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Original Studies |
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.
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Abstract |
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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.
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Introduction |
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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 
-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.
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Materials and Methods |
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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.
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Results |
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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. 1
). 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. 2).
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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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. 3 and Table 1). Thus, some microsome preparations
contained lower levels of the 100-kDa band (Fig. 3, lanes 1, 2, 3, 6,
and 8), whereas other microsome preparations contained higher levels of
the 100-kDa band (Fig. 3, lanes 4 and 7 and Table 1).
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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. 4). 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. 4). The
experiment was repeated three times with two different TPO
preparations, and the variation between assays was 6–10%.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Footnotes |
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Received February 20, 1997.
Revised April 30, 1997.
Accepted July 25, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
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, antipeptide 1
antibody binding to lower band TPO isoform; 
, mAb A4 binding to
upper band TPO isoform; 
, mAb A4 binding to lower band TPO
isoform.
, rabbit IgG
antipeptide 2 before immunodepletion of TPO-1; 
, rabbit IgG
antipeptide 2 after immunodepletion of TPO-1; 
, rabbit IgG
anti-peptide 3 before immunodepletion of TPO-1; 
, rabbit IgG
anti-peptide 3 after immunodepletion of TPO-1.