 2 antibody. Concentration of purified TPO isoform used for coating microtiter ELISA plates is shown on horizontal axis, and optical density reading is shown on vertical axis. {blacksquare}, Rabbit IgG antihuman TPO before immunodepletion of TPO-1; {square}, rabbit IgG antihuman TPO after immunodepletion of TPO-1; •, rabbit IgG antipeptide 1 before immunodepletion of TPO-1. {circ}, rabbit IgG antipeptide 1 after immunodepletion of TPO-1; {blacktriangleup}, rabbit IgG antipeptide 2 before immunodepletion of TPO-1; {triangleup}, rabbit IgG antipeptide 2 after immunodepletion of TPO-1; {blacktriangledown}, rabbit IgG anti-peptide 3 before immunodepletion of TPO-1; {triangledown}, rabbit IgG anti-peptide 3 after immunodepletion of TPO-1.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Since the cloning and identification of the presence of the mRNA coding for the alternatively spliced variant of TPO lacking exon 10 (3), experimental evidence supporting the presence of the TPO-2 protein in thyroid cells has been lacking. We have taken the approach of generating high-titer antisera directed to specific amino acid sequences in exon 10 of the larger TPO-1 form of the protein. By immunoaffinity depletion of TPO-1 from highly purified preparations of TPO, we provide evidence for the presence of the smaller, TPO-2 form in thyroid tissue. In addition, using a different set of antibody reagents than those used previously (27), we confirm that both the upper and lower bands of TPO observed under reducing SDS-PAGE contain the larger TPO-1 isoform. It is likely that differences in posttranslational processing of TPO, such as glycosylation, acylation, farnezylation, or proteolytic cleavage is responsible for the presence of the double band of TPO. However, TPO purified from thyroid microsomes prepared rapidly following thyroidectomy also gave a double band, the relative intensities of which varied in different glands, indicating that glycosylation differences may be responsible for this effect. Other studies of the carbohydrate moieties of human TPO revealed that the sugar residues in the upper and lower bands were different, and that the the lower form might represent an incompletely processed from of TPO (29). This further supports the notion that differential glycosylation is responsible for the heterogeneity of human TPO (29).

Although it is not known whether TPO-2 is enzymatically active, alignments of the amino acids at the active site regions of related peroxidases suggests that the deletion of the highly conserved distal histidine residue at position 486 in TPO-2 may lead to a nonenzymatically active protein (22, 23, 24). Deletion of the 57 amino acid residues of exon 10 in TPO-2 could also result in significant alterations in the tertiary structure of the protein, resulting in the generation of neoantigenic determinants, which may play a role in autoimmunity.

The main reason for the difficulty in conclusive identification of TPO-2 has been lack of an antibody (antiserum or mAb) specific for this isoform. The specificity of such an antibody specific for TPO-2 must depend on recognizing the polypeptide sequence joining exon 9 and exon 11, after excision of the polypeptide sequence coded by exon 10 or a conformational determinant generated by the loss of this segment. As the binding site for an antibody is typically capable of recognizing 8–10 consecutive amino acid residues in a linear sequence (30), any antibody would have to recognize, say the last four or five amino acids on either end of the exon 9/11 boundary to be specific for TPO-2. No specific antibody probe has been generated for this isoform of TPO to date.

Although crystals of human TPO have been described (31), the structure of the molecule is not known. The structure of a related peroxidase, myeloperoxidase, has shown the molecule to be a disulphide linked dimer (32). Although it is not known whether native, membrane-bound TPO exists as a dimer (33), clearly the presence of TPO-2 protein in thyroid cells introduces an added complexity in understanding the structure/function relationship of this molecule and its role in autoimmune thyroid disease.


   Footnotes
 
1 This work was supported by The Wellcome Trust and the Medical Center of Postgraduate Education (CMKP) Grant S/5 (to A.G.). Back

Received February 20, 1997.

Revised April 30, 1997.

Accepted July 25, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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P. Hobby, A. Gardas, R. Radomski, A. M. McGregor, J. P. Banga, and B. J. Sutton
Identification of an Immunodominant Region Recognized by Human Autoantibodies in a Three-Dimensional Model of Thyroid Peroxidase
Endocrinology, June 1, 2000; 141(6): 2018 - 2026.
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