From the U555 INSERM, Faculté de Médecine, Université de la Méditerranée, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France
Received for publication, September 17, 2002, and in revised form, November 19, 2002
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ABSTRACT |
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The human thyroperoxidase (hTPO) gene is composed
of 17 exons. The longest complete cDNA sequence determined so far
contains a full-length hTPO (TPO1) encoding a 933-amino acid
polypeptide. Several mRNA species encoding for hTPO isoforms are
present in normal thyroid tissues, including TPO2 with exon 10 deleted
and TPOzanelli with exon 16 deleted. In the present study, we
established the existence of two new single-spliced transcripts, TPO4
and TPO5, lacking exons 14 and 8, respectively. Upon transfecting the
TPO4 cDNA into Chinese hamster ovary cells, it was observed that
TPO4 is able to reach the cell surface, is enzymatically active, and is
able to be recognized by a panel of 12 monoclonal antibodies directed
against hTPO, whereas TPO5 does not fold correctly and is unable to
reach the cell surface. In normal tissues, the expression of TPO4
mRNA was examined by performing quantitative reverse transcription
PCR. This deleted TPO mRNA amounted to 32 ± 11% of the total
TPO mRNAs. In the same tissues, the TPO2, TPOzanelli, and TPO5
amounted to 35 ± 12%, 36 ± 14%, and ~10%,
respectively. The sum of these four species (not including TPO1) was
more than 100%, possibly due to the presence of multispliced
mRNAs. This possibility was tested, and three new variants were
identified: TPO2/3, lacking exons 10 and 16, TPO2/4, lacking exons 10 and 14, and an unexpected variant, TPO6, corresponding to the deletion of exons 10, 12, 13, 14, and 16. In conclusion, these results indicate
the existence of five new transcripts. One of them, TPO4, codes for an
enzymatically active protein, whereas TPO5 is unable to fold correctly.
The functional significance of the other newly spliced mRNA
variants still remains to be elucidated, but these results might help
to explain the heterogeneity of the hTPO purified from the thyroid gland.
Thyroperoxidase
(TPO)1 is the key enzyme in
the process of thyroid hormone synthesis. The human TPO gene is
about 150 kbp in size, is located on chromosome 2, locus 2p25, and
consists of 17 exons and 16 introns (for a review, see Ref. 1). The
complete sequence of the human TPO coding region is known (2-4). The
full-length 3048-bp transcript (TPO1) codes for a protein consisting of
933 amino acids, which have a large extracellular domain, a
transmembrane domain consisting of 60 residues, and a short
intracytoplasmic tail consisting of 60 residues. Two other transcripts
have been described, namely TPO2, in which exon 10 is spliced out, and
TPOzanelli (TPO3), in which exon 16 is spliced out. TPO2 and TPO3 have
been found to occur in normal thyroid tissues as well as in Graves' tissues (2, 5, 6). These two forms code for proteins consisting of 876 and 929 residues, respectively. TPO2 is rapidly degraded after its
synthesis, does not reach the cell surface, and does not have any
enzymatic activity (7), whereas TPO3 is able to reach the cell surface
and shows enzymatic activity (8).
After being purified from the human thyroid gland, TPO is known to show
up in SDS-PAGE under reducing conditions as a double band of 105 and
110 kDa. The relative intensity of these bands varies from one gland to
another, and it has been established that TPO2 does not correspond to
one of these bands (9, 10), certainly because it is too rapidly
degraded after its synthesis (7). The difference in molecular weight
between TPO1 and TPO3 (four amino acids) does not explain the existence
of two bands, and glycosylation is not responsible for this
heterogeneity either (11). The presence of other isoforms and/or the
occurrence of endoproteolysis might explain the existence of these
different species.
The aim of the present study was to search for the presence of new TPO
transcripts that might help to explain this heterogeneity. Reverse
transcription was carried out from the total RNAs, and PCRs were
performed with various pairs of primers. Two new single spliced species
(TPO4 and TPO5) were identified. After quantification of the four
spliced isoforms TPO2, TPO3, TPO4, and TPO5 in normal thyroid tissues,
the possible existence of multispliced isoforms was hypothesized. The
existence of two isoforms with double splicing and one isoform with
five spliced exons was established.
RNA Isolation--
Frozen normal thyroid tissue was used in
these experiments. Tissues were homogenized and prepared using the
Promega kit (SV total RNA isolation system) according to the
manufacturer's instructions, and the preparation was then treated with
DNase. The RNA concentration was determined from the spectrophotometric
absorption at 260 nm, and the RNAs were aliquoted and stored in water
at Reverse Transcription--
Depending on the experiments, reverse
transcription was carried out using either 0.5 µg of
oligo(dT)12-18, 1 µg of random hexamers, or 2 pmol of
gene-specific primer (GSP). A 40-µl reverse transcription reaction
mixture containing hexamers, GSP, or oligo(dT)12-18, 0.9 µg of RNA, 0.25 mM dNTP mix, 10 mM
dithiothreitol, and 4 units of RNase recombinant inhibitor (Invitrogen)
was incubated at 42 °C for 2 min when GSP and
oligo(dT)12-18 were used, or at 25 °C for 10 min when
random hexamers were used. Superscript II RNase H PCR, Cloning, and Sequencing--
Reaction mixtures (50 µl)
consisted of 2 units of Fast Start Taq polymerase (Roche
Molecular Biochemicals), Taq buffer, 0.3 µM
oligonucleotide primers, 300 µM dNTP, 2 mM
MgCl2, and 5 µl of GC-rich solution (Roche Molecular
Biochemicals). A DNA sample was added to this mixture, and PCR was
performed with the following profile: 5 min at 95 °C for an initial
denaturation followed by 35 cycles of 30 s of denaturation at
94 °C, 30 s of annealing at temperatures depending on the
primers, and a 45-s extension at 72 °C, ending with a 5-min final
extension at 72 °C and a soak at 4 °C. The products
obtained were electrophoresed on agarose gel in TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8.3)
buffer and stained with ethidium bromide. When necessary, the
various PCR products were extracted from the gel using Qiaquick gel
extraction kit (Qiagen) according to the manufacturer's instructions,
and sequence analysis (Genomexpress, Grenoble France) or subcloning were then performed. Subcloning of the purified PCR products was performed using the TOPO TA cloning kit (Invitrogen). Size analysis of
the fragment inserted and sequence analysis of this insert in the
vector were performed with the various clones obtained.
Quantification of TPO mRNA Variants--
To study the
relative levels of expression of the various hTPO mRNA variants,
RT-PCR was performed as described in Ref. 12 with some modifications.
PCR reactions were performed as described above except that the volume
of the mixture was 100 µl, and 9-µl aliquots were taken from the
reaction after each consecutive cycle and loaded onto 2% agarose gel.
After staining the products with ethidium bromide, bands were detected,
and their intensity was quantified using an Image Station 440 (Kodak).
To correct the difference in nucleotide length, the density of the
smaller size band was multiplied by a factor corresponding to this
difference. To determine whether the detection of a variant was really
dependent on its initial proportion within the cDNA population, we
performed PCR amplifications with various quantities of the three
cloned variants, pcDNA3-TPO2, pcDNA3-TPO3, or pcDNA3-TPO4
in relation to that of pcDNA3-TPO1 (3:1, 1:1, and 1:3,
respectively). In all the cases that were tested, the ratio between the
isoforms after their amplification corresponded to their initial proportions.
Construction of pcDNA3-TPO4 and
pcDNA3-TPO5--
Full-length 3060-kb TPO1 cDNA kindly provided
by B. Rapoport was cloned into the HindIII and
XbaI sites of the eukaryotic transfer vector pcDNA3
(Invitrogen). The internal deletion of the cDNA
corresponding to exon 14 or exon 8 was performed using a single PCR
procedure (13, 14). The primer pair used was P-TPO4F or P-TPO5F (sense)
and P-TPO4R or P-TPO5R (antisense) (see Table
I). The PCR mixture contained 160 ng of
each primer, 50 ng of the pcDNA-TPO1, 200 µM dNTPs,
10% (v/v) dimethyl sulfoxide, 2.5 units of PfuTurbo DNA
polymerase (Stratagene), and the corresponding buffer in a total volume
of 50 µl. The reaction was performed under the following conditions:
denaturation at 95 °C for 30 s followed by 17 cycles of
denaturation at 95 °C for 30 s, annealing at 55 °C for 1 min, and an extension at 68 °C for 17 min. PCR products were
incubated with 10 units of DpnI for 2 h, and 5 µl of
this solution was then transformed into 50 µl of MAX efficiency DH5 CHO Cell Cultures and Transfection--
CHO cells (ECACC no.
85050302) were kept in Ham's F-12 medium supplemented with 10% FBS,
penicillin (100 IU/ml), and streptomycin (0.1 mg/ml). Cells were
transfected using LipofectAMINE (Invitrogen) with pcDNA3-TPO4 or
pcDNA3-TPO5. Cells were incubated in a saturated atmosphere (5%
CO2/95% air) at 37 °C. Stable transfectants were selected in the presence of geneticin (400 µg/ml) and subcloned using
limiting dilutions. Positive TPO4 and TPO5 expressing cell lines were
identified by performing Western blotting or immunoprecipitation after
[35S](Met + Cys) labeling
(EXPRE35S35S protein labeling mix, PerkinElmer
Life Sciences). A significant level of TPO1, TPO4, and TPO5 expression
was obtained by growing TPO1-CHO, TPO4-CHO, and TPO5-CHO cell lines as
described previously (15).
Metabolic Labeling of TPO--
Cells were incubated in cysteine-
and methionine-free MEM supplemented with 10% FBS, 10 mM
sodium butyrate, and 100 µCi/ml [35S](Met + Cys). The
incubation was carried out for 5, 16, or 48 h. In the pulse-chase
experiments, cells were incubated for 1 h in Cys- and Met-free MEM
supplemented with 10% dialyzed FBS and 10 mM sodium
butyrate. Cells were then pulsed for 30 min in the presence of 100 µCi/ml [35S](Met + Cys). After the pulse, the labeling
medium was removed, and the cell surface was washed three times with
PBS and then replaced by Ham's F-12 medium supplemented with 10% FBS,
5 mM Met, and 5 mM Cys. Chases were performed
for 30 min and 1, 3, 5, 16, and 24 h.
Cell Lysis and Immunoprecipitation--
After being
metabolically labeled, cells were washed twice with PBS, harvested on
ice by scraping them into 1 ml of PBS, and centrifuged at 200 × g for 7 min. Cell pellets were resuspended in 600 µl of
TPO extraction buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.3% sodium deoxycholate, and
protease inhibitors (CompleteTM, Roche Molecular
Biochemicals)), vortexed every 2 min for 20 min, and centrifuged at
10,000 × g for 5 min. Radiolabeled supernatants were
incubated for 2 h at room temperature with a pair of mAbs recognizing either a sequential region (mAb 47) or a conformational epitope (mAb 15) of the TPO molecule (16). These mAbs were previously complexed with protein A-Sepharose 4B (Zymed Laboratories
Inc.) by incubating them overnight at 4 °C. Immune complexes
were then retrieved by performing a brief centrifugation (10,000 × g, 10 s) and washed 4 times with 1 ml of TPO
extraction buffer and once with 1 ml of PBS. Immunoprecipitated TPO was
recovered from mAb-protein A-Sepharose 4B complexes by boiling the
complexes for 5 min in 80 µl of electrophoresis buffer (62 mM Tris-HCl, pH 6.8, 2% SDS, 5% 2-mercaptoethanol, and
5% glycerol), and it was then analyzed by SDS-PAGE (7.5%).
Protein-associated radioactivity was detected and quantified using
a phosphorimaging device (Fudjix BAS 1000).
TPO was also immunoprecipitated with a panel of mAbs directed against
various antigenic domains of the TPO1 molecule (16). In this
experiment, [35S](Met + Cys)-radiolabeled CHO-TPO cell
lysates were immunoprecipitated for 4 h at 25 °C with 50 µg
of each of the TPO-mAbs previously complexed with protein A-Sepharose 4B.
Cell Surface Biotinylation--
TPO1-, TPO4-, and TPO5-CHO
confluent monolayers were metabolically labeled for 18 h with 100 µCi/ml [35S](Met + Cys) in the presence of 10 mM sodium butyrate, and cell surfaces were biotinylated as
described previously (15). Cells were washed twice with PBS
supplemented with 1 mM CaCl2 and 1 mM MgCl2 and exposed to a 0.5 mg/ml EZ-link
sulfo NHS-SS-Biotin (Pierce) for 20 min at 4 °C. The cross-linker
was removed, and the procedure was repeated once. The biotin reagent
was quenched by incubating the preparation with 50 mM
NH4Cl in PBS for 10 min at 4 °C. Cells were washed with
PBS and harvested. To recover the immunoprecipitated antigens, the
complexes were supplemented with 10 µl of 10% SDS, boiled for 5 min,
diluted with 600 µl of TPO-extraction buffer, and centrifuged
(10,000 × g, 3 min). Supernatant containing the total
TPO was incubated for 2 h with avidin-agarose (Pierce).
Biotinylated surface TPO and intracellular TPO were separated by
centrifugation (10,000 × g, 10 s). The beads were washed four times with TPO-extraction buffer and once with PBS, resuspended in electrophoresis buffer, and boiled for 5 min. The supernatants were analyzed by SDS-PAGE (7.5%).
TPO4 Total Enzymatic Activity--
Microsomal fraction pellets,
prepared as described previously (7), were solubilized by resuspending
them in 15 mM Tris-HCl, pH 7.5, 150 mM NaCl,
1% Triton X-100, and 0.1 mM potassium iodide. Microsomes prepared from CHO cells transfected with pcDNA3 were used as a negative control. Microsomal fractions were centrifuged (10,000 × g, 2 min), and the supernatant was used for
the enzymatic assay. Extracts containing approximately the same amount
of protein were added to 1 ml of 40 mM guaiacol (Fluka
Chimie, St. Quentin-Fallavier, France) and 67 mM sodium
phosphate buffer, pH 7.5. The reaction was performed at room
temperature and initiated by adding H2O2 to
obtain a final concentration of 0.25 mM. Guaiacol oxidation was measured by absorbance at 470 nm and monitored
spectrophotometrically every 15 s for 3 min.
TPO4 Cell Surface Enzymatic Activity--
Cell surface enzymatic
activity was assayed as in Ref. 17, with slight modifications. TPO1-
and TPO4-CHO cells were incubated in Ham's F12 medium supplemented
with 10 mM sodium butyrate and 20 µM hemin
for 48 h. CHO cells transfected with pcDNA3 alone or TPO1-CHO
cells were used as a negative or positive control, respectively. The
medium was removed, and the cells were washed twice with PBS before
being incubated with BSA (5 mg/ml in PBS) and Na125I
(106 cpm/ml), with or without 2 mM
2-mercapto-1-methylimidazole, as the control medium. The reaction was
initiated by adding H2O2 to obtain a final
concentration of 0.5 mM, and cells were incubated for 20 min at room temperature. The medium was then transferred to cold
reaction tubes; the cell surface was washed out with 0.5 ml PBS, and
the wash was added to the medium. Each tube was filled with 1 ml of
ice-cold 20% (w/v) trichloroacetic acid supplemented with
10 Identification of the Deletion Variants of Exon 8 and 14--
A
strategy was developed to search for new isoforms of hTPO. It is known
that the various transcripts resulting from alternative splicing can
have different lengths of the poly(A) tail (18), and, consequently,
some isoforms cannot be detected by using oligo(dT)12-18 during the RT experiments. In the present study, after extracting the
total RNA from normal thyroid tissues, three different kind of primers
were thus used in the first strand cDNA synthesis reaction depending on the experiment, namely random hexamers, GSPs, and oligo(dT)12-18. Random hexamers lead to the production of short cDNA fragments and can therefore be used to avoid secondary structure problems or when the mRNA has a short poly(A) tail. In
the latter case, GSP can also be used.
In the first set of experiments, which was designed to detect any
alternative splicing between exons 9 and 17, reverse transcriptions were performed using random hexamers, and several different pairs of
primers were then used in the PCR experiments. Two PCR products were
obtained with one of these pairs located in exons 12 and 15, a band
with an apparent size of 499 bp corresponding to the predicted
full-sized mRNA of 493 bp and a smaller 375-bp band (Fig.
1). Sequence analysis of the latter band
showed that this was a 362-bp species from which exon 14 had been
specifically deleted. This new variant of hTPO was named TPO4
(GenBankTM accession number AY136822). Exon 14 codes for an
extracellular part of the protein near its transmembrane domain. This
part of the protein corresponds exactly to its EGF-like domain (3). Juxtaposing exons 13 and 15 did not induce any changes in the reading
frame and the corresponding full-length cDNA codes for a protein
consisting of 889 amino acids. To detect any alternative splicing
occurring between exons 2 and 9, reverse transcription was performed
using a GSP located in exon 9 (PE9R), and a PCR experiment was then
performed using a pair of primers located in exons 2 and 9 (PE2F and
PE9R). Two bands were obtained, one with an apparent size of 1330 bp
corresponding to the predicted full-size cDNA of 1311 bp, and a
smaller one with an apparent size of 785 bp (Fig.
2). This band was purified and sequenced and found to correspond to a 793-bp species from which exon 8 had been
deleted. This variant was named TPO5 (GenBankTM accession
number AF533528). Exon 8 codes for an extracellular part of the protein
located in its myeloperoxidase-like domain. Juxtaposing exons 7 and 9 did not lead to any changes in the open reading frame, and the
corresponding full-length protein codes for a protein of 760 amino
acids. Two hypothetically crucial residues are spliced out in TPO5,
namely Arg-396, which may participate in the catalytic mechanism
underlying the formation of compound I, and Glu-399, which may
covalently bind to the heme prosthetic group through ester linkage
(19). Two potential N-glycosylation sites (Asn-307 and
Asn-342) are also spliced out. Contrary to what occurs in TPO5, the
deleted exon in TPO4 mRNA codes for a whole domain (the EGF-like
domain) that is not included in the main catalytic part of the
molecule. It therefore seemed possible that this isoform might be
active. We therefore examined the properties of the proteins
corresponding to these two transcripts, focusing in particular on
TPO4.
Expression of TPO4 and of TPO5 in CHO Cell
Line--
pcDNA3-TPO4 and pcDNA3-TPO5 were constructed from
pcDNA3-TPO1 using a one stage PCR protocol compatible with the
deletion of exon 14 or exon 8. CHO cells were transfected with
pcDNA3-TPO4 or pcDNA3-TPO5, and several clones expressing
significant levels of TPO4 or TPO5 were then isolated. After a
metabolic labeling step using [35S](Met + Cys),
immunoprecipitation was performed using the pair mAb15 + mAb47, and
TPO4 and TPO5 showed up as bands with the predicted molecular weight on
the SDS-PAGE analysis (Fig. 3,
A and B).
Stability, Immunoreactivity, and Intracellular Trafficking of
TPO4--
To determine whether TPO4 has a modified three-dimensional
structure in comparison with TPO1, we used a panel of 12 mAbs directed against hTPO. All of these mAbs except one, mAb47, were directed against conformational epitopes. TPO1- and TPO4-CHO cells were labeled
for 16 h with [35S](Met + Cys), and, after the
extraction step, immunoprecipitations were performed with each of the
12 mAbs (Fig. 4, A and
B). TPO1 as well as TPO4 immunoreactivity was observed with
all the mAbs. However, mAbs 1, 24, and 59 showed a slight decrease in
immunoreactivity with TPO4 as compared with TPO1 (Fig. 4C).
This seems to indicate that most of the TPO4 fold correctly in
comparison with TPO1 and that none of the mAbs used were directed
against the EGF-like domain. To determine whether the small differences
observed affect the global half-life of the TPO4 synthesized in CHO
cells, we performed a pulse-chase experiment. Cells were pulsed for 30 min with [35S](Met + Cys) and then chased for various
times. Immunoprecipitation of TPO was performed, and samples were
analyzed by SDS-PAGE. Quantification of these bands (Fig.
5) showed that TPO4 has a shorter
half-life than TPO1, i.e. 5 versus 7.5 h.
All of these events may affect the intracellular trafficking of TPO4
and hence its level of expression at the cell surface. To check whether
TPO4 can reach the cell surface of the CHO cells, the cell surface
expression of the two isoforms was determined after labeling CHO cells
with [35S](Met + Cys) for 48 h and performing cell
surface biotinylation (Fig.
6A). Quantification of the
bands obtained showed that 25% of the TPO1 and only 12% of the TPO4
were present at the cell surface (Fig. 6B).
When expressed in CHO cells, TPO5 showed reactivity with mAb47 but not
with mAb15, which indicates that this isoform was not able to fold
correctly. In addition, this isoform is not able to reach the cell
surface (data not shown).
Enzymatic Activity of TPO4--
The following two methods were
used to detect whether TPO4 has any enzymatic activity. In the first
step, microsomes were prepared from TPO1-, TPO4-, and pcDNA3-CHO
cells, and, after protein extraction, guaiacol oxidation was performed
(Fig. 7A). As expected, TPO1
was enzymatically active, and three times less activity was detected
with TPO4. The difference in activity was due to the fact that TPO1-CHO
cells express three times more TPO than TPO4-CHO cells. Cell surface
enzymatic activity was also determined. The TPO1 present at the cell
surface was able to catalyze the iodination of BSA. Some activity was
also detected with TPO4 (Fig. 7B). This activity was seven
times lower than that obtained with TPO1; however, this difference is
consistent with the fact that the level of TPO4 expression is three
times lower in CHO cells and only half as high as that of steady state
TPO1 at the cell surface. These results show that TPO4 is enzymatically
active and that it may be involved in thyroid hormone synthesis.
Quantification of TPO mRNA Variants--
The level of
involvement of TPO4 in thyroid hormone synthesis depends on the level
of expression of this isoform in thyroid tissues. Quantitative RT-PCR
was therefore performed to measure the level of TPO4 mRNA
expression as compared with the other transcripts. RT was performed
using random hexamers, and PCR was performed with primers PE11F and
PE15R. The 493-bp and 362-bp products obtained were separated on 2%
agarose gel (Fig. 8A). After
quantification, a correction of 1.36 was applied because of the
difference in product size. The normalized band intensities were
plotted as a function of the number of cycles (Fig. 8B). The
semi-logarithmic plot of the product accumulation versus the
number of cycles (Fig. 8C) showed that the efficiencies of
the PCRs (as given by the slopes of the lines) were the same with both
of these species. Under these conditions, the relative difference
between the original abundance of these two samples was taken to be
2n, where n is the difference between
the number of cycles necessary to reach a threshold value (12) (Fig.
8B). The threshold value was the value in the exponential
part of the curve at which a statistically significant increase in
fluorescence was detected. RNAs from 14 different thyroid tissues were
used, and the results obtained showed that TPO4 mRNA amounted to
32 ± 11% of the total TPO mRNAs. In Fig. 8, the TPO1/TPO4
transcript ratio differs from that shown in Fig. 1, because the
mRNAs used were obtained from different thyroid glands. The level
of TPO2 mRNA expression in normal thyroid tissues has never been
exactly determined, and in the case of TPO3 it is only known that in
thyroid from Graves' disease its mRNA accounts for ~50% of the
total hTPO mRNA. We also quantified these two variant mRNAs by
RT-PCR. Primers located in exons 9 and 11 and exons 15 and 17 were used
to quantify mRNA with exons 10 (Fig.
9A) and 16 deleted (Fig.
9B), respectively. TPO2 amounted to 35 ± 12%, and
TPO3 amounted to 36 ± 14%. In the case of TPO5, we did not find
in this part of the molecule any pair of primers giving a similar
reaction efficiency between TPO1- and TPO5-cDNAs. However the
quantity of TPO5 mRNA probably accounts for ~10% (Fig. 2). The
sum of the percentages of these four species (not including TPO1) was
more than 100%, possibly due to the presence of multispliced
mRNAs.
RT-PCR, Cloning, and Identification of a Multispliced Variant of
hTPO--
As it is not possible to synthesize the full-length coding
region of the hTPO mRNA efficiently, we searched for the existence of multispliced mRNAs in its 3'-terminal part corresponding
to the exon 10, 12, and 16 deletions. These TPO variant cDNAs can be amplified using primers located in exons 9 (PE9Fb) and 17 (PE17Rb). RT was performed using oligo(dT)12-18, and after an
amplification step, PCR products were analyzed on 1% agarose gel.
Three main bands migrated with apparent sizes of 1416, 1283, and 1160 bp, and faint bands with smaller sizes were observed (Fig.
10). The expected size of TPO1 (no exon
deletion) was 1413 bp, which corresponded no doubt to the widest band
obtained. As we were looking for multispliced isoforms, the smaller
bands were purified, and the cDNAs were subcloned and sequenced.
Twenty-five clones were analyzed, and three multispliced variants were
identified, namely TPO2/3 corresponding to the deletion of exons 10 and
16 (GenBankTM accession number AF533530), TPO2/4
corresponding to the deletion of exons 10 and 14 (GenBankTM
accession number AF533531), and an unexpected variant, TPO6, corresponding to the deletion of exons 10, 12, 13, 14, and 16 (GenBankTM accession number AF533529). The latter clone
certainly corresponds to one of the faint bands with a smaller size,
which can be seen in Fig. 10.
It is also worth noting that when we analyzed the intensity of the
bands obtained after RT using oligo(dT)12-18 and 35 cycles
of PCR amplification using primers located in exons 9 and 17 (Fig. 10),
the ratio between TPO1 and the various other isoforms did not correlate
with the results obtained by quantitative RT-PCR, because the major
mRNA species seems to be the TPO1 mRNA. One of the differences
between these two experiments was that oligo(dT)12-18 was
used in this experiment, and random hexamers were used in the
quantitative RT-PCR. This confirms that the choice of RT procedure is
of great importance when analyzing the level of expression of these
various isoforms.
Some years ago, five variants of hTPO were characterized,
i.e. the full-length mRNA TPO1, which consists of 17 exons (2, 3, 4), the TPO2 with exon 10 deleted (2, 5), the TPO3 with
exon 16 deleted (6), TPO I consisting of exons 1-6 plus the 5'-end of
intron 6, and TPO II consisting of exons 1-5 plus an unidentified DNA
tract 558 bp in length (20). Studies performed by expressing the
recombinant variants showed that TPO1 and TPO3 are enzymatically active
and able to reach the cell surface of CHO cells (8, 21), whereas TPO2
is rapidly degraded after its synthesis and does not have any enzymatic
activity (7).
After being purified from human thyroid glands by affinity
chromatography using anti-TPO mAbs, hTPO shows up in SDS-PAGE as a
closely migrating double band with a lower molecular weight than that
of the TPO1 and TPO3 expressed in CHO
cells.2 This difference may
be due to post-transcriptional or post-translational modifications. In
the present study, we investigated the possible existence of new
variants of hTPO mRNAs, which might help to explain the multiple
forms of hTPO obtained.
Because it is difficult to detect alternative splicing using the
full-length coding region of hTPO, we synthesized smaller cDNA
parts of hTPO that together cover the entire mRNA. For the reverse
transcription procedure, we used random hexamers, GSP or
oligo(dT)12-18. The use of random hexamers or GSP makes it
possible to detect mRNA that either does not possess any tail or
has only a very short poly(A) tail. Actually it is well known that
various transcripts from one gene can differ in their poly(A) tail
length and, in some cases, can be detected with difficulty using
oligo(dT)12-18. Using this strategy, two new monospliced variants were identified, namely TPO4 lacking exon 14, and TPO5 lacking
exon 8. We estimated by quantitative RT-PCR that TPO with exon 14 deleted accounts for 32 ± 11% of the total hTPO mRNA. Using
the same technique, the level of expression of the known transcript
with exon 10 deleted was found to be 35 ± 12%, and that with
exon 16 deleted was 36 ± 14%. The TPO5 mRNA accounts for
~10% of the total hTPO mRNAs. As the sum of these various forms,
not including TPO1, was more than 100%, the existence of multispliced
transcripts was hypothesized. The search for multispliced species with
exons 10, 14, and 16 deleted showed the existence of two transcripts
with a double splicing, namely TPO2/3, and TPO2/4. Although no variant
with triple splicing TPO2/3/4 was observed, the presence of this
species cannot be ruled out. We did not search for multispliced species
with exon 8 deleted, but these transcripts certainly exist. Moreover,
an unexpected multispliced species, TPO6, was detected with splicing of
exons 10, 12, 13, 14, and 16. It is worth noting that we never detected
the presence of TPO mRNA with a spliced exon 12 or exon 13 alone.
We then established the existence of five new hTPO mRNAs with the
other known variants; with the multiple possibilities of the
multisplicing procedure, it seems quite likely that a greater number of
hTPO mRNA variants may be found to exist.
It is not possible to quantify the proportions in which the different
multispliced transcripts are present. However, it should be noted that
although TPO5 and TPO6 mRNA are not at all abundant, the splicing
of exons 10, 14, and 16 is a very common occurrence in normal thyroid
tissues, and TPO1 mRNA is perhaps not the main transcript present
in the thyroid gland. Northern blot data obtained in previous studies
by various groups showed the presence of mRNA species of ~4.0,
3.1, 2.9, 2.1, and 1.7 kb (for a review, see Ref. 1). The 4.0-kb
transcript has been thought to be an immature mRNA precursor,
whereas the 3.1-kb transcript corresponds to TPO1, and the mRNA
species that are 2.1 and 1.7 kb in size correspond to TPO I and TPO II,
respectively. The 2.9-kb species might correspond to TPO2, TPO3, and/or
TPO4. The 3.1-kb species has been obtained by all the authors, whereas
only Kimura et al. (2) have reported the existence of the
2.9-kb species. It is difficult to understand why this is so, but
determining the mRNA size accurately in Northern blots is known to
be rather tricky, and it is also possible that the 3.1- and 2.9-kb
species may have been poorly separated because of the small difference
in size. It is therefore difficult to obtain an exact idea of the
proportion of wild type/splice variant transcripts from these Northern
blot experiments.
As to whether the RT-PCR procedure constitutes a valid means of
analyzing the expression of these various isoforms, the use of
oligo(dT)12-18 and a random hexamer or GSP clearly yielded very different results (see Figs. 8, 9, and 10). This seems
to indicate that some or all of the spliced variants of hTPO have a
shorter poly(A) tail than that of TPO1 as was also previously found to
occur in the case of TPO I and II (20). Messenger RNAs leave the
nucleus with a >200-residue poly(A) tail and are deadenylated, yielding heterogenous polymers consisting of adenosine residues. The
cytoplasmic control of poly(A) length plays a key role in activating
and repressing gene expression. A search for possible different lengths
of poly(A) tails, depending on the isoform, is now under way.
Multiple species of MPO were also produced by performing alternative
splicing. Two transcripts with deletions of 57 and 171 bp, which were
generated by partially skipping exon 9 and completely skipping exon 10 (22), and two other transcripts with modifications in their 5'-ends and
with insertions of 96 bp in exon 2 and 82 bp in exon 4 have been
described (23). Except for the splicing of exon 10, which corresponds
to TPO2, the other alternative splicing shows no similarities between
the two peroxidases.
The splicing of one exon can affect the folding of the protein
variably, depending on its location. The nucleotide and amino acid
sequences corresponding to exons 3-11 in the hTPO gene show some
significant similarities with exons 2-11 of MPO (24). The next two
exons, 13 and 14, belong to the C4b and EGF gene families, respectively. Exons 15 and 16 code for the transmembrane part of the
protein and for its cytoplasmic tail. These polypeptide parts do not
shown any similarities with other proteins. As far as TPO5 is
concerned, exon 8 codes for a large (172 amino acids) and important
part of the protein located in the middle of the myeloperoxidase-like
domain, which includes Arg-396 and Glu-399. Arg396 corresponds to the
Arg-235 of MPO, which participates in the catalytic mechanism, Glu-399
corresponds to the Glu-242 in MPO and is a possible site of the
covalent heme binding process (19). In addition, two potential
N-glycosylation sites (Asn-307 and Asn-342) are present in
this part of the protein. Therefore, as can be expected when expressed
in CHO cells, TPO5 is unable to acquire a proper three-dimensional
structure and reach the cell surface. The other monospliced species,
TPO4, has exon 14 deleted. This exon codes for a whole domain located
in the extracellular part of the molecule showing similarities with EGF
and is certainly not involved in the function of the enzyme. We
expressed this protein in CHO cells and investigated the structural and
functional aspects of TPO4 with respect to TPO1 as was previously done
with TPO2 and TPO3 (7, 8). Part of the synthesized TPO4 is
able to fold correctly and reach the cell surface, but this isoform has
a shorter half-life than TPO1 (5 versus 7.5 h). Cell
surface biotinylation showed that only 12% of the protein is present
in the steady state at the cell surface as compared with 25% in the case of TPO1. Like TPO1 and TPO3, TPO4 is enzymatically active and can
therefore be expected to play a role in thyroid hormone synthesis. Some
of these results are in agreement with the results of a very recent
study by Guo et al. (25). To localize the immunodominant region of hTPO, these authors transfected COS-7 cells with TPO cDNA
from which the EGF-like domain had been deleted. They established that
this TPO (corresponding to TPO4) is able to reach the cell surface. In
addition, they established that this protein can be recognized by human
monoclonal autoantibodies. The results of this study confirm that the
three-dimensional structure of TPO4 is very similar to that of TPO1 and
definitively exclude the possibility that the juxtamembrane EGF-like
domain is part of the TPO immunodominant region.
Our study also showed the presence of multispliced species. Based on
the results obtained after expressing recombinant monospliced species,
the effects that the absence of the same exon from multispliced species
will have are predictable. For example, we have established that
the lack of exon 10 in TPO2 leads to a rapid degradation of this
protein (7); it can therefore be expected that all isoforms with exon
10 deleted will be rapidly degraded. By contrast, the deletion of exon
14 or 16 does not have such dramatic effects, and if TPO3/4 exists, it
will therefore certainly be active.
The existence of these various transcripts certainly explains, partly,
the heterogeneity of the TPO purified from human thyroid glands. The
double bands obtained in SDS-PAGE obviously consisted of numerous
smaller bands with very similar molecular weights.2 The
existence of these various species was certainly due to the presence of
various isoforms as well as to the existence of an endoproteolytic
process, because we have established that hTPO, when expressed in CHO
cells (26) and the rat thyroid cell line PC
Cl3,3 is cleaved by
proprotein convertases in its N-terminal part.
In conclusion, the present results show that the alternative splicing
form of TPO mRNAs gives rise to a great number of different transcripts. Two new monospliced isoforms (TPO4 and TPO5) and three
multispliced isoforms (TPO2/3, TPO2/4, and TPO6) have been shown here
to exist, but the number of multispliced isoforms is certainly greater.
It was established here that TPO4 is enzymatically active and able to
reach the cell surface. Further studies will now be required to
determine the role played by the other isoforms in the process of
thyroid hormone synthesis and the true chemical structure of the
thyroperoxidase present in normal human thyroid tissues.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C until further use. The absorption ratio (260/280) was
between 1.7 and 2.0 with all the preparations.
reverse
transcriptase (0.2 units) (Invitrogen) was then added, and the mixture
was incubated at 42 °C for 50 min. The reaction was inactivated by
heating the preparation at 70 °C for 15 min. The mixture then
treated with 2 units of RNase H (Escherichia coli) at
37 °C for 20 min.
(Invitrogen). Parts of this transformant were spread onto LB agar plates. Correct pcDNA3-TPO4 and -TPO5 clones were evidenced by sequencing. pcDNA3-TPO4 and -TPO5 pure plasmid DNA preparations were obtained with the Wizard Midipreps kit (Promega, Madison, WI).
Primer sequences for RT-PCR
4 M KI, and incubated for 20 min at 4 °C
before being centrifuged (2000 × g, 6 min). The
supernatant was discarded, and the acid-insoluble iodinated material
obtained was washed three times with 2 ml of 10% (w/v) trichloroacetic
acid. The radioactivity remaining in the pellet was counted.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (41K):
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Fig. 1.
Identification of exon 14 deletion.
Reverse-transcription was performed using random hexamers, and TPO
cDNAs were amplified by performing PCR as described under
"Experimental Procedures" using PE12F and PE15R with an annealing
temperature of 70 °C. PCR products were analyzed on a 2% agarose
gel. Lane 1, RT-PCR products obtained using RNA from a
normal thyroid tissue; lane 2, negative control; lane
3, DNA size marker.
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[in a new window]
Fig. 2.
Identification of exon 8 deletion.
Reverse-transcription was performed using the gene-specific primer
PE9R, and TPO cDNAs were amplified by performing PCR as described
under "Experimental Procedures" using PE2F and PE9R with an
annealing temperature of 55 °C. PCR products were analyzed on a 1%
agarose gel. Lane 1, DNA size marker; lanes 2 and
3, RT-PCR products obtained using RNA from two different
normal thyroid tissues.
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[in a new window]
Fig. 3.
Immunoprecipitation of
[35S](Met + Cys)-labeled TPO1, TPO4, and TPO5 from stably
transfected CHO cell lines. Stably transfected cell lines TPO1,
TPO4, and TPO5 were incubated for 16 h with
[35S](Met + Cys) before being lysed. TPO1 (A
and B, lanes 1), TPO4 (A, lane
2), and TPO5 (B, lane 2) were
immunoprecipitated with the pair mAb 15 + mAb 47. Samples were run on
SDS-PAGE (7.5%), and the band was detected by phosphorimaging.
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Fig. 4.
Analysis of TPO1 and TPO4 reactivity against
a panel of TPO-mAbs. TPO1- (A) and TPO4-CHO cells
(B) were incubated for 5 h in the presence of
[35S](Met + Cys) and lysed. TPOs were immunoprecipitated
using mAb-protein A-Sepharose complexes and analyzed by SDS-PAGE
(7.5%). The bands were detected and quantified by phosphorimaging
(C). This figure gives the mean value of three
different experiments.
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Fig. 5.
Rate of degradation of TPO1 and TPO4.
Cells were pulsed for 30 min in the presence of 100µCi/ml of
[35S](Met + Cys) in a Cys- and Met-free MEM supplemented
with 10% FBS. After the pulse step, the medium was removed and
replaced by Ham's F-12 medium supplemented with 5 mM Cys
and Met. At the times indicated, after the extraction step, TPO from
radiolabeled cell lysate was immunoprecipitated using the pair mAb 15 + mAb 47. Immunoprecipitated TPO1 (A) and TPO4 (B)
were analyzed by SDS-PAGE. Bands corresponding to TPO1 ( ) and TPO4
(
) were quantified by phosphorimaging. This figure gives the results
of an experiment that is representative of four identical experiments
performed.
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Fig. 6.
Cell surface expression of TPO1 and
TPO4. TPO1- and TPO4-CHO cells were metabolically labeled for
16 h with [35S](Met + Cys), and cell surface
biotinylation was then carried out as described under "Experimental
Procedures." The cells were lysed, and TPO was immunoprecipitated
with the couple mAb 15 + mAb 47 prior to reprecipitating the TPO
present at the cell surface by adding avidin-agarose. The tagged
fraction and only one-tenth of the supernatant corresponding to the
intracellular fraction were analyzed by SDS-PAGE (7.5%). A,
supernatants corresponding to the intracellular fractions are shown in
lane 1 (TPO1) and lane 3 (TPO4). Supernatants
corresponding to cell surface fractions are shown in lane 2 (TPO1) and lane 4 (TPO4). The bands were detected and
quantified by phosphorimaging. B, the percentages of
intracellular TPO (gray) and TPO expressed at the cell
surface (black) were calculated. This figure gives the mean
value of three different experiments.
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Fig. 7.
Enzymatic activity of TPO1 and TPO4 in
TPO-CHO cells. A, guaiacol oxidation activity of TPO1
and TPO4 in microsomal fractions obtained from TPO1- and TPO4-CHO
cells. Extracts from the same quantity of microsomes containing TPO1
( ) and TPO4 (
) and extracts from cells transfected with
pcDNA3 (
) were used to oxidize guaiacol. B, enzymatic
activity of TPO1 and TPO4 at the cell surface of transfected CHO cells.
TPO1- (1, 2), TPO4- (3, 4), or CHO cells transfected with pcDNA3
alone (5) were incubated with PBS containing BSA (5 mg/ml) and
Na125I (106 cpm). Negative controls were run in
which 2 mM mercapto-1-imidazole was added (2, 4). The
reaction was initiated by H2O2 to a final
concentration of 0.5 mM. Cells were incubated for 20 min at
room temperature, the supernatant was then discarded, and the
acid-insoluble material obtained was washed three times with 2 ml of
10% trichloroacetic acid. The radioactivity remaining in the pellet
was counted. The data are means from three different experiments.
Statistically significant differences versus pcDNA3-CHO
cells as follows: ***, p < 0.001; **,
p < 0.01 (by paired Student's t
test).
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Fig. 8.
Relative abundance of TPO mRNAs with exon
14 deleted. Reverse transcription was performed using random
hexamers, and TPO mRNAs were amplified by PCR as described under
"Experimental Procedures" using primers PE12F and PE15R with an
annealing temperature of 70 °C. Aliquots of 9 µl were taken from
the reaction mixture between cycles 22 and 31. These aliquots were
analyzed with 2% gel agarose, and the bands were detected
(A) and quantified (B) using a Kodak Image
Station 440. The amplification efficiency is given by the slope of the
line in a semi-logarithmic plot of the product accumulation
versus the number of cycles (C). , TPO1;
;
TPO4.
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Fig. 9.
Relative abundance of TPO mRNAs with
exons 10 or 16 deleted. The following actions were taken to
quantify the relative abundance of TPO mRNA. A, with
exon 10 deleted, reverse transcription was performed using random
hexamers, and TPO mRNAs were amplified by performing PCR as
described under "Experimental Procedures" using primers PE9Fa and
PE11R with an annealing temperature of 55 °C. B, with
exon 16 deleted, reverse transcription was performed using random
hexamers, and TPO mRNAs were amplified by performing PCR as
described under "Experimental Procedures" using primers PE15F and
PE17Ra with an annealing temperature of 70 °C. Aliquots of 9 µl
were taken from the reaction mixture at various cycles. These aliquots
were analyzed with 2% gel agarose, and the bands were detected and
quantified using a Kodak Image Station 440.
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Fig. 10.
Amplified fragments of TPO cDNAs
obtained by performing RT-PCR between exons 9 and 17. Reverse
transcription was performed using oligo(dT)12-18, and 35 cycles of PCR were then performed using primers PE9Fb and PE17Rb with
an annealing temperature of 61 °C. Amplification products were
analyzed on 1% agarose gel. Lane 1, DNA size marker;
lanes 2-5, RT-PCR products obtained using RNA from four
different normal thyroid tissues.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank J. Lanet for expert technical assistance, C. DeMicco for generous cooperation in providing us with thyroid tissues., B. Rapoport for providing full-length hTPO-cDNA, and J. Ruf for providing hTPO mAbs.
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FOOTNOTES |
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* This work was supported by INSERM (U555) and the Ligue Nationale Contre le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY136822, AF533528, AF533530, AF533531, and AF533529.
Supported during this work by the Association pour le
Développement des Recherches Médicales.
§ To whom correspondence should be addressed. Tel.: 33-4-91-32- 43-77; Fax: 33-4-91-79-65-11; E-mail: jean-louis.franc@medecine.univ-mrs.fr.
Published, JBC Papers in Press, November 25, 2002, DOI 10.1074/jbc.M209513200
2 V. Le Fourn, M. Ferrand, and J. L. Franc, unpublished results.
3 V. Le Fourn, M. Ferrand, and J. L. Franc, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: TPO, thyroperoxidase; hTPO, human TPO; GSP, gene-specific primer; RT, reverse transcription; CHO, Chinese hamster ovary; FBS, fetal bovine serum; MEM, minimum Eagle's medium; PBS, phosphate-buffered saline; mAb, monoclonal antibody; BSA, bovine serum albumin; EGF, epidermal growth factor; MPO, myeloperoxidase.
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