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Experimental Studies |
Academic Medical Center, University of Amsterdam, Emma Children’s Hospital, Academic Medical Center, Pediatric Endocrinology (H.B., J.J.M.d.V.) and Department of Neurology (F.B.), Amsterdam
Address all correspondence and requests for reprints to: Hennie Bikker, Academic Medical Center, University of Amsterdam, Emma Children’s Hospital AMC, Experimental Pediatric Endocrinology H2-255, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands. E-mail: h.bikker{at}UVA.AMC.NL
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Generation of recombinant human TPO displaying immunological and enzymatic activity has been reported (3, 4, 5). Also, a secreted form of TPO lacking the transmembrane domain was stably expressed in Chinese hamster ovary (CHO) cells (6).
Defects in the function of TPO result in total iodide organification defects (TIODs). Complete absence of thyroid peroxidase activity has been described in patients with TIODs (7, 8, 9).
We were able to investigate thyroid tissue from three unrelated TIOD
patients (9, 10, 11), and TPO messenger RNA (mRNA) levels varied between
not detectable (9) and elevated (11). In thyroid tissue of the third
patient, the TPO-1 mRNA level was very low because of an early
termination codon, whereas alternatively spliced TPO-2 mRNA was present
(10). These three patients carried homozygous mutations in exons 2, 9,
and 10, respectively (Table 1
, mutation numbers 1, 4,
and 5).
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). All
patients but one showed mutations in both TPO alleles. We identified
eight different mutations in 25 alleles in which the TPO gene
cosegregated with the iodide organification defect (11). From the eight
mutations, it was evident that the three mutations causing frameshifts
and/or early termination signals (Table 1, mutation numbers 1, 2, and
5) could not produce active TPO. To verify whether the other five
mutations indicated by an asterisk (Table 1, mutation numbers 3, 4, and
6–8) were responsible for the organification defects, we investigated
the impact of these mutations on TPO expression and activity. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To obtain a full-length TPO complementary DNA (cDNA) probe, a
2.6-kilobase EcoRI/BglII fragment of TPO 3 cDNA
(14) was cloned in pAlter-1 (Promega, Madison, WI). After digestion
with SalI, a blunt site in the polylinker was created using
the Klenow fragment of DNA Polymerase I (15) followed by digestion with
SfiI. Thyroid cDNA generated by RT-PCR using oligo pd(N)6
(10) was amplified using primer 1F1 (5'-ACTCAGCAGTGCAGTTGGCT-3') and
8R1 (5'-ACGCGGAGCAGCCCTTCGGC-3') to generate the 5' part of the human
TPO cDNA. This cDNA fragment was treated with the Klenow fragment of
DNA polymerase I and digested with SfiI. The 875-bp fragment containing
the 5' part of TPO cDNA was cloned into pAlter-1 containing the 3' part
of TPO cDNA. Six clones were sequenced on the ABI Prism 377 DNA
sequencer using the dye terminator cycle sequencing ready reaction kit
(Perkin Elmer, Foster City, CA) for PCR errors in the first 900 bps.
Further sequencing revealed a polymorphism at position 2088 described
by Bikker et al.(11) and Kimura et al. (13).
Other single nucleotide substitutions (G 
A) at position 1169 (Gly
Asp) (not present in 25 unrelated individuals) and a (G A) at
position 1374 introducing a termination signal were also detected.
These mutations originated from the clone described by Libert et
al. (14). The original sequence (13) at 1374 was restored using
the Altered Sites II in vitro Mutagenesis System (Promega)
using primer mut8/A 1374 G (Table 2). The restored clone
pAlter/TPO 3.1 was used to generate the mutated TPO clones also by the
Altered Sites II system. Primer sequences are shown in Table 2. The
primer introducing the mutation at position 1447 was more complicated
than the other ones. To prevent severe internal hybridization of this
primer, two silent mutations were introduced creating an internal
EcoRV restriction site, which is useful for convenient
screening of mutants. The mutated cDNA clones were digested by
EcoRI/HindIII isolated from low gelling agarose.
Blunt ends were created by treatment with Klenow, and the fragments
were subcloned into the alkaline phosphatase-treated SmaI
site of pSemliki Forest Virus1 (SFV) (Gibco BRL, Life Technologies,
Gaithersburg, MD). The orientation of the fragments was tested by
SfiI/SpeI digestion, and the presence of the desired mutations was
confirmed by sequencing. All sequences generated by PCR were checked by
sequencing.
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pSFV/TPO3.1 and the clones containing the mutated sequences were linearized by digestion with SpeI. This served as a template for recombinant RNA synthesis performed according the conditions provided by the manufacturer (Gibco BRL, Life Technologies). RNA CAP Structure Analog was purchased from New England Biolabs (Beverly, MA), RNasin ribonuclease inhibitor from Promega, and SP6 RNA polymerase from Boehringer (Mannheim, Germany). Incubations were performed at 37 C for 1.5 h.
CHO-K1 cells (ATCC CCL 61) (American Type Culture Collection, Rockville, MD) were maintained in Ham’s F12 medium supplemented with 10% FCS, penicillin (50,000 IU/L), and streptomycin (10 mg/L). RNA (20 µl) and 0.8 x 107 cells in cytomix (16) were electroshocked (Easyject plus, Eurogentec, Seraing, Belgium) in a 2-mm cuvette. Electroporation was performed at 1500 microfarads and 260 V. Thereafter, the cells were plated in 10-cm dishes and for immunostaining in chamber slides (Nunc, Naperville, IL) and cultured for 24 h in the presence of 1 µg/mL hematin. For immunostaining, the cells were washed with PBS and fixed in 4% formalin in PBS for 10 min, followed by a 4-min incubation with chilled methanol (-20 C) and for 2 min in chilled acetone (-20 C). The cells were rehydrated in PBS and incubated for 15 min in PBS containing 10% FCS. Slides were incubated with polyclonal antihuman TPO for 20 h (4 C). Excess antibody was removed by washing with PBS, followed by a 1-h incubation of fluorescein isothiocyanate-conjugated goat antirabbit immunoglobulin (4 C). After washing with PBS, slides were mounted with Vectashield (Vector Labs., Burlingame, CA)
Peroxidase activity measurement
Cells were harvested with a rubber scraper in 3 mL PBS, and
protein concentration was determined on a 100-µl aliquot using the
Bio-Rad protein assay (BioRad, München, Germany). The cells were
then pelleted and subsequently suspended in 0.1% deoxycholate (0.2
mL/mg cellular protein) containing 1% aprotinin and incubated for 10
min at 4 C (4). The extract was microcentrifuged for 5 min, and the
supernatant removed to measure enzymatic activity using the guaiacol
and I3- assay (17). For the guaiacol assay, 50
µl of the supernatant was assayed in a final volume of 750 µl,
containing 35 mM guaiacol and 0.5 mM
H2O2 in 0.1 M Tris-HCl, pH 8.6. The
absorbance at 470 nm was followed and activity was expressed as
A · min-1 · mg protein-1.
For the I3- assay, 25 µl of the supernatant
was used in a final volume of 750 µl containing 0.1 M
potassium phosphate, pH 7.5, 50 mM potassium iodide, and
0.25 mM H2O2. The absorbance at 353
nm was followed, and activity was expressed as
A · min-1 · mg protein-1.
Spectrophotometric analysis was done on a Shimadzu UV-200.
Western blotting
Thirty micrograms of the deoxycholate extracted membrane protein fraction containing recombinant TPO and normal control TPO (microsomal fraction) (10) were electrophoresed on a 7.5% SDS-polyacrylamide gel under reducing conditions. The gel was blotted onto nitrocellulose using a Bio-Rad Mini Protean 2 system (Bio-Rad Labs., Richmond, CA), followed by incubation with polyclonal rabbit antihuman TPO antibody (gift of Dr. J.P. Banga). This antibody was raised against TPO isolated from thyroid tissue of a Graves’ disease patient. TPO protein was visualized using ECL Western blotting detection reagent (Amersham Life Science, Little Chalfont, U.K.).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human recombinant TPO was expressed in CHO-K1 cells using the
expression system based on the SFV replication. Cells were harvested
24, 48, and 72 h after transformation. The level of TPO expression
reached a maximum at 24 h (Fig. 1). Protein levels
declined after 24 h, and at 72 h after transfection almost no
TPO was observed (data not shown). Cells harvested 24 h after
transfection displayed TPO protein expression independent of hematin
addition. However, peroxidase activity was only observed when hematin
was added to the medium.
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and 2) was transfected and harvested
as described in Materials and Methods and analyzed by
Western blotting and immunofluorescence. All mutants expressed similar
amounts of TPO antigens of about 110 kDa on the Western blot (Fig. 1).
TPO expressed by mutant 2505+C (lane 7) was slightly shorter, caused by
the frameshift introduced by the single nucleotide insertion at
position 2505, which results in an altered stretch of 72 amino acids
and an early termination signal in exon 16 (at amino acid 879). The
predicted protein is about 6 kDa shorter.
The expression of wild-type and mutant recombinant TPO in the CHO-K1
cells was also visualized by immunofluorescence using a polyclonal
antibody to human TPO (Fig. 2). Cells expressing
wild-type TPO showed a clear fluorescein isothiocyanate signal
concentrated on the cell surface (Fig. 2A), whereas no fluorescence was
seen when pSFV1 RNA was transfected (Fig. 2C). The signal for all but
one mutant was most intense on the cell surface, comparable with the
image obtained in Fig. 2A. Only mutant 2505+C showed TPO antigens that
were not restricted to the cell surface (Fig. 2B). This was not
unexpected, because the membrane spanning part of exon 15, normally
present as a hydrophobic peak on the Kyte-Doolittle plot was, as result
of the frameshift, turned into a stretch of amino acids without
elevated hydrophobicity.
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To measure wild-type and mutant enzyme activity, both the
I3-- and guaiacol assays were carried out
(Table 3). Wild-type recombinant TPO showed enzymatic
activity in both assays. TPO generated by recombinant G 1858 A RNA
transfected CHO cells, displayed comparable enzyme activity. In the
other mutated TPO RNA transfected CHO cells, TPO activity was below
detection level. Measurement of TPO activity using the
I3- assay was only possible when 50
mM potassium iodide was present in the assay. These
reaction conditions resulted in a relatively high nonenzymatic reaction
rate, giving a high background.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Table 3). This is a strong
indication that the mutations are present at crucial positions of the
TPO gene, resulting in inactivated TPO.
The cysteine and histidine residues in the vicinity of the heme group
are located in highly conserved regions of TPO, lactoperoxidase (LPO),
myeloperoxidase (MPO), and eosinophil peroxidase (EPO), although the
overall amino acid content was identical for only about 45% (18, 19).
The mutations in exons 9 and 14 (Table 1) were located in these
conserved regions of the TPO gene. Aromatic substrate binding sites are
supposed to be essential for peroxidase activity. In LPO a tyrosine
binding site was demonstrated near the heme group (20). In MPO, a
hydrophobic pocket at the entrance of the distal heme cavity (composed
of Arg, His, and Gln) has been demonstrated. In this hydrophobic pocket
aromatic substrates such as Tyr or Phe can bind. Three Phe residues are
involved in the aromatic substrate binding site to the distal cave of
MPO (21). These amino acids are all in identical positions in TPO, LPO,
MPO, and EPO, confirming their essential function.
Kimura and Ikeda-Saito (22) suggested that in TPO the proximal His, which is linked to the ironcenter of the heme, is located at residue 407. However, x-ray crystallography at 3 Å of MPO clearly showed that His-336 is more likely to act as proximal His, because it was located within 4.5 Å of the heme (19). The corresponding His in TPO is located at position 494 in exon 9. The mutations A 1429 T and T 1447 G in exon 9, resulting in the gain (Ile 447 Phe) or loss (Tyr 453 Asp) of an aromatic ring in the neighborhood of the heme binding site, may therefore strongly interfere with heme binding, altering the hydrophobic pocket and/or influencing the electron transfer in the environment of the catalytic center.
Less is known of the structure at the carboxyl terminus of TPO. MPO,
EPO, and LPO all lack the corresponding part of the TPO protein
(encoded by exons 13–17) containing the membrane-spanning part
(encoded by exon 15). Inactivating mutations G 2486 A and 2505+C (Table 1) were both located in exon 14. Recombinant TPO containing the 2505+C
mutation, leading to TPO in which the membrane-spanning domain was
replaced by a novel stretch of amino acids, was at least partly
retained in the CHO cells as shown by immunostaining of the transfected
cells, suggesting that routing or protein folding is affected (Fig. 2B). Although a secreted form of TPO (missing the membrane-spanning
part) described by Foti et al. (6) was enzymatically active,
our mutant was inactive and retained in the cells, however, some
excretion could not be excluded. Because TPO truncated by an insertion
at -126 (2505+C) is inactive, and TPO truncated at -85 as described
by Foti et al. (6), was enzymatically active, the region
between -85 and -126 amino acids may be important for enzyme
activity.
The result of the Glu 799 Lys substitution (mutation G 2486 A), an
acidic amino acid (Glu) alteration to a basic (Lys) amino acid, is also
enzymatically inactive TPO. The cellular distribution of this mutant
TPO was comparable with wild-type recombinant TPO (Fig. 2A), indicating
that membrane binding was not affected.
Both mutations in exon 14 (G 2486 A and 2505+C) yielded enzymatically inactive TPO, suggesting that this region of the protein is important for proper folding and does not only serve as a hinge for insertion of the membrane.
Recombinant Gly 590 Ser TPO (G 1858 A) (Table 1, mutation number 6)
expressed enzymatic activity in the guaiacol and
I3- assay. This mutation was cosegregating
with TIOD in three unrelated families, homozygous in one family, and
heterozygous in two other unrelated TIOD families, in combination with
exon 9 mutations A 1429 T or T 1447 G. The possibility that mutation G
1858 A, which is present at the exon 10/intron 10 border, inactivates
the TPO genes of the patients by alternative splicing is a good
possibility. At least three other G A mutations at the 5' end of an
exon led to exon skipping, and the exon immediately preceding the
mutation was removed from the RNA transcript (23). In the case of the
mutation G 1858 A in TPO, exon 10 could be skipped, yielding inactive
TPO-2 (10). Unfortunately thyroid tissue was not available to test this
hypothesis. We cannot exclude the possibility that the G 1858 A
mutation is a linked polymorphism in a defective TPO gene carrying a
mutation we missed in the denaturing gradient gel electrophoresis
screen, however, we consider this highly unlikely.
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Acknowledgments |
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Footnotes |
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Received August 13, 1996.
Revised October 2, 1996.
Accepted October 10, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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